Sunday 13 July Industrial Biotechnology from Fundamentals to Practice (Acib Session)

New Biotechnology · Volume 31S · July 2014 SUNDAY 13 JULY INDUSTRIAL BIOTECHNOLOGY FROM FUNDAMENTALS TO PRACTICE (ACIB SESSION)

Orals

Sunday 13 July References Industrial biotechnology from fundamentals [1].Tsuji Y, Fujihara T. Chem Commun 2012;48:2365. [2].Glueck SM, Gümüs S, Fabian WMF, Faber K. Chem Soc Rev to practice (ACIB Session) 2010;39:313. [3].Matsui T, Yoshida T, Yoshimura T, Nagasawa T. Appl Microbiol Bio- ACIB-1 technol 2006;73:95. [4].Lindsey AS, Jeskey H. Chem Rev 1957;57:583. Two promising biocatalytic tools: regioselective car- [5].Wuensch C, Gross J, Steinkellner G, Lyskowski A, Gruber K, Glueck boxylation of aromatics and asymmetric hydration of SM, Faber K. RSC Adv 2014;4:9673. alkenes [6].Wuensch C, Gross J, Steinkellner G, Gruber K, Glueck SM, Faber K. Angew Chem Int Ed 2013;52:2293. Silvia M. Glueck 1,∗ , Christiane Wuensch 1, Tamara Reiter 1, http://dx.doi.org/10.1016/j.nbt.2014.05.1615 Johannes Gross 1, Georg Steinkellner 1, Andrzej Lyskowski 1, Karl Gruber 2, Kurt Faber 2

1 ACIB GmbH c/o University of Graz, Department of Chemistry, Austria ACIB-2 2 University of Graz, Austria Unconventional substrates for enzymatic reduction: car- Due to the growing demand for alternative carbon sources, boxylates and nitriles the development of CO2-ﬁxation strategies for the production M. Winkler 1,∗ K. Napora-Wijata 1 B. Wilding 1 N. Klempier 2 of valuable chemicals is a current challenge in synthetic organic , , , chemistry [1]. The enzymatic carboxylation of aromatic com- 1 ACIB GmbH, Petersgasse 14/III, 8010 Graz, Austria 2 pounds [2,3] catalyzed by various decarboxylases represents a Institute of Organic Chemistry, Graz University of Technology, Stremayrgasse promising ‘green’ alternative to the classic Kolbe-Schmitt reaction 9, 8010 Graz, Austria [4] which requires harsh reaction conditions. Depending on the type of enzyme, the carboxylation proceeded Keywords: Biocatalysis; Nitrile Reductase; Carboxylate reductase; in a highly regio-complementary fashion: Benzoic acid decarboxy- Reduction; Enzyme lases selectively catalyzed the ortho-carboxylation of phenolic substrates (conv. up to 80%) whereas phenolic acid decarboxylases The reduction of carbonyls by enzymes has been extensively exclusively catalyzed the ␤-carboxylation of styrenes (conv. up to studied and is now widely applied on industrial scale. In com- 40%) [5]. parison, enzyme catalyzed carboxylic acid/carboxylate and nitrile During these studies a promiscuous ‘hydratase-activity’ of phe- reduction are relatively young ﬁelds. Their full potential is cur- nolic acid decarboxylases was discovered, which leads to the rently being explored. The chemical reduction of nitriles and stereoselective hydration of hydroxystyrenes to yield the corre- carboxylic acids requires strong reducing agents due the low reac- sponding sec-alcohols with high conversion (up to 82%) and e.e.’s tivity of these moieties. Consequently, mild alternatives would be of up to 71% [6]. a valuable addition to the current toolbox of biocatalysis. The Acknowledgements: This work has been supported by the products of nitrile reduction are primary amines and we have Austrian BMWFJ, BMVIT, SFG, Standortagentur Tirol and ZIT recently expressed nitrile reductase enzymes (NReds), in order to through the Austrian FFG-COMET-Funding Program. apply these enzymes as biocatalysts. The substrate scopes of wild type and mutant NReds as well as their biochemical characteristics were studied [1]. The reaction mechanism of NReds involves the

1871-6784/$ — see front matter www.elsevier.com/locate/nbt S1 SUNDAY 13 JULY INDUSTRIAL BIOTECHNOLOGY FROM FUNDAMENTALS TO PRACTICE (ACIB SESSION) New Biotechnology · Volume 31S · July 2014

Acknowledgement: This project is financed by the European Union’s Seventh Framework Programme FP7/2007-2013 under grant agreement no. 289646 (Kyrobio). References [1].Winkler M, et al. Comprehensive chirality. Synthetic methods VI, vol. 7. Amsterdam: Elsevier; 2012. p. 350–71. [2].Hajnal I, et al. FEBS J 2013;280:5815–28. Figure 1 Whole cell biotransformation approach towards 3- http://dx.doi.org/10.1016/j.nbt.2014.05.1617 hydroxytyrosol – an antioxidant from olives. reaction of the substrate with an active site thiol and subsequent ACIB-4 NADPH mediated reduction of the intermediate [2], which is simi- Enzyme responsive polymers lar to the mechanism proposed for carboxylate reductase enzymes (CARs) [3]. The latter enzyme was in focus of the development of a Alexandra Rollett 1,∗ , Andrea Heinzle 2, Konstantin Schneider 2, whole cell system (Figure 1) for the production of the antioxidant Doris Schiffer 2, Gregor Tegl 3, Eva Sigl 2, Georg Guebitz 1 3-hydroxytyrosol [4]. 1 BOKU-Vienna/ACIB GmbH, Austria 2 ACIB-Austrian Centre of Industrial Biotechnology GmbH, Austria References 3 BOKU-Vienna, Austria [1].(a) Wilding B, et al. Adv Syn Catal 2012;354(11–12):2191–8; (b) Wilding B, et al. Chem Eur J 2013;19(22):7007–12. Smart polymers can change their properties depending on [2].Chikwana VM, et al. J Biol Chem 2012;387:30560–70. certain stimuli like pH, temperature, light or electrical fields. Espe- [3].Venkitasubramanian P, et al. J Biol Chem 2007;282:478–85. cially in biomedical materials, but also in technical applications, [4].Napora-Wijata K, et al. ChemCatChem, Patent application pending [in print]. like the packaging industry, enzymes are useful biomarkers for sensoring purposes. Here we present different strategies towards http://dx.doi.org/10.1016/j.nbt.2014.05.1616 enzyme responsive polymer systems for the detection of microbial contaminations in distinctive fields. Enzyme responsive hydrogel based systems were constructed ACIB-3 using methacrylated biopolymers like polygalacturonic acid [1], alginic acid [2], carboxymethylcellulose [3], peptidoglycan, Engineering of cupin hydroxynitrile lyases gelatin, collagen, alginate and agarose [4–6] while both stabil- ,∗ Kerstin Steiner 1 , Romana Wiedner 1, Bettina Kothbauer 1, Man- ity and sensitivity were tuned via the degree of UV crosslinking. dana Gruber-Khadjawi 1, Helmut Schwab 2 Theses systems were successfully used to detect contaminating 1 ACIB GmbH, Austria microorganisms based on their extracellular enzymes, and infec- 2 TU Graz, Austria tion of wounds based on enzymes from the human immune system. The latter included myeloperoxidase, cathepsin G, elas- Enantiopure cyanohydrins serve as versatile building blocks for tase and lysozyme, which were found to show an elevated activity a broad range of chemical and enzymatic reactions resulting in in infected wounds. highly valuable products for many applications. Hydroxynitrile References lyases comprise a diverse group of enzymes which catalyze the reversible cleavage of cyanohydrins to carbonyl compounds and [1].Schneider, et al. N Biotechnol 2012;29:502–9. [2].Schneider, et al. Enzyme Microbial Technol 2011;48:312–8. hydrogen cyanide [1]. For a long time HNLs were only known to [3].Schneider, et al. Process Biochem 2012;47:305–11. exist in plants, but recently (R)-selective HNL activity has been [4].Hasmann, et al. Exp Dermatol 2011;20:508–13. identified in several bacterial proteins [2]. The structure of one [5].Heinzle, et al. Wound Repair Regen 2013;21:482–9. target protein from Granulicella tundricola (GtHNL) was solved and [6].Hasmann, et al. Diag Microbiol Infect Dis 2011;71:12–23. showed high similarity to proteins of the cupin superfamily, a fold, http://dx.doi.org/10.1016/j.nbt.2014.05.1618 which has not been reported for HNLs before. Cupins are ubiquitous small beta-barrel proteins and ideal candidates for application in industrial processes and as scaffold for enzyme engineering as they are easily expressed as soluble proteins in exceptionally high yield (> 50% of total protein) in E. coli. A detailed biochemical characterisation of GtHNL revealed that the enzyme is metal-dependent. Several amino acids were identified, which are important for activity. The initial specific activity of 1.74 U/mg at pH 6 with (R)-mandelonitrile, and the 80% conversion of benzal- dehyde to (R)-mandelonitrile with 90% enantiomeric excess were significantly improved by applying random and rational protein engineering approaches.

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ACIB-5 ACIB-6

Esterases from Clostridium are involved in anaerobic Designing robust Saccharomyces cerevisiae strains degradation of synthetic polyester against stresses encountered during bioethanol fermentations from lignocellulosic biomass Veronika Perz 1,∗ , Veronika Perz 2, Armin Baumschlager 2, Klaus Bleymaier 2, Andrzej Łyskowski 2, Altijana Hromic 2, Karl Gruber 3, Vinod Kumar 1,∗ , Darren Greetham 1, Tithira Wimalasena 2 4 4 4 2 Carsten Sinkel , Ulf Küper , Melanie Bonnekessel , Doris Ribitsch , 1 The University of Nottingham, United Kingdom 5 Georg Guebitz 2 Kingston Research Limited (BP and DuPont JV), United Kingdom 1 ACIB GmbH, Austria 2 ACIB GmbH, Petersgasse 14, 8010 Graz, Austria During the pre-treatment of lignocellulosic biomass inhibitory 3 Institute of Molecular Biosciences, University of Graz, Austria compounds are released which can exert adverse effects on cellular 4 BASF SE, Carl-Bosch-Straße 38, 67056 Ludwigshafen, Germany growth, metabolism and ethanol production. In addition, osmotic 5 Institute of Environmental Biotechnology, University of Natural Resources and Life Sciences, Austria stress caused by the high concentrations of available sugars and end-stage ethanol toxicity reduce overall rates of bioethanol fer- The anaerobic degradation of synthetic aliphatic–aromatic mentation. In previous studies, F1 hybrid segregants derived from polyesters used in food packaging is of great importance during clean lineage Saccharomyces cerevisiae strains were assessed for anaerobic digestion. Several studies have proven the biodegrad- tolerance to a range of stresses encountered during industrial ability of PBAT (poly(butylene adipate-co-butylene terephthalate)) bioethanol fermentations. Using a systems biology approach (QTL- under aerobic conditions while there is only little information on Quantitative Trait Locus) chromosomal loci conferring resistance anaerobic degradation. against weak acid and osmotic stress were identified, and within In this study, imaging analysis (CLSM, SEM) and quantifica- those loci genes COX20 (acetic acid) and RCK2 (osmotic) were tion of degradation products indicated anaerobic hydrolysis of determined as important for stress response. PBAT in biogas sludge. However, the detected hydrolysis rates In the present study, strains in which COX20 or RCK2 have are still too low for efficient PBAT degradation in industrial bio- either been deleted or inserted into on tetracycline induced vec- gas plants. Consequently, hydrolysis of PBAT by enzymes from tors. Phenotypic microarray assessment (Biolog, US) of these different anaerobic organisms (Clostridium species) was investi- strains under acetic acid, formic acid, furfural, HMF, vanillin and gated. Therefore, various hydrolases from these organisms were sorbitol stress have been determined and compared against appro- successfully heterologously expressed in E. coli BL21-Gold(DE3). priate controls. Results have highlighted that presence of COX20 The kinetic parameters on the standard substrate p-nitrophenyl improves tolerance to weak acids while RCK2 gene conferred resis- acetate were determined and revealed high activity of up to 700 tance to osmotic stress. The presence of either gene also enhanced the tolerance against furanic compounds such as furfural and HMF. U/mg (vmax). Analysis of the crystal structure of one esterase from C. botulinum disclosed the presence of a Zn2+ metal ion that lies http://dx.doi.org/10.1016/j.nbt.2014.05.1620 deep beneath the protein surface. The degradation of synthesized oligomeric and polymeric model substrates was studied in order to get a deeper insight into ACIB-7 the reaction mechanisms of these new hydrolases. Esterases from C. hathewayi and C. botulinum were indeed Systems biology of Pichia pastoris able to hydrolyze aliphatic-aromatic polyesters like PBAT Brigitte Gasser and different model substrates as indicated by HPLC/MS Dep. of Biotechnology, BOKU University of Natural Resources and Life Sciences quantification of the hydrolysis products terephthalic acid, Vienna, Austria adipic acid, mono(4-hydroyxbutyl)terephthalate and bis(4- hydroxybutyl)terephthalate. We could demonstrate that the Pichia pastoris is the most frequently used yeast system for esterases show activity under mild conditions as well as in biogas heterologous protein production today, however, the toolbox of sludge. available genetic elements is rather limited. To enable more robust http://dx.doi.org/10.1016/j.nbt.2014.05.1619 and cost-effective production processes for biopharmaceutical proteins and for industrial enzymes, it is crucial to understand the molecular physiology of the host, and the specific limitations that the product may exert on expression. Instead of classical genetic approaches, we applied systems biology tools to improve several aspects of the P. pastoris production platform. Combined transcriptomics, proteomics, metabolomics and flux data were used to investigate the interplay between protein production and cell metabolism. Thereby we gained insight in key regulatory effects exerted during heterologous protein production processes. These genome scale data were then further exploited for the identification of novel regulatory elements and

www.elsevier.com/locate/nbt S3 SUNDAY 13 JULY INDUSTRIAL BIOTECHNOLOGY FROM FUNDAMENTALS TO PRACTICE (ACIB SESSION) New Biotechnology · Volume 31S · July 2014 the prediction of cell engineering targets for improved productiv- ACIB-9 ity and robustness. Additionally, the endowment of genes involved in the protein secretory pathway was analysed in a comparative Utilization of recombinase mediated cassette exchange genomics approach, as productivities are often limited at the level (RMCE) for the generation of recombinant CHO cell lines of protein folding and secretion. with defined expression properties ,∗ http://dx.doi.org/10.1016/j.nbt.2014.05.1621 Martina Baumann 1 , Elisabeth Gludovacz 2, Sabine Vcelar 1, Nicole Borth 3 1 ACIB, Austria ACIB-8 2 BOKU, Austria 3 BOKU/ACIB, Austria Cross-species comparison of recombinant protein secretion in CHO cells and Pichia pastoris For diverse applications, including generation of recombinant Nils Landes 1,∗ , Andreas Maccani 2, Christian Leitner 3, Michael production cell lines, stable integration of transgenes into the Maurer 4, Alexandra B. Graf 4, Minoska Valli 2, Clemens Gruber 5, genome of the host cell is a pre-requisite. The precise position Gerda Modarres 4, Friedrich Altmann 5, Brigitte Gasser 3, Wolfgang of the integrated transgene is a major determining factor, both Ernst 3, Renate Kunert 3, Diethard Mattanovich 3 for gene expression level and long-term stability, which makes the screening for a suitable cell clone very time-and labour inten- 1 ACIB, Austria sive. The utilization of site-specific Recombinase Mediated Cassette 2 Austrian Centre of Industrial Biotechnology (ACIB GmbH), Vienna, Austria 3 Department of Biotechnology, University of Natural Resources and Life Sci- Exchange (RMCE) opened up the possibility to transfer the gene ences, Vienna, Austria of interest (GOI) into pre-selected genomic locations with defined 4 School of Bioengineering, University of Applied Sciences FH Campus Wien, expression properties. In this study we developed a strategy sup- Vienna, Austria porting the identification of recombinant Chinese Hamster Ovary 5 Department of Chemistry, University of Natural Resources and Life Sciences (CHO) cells with integration sites favourable to persistent high Vienna, Austria transgene expression and good gene-exchangeability. A sortable reporter gene (CD4) with a leaky start codon, flanked by het- Chinese hamster ovary (CHO) cells are currently the erospecific FRT (Flippase recognition targets) sequences was stably workhorses in biopharmaceutical industry. However, yeasts such as transfected into CHO cells. The leaky start codon reduces trans- Pichia pastoris are about to enter this field. Although a vast number lation efficiency and allows sorting for the highest transcription of research studies has focused on characterization of each of the rates, while the FRT sites mediate subsequent exchange of the individual expression system, information on direct cross-species expression cassette. Two rounds of RMCE followed by FACS sor- comparative studies are limited. Here we present a comprehensive ting for top producers were performed to select for sites that allow comparison of recombinant P. pastoris strains and CHO cell lines, reliable cassette exchange besides high transgene expression. The including bioprocess engineering aspects as well as systems biology resulting master cell line can be used repeatedly for insertion of the approaches. GOI without major genetic alterations. A combination of chemi- Two model proteins of different complexity were chosen: cal selection using alternative selection markers and FACS sorting monomeric and non-glycosylated human serum albumin and a for absence of CD4 expression as criterion for successful gene more complex 3D6 single-chain Fv-Fc fusion antibody (3D6scFv- exchange enables fast establishment of recombinant cell lines with Fc), which is secreted as a homodimer and contains the Fc-specific predictable expression properties. glycosylation sites. High and low producing strains or cell lines of the two model http://dx.doi.org/10.1016/j.nbt.2014.05.1623 proteins were established and characterized in lab-scale bioreactor cultivations. To evaluate the performance of each expression system, fed batch cultivations were performed. The production ACIB-10 processes were characterized by monitoring biomass and prod- Integrated continuous refolding and precipitation of uct formation as well as product quality. The two host systems proteins in a tubular reactor were then compared regarding their biomass specific secretion rates and space-time yields of the processes. Obtained results show Siqi Pan ∗ , Monika Zelger, Rainer Hahn that P. pastoris is the preferred host system for production of HSA, Austrian Centre of Industrial Biotechnology, Austria whereas CHO cells are more suited for the production of a complex molecule like the 3D6 scFv-Fc fusion protein. Large amounts of therapeutic proteins expressed in Escherichia For transcriptomics and proteomics analysis steady state coli as inclusion bodies have created a bottleneck in downstream samples from chemostat cultures were taken. Similarities and dif- processing. Process integration of continuous processes is one way ferences between the investigated host systems as well as protein to alleviate this bottleneck. In view of this consideration, lab- specific effects will be presented. oratory scale tubular reactors for the continuous processing of http://dx.doi.org/10.1016/j.nbt.2014.05.1622 recombinant proteins were developed. Benefits include faster mixing, high design flexibility and efficient heat transfer. With these advantages in mind, refolding strategies like pulsed refolding and

S4 www.elsevier.com/locate/nbt New Biotechnology · Volume 31S · July 2014 SUNDAY 13 JULY INDUSTRIAL BIOTECHNOLOGY FROM FUNDAMENTALS TO PRACTICE (ACIB SESSION) temperature leap refolding in tubular reactors were successfully cells? Such a problem is called a (minimal) hit-ting set [(M)HS] performed. Additionally, the tubular reactor offer better integra- problem–a classical problem in combinatorial optimization. The tion between processing steps while the ability to reach steady problem of calculating MHSs is often encountered in computa- state ensured consistent product quality. In an exemplary exam- tional biology with various applications in shotgun proteomics, ple, a fully continuous process for an autoprotease fusion protein gene expression analysis, multi-genome alignment and metabolic was established. This includes inclusion body dissolution, refol- engineering. ding and autocleavage, followed by purification by precipitating Here we present MHScalculator, which allows for fast calcula- out impurities. Process runs were robust over extended periods tion of MHSs in large systems. MHScalculator was developed at acib while reaction rates, product quality and yields were equal to batch and is freely available. We illustrate the power of MHScalculator processing. In an advanced setup, oxidative refolding by aeration for genomics, systems pharmacology, and systems biotechnology in loop configuration, equipped with static mixers, sparging ele- by aligning multiple genomes, by predicting minimal drug cock- ments and air-traps was successfully performed. The integration tails, and by finding minimal metabolic intervention strategies to of inline sensors also allowed the monitoring of critical process turn microorganism into optimal cell-factories for bio-production parameters. Process runs confirmed that productivity with the of chemical commodities. MHScalculator is able to process large tubular reactor were more than twice of the batch reactor. With a input data sets typically within a minute and allows for a fast growing interest in integrated continuous biomanufacturing, the and comprehensive analysis on standard computer infrastructure clear advantages of the tubular reactor would be essential to this outperforming alternative solvers. field. http://dx.doi.org/10.1016/j.nbt.2014.05.1625 http://dx.doi.org/10.1016/j.nbt.2014.05.1624

ACIB-11

From multi-sequence alignment to metabolic engineering to minimal drug cocktails: a new hitting set calculator

Juergen Zanghellini ∗ , Christian Jungreuthmayer University of Natural Resources and Life Sciences, Austria

Suppose you are given a set of drugs for the treatment of malignant cell lines. Typically a single drug does not treat all malignant cells. So what is the (smallest) set of drugs killing all malignant

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Biomolecular technology of proteins penetratin. Flow cytometry measurements and confocal imaging show that human Oct4-PTD internalizes into live cells slightly BIOTOP-1 more efficiently than penetratin. However, the cellular distribution differs greatly, with Oct4-PTD showing diffuse cytosolic and Design and engineering of next generation mammalian nuclear staining, whereas penetratin is strictly localized to a punc- cell factories tuate pattern in the cytoplasm. By using a Cre/loxP-based reporter David James system, we further show that this peptide also drives translocation of a functionally active Oct4-PTD-Cre-fusion protein into reporter The University of Sheffield, United Kingdom cells. Finally, recombinant full length Oct4 is shown to translo- cate into human and mouse fibroblasts even without addition of The majority of new biopharmaceuticals are made in Chinese any kind of cationic fusion tag. Our data therefore support the idea hamster ovary cells, a transformed cell type originally isolated that transcription factors might be part of an alternative signalling in the 1950s. The adaptability and utility of CHO cell factories pathway, where the proteins are transferred to neighbouring cells derives from our exploitation of their acquired genetic/functional to actively change the behaviour of the recipient cell. variation using high-throughput functional screening and selection processes which enable industry to isolate and maintain cell http://dx.doi.org/10.1016/j.nbt.2014.05.1627 factories with unusual and desirable properties. However, we still have a limited understanding of enabling cellular mechanisms that underpin the ideal manufacturing phe- BIOTOP-3 notype. This knowledge would permit design and construction of From slow to fast: effects of growth rate on global gene intrinsically better cell factories using the new concepts and tools expression and recombinant protein secretion in Pichia of systems and synthetic biology. pastoris We now have the potential to provide disruptive new solutions ,∗ for cell and process engineering, where we will be able to create Corinna Rebnegger 1 , Alexandra B. Graf 2, Minoska Valli 3, Brigitte bespoke cell factories with a predictable ability to manufacture Gasser 3, Michael Maurer 2, Diethard Mattanovich 1 complex biopharmaceutical proteins. 1 Department of Biotechnology, University of Natural Resources and Life Sci- http://dx.doi.org/10.1016/j.nbt.2014.05.1626 ences, Vienna, Austria 2 School of Bioengineering, University of Applied Sciences FH-Campus Vienna, Vienna, Austria 3 Austrian Centre of Industrial Biotechnology (ACIB GmbH), Vienna, Austria BIOTOP-2 In Pichia pastoris recombinant protein production is related to Characterization of a novel cell penetrating peptide the specific growth rate ␮. To investigate the impact of varying derived from human Oct4 ␮ on productivity in more detail, we studied secretion rates and Eva Harreither 1,∗ , Hanna Rydberg 2, Helene Amand 2, Vaibhav the transcriptome of a recombinant P. pastoris strain producing Jadhav 1, Lukas Fliedl 3, Christina Benda 4, Miguel Esteban 5, human serum albumin (HSA) at ␮ ranging from 0.015 to 0.15 h−1 Duanqing Pei 5, Nicole Borth 1, Regina Grillari-Voglauer 1, Oliver in glucose-limited chemostat cultures. Production rates of HSA Hommerding 6, Frank Edenhofer 7, Bengt Nordén 2, Johannes correlated positively with ␮ while product quality remained unaf- Grillari 1 fected. Analysis of global gene expression showed that ribosomal 1 Department of Biotechnology, BOKU University Vienna, Austria genes and other genes involved in gene expression and translation 2 Department of Chemical and Biological Engineering/Physical Chemistry, as well as mitochondrial genes were upregulated with increasing Chalmers University of Technology, Gothenburg, Sweden growth rate, while many transcriptional regulators, carbon source 3 ACIB GmbH, Austrian Center of Industrial Biotechnology, Austria responsive genes, autophagy and other proteolytic processes were 4 Key Laboratory of Regenerative Biology, Chinese Academy of Sciences, downregulated at higher ␮. Expression of mating and sporula- Guangzhou, China ␮ −1 5 Chinese Academy of Sciences, Guangzhou, China tion genes peaked at intermediate of 0.05 and 0.075 h , and −1 6 Stem Cell Engineering Group, Institute of Reconstructive Neurobiology, Uni- at very slow growth (␮ = 0.015 h ) many transporter genes were versity of Bonn, Germany differentially expressed. Analysis of a subset of genes related to pro- 7 Stem Cell and Regenerative Medicine Group, Institute of Anatomy and Cell tein folding and secretion revealed that unfolded protein response Biology, Julius-Maximilians-University Würzburg, Germany targets such as translocation, endoplasmic reticulum genes, and cytosolic chaperones were upregulated with increasing ␮ while Oct4 is a transcription factor that plays a major role for the proteolytic degradation of secretory proteins was downregulated. preservation of the pluripotent state in embryonic stem cells as We therefore conclude that a high ␮ positively affects specific pro- well as for efficient reprogramming of somatic cells to induced tein secretion rates by acting on multiple cellular processes. pluripotent stem cells (iPSC) or other progenitors. We here report that a 16 amino acid peptide representing the third helix of the http://dx.doi.org/10.1016/j.nbt.2014.05.1628 human Oct4 homeodomain can internalize in mammalian cells upon conjugation to a fluorescence moiety thereby acting as a cell penetrating peptide (CPP). This Oct4-protein transduction domain (PTD) was characterized by comparison to the well studied CPP

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BIOTOP-4 enzyme system which efficiently catalyzes O-O bond formation beside photosystem II. Moreover, Clds are phylogenetically related The human anti-HIV antibodies 2F5, PG9 and 2G12 differ with a recently discovered new heme peroxidase family called in their proteolytic susceptibility dye-decolorizing peroxidases (Dyps). They were shown to have a Melanie Niemer 1,∗ , Melanie Niemer 2, Ulrich Mehofer 2, Juan Anto- unique tertiary structure with a distal heme region that is differ- nio Torres Acosta 2, Maria Verdianz 2, Theresa Henkel 2, Andreas ent from conventional peroxidases from plants and animals [3] but Loos 2, Richard Strasser 2, Daniel Maresch 2, Thomas Rademacher 3, similar to Cld. Here, we show an up-to-date phylogenetic analysis Herta Steinkellner 2, Lukas Mach 2 of Clds and Dyps and point out their structural relationship as well as their functional differences [4]. 1 University of Natural Resources and Life Sciences Vienna, Austria 2 University of Natural Resources and Life Sciences, Austria References 3 Institute of Molecular Biotechnology, RWTH Aachen University, Germany [1].van Ginkel CG, Rikken GB, Kroon AG, Kengen SW. Arch Microbiol 1996;166:321–6. The tobacco-related species Nicotiana benthamiana has recently [2].Maixner F, Wagner M, Lücker S, Pelletier E, Schmitz-Esser S, Hace K, emerged as a promising host for the manufacturing of protein et al. Environ Microbiol 2008;10:3043–56. therapeutics. However, the production of recombinant proteins [3].Sugano Y, Muramatsu R, Ichiyanagi A, Sato T, Shoda M. J Biol Chem 2007;282:36652–8. in N. benthamiana is frequently hampered by undesired proteo- [4].Hofbauer S, Schaffner I, Furtmüller PG, Obinger C. Biotechnol J 2014 lysis. Here we show that the expression of the human anti-HIV [in press]. antibodies 2F5, PG9 and 2G12 in N. benthamiana leaves leads http://dx.doi.org/10.1016/j.nbt.2014.05.1630 to the accumulation of discrete heavy-chain derived degradation products of 30–40 kDa. Incubation of purified 2F5 with N. benthamiana intercellular fluid resulted in rapid conversion into the BIOTOP-6 40-kDa fragment, whereas 2G12 proved largely resistant to degradation. Such a differential susceptibility to proteolytic attack was Enzymatic oxidation of plant polysaccharides adsorbed also observed when these two antibodies were exposed to various to cellulose surfaces types of proteinases in vitro. While serine and cysteine proteinases 1,∗ 2 3 2 are both capable of generating the 40-kDa 2F5 fragment, the Filip Mollerup , Matti Häärä , Kirsti Parikka , Chunlin Xu , 3 4 30-kDa polypeptide is most readily obtained by treatment with Maija Tenkanen , Emma Master the latter class of enzymes. The principal cleavage sites reside 1 Department of Biotechnology and Chemical Technology, Aalto University, within the antigen-binding domain, the VH-CH1 linker segment Finland 2 and the hinge region of the antibodies. Collectively, these results Laboratory of Wood and Paper Chemistry, Åbo Akademi University, Finland 3 indicate that down-regulation of endogenous serine and cysteine Department of Food and Environmental Science, University of Helsinki, Finland proteinase activities could be used to improve the performance of 4 Department of Chemical Engineering and Applied Chemistry, University of plant-based expression platforms destined for the production of Toronto, Canada biopharmaceuticals. http://dx.doi.org/10.1016/j.nbt.2014.05.1629 The development of renewable energy and materials depends on intelligent utilization of abundant plant resources. While biotechnology for biofuels has received considerable attention, the BIOTOP-5 potential of enzymes to tailor plant polymers for novel biopolymer synthesis is comparatively unexplored. Chlorite dismutases and dye-decolorizing peroxidases – In particular, enzymes that catalyze selective oxidation of similarities and differences within a structural super- specific hydroxyls to carbonyl and carboxyl groups can facili- family of heme proteins tate targeted chemical derivatizations of polysaccharides to create ∗ renewable polymers with preferred physical and chemical proper- Irene Schaffner , Stefan Hofbauer, Paul Furtmüller, Christian Obinger ties [1]. In this regard, galactose oxidase (GaOx) continues to be a promising catalyst for specific delivery of new functionality to BOKU Vienna, Austria plant polysaccharides. Current chemo-enzymatic protocols with GaOx begin by oxidizing galactose substituents in polysaccharides Chlorite dismutases (Clds) are heme b containing enzymes (e.g. galactoglucomannan, xyloglucan) that are suspended in solu- which were discovered in chlorate- and perchlorate-reducing tion. Oxidized polysaccharides can then be adsorbed to cellulose bacteria (PCRBs) [1] but are found in many other bacterial and surfaces to modify the surface reactivity and/or barrier properties archaeal phyla [2]. Cld protects PCRBs from the accumulation of of cellulose-based materials. Although this is an elegant approach the harmful metabolic intermediate chlorite by degrading it to to expanding the range of cellulosic bioproducts, oxidation and chloride and dioxygen [1]. This catalytic function turns Cld into a derivatization of poly and oligo-saccharides can reduce their affin- highly interesting enzyme for bioremediation as the environmen- ity to cellulose surfaces. tal pollutants chlorate and chlorite are used as bleaching agents, as disinfectants, in pesticides etc. Additionally, Cld is extremely interesting from a biochemical point of view as it is the only known

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Accordingly, the aim of this study was to investigate the poten- Reference tial to oxidize xyloglucan and galactoglucomannan following [1].XuC, Spadiut O, Araújo AC, Nakhai A, Brumer H. Chemo-enzymatic adsorption of these polysaccharides to cellulose (Whatmann ﬁlter assembly of clickable cellulose surfaces via multivalent polysaccha- paper no. 1). Wild-type GaOx along with GaOx fused to a family rides. ChemSusChem 2012;5:661–5. 29 CBM from Piromyce equi were used in the analysis. Recent char- http://dx.doi.org/10.1016/j.nbt.2014.05.1631 acterization of the GaOx RQW mutant fused to a cellulose-bind CBM3 will also be presented.

S8 www.elsevier.com/locate/nbt New Biotechnology · Volume 31S · July 2014 OPENING CEREMONY

Opening Ceremony atively high reliability), synthetic biologists will soon design and build engineered biological systems. PL1–1 We have used synthetic biology to create inexpensive, effective, anti-malarial drugs. Currently, malaria infects 300–500 million How to get biotechnology to work for us? people and causes 1-2 million deaths each year, primarily children Anne Glover (CBE) in Africa and Asia. One of the principal obstacles to addressing this global health threat is a lack of effective, affordable drugs. CSA to President Barroso, European Commission The chloroquine-based drugs that were used widely in the past have lost effectiveness because the Plasmodium parasite which One thing scientists are not short of is imagination and our causes malaria has become resistant to them. The faster-acting, endeavours in biotechnology are an example of that. At this more effective artemisinin-based drugs – as currently produced conference we will hear about synthetic polymers, plants as from plant sources – are too expensive for large-scale use in the bio-factories, stem cell therapies, climate change mitigation and countries where they are needed most. The development of this biofuels to mention just some of the topics. This harnessing of technology will eventually reduce the cost of artemisinin-based cellular and biomolecular processes to develop new products and combination therapies signiﬁcantly below their current price. To processes and safeguard our planet requires more than just sci- reduce the cost of these drugs and make them more widely avail- entists to make sure it is the success that we hope for. We need able, we have used synthetic biology to engineer microorganisms communicative scientists who can talk about the opportunities as to produce artemisinin from renewable resources. well as the threats of new technologies; we need citizens to engage Having successfully completed the artemisinin work, we are and question what we do; we need honest and transparent busi- now engineering the metabolism of the same microorganisms nesses to translate knowledge into the economy and offer new (Escherichia coli and Saccharomyces cerevisiae) for production of possibilities; we need imaginative policymakers to develop smart advanced biofuels and chemicals that might otherwise be pro- options to allow exploitation of the knowledge that we produce duced from petroleum. Unlike ethanol, the advanced biofuels and we need effective and transparent politicians who make sure have the full fuel value of petroleum-based biofuels, will be trans- that citizens realise the maximum beneﬁt of the research that they portable using existing infrastructure, and can be used in existing have funded. We are living in an era dominated by biology and the automobiles and airplanes. Similarly, the microbially sourced bioeconomy has never held out so many possibilities. Only if we chemicals can be dropped into existing processes used to pro- all work together will we be able to achieve the best for citizens, duce existing materials. These chemicals will be produced from our lifestyle and our planet. natural biosynthetic pathways that exist in plants and a variety http://dx.doi.org/10.1016/j.nbt.2014.05.1632 of microorganisms as well as from pathways that have no rep- resentation in the natural world. Large-scale production of these chemicals and fuels will reduce our dependence on petroleum and PL1–2 reduce the amount of carbon dioxide released into the atmosphere, while allowing us to take advantage of our current transportation Synthetic biology for synthetic chemistry infrastructure and products supply chains. , , Jay Keasling a b c http://dx.doi.org/10.1016/j.nbt.2014.05.1633 a Departments of Chemical Engineering and Bioengineering, University of California, Berkeley, CA 94720, United States b Synthetic Biology Department, Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, United States c Joint BioEnergy Institute, Emeryville, CA 94608, United States

Synthetic biology is the design and construction of new biological entities such as enzymes, genetic circuits, and cells or the redesign of existing biological systems. Synthetic biology builds on the advances in molecular, cell, and systems biology and seeks to transform biology in the same way that synthesis transformed chemistry and integrated circuit design transformed computing. The element that distinguishes synthetic biology from traditional molecular and cellular biology is the focus on the design and construction of core components (parts of enzymes, genetic circuits, metabolic pathways, etc.) that can be modeled, understood, and tuned to meet speciﬁc performance criteria, and the assembly of these smaller parts and devices into larger integrated systems that solve speciﬁc problems. Just as engineers now design integrated circuits based on the known physical properties of materials and then fabricate functioning circuits and entire processors (with rel-

www.elsevier.com/locate/nbt S9 MONDAY 14 JULY BIOCHEMICAL ENGINEERING JOURNAL YOUNG INVESTIGATOR AWARD LECTURE New Biotechnology · Volume 31S · July 2014 Monday 14 July biotherapeutic, both from regulatory and economic standpoints, which has implications for commercial manufacturing. Increased Biochemical Engineering Journal Young regulatory clarity around biosimilars has intensiﬁed efforts in Investigator Award Lecture this space and matching product quality of molecules developed decades ago with current processes is most effectively done with YI–1 robust mechanistic understanding of cellular processes rather than by purely empirical approaches. In this changing landscape, A glimpse into the future of mammalian cell culture pro- recent advances in systems biology and a renewed interest in cess development: innovative approaches to impact time continuous perfusion cultivation offer avenues to bring about to clinic, product quality, and cost of process develop- paradigm shifts in cell culture process development and commer- ment and commercial manufacturing cial manufacturing. An approach for streamlined cell line and Chetan Goudar early-stage cell culture process development will be presented which can accelerate time to human clinical testing by as much as Amgen Inc., United States 8 months. Application of a poly-omics systems biology approach for identifying modulators for a critical product quality attribute There has been remarkable progress in cell line generation and will be presented followed by validation of these targets using cell cell culture processes over the past 2 decades. Expression levels line engineering. Recent dramatic productivity enhancements have increased by orders of magnitude and a broad spectrum of from continuous perfusion cultures will be presented and the complex biotherapeutics have been successfully manufactured implications of these advances on next-generation commercial at commercial scale. Despite these advances, it is important to manufacturing facilities will be discussed. recognize that the future poses fundamentally different challenges. While development costs and speed to in vivo validation http://dx.doi.org/10.1016/j.nbt.2014.05.1634 of biology will face increasing pressure, decentralized manufacturing is increasingly becoming a necessity for global reach of a

S10 www.elsevier.com/locate/nbt New Biotechnology · Volume 31S · July 2014 SYMPOSIUM 1: STEM CELL APPLICATIONS AND GENE THERAPIES: WHERE ARE WE?

Symposium 1: Stem cell applications and gene to a strong viral enhancer for signal amplification. Human pri- therapies: where are we? mary ASC were co-transfected with luciferase based reportergenes pCMVE/mOCP-MetLuc (osteocalcin) or pCMVE/mACDC-MetLuc O1-1 (collagen type II) together with renilla control plasmid. For transfection, a mild lipofection was applied and a green fluorescence Engineering synthetic stem cell niches protein (GFP) control was included to assess transfection efficiency. Secreted metridia luciferase allows to measure promoter Matthias P.Lutolf activation in the supernatant without need of cell lysing. The activ- Laboratory of Stem Cell Bioengineering and Institute of Bioengineering, School ity of the reportergenes was enhanced in transfected ASC treated of Life Sciences, Ecole Polytechnique Fédérale de Lausanne, CH-1015 Lausanne, with osteogenic or chondrogenic differentiation media compared Switzerland to control condition. Osteogenic differentiation was confirmed with Alizarin red quantitative and histological stainings whereas In vivo, the behavior of stem cells in a tissue is governed chondrogenic differentiation was confirmed with collagen type by the three-dimensional (3D) microenvironment, or ‘niche’, II staining. These findings were further verified by qRT-PCR. which involves a dynamic interplay between biochemical and Although osteocalcin and collagen type II are late markers, the mechanical signals provided by the extra-cellular matrix (ECM), activation of the signal-amplified and sensitive reportergenes can cell-cell interactions and soluble factors. The complexity of niches already be determined after 3 days of differentiation in vitro. and the context-dependent cell-responses that arise from these interactions have posed a major challenge to understanding the http://dx.doi.org/10.1016/j.nbt.2014.05.1636 underlying regulatory mechanisms and to identify artificial niches controlling the behaviour of specific stem cell types. To systematically dissect the role of the various interacting factors that O1-3 determine stem cell fate in a 3D context, we have developed experimental paradigms to simultaneously generate hundreds to Repeated systemic administration of human adipose- thousands of unique microenvironments and probe their effects derived stem cells attenuate diabetic nephropathy in the on cell fate. In this talk I will discuss our approach by way of sev- rats eral experiments in which we have measured the combined effects Xue-Yuan Bai ∗ , Yuxiang Ma, Xiangmei Chen of microenvironment stiffness, proteolytic degradation, and three Department of Nephrology, Chinese PLA General Hospital, China classes of signaling proteins on pluripotent stem cell fate, unveil- ing a comprehensive map of the interactive involvement of these Diabetic nephropathy (DN) is a major complication of diabetes parameters in regulating self-renewal and early differentiation. and represents the leading cause of end-stage renal disease world- Our approach is broadly applicable to gain a systems-level under- wide. Unfortunately, once the patients develop overt proteinuria, standing of multifactorial 3D cell-matrix interactions and opens there is no cure for DN. Therefore, the availability of a strategy the door for discovering unique artificial niches that control the aimed to delay or revert DN would be highly desirable. behavior of difficult-to-culture mammalian stem cells. To investigate the role of human adipose-derived stem cells http://dx.doi.org/10.1016/j.nbt.2014.05.1635 (ASCs) in treatment of diabetic nephropathy, Sparague-Dawley rats were made diabetes by intraperitoneal injection of streptozotocin after uninephrectomy. After 12 weeks proteinuria was O1-2 well-established. The rats received injection of human ASCs via tail vein at 5 × 106 every 4 weeks for five times. Reduction of protein- A quick potency assay for osteogenic and chondrogenic uria was not observed in diabetic rats until 24 weeks after three differentiation of adipose derived stem cells doses of ASCs. However, since 28 weeks, urinary protein excre- Eleni Oberbauer 1,∗ , Florian Hildner 2, Ara Hacobian 1, Susanne tion was significantly suppressed, and persisted up to 32 weeks Wolbank 1, Carolin Steffenhagen 1, Georg Feichtinger 1, Anja after streptozotocin. Hypoalbuminemia and hyperlipidemia were Peterbauer 2, Christian Gabriel 2, Heinz Redl 1 also improved in ASCs treated group. ASCs significantly attenuated glomerulus hypertrophy and renal tubular interstitial injury 1 Ludwig Boltzmann Institute for Experimental and Clinical Traumatology, Austria as well as downregulation of podocyte markers, WT-1 and snap- 2 Red Cross Blood Transfusion Service of Upper Austria, Austria topodin. Hoechst labeled ASCs were injected into DN rat via tail vein. Cells were detected in lung and spleen, and peritubular The field of bone- and cartilage regeneration has evoked strong regions, but rarely in glomeruli and pancreas within 48 hours after interest in tissue engineering. Osteogenic and chondrogenic dif- injection. Gene expression of human Alu could be detected in ferentiation potential of adipose derived stem cells (ASC) are rat lung and spleen till 4 weeks after injection. The ASCs did not challenging and promising for bone and cartilage repair. Current improve hyperglycemia and pancreatic damage. in vitro methods to analyze the differentiation capacity are time These findings indicate that repeated intravenous ASCs can consuming and thus we designed and established novel enhancer reduce diabetic kidney damage in rats even at the progressive stage. and tissue-specific promoter for osteogenic and chondrogenic http://dx.doi.org/10.1016/j.nbt.2014.05.1637 differentiation of ASC together with a quick potency bioassay. For this, osteocalcin or collagen type II promoter was coupled

www.elsevier.com/locate/nbt S11 SYMPOSIUM 1: STEM CELL APPLICATIONS AND GENE THERAPIES: WHERE ARE WE? New Biotechnology · Volume 31S · July 2014

O1-4 suitable clinical tool to improve regeneration of a variety of tissues, however, the mechanisms underlying these beneficial effects Producing and harvesting culture-derived platelets with still remain widely unknown. In this study we address the effects functional activity from blood stem cells of ESWT onto ASCs and their stemness as well as differentiation William Miller 1,∗ , Alaina Schlinker 1, Katherine Radwanski 2, potential in high passages. Christopher Wegener 2, K. Augustine Min 2 In our study we show that human and rat ASCs respond strongly to repetitive shockwave treatment in vitro, resulting in 1 Northwestern University, United States maintenance and significant elevation of mesenchymal mark- 2 Fresenius Kabi, USA ers (flow cytometry: CD73, CD90, CD105), while cell viability and proliferation remain at a comparable level to control group. Platelet production in culture by megakaryocytic cells (Mks) Another effect observed was a significant increase in differ- derived from hematopoietic stem and progenitor cells (HSPCs) entiation capacity into osteogenic (von Kossa staining; PCR: would supplement donations, but it is very challenging to purify osteocalcin, biglycan) and adipogenic lineage (Oilred O staining) the platelets. As an alternative to multi-step centrifugation, we as well as into Schwann like cells (flow cytometry: P75, S100, P0) used a polycarbonate spinning membrane with 4-␮m cylindri- in high passages. cal pores. Because platelet release is asynchronous, we explored Our results indicate that ESWT preserves stemness and multipo- whether we could harvest platelets generated early in culture and tency of ASCs even in higher passages after extensive expansion. reseed immature Mks to generate platelets later in culture. Exper- Hence, ESWT might be a promising tool to improve the quality iments with immortalized Mks and apheresis platelets showed and consistency of ASCs for cell therapy in tissue engineering and efficient platelet recovery and the exclusion of Mks. Recovered regenerative medicine. platelets had little pre-activation and the Mks retained viabil- Acknowledgement: Financial support from FFG (#818412) ity and proplatelet formation. Next, primary CD34+ HSPCS were and the “City of Vienna Competence Team Tissue Engineering differentiated to Mks that released platelets. As for the Mk line, Bioreactors” is gratefully acknowledged. primary Mks were excluded from the platelet fraction and the viability and ploidy distribution of recovered Mks were similar to http://dx.doi.org/10.1016/j.nbt.2014.05.1639 those of input Mks. Recovered platelets expressed surface markers and spread after activation by thrombin in a similar manner to unprocessed platelets. However, culture-derived platelet recovery O1-6 was lower than for apheresis platelets (70 vs. 90% at 3000 rpm). This is likely due to the larger size of culture-derived platelets Engineering bacteria for the discovery of potential ther- and preplatelets (5.6 vs. 3.3 ␮m). After screening experiments apeutic compounds against protein misfolding diseases with even larger red blood cells (7.3 ␮m), we increased culture- Georgios Skretas 1,∗ , Dafni Delivoria 2, Ilias Matis 2, Nikoletta derived platelet recovery with essentially no Mk contamination Papaevgeniou 2, Niki Chondrogianni 2 by decreasing the rotation rate to 2000 rpm. Recovered platelets 1 National Hellenic Research Foundation, Greece showed minimal pre-activation and were activated by thrombin. 2 Institute of Biology, Medicinal Chemistry & Biotechnology – National Hellenic Importantly, Mks in the cell fraction that were returned to culture Research Foundation, Greece released platelets that were harvested two days later. http://dx.doi.org/10.1016/j.nbt.2014.05.1638 It has now been widely recognized that many incurable diseases with enormous socioeconomic impact, such as Alzheimer’s disease, Parkinson’s disease, type 2 diabetes, etc., are initiated by O1-5 a common mechanism: the misfolding of specific proteins. Here, we describe the use of engineered bacterial cells as a platform for Adipose derived stem cells respond to in vitro extracorpo- the discovery of potential therapeutics against such protein mis- real shockwave treatment with increased stemness and folding diseases (PMDs). The topic of the described research is the multipotency application of molecular evolution approaches for the discovery ,∗ of compounds that rescue the misfolding of PMD-associated pro- Christina Schuh 1 , Philipp Heher 1, Anna Weihs 2, Asmita teins. To achieve this, Escherichia coli cells are first engineered to Banerjee 1, Susanne Wolbank 1, Rainer Mittermayr 1, Heinz Redl 1, biosynthesize large libraries of test compounds with high struc- Dominik Rünzler 2, Andreas Teuschl 2 tural diversity. Then, the same cells are modified further so that 1 LBI for Experimental and Clinical Traumatology, Austria they allow the identification of the rare molecules that correct the 2 University of Applied Sciences Technikum Wien – Department of Biochemical folding of particular misfolding-prone and PMD-associated pro- Engineering, Austria teins (MisPs) with the use of a genetic screen. Lead compounds identified by this initial screen, are then subjected to more detailed Tissue resident adipose derived progenitor/stem cells (ASCs) are evaluation by biochemical and biophysical methods of protein a promising tool for tissue engineering, addressing the problem of analysis, and their ability to inhibit MisP-induced pathogenicity tissue and organ shortage. Limiting factors for the use of ASCs are is tested using appropriate human cell assays or in vivo models donor variation and senescence, loss of differentiation capacity as of the disease of interest. The molecules capable of rescuing the a consequence to loss of multipotency. For several decades extra- misfolding of the target MisP and of antagonizing its associated corporeal shockwave treatment (ESWT) has been proven to be a

S12 www.elsevier.com/locate/nbt New Biotechnology · Volume 31S · July 2014 SYMPOSIUM 1: STEM CELL APPLICATIONS AND GENE THERAPIES: WHERE ARE WE? pathogenicity become drug candidates against the specific dis- A comparison of gene expression data from five independent ease. We will describe our efforts to identify such “pharmacological studies, obtained from Gene Expression Omnibus has been per- chaperones” against the misfolding of the amyloid ␤ (A␤) peptide formed. Each dataset was statistically analyzed in order to identify and of certain carcinogenic misfolded variants of human p53, with differentially expressed genes (DEGs). Proteins encoded by DEGs the aim of developing potentially therapeutic compounds against were determined and integrated with protein-protein interaction Alzheimer’s disease and cancer, respectively. (ppi) data for further analyses and to identify hub proteins. Enrich- http://dx.doi.org/10.1016/j.nbt.2014.05.1640 ment analyses were performed to map the interconnectivities between diseases and biological pathways. Comparative analyses indicated that six DEGs were common O1-7 between all three diseases, which are immune related and have associations to cardiovascular diseases. Enrichment analyses lead Computational prediction of associations between psori- to a significant association with PPAR signaling pathway. There are asis, rheumatoid arthritis and osteoarthritis 34 Transcriptional Factors regulating these DEGs. The hub proteins in the disease ppi network are: CEBPD and MMP1. Tuba Sevimoglu ∗ , Kazim Yalcin Arga This study suggests a genetic cause common between psoriasis, Marmara University, Turkey OA and RA. The commonality between psoriasis and RA comes from the fact that they are both autoimmune diseases. In the Microarray technologies have enabled rapid and efficient case of OA and psoriasis, Psoriatic Arthritis might be the link that expression profiling of thousands of genes simultaneously. connects psoriasis to OA. This study will elucidate molecular and Microarray studies on disease datasets have already revealed genes cellular mechanisms which will lead to a better understanding and that could be implicated in the pathogenesis of that disease thus better drug design for these diseases. opening the path to accurate diagnosis and potential biomarkers. http://dx.doi.org/10.1016/j.nbt.2014.05.1641 This study aims at comparative analysis of three common diseases with important genetic origin: Osteoarthritis (OA), rheumatoid arthritis (RA) and psoriasis.

www.elsevier.com/locate/nbt S13 SYMPOSIUM 2: PLANTS FOR THE PRODUCTION OF HIGH VALUE CHEMICALS New Biotechnology · Volume 31S · July 2014

Symposium 2: Plants for the production of selection of four markers had significant genetic effect. In the two high value chemicals groups of ++/+ genotype, the average fiber strength of individuals were 31.21-32.62 cN/tex, and 30.77-32.50 cN/tex for the +/− geno- O2-1 type, selection effects of single marker were 0.80-1.51cN/tex; In the groups for QTL-1 × QTL-3 and QTL-2 × QTL-3Combinations, Cultured plant cambial meristematic cells as a chassis for the average fiber strength of individuals while pyramiding two the production of pharmaceuticals QTLs were 33.40-34.08 cN/tex, the selection effects were 2.73-3.56 cN/tex which compared to the plants without the two QTL, and Susan Howat 1 , Marisol Ochoa Villarrea 1, Rabia Amir 1, Eunjung the selection effects of 1.12-3.02 cN/tex compared to the plants Kwon 1, Zejun Yan 1, Young-Woo Jin 2, Eun-Kyong Lee 2, Gary J. ,∗ with one of the two QTL. In addition, QTL-1 and QTL-2 might be Loake 1 the same QTL, QTL-2 and QTL-3 had obvious epistasis effect. So, 1 Institute of Molecular Plant Sciences, School of Biological Sciences, University This shows it is feasibility to use the molecular marker for assisting of Edinburgh, King’s Buildings, Edinburgh EH9 3JR, UK the pyramiding selection of fiber strength. 2 Unhwa Corp., 874-1, 2Ga, Wooah-Dong, Dukjin-gu, Jeonju, South Korea http://dx.doi.org/10.1016/j.nbt.2014.05.1643 A plethora of important, chemically diverse natural products are derived from plants. In principle, plant cell culture offers an attractive production platform for some natural products but O2-3 often is not a commercially viable strategy because of difficulties associated with culturing dedifferentiated plant cells (DDCs) The CO2 microalgae biorefinery: high value products and on an industrial scale. To address this issue we have isolated biofuels using halophilic microalgae in the “D-Factory” and cultured innately undifferentiated cambial meristematic cells Patricia Harvey 1,∗ , David Bailey 1, Ami Ben-Amotz 2, Vitor (CMCs). Utilizing a combination of deep sequencing technologies, Verdelho 3, Guy Harris 4, David Rooke 4, Herre Hoekstra 5, Paul we identified marker genes and transcriptional programs consis- Goacher 6, Joao Crespi 7, Guido Reinhardt 8, Laura Martinelli 9, tent with a stem cell identity. Declan Schroder 10, Richard Pipe 10, Nadine Igl-Schmid 11, Antonis CMCs derived from Taxus cuspidata and Panax ginseng, sources Kokossis 12, Karin Perrson 13 of the key anticancer drug, paclitaxel and neuroprotective ginseno- 1 University of Greenwich, United Kingdom sides, respectively, circumvented obstacles routinely associated 2 Nature Beta Technologies Ltd., (NBT), Israel with the commercial growth of DDCs. Subsequent molecular 3 A4F AlgaFuel, Portugal strategies uncovered a network of regulators that control the 4 Dynamic Extractions, United Kingdom 5 expression levels of genes integral to the biosynthesis of these key Evodos, Germany 6 Hafren Investments, United Kingdom natural products. We envisage both natural and where appropriate, 7 Instituto de Biologia Experimental e Tecnológica (IBET), Portugal engineered CMCs, will provide a cost-effective and environmen- 8 IFEU – Institute for Energy and Environmental Research, United States tally friendly platform for the sustainable production of a wide 9 IN SRL Italy, Italy variety of important plant-derived pharmaceuticals. 10 Marine Biological Association of the UK, United Kingdom 11 NATECO2 GmbH & Co. KG, Germany http://dx.doi.org/10.1016/j.nbt.2014.05.1642 12 National Technical University of Athens, Greece 13 SP Technical Research Institute of Sweden AB, Sweden

O2-2 Fuel-only algal systems are not economically feasible because yields are too low and costs too high for producing microal- Molecular marker-assisted selection and pyramiding gal biomass compared to using agricultural residues e.g. straw. effect of major QTLs for cotton fiber strength Biorefineries which integrate biomass conversion processes and Youlu Yuan ∗ , Tiankang Wang, Yuzhen Shi, Haihong Shang, Aiy- equipment to produce fuels, power and chemicals from biomass, ing Liu, Junwen Li, Juwu Gong, Tao Wang, Wan-kui Gong, Tingting offer a solution. The CO2 microalgae biorefinery (D-Factory) is a 10 Chen, Botao Li million Euro FP7-funded project which will cultivate the microalga Dunaliella in highly saline non-potable waters in photobioreactors Institute of Cotton Research of Chinese Academy of Agricultural Sciences/State Key Laboratory of Cotton Biology/Key Laboratory of Biological and Genetic and open raceways and apply biorefinery concepts and European Breeding of Cotton, The Ministry of Agriculture, China innovations in biomass processing technologies to develop a bas- ket of compounds from Dunaliella biomass, including the high Two elite fiber quality materials 0-153 and Xinluzao 24, and value nutraceutical, ␤-carotene, and glycerol. Glycerol now finds two commercial cotton cultivars (lines) Lumianyan 28 and Jimian markets both as a green chemical intermediate and as a biofuel in 516 were used as parents to develop two double-cross com- CHP applications as a result of novel combustion technology. Driv- binations (Jimian 516 × 0-153) × (Jimian 516 × Xinluzao24)Pop1, ing down costs by recovering the entire biomass of Dunaliella cells (Lumianyan 28 × 0-153) × (Lumianyan 28 × Xinluzao 24)Pop2, from saline cultivation water poses one of the many challenges Four SSR markers, which were linked to three QTLs of fiber strength for the D-Factory because Dunaliella cells are both motile, and do and derived from 0-153 or Xinluzao 24, were used respectively not possess an external cell wall, making them highly susceptible to study the efficiency of molecular marker-assisted selection and to cell rupture. Controlling expression of desired metabolic path- pyramiding effect of fiber strength. The result showed that assisted ways to deliver the desired portfolio of compounds flexibly and

S14 www.elsevier.com/locate/nbt New Biotechnology · Volume 31S · July 2014 SYMPOSIUM 2: PLANTS FOR THE PRODUCTION OF HIGH VALUE CHEMICALS sustainably to meet market demand is another. The first proto- consortium is looking into financially incentivising land decon- type D-Factory in Europe will be operational in 48 months, and tamination by producing high-value products from the waste, with will serve as a robust manifestation of the business case for global our initial case studies focusing on arsenic, platinum and palla- investment in algae biorefineries and in large-scale production of dium. CL4 W is following a number of research tracks, including microalgae. investigating the optimal plant species for phytoremediating a http://dx.doi.org/10.1016/j.nbt.2014.05.1644 number of different metals and developing cost-effective methods of processing the phytoremediation waste. We are also using synthetic biology tools to employ micro-organisms in harvest- O2-4 ing heavy metals from the waste, engineering bacteria to convert these metals into high-value nanoparticles for use in medicine and Exploiting nature’s chemists: high value bioactive com- industry, and looking into extracting energy from the left-over pounds from algae plant material. The data presented here will detail the progress made so far on Christine Edwards ∗ , Linda Lawton the CL4 W project, highlighting our work in evaluating contami- IDEAS/Robert Gordon University, United Kingdom nated industrial sites and our current model of assessing the costs and benefits of the project. As part of the nanoparticle production Algae are an essential component of global ecosystems using research track we are working with bacteria able to reduce plat- light and carbon dioxide to produce organic carbon and oxygen. inum and palladium, and have identified a number of the genes Consequently they are found in diverse habitats including ter- involved in these pathways. We have also engineered a strain of restrial and aquatic habitats, hot springs to Antarctic mats. The E. coli able to tolerate high levels of arsenic, and which at the ecological diversity has resulted in chemical diversity and they same time produces nano-scale structures when in the presence increasingly investigated as sources of new chemical entities in of arsenic. drug discovery programs, in particular anti-cancer. http://dx.doi.org/10.1016/j.nbt.2014.05.1646 In order to exploit the chemical diversity in target algae, 400–500 L algae per month, are grown in parallel batch cultures. Cells are harvested and bioactive compounds are extracted O2-6 using methanol, cleaned up by automated flash chromatography with final polish by preparative high performance liquid Gene isolation and its identification of salinity stress on chromatography (HPLC). Throughout the process quality and G. hirsutum L. quantity of compounds in extracts/fractions are tracked by ultra Wuwei Ye performance liquid chromatography–photodiode array-mass spectrometry (UPLC-PDA-MS) to ensure high purity is achieved. Due Institute of Cotton Research, CAAS, China to the highly toxic nature of the compounds, extreme precautions must be exercised throughout processing, with frequent review. Soil salinization has become a serious global problem affect- In addition, shipping is a challenge as they are categorised as ing the agricultural development and the ecological environment. extremely dangerous according to transport regulations so they Salinityas one of the most important abiotic stresses in the world, usual couriers such as FedEx will not take them. In addition, as they severely limits the production of crop. Saline-alkali land in our are considered as potential weapons of mass destruction, export country is widely distributed with the character of multi types licence and control is essential. and serious salt-deposition. In order to carry out the utilization Despite all the challenges 14 products are globally available via of saline-alkali land efficiently, it is necessary to develop the agri- a fine biochemical company, generating in excess of £120 K per culture on the saline-alkali land. Cotton, the major cash crop in annum. China, is playing a crucial role in national economic development. http://dx.doi.org/10.1016/j.nbt.2014.05.1645 China, with less cultivated lands and more people, faces the con- tradiction between food and cotton, which seriously affects the cultivation and production of cotton. Therefore, it is an effective O2-5 way to farm saline land and to enhance the sustainable agricultural development by develop the salinity-tolerant varieties of cotton. Use of synthetic biology in creating high-value metal Identification of salinity-tolerance also plays a vital role on cot- nanoparticles from phytoremediated waste ton breeding. 11 salt-tolerance related genes, H+-pyrophosphatase gene and S-adenosylmethionine synthetase gene and others, were Matthew Edmundson ∗ , Michael Capeness, Louise Horsfall cloned from the salt-tolerance material on Gossypium hirsutum, Edinburgh University, United Kingdom which were named GhVP and GhSAMS, respectively. The bioinformatics analysis and their transformed accessions were tested Metal and metalloid contamination of land is a major prob- and identified. lem on former industrial sites in the UK and in the wider world. http://dx.doi.org/10.1016/j.nbt.2014.05.1647 Phytoremediation has been used as a method to clean up this contamination in the past but disposal of the contaminated plant waste is still a problem. The Cleaning Land for Wealth (CL4 W)

www.elsevier.com/locate/nbt S15 SYMPOSIUM 3: GLYCOBIOTECHNOLOGY New Biotechnology · Volume 31S · July 2014

Symposium 3: Glycobiotechnology Although some tools are available to produce proteins with tailored N-glycans in P. pastoris (GlycoSwitch®), such engineering O3-1 process is costly and time-consuming. We developed a method that enables the in vivo removal of Development of new synthetic and analytical tools in N-glycans by inducible co-expression of a fungal endoglycosi- glycobiotechnology dase, removing N-glycosylation associated heterogeneity without Sabine L. Flitsch impairing strain viability and growth. This approach, called Pichia GlycoDelete, drastically reduces sample heterogeneity, facilitates The University of Manchester, United Kingdom protein purification and alleviates many of the problems that are associated with yeast-type N-glycosylation. Carbohydrates provide the largest biomass on Earth and are central to many aspects of biotechnology with applications in http://dx.doi.org/10.1016/j.nbt.2014.05.1649 biofuels, biomaterials, food and medicine. Carbohydrates are complex biomolecules and there is an urgent need to develop robust synthetic and analytical methodologies to fully exploit oppor- O3-3 tunities presented by glycobiotechnology. We have developed a Enzymatic remodelling of chitin for agrochemical appli- toolbox for the synthesis and analysis of complex carbohydrates cations and their function with a focus on (i) chemoenzymatic synthesis of ,∗ glycoconjugates such as glycopeptides, (ii) glycoarrays to study gly- Rémi Chambon 1 , Guillaume Despras 2, Dominique Urban 2, Boris coenzyme activity and discover carbohydrate-protein interactions Vauzeilles 3, Jean-Marie Beau 3, Sébastien Fort 4, Sylvie Armand 4, and (iii) ion mobility mass spectrometry for high resolution struc- Sylvain Cottaz 4 tural analysis of carbohydrates and (iv) mass spectrometry for the 1 CERMAV-CNRS, Grenoble label free identification of carbohydrate-binding proteins [1–5]. 2 Université Paris Sud and CNRS – Institut de Chimie Moléculaire et des Matéri- aux d’Orsay, France References 3 Université Paris Sud and CNRS – Institut de Chimie Moléculaire et des Matéri- [1].Both, et al. Nat Chem 2014;6(1):65. aux d’Orsay – Centre de Recherche de Gif, Institut de Chimie des Substances [2].Noble, et al. J Am Chem Soc 2012;134(31):13010–7. Naturelles, France 4 [3].Sardzík,ˇ et al. J Am Chem Soc 2012;134(10):4521–4. Centre de Recherches sur les Macromolécules Végétales – CNRS, France [4].Castangia, et al. Angew Chem 2012;51(52):13016–8. [5].Rannes, et al. J Am Chem Soc 2011;133(22):8436–9. Lipo-chitinoligosaccharides (LCOs) are produced by bacteria http://dx.doi.org/10.1016/j.nbt.2014.05.1648 (genus Rhizobium) and arbuscular mycorrhizal fungi, and are involved in the establishment of symbiosis with plants. They promote the assimilation of atmospheric nitrogen in legumes (Nod O3-2 factors [1]) and intake of water and nutrients in 80% of plant species (Myc factors [2]). These Nod and Myc factors consist of a Pichia pastoris GlycoDelete: the way out when N-glycans backbone of four or five N-acetylglucosamine residues modified by are a burden a fatty acid on the non-reducing unit and have various decorations on some hydroxyl groups. Bram Laukens 1,2,∗ , Katrien Claes 1,2, Charlot De Wachter 1,2, Nico The LCOs, which are active at subnanomolar concentra- Callewaert 1,2 tion, are produced naturally in extremely small quantities. The 1 Unit for Medical Biotechnology, Inflammation Research Centre (IRC), VIB- detailed analysis of their recognition mechanisms as well as their UGent, Technologiepark 927, B-9052 Ghent-Zwijnaarde, Belgium agrochemicals applications requires the development of efficient 2 Department of Biochemistry and Microbiology, Laboratory for Protein Bio- chemistry and Biomolecular Engineering, UGent, K.L.-Ledeganckstraat 35, synthetic methods. B-9000 Ghent, Belgium In this context we developed a chemo-enzymatic chitin remodeling approach to access LCOs via combined use of the In order to simplify bio-production of glycoproteins pro- chitin-N-deacetylase NodB (Sinorhizobium meliloti) and chitin duced in Pichia pastoris, new approaches need to be developed to oligomers chemically functionalized on the reducing unit [3]. deal with the heterogeneity that arises from the fungal-type N- References glycosylation. Here, we present a method that enables the in-vivo [1].Lerouge P, Roche P, Faucher C, Maillet F, Truchet G, Prome JC, et al. removal of N-glycans during production of recombinant glyco- Nature 1990;344(6268):781–4. proteins in Fungi. [2].Maillet F, Poinsot V, Andre O, Puech-Pages V, Haouy A, Gueunier M, The methylotrophic yeast P. pastoris is an excellent host for et al. Nature 2011;469(7328):58–63. recombinant protein production thanks to the strong, tightly [3].This project is supported by the ANR (ANR-10-CD2I-0008, BioCOS controlled methanol-inducible promoter system and the well- 2010-2014). established upscaling processes. Moreover, Pichia does not secrete http://dx.doi.org/10.1016/j.nbt.2014.05.1650 a lot of endogenous proteins which facilitates downstream processing. However, the sometimes very extensive oligomannose- type N-glycosylation pattern can be of concern during downstream processing and characterization of the recombinant product.

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O3-4 lating alternative acceptors [2]. Lsc3 has very high catalytic activity

(kcat ∼500 1/s), stability and therefore a high biotechnological Polysaccharides production by autotrophic cultures of potential. microalgae In this study, we optimized levan and FOS production by Lsc3 Giuseppe Olivieri 1 , Renato S. Coellho 2, Telma T. Franco 2, Antonino protein. High-performance liquid chromatography system cou- Pollio 3, Antonio Marzocchella 1,∗ pled with ELS detector was used to detect and quantify saccharides, levan was quantiﬁed spectrophotometrically. 1 DICMaPI – Università degli Studi di Napoli Federico II, Italy We showed that Lsc3 produced up to 15.4 g of FOS per mg of 2 Faculdade de Engenharia Química – University of Campinas, Brazil 3 Biological Science Department – Università degli Studi di Napoli “Federico II”, protein under optimized conditions. Product yield and spectrum Italy depended on reaction conditions. Also, permeabilized bacterial cells expressing levansucrase were shown to serve as effective cat-

Light energy and CO2 may be considered as the most abun- alyst for FOS production. dant and cheap feedstock for bioprocesses aimed to produce energy We developed a method for enzymatic production of levan-type vectors as well as green chemical building blocks. Microalgae can FOS from sucrose and a simple yeast-based method for the removal be cultivated on waste streams of gas (CO2 polluted) and liquids of monosaccharides that form as by-products of the reaction. (industrial effluents and salt supplements) for producing biofuels Acknowledgements: This work was supported by ERF grant and carbohydrates, a building block for bioplastics production. 3.2.0701.12-0041 (SLOMR12215T) managed by Archimedes Foun- The aim of this contribute is to report about recent joint dation and an ETF grant GLOMR9072. progress of a research activity carried out in Campinas (BR) and in Napoli (IT). The study regards autotrophic cultures of microalgae References selected to produce a significant amount of carbohydrates coupled, [1].Marx SP, et al. FEMS Microbiol Lett 2000;182:163–9. if possible, with lipids as biofuel resource. [2].Visnapuu T, et al. J Biotechnol 2011;155:338–49. Selected strains were autochthon in Brazil and from http://dx.doi.org/10.1016/j.nbt.2014.05.1652 the collection available at the University of Napoli ACUF (http://www.biologiavegetale.unina.it/acuf.html). Microalgae were grown in flasks and then inoculated in the photobioreactors. O3-6 Both the broth and the cells have been characterized for cultures carried out under a wide interval of operating conditions. Main 7-Hydroxydehydronuciferine induces human melanoma measured data were cell concentration, total organic carbon (TOC) A375.S2 autophagy and apoptosis and inhibits metasta- in the liquid phase, polysaccharide concentration and protein sis in vitro and in vivo concentration. The attention was paid on both the extracellular Hui Min Wang and intracellular polysaccharides. Kaohsiung Medical University, Taiwan http://dx.doi.org/10.1016/j.nbt.2014.05.1651

Melanoma is the deadliest cancer. We identified 7- hydroxydehydronuciferine (7-HDNF) isolated from the O3-5 leaves of Nelumbo nucifera Gaertn cv. Rosa-plena to be a Synthesis of potential prebiotics using Pseudomonas bioactive agent against human melanoma A375.S2 cells. 7- syringae DC3000 levansucrase Lsc3 hydroxydehydronuciferine (7-HDNF) was known to induce autophagy and apoptosis response mechanisms, and anti- Triinu Visnapuu 1,∗ Anneli Aasamets 1 Karin Mardo 1 Eerik Jõgi 1 , , , , migratory activity of melanoma in vitro and in vivo. Cell Heiki Vija 2 Tiina Alamäe 1 , proliferation assay was used to test cell viability. Acridine orange 1 University of Tartu, Estonia (AO) staining and flow analysis were applied to observe cell 2 National Institute of Chemical Physics and Biophysics, Estonia morphology. The apoptotic cell death ratio was measured via two-dimensional flow cytometry by annexin V-fluorescein iso- Plant-derived inulin-type oligofructans are considered to be thiocyanate (FITC)/propidium iodide (PI) double stained. Western effective prebiotics that function as specific growth substrates blot was applied to examine protein expressions whereas wound for beneficial gut bacteria. The effects of levan-type FOS (fruc- healing assay was to examine cell activity. Strong anticancer effects ␤ tooligosaccharides) containing -2,6 linkages are poorly studied of 7-HDNF exhibited in a dose-dependent manner, and displayed as they are not commercially available. Some studies prove their minor cytotoxicities on normal human skin cells.7-HDNF induced enhanced prebiotic effect [1]. Polymeric levan has potential appli- the formation of intracellular vacuoles and the augmentation of cations as anti-cancer, anti-inflammatory and immune stimulating acidic vesicular organelles (AVO). 7-HDNF increased the cellular agent. arrest in cell cycle at G2/M phase. Cellular membrane asymme- Levansucrases (EC 2.4.1.10) are bacterial enzymes belonging try lost was confirmed. Protein expressions were discovered to to GH68 family of glycoside hydrolases. Pseudomonas syringae verify autophagy and apoptosis response mechanisms sharing DC3000 encodes three levansucrases (Lsc1, Lsc2, Lsc3). Recom- the associated pathways. 7-HDNFpresented the high-quality ␤ binant Lsc3 splits sucrose and synthesizes -2,6-linked FOS, anti-migratory activity. 7-HDNF inhibited melanoma tumor polymeric levan and also heterooligofructans when transfructosy- growth in mice xenograft model, accompanied with a decrease

www.elsevier.com/locate/nbt S17 SYMPOSIUM 3: GLYCOBIOTECHNOLOGY New Biotechnology · Volume 31S · July 2014 of phosphorylation of AKT. We demonstrated the mechanism anti-migratory bio-functions and inhibited melanoma tumor of this compound starting with the formation and accumula- growth in mice xenograft model. tion of AVO leading to autophagy. 7-HDNF caused the cellular http://dx.doi.org/10.1016/j.nbt.2014.05.1653 membrane asymmetry lost, triggering the G2/M cell cycle arrest in caspase-dependent apoptosis. 7-HDNF presented high-quality

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Symposium 4: Robust biocatalysts for the pro- We have solved the structures of TAs of the Pfam classes, III, duction of novel bio-based products IV and V in order to further understand their mechanism and substrate specificities. O4-1 The class III ␻-amino acid TAs catalyse transamination of ␻- amino acids such as ␤-alanine or ␥-aminobutyric acid where the Molecular design of transglucosidases for polysaccharide transferred amino group is not adjacent to the carboxyl group. and oligosaccharide synthesis The crystal structures and inhibitor complexes of two ␻-TAs from , , , , , Pseudomonas aeruginosa and Chromobacterium violaceumhave been P.Monsan a b c d e f determined to understand differences in their substrate specificity. a Université de Toulouse; INSA, UPS, INP,LISBP,135 Avenue de Rangueil, F-31077 The class V TA enzymes include the serine:pyruvate transami- Toulouse, France nases having broad substrate specificity including a reaction with b CNRS, UMR 5504, F-31400 Toulouse, France ␤ c INRA, UMR 792 Ingénierie des Systèmes Biologiques et des Procédés, F-31400 a -hydroxyl substrate. The structure of the thermophilic archaeal Toulouse, France Sulfolobus solfataricus serine TA has been determined to 1.8 A˚ res- d Toulouse White Biotechnology, 3 rue des Satellites, F-31400 Toulouse, France olution and in complex with the inhibitor, gabaculine. These e INRA, UMS 1337TWB, F31400 Toulouse, France structures have shown the conformational changes in the enzyme f CNRS 3582, F-31400 Toulouse, France active site during the course of the catalytic reaction. The structure of the class IV Nectria haematococca transam- Among the diversity of transglucosidase enzymes, a specific inase enzyme has been determined in the holo and inhibitor interest is given to the glucansucrases of the GH70 family, which bound form which offers a detailed insight into the structural are produced extracellularly by different lactic acid bacteria: Leu- basis for substrate specificity and enantioselectivity of (R)-selective conostoc sp., Lactococcus sp., Streptococcus sp., Weissella sp. These amine:pyruvate transaminases [1–4]. enzymes are able to use the energy of the osidic linkage of the sucrose molecule (27.6 kJ/mol) to catalyse the efficient transfer of its ␣-D-glucopyranosyl unit. This results in the synthesis of a wide References variety of products, polysaccharides, oligosaccharides and gluco- [1].Sayer S, et al. Acta Cryst 2007;F63:117–9. conjugates, which contain a diversity of linkages: ␣-1,2; ␣-1,3; [2].Sayer S, et al. Acta Cryst 2012;D68:763–72. ␣-1,4; ␣-1,6. [3].Sayer S, et al. Acta Cryst 2013;D69:564–76. It is possible to combine the biochemical and structural char- [4].Sayer S, et al. FEBS J 2014 [in press]. acterization of these transglucosidases with gene sequence data http://dx.doi.org/10.1016/j.nbt.2014.05.1655 and alignement analysis, to develop structure-function relationship studies and molecular engineering based on both rational in silico and combinatorial in vitro approaches. The resulting variants O4-3 present new and improved catalytic properties, resulting in the synthesis of new products. Current developments on the engineering of Escherichia In addition, original transglucosidases have been obtained from coli biofilms for enzymatic biosynthesis of halotrypto- sequencing the genome of several lactic acid bacteria and isola- phans tion of the corresponding genes. They are able to decorate dextran Isaac Vizcaino-Caston 1,∗ , James Thomas Leech 1, Tania Triscari ␣ ␣ ␣ polysaccharides ( -1,6 main backbone linkage) with -1,2 and - Barberi 2, Rebeca J.M. Goss 2, Mark J.H. Simmons 1, TimW.Overton1 1,3 osidic branching and generate a new series of polysaccharides 1 The University of Birmingham, United Kingdom and oligosaccharides. 2 University of St. Andrews, United Kingdom http://dx.doi.org/10.1016/j.nbt.2014.05.1654 Halogenated molecules are being utilized on a daily basis by the fine chemistry and pharmaceutical industries in diverse reac- O4-2 tions for the production of enantiomerically pure compounds. The chemical synthesis of such molecules requires harsh solvents Structural studies on transaminase enzymes and appli- and conditions which result in expensive and environmen- cations in biocatalysis tally unfriendly processes. We propose an alternative to such Jenny Littlechild 1,∗ , C. Sayer 1, M. Isupov 1, J. Ward 2, J. Littlechild 1 methods utilizing recombinant bacterial biofilms to synthesise 5-halotryptophans. The plasmid pSTB7 expressing recombinant 1 University of Exeter, United Kingdom 2 University College London, United Kingdom tryptophan synthase TrpBA was transformed into diverse strains of E. coli and used to generate engineered biofilms [1,2]. These The transaminases (TAs) catalyse the transfer of an amino group biofilms were used as biocatalysts for the bioconversion of 5- from an amino acid to a keto acid using the cofactor pyridoxal 5’- haloindoles into 5-halotryptophans. Biofilm cultures showed phosphate (PLP) and are important for the production of optically higher yield than planktonic bacteria, bacterial lysates or immo- pure amines and amino alcohols used in the synthesis of many bilised TrpBA enzyme [3]. Biofilm topology and viability were important drugs. assessed using confocal microscopy before and after reactions. In order to enable scale-up, a novel method to adhere bacteria to supports was devised. We will discuss the ability of different cell

www.elsevier.com/locate/nbt S19 SYMPOSIUM 4: ROBUST BIOCATALYSTS FOR THE PRODUCTION OF NOVEL BIO-BASED PRODUCTS New Biotechnology · Volume 31S · July 2014 immobilization methods to develop biofilm communities as well O4-5 as their ability to perform biotransformations. Lessons on directed evolution of hydrolases and glucose References oxidase [1].Tsoligkas AN, Winn M, Bowen J, Overton TW, Simmons MJH, Goss RJM. Engineering biofilms for biocatalysis. ChemBioChem Ulrich Schwaneberg 2011;12:1391–5. RWTH – Aachen University, Chair of Biotechnology, Germany [2].Tsoligkas AN, Bowen J, Winn M, Goss RJM, Overton TW, Simmons MJH. Characterisation of spin coated engineered Escherichia coli biofilms using atomic force microscopy. Colloids and Surfaces B: Bioin- Protein engineering by directed evolution and semi-rational terfaces 2012;89:152–60. design has become a standard method to tailor enzyme properties [3].Perni S, Hackett L, Goss R, Simmons M, Overton T. Optimisation to industrial demands. Improving thermal stability and activ- of engineered Escherichia coli biofilms for enzymatic biosynthesis of ity simultaneously is often challenging since high activity often L-halotryptophans. AMB Express 2013;3:66. requires flexibility whereas thermal resistance relies and ‘strong’ http://dx.doi.org/10.1016/j.nbt.2014.05.1656 interactions within a protein. On the example of proteases (BgAP [1,2], S41 [3]) and a phytase [4,5], lessons learned from improving both properties individually and simultaneously will be presented. O4-4 Subsequently, lessons on improving detergent and salt (ionic liquid) resistance of a protease (subtilisin E [6,7]) and a cellulase Structural and biochemical characterization of two (CelA2 [8,9]) will conclude the hydrolase reengineering examples. novel enzymes with promiscuous ene-reductase activity As a highlight, the generation of oxygen independent and Tea Pavkov-Keller 1,∗ , Alexandra Binter 2, Steinkellner Georg 1, highly active glucose oxidase variants (GOx from A. niger) [10,11] Christian C. Gruber 1, Kerstin Steiner 2, Christoph Winkler 3, Helmut will conclude the presentation. Schwab 4, Kurt Faber 3, Peter Macheroux 5, Karl Gruber 6 References 1 ACIB GmbH, C/o ZMB, Austria 2 ACIB GmbH, Austria [1]. Martinez R, et al. Biotechnol Bioeng 2013;110:711–20. 3 Department of Chemistry, University of Graz, Austria [2]. Jakob F, et al. Appl Microbiol Biotechnol 2013;52:2359–63. 4 Institute of Molecular Biotechnology, Graz University of Technology, Austria [3]. Martinez R, et al. Protein Eng Des Sel 2011;24:533–44. 5 Institute of Biochemistry, Graz University of Technology, Austria [4]. Shivange A, et al. J Biotechnol 2014;170:68–72. 6 Institute of Molecular Biosciences, University of Graz, Austria [5]. Shivange AV, et al. Appl Microbiol Biotechnol 2012;95:405–18. [6]. Li Z, et al. ChemBioChem 2012;13:691–9. [7]. Li Z, et al. J Biotechnol 2014;169:87–94. An approach using three-dimensional motifs reflecting specific [8]. Lehmann C, et al. Green Chem 2012;14:2719–3272. active site arrangements (catalophore) was developed in our group. [9]. Pottkämper J, et al. Green Chem 2009;11:691–7. It does not depend on overall protein similarity and therefore [10].Arango Gutierreza E, et al. Biosens Bioelectron 2013;50:84–90. enables the search across enzyme families and the detection of [11].Prodanovic R, et al. Anal Bional Chem 2012;404:1439–47. potential catalytic promiscuity. This catalophore approach led to http://dx.doi.org/10.1016/j.nbt.2014.05.1658 the discovery of two novel enzymes with ene-reductase activity. Enzymes of this family have recently been shown to possess a great potential for (industrial) biotransformations. Neither the amino O4-6 acid sequence of these two enzymes nor their overall structure is related to those of the well-known old yellow enzymes (OYE). Immobilization of carbonic anhydrase for biomimetic These two flavoproteins contain FMN as a cofactor and exist as CO2 capture in slurry absorber homodimers. We cloned, expressed and purified both enzymes ,∗ Sara Peirce 1 , Maria Elena Russo 2, Viviana De Luca 3, Clemente and subjected them to crystallization trials. Obtained crystals Capasso 3, Mosè Rossi 3, Giuseppe Olivieri 4, Piero Salatino 4, Anto- were soaked with putative substrates/inhibitors. The enzymatic nio Marzocchella 4 characterization was pursued by stopped-flow and difference titra- 1 tion experiments. Additionally, several typical OYE substrates (i.e. Dipartimento di Ingegneria Chimica dei Materiali e della Produzione Industri- ale – Università degli Studi di Napoli Federico II, Italy alkenes bearing an electron-withdrawing activating group) were 2 Consiglio Nazionale delle Ricerche – Istituto di Ricerche sulla Combustione, tested to assess the biocatalytic performance. The analysis showed Italy some salient features of typical OYEs as well as some striking 3 Consiglio Nazionale delle Ricerche – Istituto di Bioscienze e Biorisorse, Italy differences, i.e. a stereocomplementary behaviour. In conclusion, 4 Dipartimento di Ingegneria Chimica, dei Materiali e della Produzione Indus- the two novel enzymes can be described as NADPH-dependent triale – Università degli Studi di Napoli Federico II, Italy quinone reductases with significant OYE-like side activities. Novel post-combustion treatments include biomimetic Carbon http://dx.doi.org/10.1016/j.nbt.2014.05.1657 Capture and Storage (CCS) processes based on CO2 absorption into aqueous solution assisted by enzyme catalysis. Carbonic anhydrase

(EC 4.2.1.1) catalyzes CO2 hydration and it has been proposed as industrial biocatalyst for biomimetic CCS processes [Lacroix and Larachi, 2008, Recent Patent Chem Eng; Russo et al., 2013, Sep Pur Technol].

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The present contribution reports results of immobilization of ages to the enzyme itself, the surrounding environment and a carbonic anhydrase (CA) on fine solids. The aim of the study is more effective electron transfer to the redox mediator or elec- twofold: the improvement of the biocatalyst stability at the oper- trode surface. In this study we aimed to identify amino acid ating conditions typical of CCS processes; the confinement of the residues involved in oxygen reactivity. We applied a semi-rational biocatalyst in the CO2 absorption reaction volume during contin- protein engineering approach. Eleven amino acids around the uous operations. Studies reported in the literature suggest the use active site were chosen as target positions for site-saturation muta- of slurry reactors as an effective technology for the maximization genesis. Using a screening assay in 96-well plates with ABTS of the absorption rate enhancement in the presence of heteroge- and 2,6-Dichlorophenolindophenol (DCPIP) [4] five variants with neous catalysis [Alperet al., 1980, Chem Eng Sci; Penders-van Elk decreased oxidase activity (0.1 to 39%) and maintained dehydroge- et al., 2012–2013, Int J Greenhouse Gas Control; Russo et al., 2013]. In nase activity were identified. Our results show that the exchange of agreement with observation reported in the literature, a covalent one amino acid can reduce the unfavourable production of H2O2 CA immobilization technique has been investigated. The carriers of POx without losing activity with alternative e− acceptors. have been fine paramagnetic, non-porous and polyglutaraldehyde References functionalized particles. Bovine CA was used as model enzyme to optimize immobilization procedure and solid biocatalyst activity [1].Leitner C, Haltrich D, Nidetzky B, et al. Appl Biochem Biotechnol 1998;70–72:237–48. assay. [2].Spadiut O, Brugger D, Vasile C, et al. Electroanalysis 2010;22:813–20. Results have pointed out that the activity of the immobilized [3].LeitnerC, Volc J, Haltrich D. Appl Environ Microbiol 2001;67:3636–44. enzymes is satisfactory. The immobilized enzymes may be effec- [4].Brugger D, Krondorfer I, Zahma K, et al. Biotech J 2013, tively used in biomimetic CCS processes. http://dx.doi.org/10.1002/biot.201300336. Further investigation will be focused on the immobilization http://dx.doi.org/10.1016/j.nbt.2014.05.1660 of thermostable recombinant carbonic anhydrase [Capasso et al., 2012, Chem Eng Trans]. http://dx.doi.org/10.1016/j.nbt.2014.05.1659

O4-7

Engineering of pyranose 2-oxidase for modiﬁed oxygen reactivity

Dagmar Brugger 1,∗ , Dietmar Haltrich 2, Clemens Karl Peterbauer 2 1 Food Biotechnology, BOKU Vienna, Austria 2 BOKU Vienna, Austria

The ﬂavoprotein pyranose 2-oxidase (POx) [1] attracts interest as potential component in electrochemical devices [2]. POx catalyses the oxidation of aldopyranoses, preferentially D-glucose, using molecular oxygen, benzoquinone, radicals or chelated metal ions [3] as e− acceptors. Reduced oxygen reactivity is a desirable property for applications in biosensors/biofuel cells because of reduced H2O2 production and consequently reduced dam-

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Symposium 5: Synthetic biology ated genes, so that optimising and testing each control element step by step to achieve desired outcomes is laborious. We have O5-1 engineered chimaeric activators of the ␴54 RNA polymerase to control transcription of the bacterial ntr regulon, comprising over Design and production of new-to-nature antimicrobials 100 genes involved in the regulation, metabolism, scavenging and by synthetic biology transport of nitrogen compounds. Oscar P. Kuipers ∗ , Auke van Heel, Dongdong Mu, Liang Zhou, We functionally combined the signal input and functional out- ␴54 Andrius Buyvidas, Manuel Montalbán-López put domains from various RNA polymerase activators with the dimerisation/promoter recognition domains of the master Ntr reg- Department of Molecular Genetics, University of Groningen, Germany ulator NtrC and established orthogonal signal input/transcription output transfer functions. Such transcription activators allow to Antibiotic resistance in human pathogens is on the rise and activate transcription ‘against the grain’ of the cell physiology. this rise is not met with new approved antibiotics to combat By force driving transcription of dozens of genes and measuring these resistant bacteria. To solve this growing problem of resis- systems changes (transcriptomic, metabolomic and proteomic) we tance, alternative sources of antibiotics should be explored. One could observe which aspects of the cell system resisted or gave way of these sources could be the class of ribosomally synthesized, post- to the synthetic perturbation and at which level along the central translationally modified peptides called lantibiotics. We’ll describe dogma of molecular biology. This revealed inter-network connec- four different approaches to develop novel antimicrobials: tions and control hierarchies between these levels and how they (1) Developing a synthetic biology approach to efficiently use the contribute to cellular robustness. We found that in the synthetic large amount of sequenced genomes (>4000) as a resource for cell system, the intracellular carbon/nitrogen levels can be shifted novel lantibiotics. Here we present the results of the first 25 towards higher nitrogen assimilation compared to the wild type candidates. E. coli. (2) Use of heterologously expressed post-translational modification enzymes to hypermodify lantibiotics. Various docu- http://dx.doi.org/10.1016/j.nbt.2014.05.1662 mented posttranslational modifications have been introduced into lantibiotic peptides broadening the antimicrobial spectrum O5-3 (3) Design and production of novel lantibiotics by ring module- Cofactor uptake in 1,2-propanediol metabolising micro- and hinge-variation. Specific lantibiotic modules have been compartments randomly fitted in a defined architecture, and the nisin ,∗ induction and modification machinery are exploited for the Matthias Mayer 1 , Stefanie Frank 2, Evelyne Deery 2, Andrew required modifications. The screening of more than 10,000 Lawrence 2, Mark Smales 2, Martin Warren 2 chimeric molecules for biological activity has already ren- 1 University of Kent/Warren Group, United Kingdom dered several more active antimicrobials. 2 University of Kent, United Kingdom (4) Biomodules for introducing various types of circular and heterocyclic modifications in lantibiotics. Purpose is to Propanediol utilising microcompartments are proteinaceous design three unique biomodules that introduce heterocyclic, structures consisting of a shell that incorporates enzymes necessary cyclic and circular (head-to-tail) modifications in lantibiotics for 1,2-propanediol degradation. The shell is formed by 7 different (already containing lanthionine rings types of proteins that form hexamers (assemble to form the faces of http://dx.doi.org/10.1016/j.nbt.2014.05.1661 the shell) or pentamers (vertices of the shell) with central pores and is thought to protect the cell from the toxic propionaldehyde intermediate as well as to transport substrate and cofactors across the O5-2 protein shell. The dehydration of 1,2-propanediol to propionaldehyde, which is catalysed by the protein complex PduCDE, is B12 Synthetic transcription factors allow regulon wide (adenosylcobalamin) dependent. Previously it was shown that the control and shifting the nitrogen/carbon balance in pdu microcompartment houses enzymes to regenerate B12. Uptake bacteria of B12 into enteric cells and reactivation are well described, but it still remains unclear if B is transported or accumulated inside Jorg Schumacher 1,∗ , Baojun Wang 2, Ana Claudia Bonatto 3, Martin 12 Pdu microcompartments. Buck 1 We investigated if Pdu microcompartments take up or 1 Imperial College London, UK accumulate B12. Recombinantly expressed and purified empty 2 University of Edinburgh, UK microcompartments (shell proteins) and full Pdu microcompart- 3 Universidade Federal do Paraná, Curitiba, PR, Brazil ments (fully expressed pdu operon) showed a higher content of B12 than the crude cell lysate, suggesting that Pdu microcompartments Synthetic approaches to enhance the production of a desired have an inherent ability to bind and gather B . Different cobal- product commonly involve altering the DNA control sequences 12 amins were found to be co-purified with both the empty and the regulating expression of the enzymes required. However, many fully expressed microcompartments. This finding was confirmed metabolic pathways involve many core and functionally associ- by in vitro co-localisation studies of purified microcompartments

S22 www.elsevier.com/locate/nbt New Biotechnology · Volume 31S · July 2014 SYMPOSIUM 5: SYNTHETIC BIOLOGY with fluorescently labelled B12. The uptake mechanism remains O5-5 unclear and is part of our current research. An understanding of transport along the protein shell and its Novel tuneable gene expression systems based on ortho- selectivity is an important landmark in our efforts to generate engi- gonal riboswitches neered compartments capable of sequestering complex synthetic Neil Dixon metabolic pathways. MIB – Manchester, United Kingdom http://dx.doi.org/10.1016/j.nbt.2014.05.1663 Strategies that permit precisely controlled, differential, and simultaneous expression of multiple genes would be extremely O5-4 useful for a broad range of metabolic engineering, protein expression and synthetic biology applications. Use of transporter plug-ins in building effective micro- A new paradigm in genetic regulation emerged with the dis- bial cell factories for chemical and fuel production covery of novel genetic regulatory elements within the 5 UTR Christopher Grant ∗ , Phattaraporn Morris, Frank Baganz of bacterial mRNA. Upon binding to a specific metabolite, these University College London, United Kingdom so-called ‘riboswitches’ change conformation, permitting differential gene regulation to occur. As these switches operate via a Synthetic biology hopes to predictably build cellular factories small molecule-dependent, protein-free mechanism, they present for the production of chemicals, pharmaceuticals and fuels from themselves as attractive targets for use as novel genetic control cheap renewable feedstocks. A critical, yet understudied, element elements. of this is control of compound transport both into and out of Previously, we showed that it is possible to re-engineer the cell as a means of avoiding issues such as substrate access riboswitches so that they no longer bind to their original cognate limitations, substrate and product inhibition and aiding product ligands, but are instead activated by synthetic ligands, and further recovery. that these ‘orthogonal’ riboswitches can be used to control het- We report here a successful strategy for the contextual evalua- erologous gene expression in vivo [Dixon et al., PNAS 2010]. We tion of this topic using a library of transport modifying plug-ins have further developed these into multi-component systems that combined with multifactorial characterisation. Auxiliary plas- permit fine-tuning over co-expression output stoichiometry, with mids (pUMP) were used to express a library of transporters as wide ranging potential applications in functional and structural plug-ins alongside two biosynthesis plasmids pGEC41, which oxi- analysis [Dixon et al., Angew Chem, 2012]. dises hydrocarbons into fatty alcohols and pADAR7942 which Finally, I will discuss the development of these cellular systems synthesizes bioalkanes from metabolic fatty acid precursors. We and molecular devices from simply proof-of-principle studies, into demonstrate here benefits of the transporter plug-in approach for: the tuneable modular expression system RiboTite, and demonstrate (i) Facilitated delivery hydrophobic substrates to improve the application of this expression technology for the production whole-cell biocatalysis rates by up to 70 fold of proteins of biotechnological interest.

(ii) Industrially relevant product yields of over 40 g/Lorganic phase http://dx.doi.org/10.1016/j.nbt.2014.05.1665

(8 g/Ltotal) (iii) Reducing byproduct formation in whole-cell bioconversion of alkanes to alkanols O5-6 (iv) Improving bioalkane synthesis yields from glycerol by >5 fold (v) Reducing alkanol and aldehyde intermediate formation in Signal transduction engineering: a powerful platform biosynthesis of bioalkanes by > 10 fold technology for enhancing secondary metabolite produc- (vi) The integration with in situ product removal strategies to tion improve bioalkane yields by 10 fold compared to the starting Jian-Jiang Zhong ∗ , Yi-Ning Xu, Gao-Yi Tan, Linquan Bai process. Shanghai Jiao Tong University, China This library and plug-in approach is of broad appeal for biological production of hydrophobic compounds and could be a key Streptomycetes and higher fungi produce many bioactive enabling technology for biological routes for producing a wider secondary metabolites. Ganoderic acids (GAs) produced by Gan- range of hydrophobic compounds such as biofuels, fine and spe- oderma lucidum, a higher fungus, have significant anti-tumor and cialty chemicals and pharmaceutical intermediates. anti-metastasis activities. Validamycin, an anti-fungal antibiotic http://dx.doi.org/10.1016/j.nbt.2014.05.1664 produced by Streptomyces hygroscopicus 5008, is an efficient rice sheath blight controller, and can be used as the source for chemical synthesis of voglibos, an antidiabetic drug. Engineering of regulatory mechanism was done to enhance the production of target secondary metabolites. The effects of metal ions on the GAs biosynthesis in liquid cultures of G. lucidum were investigated, and the increased enzyme activities and up-regulation of transcriptional levels of genes in the triterpene biosynthesis were

www.elsevier.com/locate/nbt S23 SYMPOSIUM 5: SYNTHETIC BIOLOGY New Biotechnology · Volume 31S · July 2014 observed. The regulation mechanism of Mn2+ on the GA biosyn- so as to improve the engineering efficiency, we devised two thesis was found to be via calcineurin signaling transduction. For novel methods termed as “Genome Replication Engineering the validamycin biosynthesis, our previous study indicated the Assisted Continuous Evolution (GREACE)” and “Stress-Induced- involvement of A-factor-like cascade. A recent genome-wide anal- Mutagenesis Based Adaptive Evolution (SIMBAE)”. Both methods ysis reveals three pairs of afsA-arpA in S. hygroscopicus 5008. This implant controllable in vivo mutagenesis machineries into the cell. work aims to decipher the regulatory role of the multiple afsA-arpA Starting the mutagenesis machineries under stressful conditions homologs in the A-factor-like cascade, and then to improve the will trigger continuous mutation generation andsynchronous validamycin production by engineering the regulatory cascade. By selection process, and then result a continuous and efficient evolu- double deletion of shbR1/R3, the transcriptions of adpA-H and the tion process which enabled “Mutagenesis coupled-with Selection”. validamycin biosynthetic genes were up-regulated, and the vali- Specifically, GREACE uses a library of activity-compromised proof- damycin production and productivity were enhanced significantly reading elements of the main DNA polymerase during genome for both the wide-type and a high-producing industrial strain. The replication as the mutagenesis machinery. In SIMBAE, the muta- transcriptomic analysis revealed that the engineering of A-factor- genic state is generated by constructing a SIM module which like signaling cascade caused a shift from primary to secondary re-produces complex cellular stress-responses implemented by up- metabolism. The signal transduction engineering proposed here is regulation and down-regulation of various genes. Either method very useful for efficient production of those secondary metabolites is capable of increasing genomic mutation rate up to 3000-fold. in cultivation processes. Significantly improved chemical tolerance (n-butanol, acetate, kanamycin), themotolerance, and osmotic tolerance of E. coli were http://dx.doi.org/10.1016/j.nbt.2014.05.1666 achieved within 9–90 days using the two methods. http://dx.doi.org/10.1016/j.nbt.2014.05.1667 O5-7

Development of two continuous genome engineering strategies for efﬁcient microbial evolution

Zhen Cai ∗ , Guodong Luan, Linjiang Zhu, Yin Li Institute of Microbiology, Chinese Academy of Sciences, China

Engineering complex phenotypes of microbes, for instance stress tolerance, remains to be a big challenge in this field. Current evolutionary engineering methods including physical and chemical mutagenesis, global transcription machinery engineering, and artificial transcription factors engineering, use “Mutagenesis followed-by Selection” as the core principle. That is, mutations or genetic perturbations are firstly introduced by exogenous mutagens or genetic manipulations, followed by selection of desired phenotypes. Thus iterative rounds of mutagenesis-selection and frequent manual interventions are often required, resulting in discontinuous and inefficient strain improvements. To address the discontinuity of the existing evolutionary engineering approaches

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Symposium 6: Assimilation of CO2, CO and production. Microalgal strains M. afer PKUAC 9 and S. abun- CH4 into biobased products dans PKUAC 12 isolated from coastal waters of Pearl River Delta region were used for saccharification and subsequent fermenta- O6-1 tive bioethanol production where as Hindakia sp. PKUAC 169 and Chlorella sp. PKU AC 102 were optimal for biodiesel production. Microbial fixation of CO2 in water bodies and in drylands Hindakia sp. PKUAC 169 isolate was successfully cultivated in two to combat climate change, soil loss and desertification derivatives of BG11 medium: nitrogen starved and salt induced. Both methods yielded improved lipid accumulation, but only salt Roberto De Philippis induction resulted in increased overall lipid productivity. Derivati- Department of Agrifood Production and Environmental Sciences, University of sation of algal lipids to FAMEs showed different lipid profiles under Florence, Florence, Italy selected growth conditions and suggest that salt induced medium is better suited for biodiesel production than nitrogen starved The growing concern for the increase of the global warm- medium. Optimisation of heterotrophic cultivation of algal strain ing effects raises the challenge of finding novel technological Chlorella sp. PKUAC 102 resulted in tenfold higher lipid produc- approaches to stabilize CO emissions in the atmosphere. 2 tivities than autotrophically grown strains. Biological-CO2 mitigation, triggered through biological fixation, is considered a promising and eco-sustainable method. Microor- http://dx.doi.org/10.1016/j.nbt.2014.05.1669 ganisms such as cyanobacteria, green algae and some autotrophic bacteria could potentially fix CO2 more efficiently than higher plants, due to their faster growth. Some examples of the poten- O6-3 tial of Biological-CO mitigation will be reported and discussed in 2 Exploring the potential of microalgae for bioenergy pro- this lecture. duction In arid and semiarid environments, soil carbon sequestra- ∗ tion (CO2 fixation) by cyanobacteria and Biological Soil Crusts Frank Baganz , Yanan Xu, Paul Hellier, Nicos Ladommatos, Saul is considered an eco-friendly and natural process to increase soil Purton C content and a viable pathway to soil restoration after one University College London, United Kingdom disturbance event. Another way for Biological-CO2 mitigation intensively studied in the last few years is related to the possibility Intensive research is being applied to biofuel production from to perform carbon dioxide sequestration using microalgae, obtain- algal biomass owing to their fast growth rates and high lipid con- ing at the same time bioproducts of industrial interest. Another tent. This work explores key steps in algal bioprocessing focusing possibility under study is the exploitation of specific chemotrophic on algal biomass harvesting through flocculation with chitosan; bacteria for CO2 fixation coupled with the production chemicals and the use of algal biomass for engine combustion. such as polyhydroxyalkanoates. In spite of the potential of these Initially flocculation with chitosan to harvest algal biomass of processes, multiple factors still have to be optimized in order to the green alga Chlorella sorokiniana was explored. Chitosan proved achieve a cost-effective CO2 sequestration. to be highly efficient in the induction of flocculation with the clar- http://dx.doi.org/10.1016/j.nbt.2014.05.1668 ification efficiency reaching >99% below pH 7 at optimal dosage. Influencing factors of flocculation efficiency and its effect on the subsequent dewatering process were also evaluated. It was shown O6-2 to reduce the volume to be processed by 20–50 folds, and significantly reduce energy input and material costs of centrifugation Microalgal biofuels from native biological resource of or filtration operations. In order to reduce the complicated and Pearl River Delta costly downstream processes of algal biofuel production, this work Maurycy Daroch ∗ , Zongchao Jia, Cong Shao, Hui Guo, Ying Liu, Jay explores an alternative way of utilizing energy from algal cells by J. Cheng blending algal slurry into fossil diesel using specific combinations of surfactants. We will show that this approach provides benefits by Peking University School of Environment and Energy, China reducing the consumption of fossil fuel and also reducing exhaust emissions particularly NOx thus benefiting the environment. Algal biofuels are seen as promising solutions of global energy crisis and climate change for the years to come. Major advantages http://dx.doi.org/10.1016/j.nbt.2014.05.1670 of algae are potentially high yield and no competition with food crops for arable land and fresh water. Although the coastal areas of Pearl River Delta have long been known for huge diversity of aquatic life little work has been done to assess the possibility of using local microalgae resources for biofuel production. Our study fills this niche and tests the feasibility of producing renewable fuels ethanol and biodiesel from local algal strains. A total of 89 unique algal strains from Peking University Algae Collection were isolated and screened as feedstocks for biofuel

www.elsevier.com/locate/nbt S25 SYMPOSIUM 6: ASSIMILATION OF CO2, CO AND CH4 INTO BIOBASED PRODUCTS New Biotechnology · Volume 31S · July 2014

O6-4 exclusion chromatography. Individual fractions were analyzed for the capability to increase the activity of a commercial cellulase Development of luminescent photobioreactors for mix. Fractions showing the highest cellulolytic activity were ana- improved microalgae cultivation lyzed by LC GC/MS. Two new polysaccharide monooxygenases Seyedeh Fatemeh Mohsenpour ∗ , Nik Willoughby (PMO, former GH61) and one xylanase were identified, amongst other hydrolytic and oxidative enzymes. Corresponding genes Heriot-Watt University, United Kingdom were cloned by reverse transcription and PCR, and sequenced. Pro- teins GtLPMO9A, FoLPMO9A and GtXyl1 were produced in the Effect of light conditions on the growth, photosynthetic pig- heterologous host Pichia pastoris and purified. Different combina- ments and lipid production of green algae Chlorella vulgaris tions of these enzymes with cellulases, in reactions with wheat and cyanobacteria Gloeothece membranacea was investigated. The straw as substrate, were tested for production of glucose and xylose. microalgae were cultivated in luminescent acrylic bubble column Results indicate that GtLPMO9A, FoLPMO9A and GtXyl1 increase photobioreactors (PBR) under varying conditions. Luminescent the production of monomer sugars, suggesting that they could be acrylic PBR in blue, green, yellow, orange, and red ranges, capa- useful tools for improvement of lignocellulose degradation. ble of spectral conversion of simulated full-spectrum daylight were used. The results showed significant variations in culture Acknowledgement growth and photosynthetic pigmentation dependent on culture conditions and illumination. The highest biomass productivity of This work was financed by Fondecyt, Project 1121088. 184 mg L−1 day−1 was obtained in red luminescent PBR. Increas- ing the initial culture density reduced the rate of productivity http://dx.doi.org/10.1016/j.nbt.2014.05.1672 although lipid accumulation was significantly induced. Blue and yellow PBR doubled lipid accumulation up to 11% in high density cultures. However, blue PBR provided the least efficient light O6-6 condition for bio-pigment production. Red PBR was the most effec- Engineering biofuel producing microbes for efficient tive portion of PAR ranges to enhance chlorophyll production hemicellulose utilisation using synthetic biology up to 0.75% in G. membranacea whilst green PBR induced pigmentation in C. vulgaris. Phycobiliproteins were the dominant Gavin Thomas 1,∗ , Rosanna C. Hennessy 1, Henrique I. Neves 1, pigments in G. membranacea and red and green light favoured Preben Krabben 2, Elizabeth Jenkinson 2 synthesis of these pigments. The average production of phyco- 1 University of York, United Kingdom erythrin and phycocyanin was slightly improved in high density 2 Green Biologics Limited, United Kingdom cultures. The proposed illumination strategy offers improved microalgae Rapid depletion of fossil fuels and oil reserves is driving the growth without resorting to artificial light sources, reducing energy development of biofuels to replace reliance on petroleum usage. use and costs of cultivation. Hemicellulose is the second most abundant renewable polymer http://dx.doi.org/10.1016/j.nbt.2014.05.1671 on Earth, and thus an attractive resource for the production of bulk chemicals and fuels (e.g. biobutanol). Development of biomass-derived biofuels is however challenged by the recalci- O6-5 trance of lignocellulosic materials to degradation. Harsh and costly pretreatment methods are required to break down struc- Identification of new auxiliary enzymes for the hydrol- turally robust plant biomass to fermentable sugars, which can ysis of lignocellulose release inhibitors and retard subsequent fermentations. An alter- ∗ native method is microbial-based enzymatic degradation. While Oriana Salazar , Gonzalo Carvajal, Javier Devia, Tania Quiroz diverse hemicellulolytic bacteria have been identified, many are University of Chile, Chile poorly characterised with limited genetic manipulation tools and importantly lack industrially relevant pathways. A synthetic biol- Despite important technological advances, the cost of lignocel- ogy approach is therefore being used to engineer hemicellolytic lulosic bioethanol as an energy source is still high compared to ability into butanol producing bacteria. Synthetic gene clusters fossil fuels. Cellulose hydrolysis is carried out by the action of cel- comprising a minimal enzyme system required to catalyse the lulases and, given the recalcitrance of the biomass high loading common hemicellulose xylan will enable such bacteria to trans- of enzymes is required, impacting significantly the total cost of port and metabolise hemicellulose-derived sugars more efficiently the process. Efficient hydrolysis of lignocellulose depends on the to butanol. Synthetic gene clusters will include endoxylanases and concerted action of cellulases and auxiliary proteins, which can beta-xylosidases to hydrolyse xylan into xylose monomers but also help to reduce cellulase loading in the conversion to monomer enzymes that hydrolyse side chains or modifications. As the rate of sugars. substrate uptake is a key factor in determining the rate of product In order to identify auxiliary proteins from rot fungi and formation, transporters will be an important design consideration. improve the action of cellulases on pretreated wheat straw, extra- At the bioprocess level, milder pretreatment procedures will enable cellular proteins from Gloeophyllum trabeum, Fusarium oxysporum deployment of a cost-competitive technology with reduced envi- and Trametes versicolor cultured in wheat straw were fractioned by

S26 www.elsevier.com/locate/nbt New Biotechnology · Volume 31S · July 2014 SYMPOSIUM 6: ASSIMILATION OF CO2, CO AND CH4 INTO BIOBASED PRODUCTS ronmental impact for the production of biofuels from renewable cell mass yield of glucose and wood hydrolyzates were 0.5 g/g. resources. Lipid contents from 10 to 90% of cell mass, bioreactor productivity http://dx.doi.org/10.1016/j.nbt.2014.05.1673 of 0.5–64 g/L/h and plant capacity of 20,000–1,000,000 tons/year were assumed. A $0.902/kg-lipid was predicted with $0.2/kg wood hydrolyzates and $0.1/kg VFAs with productivity, 12 g/L/h and O6-7 capacity, 100,000 tonnes/year and 75% lipids content. The raw materials accounted for 55.7% of total operating costs followed Economic assessment of microbial lipids for biodiesel by direct ﬁxed cost dependent 17.4%. A 50% Chemical oxygen production: Competitiveness with microalgae and agri- demand (COD) recycle of waste empty cell mass to VFAs further cultural plant oils reduced the cost to $0.886.A biodiesel from microbial lipid has a potential to become competitive with diesels from other sources. Ho Nam Chang ∗ , Gwon Woo Park http://dx.doi.org/10.1016/j.nbt.2014.05.1674 KAIST, Republic of Korea

For economic assessment of microbial lipids volatile fatty acids (VFAs) derived from waste organics was used as low cost carbon source and multi-stage continuous high cell density culture (MSC- HCDC) process for high bioreactor productivity and titer. The experimental lipid yield was 0.27 g/g-VFA, and the experimental

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Symposium 7: Applications of metabolic mod- [8].Driouch H, Melzer G, Wittmann C. Metab Eng 2012;14:47. elling [9].Becker J, Zelder O, Häfner S, Schröder H, Wittmann C. Metab Eng 2011;13:159.

O7-1 http://dx.doi.org/10.1016/j.nbt.2014.05.1675

Making use of metabolic models – in silico driven design and engineering of industrial microorganisms O7-2 Christoph Wittmann Design of optimally constructed metabolic networks of Institute of Systems Biotechnology, Saarland University, 66123 Saarbrücken, minimal functionality Germany David Ruckerbauer ∗ , Christian Jungreuthmayer, Jürgen Zanghellini Superior microorganisms that produce chemicals, materials ACIB GmbH, Austria and fuels from renewables are major drivers of the developing bio-based economy. Firstly, they hold the key to the optimized Background: Metabolic engineering aims to design microor- production of traditional bio-based products regarding key perfor- ganisms that will generate a product of interest at high yield. Thus, mance indicators such as titer, yield and productivity [1]. Secondly, a variety of in silico modeling strategies has been applied success- they enable future production of important petrochemicals from fully, including the concepts of elementary flux modes (EFMs) and green resources rather than from fossil fuels [2]. Without doubt, constrained minimal cut sets (cMCSs). systems metabolic engineering is changing the way to design and The EFMs (minimal, steady state pathways through the system) optimize such microbial cell factories for industrial production: can be calculated given a metabolic model. cMCSs are sets of reac- the integration of systems biology and systems biotechnology with tion deletions in such a network that will allow desired pathways new concepts from synthetic biology enables the global analysis to survive and disable undesired ones (e.g., those with low product and engineering of microorganisms–and bioprocesses at efficiency secretion or low growth rates). Grouping the modes into desired and versatility otherwise not accessible. In this regard, the lec- and undesired categories had to be done manually until now. ture will highlight the integration of model-based prediction and Results: Although the optimal solution for a given set of design into industrial strain engineering: driven by massively pathways will always be found with the currently available tools, increasing genomic information, stoichiometric models provide manual selection may lead to a sub-optimal solution with respect valuable insights into the properties of metabolic networks and to a metabolic engineering target. A small change in the selection give access to theoretical maximum yields, optimal pathways and of modes can reduce the number of necessary deletions while only preferred production hosts [3]. Most importantly, metabolic mod- slightly reducing production. Based on our recently introduced for- els can even guide smart and straightforward engineering of cells mulation of cut set calculations using binary linear programming, and processes [4]. The use of models for decision-making and we suggest an algorithm that does not require manual selection of strategic developments appears particularly useful for industrial the desired pathways. application. Model-based approaches are fast, feasible at limited Conclusions: We demonstrated the principle of our algorithm level of knowledge in early project stages and allow the investiga- with the help of a small toy network and applied it to a model of tion of different scenarios, all relevant for short development time E. coli using different design objectives. Furthermore we validated and high performance required. Different examples from indus- our method by reproducing previously obtained results without trial biotechnology will demonstrate the power of using metabolic requiring manual grouping of modes. models: bio-based production of chemicals [5,6], polymers [7] and http://dx.doi.org/10.1016/j.nbt.2014.05.1676 recombinant proteins [8]. Regarding industrial lysine production model-based design has provided synthetic strains, which reach the high performance of classical producers derived over the past O7-3 fifty years [9]. References What is the relationship between intracellular and extracellular metabolites? The theory of “metabolic [1].Becker J, Wittmann C. Curr Opin Biotechnol 2012;23:718. overflow” put into test [2].Becker J, Wittmann C. Curr Opin Biotechnol 2012;23:631. [3].Krömer JO, Wittmann C, Schröder H, Heinzle E. Metab Eng Silas Villas-Boas 1,∗ , Ting-Li Han 1, Tim Liu 1, Sang Kim 1, Sonia 2006;8:353. Carneiro 2, Eugenio Ferreira 2, Isabel Rocha 2 [4].Melzer G, Eslahpazir M, Franco-Lara E, Wittmann C. BMC Syst Biol 2009;3:12. 1 The University of Auckland, New Zealand [5].Buschke N, Becker J, Schäfer R, Kiefer P, Biedendieck R, Wittmann C. 2 University of Minho, Portugal Biotechnol J 2013;8:557. [6].Kind S, Neubauer S, Becker J, Yamamoto M, Völkert M, Weong WK, Compared to our knowledge on metabolic pathways and the et al. Metab Eng 2014 [in press]. [7].Poblete-Castro I, Binger D, Rodrigues AL, Becker J, Dos Santos V, establishment of tools to manipulate these pathways, we know Wittmann C. Metab Eng 2013;15:113. very little about the mechanisms behind the secretion of metabolic intermediates. Microorganisms secrete a wide range of metabolic intermediates, and many of them are of great industrial interest.

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Despite the cellular process of metabolite efflux being ubiquitous E. coli. For instance, our analysis confirms the inactivity of gluta- to all microbial cells, we still do not know clearly how this process mate dehydrogenase in E. coli grown on glucose. works and how it is regulated. It is believed that small metabolites, Conclusion: Thermodynamic EFM analysis successfully inte- mainly those end products of fermentation are excreted through grates the metabolome into an EFM analysis. It is solely based on the plasma membrane passively, or they are secreted through first principles, and allows for an unbiased characterization of the specialised mechanisms such as vectorial reaction in response to metabolic capabilities in genome-scale metabolic networks. hypo-osmotic stress, or uniport and synport transport systems. http://dx.doi.org/10.1016/j.nbt.2014.05.1678 However, all of these mechanisms are based on the concept of “metabolic overflow”, which under specific metabolic conditions it is observed a massive excretion of some metabolic intermedi- O7-5 ates due to their intracellular accumulation. Although this concept seems appropriate to explain the secretion of some intracellu- Genome scale metabolic modeling of recombinant pro- lar metabolites, it does not apply to many cases studied during tein producing yeasts: prediction of process parameters continuous culture. Our metabolomics data obtained during dif- and metabolic engineering targets for efficient produc- ferent time-series studies of microbial growth under continuous tion and batch cultures confirm that the concept of “metabolic over- Diethard Mattanovich flow” cannot be applied to explain the efflux of several intracellular metabolites found in the extracellular medium. Most of our studies University of Natural Resources and Life Sciences Vienna, Austria indicate that microbial cells very often get rid of some intracellular metabolites in response to an environmental stimulus, Genome scale metabolic models have been successfully even if these metabolites are key intermediates of central carbon applied to predict genetic interventions to redirect metabolic metabolism. fluxes towards desired products of the primary and secondary metabolism. Complex polymeric products like heterologous pro- http://dx.doi.org/10.1016/j.nbt.2014.05.1677 teins equally demand redistributions of primary metabolic fluxes, their rational design however is far less obvious. Therefore cell engineering for protein overproduction has concentrated mainly O7-4 to transcription, codon usage, protein folding and secretion. Towards genome-scale metabolic pathway analysis: We have developed the first genome scale metabolic model for metabolome integration allows efficient enumeration of the yeast Pichia pastoris (PipMBEL1254), and integrated the synthe- elementary flux modes in metabolic networks sis of heterologous protein. The model could successfully predict the increase of protein production in oxygen limited conditions, as Jürgen Zanghellini ∗ Matthias P. Gerstl David Ruckerbauer Chris- , , , well as changes of central metabolic fluxes in production strains, tian Jungreuthmayer as measured by 13C flux analysis. Metabolic engineering targets Austrian Centre of Industrial Biotechnology, Austria for enhanced protein productivity were predicted with FSEOF for gene overexpression and MOMA for gene deletion. After deleting Background: Elementary flux mode (EFM) analysis is used to or overexpressing the respective genes as predicted more than 50% identify all non-decomposable steady state pathways in metabolic of the interventions led to an enhanced production of cytosolic networks. EFMs represent minimal functional building blocks. human superoxide dismutase (hSOD), and similarly of other pro- They can be used to characterize metabolic phenotypes and cellu- teins. Beneficial mutations were mainly related to reduction of the lar robustness or identify targets in metabolic engineering. Despite NADP/H pool and the deletion of fermentative pathways. its potential EFM analysis is currently restricted to medium scale We demonstrate that genome scale metabolic modeling is suit- models as the number of EFMs in a network explodes with the able to describe metabolic changes in recombinant strains and network’s size. can be successfully applied to design genetic interventions to the Method: We present a novel computational method that inte- primary metabolism to increase recombinant protein production. grates the cellular metabolome into an EFM analysis. As the http://dx.doi.org/10.1016/j.nbt.2014.05.1679 metabolome directly maps to the Gibbs free energy surface of the reaction network, we are able to identify and remove thermodynamically infeasible EFMs during the runtime of an analysis O7-6 without impacting any biologically relevant EFMs. Thermody- namic infeasibility is determined in the framework of network Use of a novel combinatorial genetics platform to rapidly embedded thermodynamics. In this way we curb the explosion clone, express and select target biocatalytic activities for of the number of EFMs in large networks. multigenic metabolic pathway optimization Results: We present details of our implementation and demon- Ian Fotherigham strate that our new approach successfully tackles the major bottleneck strongly reduce the memory consumption. Thus an Ingenza, Ltd., United Kingdom unbiased characterization of large-scale metabolic models is made possible. In fact, we present the first thermodynamically consis- Replacement of petroleum-based products and manufactur- tent EFM analysis of iJE660a, a genome-scale metabolic model of ing processes with competitive bio-based alternatives is attracting

www.elsevier.com/locate/nbt S29 SYMPOSIUM 7: APPLICATIONS OF METABOLIC MODELLING New Biotechnology · Volume 31S · July 2014 increased attention due to environmental concerns surrounding For implementation of the RuMP pathway, three heterol- petroleum sustainability and supply. Replacement of conventional ogous genes encoding methanol dehydrogenase, 3-hexulose-6- processes for manufacturing valuable industrial products and the phosphate synthase, and 6-phosphate-3-hexuloisomerase were selection of optimal biosynthetic routes requires the construc- expressed in C. glutamicum wild type. All other enzymes required tion, and in most cases subsequent context-dependent evaluation, for conversion of fructose-6-phosphate and regeneration of and optimization of multicomponent biosynthetic pathways to ribulose-5-phosphate as C1-acceptor can be recruited from endoge- generate intermediates and end products. This talk will present nous metabolism. The recombinant strain metabolized methanol the use of Ingenza’s proprietary combinatorial genetics platform at a rate of 500 ␮mol h−1 (g CDW)−1 during growth on glu- (inABLE®) to rapidly clone, express, select and optimize target cose/methanol mixtures. activities for many separate enzymatic reactions, from thousands Further studies revealed, however, that C. glutamicum wild type of independent genes derived from metagenomic and phyloge- is able to oxidize methanol via formaldehyde and formate to CO2 netic discovery approaches. Multiple gene variants comprising with a rate of 200 ␮mol h−1 (g CDW)−1. The alcohol dehydroge- of up to ten individual genetic elements are combined in sin- nase AdhA is mainly responsible for the oxidation of methanol to gle reactions, generating expression libraries with hundreds or formaldehyde, whereas the oxidation of formaldehyde is predomi- thousands of members in diverse heterologous configurations for nantly catalyzed by two enzymes, the acetaldehyde dehydrogenase HTP interrogation. Obvious synergy exists between this approach Ald and the mycothiol-dependent formaldehyde dehydrogenase and versatile, solid phase screening and selection methods using AdhE. The resulting formate is oxidized via formate dehydroge- growth-based, crossfeeding or colorimetric methods to identify nase FdhF to CO2 [1,2]. A C. glutamicum mutant lacking Ald and colonies of interest. This is illustrated through the rapid identifica- AdhE activity is severely impaired in its ability to oxidize formalde- tion of critical pathway enzymes, optimal gene coding sequences hyde to CO2 and thus, represents a promising strain to enforce and enzyme variants from inABLE®-derived high quality variant methanol assimilation and prevent dissimilation. libraries for bio-based polymer production. The technology aims References to bring increasing predictability and overcome limitations associated with iterative and empirical processes for microbial strain [1].Witthoff S, Eggeling L, Bott M, Polen T. Microbiology 2012;158:2428–39. improvement. The successful realization of optimal target reac- [2].Witthoff S, Mühlroth A, Marienhagen J, Bott M. Appl Environ Micro- tions enables rapid pathway definition and progression to process biol 2013;79:6974–83. optimization and scale-up. http://dx.doi.org/10.1016/j.nbt.2014.05.1681 http://dx.doi.org/10.1016/j.nbt.2014.05.1680

O7-7

Methanol – a potential carbon source for Corynebac- terium glutamicum

Sabrina Witthoff ∗ , Jan Marienhagen, Michael Bott Forschungszentrum Jülich, Germany

Methanol represents an interesting carbon source for microbial production processes, but cannot be assimilated by Corynebac- terium glutamicum, an industrial producer of >4 million tons of amino acids and a model organism in white biotechnology. We therefore attempted to introduce the ribulose monophosphate (RuMP) pathway for methanol assimilation in this species.

S30 www.elsevier.com/locate/nbt New Biotechnology · Volume 31S · July 2014 SYMPOSIUM 8: INDUSTRIAL BIOTECHNOLOGY OF NATURAL AND SYNTHETIC POLYMERS

Symposium 8: Industrial biotechnology of by open mixed microbial cultures. Lab-scale bioreactors, inocu- natural and synthetic polymers lated with soil as a source of microorganisms, were operated under anaerobic conditions and fed with either glucose or cellulose (two O8-1 different particle sizes) as only carbon sources. Ethanol yield up to approx. 20% of the theoretical value was obtained with glucose. Green polymer processing with enzymes In the cellulose-fed reactors, cellulose hydrolysis occurred and it was found to occur faster with the smaller particle size. Enrique Herrero Acero a , Doris Ribitsch a, Veronika Perz a, Alessan- dro Pellis b, Antonino Biundo b, Katrin J. Greimel a,b, Gibson S. http://dx.doi.org/10.1016/j.nbt.2014.05.1683 Nyanhongo b, Georg M. Guebitz a,b,∗ a Austrian Centre of Industrial Biotechnology, Konrad Lorenz Strasse 20, 3430 Tulln, Austria O8-3 b University of Natural Resources and Applied Life Sciences, Vienna, Konrad Lorenz Strasse 20, 3430 Tulln, Austria Strategies for enzymatic functionalization of synthetic polymers

Environmentally friendly reaction conditions and the highly Enrique Herrero Acero 1,∗ , Caroline Gamerith 1, Andreas Ortner 1, specific mode of action are among the advantages of enzyme based Doris Ribitsch 1, Georg Steinkellner 1, Karl Gruber 2, Helmut processing of (bio)synthetic polymers when compared to chemi- Schwab 3, Georg M. Guebitz 4 cal processes. In coatings, enzymes can replace toxic metal based 1 ACIB GmbH, Austria catalysts or improve dispersing properties of lignins. Their surface 2 University of Graz, Austria specific action is exploited in functionalization of synthetic mate- 3 Graz University of Technology, Austria rials to impart e.g. antimicrobial properties. On the other hand, 4 University of Natural Resources and Applied Life Sciences, Vienna, Austria enzymes have a potential for specific recovery of polymer building blocks from composite materials or mixed plastics waste. To Polymers have a wide range of uses because of their outstanding improve the efficiency of enzymes on non-natural polymeric sub- bulk properties, but their surfaces are lacking of desired reactiv- strates, engineering enzyme surface properties and attachment of ity or properties. Current activation processes rely in the use of binding modules are useful strategies together with engineering of concentrated acids/alkali solution or plasma approaches. While the active site architecture [1–4]. harsh chemicals typically decrease the polymer properties, plasma treatment are very energy demanding and unable to function- References alize complex geometries like tubes. Enzymes have revealed as [1].Herrero Acero E, et al. Biotechnol Bioeng 2013;110(10):2581–90. powerful catalyst able to overcome all these limitations. Enzymes [2].Ribitsch D, et al. Biomacromolecules 2013;14(6):1769–76. are able to partially hydrolase the upper most layers of polymers [3].Greimel KJ, et al. Green Chem 2013;15(2):381–438. in a controlled manner, introducing anchor groups for subse- [4].Nugroho Prasetyo E, et al. Bioresour Technol 2010;101(14):5054–62. quent grafting. Following this approach it was possible to graft http://dx.doi.org/10.1016/j.nbt.2014.05.1682 Human Serum Albumin (HSA) to polylactic acid (PLA) membranes in which the polymer was activated in a first step with a cutinase [1]. The functionalized material had a higher hydrophilicity, O8-2 antioxidant capacity, which improved its biocompatibility. Using a more elaborated double enzymatic reaction it was possible to Open mixed cultures for bioethanol production from lig- covalently graft ferulic acid on polyamide (PA) fibers [2]. In order nocellulosic materials to improve the enzymatic process we have improved the polymer- Davide Dionisi ∗ , Tom Adams, Craig Dempster enzyme interaction via rationale design of the enzyme surface [3] and by fusion of binding modules leading to increased adsorption University of Aberdeen, United Kingdom as measured via Quartz Crystal Microbalance [4].

This study investigates the feasibility of a new process for References the production of bioethanol from lignocellulosic wastes. In the [1].Nyanhongo GS, et al. React Funct Polym 2013;73(10):1399–404. investigated process, conversion of lignocellulosic materials to [2].Herrero Acero EJ. Mol Catal B: Enzym 2012;79:54–60. ethanol is obtained by using microorganisms for all the required [3].Herrero Acero E. Biotechnol Bioeng 2013;110(10):2581–90. [4].Ribitsch D, et al. Biomacromolecules 2013;14(6):1769–76. process stages, i.e. lignin and cellulose hydrolysis and carbohydrates conversion to ethanol. The aim is therefore to avoid the http://dx.doi.org/10.1016/j.nbt.2014.05.1684 use of expensive chemical-physical pretreatments for lignin and cellulose hydrolysis, replacing them with cheaper and more environmentally sustainable microbial processes. A key aspect of the investigated process is the use of open mixed microbial cultures, instead of pure cultures of selected microorganisms. This presentation will discuss the main challenges to be overcome for the development of the investigated process and will present experimental data on glucose and cellulose fermentation

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O8-4 lum tricornutum is able to accumulate large amounts of specialty carotenoids. Among the valuable carotenoids present in diatoms, Influence of nutritional and physicochemical variables fucoxanthin has health promoting effects in humans, including in PHB production from raw glycerol by a wild Bacillus anti-cancer, anti-obesity, and anti-diabetic effects as well as anti- megaterium strain malarial activity. Fucoxanthin has also been observed to be more ,∗ ␤ Carolina Guzmán Luna 1 , Paalo Andrea Moreno Yanez˜ 1, Camilo Jose potent than -carotene and astaxanthin in terms of anti-obesity Yanez˜ Diaz 1, Nilo Sérgio Medeiros Cardozo 2, Humberto Escalante 1, and anti-proliferative effects on adult T-cell leukemia cells. The Marianny Y. Combariza 1 demand for fucoxanthin in the global market has been increasing dramatically. In this study, we characterized the biomass produc- 1 Universidad Industrial de Santander, Brazil tivity and biomass yield of P. tricornutum with light emitting diode 2 Federal University of Rio Grande do Sul, Brazil (LED)-based photobioreactors with different light intensities and wavelengths. Under red LED illumination, increasing the photon Reducing production costs and increasing strain productivity flux from 85 to 255 ␮E/m2/s caused photoinhibition of Phaeodacty- in biosynthetic processes are decisive factors linked to biodegrad- lum cells when there was either 3.0 mM or 0.3 mM of metasilicate able polymers worldwide production and usage. Agro-industrial in the medium. In contrast, combined red and blue (50:50) LED byproducts as alternative carbon sources and optimization of cul- illumination produced a higher biomass yield and growth rate. ture media composition are some of the strategies used to address Further, the presence of blue light enhanced the accumulation of these issues. fucoxanthin in comparison to illumination with red light only. Amongst many available biopolymers polyhydroxybutyrate These results demonstrated the feasibility of fucoxanthin produc- (PHB), intracellularly produced and accumulated by abundant tion in P. tricornutum, and a method combining LED technology bacteria species, could become a substitute for synthetic plastics and synthetic biology approaches was proposed to facilitate its due to its thermoplastic and mechanical properties. In this con- development. tribution we report on the influence of five nutritional and two physicochemical variables in PHB production by Bacillus mega- http://dx.doi.org/10.1016/j.nbt.2014.05.1686 terium B2, a recently isolated and characterized bacterium with PHB accumulating ability using raw glycerol, from the biodiesel industry, as carbon source [1]. O8-6 According to Plackett Burman and central composite designs Antibacterial and antifungal activity of charcoal mate- temperature, glycerol and Na2HPO4 concentrations are the most significant variables on PHB production by B2, with optimal values rials and microwave radiation ◦ of 34 C, 7.6 g/L and 3 g/L, respectively. After 14 hours of fermenta- Hee Jin Yang 1,∗ , Yun Jeong Cha 2, Hern Kim 2, Shin Sik Choi 2,3 tion, in shake flasks with optimized medium, B2 produced 0.37 g/L 1 Myoungji University, Republic of Korea of PHB with an 18% w/w accumulation. This corresponds to an 2 Department of Energy and Biotechnology, Myongji University, Republic of 85% increase in PHB production when compared to initial culture Korea conditions. These results suggest the potential of B. megaterium B2 3 Department of Food and Nutrition, Myongji University, Republic of Korea as PHB producer using raw glycerol an inexpensive, abundant and readily available carbon source. Suppression of microbial contamination or growth using antibacterial and antifungal materials has been required in vari- Reference ous commercial products including foods and cosmetics. In this study, we have investigated the inhibitory effect of charcoal poly- [1].Tarazona N, Moreno P, Yanez˜ C, Combariza MY, Guzmán C. Micro- bial production of polyhydroxybutyrate by native Bacillus strains using a mers and microwave radiation on bacterial and fungal cell growth biodiesel by-product as carbon source. Lisbon, Portugal: Presented at the or survival rate. When bacterial and fungal cells were cultured with European Symposium on Biopolymers; October 7–9, 2013. PS 2.1. charcoal polymers, the growth of some microbes was significantly http://dx.doi.org/10.1016/j.nbt.2014.05.1685 inhibited by nano and micro sized charcoal particles protruded from the surface of polymers. Four species of fungi were also dramatically killed by microwave radiation for less than 5 min. These O8-5 results suggest that the use of charcoal plastics and microwave has a great potential for reducing microbial contamination in multiple Value-added carotenoid production in the pennate areas. diatom Phaeodactylum tricornutum with light emitting Acknowledgements: This work was supported by NGV of diode based photobioreactors Hyundai Motors Company and BK21 Plus of Ministry of Education (22A20130012051). Weiqi Fu 1,∗ , Sigurður Brynjólfsson 1, Bernhard Palsson 2 http://dx.doi.org/10.1016/j.nbt.2014.05.1687 1 University of Iceland, Iceland 2 University of California, San Diego, United States

Diatoms are one of the most promising feedstocks for producing feed supplements, bioactive pharmaceuticals and biofuels in a bio-based economy. The marine pennate diatom Phaeodacty-

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O8-7 has able to accumulate up to 47% PHB of cell dry weight with a productivity of 0.24 g HA/L d. The overall PHA yield on total sub- Polyhydroxyalkanoates production by aerobic mixed strate consumed (0.32 g COD HB/g COD crude glycerol) was in microbial cultures using crude glycerol the middle range of those reported in literature (0.08–0.58 g COD Paulo Costa Lemos ∗ , Rita Moita, André Freches, Rita Pontes PHA/g COD real waste). Molecular biology methods (FISH, DGGE) were used to characterize the microbial population. REQUIMTE/CQFB, Portugal As opposed to many other reports with real wastes, crude glycerol can be directly used to produce PHA. Its use avoids an Due to the prospective partial replacement of fossil fuels additional pre-fermentation step, making the overall production by biodiesel, its production has continuously grown in the process economically more competitive, reflected in a reduction last decade. In order to biodiesel production to be carried out of the polymer final cost. This was the first study that demon- under sustainable conditions, new application to their wastes/by- strates the valorisation of the glycerol fraction present in the crude products need to be investigated, especially concerning their major glycerol into PHA using an aerobic mixed microbial consortium. by-product – crude glycerol. In this study the feasibility of producing polyhydroxyalkanoates (PHA) by mixed microbial community http://dx.doi.org/10.1016/j.nbt.2014.05.1688 using crude glycerol as feedstock was investigated. The microbial population selected under aerobic dynamic feeding conditions had the ability to consume both major carbon fractions present in the crude, glycerol and methanol. Two biopolymers were stored, poly-3-hydroxybutyrate (PHB) and glucose biopolymer (GP), apparently using glycerol as the only carbon source for their production. The microbial enrichment obtained

www.elsevier.com/locate/nbt S33 BIOECONOMY New Biotechnology · Volume 31S · July 2014

Bioeconomy The supply chain of biotech products involves many stakehol- ders, organizations, activities and resources, some of which have SP3-1 a direct and often signiﬁcant inﬂuence on consumers purchase behaviors. One of them is the retailers, who are a key players in Asian bioeconomy and biobusiness: current scenario and the supply chain by providing link between the end-consumers future prospects and the manufacturers as well as suppliers. Satyahari Dey 1,2,∗ Our study took place in 2012 and 2013 and involved more than 250 visits in various retail outlets in Poland and conducting 1 Indian Institute of Technology Kharagpur, India standardized interviews with merchants concerning their views 2 Deputy Secretary General, Asian Federation of Biotechnology, India and knowledge of products referring to innovative biotechnology. Within this study we were able to identify goods (manufactured in The 20th century witnessed exciting inventions in biology different EU countries) that were either direct applications of inno- that has resulted in innovations today. The recombinant DNA vative biotechnology or unduly usurped to be such products The technology, animal cell culture, bioprocess engineering of recom- study revealed a positive correlation between the level of knowl- binant cells and the deciphering of human genome are notable. edge and the opinion on biotech products. The results helped A plethora of ‘omics’ related platform technologies have enabled to gain an understanding of reasons and motivations underlying precise and rapid probing of cellular processes facilitating huge retailers’ attitudes towards biotech products. applications in 5 Fs (food, fuel, f(ph)armaceuticals, fabric and fod- der). http://dx.doi.org/10.1016/j.nbt.2014.05.1690 The revenue from bio-industries in some countries, including a few in Asian regions, was as high as 2.5% of GDP. The developing regions in Asia could not experience such a growth uniformly. The sluggish global market over the last 5 years was also a bottleneck to this aspiration. The world trade trends now to recover from slowdown. It is important to set a realistic goal for biobusiness in Asia for 2025. Asia in one hand is blessed with the highest number of biotechnologists of the world in the younger age group, and the largest population of underprivileged people (having lower human development indices) on the other. The bountiful bioresources in Asia if exploited in bioentrepreneurship could eliminate the disparities. Inspiring young biotechnologists towards this goal is essential. A strong academia-industry partnership could accelerate the success. The European Federation of Biotechnology and Asian Federa- tion of Biotechnology together can mobilize their members, and interface with respective Governments facilitating favourable regulatory and policy frameworks. http://dx.doi.org/10.1016/j.nbt.2014.05.1689

SP3-2

Biotech products on the market as a main driver for knowledge based bio-economy

Aleksandra Małyska ∗ , Tomasz Twardowski Institute of Bioorganic Chemistry Polish Academy of Sciences, Poland

As biotechnology applications are present in broad variety of sectors it has been the key technology for realising the KBBE providing a more cost-effective option and better quality products. At the same time some of its products arouse great public controversy, in particular, the products of genetic engineering in the agri-food sector. Poland is no exception and international studies revealed that attitudes of Poles towards biotech products reﬂect the views of other Europeans. It is understandable because people are not aware in how many everyday products biotechnology is already applied.

S34 www.elsevier.com/locate/nbt New Biotechnology · Volume 31S · July 2014 TUESDAY 15 JULY PLENARY LECTURE: PROSTHETIC GENE NETWORKS FOR BIOMEDICAL APPLICATIONS Tuesday 15 July provide the dynamic interventions, the immediate pre-disease action and the multi-target capacity required to meet with the Plenary lecture: Prosthetic gene networks for treatment challenges of the future. Prosthetic networks consist biomedical applications of synthetic sensor-effector gene circuits that (i) seamlessly operate in implanted designer cells, (ii) constantly sense, monitor and PL2-1 score metabolic disturbances in peripheral circulation, (iii) process OFF-level concentrations of pathologic metabolites, and (iv) Prosthetic networks – synthetic biology-inspired treat- coordinate an adjusted therapeutic response in an (v) automatic ment strategies for metabolic disorders and self-sufﬁcient manner. We will present our latest generation Martin Fussenegger of synthetic mammalian gene circuits and provide a few examples of prosthetic networks operating in animal models of prominent ETH Zurich, Department of Biosystems Science and Engineering, Basel, Switzerland human diseases to highlight the challenges and impact of synthetic biology on future biomedical applications. Since Paracelsus’ (1493–1541) deﬁnition that the dosing makes http://dx.doi.org/10.1016/j.nbt.2014.05.1691 the drug the basic treatment strategies have largely remained unchanged. We continue to use a precise prescribed dose of a small-molecule drug, a protein therapeutic or a therapeutic transgene to constitutively modulate or complement the activity of a disease-relevant target. However, this treatment concept does neither consider the metabolic dynamics nor the interdependence of the most important pathophysiology’s of the 21st century such as obesity, diabetes and cardiovascular disorders. Synthetic biology- inspired prosthetic networks may act as metabolic prostheses that

www.elsevier.com/locate/nbt S35 SYMPOSIUM 9: BIOMARKERS AND DIAGNOSTIC TOOLS New Biotechnology · Volume 31S · July 2014

Symposium 9: Biomarkers and diagnostic recognize sequence epitopes that are difﬁcult to target using con- tools ventional antibodies, and that these novel antibodies are potent inhibitors of protein aggregation. O9-1 http://dx.doi.org/10.1016/j.nbt.2014.05.1693 Designing nanomaterials for ultrasensitive biosensing

Molly Stevens O9-3 Imperial College London, UK Engineering cofactor speciﬁcity of methyltransferases

Bio-responsive nanomaterials are of growing importance with Martin Tengg 1,∗ , Yu Zheng 2, Mandana Gruber-Khadjawi 3, Elmar potential applications including drug delivery, diagnostics and tis- Weinhold 1 sue engineering. This talk will provide an overview of our recent 1 Institute of Organic Chemistry, RWTH Aachen University, Germany developments in the design of materials for ultrasensitive biosens- 2 New England Biolabs Inc., Ipswich, MA, USA ing. Our recent simple conceptually novel approaches to real-time 3 ACIB GmbH, Austrian Centre for Industrial Biotechnology, Graz, Austria monitoring of protease, lipase and kinase enzyme action using modular peptide functionalized gold nanoparticles and quantum Biological methylations of various substrates occur in every dots will be presented. Furthermore we have recently developed living cell and are essential for cell survival. These transforma- a new approach to ultrasensitive biosensing through plasmonic tions are catalyzed by methyltransferases (MTases) which generally nanosensors with inverse sensitivity by means of enzyme-guided transfer the activated methyl group from S-adenosyl-l-methionine crystal growth as well as a “Plasmonic ELISA” for the ultrasensitive (AdoMet) to DNA, RNA, proteins and small biomolecules. Recent detection of disease biomarkers with the naked eye. We are apply- studies demonstrated that many MTases can accept AdoMet ana- ing these biosensing approaches both in high throughput drug logues for transfer of extended carbon chains to their substrates screening and to diagnose diseases ranging from cancer to global [1,2]. The ability to accept a broad range of cofactor analogues is health applications. of great interest both in terms of DNA diagnostics and biocatalytic http://dx.doi.org/10.1016/j.nbt.2014.05.1692 synthesis. In order to increase transfer rates of extended groups from AdoMet analogues protein engineering approaches are performed. O9-2 High-throughput directed evolution of the DNA MTase M.SssI is performed by in vitro compartmentalization. This method links Antibodies by design genotype and phenotype for an effective selection of active vari- Peter Tessier ants. The bacterial enzyme M.SssI is of particular interest because Rensselaer Polytechnic Institute, United States it performs the same reaction as mammalian DNA MTases by targeting 5-CG-3 (CpG) DNA sequences. CpG modifications are key The ability of antibodies to recognize target molecules (anti- epigenetic signatures in transcriptional regulation and genome gens) with high affinity and specificity is central to their imprinting. The transfer of functional groups will provide new widespread use in diagnostic and therapeutic applications. The tools for DNA labelling as well as DNA modification detection. binding activity of antibodies is encoded in up to six of their M.SssI evolution will also guide rational protein engineering of the solvent-exposed peptide loops that directly contact antigens. Anti- homologous small molecule MTase NovO, for the synthesis of new bodies are generated by randomly varying the sequences of their fine chemicals as well as bioactive intermediates and products. antigen-binding loops and selecting rare variants that are com- References plementary to target antigens. Due to the daunting number of possible antibody sequences with variation only within their [1].Dalhoff C, Lukinavicius G, Klimasauskas˘ S, Weinhold E. Nat Chem antigen-binding loops (>1030ˆ variants), it seems unlikely that Biol 2006;2:31–2. [2].StecherH, Tengg M, Ueberbacher BJ, Remler P, Schwab H, Griengl H, the needles (antibodies with desired binding activity) in the Gruber-Khadjawi M. Angew Chem Int Ed 2009;48:9546–8. haystack (all possible antibody variants) can be predicted instead of being selected. We have challenged this conventional wisdom by http://dx.doi.org/10.1016/j.nbt.2014.05.1694 reducing the seemingly intractable problem of designing multiple antibody loops to cooperatively bind antigens to a tractable one in which we design individual antibody loops with binding activity. O9-4 Using this simplified design strategy that is inspired by natural bio- Synthetic bioreporters for detection of environmental logical interactions, we find that antibody fragments can be readily pollutants engineered to recognize diverse aggregated proteins linked to neu- ∗ rodegenerative disorders (e.g., Alzheimer’s disease) by targeting Jan Roelof van der Meer , Davide Merulla, Siham Beggah unique structural features within such proteins. Our innovative University of Lausanne, Switzerland approach generates single- and multidomain antibodies that recognize misfolded proteins not only based on their sequence, but One of the main immediate application areas for synthetic also based on their conformation. We also find that our antibodies biology are bioreporters, living cells with simple designed genetic

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circuits that permit detection of a specific chemical or group of GTP hydrolysis assay was used to screen 1280 compound small chemicals, under the concomitant production of an easily but molecule library whereof twelve inhibitors were found (average Z- accurately quantifiable reporter signal. Bioreporters have attracted factor 0.78). The GTP hydrolysis assay can simultaneously detect considerable interest because they offer cheap alternatives for inhibitors affecting either nucleotide exchange or GTP hydrolysis. chemical analysis in remote areas where high-end instruments are Therefore, same screening was performed by monitoring Eu3+-GTP unavailable. Such demands on bioreporters, however, require an association to H-RasWt (average Z-factor 0.78). From the screening almost fail-proof and robust technology that goes much beyond assays, seven same inhibitor hits were found. Additionally, five what traditional research “proof-of-principles” have been able to inhibitors were found only in GTP hydrolysis assay. demonstrate. Conclusions: These novel QRET assays for GTPase research We will show on the example of a bioreporter for arsenic, how can be performed using nanomolar protein concentrations. The important improvements can be made in the circuit design and presented QRET assays enable the study of whole small GTPase in the optimization of the assay technology. Arsenic is a recurring cycle by monitoring the guanine-nucleotide exchange factor (GEF) noxious contaminant of drinking water in large areas of our planet, induced nucleotide exchange and/or GTPase-activating proteins and bioreporter technology can provide the means to rapidly (GAP) catalyzed GTP hydrolysis. ␮ quantify its presence in the concentration range of 1–10 g/L. First, http://dx.doi.org/10.1016/j.nbt.2014.05.1696 we demonstrate how designing a feedback or an uncoupled circuit affects the signal output and detection sensitivity for arsenic. Sec- ondly, we show a new system to better control residual background O9-6 expression in the circuit while maintaining optimal induction, and we also provide experimental evidence to improve the circuit Comparative large scale microRNA expression profiles behaviour by avoiding cross-interference from the host. Finally, we of cynomolgus monkeys, rat and human reveal miR-182 present the use of micro-engineered structures that would permit associated with Type 2 diabetes continuous remote operation of a bioreporter sensor. Hongli Du ∗ , Jinghui Zhou, Yuhuan Meng, Xiaoning Wang http://dx.doi.org/10.1016/j.nbt.2014.05.1695 South China University of Technology, China

Type 2 diabetes (T2D) is a prevalent disease that is present O9-5 throughout the world, and is usually associated with insulin resis- A homogeneous quenching resonance energy trans- tance. MicroRNAs (miRNAs) play important role in the suppression fer assay for H-Ras activation cycle monitoring and of gene expression and have been shown to be implicated in inhibitor screening human diseases. We used a novel animal model, cynomolgus monkey fed with normal and high fatty diet (HFD) respectively, to Kari Kopra 1,∗ Arjan van Aldrichem 2 Markku Syrjänpää 1 , , , analyze the miRNA expression profile in whole blood by deep- Stefan Veltel 3 Pekka Hänninen 1 Daniel Abankwa 4 Urpo , , , sequencing. Finally in total 24 miRNAs with differential expression Lamminmäki 5 Harri Härmä 1 , were filtered. Among them, mir-182 and mir-183, related to insulin 1 Laboratory of Biophysics, University of Turku, Finland resistance by modulating FOXO1 and PI3 K/AKT cascade, had the 2 Institute for Molecular Medicine Finland, University of Helsinki, Finland greatest copy number in the whole blood. Decrease of mir-182 3 University Hospital Hamburg-Eppendorf, Finland in T2D cynomolgus individuals is completely consistent with the 4 Turku Centre for Biotechnology, University of Turku and Åbo Akademi Univer- sity, Finland previous studies in human and rat. Integrating mir-182 tissue 5 Department of Biotechnology, University of Turku, Finland expression profile, target genes and copy number in blood revealed that mir-182 plays a key role in FOXO1 modulation that leads Background: Recently, we have developed a homogeneous to potential hyperglycemia and modulates the insulin secretion. single-label signaling technique, the Quenching Resonance Energy In addition, the possible miRNA regulation system of T2D under Transfer (QRET). In this study, the homogeneous QRET technol- the influence of different diet conditions was also interpreted in ogy was applied to monitor GTPase activation cycle and activation the present study. The cholesterol content influences mir-182 and cycle inhibition. potentially other miRNAs expression level that causes the insulin Methods: The QRET system is based on the protection of tar- resistance and the various miRNA regulation systems between nor- get protein bound Eu3+-GTP from a soluble quencher molecule. (1) mal diet and HFD. The small GTPase cycle (nucleotide exchange and hydrolysis) can http://dx.doi.org/10.1016/j.nbt.2014.05.1697 be monitored in the competitive assay by tracking GTP hydrolysis in the presence of nanomolar concentrations of H-RasWt, SOScat, and p120GAP. (2) The nucleotide exchange can be monitored using a second assay with H-RasWt and SOScat. The increased time- resolved luminescence (TRL) signal is monitored when either GTP hydrolysis (1) or Eu3+-GTP association (2) occurs. Results: We have proven the suitability of the QRET system to monitor GTP hydrolysis (1) and nucleotide exchange (2). The

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O9-7 rs429358 (ApoE4) and rs7412 (ApoE2). The genotype frequencies were: E3/E3 (75%), E2/E3 (4.1%), E3/E4 (20%), E4/E4 (0%), E2/E4 Association of apolipoprotein E gene polymorphism (0%), and E2/E2 (0.45%). The allele frequencies were 0.025, 0.875 with serum lipid levels and 0.1 for ␧2, ␧3 and ␧4, respectively. In control group allele fre- ∗ ␧ ␧ ␧ Sehrish Fatima , Syed Shahid, Obaid Khan, Abid Azhar quencies for 2, 3 and 4 were 0.045, 0.83 and 0.127, respectively, whereas in dyslipidemic patients group allele frequencies for ␧2, ␧3 University of Karachi, Pakistan and ␧4 were 0.045, 0.682 and 0.273 respectively. Total cholesterol (TC) and low density lipoprotein cholesterol (LDL-C) levels were Dyslipidemia has been marked to play an integral role in the significantly high in ␧4 allele carriers (p < 0.001) and high density development of cardiovascular diseases. Various environmental lipoprotein cholesterol (HDL-C) level was high in ␧2 allele carriers and genetic factors may influence dyslipidemia. Polymorphism of (p < 0.05) indicating protective effect of ␧2 allele. Apo E polymor- the apolipoprotein E (apo E) gene may influence lipid metabolism phism has significant influences on lipid profile in dyslipidemic by modulating lipid levels. This study was designed to investi- patients group as frequency of ␧4 allele was found to be prevalent gate the role of genetic variants of apo E in dyslipidemic patients. in patients group which indicates a relationship of ␧4 allele in apo The study was carried out in equal number (n = 110) of normal E gene with dyslipidemia. subjects and dyslipidemic patients. Genotyping was performed by Polymerase Chain Reaction and Restriction Fragment Length Poly- http://dx.doi.org/10.1016/j.nbt.2014.05.1698 morphism (PCR- RFLP) at Single Nucleotide Polymorphisms (SNPs)

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Symposium 10: Biowaste biorefinery for a occurs under high pressure and low pH conditions. SSL diges- more sustainable bioeconomy tion leads to hemicellulose degradation to C6 and C5 sugars and lignin depolymerisation by sulfonation and hydrolysis. SSL O10-1 was processed via ultrafiltration to produce a sugar-rich permeate that is suitable as carbon source for fermentative production Biowaste biorefinery for a more sustainable and of succinic acid. The retentate contained a high concentration of biobased food industry lignosulfonates. An antioxidant-rich fraction was extracted from the permeate stream via solvent extraction with ethyl acetate. Fabio Fava The total phenolic content, the antioxidant activity and specific DICAM, School of Engineering, Alma Mater Studiorum-University of Bologna, phenolic compounds were analysed in this stream. Production Bologna, Italy of succinic acid was evaluated with the bacterial strains Acti- nobacillus succinogenes and Basfia succiniciproducens in batch and Modern biobased industry is giving its close attention on fed-batch bioreactor cultures using either free or immobilized organic waste as a new bioresource. Specific biowaste valorization cells. SSL valorisation could lead to a sustainable biorefinery pathways are focusing on food processing waste, being food sector concept. the first manufacture in Europe. Anyway they need to become inte- Acknowledgements: The authors gratefully acknowledge grated, combining biomass pretreatments and recovery of biogenic the financial support by the FP7 research project BRIGIT (KBBE- chemicals with bioconversion processes in order to obtain a large 2012-6-311935, www.brigit-project.eu). class of chemicals. This will help to (a) use the whole biowaste, by avoiding producing residues and providing to the approach http://dx.doi.org/10.1016/j.nbt.2014.05.1700 the required environmental sustainability, and (b) producing different biobased products that enter different markets, to get the possible economical sustainability of the whole biorefinery. How- O10-3 ever, the costs of the developed integrated processes might be high, Biotechnological conversion of spent coffee grounds into mostly for the fact that the industry dealing with such issues is polyhydroxyalkanoates still underdeveloped and therefore dominated by high processing costs. Such costs can be significantly reduced by intensifying Stanislav Obruca ∗ , Pavla Benesova, Sinisa Petrik, Dan Kucera, research & innovation on process integration and downstream Ivana Marova processing. The low or no cost of starting material along with the Faculty of Chemistry, Brno University of Technology, Czech Republic environmental benefits coming from the concomitant biowaste disposal would offset the high capital costs for initiating such a Coffee is one of the world’s most popular beverages and has biorefinery. been growing steadily in commercial importance. Nowadays, cof- http://dx.doi.org/10.1016/j.nbt.2014.05.1699 fee is, after petroleum, the second largest traded commodity in the world. Hence, coffee industry is responsible for the generation of large amounts of waste, above all, spent coffee grounds (SCG). O10-2 The aim of this work was to study conversion of SCG into valuable product–polyhydroxyalkanoates (PHAs). These polyesters Development of an advanced biorefinery concept based are accumulated by various bacteria as carbon and energy storing on valorization of pulp and paper industry waste materials. Due to their mechanical properties, they are considered streams being an alternative to petrochemical plastics. Apostolis Koutinas , Mary Alexandri ∗, Chrysanthi Pateraki, Anestis At first, oil extracted from SCG (approx. 15 wt% oil in SCG) was Vlysides, Harris Papapostolou efficiently (YP/S = 0.82) converted into PHA employing Cupriavidus necator H16. Further, the solid residues after oil extraction were Agricultural University of Athens, Greece hydrolysed (combination of chemical and enzymatic hydrolysis) yielding fermentable sugars, which were further used as a sub- The biorefinery concept is based on the efficient fractionation strate for production of PHAs employing Bacillus megaterium and of a renewable resource followed by the conversion of individual Burkholderia cepacia. B. cepacia revealed significantly higher PHAs fractions into marketable products in a way that exploits the full yields and; moreover, it was capable of direct accumulation of potential of the resource. Research at the Agricultural University copolymer of 3-hydroxybutyrate and 3-hydroxyvalerate without of Athens focuses on restructuring conventional industrial plants addition of precursors. into integrated biorefineries through the valorization of waste or Finally, solids after SCG hydrolysis possess high calorific value by-product streams. and can be used as a fuel to at least partially cover energetic This study evaluates the potential restructuring of a pulp and demands of the process. Hence, entire biomass of SCG can be paper process employing the sulfite process through fraction- used for sustainable production of PHAs employing bio-refinery ation of spent sulfite liquor (SSL) for the production of phenolic approach. compounds as antioxidants, lignosulfonates and succinic acid. Acknowledgement: This work was supported by SSL is a complex waste stream generated via wood chip diges- projects “Centre for Materials Research at FCH BUT” No. tion with calcium or magnesium sulfite where delignification

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CZ.1.05/2.1.00/01.0012 from ERFD and “Excellent young obtain extracts rich in sugars, containing around 12.5 g/L sucrose, researcher at BUT” No. CZ.1.07./2.3.00/30.0039. 7 g/L glucose and 2.5 g/L fructose. All sugars were consumed and http://dx.doi.org/10.1016/j.nbt.2014.05.1701 SA yield of 0.4–0.5 g SA/g sugar could be obtained, depending on extract concentration. These results show that carob pulp water extracts or glycerol are promising substrates for SA production, O10-4 demonstrating the high versatility of A. succinogenes. References Extraction of the protein fraction of dry distillers grains with solubles, implementing biocatalytic and chemical [1].McKinlay, et al. Appl Microbiol Technol 2007;76:727–40. [2].Carvalho, et al. Nat Biotechnol 2014;31:133–9. methods http://dx.doi.org/10.1016/j.nbt.2014.05.1703 Maria Villegas Torres ∗ , Gary Lye, John Ward UCL, United Kingdom O10-6 Distillers dried grain and solubles (DDGS) is a by-product from distilleries and breweries, currently used as animal feed. It con- Enhanced welan gum production using cane molasses as tains mainly protein, fats and residual carbohydrates, making it substrate by Alcaligenes sp. ATCC31555 an ideal feedstock for biocatalytic processes. In addition, due to Jufang Wang ∗ , Hongxia Ai, Min Liu the current expansion of the biofuel industry in the UK, a dra- South China University of Technology, China matic increase in DDGS production is expected. Therefore, it is of industrial interest to develop manufacturing processes of indus- Welan gum is a new high molecular microbial exopolysaccha- trially relevant compounds implementing DDGS as feedstock. In ride with a wide range of potential industrial applications in food, this work, we focused on the extraction of the protein fraction (up concrete, petroleum, ink and for enhancing oil recovery. Welan to 40%), which is expected to have a high frequency of specific gum is more effective than any other polysaccharides in enhancing amino acids, as wheat grain is mostly gluten -a highly elastic pro- oil recovery and expected to become a novel oil recovery agent for tein mainly composed of glutamine and proline. Glutamine has its excellent stability at high temperature. For new polymers to be a been extensively used as injury treatment, and muscle growth, and commodity product in the near future, it is crucial to enhance the because of its physical characteristics can be an ideal source for bio- productivity and lower their production costs. In this work, cane materials manufacture. We optimized a protein extraction process molasses was selected for welan gum production by Alcaligenes sp. involving both chemical steps combined with enzyme hydrolysis. ATCC31555 in 5L fermentor. The pre-treatment of cane molasses, The optimized extraction method is a novel approach that can be agitation and the additives for improving dissolved oxygen were applied at industrial scale. investigated for process optimization. Sulfuric acid hydrolysis was http://dx.doi.org/10.1016/j.nbt.2014.05.1702 the optimal molasses pretreatment for welan gum production with a maximum welan gum concentration (33.5 g/L), yield (0.62 g/g), productivity (0.28 g/L/h), and broth viscosity (3.375 Pa s), which O10-5 are 50%, 138%, 47%, and 67% higher, respectively, compare to those without treatment. The process was subsequently optimized Succinic acid production from raw materials by Acti- by agitation and adding additives for improving the dissolved oxy- nobacillus succinogenes gen at special time point during the fermentation. Optimal welan Christophe Roca 1 , Margarida Carvalho 2,∗, Maria A.M. Reis 2 gum production (41.0 ± 1.41 g/L) was found at 600 rpm under ◦ 1 REQUIMTE, Portugal 30 C with addition of 10% n-dodecane at 36 h in 5L fermentor, 2 REQUIMTE/Universidade Nova de Lisboa, Portugal which is the highest welan gum concentration so far. Moreover, welan gum from molasses displayed similar rheological properties Succinic acid (SA) is currently considered a key platform chemi- and thermal stability than that from glucose. It indicated that cane cal as it is used in the production of a wide range of products, from molasses may be an economical industrial substrate for welan gum pharmaceuticals to green solvents, fibers and bioplastics [1]. The fermentation. use of renewable feedstocks as carbon source for the production of http://dx.doi.org/10.1016/j.nbt.2014.05.1704 SA via fermentation has been suggested as a solution to reduce production costs and develop a sustainable SA production process [2]. In this work, Actinobacillus succinogenes was used as biocatalyst for the conversion of two raw materials into SA: i) glycerol, byproduct of biodiesel industry, is usually poorly metabolized to SA because of a redox imbalance but here, it could be efficiently converted to SA, reaching concentration as high as 49 g/L SA using DMSO as electron acceptor; ii) extracts from carob pods, a by-product of carob locust bean gum industry were used as carbon source in small scale batch cultivations. A simple aqueous extraction was used to

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O10-7 The type of application usually defines the conditions where the reactions should take place. Thus, novel enzymes with variable Investigating the biomass modifying and degrading combined properties, such as different thermotolerance, pH range enzymatic toolbox of Aspergillus japonicus var aculeatus of activity, substrate specificity and solvent tolerance, still need FEC 156 with quantitative proteomics and New Genera- to be discovered and developed to achieve the highest possible tion Sequencing tools efficiency in each case. George Anasontzis 1,∗ , Thuy Nguyen Thanh 2, Thanh Vu Nguyen 2, We isolated an Aspergillus japonicus var aculeatus strain and Lisbeth Olsson 1 evaluated its cellulase and hemicellulases activities. It was then cultivated in bioreactors with different carbon sources, such as 1 Chalmers University of Technology, Sweden wheat bran, spruce and Avicel and its biomass degrading capac- 2 Food Industries Research Institute, Sweden ity was determined and semiquantified with TMT (Tandem Mass Tags), through cross species protein identification of its secretome. In the bio-based economy concept, the current hydrocarbon Information on the genes involved in the different stages of the fuels and non-biodegradable plastics will be replaced by new prod- fermentation and carbon sources have been acquired with next ucts which will derive from natural and renewable resources. The generation sequencing of its total transcriptome. synthesis of such biofuels and biochemicals is still challenged by Interesting transcripts are being heterologously cloned and the difficulties to cost efficiently degrade lignocellulosic materials expressed in order to identify their role and potential use in the to fermentable sugars or to isolate the intact polymers. Biomass biorefinery concept. degrading and modifying enzymes play an integral role both in the separation of the polymers from the wood network, as well as in http://dx.doi.org/10.1016/j.nbt.2014.05.1705 subsequent modifications, prior to further product development.

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Symposium 11: Nanotechnology: new biologi- of VLP assembly and release from cells. Transient gene expression cal applications (TGE) offers the flexibility to rapidly screen a number of product variants with sufficient quantity and quality for preclinical proof O11-1 of concept studies. In this work, mammalian cells are used for the production of a fluorescent variant Gag-based VLP using a TGE Design, structure and assembly of superparamagnetic approach. A method based on GFP fluorescence has been devel- core–shell nanoparticles oped to quantify both the transfection efficiency and the Gag-GFP VLP production. Additionally, the properties and purity of the Erik Reimhult obtained VLPs are further characterized by other techniques such Institute for Biologically inspired materials, Department of Nanobiotechnology, as transmission electron microscopy, nanoparticle tracking analy- University of Natural Resources and Life Sciences Vienna, Vienna, Austria sis and size-exclusion chromatography. Discussion will be focused on the characterization of the process as well as the obtained prod- Nanoparticles with carefully controlled core–shell structures uct. can be used in biomedical applications, for example, for separation, as contrast agents, for hyperthermia and in drug delivery http://dx.doi.org/10.1016/j.nbt.2014.05.1707 [1,2]. Exquisite control allows further applications through assembly into biomimetic membrane and vesicular structures for which permeability externally can be controlled by applied magnetic O11-3 fields. Bacterial microcompartments moving into the world of I will briefly describe a new synthetic toolkit based on nitro- biotechnology catechol dispersants, monodisperse synthesis of Fe3O4 cores using capping agents and new approaches to ligand grafting into bio- Stefanie Frank 1,∗ , Andrew Lawrence 1, Allan Pang 2 compatible shells on such nanoparticles; these developments 1 University of Kent, United Kingdom allow the synthesis of a range of new magnetic nanoparticles 2 University of California, United States for defined biotechnological applications and for fundamental research. The emphasis is on the purification and characterization Bacterial microcompartments (BMCs) are cytosolic protein of such nanoparticles and further on the assembly of nanopar- structures that are composed of a shell housing sequentially act- ticles into membrane superstructures that can be magnetically ing enzymes associated with particular metabolic pathways.BMCs controlled. The relationship between nanoparticle structure, the permit the enhancement of metabolic processes, which is why assembled membrane structure and the release of a model drug they are expected to have great potential for biotechnological from nanoparticle actuated vesicles will be highlighted. applications. One of the most complex BMCs is associated with References 1,2-propanediol utilisation (pdu) and the focus of our efforts for microcompartment engineering. The shell of the native Pdu [1].Amstad E, Reimhult E. Nanomedicine 2012;7:145–64. microcompartment is composed of seven proteins (PduA, PduB, [2].Amstad E, Textor M, et al. Nanoscale 2011;3:2819–43. PduJ, PduK, PduN, PduT and PduU); only five are required for http://dx.doi.org/10.1016/j.nbt.2014.05.1706 the self-assembly of empty heterologous microcompartments in E. coli. PduA is one of the most abundant shell proteins and has the ability to self-assemble into higher-order structures which we O11-2 found can be manipulated by strategic intervention at the molecular level. Targeting of enzymes to the shell is mediated by short Production and characterization of HIV-1 virus-like par- peptide sequences. We have solved the structure for the N-terminal ticles using transient gene expression in mammalian targeting peptide (P18) of PduP, an enzyme associated with the cells Pdu microcompartment and identified a main interaction part- Sonia Gutiérrez-Granados ∗ , Laura Cervera, Segura Maria de las ner PduK, a protein of the outer shell. We have also fused two Mercedes, Francesc Gòdia heterologous enzymes required for ethanol production (pyruvate Universitat Autònoma de Barcelona, Spain decarboxylase and alcohol dehydrogenase) with targeting peptides and found it has been possible to direct these enzymes to empty New vaccine strategies based on virus-like particles (VLPs) are BMCs in vivo and to generate a functional ethanol bioreactor. rapidly evolving. Upon expression in a number of heterologous Ongoing research is striving to further understand protein- host systems, Gag polyprotein of HIV-1 spontaneously assembles protein interactions and assembly of microcompartments and in the vicinity of the plasma membrane and it is released by bud- related higher order protein structures in molecular detail in order ding producing enveloped particles that resemble immature HIV-1 to control and manipulate these structures for the development of virions. These particulate immunogens have proven to be potent ‘optimized’ bioreactors. stimulators of both cellular and humoral immune responses in http://dx.doi.org/10.1016/j.nbt.2014.05.1708 animal models. Besides, Gag-based VLPs do not contain the viral genome, minimizing biosafety concerns. Thus, VLPs offer great promise as HIV-1 vaccines. Mammalian cells are the preferred system as a cell factory for this kind of vaccines due to the complexity

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O11-4 precipitation. Biological copper ion removal from effluents has been shown to be quite effective. Protein supramolecular engineering – applications in There are two biological methods to remove copper from biotechnology effluent which involve biosorption and reduction. Biosorption Patrick Shahgaldian 1,∗ , Maria Rita Correro 2, Alessandro Cumbo 3, involves bacteria binding to copper via the cysteine-rich transport Philippe Corvini 2 proteins that are associated with the cell membrane to precipitate it. Some bacteria are also able to reduce higher valency 1 University of Applied Sciences and Arts Northwestern Switzerland, insoluble copper ions into zero valency insoluble forms of the Switzerland 2 University of Applied Sciences Northwestern Switzerland, Switzerland metal. 3 INOFEA AG, Switzerland Here we present a metal-reducing bacterium Morganella psychrotolerans, which is able to reduce Cu2+ to insoluble Cu0 A large number of living organisms possess the capability nanoparticles. The copper nanoparticles are produced as part of to produce intricately patterned and hierarchically structured the bacterium’s defence mechanism against the toxic effects of biogenic silica. Imitating natural systems’ ability to produce hier- metal ions. M. psychrotolerans grows at low a temperature which archical silica structures may provide the possibility to design makes it ideal for industrial applications as energy for heating is functional (nano)materials with the same degree of complexity. In not required; hence it can be potentially cheaper than current this lecture, two novel design strategies of nanomaterials capable methods of copper removal. The copper nanoparticles produced of either molecular recognition or biocatalysis will be discussed; during the distillery co-product retreatment can be isolated and both approaches are based on the self-assembly of organo-silica subsequently used for purposes such as optics, catalysts, antimi- precursors using protein templates [1]. crobials or recycled in order to make new copper stills for whisky The first part of this presentation will be dedicated to the devel- distilleries. opment of a novel class of nanomaterials possessing selective http://dx.doi.org/10.1016/j.nbt.2014.05.1710 molecular recognition properties of viruses. The synthetic strategy to produce those nanoparticles is based on the formation of a chemical imprint of the template virion at the surface of silica O11-6 nanoparticles. It is demonstrated that the so-produced particles possess enhanced molecular recognition properties for their tar- Insight into the physiological role of a compartmen- get, even in complex media (e.g. human serum). The second part talised ferritin like protein in Rhodospirillum rubrum of this lecture will be dedicated to the development of a chemical Jon Marles-Wright ∗ , Didi He, Atanas Georgiev, David Clarke strategy to produce nanobiocatalysts with enhanced biochemical, physical and chemical stabilities. It is based on the formation of a University of Edinburgh, United Kingdom protective shell at the surface of enzyme proteins that provides a comfortable medium that allows for enzyme stabilization. It will All domains of life have evolved systems for the com- be demonstrated that those systems can find a number of biotech partmentalisation of enzymes and metabolic pathways. These applications. include the lipid-bounded organelles in eukaryotes and extend to protein-shelled microcompartments, such as carboxysomes, in prokaryotes. Rhodospirillum rubrum, which is a model organism Reference for the study of bacterial nitrogen fixation, possesses two distinct metabolic compartments. We have characterised the simplest [1].CumboA, Lorber B, Corvini PF-X, Meier W, Shahgaldian P. Nat Com- mun 2013;4:1503. of these compartments, which is encoded in a two-gene locus and comprises a linocin-family shell protein and a ferritin-like http://dx.doi.org/10.1016/j.nbt.2014.05.1709 protein. Using a combination of structural biology techniques, including X-ray crystallography, native mass-spectrometry, and electron microscopy, in combination within vivostudies we have O11-5 determined the structure and function of this intriguing metabolic Biological production of stable copper nanoparticles compartment. The compartment shell is build from 60 subunits of the linocin ∗ Nikolaos Pantidos , Louise Horsfall protein and forms a 25 nm icosahedron, much like the capsid of a University of Edinburgh, United Kingdom virus. The ferritin is encapsulated co-translationally through the interaction of a short signal sequence at its terminus with the Many nonferrous industries such as mining and surface treat- linocin. It forms a donut shaped structure and presents a unique ment plants produce co-products that are high in heavy metals inverted ferroxidase site, which is formed through the interaction and therefore toxic to the environment. A less obvious producer of of two protein monomers. The activity of this ferritin is mediated heavy metal containing co-products is the whisky industry. Cur- by encapsulation within the linocin shell and is distinct to the fer- rent methods of copper removal from such co-products include ritins, bacterioferritins and DPS proteins that make up the rest of electrolysis and membrane filtration which are reported to be the family of ferritin-like proteins. impractical and costly. Alternatively, when copper is found as a This metabolic compartment is widely distributed across bac- salt, current methods of removal include settlement, filtration and terial groups and is highly conserved, indicating a common

www.elsevier.com/locate/nbt S43 SYMPOSIUM 11: NANOTECHNOLOGY: NEW BIOLOGICAL APPLICATIONS New Biotechnology · Volume 31S · July 2014 physiological purpose. The promiscuous metal ion binding sites we fused the nitrilase from Alcaligenes faecalis with an amphipathic in the ferritin and localisation mechanism give hints into the peptides. Results showed that approximately 90% of nitrilase were potential for engineering these compartments. produced as active inclusion bodies (aggregrates) in Escherichia coli http://dx.doi.org/10.1016/j.nbt.2014.05.1711 cells. The thermal stability of the self-assembly induced nitrilase was increased by 6.8 and 4.3 folds at 45◦C and 50◦C, respectively. When the nitrilase aggregrates are purified by differential cen- O11-7 trifugation with sucrose density gradient and negatively stained, a highly ordered amyloid fibrils structure is observed under TEM. Catalytic properties improvements of Alcaligenes faecalis After two steps of washing with buffers, the nitrilase aggregates nitrilase by self-assembly induced aggregation were purified and further immobilized with sodium alginate as the carrier, then designated as iSEA. Thermal stability was fur- Shuang Li ∗ , Xiaofeng Yang ther increased by 1.5-fold compared to the aggregrates. Also the School of Bioscience and Bioengineering, South China University of Technol- iSEA showed good progress in the tolerance of mandelonitril from ogy, China 30 mM to 200 mM. High expression yields, simple recovery steps of aggregates from the host cells and the favorable catalytic char- A large number of studies have been devoted to improve acteristics are attractive features industrially. catalytic properties of enzymes by protein engineering. How- http://dx.doi.org/10.1016/j.nbt.2014.05.1712 ever, these conventional methods, i.e. site directed, random and saturated mutagenesis, always rely on the correlation between structure and function or high-throughput screening approaches, which make it hard to obtain positive mutants in a short time. Here

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Symposium 12: Stress responses in microbial References bioprocessing [1].Wang B, Kitney R, Joly N, Buck M. Engineering modular and orthogonal genetic logic gates for robust digital-like synthetic biology. Nat O12-1 Commun 2011;2:508. [2].Wang B, Buck M. Customizing cell signalling using engineered Mechanisms of protein folding and quality control in genetic logic circuits. Trends Microbiol 2012;20(8):376–84. [3].Wang B, Barahona M, Buck M. A modular cell-based biosensor using bacteria engineered genetic logic circuits to detect and integrate multiple Bernd Bukau environmental signals. Biosens Bioelectron 2013;40:368–76. Center for Molecular Biology of the University of Heidelberg, German Can- http://dx.doi.org/10.1016/j.nbt.2014.05.1714 cer Research Center, DKFZ-ZMBH Alliance, Im Neuenheimer Feld 282, D-69120 Heidelberg, Germany O12-3 Protein homeostasis is established by a complex cellular machinery which assists and regulates regular folding pathways Stochastic activation of the GlpR-controlled glp gene and counteracts protein misfolding and aggregation. A particularly cluster in Pseudomonas putida KT2440 results in a critical process in the life of a protein is the native folding of newly bistable growth pattern on glycerol synthesized proteins, which therefore is tightly controlled. Already Pablo Ivan Nikel ∗ , Victor de Lorenzo during ongoing synthesis by the ribosome nascent polypeptides Centro Nacional de Biotecnologia (CNB-CSIC), Spain are subject to enzymatic processing, chaperone-assisted folding to the native state or targeting to translocation pores at membranes. The ribosome itself plays a key role in these different tasks by serv- Phenotypic variation is a widespread trait among prokary- ing as platform for the regulated association of enzymes, targeting otes, and the molecular mechanisms underlying the phenomenon factors and chaperones that act upon the nascent polypeptides include genetic changes (e.g., genomic inversions and strand- emerging from the exit tunnel. The molecular mechanisms inte- slippage processes), epigenetic variations (e.g., distinct patterns of grating the different co-translational processes leading to the DNA methylation), as well as feed-back-based multi-stability. All maturation and native folding of nascent chains will be described. these mechanisms are known to ultimately lead to the appearance of at least two distinct phenotypes within an otherwise isogenic http://dx.doi.org/10.1016/j.nbt.2014.05.1713 population. However, this complex trait has been scarcely explored in microorganisms that have environmental and industrial interest. The soil bacterium Pseudomonas putida exhibits promising O12-2 biotechnological potential, together with its generally regarded- as-safe certificates, resistance to endogenous and exogenous stress, Engineering customised cell signalling circuits and their amenability to genetic manipulation, and suitability as a host biotechnological applications for heterologous gene expression. Growth of P. putida KT2440 Baojun Wang on glycerol is characterized by an unexpectedly long lag phase University of Edinburgh, United Kingdom (Nikel PI, Kim J, de Lorenzo V. Metabolic and regulatory rearrangements underlying glycerol metabolism in Pseudomonas putida Cells live in an ever-changing environment and continuously KT2440. Environ Microbiol 2014;16:239-54). In the present con- sense, process and react to environmental signals using their inher- tribution, the growth of individual P. putida KT2440 cells was ent signalling and gene regulatory networks. Here I will present assessed on glycerol to further explore this phenotypic behavior. the construction of synthetic gene circuits to customise cellular It was found that the physiological properties of cells growing information processing and responses by harnessing the inher- on this carbon source resulted in the appearance of two distinct ent modularity of signalling networks [1–3]. In particular, a set of cell sub-populations which significantly differed in their metabolic modular and orthogonal genetic logic gates, e.g. AND and NAND, activity. These macroscopic properties are wired to the dual logic of and analogue circuits such as a gain-tunable genetic amplifier were the GlpR regulator, repressing the transcription of the cognate glp engineered to modulate multiplein vivo transcriptional signals in genes, encoding the enzymes needed for glycerol catabolism. Ana- either digital-like or bespoke analogue manner. I will then show lyzed in perspective, these results suggest that P. putida is subjected that how these gene circuits can be used to enhance the specificity to a carbon-source-dependent bet-hedging strategy that might be and sensitivity of synthetic cell-based biosensors for detecting relevant in the natural niches it inhabits. heavy metal ions and bacterial signalling molecules in an aqueous http://dx.doi.org/10.1016/j.nbt.2014.05.1715 environment, and to realise robust gene expression control and sensing in single cells over a range of abiotic conditions. Further- more, we are engineering modular genetic controllers that can act as dynamic stress sensor-regulators to achieve adaptive control of cellular pathway gene expression flows for optimised biomolecule production.

www.elsevier.com/locate/nbt S45 SYMPOSIUM 12: STRESS RESPONSES IN MICROBIAL BIOPROCESSING New Biotechnology · Volume 31S · July 2014

O12-4 fact that the active recovery is a key stress response, the nature of these changes is not understood. The biotechnology indus- The adaptation of the intestinal sulphate reducing bac- try is heavily involved in engineering bacteria for production of terium, Desulfovibrio desulfuricans, to nitrosative stress useful compounds. High-level excretion of substrate feed during induced by nitric oxide biotechnological applications constantly increases medium con- Matthew Faulkner ∗ , Jeff Cole centration. As a result, limited cell growth and low volumetric productivity has been reported. Here, we use fluorescence imaging The University of Birmingham, United Kingdom of single cells during hyperosmotic shocks, combined with custom made microfluidic devices, to show that cells fully recover their Desulfovibrio desulfuricansis an environmentally important volume to the initial value and continue to grow slower immedi- organism that causes widespread bacterial corrosion to metal struc- ately after the recovery. We also show that turgor pressure recovers tures. It is useful for the bioremediation of heavy metals in the to the initial value along with the cell volume and, unlike previ- environment. The presence of D. desulfuricans populations in the ously thought, does not cause the reduction in growth rates. We human gut has been correlated with gastro-intestinal disease: its present results that point to changes in cellular energetics as the metabolism is thought to form inflammatory products. Unlike main cause of growth slow down at high external osmolarities. Desulfovibrio vulgaris, D. desulfuricans reduces both sulphate and nitrate as terminal electron acceptors. Nitrate is reduced via nitrite http://dx.doi.org/10.1016/j.nbt.2014.05.1717 to ammonia, a process that generates nitric oxide as a side product. A strong adaptive nitrosative stress response is essential for organisms living in nitrate-rich, micro-oxic environments. Nitric O12-6 oxide is generated during nitrate reduction in this, and other, orga- Engineering global regulator cAMP receptor protein nisms. Nitrosative stress, caused by NO production, is induced (CRP) of E. coli for improved biobutanol tolerance by macrophages to kill pathogens during human infection. It is therefore important to understand how D. desulfuricans responds Rongrong Jiang ∗ , Hefang Geng, Huiqing Chong to nitrosative stress. Nanyang Technological University, Singapore The tolerance of D. desulfuricans ATCC 27774 to nitric oxide was defined for bacteria with various histories of exposure. Cultures The toxicity of biofuel, including isobutanol and 1-butanol, grown with nitrate as terminal electron acceptor showed a 10-fold to microbial host is a major drawback during fermentative bio- increase in tolerance to NO compared to sulphate grown cultures. fuel production. Previously, UV/chemical mutagens and metabolic Nitrate grown cells also reduced nitric oxide at a 5-fold higher rate engineering have been used for strain engineering. However, these than sulphate grown cells. These phenotypic changes were inves- approaches are either labor intensive or time consuming, and tigated in the transcriptome of cells from stressed and unstressed require comprehensive metabolic information. In this work, we conditions by qPCR. The data reveal that exposure to nitrosative aimed to improve E. coli tolerance towards isobutanol and 1- stress induced a global response, that includes the induction of butanol by rewiring its global transcription factor cAMP receptor genes encoding proteins of previously unknown function. protein (CRP). Error-prone PCR was performed to mutate crp, and http://dx.doi.org/10.1016/j.nbt.2014.05.1716 the random mutagenesis libraries were subjected to isobutanol or 1-butanol stress for screening. After 3–5 subcultures with increased stressor concentration, the mutants with improved tolerance were O12-5 enriched in cell culture. Mutant IB2 (S179P, H199R) and MT5 (G71D, T127N, D138V, T208N) exhibited enhanced isobutanol/1- Origins of Escherichia coli growth rate and cell shape butanol tolerance respectively. In the presence of 1% (v/v, 9.6 g/L) changes at high external osmolality isobutanol, IB2 had the growth rate of 0.18 hour−1, 3.6-times −1 Teuta Pilizota 1,∗ , Joshua Shaevitz 2 faster than the control (0.05 hour ). Similarly, when challenged with 1.2% (v/v, 9.6 g/L) 1-butanol the growth rate of MT5 was 1 University of Edinburgh, United Kingdom 0.18 hour−1, double that of the control (0.09 hour−1). Genome 2 Princeton University, United States wide DNA microarray revealed that IB2 had 366 differentially expressed genes (>2 fold, p-value < 0.05) under isobutanol stress, In line with the challenge of designing robust synthetic sys- including acid resistance genes (gadABCE, hdeABD) and trans- tems, there has been an increased interest in understanding porters (proVWX, manXYZ). Quantitative real-time PCR showed bacterial growth rates. Growth can be modulated in different ways, that 1-butanol stress response genes (rpoH, ompF, sodA, manX, including temperature, antibiotics, toxins, nutrients and osmolar- marA) in MT5 demonstrated differential expression compared to ity. While the effects of nutrient availability have been extensively the control. Therefore, we believe that engineering CRP can serve characterized, an almost equally common modulation of bacte- as an effective alternative for E. coli engineering. rial growth occurs in response to changes in external osmolarities. In Escherichia coli, a sudden increase in external concentration http://dx.doi.org/10.1016/j.nbt.2014.05.1718 causes a pressure drop across the cell envelope, followed by an active recovery. Post recovery E. coli cells have been shown to grow slower, smaller and at a reduced turgor pressure. Despite the

S46 www.elsevier.com/locate/nbt New Biotechnology · Volume 31S · July 2014 SYMPOSIUM 12: STRESS RESPONSES IN MICROBIAL BIOPROCESSING

O12-7 measles virus hemagglutinin (MeH) is inefficient mostly due to bottleneck in the translocation of precursors of viral protein into Heat shock at higher cell densities improves the translo- the ER of the yeast cells. The aim of this study was to improve cation of measles virus hemagglutinin into the yeast translocation of MeH in S. cerevisiae by manipulating cell culture endoplasmic reticulum conditions. Induction of MeH expression under various conditions Rimantas Slibinskas ∗ , Edita Bak¯unaite˙, R¯uta Zinkeviciˇ ¯ute˙, was tested to establish factors that influence the efficiency of MeH Raimundas Razanskasˇ , Evaldas Ciplysˇ translocation. We found that heat shock with subsequent induction of MeH expression at 37◦C improved translocation of MeH Vilnius University Institute of Biotechnology, Lithuania precursors at the higher cell densities. The amount of MeH gly- coprotein increased about three-fold after the heat shock in the The yeast Saccharomyces cerevisiae is a widely used cell factory late-log phases of both glucose and ethanol growth. Our results for the production of heterologous proteins. However, secretory suggest that the heat shock may be employed for the improve- expression of the heterologous proteins in yeast is often subject ment of expression of the heterologous proteins in the S. cerevisiae to several bottlenecks that limit yield. Translocation of newly syn- secretory pathway. thesized proteins into endoplasmic reticulum (ER) is the first stage of the secretion pathway. It was earlier shown that synthesis of http://dx.doi.org/10.1016/j.nbt.2014.05.1719

www.elsevier.com/locate/nbt S47 FUTURE COLLABORATIONS OF AFOB-EFB AND MOU SIGNING CEREMONY New Biotechnology · Volume 31S · July 2014

Future collaborations of AFOB-EFB and MOU (3) Biopharmaceutical and Medical Biotechnology, (4) Biocatal- signing ceremony ysis and Protein Engineering, (5) Bioprocess and Bioseparation Engineering, (6) Bioenergy and Biorefinery, (7) Environmental SP6-1 Biotechnology, (8) Marine Biotechnology, (9) Nanobiotechnology, Biosensors and Biochips, (10) Systems and Synthetic Biotech- Asian Federation of Biotechnology (AFOB) as a driving nology, (11) Tissue Engineering and Biomaterials, and (12) Asia force for collaborative growth of biotechnology in Asia Bioeconomy and Biobusiness. Currently, some AFOB Divisions and beyond have started to work. For example, the Bioenergy and Biorefin- ∗ ery Division will have the annul division meeting and summit in Ho Nam Chang , Jian-Jiang Zhong , Tai Hyun Park, Hyun Jung Kim coming August, in which the leader of EFB Environmental Biotech- Asian Federation of Biotechnology, 508 Meet-You-All Tower Annex B, 12 nology Section has been invited to join as an invited speaker. Gaetbeol-ro, Yeonsu-gu, Incheon, Republic of Korea The division meeting of Bioprocess and Bioseparation Engineer- ing Division is to be held in this September. AFOB and its Divisions Asian Federation of Biotechnology (AFOB, homepage: have strong interest in (co-)hosting various seminars or symposia www.afob.org) is a non-profitable organization established in with EFB and other societies. In addition to collaborations on 2008. Since then, 13 Asian regions have joined AFOB as member congresses of AFOB and EFB, the collaborations between similar regions and currently 3000 individual members from Asia are divisions from two parties will also be beneficial. For collaboration actively involved in AFOB activities. Of course, the number of in Division/Section level, we are expecting academic Divisions of both member regions and individual members is still increasing. AFOB and their counterpart Sections of EFB to jointly organize Following the mission to promote co-operation and to expand academic activities and establish close collaborations. network between academia and industry of biotechnology, AFOB holds various events including congresses and symposia for scien- http://dx.doi.org/10.1016/j.nbt.2014.05.1721 tists and engineers in all areas of biotechnology. Especially, AFOB has established a tradition in Asian Congress on Biotechnology (ACB) since 1990 when APBioChEC was its predecessor, Young SP6-3 Asian Biochemical Engineers’ Community (YABEC) since 1995, Advances in Biotechnology in Asia and Future Collabo- AFOB Regional Symposium (ARS) and AFOB International Sym- ration with EFB posium. AFOB also organizes Annual Meetings and Summits for each Tai Hyun Park ∗ , Teruyuki Nagamune, Jung-Keug Park, Jian-Jiang division to promote their activities and collaboration with related Zhong, Ho Nam Chang divisions in partner organizations. Asian Federation of Biotechnology, 508 Meet-You-All Tower Annex B, 12 In addition, Biotechnology Journal publishes AFOB special issues Gaetbeol-ro, Yeonsu-gu, Incheon, Korea twice a year as an official partnership between AFOB and the Wiley publisher, enhancing the international status of Asian regions in The fast advancement of biotechnology around the world biotechnology and exhibiting top biotechnology achievements provides us with new products, ideas, methods, and tools. from member institutions and organizations to the world beyond Biotechnology in Asia is also growing very fast, and abundant bio- the Asia. All the members of AFOB are working hard for innovative diversities and biomass resources are located in Asian region. AFOB and practical biotechnology for the benefits of our human beings and EFB are able to develop a partnership to provide their members for better life, clean environment and sustainable development of with the opportunities for the collaborative research. In this sense, the society. academic societies can play an important role. As an example, dur- http://dx.doi.org/10.1016/j.nbt.2014.05.1720 ing last three years the American Institute of Chemical Engineers (AIChE) has been organizing an International Forum: Biotechnol- ogy in Asian Countries. The topics of the International Forum at SP6-2 AIChE annual meetings were as follows: Biotechnology in China (2011), Biotechnology in Taiwan and Southeastern Asia (2012), Academic Division of AFOB and Possible Collaboration Biotechnology in South Korea and Japan (2013). In this presenta- with EFB tion, we are going to introduce some activities of AFOB and the ∗ biotech researches in Asian countries. We would also like to sug- Wen-Chien Lee , Hyung Joon Cha, Jian-Jiang Zhong, Ho Nam gest some possible strategies for strengthening the collaboration Chang between AFOB and EFB, and any feedback from EFB colleagues is Asian Federation of Biotechnology, 508 Meet-You-All Tower Annex B, 12 well expected. Gaetbeol-ro, Yeonsu-gu, Incheon, Korea http://dx.doi.org/10.1016/j.nbt.2014.05.1722 Asian Federation of Biotechnology (AFOB) has set up an academic committee to coordinate and support its academic Divi- sions. Totally twelve academic Divisions are established to cover all areas of applied biotechnology. These are Divisions of (1) Agricultural and Food Biotechnology, (2) Applied Microbiology,

S48 www.elsevier.com/locate/nbt New Biotechnology · Volume 31S · July 2014 ADVANCES IN OMIC TECHNOLOGIES

Advances in omic technologies Syst Biol 10,721). iAdipocytes1809 was employed for gaining further insights from transcriptomics data obtained from Swedish SP4-1 Obese Subjects (SOS) Sib Pair study whereas iHepatocytes2322 was employed for the analysis of transcriptomics and metabolomics Integrating omics to study human biology and disease data obtained from NAFLD patients, and thereby we identified Mathias Uhlen both putative biomarkers as well as therapeutic targets. Person- alized GEMs for HCC patients were employed for identifying Science for Life Laboratory, Sweden anticancer drugs that are specific to individual patients using the concept of antimetabolites i.e. drugs that are structural analogs to The human proteins constitute the major building blocks for metabolites. The toxicity of each antimetabolite was predicted by the function of the various processes necessary for human life. assessing the in silico functionality of 83 healthy cell type specific The mapping of the human genome has allowed us to predict that GEMs. Finally, I will present the integration of data from RNA- each human has approximately 20 000 genes encoding for pro- Seq and antibody-based immunohistochemistry across all major teins. In the field of proteomics, these proteins are studied using human tissues and organs to explore the human tissue proteome various tools such as mass spectrometry, antibody-based profiling, with enriched expression (Mol Cell Proteomics 13, 397;Faseb chromatography, bioimaging, crystallography and spectroscopy. J, doi:10.1096/fj.14-250555) and its use for the validation and In addition, the new tools in genomics based on next generation improvement of the GEMs. sequencing have open up the possibility to study human variation and expression levels in a quantitative manner not possible only http://dx.doi.org/10.1016/j.nbt.2014.05.1724 a few years ago. We have classified all the protein coding genes in humans using a combination of genomics, transcriptomics, proteomics and antibody-based profiling. We have used this data to study the global protein expression patterns in human cells, tissues and organs as well as a discovery tool to find potential biomarkers for disease, such as cancer [1–9].

References [1].Mardinoglu, et al. Nat Commun 2014;5:3083. [2].Agren, et al. Mol Syst Biol 2014;10:721. [3].Stadler, et al. Nat Meth 2013;10(4):315–23. [4].Danielsson, et al. Proc Natl Acad SciUSA2013;110(17):6853–8. [5].Mardinoglu, et al. Mol Syst Biol 2013;9:649. [6].Paik, et al. Nat Biotechnol 2012;30(3):221–3. [7].Uhlen, et al. Nat Biotechnol 2010;28(12):1248–50. [8].Bourbeillon, et al. Nat Biotechnol 2010;28(7):650–3. [9].Vashisht, et al. Science 2009;326:718–21. http://dx.doi.org/10.1016/j.nbt.2014.05.1723

SP4-2

The use of Genome-scale metabolic models for drug target and biomarker identiﬁcation

Adil Mardinoglu 1,∗ , Mathias Uhlen 2, Jens Nielsen 1 1 Chalmers University of Technology 2 Royal Institute of Technology

The elucidation of diverse disease mechanisms for identiﬁca- tion of novel drug targets and discovery of biomarkers has been a major focus in medicine, and further research efforts are still needed for developing efﬁcient diagnostic and treatment strategies. Genome-scale metabolic models (GEMs) can aid in this by providing a scaffold for integration of omics data and revealing the molecular mechanisms involved in the occurrence of the disease (J Intern Med 271, 142;Biotechnol J 8,985). My presentation will cover the reconstruction and the use of functional GEMs for adipocytes (iAdipocytes1809) (Mol Syst Biol 9, 649), hepatocytes (iHepatocytes 2322) (Nat Commun 5,3083) and per- sonalized GEMs for Hepatocellular carcinoma (HCC) patients (Mol

www.elsevier.com/locate/nbt S49 SPONSORED SYMPOSIUM New Biotechnology · Volume 31S · July 2014

Sponsored Symposium strain C. glutamicum DM1933. Within oxygen-limited plug-flow- compartment coupled to aerobic stirred-tank-reactor, primary (i.e. SS-1 deliberately induced) and secondary (resulting from primary) oscillations of substrate and dissolved oxygen concentration as Recent developments in scaling down and using single well as pH and metabolic side product concentrations are studied. use probes for measuring the live cell concentration by With anaerobic residence times from 40 s up to several minutes, dielectric spectroscopy robustness and performance of microbial metabolism is challenged John Carvell , Matt Lee, Pardip Sandhar ∗ at continuously oscillating microenvironments. Carbon flux, respiratory activity and side product concentra- Aber Instruments Ltd, United Kingdom tions show fundamental differences under oscillating conditions. Most strikingly, Omics-data from the metabolome, proteome and Real-time bioprocess monitoring is fundamental for maxi- transcriptome show only marginal effects with respect to oscil- mizing yield, improving efficiency and process reproducibility, lation. This indicates that the regulon of C. glutamicum is very minimizing costs, optimizing product quality, and full under- robust against oscillatory conditions, which constitutes an impor- standing of how a system works. Bioreactors that are monitored tant mechanism for robustness in industrial processes. continuously and in real-time offer the advantage of meeting current and future supply demands with biological product of the http://dx.doi.org/10.1016/j.nbt.2014.05.1726 utmost quality and safety, achieved at the lowest overall cost and with least risk. This paper will focus on the latest developments in dielectric spectroscopy for live cell concentration measure- SS-3 ment and how the technology has been scaled down allowing Automated development of recombinant bioreactors with less than 100 ml working volume to be mon- bioprocesses–from vision to mission itored in real time. The presentation will also focus on how ,∗ dielectric spectroscopy can also be applied to single use biore- Florian Glauche 1 , Andreas Knepper 1, Michael Heiser 1, Lorenz actors in a cGMP environment and on samples down to 100 ␮l Theuer 1, Fabian Wollny 1, Susan Bigesse 1, Antje Neubauer 2, Sarina volume. Arain 3, Gernot Thomas John 3, Joachim Aschoff 4, Birgit Stehlik 4, Ingrid Schmidt 4, Rudibert King 5, Norman Violet 5, Detlef Goelling 6, http://dx.doi.org/10.1016/j.nbt.2014.05.1725 Andreas Raab 6, Gregor Kiesewetter 6, Rick Nolte 6, Peter Neubauer 1 1 Chair of Bioprocess Engineering, TU Berlin 2 SS-2 BioSilta Europe GmbH 3 PreSens Precision Sensing GmbH 4 Scale-down study of oscillations in oxygen and substrate Infoteam Software AG 5 Chair of Measurement and Control, TU Berlin supply for Corynebacterium glutamicum 6 Organobalance GmbH Marco Oldiges 1,∗ , Friedrich Käß 1, Ioanna Hariskos 1, Andrea Michel 1, Robert Spann 2, Peter Neubauer 2, Stefan Junne 2, Wolf- The development of recombinant protein production processes gang Wiechert 1 is a time and labor-intensive task. Implementing Quality by Design (QbD) and Design of Experiments (DoE) principles, already at the 1 Forschungszentrum Jülich/Institute of Bio- and Geosciences – IBG-1: Biotech- nology, Germany screening stage can significantly reduce development risk, time 2 Chair of Bioprocess Engineering/TU Berlin, Germany and costs. With the recent advances in laboratory automation, most steps of up- and downstream bioprocess development can be The platform organism Corynebacterium glutamicum has a suc- automated with implementation of DoE and QbD. At TU Berlin, in cessful history of biotechnological application and prevailing close collaboration with industrial partners, a reference laboratory, significance in industrial biotechnology with applications at biore- which focuses on the automation of recombinant protein pro- actor scales of several hundred cubic meters. Studies in metabolic duction process development, was established. Key features of the and bioprocess engineering, however, are mostly limited to cul- system are (i) development of software solutions in order to apply tivation at laboratory scale. An often neglected upscaling effect DoE for screening and optimization procedures, (ii) automated at- is that physical transport and metabolic uptake processes of, for line and on-line analyses by the use of analytical procedures and example oxygen and substrate lead to stronger concentration gra- sensors, (iii) software-supported model creation and development dients with increasing bioreactor working volume. This causes of process models for advanced process control at the bioreac- oscillating microenvironments for individual cells. The metabolic tor scale. Microbial cultivations are performed in 96 and 24 well impact of such oscillations can be monitored in specialized scale- plates as well as in a 10 mL 48-bioreactor system. The application down bioreactor setups, where large scale effects are simulated in of EnBase® technology to maintain controlled growth is one of laboratory-scale experiments. the key technologies in this concept. Growing cells in fed-batch In this work, two-compartment reactor systems (stirred-tank- cultures over the whole developmental line ensures consistency reactor connected to plug-flow-reactor) have been used to generate in operation procedures from microliter and milliliter scales to defined oscillations in oxygen and substrate supply during cul- pilot plant scale. The overall strength of the automation concept tivation of C. glutamicum ATCC13032 and lysine producing

S50 www.elsevier.com/locate/nbt New Biotechnology · Volume 31S · July 2014 SPONSORED SYMPOSIUM is demonstrated on a number of difﬁcult to produce proteins in SS-5 Escherichia coli and Saccharomyces cerevisiae. Massive protein superfamily data integration applied to http://dx.doi.org/10.1016/j.nbt.2014.05.1727 smart library design

Henk-Jan Joosten SS-4 Bio-Prodict, Nijmegen, Netherlands

Scale up of chito-oligomer production via bacterial fer- Nature offers a wide variety of enzymes that can be utilized mentation in many different processes, but often these enzymes need to be Hendrik Waegeman 1,∗ , Ko Wisse 2, Saskia Vander Meeren 2, An optimized by introducing mutations to meet the requirements of Vermeulen 2, Brecht Vanlerberghe 2 these (biotechnological) processes. In many cases a combination 1 Bio Base Europe Pilot Plant vzw, Belgium of multiple mutations are needed to reach these goals, but finding 2 Bio Base Europe Pilot Plant, Belgium the right combination of mutations still is problematic. More and more protein engineers use a strategy referred to as “smart library Chito-oligomers constitute an interesting class of specialty car- design”. Smart mutant libraries contain only a small number of bohydrates, among other applications used in plant protection mutants and are designed such that they contain a high number of and wound healing products. Today’s commercially available chi- active clones with mutations at positions (hotspots) that are likely tosans are produced chemically from chitin isolated from shrimp to show the desired effect. The quality of a smart library depends shell wastes. They can be well defined concerning their degree of on: 1. The selection of hotspots and 2. The prediction of the best polymerisation and degree of acetylation, but they are invariably amino acid changes at these hotspots. Recently it was shown that characterised by a random pattern of acetylation (PA), despite this the vast amounts of data nowadays available for protein super- influences the activity greatly. families can be used for the prediction of both these steps and Together with other partners within the ERA-IB ChitoBioEngi- therefore for the design of high quality smart mutant libraries. neering and FP7 Nano3Bio project, Bio Base Europe Pilot Plant Here we present how 3DM, a protein superfamily analysis plat- aims at establishing, through genetic, metabolic and enzyme form that integrates many different data types for complete protein engineering, biotechnological ways of producing fully defined, superfamilies, can be used to design smart libraries. Using this strat- partially acetylated chitosans, of which no production methods egy different enzymes features, such as enantioselectivity, activity are available to date. Our specific goal, is to employ the strains and thermostability have been optimized. Comparisons with ran- tested at laboratory scale, to develop an industrially viable fer- dom designed libraries show that, by using 3DM when designing a mentation and downstream purification process for every specific smart library, libraries are of high quality, which reduces not only chito-oligomer. Processes, meeting defined requirements in terms the number of clones that need to be screened but it also increases of titer and production rate are scaled up in our facilities. This pre- the chance of finding an enzyme with the desired properties. sentation will show the results of the successful scale up of the first http://dx.doi.org/10.1016/j.nbt.2014.05.1729 product developed by BBEPP and its partners. http://dx.doi.org/10.1016/j.nbt.2014.05.1728

www.elsevier.com/locate/nbt S51 SYMPOSIUM 13: BIOMEDICAL RESEARCH New Biotechnology · Volume 31S · July 2014

Symposium 13: Biomedical research polymers namely, poly urethane and polycaprolactam, which are approved by FDA for medical applications. The enzyme is immo- O13-1 bilised using glutaraldehye as the fixing agent. Presence of the enzyme is confirmed using FTIR. The conditions namely, temper- Bacteria fabricate 3D scaffolds for organ regeneration ature, pH and time of reaction for immobilization are optimized Paul Gatenholm ∗ , Hector Martinez, Johan Sundberg, Sara to achieve maximum enzyme activity. The storage stability of the Johannesson, Daniel Hägg surfaces is tested successfully. The formation of Staphylococcus aureus and Escherichia coli biofilm on these surfaces are tested. BBV Laboratory and WWSC, Department of Chemical and Biological Engineer- These organisms are widely found in implant associated infection. ing, Biopolymer Technology, Chalmers University of Technology, Göteborg, Sweden Both the surfaces reduced the number of live colonies (by about 30 times), amounts of biomass, protein and carbohydrate content (by In recent years, stem cells and biomaterials have emerged as 6 times) in the bacterial biofilm. The composition of the biofilm tools to regenerate damaged or diseased tissue. Typically, a bio- was probed using FT-Raman spectroscopy. This study indicates that compatible material that can be engineered to match the shape of stable antimicrobial surfaces could be prepared using this enzyme. the defect and can function as a vehicle to deliver cells and/or bio- Such surfaces could find applications in the design of implantable logically active factors that promotes tissue regeneration is used. or topical biomaterials which require prevention of biofilm. Bacterial nanocellulose is emerging biomaterial which combines http://dx.doi.org/10.1016/j.nbt.2014.05.1731 the nanofibril network with hydrogel like behavior which makes it ideal for Tissue Engineering applications. We have previously used bacteria to fabricate artificial blood vessels and menisci substitutes, O13-3 and excellent biocompatibility has been shown in rat, hamster, pig and sheep. In the present study we describe a novel biofabrica- Osteoblast cell proliferation on magnesium-substituted tion method, which uses the Gluconoacetobacter Xylinus machinery hydroxyapatite (Mg-HA) coatings to engineer 3D scaffolds with features at different length scales – Fatma Nese Kok 1,∗ , Sakip Onder 2, Ayse Ceren Calikoglu 3, Kursat ranging from the nano, subcellular to the macroscale. Examples Kazmanli 4, Mustafa Urgen 4, Gamze Torun Kose 3 of 3D Bacterial nanocellulose scaffolds prepared in our laboratory 1 Istanbul Technical University, Turkey include multichannel scaffolds for preparation of microvascular 2 Istanbul Technical University, Molecular Biology Genetics and Biotechnology structures, highly porous 3D structures for growth of cartilage tis- Programme, Turkey sue and combination of 3D multichannel scaffold with highly 3 Yeditepe University, Genetics and Bioengineering Department, Turkey porous architecture for growth of vascularized tissue such as 4 Istanbul Technical University, Department of Metallurgical and Materials Engi- bone and adipose tissue. 3D Bacterial nanocellulose scaffolds have neering, Turkey shown to support neural network formation and enable stem cell differentiation. Human tissues grown on 3D Bacterial nanocel- Hydroxyapatite (HA; Ca10(PO4)6(OH)2) is a well-known bio- lulose scaffolds show great potential of this new biomaterial-cell compatible material commonly preferred in surface modification constructs for applications in reconstructive surgery and as in vitro of hard tissue implants to ensure better osteointegration with the model of diseases such as Alzheimer and Osteoarthritis. surrounding bone tissue. Properties of HA may also be improved via doping different ions into HA structure for better osteoin- http://dx.doi.org/10.1016/j.nbt.2014.05.1730 tegration and cell proliferation properties. In our study, cell proliferation studies on magnesium substituted HA coatings (Mg- HA) that were deposited on different Mg2+ containing (Ti,Mg)N O13-2 thin film coatings (0, 4.24 and 10.42% at) were studied to deter- 2+ Enzyme immobilized polymeric biomaterials mine the effect of Mg on cell proliferation. Mg-HA coatings and characterization studies were conducted as our previous study [1]. ∗ Mukesh Doble , Prabhawathi Veluchamy Then, osteoblast cells were seeded on HA and Mg-HA coatings IIT Madras, India and MTS studies were conducted for 1, 4 and 7 days. Results showed that cell proliferation was better on Mg-HA coatings that 2+ Polymers are widely used in the biomedical technology for were deposited on low Mg (4.24 at%) containing surfaces com- 2+ the fabrication of medical implants and devices. Implant based pared with the HA coatings that were deposited on Mg free infection is highly prevalent in biomaterial which leads to its surfaces. Cell proliferation slowed down on Mg-HA coatings that 2+ early rejection. Several physical, chemical and biological meth- were deposited on high Mg (10.42 at%) containing surfaces. High ods are practiced to overcome the bacterial attachment and the Mg content also accelerated the corrosion so this was taught to be subsequent formation of biofim. Antibiotics are used as main the major reason for the decrease in cell survival. Studies on the form of therapy, but emergence of antibiotic resistance bacteria biocompatibility of (Ti,Mg)N thin film coatings prepared in differ- is forcing the researches to search for other methods. Biomateri- ent conditions are being tested to better understand the effect of als are hydrophobic, with no labile chemical groups that might Mg presence in the coatings. The grant from TUBITAK (112M339) be used with conventional immobilization strategies. Papain, an is gratefully acknowledged. antimicrobial and biocompatible enzyme is immobilized on two

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Reference Current practice is to refrigerate blood (from donor centres), [1].Onder S, et al. Mater Sci Eng C 2013;33(7):37–42. with an anticoagulant, where it is retained for a mere 42 days. To ensure a readily available safe blood supply, preservation tech- http://dx.doi.org/10.1016/j.nbt.2014.05.1732 niques and methods for long-term storage need to be investigated. Cryopreservation in glycerol has shown some success in extending the shelf life of blood. However, prior to use, the glycerol O13-4 must be removed. This is a demanding, labour intensive and costly process resulting in a limited 24-hour shelf life of deglycerolised Bone regeneration through facile binding of bone graft sample making it an unfeasible procedure. substitute particles using mussel adhesive protein We present the use of freeze-drying with intracellular sugars as a ,∗ Hyung Joon Cha 1 , Bong-Hyuk Choi 1, Hogyun Cheong 1, Yun Kee method for stabilisation. A two part process; freeze-drying involves Jo 1, Jin-Soo Ahn 2, Sang-ho Jun 3 a freezing step followed by a sublimation process that takes place 1 Pohang University of Science and Technology, Republic of Korea with the sample held in a vacuum. The ideal product would be 2 Seoul National University, Republic of Korea a dried powder capable of room temperature storage. With the 3 Korea University Anam Hospital, Republic of Korea appropriate additives, the hydrated blood would be suitable for direct transfusion. While the process is still in its infancy; freeze- To date, hydroxyapatite and related calcium phosphates have drying has the potential to change the future of blood banking. been intensively investigated as the bone substitutes for bone http://dx.doi.org/10.1016/j.nbt.2014.05.1734 tissue engineering. Among these, inorganic bovine bone is the most commonly used bone graft material. However, lack of cell recognition motifs and/or biochemical factors has been consid- O13-6 ered a limitation. Mussel adhesive proteins (MAPs) are one of most remarkable and powerful adhesive materials in nature. Pre- Stable aqueous solutions of keratin polypeptides: their viously, recombinant MAPs were successfully demonstrated to be properties in solution and at interfaces functional cell adhesion materials on various surfaces due to their Fang Pan ∗ Jian Lu peculiar adhesive properties. Herein, MAPs were applied as surface , coating and functionalization biomaterials to xenograft materials. The University of Manchester, United Kingdom We successfully coated MAPs onto xenograft surfaces by simply mixing xenografts with the MAP solution. Through in vitro Keratins are important structural fibrous proteins and are the study using mouse osteoblast cell line MC3T3-E1, significant main constituting components in skin, hair, wool, feathers, nails, enhancement of cellular activities such as attachment, prolifer- horns and connecting tissues. Their physical and biological studies ation, spreading, and osteogenic differentiation was observed on have direct relevance to health and disease and also bear impor- MAP-coated xenografts. In addition, we found that in vivo implan- tant implications to hair and skin care. In addition to personal tation of MAP-coated xenografts enhanced bone regeneration in care, there are many other reasons that require us to understand a rat calvarial defect model. These results collectively demonstrate how keratins behave physically and how they interact with other that facile coating of xenografts using biofunctional MAP would be molecules. Wool is predominantly composed of keratin proteins a promising strategy for successful bone tissue engineering appli- that provide desirable properties. The purpose of this work is cations. to develop water-soluble keratin polypeptides from sheep wool, http://dx.doi.org/10.1016/j.nbt.2014.05.1733 which can be used to form smooth molecular layers and optically flat thin films. These model biointerfaces can facilitate various physical and biological measurements that would otherwise be too O13-5 difficult to contemplate. Successful preparation of keratin samples was demonstrated by identification of molecular weights of the The stabilisation of red blood cells in a powdered form key components from gel electrophoresis and measurements of their surface tension and basic solution properties. Zeta poten- Krishnaa Mahbubani ∗ , Nigel K.H. Slater tial measurements from keratin samples prepared demonstrated Department of Chemical Engineering and Biotechnology, University of almost identical pH dependent surface charge distributions with Cambridge, United Kingdom isoelectric points around pH 3.5, showing that during purification by dialysis has completed removal of SDS used during wool fibre In recent years apprehension surrounding blood transfusions dissolution. have significantly reduced. However, fears around the supply of http://dx.doi.org/10.1016/j.nbt.2014.05.1735 blood have become a concern. In metropolitan areas, managing the blood inventory is critical to ensure emergency and elec- tive surgeries can take place without shortages while military blood banking faces a slightly different challenge, in obtaining the required amount and type of blood products at specific times and often in remote areas.

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O13-7 city of producers and high cytotoxicity of produced TL limit their biomedical applications. Therefore, the search for TL pro- Trehalolipid biosurfactants from Rhodococcus ruber ducers among nonpathogenic actinobacteria is essential. In in vitro with anti-adhesive and immunomodulatory activities experiments, TL biosurfactants from Rhodococcus ruber IEGM 231 Maria Kuyukina 1,∗ , Irena Ivshina 1, Tatiana Baeva 1, Olesia stimulated both proinﬂammatory (interleukin (IL)-1b, IL-6, tumor ␣ Kochina 1, Sergey Gein 2 necrosis factor-a (TNF- ) and anti-inﬂammatory (IL-12, IL-18) cytokine production of human monocytes, depending on cell 1 Institute of Ecology and Genetics of Microorganisms, Russia culture composition and induction. Also, diverse anti-adhesive 2 Perm State University, Russia effects of TL towards human monocytes and bacterial species were revealed. Since TL from R. ruber displayed no cytotoxicity against In recent years, glycolipid biosurfactants traditionally con- human lymphocytes or bacterial cells, they could be proposed sidered as emulsifying and solubilizing agents are attracting as potential immunomodulatory, antitumor and anti-adhesive an increasing attention as possible biomedical agents with agents. expressed biological activities [1]. Trehalolipids (TL) produced by Research was funded by the RAS MCB and RF President Leading members of closely related actinobacterial genera Rhodococcus, Science School programs. Nocardia, Corynebacterium, Gordonia, Mycobacterium, Tsukamurella, and Arthrobacter include ␣,␣-D-trehalose, a nonreducing disac- References charide, which is linked by an ester bond to long-chain fatty [1].Kitamoto, et al. J Biosci Bioeng 2002;94:187–201. acids [2]. Well-known TL of pathogenic Mycobacterium tuberculo- [2].Kuyukina, Ivshina. Microbiol Monographs 2010;16:292–313. sis, Corynebacterium diphtheriae play a key role in the infections [3].Ryll, et al. Microbiol Immunol 2001;45:801–11. caused by these actinobacteria, and they are characterized by http://dx.doi.org/10.1016/j.nbt.2014.05.1736 high immunomodulatory activity [3]. However the pathogeni-

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Symposium 14: Plant genetic engineering After comparing the transcriptome data of Ari1327 with Ari971 and Ari3697, 13919 differentially expressed unigenes (DEGs) were O14-1 identified, of which5406 up-regulated while 8513 down-regulated in Ari1327. The 16 genes in plant hormone signal transduction Genetic engineering of secondary metabolism for plant pathway was validated and analysed using quantitative real time protection PCR (qRT-PCR). After analysis of the differential expression of J.A. Pickett miRNA families in each sample, 16 miRNA families were found to up-regulate in Ari1327 and down-regulated in Ari3697. In three Rothamsted Research, Harpenden AL5 2JQ, United Kingdom miRNAs libraries, we found 25 miRNAs only expressed in Ari1327 and 13 miRNAs only expressed in Ari3697. These 16 miRNA fami- Only recently has secondary plant metabolism become a target lies and 38 specific expressed miRNAs may play significant role in for genetic modification, initially to investigate the role of metabo- regulating plant height of Ari1327. Some miRNAs were selected to lites and now in crop plants for pest management. By generation in validate the reality of small RNA sequencing by using stem-loop the plant, chemically unstable and even highly volatile metabo- RT-PCR. The target genes of these differentially expression miRNAs lites can be exploited beyond the toxicants traditionally used to were also annotated in A and D genomes in cotton. control pests. Expression of the aphid alarm pheromone, (E)-␤- farnesene, in wheat will be described leading on to the need for http://dx.doi.org/10.1016/j.nbt.2014.05.1738 induced or primed expression. Examples of other semiochemicals, in addition to pheromones, such as stress related homoterpenes, that are already exploited in crop protection, demonstrate also O14-3 induction or priming as a consequence of stress related plant Can plant still be a major and cost-effective source for signalling involving secondary metabolism. Such signals them- the supply of artemisinin, the most potent anti-malaria selves then provide alternatives to constitutive expression for use drug in GM crops and already provide evidence of likely success as ∗ well as offering new types of sentinel plants and potentially solu- Kexuan Tang , Qian Shen, Fangyuan Zhang tions for problems of perennialisation of arable crops. Secondary Shanghai Jiao Tong University, China metabolites, exploited in these ways, also show promise for not only disease and weed management, but in dealing with other Artemisinin isolated from Artemisia annua L., is most potent requirements of more sustainable agriculture relating to non-CO2 in treating malaria. Since the discovery of artemisinin in 1970s, greenhouse gas production. A. annua has been the only source for artemisinin supply. Recent http://dx.doi.org/10.1016/j.nbt.2014.05.1737 breakthrough in transgenic yeast has broken the traditional supply way of artemisinin. According to the report, transgenic yeast system can supply 50–60 tons of artemisinin annually at the sale O14-2 price of $350–400/kg, which is lower than the current artemisinin production cost by using traditional plant varieties. Data showed Transcriptome and small RNA sequencing analysis of a in this year A. annua plantation has been reduced by over 60%. new dwarf mutant in Gossypium hirsutum L It brings concerns if farmers are not willing to grow A. annua Xiongming Du 1,∗ , Wen-yan An 2, Jun-ling Sun 1, Wen-fang Gong 1, anymore, is the yeast system ready for providing all the amount Shou-pu He 1, Zhao-e Pan 1 of artemisinin the world needs and when? Will plant system to produce artemisinin be out of the stage? 1 Institute of Cotton Research of Chinese Academy of Agricultural Sciences (ICR, Here, we report the development of three strategies, includ- CAAS), China 2 Institute of Cotton Research of Chinese Academy of Agricultural Sciences ing spraying fertilizer technology, developing high artemisinin (ICR, CAAS)/College of Life Science and Technology, Huazhong Agricultural content varieties, and transgenic plant technology to increase University, China artemisinin content in A. annua, which dramatically reduces production cost, and could supply artemisinin in large quantity at Plant height is an important trait in upland cotton. Ari1327 the sale price of $250 in 3 years. Transgenic lines have been was a dwarfed mutant derived from an American upland cot- approved for environmental release, the first GM medicinal plants ton varieties-Ari971 by 60Co ␥-ray irradiation. Ari1327 exhibited to be approved in the world. Environmental test and animal test dwarf trait during germination and cotyledon periods. Using the demonstrate transgenic lines are safe and artemisinin extracted next generation high-throughput sequencing technology, three from transgenic lines has same function with that extracted from cDNA and small RNA libraries of the dwarfed mutant Ari1327, non-transgenic varieties. We believe these strategies, together with higher plant mutant Ari3697 and their wild type Ari971 were transgenic yeast system, will secure artemisinin supply enough at constructed and sequenced with Illumina HiSeqTM2000 system. affordable price to combat malaria. Through comprehensive analysis with transcriptome and miRNA http://dx.doi.org/10.1016/j.nbt.2014.05.1739 regulation level for the plant high mutant, it will be beneficial to reveal the molecular mechanism dwarf mechanism and clone the related genes for dwarf plant breeding in cotton.

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O14-4 protein expression and fluorescence levels could be improved by: (i) expressing VFP under the control of the psaA promoter/5UTR; Engineering barley for increased drought resistance (ii) fusing the VFP coding sequence to that of an endogenous gene Ivo Frébort ∗ , Hana Pospísilováˇ , Petr Galuszka (rbcL); (iii) co-expressing a bacterial chaperone in the chloroplast, obtaining increased protein expression for (i) and (iii). Palacky University in Olomouc, Czech Republic These constructs have been grown using different media and temperatures, in order to evaluate growth rate together with level Barley is an agriculturally important crop and the production of of protein expression and fluorescence. plants with enhanced stress tolerance is one of the important goals This approach merges both cell engineering and bioprocess in barley breeding. New techniques of molecular cloning and plant optimisation of a strain of algae widely used in research and has the transformation accelerate classical breeding techniques helping to potential to be applied to other recombinant proteins expressed in produce barley plants with required enhanced traits. Morphology C. reinhardtii. and development of the barley plants have been altered by genetic manipulation with genes coding for cytokinin dehydrogenase (EC http://dx.doi.org/10.1016/j.nbt.2014.05.1741 1.5.99.12; CKX), a principal enzyme controlling cytokinin levels in plants. Three unique transgenic barley lines (Hordeum vulgare cv. O14-6 Golden Promise) transformed with an expression cassette consist- Comparative evaluation of bacterial diversity from gm ing of ␤-glucosidase root specific promoter, modified CKX gene and Non-GM maize rhizosphere from Arabidopsis thaliana with engineered protein targeting to cytoplasm, vacuoles or apoplast, and NOS terminator were pre- Naseer Ahmad 1,∗ , Naseer Ahmad 2, Zabta Khan Shinwari 3 pared and T2 generations of homozygous plants were analyzed. 1 Department of Environmental Sciences, COMSATS Institute of Information Selected transgenic lines, distinctive with altered morphology of Technology (CIIT), Pakistan the root system, showed higher resistance to drought conditions 2 COMSATS Institute of Information Technology (CIIT), Pakistan 3 than wild type plants, both when grown in soil or in hydro- Quaid-i-Azam University, Islamabad, Pakistan ponic culture. This method of conveying drought resistance may be further exploited in order to create a usable trait that can be The rhizosphere is a critical interface supporting the exchange transferred to commercial cultivars of barley. of resources between plants and their associated soil environment. Rhizosphere microbial diversity is influenced by the physical and http://dx.doi.org/10.1016/j.nbt.2014.05.1740 chemical properties of the rhizosphere, some of which are determined by the genetics of the host plant. However, within a plant species, the impact of genetic variation in the composition of the O14-5 bacterial biota of GM and Non-GM maize rhizosphere is poorly Recombinant protein expression in the chloroplast of understood. Here, we studied the bacterial diversity and popula- the green microalga Chlamydomonas reinhardtii: A case tion dynamics in the rhizosphere of one GM and two Non-GM study using a novel green fluorescent protein as a maize cultivars (IG and IW) grown under field conditions, by tradi- reporter tional cultivation techniques and 16S rRNA gene-based molecular analysis of DNA directly extracted from pre cultivated soil and ∗ Stephanie Braun Galleani , Frank Baganz, Saul Purton rhizosphere samples. Rhizosphere and pre cultivated soil samples UCL, United Kinglom were taken at three different plant growth stages. The isolated bacterial strains were further screened for different functional char- Unicellular algae such as Chlamydomonas reinhardtii are attract- acterization. From this study, we find that the transgenic crop has ing increasing interest as low cost, GRAS platforms for the no positional impact on the rhizospheric bacterial community. synthesis of high-value heterologous proteins. Foreign genes http://dx.doi.org/10.1016/j.nbt.2014.05.1742 introduced into its chloroplast genome can be targeted to a precise locus and expressed without suffering gene silencing issues. Fluorescent proteins such as green fluorescent protein (GFP) provide a simple tool for monitoring protein synthesis in vivo. However, the level of fluorescence from GFP expressed in the C. reinhardtii chloroplast has proved disappointingly low. A newly discovered protein called Verde Fluorescent Protein (VFP) has showed superior fluorescence in other organisms, so we are currently investigating its utility as reporter. A codon-optimised gene encoding VFP was synthesised and successfully introduced into the chloroplast genome under the control of the atpA promoter/5UTR. Protein expression was confirmed by western blotting, and fluorescence was demonstrated by confocal microscopy and flow cytometry. We explored whether

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O14-7 ods require plant and pathogen cells to be physically separated. However, probe-independent RNA sequencing method allows Understanding the interactions between plant biotic plant and pathogen transcripts to be analyzed simultaneously. and abiotic stress through characterization of microRNA MicroRNA effectors serve the conserved gene regulatory mech- effectors in jute (Corchorus spp.) – Macrophomina phase- anisms which have been reported to control plant–pathogen olina interaction system interactions. Most works on miRNAs have focused only on ini- Lalit Kharbikar ∗ , Pran Gobinda Karmakar tial pathogen infection processes under controlled environment. However, miRNA-mediated regulation of disease development Central Research Institute for Jute and Allied Fibres (Indian Council of Agricul- throughout life cycle of plants under the influence of abiotic tural Research), India stresses, such as drought has not been studied so far. Since, stem rot becomes severe at maturity under hot and humid weather, Jute (Corchorus spp.) is an important fibre crop in India. This despite the colonization of roots by M. phaseolina at early growth smallholder’s crop is vulnerable to most devastating pathogen, stages of jute, under high soil temperature and low soil moisture Macrophomina phaseolina. It causes damping off, root rot and col- (drought) conditions, the jute–M. phaseolina interaction system lar rot collectively known as stem rot, which results in up to could provide a good model to study whether physiological 30% yield loss and low fibre quality. The pathogen first causes alterations induced by pathogen colonization or drought stress root rot upon drought stress and then colonizes physiologically facilitate the development of disease. Sequencing of miRNAs dur- altered stem causing stem rot at maturity, under favorable weather. ing this could elucidate molecular interactions between plant However, the molecular mechanisms underlying are unknown. biotic and abiotic stress. Plant–pathogen–weather interface is regulated by a number of genes. Existing, probe-dependent gene regulation analysis meth- http://dx.doi.org/10.1016/j.nbt.2014.05.1743

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Symposium 15: Recombinant protein produc- interest in the field and as MPs are extremely important drug tar- tion gets they attract the attention of the pharmaceutical industry. For characterization of MPs and acquisition of information for the O15-1 rational design of drugs large amounts of protein are required. As their natural abundance is usually very low, MPs are produced by Getting a grip on complexes: tools and technologies for overexpression in heterologous hosts. However, they are notori- multiprotein complex research ously difficult to express as the yields per cell are typically low and MP production is highly toxic to cells. The aim of the present Imre Berger work is to engineer strains of Escherichia coli to overcome MP- EMBL, Grenoble overexpression toxicity. The ultimate goal is to use these strains for large-scale production of MPs. Also, our goal is to investigate the Most eukaryotic proteins exist as large multicomponent assem- mechanism behind the observed cytotoxicity. For this purpose we blies with many subunits, which act in concert to catalyze specific have generated libraries of mutant bacteria carrying different types cellular activities. Many of these are only present in low amounts of genetic modifications and used appropriate genetic screens to in their native hosts, impeding purification from source material. isolate the desired clones. The human GPCR bradykinin recep- Unraveling their mechanisms of action will therefore often depend tor 2 that shows very high overexpression toxicity in E. coli was on heterologous overproduction. used as our model MP. We have identified a number of strains that Complex proteins, involved in disease causing processes, are overcome the toxicity upon BR2 overexpression while more over- also entering center-stage as key drug targets of the future. In expressed protein is produced. The strains have also been shown addition to their essential role as tools for drug discovery, such to have a more general application for several GPCRs in E. coli. The complex biologics themselves are increasingly being employed as clones are being characterized using various methods. A potential medicines, for example in the form of multicomponent vaccines. physiological role of the identified mutations is proposed. These are predicted to dominate the next generation of drugs. http://dx.doi.org/10.1016/j.nbt.2014.05.1745 My laboratory develops advanced methods for producing multicomponent protein biologics for studying their structure and function at molecular resolution. We have created MultiBac, a O15-3 baculovirus/insect cell system designed for high-quality multiprotein complex production, and have installed MultiBac as a Population heterogeneity in Pseudomonas putida ana- platform technology at the Eukaryotic Expression Facility (EEF) in lyzed on the single cell level using proteomics and digital our laboratory. More recently, in the context of the EC FP7 project PCR ComplexINC, we have extended our technology concept to mul- 1,∗ 1 1 tiprotein production in other host systems including mammalian Michael Jahn , Carsten Vorpahl , Dominique Türkowsky , Martin 2 2 1 1 cells. Our technologies and successful applications in academic Lindmeyer , Bruno Bühler , Hauke Harms , Susann Müller and industrial R&D will be presented [1–3]. 1 Helmholtz-Centre for Environmental Research - UFZ Leipzig, Germany 2 TU Dortmund University, Germany References

[1].Bieniossek, et al. The architecture of human general tran- Recombinant protein production using microbial cells is a key scription factor TFIID core complex. Nature 2013;493(January player for the chemical and pharmaceutical industry of the future. (7434)):699–702. The efficiency of such biotechnological processes is usually bench- [2].Barford D, Takagi Y, Schultz P, Berger I. Baculovirus expression: tackling the complexity challenge. Curr Opin Struct Biol marked using bulk parameters. However, the catalytic unit driving 2013;23(3):357–64. a process is the single cell. Even clonal populations show high cell- [3].Bieniossek C, Imasaki T, Takagi Y, Berger I. MultiBac: expanding to-cell variability, e.g. caused by genetic mutations, cell cycling or the research toolbox for multiprotein complexes. Trends Biochem Sci regulatory decisions. 2012;37(February (2)):49–57. As a model system for population heterogeneity, we use Pseu- http://dx.doi.org/10.1016/j.nbt.2014.05.1744 domonas putida with plasmid-based production of an EGFP fused recombinant protein (StyA). With EGFP as a marker, flow cytometry shows large proportions of cells (30–60%) not able to O15-2 produce functional protein even under high induction regimes. We used cutting edge cell sorting of fluorescent (EGFP+) and Making life better for Escherichia coli cells that produce non-fluorescent cells (EGFP−) to identify the cause for impaired toxic membrane proteins protein production. Sorted sub-populations were analyzed by pro- Dimitra Gialama 1,∗ , Fragiskos Kolisis 2, Georgios Skretas 1 tein mass spectrometry to reconstruct pathways, and by digital PCR to determine the plasmid copy number (PCN) with extraor- 1 National Hellenic Research Foundation, Greece 2 National Technical University of Athens, Greece dinary precision. In summary, only minor changes in the protein inventory were Membrane proteins (MPs) constitute an exciting world that found, most of them related to stress as a result of protein aggre- includes protein families such as G protein-coupled receptors gation. In contrast to that, we identified unequal distribution of (GPCRs) and transporters. Currently, there is increasing scientific plasmids as a major cause for heterogeneity: The non-fluorescent

S58 www.elsevier.com/locate/nbt New Biotechnology · Volume 31S · July 2014 SYMPOSIUM 15: RECOMBINANT PROTEIN PRODUCTION sub-population was almost plasmid-free, although some cells important biopharmaceutical cell factories. In this regard, recently refused gene expression despite presence of the plasmid. we have developed stable engineered miR-17 overexpressing CHO http://dx.doi.org/10.1016/j.nbt.2014.05.1746 cells, which exhibit both enhanced growth performance and increase in specific productivity. This lead to overall 3-fold increase in recombinant protein (EpoFc) titers, this parallel enhancement O15-4 of both cell-specific growth rate and productivity is very unique. The underlying molecular mechanism that controls such pheno- Synthesis of antibacterial bacteriophage proteins in type is generated by co-regulations various biological networks microalgae and understanding such molecular regulation is hindered by the lack of a miRNA-mRNA interaction database for CHO cells. For Laura Stoffels 1,∗ , Bambos Charalambous 2, Saul Purton 3 this reason, we applied transcriptomics (mRNA and miRNA) using 1 University College London, UK microarray platform and quantitative proteomics using iTRAQ 2 Research Department of Infection, University College London Medical School, technology. These data sets are ultimate readout of miRNA activity UK and developed a bioinformatics pipeline to handle such datasets 3 Institute of Structural and Molecular Biology, University College London, UK where the rules of interaction are not a priori known. This novel inter-omics approach is described as well as an interpretation of the Widespread antibiotic resistance among pathogenic bacteria effects caused by miR-17 overexpression that led to the interesting and the low specificity of these drugs cause a pressing need for the phenotype described above. development of novel antibacterial agents. Endolysins are antibacterial proteins that are produced by bacteriophages to digest the http://dx.doi.org/10.1016/j.nbt.2014.05.1748 bacterial cell wall for phage progeny release at the end of the lytic cycle. These efficient enzymes are highly specific for the cell wall of the target bacteria without affecting other species. Development O15-6 of resistance against endolysins is very rare, because they evolved to target molecules in the cell wall that are essential for bacte- Investigating the physiological effect of increased rial viability. Taken together, this makes them promising novel heterologous gene dosage in Pichia pastoris using tran- antibacterial agents. The eukaryotic microalga Chlamydomonas scriptomics reinhardtii offers already established techniques for the expression Elena Camara 1,∗ , Landes Nils 2, Lluís Revilla Sancho 3, Joan Albiol 4, of foreign genes in the chloroplast and is an attractive expression Diethard Mattanovich 2, Pau Ferrer 4 platform for therapeutic proteins, due to the lack of endotoxins 1 UAB, United States and potentially infectious agents. Furthermore it can be inexpen- 2 Department of Biotechnology, University of Natural Resources and Life Sci- sively cultivated in full containment and under sterile conditions ences Vienna/Austrian Centre of Industrial Biotechnology (ACIB GmbH), Austria in simple photobioreactors. Two bacteriophage endolysins tar- 3 School of Bioengineering, University of Applied Sciences FH Campus Vienna, geting two major human pathogens were successfully expressed Austria 4 Department of Chemical Engineering, Autonomous University of Barcelona, in the chloroplast of C. reinhardtii, purified and their specificity Spain and efficiency in killing the target bacteria was assayed in vitro. Furthermore the cyanobacterium Synechocystis sp. PCC6803 was The alcohol oxidase (AOX1) promoter of Pichia pastoris is one investigated as an alternative expression platform for endolysins. of the strongest promoters for heterologous gene expression in http://dx.doi.org/10.1016/j.nbt.2014.05.1747 methylotrophic yeast, allowing the methanol-regulated expression of the gene of interest. Previous studies have shown that increased heterologous gene dosage often leads to the overload O15-5 of the secretory pathway and metabolic burden. The expression of Rhizopus oryzae lipase (Rol) in P. pastoris has Integrative ‘-omic’ approach to explore molecular mech- been previously shown to trigger the unfolded protein response anism of miRNA engineered Chinese hamster ovary cell (UPR) [1]. To assess that physiological response in P. pastoris is Vaibhav Jadhav 1,∗ , Matthias Hackl 1, Deniz Baycin-Hizal 2, Gerald related to the increased ROL gene dosage, a subset of strains with 1, Klanert 3, Michael Betenbaugh 2, Johannes Grillari 1, Nicole Borth 3 2, 4, 8 and 15 copies was constructed and tested in carbon-limited chemostat cultures, using a mixed glycerol:methanol substrate 1 University of Natural Resources and Life Sciences, Vienna, Austria 2 Johns Hopkins University, Baltimore, USA feed. Once the system was at steady state a transcriptomic analysis 3 Austrian Center for Industrial Biotechnology (ACIB), Austria was performed using DNA microarrays. The macroscopic physiological parameters revealed that growth MicroRNAs (miRNA) are noncoding regulators of translation, yield and carbon uptake rate are gene dosage dependent, and the controlling a broad range of physiological functions. They have highest productivity was found at 2 copies of ROL. These results been shown to regulate large number of mRNA targets simulta- were supported by the transcriptomic data, showing a correlation neously, thus acting as global regulators to alter cellular functions. between the regulation of central carbon metabolism and the gene The application of miRNAs to engineer Chinese hamster ovary copy number. The number of regulated genes increased with gene (CHO) cells is an emerging strategy to improve the bioindustrially dosage. In addition to carbon metabolism, transcriptional changes relevant characteristics of CHO cells, which are one of the most

www.elsevier.com/locate/nbt S59 SYMPOSIUM 15: RECOMBINANT PROTEIN PRODUCTION New Biotechnology · Volume 31S · July 2014 in other cellular processes such as peroxisome biogenesis and stress modate recombinant membrane proteins. Hereto, we inactivated responses involving the UPR were observed. the phosphatidic acid phosphatase gene, PAH1, which codes for a key enzymatic and regulatory factor in lipid biosynthesis. The Reference result is a shift of lipid metabolism away from triacylglycerol- and [1].Resina D, Bollók M, Khatri NK, Valero F, Neubauer P, Ferrer P. Trans- sterylester-storage towards membrane phospholipid synthesis. We criptional response of Pichia pastoris in fed-batch cultivations to chose Yarrowia lipolytica as a test organism, as this yeast prefer- Rhizopus oryzae lipase production reveals UPR induction. Microb Cell Fact 2007;6:21. entially grows on fatty acids and a compatible strong inducible protein expression system is available. http://dx.doi.org/10.1016/j.nbt.2014.05.1749 Electron microscopic imaging of the knock-out revealed strong membrane proliferation upon growth on oleic acid, without any signs of ER-phagy. Guided by this, we analyzed membrane protein O15-7 expression in the PAH1 knock out strain of Yarrowia lipolytica. Enhanced membrane protein expression by engineering Analysis of eight representatives of different integral membrane increased intracellular membrane production protein families showed enhanced protein accumulation levels for all of them and in some cases also reduced proteolysis. Compli- 1,2,∗ 1,2 1,2 Katrien Claes , Mouna Guerfal , Nico Callewaert mentary to this improvement, UPR co-induction further enhances 1 Unit for Medical Biotechnology, Inﬂammation Research Center (IRC), VIB, the quality of the membrane proteins in terms of proper folding Belgium and biological activity. 2 Laboratory for Protein Biochemistry and Biomolecular Engineering, Depart- These results allow to conclude that re-routing of lipid ment of Biochemistry and Microbiology, Ghent University, Belgium metabolism is a valid strategy to increase membrane protein production and quality. As this pathway is conserved in eukaryotes, Membrane protein research is frequently hampered by low nat- similar strategies can be explored in other frequently used expres- ural abundance of these proteins in cells and typically requires sion organisms. recombinant expression. Different expression systems have been used to date, but only little research is directed towards the speciﬁc http://dx.doi.org/10.1016/j.nbt.2014.05.1750 customization of the host cell itself. We hypothesized that increasing the intracellular membrane content would increase the membrane area available to accom-

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Symposium 16: Bioprocessing tion. It was shown that the extent of heterogeneity is strongly linked to frequency and amplitude of physicochemical changes O16-1 in the extracellular environment. Hence not only the identification of individual physiological phenotypes, but also the ability Bacterial enzymes for lignin degradation: production of to analyze single cells in a controlled and steady environment is a aromatic chemicals from lignocellulose fundamental requirement for the accurate description of cellular Timothy D.H. Bugg features. We tackle the challenge of creating controlled physicochemical microenvironments for a precise analysis of individual University of Warwick, Department of Chemistry, Coventry CV4 7AL, UK cellular physiology with the Envirostat single cell analysis system [1,2]. In contrast to most other microfluidic systems for SCA, The lignin content of lignocellulose and lignin-containing which mostly rely on the mechanical trapping of cells, the Envi- wastes represents a possible resource for production of aromatic rostat uses contactless retention via negative dielectrophoresis, chemicals, if efficient biocatalytic routes for lignin degradation which excludes cell-surface interaction and resultant changes in can be found. The enzymology of fungal lignin degradation is the cellular phenotype. Furthermore, the immediate removal of well studied, but the enzymology of bacterial lignin degradation potentially inhibiting metabolites and unlimited availability of is much less well known. In Rhodococcus jostii RHA1 we have iden- nutrients and dissolved oxygen is guaranteed by a continuous tified a dyp-type peroxidase DypB as a lignin peroxidase that is medium flow. CFD simulations indicate that the microenviron- 2+ ␤ activated by Mn , and shows activity with a -aryl ether lignin ment of the target cell has indeed a virtually static composition model compound, with Kraft lignin, and with lignocellulose. [1]. We experimentally validated the Envirostat principle with sin- Using a colorimetric assay as a screen, we have also identified gle cell growth studies [3]. We observed significant differences a number of novel lignin-degrading bacteria from soil samples, between specific growth rates of single cells and populations in which show higher activity for lignin degradation, including three bulk cultivations. Two yeast species and one bacterial strain con- strains of Microbacterium and a thermotolerant strain of Sphingob- sistently exhibited increased specific growth rates of up to 120% acterium, in which strains we are currently investigating further when cultivated with the Envirostat system [3]. Our results imply enzymes for lignin degradation. Deletion of the gene encoding that the extracellular environment can dictate the specific growth vanillin dehydrogenase in Rhodococcus jostii RHA gives a mutant rate of unicellular microbial eukaryotes and prokaryotes and a strain which, when grown on minimal media containing wheat constant extracellular environment diminishes the influence of straw lignocellulose, accumulates up to 96 mg/L of vanillin, a high physiological cell-to-cell differences. Moreover, our experiments value chemical, highlighting the potential for pathway engineer- demonstrate the Envirostat as a platform for systems biology that ing to be used for conversion of lignin into renewable aromatic may be used for disclosing the impact of controlled perturbations chemicals [1–4]. on cellular physiology unbiased by population activity.

References References [1].Ahmad M, Taylor CR, Pink D, Burton K, Eastwood D, Bending GD, [1].Kortmann H, et al. Lab Chip 2009;9:576. et al. Mol Biosyst 2010;6:815–21. [2].Fritzsch F, et al. Lab Chip 2013;13:397. [2].Ahmad M, Roberts JN, Hardiman EM, Singh R, Eltis LD, Bugg TDH. [3].Dusny C, et al. Appl Environ Microbiol 2012;78:7132. Biochemistry 2011;50:5096–107. [3].Taylor CR, Hardiman EM, Ahmad M, Sainsbury P, Norris PR, Bugg http://dx.doi.org/10.1016/j.nbt.2014.05.1752 TDH. J Appl Microbiol 2012;113:521–30. [4].Sainsbury PD, Hardiman EM, Ahmad M, Otani H, Seghezzi N, Eltis LD, et al. ACS Chem Biol 2013;8:2151–6. O16-3 http://dx.doi.org/10.1016/j.nbt.2014.05.1751 Continuous precipitation of recombinant antibodies from CHO cell culture supernatant by calcium- O16-2 phosphate flocculation and cold ethanol precipitation Nikolaus Hammerschmidt 1,∗ Anne Tscheließnig 2 Bernhard Helk 3 Quantitative single cell analysis of isolated microbes in , , , Alois Jungbauer 2 controlled microenvironments 1 ∗ Centre of Industrial Biotechnology (ACIB), Austria Christian Dusny , Frederik Fritzsch, Katrin Rosenthal, Andreas 2 University of Natural Resources and Life Sciences Vienna, Austria ∗ Schmid 3 Novartis Pharma, Austria TU Dortmund University, Laboratory of Chemical Biotechnology, 44227 Dort- mund, Germany We have developed two precipitation steps that could replace the costly protein A affinity chromatography as capture step in the Single cell analysis (SCA) has been recognized as the key tech- purification of recombinant antibodies, relying solely on cheap

nology for an unbiased disclosure of cellular functionality. In mineral salts (CaCl2) and organic solvents (ethanol). Both steps contrast to conventional bulk analysis strategies, SCA grants access can be performed in continuous mode using tubular reactors with- to mechanistic data of individual cells–the minimal functional out changes in performance compared to batch mode. The startup unit of cell based bioprocesses. These data are usually masked time until steady state conditions were reached was very short and behind an average value of a clonal, but heterogeneous popula- both reactors were operated for several hours at steady state with-

www.elsevier.com/locate/nbt S61 SYMPOSIUM 16: BIOPROCESSING New Biotechnology · Volume 31S · July 2014 out manual intervention, delivering antibody at constant yield O16-5 and purity. An overall yield of >90%, a host cell protein reduction to 9000 ppm and a DNA reduction to 7 ppm (for a titer of Bioprocess strategies for production of xylanase on agro- 7.7 g/L) could be achieved for the antibody investigated. The cold residual products with Aureobasidium pullulans ethanol precipitation step can be used for concentrating the anti- Sirma Yegin 1,∗ , Sayit Sargin 2, Yekta Goksungur 1 body to >45 g/L, thereby making a subsequent concentration step 1 Ege University, Food Engineering Department, Turkey unnecessary. Furthermore, the antibody can be readily dissolved 2 Ege University, Bioengineering Department, Turkey in a variety of buffers of high and low ionic strength optimized for the next purification step. Cell culture supernatants with high anti- Xylanases are mainly used in pulp and paper industry and body titer can be processed with constant tubular reactor size and recently found widespread applications in food and feed indus- without changing any parameters or increasing precipitant con- tries. They are also used in waste clarification processes and sumption. Continuous precipitation allows the use of disposable bioethanol production. reactors, allowing flexible operation. The aim of this study was to enhance production of xylanase http://dx.doi.org/10.1016/j.nbt.2014.05.1753 with A. pullulans by evaluating the effects of different fermentation parameters. Among the A. pullulans strains tested, NRRL Y2311-1 provided the highest activity on both xylose and xylan O16-4 grown cultures. Maximum activity was observed after 96 h of cultivation at 28 ◦C. The optimized initial medium pH and shaking Modelling of mixing and microbial growth in bubble col- speed for cultures grown on xylan were 3.0 and 200 rpm, respec- umn bioreactors using computational fluid dynamics tively. After elucidating the effects of fermentation parameters on Dale McClure ∗ , John Kavanagh, David Fletcher, Geoffrey Barton xylanase production in synthetic medium, the potential of several agro-residual products were examined for the production of The University of Sydney, Australia xylanase. The highest activity was obtained when wheat bran was utilized. Further optimization studies were performed by response Bubble columns are widely used in the bio-processing indus- surface methodology for cultures grown on wheat bran. By fit- try for large-scale aerobic fermentations. In order to maximise ting the experimental data to second-order polynomial equation, the yield of many bioprocesses, it is desirable to achieve a homo- the optimum levels of initial medium pH (4.2), temperature (30.3) geneous distribution of substrate; a task of some complexity at and incubation time (126.7 hours) were determined. The maxi- the industrial scale. Computational Fluid Dynamics (CFD) offers a mum xylanase activity at optimum process conditions reached to promising approach as a tool to model such processes, as it enables 851.95 ± 28.70 U/ml. The model obtained satisfactorily explained flow patterns inside the reactor to be readily visualised, something the effect of process variables on enzyme production. which is very difficult to achieve experimentally. A further advan- Acknowledgements: This study was supported by TUBITAK- tage of a CFD model is that it allows different designs and operating TOVAG 112O521 and COST TD1203. conditions to be evaluated in silico, minimising the need for costly and time-consuming experimentation. By including growth kinet- http://dx.doi.org/10.1016/j.nbt.2014.05.1755 ics into the CFD model, it becomes possible to quantify the effect of poor mixing on the overall yield of the process. As part of a long- term industrial collaboration we have developed and validated O16-6 a CFD model of the complex and time-varying hydrodynamics Bioconversion of lignocellulosic hydrolysates: strategies (including mixing) inside bubble columns operating at industri- to overcome the inhibitory effects at high gravity pro- ally relevant superficial velocities. Here, we extend this model by cesses including the growth kinetics for two processes of industrial interest; namely the production of baker’s yeast and the production of Charilaos Xiros ∗ , Lisbeth Olsson a recombinant protein using Escherichia coli. The impact of sub- Chalmers University of Technology, Sweeden strate gradients on the yield of each process is examined, as well as the potential for employing CFD as a tool to model industrial High-gravity (HG) technology aims at generating final ethanol bio-processes. − concentrations above 50 kg m 3 in order to reduce the cost of the http://dx.doi.org/10.1016/j.nbt.2014.05.1754 distillation step. The generation of higher amounts of inhibitors during the pretreatment step is one of the challenges that accom- pany the increase in initial dry matter. Detoxification of spruce hydrolysate, adaptation of the cells before fermentation, supplementation with nutrients, and washing of solids were the strategies compared in this study. They represent different approaches to cope with the inhibitory effects, and we compared their efficiencies using a thermotolerant strain of Saccharomyces cerevisiae at temperatures from 30 ◦Cupto40◦C.

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The dilute acid-pretreated spruce used as substrate in this nutrients supplementation gave better final ethanol yields than study was not fermentable under HG conditions (200 g kg−1 detoxification of the material, reachingan ethanol yield of about water-insoluble solids) when no improvement method was 60% of the theoretical (on total sugars). The results obtained, applied. In HG simultaneous saccharification and fermentation showed an increase in severity of inhibitory effects with tem- at 30 ◦C combined with a 24 h pre-hydrolysis step, the detoxifi- perature increase. Improved cell viability was observed when cation of pretreated spruce with reducing agent (Na2S2O4) gave detoxified material was used and also when yeast extract addition the best result with an ethanol yield of 57% (on total sugars) was coupled with adaptation of the cells to the hydrolysate. of the maximum theoretical and a volumetric productivity of http://dx.doi.org/10.1016/j.nbt.2014.05.1756 1.58 g dm−3 h−1. In HG separate hydrolysis and fermentation,

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Symposium 21: Biodegradation and bioreme- quencing. The result of PLFA revealed that the application of nZVI diation stimulated significantly growth of autochthonous microorganisms indicating general reduction of toxicity of the site in the first phase. O21-1 The consequent injection of an organic substrate enabled further transformation of Cr(VI) below the respective quantification limit Bioremediation for resource recovery even after 9 months of the single organic substrate injection. The results of the pilot test indicated a synergic effect of both of the Louise Horsfall abiotic and biotic phases. nZVI that had been oxidized during the University of Edinburgh, United Kingdom abiotic phase was afterwards partially recovered during the biotic phase due to the substantial decrease of the redox potential. The In order to move towards a sustainable and circular economy immobilized Fe (III) was probably microbially reduced to Fe(II) that we need to start viewing waste differently. Through biotechnol- acted further as the reducing agent for Cr(VI) even when microbial ogy research we have the potential to use waste as a feedstock density was already low due to depletion of the organic substrate. rather than it being an end product. A limiting factor in using http://dx.doi.org/10.1016/j.nbt.2014.05.1758 waste in this way is the presence of minor amounts of metal contaminants, which inhibit bioprocesses and kill bioremediat- ing microorganisms. There are, however, bacteria that are tolerant O21-3 to high concentrations of metal ions due to a resistance mechanism that involves metal reduction and nanoparticle formation. Isolation of PAH dwelling Penicillium for application in We are investigating this pathway to enable the bioremediation of bioremediation processes waste, water and land; employing the techniques, tools and prin- 1,∗ 1 1 ciples provided by synthetic biology to increase the value of the Elisabet Aranda , Patricia Godoy , Rocio Reina , Marina Badia- 2 3 1 metal recovered. To achieve this we are taking a modular approach Fabregat , Mónica Rosell , Regina Michela Wittich , Ernest Marco- 2 1 to identify and optimise genetic elements that have transferable Urrea , Inmaculada García-Romera metal ion use, with an aim to control production, size, shape 1 Spanish National Research Council (CSIC), Spain and homogeneity, tailoring the nanoparticles to their ascribed 2 Universitat Autònoma de Barcelona, Spain 3 use. Our particular case studies under investigation are platinum Universitat de Barcelona, Spain group metals, nickel, copper and arsenic. However, since we are attempting to develop a flexible process, there is huge potential to Fungi represent the living dominant biomass in soils and are transfer the knowledge gained in our biomineralisation studies to abundant in aqueous systems. In addition, they possess a high other metal waste streams. potential for degrading environmental organic chemicals. The aim of this study is to find polycyclic aromatic hydrocarbon (PAH) http://dx.doi.org/10.1016/j.nbt.2014.05.1757 degrading fungi, which are adapted to polluted environments, using culturing-based techniques. In this study, a total of 12 fungal cultivable species have been isolated from a PAH contaminated O21-2 pond. The isolated fungi were genetically identified by amplifying, Reduction of hexavalent chromium using combination cloning and further sequencing fragments corresponding to the of nanoscale zero-valent iron and biological treatment ITS1-5.8S-ITS2 (internal transcribed spacer ITS) region of each cul- in situ tivable fungal strain. We tested their ability to convert anthracene, in time courses of 42 days. Among the 12 screened fungal species, 1,∗ 2 2 3 Tomásˇ Cajthaml , Jan Nemeˇ cekˇ , Petr Pokorný , Lenka Lacinová , Penicillium oxalicum showed remarkable conversion ability, degrad- ˇ 3 3 4 Miroslav Cerník , Zuzana Masopustová , Ondrejˇ Lhotský ing 100 ␮M in 5 days in a rich carbon source medium. The use of 1 Institute of Microbiology v.v.i., ASCR, Czech Republic a defined mineral medium with 13C-labelled anthracene showed 2 ENACON s.r.o, Czech Republic that P. oxalicum can cometabolize anthracene, leading to the for- 3 Technical University of Liberec, Czech Republic mation of anthraquinone, anthrone and hydroxyderivatives, as 4 DEKONTA a.s., Czech Republic revealed by nuclear magnetic resonance (NMR) analysis. The last metabolites could indicate the further ring cleavage of anthracene Hexavalent chromium is considered as a priority pollutant due that could be mediated by hydrolase or dioxygenase enzymes. The to its high toxicity and mobility. The aim of this study was to set up conversion level of anthracene was reduced to 50% in presence of a pilot-scale in situ remediation experiment in the saturated zone an inhibitor of cytochrome P450 monooxigenase, suggesting its of a historically Cr(VI)-contaminated site. Two geofixation meth- participation in the first oxidation step. Our results show the high ods were used – chemical reduction of Cr(VI) with commercially effectiveness in PAHs conversion by the isolated fungi P. oxalicum available nanoscale zero-valent iron (nZVI) and consequent bio- by cometabolic strategies, indicating its potential application in logical reduction of Cr(VI). Combination of the methods resulted biotechnological pollutant removal processes. in a rapid decrease of Cr(VI) concentrations in the groundwater. The process was monitored using standard chemical analyses, http://dx.doi.org/10.1016/j.nbt.2014.05.1759 measurement of redox potential, standard ecotoxicity tests and phospholipid fatty acid analysis (PLFA) supported with 454 pyrose-

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O21-4 O21-5

Potential of ectomycorrhizal fungus Pisolithus tincto- Evaluation of the biological sulfamethoxazole degrada- rius to tolerate and to degrade trifluoroacetate into tion mechanism for biotechnological applications fluoroform Benjamin Ricken 1,∗ , Markus Lenz 1, Danuta Cichocka 1, Hans-Peter Paula Castro 1,∗ , Albina Franco 2, Miguel Ramos 2, Sara Cravo 3, Car- Kohler 2, Boris Kolvenbach 1, Philippe Francois-Xavier Corvini 1 3 los Afonso 1 Institute for Ecopreneurship/University of Applied Sciences and Art North- 1 CBQF – Centro de Biotecnologia e Química Fina – Laboratório Associado, western Switzerland, Switzerland 2 Escola Superior de Biotecnologia, Universidade Católica Portuguesa/Porto, Eawag, Swiss Federal Institute of Aquatic Science and Technology, Depart- Portugal ment of Environmental Microbiology, Switzerland 2 CBQF – Centro de Biotecnologia e Química Fina – Laboratório Associado, Escola Superior de Biotecnologia, Universidade Católica Portuguesa, Portugal Sulfonamide antibiotics are of rising concern as their release 3 2CEQUIMED-UP, Laboratório de Química Orgânica e Farmacêutica, Centro into the environment has been suspected to enhance the for- Interdisciplinar de Investigação Marinha e Ambiental (CIIMAR/CIMAR), Depar- tamento Ciências Químicas, Faculdade de Farmácia, Universidade do Porto, mation of resistant pathogenic bacterial strains [1]. Among Portugal sulfonamides, antibiotics such as sulfamethoxazole (SMX) are arousing public interest since they are photo- and thermostable as Trifluoroacetate (TFA) is a persistent fluorinated organic com- well as recalcitrant to traditional, biological waste water treatment pound originated from the degradation of fluorinated compounds, processes [2]. such as HCFC and isoflurane, or as a side product from the ther- Our studies focused on SMX as it is the most used sulfonamide molysis of fluoropolymers, like Teflon. TFA can reach soil through antibiotic in human medicine. Even though extensive research has precipitation, where it persists in water and soil, and may con- been done on SMX degradation by bacterial consortia or isolates, tribute to forest decline. In this study, we assessed the capacity the complete pathway is not yet understood, let alone the proteins of P. tinctorius, an ectomycorrhizal fungus (ECMF), to tolerate involved. and/or degrade TFA. In vitro studies in glucose-supplemented solid We were able to identify the first crucial degradation step of medium showed that the fungus tolerated up to 8.77 mM TFA. P. several sulfonamide antibiotics [3] by means of isolating Microbac- tinctorius also degraded 88.3%, 89.9%, and 42.1% of 0.88, 2.39, terium sp. strain BR1, proven to partially mineralize SMX for and 4.39 mM TFA, respectively, in liquid cultures. No TFA accu- the first time [4]. Concerning the pathway, molecular oxygen is mulation was detected on the fungus mycelium, suggesting that incorporated into the sulfonamide antibiotic at its aniline moi- TFA depletion was due to fungal degradation. Defluorination was ety through ipso-substitution, which is an uncommon process for not detected. A volatile compound with a structure and behav- biodegradation. This leads to the release of the verified metabolites: ior compatible with fluoroform (CHF3), a potent greenhouse gas, 3-amino-5-methylisoxazole, p-aminophenol and sulfite. was detected using GC-MS/MS, only in the gas phase of sealed P. Further investigations are currently under way in order to iden- tinctorius cultures supplied with TFA. Further confirmation of this tify the involved protein and its cofactor dependencies. compound is needed. Nevertheless, the study shows that P. tincto- To conclude, it is postulated that there is one common sul- rius was capable to degrade TFA possibly through a similar pathway fonamide degradation mechanism among various bacteria [3]. Its to that found on marine sediments. The results evidence the role elucidation and the determination of involved proteins are essen- of ECMF may play in the degradation of fluorinated organic com- tial for upcoming biological sulfonamide removal approaches. pounds, enhancing their potential contribution on establishing References tree growth in soils exposed to organic contamination. [1].Hruska, Franek. Vet Med 2012;2012:1–35. Acknowledgements: A. Franco thanks FCT the grant [2].Gros, et al. Environ Int 2010;36:15–26. SFRH/BD/47722/2008. This work was supported by FCT Project [3].Ricken, et al. Appl Environ Microbiol 2013;79:5550–8. -PTDC-AGR-CFL-111583-2009 and PEst-OE/EQB/LA0016/2011. [4].Bouju, et al. Appl Environ Microbiol 2012;78:277–9. http://dx.doi.org/10.1016/j.nbt.2014.05.1760 http://dx.doi.org/10.1016/j.nbt.2014.05.1761

www.elsevier.com/locate/nbt S65 WEDNESDAY 16 JULY SYMPOSIUM 17: DEVELOPMENT OF NEW VACCINES AND ANTIMICROBIALS New Biotechnology · Volume 31S · July 2014 Wednesday 16 July O17-2

Symposium 17: Development of new vaccines Engineering of factor H binding protein, a key vaccine and antimicrobials antigen for the prevention of meningococcal disease Hayley Lavender ∗ , Susan Lea, Christoph Tang O17-1 University of Oxford, United Kingdom Insect cell technology as a vaccine producing platform Neisseria meningitidis is a leading cause of bacterial sepsis and Paula Marques Alves meningitis in children. There are effective vaccines available to IBET/ITQB-UNL, Apartado 12, Oeiras, Portugal prevent meningococcal disease caused by strains expressing certain polysaccharide capsules. However, capsule-based approaches The insect cell-baculovirus system is today a well-accepted uni- cannot be used against serogroup B N. meningitidis because its cap- versal manufacturing platform as demonstrated by the number of sule is identical to a molecule found in the developing human approved veterinary and human vaccines. The key advantage of brain. Therefore there have been intense efforts to identify protein this platform is that a universal “plug and play” process may be antigens that provide protective immunity against this important used for producing a broad range of biologicals while offering the human pathogen. potential for low manufacturing costs. Its major downside resides Meningococcal factor H binding protein (fHbp) is a surface on the quality and quantity of the generated product, two critical lipoprotein that elicits serum bactericidal responses in mice and variables that seldom pose problems to the translation of the pro- human volunteers. This antigen is a key component of Bexsero, a duction process from lab to industrial scale. Aiming at closing this licensed vaccine for the prevention of meningococcal disease. gap, our group has been focusing on (1) the application of systems There is considerable sequence variation of fHbp among strains biology tools and (2) the development of stable insect cell lines to of N. meningitidis, affecting predicted coverage of fHbp-based vac- better understand and optimize this producing platform. cines. Also fHbp binds the human complement regulator factor H Taking advantage of recent advances in systems biology tools (fH); there is evidence that fH binding impairs immunogenicity of for insect cells we have conducted a metabolomics study with the fHbp. Finally, certain fHbps display inherent instability, making two most used cell lines, Sf9 and Hi5 cells, with the objective of them unsuitable as vaccine antigens. fine-tuning the insect cell-baculovirus system for production of Based on the structure of the fHbp:fH complex and muta- enveloped virus-like particles (VLPs) and retrovirus like particles genesis studies, we have identified non-functional fHbps for all (unpublished data). Results demonstrate that although Sf9 cells three variant groups, and generated stable versions of the proteins. have improved growth and metabolic efficiency, Hi5 cells were Additionally, we found that a homologue of fHbp in Neisseria gon- better recombinant protein producers for all tested targets with orrhoeae fails to bind fH. Given the specificity of fHbp for human productivities 3- to 4-fold higher. In addition, data points out fH, the vaccine candidacy of these antigens has been evaluated in towards the potential of media manipulation strategies as a way to a transgenic mouse model. reinforce specific cellular pathways associated with higher produc- In conclusion, we have employed structure-based design to tivity and thus optimize the production of complex biologicals. modify and characterise novel fHbps as antigens in next gener- In parallel, we have been developing stable insect cell lines ation vaccines against meningococcal disease. using targeted approaches based on the flp recombinase-mediated http://dx.doi.org/10.1016/j.nbt.2014.05.1763 cassette exchange (RMCE) system. Sf9 and Hi5 cells populations after flp-mediated cassette exchange were compared for complex proteins expression, including influenza VLPs. Specific protein O17-3 productivities in Hi5 cells were markedly higher than in Sf9 cells for all proteins tested. Noteworthy, the metabolic efficiency of Antimicrobial properties of sophorolipids produced by Sf9 cells allowed to extend the production phase and to deliver Candida Bombicola ATCC 22214 against gram positive similar protein titers at the end of the process. Overall, a compar- and Gram-negative bacteria ison of Sf9 and Hi5 cells expression capabilities with our flexible 1,∗ 1 plug-and-play Flp-RMCE platform will be discussed. Mayri Alejandra Diaz De Rienzo , Ben Dolman , Fernando Guzman 1, Candice Kaisermann 1, James Winterburn 1, Ibrahim M. http://dx.doi.org/10.1016/j.nbt.2014.05.1762 Banat 2, Peter Martin 1 1 University of Manchester, United Kingdom 2 University of Ulster, United Kingdom

Biosurfactants are amphipathic, surface-active molecules of microbial origin which accumulate at interfaces, decreasing surface and interfacial tensions and forming aggregated micellular structures in solution. Some are reported to have antimicrobial properties and anti-adhesive/disruption abilities on bioﬁlm formation. Sophorose lipids are glycolipids Biosurfactant con-

S66 www.elsevier.com/locate/nbt New Biotechnology · Volume 31S · July 2014 WEDNESDAY 16 JULY SYMPOSIUM 17: DEVELOPMENT OF NEW VACCINES AND ANTIMICROBIALS sisting of a dimer sugar, sophorose and a long-chain fatty acid O17-5 that are produced by yeasts belonging to the Candida genus. Antimicrobial properties and the ability to disrupt biofilms using Progesterone biosynthesis by combined action of adrenal sophorolipids produced by Candida bombicola ATCC 22214 during steroidogenic and mycobacterial enzymes in fast grow- a fed-batch fermentation, using glucose 100 g/L as carbon source ing mycobacteria and glucose/rapeseed oil as feed at 47 and 120 h and sodium Nicolai Strizhov 1,∗ , Victoria Fokina 1, Galina Sukhodolskaya 1, dodecyl sulfate (SDS) at different concentrations were investi- Dmitry Dovbnya 1, Mikhail Karpov 1, Andrey Shutov 1, Ludmila gated. Growth of Ralstonia eutropha ATCC 17699 was inhibited Novikova 2, Marina Donova 1 by sophorolipids and SDS at concentrations > 1% (v/v) and the 1 Institute of Biochemistry & Physiology of Microorganisms, Russian Academy growth of B. subtilis BBK006 was inhibited by sophorolipids and of Sciences, Russia SDS at concentrations > 0.5%, v/v. Sophorolipids were able to dis- 2 Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State rupt biofilm formation (at concentrations higher than 1%, v/v) University, Russia and the effect was evidenced through fluorescence microscopy. It is concluded that sophorolipids are promising compounds for the We have for the first time demonstrated the use of mycobac- inhibition/disruption of biofilms formation by Gram-positive and teria as a harbor for synthesis of C21-steroids by heterologous Gram-negative microorganisms which could be enhanced by the expression of mammalian steroidogenesis key proteins. Proges- presence of a combination of biosurfactants. terone biosynthesis was successfully reconstructed in recombinant http://dx.doi.org/10.1016/j.nbt.2014.05.1764 mycobacterial cells. The first stage of mammalian steroidogenesis, cholesterol conversion to pregnenolone is carried out by heterologously expressed cytochrome P450scc cleaving the sub- O17-4 strate side chain. cDNA copies of the CYP11A, Adx and AdR genes encoding mature forms of cytochrome P450 cholesterol hydrox- Tailoring Streptomyces: producing novel minor groove ylase/20, 22-lyase (P450scc), adrenodoxin (Adx) and adrenodoxin binder antibiotics reductase (AdR) from bovine adrenal cortex were transferred with pNS11 construct to Mycobacterium smegmatis mc2155. Acetamide Emilio Cortes Sanchez 1,∗ , Sara De Ornellas 2, John May 2, Glenn chemoinduction resulted in high expression level of all three Burley 2, Paul Hoskisson 3 proteins. Pregnenolone oxidation to progesterone in the subse- 1 University of Strathclyde, United Kingdom quent step is mediated by innate mycobacterial 3␤-hydroxysteroid 2 University of Strathclyde, Chemistry, United Kingdom dehydrogenase (3␤-HSD). However, no progesterone synthesis 3 University of Strathclyde, SIPBS, United Kingdom was detected in mycobacteria with pNS10 construct expressing P450scc alone without Adx and AdR redox partners. We concluded The rise of antibiotic resistant strains and the decline of antibi- that bacterial ferredoxins and ferredoxin reductases ubiquitously otic discovery have resulted in an urgent need to develop and present in mycobacteria fail to deliver electrons to P450scc. Pro- discover novel antimicrobials. Minor groove binders (MGBs) are gesterone was also produced by recombinant pNS11 mycobacterial compounds that shown antibiotic, anticancer and antiviral activ- cells with sitosterol as a substrate for bioconversion though with ity by binding to the minor groove of the DNA helix. lower yields. MGBs are naturally produced by Streptomyces, and recently The highest progesterone production level achieved is 25 mg/l some of the clusters encoding for those antibiotics have been char- at 7.5 mM cholesterol load. This yield is 60 fold higher than acterised. In order to produce a new drug, we are using synthetic the maximum pregnenolone concentration we obtained earlier biology and combinatorial biosynthesis to modify a pathway of a with recombinant Escherichia coli (pBar Triple). The level difference Streptomyces strain, forcing the assembly of a new antibiotic. between two hosts clearly reveals prospects of the mycobacterial For this we transformed several Streptomyces strains with an progesterone biosynthesis for biotechnological purposes. Our find- MGB producing biosynthetic cluster containing four independent ings pave the way for future exploration of these lipophilic bacteria non-ribosomal peptide synthetases. These strains were fermented for production of valuable active pharmaceutical ingredients. and sampled at different time points and the organic extractions were analysed looking for the production of novel compounds. http://dx.doi.org/10.1016/j.nbt.2014.05.1766 The HPLC, LCMS and HRMS data suggest the assembly of a novel compound, which exhibits antibiotic activity according to our bioassays. Further characterisation of the molecule will be done by O17-6 NMR .We have also cloned one of the non-ribosomal peptide syn- DNA vaccine expressing ubiquitin-conjugated multi- thetases in order to engineer this enzyme to broaden its substrate fragments antigens protects BALB/c mice against Toxo- specificity. plasma gondii infection http://dx.doi.org/10.1016/j.nbt.2014.05.1765 Hua Cong ∗ , Quan Yuan Shandong University, China

Toxoplasma gondii, an intracellular parasite with a complex life cycle, is highly prevalent in humans and animals and causes

www.elsevier.com/locate/nbt S67 WEDNESDAY 16 JULY SYMPOSIUM 17: DEVELOPMENT OF NEW VACCINES AND ANTIMICROBIALS New Biotechnology · Volume 31S · July 2014 zoonotic toxoplasmosis. In order to prevent from I infection, cuit and sporulation regulator Spo0A while negatively regulated by an ideal vaccine that can elicit protective cell-mediated immune transition regulators CodY, ScoC and AbrB. In this study, a compar- responses is greatly needed. In our study, we selected seven frag- ative proteomic analysis of the bacilysin producer B. subtilis PY79 ments derived from seven T. gondii surface antigens (SAG3, ROP18, and its bacilysin non-producer derivative OGU1 (bacA::lacZ::erm) MIC6, GRA7, MAG1, BAG1, SPA), which was linked to ubiquitin was performed in order to gain a deeper insight into the func- protein. DNA vaccines encoding 7 fragments and ubiquitin pro- tional roles of bacilysin biosynthesis in its producer. 2-DE gel tein were constructed. BALB/c mice were immunized with p-Ub electrophoresis coupled to MALDI-TOF/MS within different pH p-Tgag, pVAXI, or PBS and challenged with highly virulent T. gondii ranges separated more than 1900 protein spots. Of these, 159 RH strain or the cyst of T. gondii genotype II strain of PRU. Vaccina- protein spots were identified which corresponded to 123 distinct tion with p-Ub and p-Tgag both showed high ability to generate a proteins. 60 out of 123 distinct proteins were down-regulated in strong Th1 cell response with significant production of IFN-␥, IL-2 OGU1 strain as compared to the wild-type. Thirty-five of 123 pro- and low levels of IL-10, and protected mice against high parasite tein spots were expressed more abundantly while 19 protein spots burden when challenged with T. gondii compared with PBS and were found to be absent and one protein spot was newly induced in pVAX1. Furthermore, the result of vaccination with p-Ub demon- the mutant strain. To avoid any experimental limitations, we also strated that it was more effective than p-Tgag in protection of T. performed 1-DE gel electrophoresis coupled to LC–MS/MS analy- gondii infection. We conclude that the DNA vaccine we constructed sis which led to the identification of 1282 proteins from the total in this study is efficacious and in particular, ubiquitin-conjugated soluble proteome of PY79 and OGU1. 76 of those proteins were vaccines showed a stronger Th1-type immunity and significant found to be differentially expressed as deduced from their relative resistance to parasite challenge. abundance. http://dx.doi.org/10.1016/j.nbt.2014.05.1767 The analysis of the results obtained from 2-DE gel electrophoresis coupled to MALDI-TOF/MS and GeLC–MS/MS revealed the impact of the absence of bacilysin biosynthesis on the expres- O17-7 sion of sporulation, two component-regulatory system, peptide transport, stress and global repressor CodY-regulated proteins. Proteome-wide analysis of the functional roles of http://dx.doi.org/10.1016/j.nbt.2014.05.1768 bacilysin biosynthesis in Bacillus subtilis

Gulay Ozcengiz 1,∗ , Asli Aras Taskin 2, Mustafa Demir 1, Ayten Yaz- gan Karatas 3 1 Department of Biol. Sci., METU, Turkey 2 University of Freiburg, Germany 3 Mol. Biol. Gen. Department, Istanbul Technical University, Turkey

Bacilysin, being produced and excreted by certain strains of Bacillus subtilis, is a dipeptide antibiotic composed of L-alanine and L-anticapsin. We previously showed that the biosynthesis of bacilysin is positively regulated by quorum sensing regulatory cir-

S68 www.elsevier.com/locate/nbt New Biotechnology · Volume 31S · July 2014 SYMPOSIUM 18: EXPLOITATION OF METAGENOMICS FOR ENVIRONMENTAL AND BIOCATALYTIC APPLICATIONS

Symposium 18: Exploitation of metagenomics cosms with topsoil and rhizosphere soil, respectively. Several PCB for environmental and biocatalytic applica- degradation intermediates were detected (i.e. chlorobenzoates, tions hydroxylated and methoxylated PCBs) and the initial acute toxicity was reduced mainly in the rhizosphere soil. Furthermore, O18-1 to gain new insights into the biota composition throughout the remediation processes, the diversity and dynamics of both bacte- Metagenomics: mining for novel catalysts rial and fungal communities were estimated via high-throughput 454-pyrosequencing method. Metagenomic analysis showed that Elizaveta Bonch-Osmolovskaya Firmicutes relative abundance increased when the bulk and rhizo- Winogradsky Institute of Microbiology, Russian Academy of Sciences, Moscow, sphere soils were treated with P. ostreatus. Conversely, in all soil Russia samples augmented with I. lacteus an initial rise in the relative abundance of the Proteobacteria phylum was observed, whereas About three decades back the analysis of environmental 16S Bacteroidetes predominate in the topsoil and rhizosphere soil at rRNA clone libraries revealed that roughly 95% of microorgan- the end of incubation. P. ostreatus was able to compete with the isms in natural microbial communities had never been obtained autochthonous fungi, its relative abundance being higher than in laboratory cultures, and so their catalytic capacities were not 90% along the whole incubation period. By contrast, the abun- known. The metagenomic approach, actively developing over the dance of I. lacteus sequences tended to decline during the last last 10 years, provides access to genes of microorganisms that 6 weeks of incubation. In biostimulated microcosms, the large might never be isolated. This became possible as a result fast majority of detected sequences belonged to the phyla Ascomycota progress of the sequencing technologies, leading to their signifi- and Zygomycota. cant cost reduction. The contemporary state of technology makes feasible efficient sequencing of environmental DNA with multiple http://dx.doi.org/10.1016/j.nbt.2014.05.1770 coverage, allowing consequent alignment and assemblage of large genome fragments. Metagenomic libraries became an object for gene mining, both for predicting novel metabolic pathways, not O18-3 known in cultured prokaryotes, and for searching new catalysts Mining alginate lyases in sediment metagenomes from for biotechnology. For the latter purpose, microbial communities four geographically distant cold coastal environments of extreme environments have been investigated, including hot ,∗ springs, acidic mines, soda lakes, and polar soils; and new enzymes Hebe Dionisi 1 , Marina Matos 1, Luciano Anselmino 1, Mariana – highly stable hydrolases, esterases, DNA polymerases with new Lozada 1, Walter Mac Cormack 2, Jolynn Carroll 3, Leif Lundgren 4, capacities have been cloned and expressed. One example of such Sara Sjöling 5, Krystle Chavarría 6, Bernard Henrissat 7, Janet work is a current FP7 project “HotZyme – Systematic screening of Jansson 6 novel hydrolases from hot environments”, in which an interna- 1 Patagonian National Research Center (CENPAT-CONICET), Argentina tional team covers ground from environmental samples to purified 2 Argentinean Antarctic Institute and National University of Buenos Aires, and crystallized new proteins with target activities. Argentina 3 University of Tromsø, Norway and Akvaplan-niva AS, FRAM – High North http://dx.doi.org/10.1016/j.nbt.2014.05.1769 Research Centre for Climate and the Environment, Tromsø, Norway 4 Stockholm University, Sweden 5 Södertörn University, Sweden 6 O18-2 Lawrence Berkeley National Laboratories, USA 7 Centre National de la Recherche Scientifique, Marseille, France Metagenomics unveils bacterial and fungal communities response to mycoremediation of polychlorinated Brown macroalgae are considered an attractive option as sus- biphenyl-contaminated soil tainable feedstock for the production of biofuels and commodity chemicals due to their high carbohydrate content. Microbial com- Tatiana Stella 1,∗ Stefano Covino 1 Monika Cvanˇ carováˇ 1 Maurizio , , , munities from cold coastal environments represent promising Petruccioli 2 Alessandro D’Annibale 2 Tomas Cajthaml 1 , , sources of novel enzymes depolymerizing brown algal polysac- 1 Institute of Microbiology AS CR, v.v.i., Czech Republic charides such as alginates, as these organisms constitute a large 2 Tuscia University, Italy primary biomass in these environments. We used a nested sampling strategy to obtain sediment samples from four high-latitude The remediation of sites contaminated by polychlorinated coastal environments (Svalbard Archipelago, Norway; Baltic Sea, biphenyls (PCBs) is a major priority due to the teratogenic, car- Sweden; Ushuaia Bay, Argentina and Potter Cove, Antarctica). cinogenic and endocrine-disrupting features of these xenobiotics. Twenty-three samples were sequenced using Illumina HiSeqTM Within this frame, the present work was aimed at assessing the 1500, assembled and annotated using the IMG/M pipeline. The technical feasibility of both bioaugmentation (white-rot fungi complete assembled metagenome dataset contains 5.6 Gb and Irpex lacteus and Pleurotus ostreatus) and biostimulation treatments 1.4 × 107 protein coding genes. With the goal of identifying algi- of three different samples of a historically PCB-contaminated soil nate lyase homologs in the metagenomes, we mined this dataset (bulk soil, topsoil and rhizosphere soil). The highest PCB depletion using both blastp searches and product names assigned in the yields (41 and 51%) were observed in P.ostreatus-augmented micro- functional annotation. We retrieved 2,705 sequences between

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100 and 1,166 amino acids in length, mostly belonging to the of microbial colonies on solid plates for the existence of target CAZy polysaccharide lyase families PL17 (30.4%), PL7 (28.2%) enzyme activity using designed sensor cells. When an effector, or PL6 (22.1%). When normalized with the protein coding gene such asp-nitrophenol, is released from a microbial colony, it will numbers of the assembled metagenomes, Antarctic samples con- diffuse out to the vicinity surrounding the colony and the sensor tained the highest abundance of identified sequences, and Baltic cells in the vicinity turn on the expression of a fluorescence and Sea samples contained a larger proportion of novel sequences an auxotrophic genes. The expression of these reporters enables (Kruskal–Wallis test, p < 0.05). Different levels of gene order con- the sensor cells to grow rapidly and to display strong fluores- servation were found among scaffolds containing these genes, and cence signals near the original colonies. So, by simply co-culturing with genomes of isolated alginate-degrading bacteria. This study these sensor cells with any microbes from nature, the sensor revealed a large diversity of alginate lyase homologs from yet-to-be cells will directly mark the locations of microbial colonies with cultured marine microorganisms, which could aid in the engineer- target enzyme activity, which is responsible for the buildup of p- ing of microbial platforms for biorefineries. nitrophenol. We tested this proof-of-concept using Escherichia coli http://dx.doi.org/10.1016/j.nbt.2014.05.1771 sensor cells with a phenol-sensitive promoter, dmpR, to detect Cit- robacter freundii cells expressing tyrosine phenol-lyase. Also, the sensor cell was applied to examine diverse microbial cells from O18-4 soils, to find a cellulase activity using p-nitrophenyl-cellobioside as a substrate. We isolated seven cellulase producing bacteria and Designed sensor cells for direct detection of microbial identified one of them identified as a novel species of Pseudomonas, colonies with target enzyme activities on solid plates based on 16s rRNA/NGS analysis along with morphological, biochemical, and physiological properties. Lastly, the sensor cell was Haseong Kim ∗ , Eugene Rha, Bong-Hyun Sung, Seung-Goo Lee used to isolate new cellulases from metagenome libraries, which KRIBB, Republic of Korea were confirmed to have the hydrolyzing activity and specificity on cellulose by conventional methods. Screening of new enzymes from vast genetic resources is indis- http://dx.doi.org/10.1016/j.nbt.2014.05.1772 pensable for environment-friendly, cost-effective, and sustainable chemical industry. Here is a new method to explore the variety

S70 www.elsevier.com/locate/nbt New Biotechnology · Volume 31S · July 2014 SYMPOSIUM 19: EVOLUTIONARY STRATEGIES FOR CELL FACTORY DEVELOPMENT

Symposium 19: Evolutionary strategies for ing techniques in order to find the genetic changes. Four strains cell factory development were selected from the evolution of BY4741. The mutations found, pointed to the Rsp5p-Bul1/2p ubiquitin ligase complex as the pre- O19-1 ferred evolutionary target under these experimental conditions. Rsp5p is a multifunctional enzyme able to ubiquitinate target pro- Evolutionary and reverse metabolic engineering of Sac- teins participating in different cellular processes, while Bul1p is charomyces cerevisiae an Rsp5p substrate adaptor involved in the ubiquitin-dependent internalization of Gap1p and other plasma membrane permeases. Jack T. Pronk Our results might be related to increased halftime of plasma mem- Department of Biotechnology, Delft University of Technology, Delft, The brane amino acid permeases. Apparently the genetic background Netherlands of the laboratory strain is conditioning the result. However, we have shown the strength of this approach. The evolution of the Laboratory evolution is a powerful, versatile approach for haploid segregant of EC1118 was run for 250 generations, three improving and expanding the capabilities of industrial micro- adapted strains were selected. Preliminary bioinformatic analysis organisms which, in contrast to targeted genetic modification, highlighted changes in chromosome numbers in mutant strains. does not require a detailed a priori understanding of the molec- We are currently testing these results by karyotyping, qPCR, flow ular basis for the trait of interest. Cultivation in bioreactors offers cytometry and other techniques. many options to design and implement culture conditions that maximize the selective advantage of spontaneous mutations that http://dx.doi.org/10.1016/j.nbt.2014.05.1774 confer a specific, industrially relevant trait. I will briefly discuss applications of this ‘evolutionary engineering’ approach to the yeast Saccharomyces cerevisiae for optimizing its sugar fermentation O19-3 kinetics and for increasing its robustness to industrially relevant Genome dynamics of the human embryonic kidney 293 stresses. (HEK293) lineage in response to cell biology manipula- Until recently, expression profiling with DNA micro-arrays tions was the only cost-affordable genome-wide analytical technique for analysing the molecular basis for improved performance of Morgane Boone 1,2,∗ , Yao-Cheng Lin 3,4, Leander Meuris 1,2, Irma yeast strains derived from evolutionary engineering experiments. Lemmens 5,6, Nadine Van Roy 7, Arne Soete 8, Joke Reumers 9, However, such transcriptome analyses tended to generate large Matthieu Moisse 9,10, Stephane Plaisance 11, Radoje Drmanac 12, numbers of targets, often without a clear lead to the responsible Jason Chen 12, Frank Speleman 7, Diether Lambrechts 9,10, Yves Van mutation(s). Based on recent studies from the Delft yeast group, de Peer 3,4,13, Jan Tavernier 5,6, Nico Callewaert 1,2 I will illustrate how the advent of whole-genome sequencing has 1 Unit for Medical Biotechnology, Inflammation Research Center (IRC), VIB, transformed the molecular analysis of evolved genotypes. Analysis Belgium of multiple, independent evolution experiments and integration 2 Laboratory for Protein Biochemistry and Biomolecular Engineering, Depart- of classical genetics approaches were shown to greatly amplify the ment of Biochemistry and Microbiology, Ghent University, Belgium 3 Department of Plant Systems Biology, VIB, Belgium power of whole-genome resequencing of evolved strains. 4 Department of Plant Biotechnology and Bioinformatics, Ghent University, http://dx.doi.org/10.1016/j.nbt.2014.05.1773 Belgium 5 Department of Medical Protein Research, VIB, Belgium 6 Department of Biochemistry, Faculty of Medicine and Health Sciences, Ghent University, Belgium O19-2 7 Center for Medical Genetics, Ghent University Hospital (MRB), Belgium 8 Bioinformatics Core Facility, Inflammation Research Center (IRC), VIB, Belgium Adaptive evolution of saccharomyces cerevisiae to early 9 Laboratory for Translational Genetics, Department of Oncology, KULeuven, stage of an alcoholic fermentation Belgium 10 ∗ Vesalius Research Center, VIB, Belgium Ana Mangado , Pilar Morales, Jordi Tronchoni, Ramon Gonzalez 11 VIB Bioinformatics Training and Services (BITS), VIB, Belgium 12 ICVV/CSIC, Spain Complete Genomics, USA 13 Genomics Research Institute, University of Pretoria, South Africa

Experimental evolution was used to identify genes involved in The HEK293 human cell lineage is widely used in cell biol- the adaptation to the early stages of wine fermentation. Evolution ogy and biotechnology. We used whole genome resequencing experiments were performed in continuous culture for 150-250 methods in six 293 cell lines to study the dynamics of this ane- generations, in conditions emulating the initial stages of alcoholic uploid genome in response to the cell biology manipulations that fermentation. We performed three independent experimental evo- were used to generate common derivatives of 293 cells, such as lution experiments of a haploid laboratory strain BY4741 and transformation and stable clone generation (293T); suspension one evolution with a haploid strain (not adapted to winemaking growth adaptation (293S) and cytotoxic lectin selection to isolate growth conditions) obtained by meiotic segregation of the wine a glycosylation-homogenous clone (293SG). While the chromo- yeast EC1118. By the end of the experiments, colonies were pheno- somal structure of single 293 cells within a culture appears to be typically characterized, and strains that showed improved initial extremely diverse, our analysis suggests that standard cell culture growth rates were selected. We used next generation sequenc-

www.elsevier.com/locate/nbt S71 SYMPOSIUM 19: EVOLUTIONARY STRATEGIES FOR CELL FACTORY DEVELOPMENT New Biotechnology · Volume 31S · July 2014 procedures (passaging and cell banking) do not affect the ‘average’ O19-5 genome structure and sequence to a great extent. The extraordi- nary chromosomal plasticity of this genome, however, seems to be Novel human kidney epithelial cell line in pharmaceuti- the driving adaptive force when cells are put through a bottleneck. cal biotechnology This feature underlies a novel application for which we provide Lukas Fliedl 1,∗ , Matthias Wieser 1, Gabriele Manhart 1, Matthias proof of concept here: selection of 293 clones surviving stringent P. Gerstl 1, Florian Kast 1, Abdulhameed Khan 2, Renate Kunert 2, selective conditions (eg ricin toxin), followed by whole-genome Johannes Grillari 2, Regina Grillari-Voglauer 2 analysis of copy number alterations, can effectively pinpoint the 1 ACIB, Austria genomic region(s) that contain the gene(s) required for adaptation 2 Department of Biotechnology, University of Natural Resources and Life Sci- to those selective conditions. Furthermore, up to the level of sen- ences Vienna, Austria sitivity afforded here (single copy plasmid insertions were easily detected), these cell lines have no inadvertent virus insertions. In Mammalian cells are used as model systems, products them- terms of tools, we optimized a workflow to detect human/vector selves and as producers of recombinant proteins and vaccines. genome breakpoints, and enabled visualization of the 293 genome In these different applications a variety of different cell lines data both through a user-friendly visualization web page, as well are used and although all have proven valuable for their spe- as through the Integrative Genome Browser (IGV) for rich data cific application, there is still room for improvement in terms of mining. posttranslational modifications. Especially novel human cell lines http://dx.doi.org/10.1016/j.nbt.2014.05.1775 are of ever increasing importance since they ideally represent the in vivo situation and might produce high quality biopharmaceuticals as similar to endogenous proteins as possible. O19-4 Therefore we established a novel human continuously growing renal proximal tubular epithelial cell line (RPTEC) that has Versatile and stable vectors for efficient gene expression maintained many differentiated characteristics of the normal non- in Ralstonia eutropha H16 transduced counterpart and tested its performance in the different Steffen Gruber , Jeremias Hagen, Helmut Schwab, Petra Koefinger ∗ fields of pharmaceutical biotechnology. A complex model protein, erythropoietin, was stably produced Graz University of Technology, Austria in this cell line and the quality of the recombinant protein was compared to CHO derived product by analysis of isoforms as well ␤ The gram-negative -proteobacterium Ralstonia eutropha H16 is as specific non-human glycopatterns. Additionally, we used our primarily known for polyhydroxybutyrate (PHB) production and cell line to produce influenza virus and proved high capabilities of its ability to grow chemolithoautotrophically by using CO2 and our cell line in this application. H2 as sole carbon and energy sources. Up to now some basic sys- Finally, we used our cell line to get insights into nephrotoxi- tems for targeted genetic manipulation of this bacterium were city induced by cisplatin, which has important implications as a already established. However, the majority of metabolic engineer- chemotherapeutic drug and hypothesize that especially epithelial ing and heterologous expression studies conducted so far rely on a barrier formation and polarity of RPTECs need to be considered in small number of suitable expression systems. Particularly the plas- toxicity models to validly predict the in vivo situation. mid based expression systems already developed for the use in R. Therefore, the here established kidney epithelial cells combine eutropha H16 suffer from high segregational instability and plas- applicability in various fields of pharmaceutical biotechnology, as mids loss after a short time of fermentation. In order to develop they are capable of production as well as of pre-clinical testing of efficient and highly stable plasmid expression vectors for the use biopharmaceuticals. in R. eutropha H16 a new plasmid design was created including the RP4 partitioning system, as well as various promoters and http://dx.doi.org/10.1016/j.nbt.2014.05.1777 origins of replication. The application of minireplicons derived from broad-host-range plasmids RSF1010, pBBR1, RP4 and pSa for the construction of expression vectors and the use of numerous, O19-6 versatile promoters extend the range of feasible expression levels De novo production of geranic acid with Pseudomonas considerably. Moreover, the implementation of the RP4 partition putida sequence in plasmid design increased plasmid stability signifi- ∗ cantly and enables fermentations with marginal plasmid loss of Jens Schrader , Jia Mi, Daniela Becher, Patrice Lubuta, Markus recombinant R. eutropha H16 for at least 96 hours. The utility of Buchhaupt, Dirk Holtmann the new vector family is demonstrated by providing expression DECHEMA Research Institute, Germany data with different model proteins. http://dx.doi.org/10.1016/j.nbt.2014.05.1776 Production of plant terpenes by engineered microbes has become a prime example of applied synthetic biology with tremendous progress being made during the last decade. Whereas sesquiterpene titers reported have already reached g/L values in the bioreactor, efficient monoterpene production seems to be more

S72 www.elsevier.com/locate/nbt New Biotechnology · Volume 31S · July 2014 SYMPOSIUM 19: EVOLUTIONARY STRATEGIES FOR CELL FACTORY DEVELOPMENT difficult with conventional host strains due to product toxicity. tions despite an indisputable potential. While peptide engineering Hence, we set out to investigate to potential of solvent tolerant protocols allow for the generation of thousands of novel, putative Pseudomonas putida for monoterpene production. P. putida DSM active antimicrobial peptides at ease, the assessment of their activ- 12264 shows a pronounced robustness in the presence of monoter- ity is a rather laborious procedure limiting the assay throughput penes. Recent results revealed that the wildtype strain efficiently to 102 variants per day. converts geraniol to geranic acid. The monoterpenoic acid shows We will present a platform based on nL-sized reaction vessels interesting properties as a fungicidal agrochemical and may be (nL-reactors) that are used for peptide production and activity- used as a natural preservative for foods and cosmetics. Comparing screening in a single step and at rates of 105 variants per day. the growth pattern with E. coli and S. cerevisiae, P. putida revealed During screening, library cells are grown to microcolonies within a several times higher tolerance towards geranic acid. Following nL-reactors along with a sensor strain serving as a model for a the cell factory idea, we functionally expressed plant geraniol syn- pathogen. Library cells secreting an active antimicrobial peptide, thase in P. putida, which led to the production of small amounts of will deactivate the sensor cells within the encircling nL-reactor. the desired geranic acid from glycerol as the C-source indicating Clearance of an nL-reactor from the sensor thus indicates the pres- exploitation of terpene precursors derived from the endogenous ence of a strain secreting a highly active peptide. We use large DXP pathway. The cellular precursor supply was improved by particle flow cytometry and fluorescently labeled cells in order to expression of the mevalonate (MVA) pathway from Myxococcus isolate promising candidates. xanthus. With this P. putida strain, a product concentration of ca. The power of the technology will be demonstrated by screening 230 mg/L was obtained in a fed-batch bioreactor. This is the first of libraries containing ∼105 peptide variants, generated by site- example of de novo monoterpenoic acid production with an engi- saturation mutagenesis or synthetic biology approaches based on neered microbe. We intend to further improve the product titers the blueprint of natural antimicrobial peptides. We show that by pathway engineering to eventually take advantage of the host’s the assay has not only a high throughput but also a high accu- monoterpene tolerance. racy, making this technique a valuable contribution for future http://dx.doi.org/10.1016/j.nbt.2014.05.1778 approaches targeting the design of highly active antimicrobial compounds. http://dx.doi.org/10.1016/j.nbt.2014.05.1779 O19-7

High-throughput nL-reactor screening for antimicrobial peptides

Steven Schmitt 1,∗ , Manuel Montalban Lopez 2, Oscar Kuipers 2, Sven Panke 1, Martin Held 1 1 ETH Zürich, Department of Biosystems Science and Engineering, Switzerland 2 University of Groningen, Molecular Genetics Group, Netherlands

The number of multi drug resistant pathogens is constantly growing and novel antibiotic substances are desperately needed in order to at least maintain the status quo. Ribosomally synthesized antimicrobial peptides are not yet exploited for human applica-

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Symposium 20: Systems biocatalysis [4].BaerK, Kraußer M, Burda E, Hummel W, Berkessel A, Gröger H. Angew Chem 2009;121:9519–22; Angew Chem Int Ed 2009;48:9355–8. O20-1 [5].Rulli G, Duangdee N, Baer K, Hummel W, Berkessel A, Gröger H. Angew Chem 2011;123:8092–5; Combination of the two ‘worlds’ chemo- and biocatalysis Angew Chem Int Ed 2011;50:7944–7. towards multi-step one-pot processes [6].Burda E, Ress T, Winkler T, Giese C, Kostrov X, Huber T, et al. Angew Chem 2013;125:9493–6; Harald Gröger Angew Chem Int Ed 2013;52:9323–6. Faculty of Chemistry, Bielefeld University, Universitätsstr. 25, 33615 Bielefeld, http://dx.doi.org/10.1016/j.nbt.2014.05.1780 Germany

Multi-step one-pot processes represent an attractive synthetic O20-2 concept for the improvement of overall process efficiency by decreasing the required number of work up and purification steps. Engineering artificial metabolisms in vitro By avoiding such time-, capacity- and solvent-intensive process steps, multi-step one-pot syntheses contribute to a significantly Wolf-Dieter Fessner improved process economy as well as to more sustainable syn- Technische Universität Darmstadt, Institut für Organische Chemie und Bio- thetic routes. A key criterion for multi-step one-pot processes is chemie, Alarich-Weiss-Str. 4, 64287 Darmstadt, Germany the compatibility of the individual reaction steps with each other. Accordingly, most of today’ known multi-step one-pot processes Systems Biocatalysis is a new concept [1] of organizing best are based on either chemocatalytic multi-step reactions or ‘pure’ enzymes in vitro to construct novel artificial metabolisms for an biotechnological processes such as, for example, fermentation. In efficient, sustainable synthesis of valuable chemical products [2]. contrast, successful combinations of chemo- and biocatalytic reac- The strategy merges the synthetic focus of chemistry with the tions, in particular in aqueous reaction media, are much less widely modular design of biological systems, which is similar to Synthetic known. Biology but can be realized at a far lower level of complexity from In this contribution strategies for the combination of chemo- a true reductionist approach. and biocatalysts towards the development of multi-step one- A particular advantage of the concept arises from the inherent pot processes in aqueous reaction media are presented. potential to construct novel biocatalytic reaction systems for the Since palladium-catalyzed cross-coupling reactions are of partic- efficient synthesis of non-natural products by using enzymes engi- ular importance in the field of metal catalysis, as enzymatic neered for non-natural substrate promiscuity. Such operations are reductions are in the field of biocatalysis, we were interested in free from material erosion by competing metabolic pathways and the investigation of the compatibility of these types of reactions from kinetic restrictions by regulating circuits, which are notorious with each other in water. As an example for such a one-pot process problems in cellular production systems (cell factories). the synthesis of chiral biaryl-containing alcohols via Suzuki- Examples illustrating the technology will be discussed. cross-coupling reaction and subsequent asymmetric enzymatic reduction is shown [1]. A further example for the combination of References palladium catalysis and a biotransformation is a one-pot process [1].http://www.cost.eu/domains actions/cmst/actions/CM1303. comprising a Wacker oxidation and subsequent enzymatic reduc- [2].Fessner W-D, Walter C. “Artificial metabolisms” for the asymmetric tion [2]. Very recently we could also demonstrate the compatibility one-pot synthesis of branched-chain saccharides. Angew Chem Int Ed of a metal-catalyzed cross-metathesis reaction with a biotransfor- Engl 1992;31:614–6. mation [3]. A further research focus is on the combination of http://dx.doi.org/10.1016/j.nbt.2014.05.1781 enzyme-compatible organocatalytic reactions with biotransformations towards multi-step one-pot syntheses. It turned out that a reaction mixture resulting from an asymmetric organocatalytic O20-3 aldol reaction is compatible with a direct subsequent enzymatic reduction without the need for a work-up step of the aldol reac- Expanding the diversity of diketopiperazines biosynthe- tion [4,5]. In addition, an organocatalytic nitroalkene synthesis sized by cyclodipeptide synthases has been successfully combined with its subsequent ene reductase- Isabelle Jacques ∗ , Jérôme Seguin, Mireille Moutiez, Emmanuel catalyzed asymmetric reduction, leading to the corresponding Favry, Muriel Gondry, Pascal Belin nitroalkane with high enantioselectivity [6]. CEA, iBiTec-S, Service d’Ingénierie Moléculaire des Protéines (SIMORPO), France

Cyclodipeptides and more complex diketopiperazines (DKPs) References are secondary metabolites mostly synthesized by microorganisms. They are well known for their wide range of noteworthy biological [1].Burda E, Hummel W, Gröger H. Angew Chem 2008;120:9693–6; activities including antibacterial, antifungal, antiviral and anti- Angew Chem Int Ed 2008;47:9551–4. [2].Schnapperelle I, Hummel W, Gröger H. Chem Eur J 2012;18:1073–6. cancer effects [1]. In the last decade, an important effort has been [3].Tenbrink K, Seßler M, Schatz J, Gröger H. Adv Synth Catal made to elucidate the biosynthesis pathways of these compounds. 2011;353:2363–7. Our group and others characterized ﬁve novel pathways [2–4] that

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are dependent on cyclodipeptide synthases (CDPSs) [5,6], enzymes O20-5 that catalyze the formation of the DKP scaffold by condensing two aminoacyl-tRNAs. Herein, I will describe our medium-high Artificial enzyme cascade to the polymer building block ␻ throughput method to characterize 46 putative CDPSs identified -amino caproic acid by bioinformatics. Our work confirms thereby 40 new CDPSs and Wolfgang Kroutil 1,∗ , Johann Sattler 2, Michael Fuchs 1, Verena reveals a large variety of produced cyclodipeptides. This study Resch 1, Joerg Schrittwieser 1 opens the ways to a future characterization of the CDPS-dependent 1 University of Graz, Austria pathways, enabling combinatorial biosynthesis to produce unnat- 2 ACIB GmbH, Austria ural natural DKPs. References The monomer units for polyamides may be ␻-amino-carboxylic [1].Borthwick AD. Chem Rev 2012;112:3641–716. acids, lactams or diamines and dicarboxylic acids. Caprolactam, for [2].Belin P, et al. Nat Prod Rep 2012;29:961–79. example, is produced chemically at 4.2 million ton/year mainly [3].Giessen TW, et al. Chem Biol 2013;20:828–38. from cyclohexanone via its oxime and Beckmann rearrangement. [4].Giessen TW, et al. Biochemistry 2013;52:4274–83. Biocatalytic cascades involving redox steps [1] allow to perform [5].Gondry M, et al. Nat Chem Biol 2009;5:414–20. oxidation and reduction reaction in the linear sequence simulta- [6].Seguin J, et al. Chem Biol 2011;18:1362–8. neously. [2,3] Oxidation and reduction reactions might be coupled http://dx.doi.org/10.1016/j.nbt.2014.05.1782 in that way that the electrons gained in the oxidation step are consumed in the reduction step turning the overall process redox neutral and cost efficient. O20-4 Here we report a biocatalytic redox cascade starting from cyclohexanol to yield ␻-amino caproic acid, thus the hydrolysed Improving the performance of coupled race- equivalent to caprolactam. The designed cascade involves four mase/acylase systems: new structural insights and redox steps and additional hydrolytic steps, whereby the cas- novel tools for high-throughput screening cade was designed in that way, that no external redox reagents Guiomar Sanchez Carron 1,∗ , Dominic Campopiano 1, Toni Fleming 2 were required, thus the cascade was redox neutral or redox self- 1 University of Edinburgh, United Kingdom sufficient. Since an intermediate in the reaction sequence caused 2 Dr. Reddys Laboratories, India inhibition for a later enzyme in the cascade an in situ functional group protection strategy was established to avoid the formation A coupled enzymatic dynamic kinetic resolution (DKR) sys- of the inhibiting compound. tem to allow the preparation of synthetically useful amino acids has been developed and improved. We combined a previously References engineered N-acetyl amino acid racemase (NAAAR G291D/F323Y) [1].Schrittwieser H, Sattler J, Resch V, Mutti FG, Kroutil W. Curr Opin with an L-orD-specific acylase and used them in a preparative Chem Biol 2011;15:249–56. scale DKR to generate enantiomerically pure amino acids. In this [2].Staudt S, Burda E, Giese C, Müller CA, Marienhagen J, Schwaneberg U, et al. Angew Chem Int Ed 2013;52:2359–63. work we describe the latest efforts to expand the synthetic utility [3].SattlerJH, Fuchs M, Tauber K, Mutti FG, Faber K, Pfeffer J, et al. Angew of this process. We present two novel spectrophotometric assays Chem Int Ed 2012;51:9156–9. to measure NAAAR activity towards different substrates, replac- http://dx.doi.org/10.1016/j.nbt.2014.05.1784 ing the existing HPLC and In vivo selection assays. One of them links the NAAAR/acylase couple to an L-amino acid oxidase and peroxidase. The other uses a D-amino acid dehydrogenase with O20-6 broad substrate specificity. These assays allow us to identify novel NAAAR substrates and facilitate the high-throughput screening of Uncovering the broader roles of redox partner proteins NAAAR saturation mutagenesis libraries. X-ray structural analysis for cytochrome P450 enzymes of NAAAR mutants in complex with N-acetyl-D-naphthylalanine 1,∗ 1 1 2 2 reveals active site residues involved in the accommodation of Shengying Li , Wei Zhang , Yi Liu , Yojiro Anzai , Fumio Kato , 3 4 bulkier substrates. These assays, combined with structural insights, Larissa Podust , David Sherman guide the engineering of new NAAARs with catalytic potential 1 Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy across a larger range of amino acids. of Sciences, China 2 Acknowledgements: This work has been funded by the Toho University, Japan 3 University of California, San Francisco, USA BBSRC and Dr Reddys Laboratories. 4 University of Michigan, Ann Arbor, USA http://dx.doi.org/10.1016/j.nbt.2014.05.1783 The superfamily of cytochrome P450 enzymes is one of the most versatile biocatalytic systems in nature. P450 enzymes are capable of catalysing many distinct types of industrially important reactions such as regio- and stereoselective oxidation of unactivated C−H bonds, dealkylation, decarboxylation, and aromatic coupling. For most P450 enzymes, redox partner proteins

www.elsevier.com/locate/nbt S75 SYMPOSIUM 20: SYSTEMS BIOCATALYSIS New Biotechnology · Volume 31S · July 2014 are required for the electron transfer during catalysis. Conven- partner proteins are able to support the activity of a biofuel related tionally, it has been thought that these auxiliary proteins only P450 peroxygenase from Jeotgalicoccus sp. ATCC 8456 that nor- inﬂuence catalytic efﬁciency and/or product distribution, but not mally uses hydrogen peroxide as cofactor (Biotechnol. Biofuels the type and selectivity of reactions catalysed by P450s. However, 2014, 7, 28). These results highlight broader roles of redox part- our recent study (J. Am. Chem. Soc. 2014, 136, 3640) on MycG, ner proteins for modulating the catalytic activity of P450 enzymes which is the multifunctional P450 monooxygenase involved in the via alternative protein-protein interactions that have not been well biosynthetic pathway of 16-membered ring macrolide antibiotics understood so far. mycinamicins in the rare actinomycete Micromonospora griseoru- http://dx.doi.org/10.1016/j.nbt.2014.05.1785 bida, has provided solid evidence to challenge this generally accepted “postulate”. Moreover, we have found that some redox

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Bio-based production of chemicals, fuels developing bio-based processes for the production of chemicals, and materials by metabolically engineered fuels and materials from renewable non-food biomass. In order microorganisms to maximize the efficiencies of bioconversion process, metabolic engineering has become an essential practice. Metabolic engineer- PL3-1 ing has recently become more powerful through the integration with systems biology, synthetic biology and evolutionary engi- Bio-based production of chemicals, fuels and materials neering, which led to the birth of systems metabolic engineering. by metabolically engineered microorganisms In this lecture, systems metabolic engineering approaches taken to develop strains capable of efficiently producing various chemicals, Sang Yup Lee fuels and materials will be described together with several exam- Department of Chemical and Biomolecular Engineering (BK21+ Program), ple cases. Through systems metabolic engineering, it is possible BioProcess Engineering Research Center, Center for Systems and Synthetic to achieve cost-effective production of desired bioproducts, which Biotechnology, Institute for the BioCentury, KAIST, 291 Daehak-ro, Yuseong-gu, Daejeon, Republic of Korea are either natural or nonnatural products. Acknowledgements: This work was supported by the Tech- Many chemicals, fuels and materials we use every day are nology Development Program to Solve Climate Changes on derived from fossil resources. Relying on the current produc- Systems Metabolic Engineering for Biorefineries of Ministry of Sci- tion system is not sustainable and has been raising concerns ence, ICT & Future Planning. due to the climate change and other environmental problems. http://dx.doi.org/10.1016/j.nbt.2014.05.1786 To address this issue, there has recently been much interest in

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Posters

Biocatalysis PA-02

PA-01 Simultaneous improvement of speciﬁc catalytic activity and thermal stability of MBP fusion heaprinase II by Enhancing of enzymatic palmitoylation of racemic 9- modifying amino acid residues in 758 site (2,3-dihydroxypropyl)adenine in co-solvent mixture as ∗ the reaction media Nan Su , Jingjun Wu, Chong Zhang, Xin-Hui Xing

Jana Brabcova 1,∗ , Jiri Blazek 1, Marcela Krecmerova 1, Marie Key Laboratory for Industrial Biocatalysis, Ministry of Education of China, Department of Chemical Engineering, Tsinghua University, Beijing, China Zarevucka 1, Jose M. Palomo 2

1 Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Heparinase II (HepB) is an important polysaceharide lyase, Czech Republic, Prague which plays an vital role in the development of anticancer drugs, 2 Departamento de Biocatálisis. Instituto de Catálisis (CSIC).Campus UAM Can- toblanco, Madrid, Spain production of low molecular weight heparin (LMWH) and quality control of heparins. At present, the main technical bottleneck A comparative study of racemic 9-(2,3-dihydroxypropyl) which has restrained its industrial application is the high produc- adenine (DHPA) palmitoylation, a potential prodrug, catalysed by tion cost, the poor level of heterologous recombinant expression several lipases in DMF/co-solvent mixtures and in pure organic and lack of the thermal stability. In a systematic design for con- solvent was performed. The optimal conditions were investigated struction of fusion expression system with the MBP tag in E.coli, we found that heparinase II could be expressed efficiently with as follows: optimal co-solvent mixture, initial aw (water activity), vinyl palmitate/DHPA molar ratio, temperature, enzyme dosage, high activity in the soluble form, and firstly observed that the and chemical modification of biocatalysts. It was shown that an amino acid residues of the 758 site in the HepB sequence greatly enhancement in the substrate conversion could be achieved with affected the catalytic activity and thermal stability of this enzyme, DMF/hexane (4:1) co-solvents as the reaction medium instead of which were closely related with the protein dimmer formation pure organic solvent with immobilized Candida antarctica B (CALB) of HepB. Furthermore, we systematically studied the fusion pro- lipase, although in very low yield. The chemical modification of tein design by changing the linker, indicating that linker property the lipase had strong positive effect on its activity. The reaction could significantly affect both the fusion enzyme activity during yield was successfully increased (50% after 24 hours) when CALB the expression and the thermo stability. As a result, the total MBP- glycosylated with dextran polymer was used under optimal con- HepB activity reached 3674.82IU/L, and half-life of the enzyme in ◦ ditions. The results described further highlight the versatility of 30 C reached more than 250 h which increased 25 times compared lipases and the potential of rational substrate, solvent and enzyme with the wild HepB. engineering for modulating enzymatic reactions. http://dx.doi.org/10.1016/j.nbt.2014.05.1788 Acknowledgments: The authors are grateful for the financial support of the Academy of Sciences of the Czech Republic (project No. M200551203). http://dx.doi.org/10.1016/j.nbt.2014.05.1787

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PA-03 fer of substrate, which have important role in immobilized enzyme activity. Only few MPS sheet have been reported because they are Effective encapsulation of biocatalysts in sol–gel silica quite difficult to prepare. On these bases, we introduce a new syn- nanosheet prepared by peptide catalysts thetic approach to MPS sheets (thickness under 100 nm) by using Katsuya Kato ∗ , Hitomi Nakamura, Fukue Nagata the carboxylate surfactant (N-palmitoyl-L-alanine) and Pluronic P123 as dual-templating agents. Cytochrom c (cyt c, molecular size National Institute of Advanced Industrial Science and Technology (AIST), 2266- of 2.5 × 2.5 × 3.7 nm) immobilised on the MPS sheet exhibited a 98 Anagahora, Shimoshidami, Moriyama-ku, Nagoya 463-8560, Japan remarkably higher catalytic oxidation activity than native cyt c. Encapsulation restricts the mobility of the biomolecules con- Reference tained within a confined space and can prevent denaturation [1].Nakanishi K, et al. Mater Chem B 2013;1:6321. even in harsh environmental conditions related to temperature, http://dx.doi.org/10.1016/j.nbt.2014.05.1790 pH, and solvents. For this reason, many biomolecules have been trapped within sol–gel silica, and this research has led to some of the most interesting and important applications of these materi- PA-05 als, paving the way for their use in the design and fabrication of biocatalysis and biosensing devices [1]. Very recently, we reported Physiological responses of Pinus sylvestris var. mongolica [2] the mesoporous silica (MPS) sheet (thickness < 100 nm) was syn- seedlings to the interaction between Suillus luteus and thesized using a novel approach for the immobilization of enzyme. Trichoderma virens Enzyme immobilized on the MPS sheet exhibited a remarkably 1,∗ 1 2 higher catalytic oxidation activity than native enzyme. From these Ruiqing Song , Dachuan Yin , Xun Deng results, the sheet morphology significantly influences the activity 1 Northeast Forestry University of immobilised enzyme. In this report, we present the details of our 2 Forestry Protection Institute, Heilongjiang Academy of Forestry investigation on the immobilization of the enzyme, glucose oxidase (GOX), in a sol–gel silica matrix using poly-L-lysine assisted The effects of the interaction between Suillus luteus (L.) Roussel direct condensation reactions of silicon oxide, trimethoxysilane. and Trichoderma virens (J.H. Mill., Giddens & A.A. Foster) Arx on The resulting GOX-silica composites with hexagonal sheet mor- Pinus sylvestris var. mongolica Litv. were studied using plant phys- phologies were thoroughly characterized in terms of morphology, iology, mycorrhizal science, forest pathology and biochemistry. size and surface structure. The activity and stability of the immo- Seedling growth and physiological parameters were determined, bilized GOX were also investigated in details. including the colonization rate of mycorrhizal fungi, biomass, root activity, photosynthetic pigment content, soluble protein content, References antioxidant enzyme activities, rhizosphere soil enzyme activities and protective enzyme activities. In addition, an optimal resis- [1].Kato K, et al. J Asian Ceram Soc 2014. DOI: 10.1016/j.jascer.2013.12.004. tance system involving T. virens, mycorrhizal fungus (Suillus luteus) [2].Nakanishi K, et al. RSC Adv 2014;4:4732. and P. sylvestris var. mongolica seedlings was constructed. Syner- gies between S. luteus and T. virens were observed and most of the http://dx.doi.org/10.1016/j.nbt.2014.05.1789 parameters of Pinus sylvestris var. mongolica seedlings inoculated with S. luteus 30 days + T. virens were higher than other treatments. After three months, when compared the control, the S. luteus PA-04 30 days + T. virens treatment gave increases in: height (42.3%); Novel mesoporous silica sheet as a protein carrier for collar diameter (66.7%); fresh weight (54%); dry weight (50%); enhancement of catalytic activity soluble protein content (69.86%); root activity (150%); chlorophyll a (77.6%); chlorophyll b (70.5%); carotenoids (144%); CAT 1,2,∗ 1 2 Kazuma Nakanishi , Masahiro Tomita , Katsuya Kato activity (876.9%); POD activity (268.3%); SOD activity (66.18%); 1 Department of Chemistry for Materials, Mie University, 1577 Kurimamachiya- ␤-1,3-glucanase activity (125.8%); chitinase activity (40%); rhi- cho Tsu city, Mie 514-8507, Japan zosphere soil catalase activity (97.8%); and phosphatase activity 2 National Institute of Advanced Industrial Science and Technology (AIST), (266.7%). These results indicate that there may be a stimulating 2266-98 Anagahora, Shimoshidami, Moriyama-ku, Nagoya 463-8560, Japan factor between S. luteus and T. virens when they are inoculated together (S. luteus 30 days + T. virens) Porous materials have much attention because they can interact Keywords: Suillus luteus; Trichoderma virens; interaction; with atoms, ions, molecules, and nanoparticles, at their surfaces synergies; Pinus sylvestris var. mongolica as well as throughout the bulk of the material. Mesoporous silica (MPS) (pore size 2–50 nm) with great mechanical stability has been http://dx.doi.org/10.1016/j.nbt.2014.05.1791 applied in many fields including enzyme immobilization, catalysis and chromatography. Recently, we reported the enhanced activity and stability of enzymes and proteins on MPS as an effective carrier. [1] The presence of pore in nanostructured materials for biological applications must be promoted. Then we focused on the morphologies of materials in order to increase the mass trans-

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PA-06 naphthoic acids. [1]. Furthermore, CYP199A2 was able to catalyze the regioselective hydroxylation of hydroxy-2-naphthoic acids, ␤ Purification and characterisation of a -galactosidase indolecarboxylic acids, and a quinolinecarboxylic acid [2,3]. These from the thermoacidophilic bacterium Alicyclobacillus results indicate that CYP199A2 should be an efficient biocata- vulcanalis DSM 16176 lyst for the hydroxylation of various aromatic carboxylic acids Gary Walsh ∗ , Jayne Murphy [4]. The crystal structure of CYP199A2 determined by Bell et al. (J. University of Limerick Mol. Biol., 383, 561 (2008)) shows that Phe at the 185 position is situated directly above the heme iron. When this Phe185 residue Thermoacidophiles are microorganisms capable of optimum was replaced with hydrophobic or hydroxylated amino acids, growth under a combination of high temperature and low pH. several mutants predominantly produced 5-hydroxy-2-naphthoic These microorganisms are a rich source of thermo- and acid- acid from 2-naphthoic acid. Interestingly, these Phe185 mutants active/stable glycosyl hydrolases. Such enzymes could find use also exhibited novel substrate specificities. In particular, the as novel biocatalysts in some industrial processes, as operation Phe185Leu mutant exhibited high hydroxylation activity for cin- at elevated temperature can increase substrate solubility, decrease namic and p-coumaric acids [5]. The Phe185Leu whole-cell catalyst viscosity and reduce the risk of microbial contamination [1,2]. achieved gram-per-liter-scale production of caffeic acid from p- We report the purification and characterisation of an intracel- coumaric acid. lular ␤-galactosidase from the thermoacidophile Alicyclobacillus vulcanalis DSM 16176. The enzyme was purified 110-fold, with a 5% yield. Denatured (84 kDa) and native (179 kDa) molecular References masses were determined by SDS-PAGE and gel filtration, respec- [1].Furuya T, Kino K. ChemSusChem 2009;2:645–9. tively and suggest the enzyme functions as a homodimer. Highest [2].Furuya T, Kino K. Biosci Biotechnol Biochem 2009;73:2796–9. ◦ activity was measured at 70 C and pH 6.0. The Km on the sub- [3].Furuya T, Kino K. Appl Microbiol Biotechnol 2010;85:1861–8. strates ONPG and lactose were, respectively, 3.8 and 425.3 mM. [4].Furuya T, Kino K. Appl Microbiol Biotechnol 2010;86:991–1002. This enzyme is thermostable, retaining 76, 50 and 42% rela- Review. [5].Furuya T, et al. Appl Environ Microbiol 2012;78:6087–94. tive activity after 30, 60 and 120 min, respectively, at 70 ◦C. This property could lend its use to high-temperature industrial pro- http://dx.doi.org/10.1016/j.nbt.2014.05.1793 cesses requiring a thermo-active ␤-galactosidase, including the production of lactulose and in the manufacture of lactose-free milk and dairy products. To date, there have been no reports PA-08 in the literature on the characterisation of a glycosyl hydro- New biotransformation process for production of ␥- lase from A. vulcanalis DSM 16176. Funded by IRCSET EMBARK lactones from hydroxy fatty acids by permeabilized Initiative. Waltomyces lipofer cells induced with oleic acid Key-words., ␤-galactosidase, thermoacidophile, protein purification. Jung ung An ∗ , Deok kun Oh

References Konkuk university [1].Niehaus F, Bertaldo C, Kahler M, Antranikian G. Applied Microbiology and Biotechnology 1999;51(6):711–29. The production of the flavor lactone was developed by using [2].Turner P, Mamo G, Karlsson EN. Microbial Cell Factories 2007;6:9. permeabilized Waltomyces lipofer, which was selected as an efficient ␥-lactones producing yeast among 10 oleaginous yeast strains. http://dx.doi.org/10.1016/j.nbt.2014.05.1792 The cells were permeabilized using 50% ethanol and 0.5% Tri- ton X-100, sequentially. Among several fatty acids tested, oleic acid was selected as the most efficient inducer for the produc- PA-07 tion of ␥-lactones. The cells were induced by incubation for 12 h −1 −1 Regioselective hydroxylation of aromatic carboxylic in a medium containing 10 g l yeast extract, 10 g l peptone, −1 −1 acids by cytochrome P450 CYP199A2 and its mutants 5g l oleic acid, 1 g l glucose, and 0.05% (w/v) Tween 80. The ␥ ∗ optimal reaction conditions for -lactones production by whole Toshiki Furuya Kuniki Kino ◦ , Waltomyces lipofer were pH 6.5, 35 C, 200 rpm, 0.71 M Tris, 60 g Waseda University l−1 hydroxy fatty acid, and 20 g l−1 cells. Under these conditions, non-treated cells produced 30 g l−1 ␥-dodecalactone from 60 g l−1 Hydroxy-aromatic carboxylic acids are practically or poten- 10-hydroxystearic acid after 30 h, with a conversion yield of 63% tially important industrial chemicals since the functional groups (w/w) and a productivity of 1.3 g l−1 h−1, whereas treated cells pro- play key roles in biological activities and physical properties duced 51 g l−1 ␥-dodecalactone from 60 g l−1 10-hydroxystearic and also can be utilized for further modification of the chemi- acid after 30 h, with a conversion yield of 85% (w/w) and a pro- cals. Cytochrome P450 monooxygenases are promising catalysts ductivity of 1.7 g l−1 h−1. The conversion yield and productivity for use in the hydroxylation of chemicals. We found that of treated cells were 22% and 1.3-fold higher, respectively, than CYP199A2 from Rhodopseudomonas palustris has catalytic activity those of non-treated cells. The treated cells also produced 28 g l−1 for the hydroxylation of 2-naphthoic acid to 7- and 8-hydroxy-2- ␥-decalactone and 12 g l−1 ␥-butyrolactone. These are the highsest

S80 www.elsevier.com/locate/nbt New Biotechnology · Volume 31S · July 2014 BIOCATALYSIS reported concentration, conversion yield, and productivity for the plemented with 4.5 U ml−1 SA-bglu. Under optimum conditions, production of bioﬂavor lactone. major protopanaxadiol ginsenosides in ginseng root extract were http://dx.doi.org/10.1016/j.nbt.2014.05.1794 completely converted to compound K after 20 h with the respective productivities of 144 mg l−1 h−1. http://dx.doi.org/10.1016/j.nbt.2014.05.1796 PA-09

Bioconversion of linoleic acid to 5,8-dihydroxy- PA-11 9,12(Z,Z)-octadecadienoic acid by 5,8-diol synthase from Aspergillus nidulans Structural conformation of enzyme in ionic liquids: molecular dynamic simulation study Seo Min-Ju ∗ , Shin Kyung-Chul, Oh Deok-Kun Konkuk university Yoon-Mo Koo Inha University The fungus diol synthase from Aspergillus nidulans was cloned and expressed in Escherichia coli. Recombinant E. coli cells con- Enzymatic reactions in ionic liquids have been gaining increas- verted linoleic acid to 5,8-dihydroxy-9,12(Z,Z)-octadecadienoic ing interests during the last decade due to their unique properties. acid, which was identified by LC-MS/MS. The recombinant cells It is well documented that, in some cases, enzymes showed and the purified enzyme showed activity for linoleic acid. The reac- enhanced activity, selectivity and stability in ionic liquids. How- tion using purified diol synthase stoppedafter 15 min, whereas the ever, there were very few studies investigating the structure of reaction using recombinant cells expressing diol synthase from enzymes in ionic liquids. In this study, the conformational struc- A. nidulans continued over 60 min, indicating that recombinant ture changes of Candida antarctica lipase B (CALB) in different cells expressing diol synthase were more stable than the puri- imidazolium-based ionic liquids observed by molecular dynamic fied enzyme. The optimal reaction conditions for the production simulation are discussed. The results showed that two isoleucines, of linoleic acid to 5,8-dihydroxy-9,12(Z,Z)-octadecadienoic acid ILE-189 and ILE-285, in CALB played critical role in the open- ◦ using whole recombinant E. coli cells were pH 7.5, 35 C, 250 rpm, closed conformations of the catalytic cavity. The ILE-285 situated − − 23 g l 1 cells, 5 g l 1 linoleic acid, and 20% (v/v) dimethyl sulfoxide on ␣-10 helix region (residues 268-287) where its conformation in a 250 ml-baffled flask. Under these optimized conditions, whole can be significantly changed in different solvents. For example, − recombinant cells produced 4.98 g l 1 5,8-dihydroxy-9,12(Z,Z)- in 1-butyl-3-methylimidazolium chloride ([Bmim][Cl]) conforma- octadecadienoic acid for 150 min, with a conversion yield of 99% tion of ␣-10 helix changed to a turn as a result of direct interactions − − (w/w) and a productivity of 2.5 g l 1 h 1. This is the biotechnolo- with chlorine anions gave rise to a closed conformation of the gical production of dihydroxy fatty acid using whole recombinant catalytic cavity, corresponding to lower enzyme activity. cells expressing diol synthase. http://dx.doi.org/10.1016/j.nbt.2014.05.1797 http://dx.doi.org/10.1016/j.nbt.2014.05.1795

PA-12 PA-10 Conjugation of chitooligosaccharides and glucosamine Production of compond K from major protopanaxadiol to bovine trypsin. Effects on stability and functionality ginsenosides by combined enzymes, including ␣-L- Hörður Filippusson ∗ , Jóhann G.K. Gizurarson arabinofuranosidase and ␤–galactosidase from Caldicel- lulosiruptor saccharolyticus and ␤–glucosidase from University of Iceland Sulfolobus acidocaldarius The improvement of protein stability is important in relation to Kyung-Chul Shin ∗ , Hye-Jin Oh, Baek-Joong Kim, Deok-Kun Oh the use of enzymes as practical biocatalysts and in the use of pro- Konkuk university teins as pharmaceuticals. All proteins are unstable, especially when in solution. Among the processes that can affect protein stability The ginsenoside compound K has diverse pharmaceutical activ- are proteolytic degradation in vitro or in vivo, thermal denaturation, ities such as anti-tumor, anti-inflammatory, anti-allergic, and and antigenicity after injection. Attempts to improve protein hepatoprotective effects. To increase the production of com- stability include protein engineering, immobilization, chemical pound K from major protopanaxadiol ginsenosides in ginseng root modification and the use of cosolutes. extract, ␣-l-arabinofuranosidase (CS-abf) and ␤–galactosidase (CS- In this study bovine trypsin was modified by coupling bgal) from Caldicellulosiruptor saccharolyticus were mixed with the enzyme to either D-glucosamine or a partially acetylated ␤–glucosidase (SA-bglu) from Sulfolobus acidocaldarius. The opti- chitoologosaccharide via binary carbodiimide/succinimide ester mum conditions for the production of ginsenoside compound K conjugation. D-glucosamine was found to conjugate, on average, from major protopanaxadiol ginsenosides in ginseng root extract to 12 residues on trypsin. The oligosaccharide coupling produced ◦ were determined to be pH 6.0 and 75 C with 10% (w/v) ginseng cross-linked polydisperse complexes with hydrodynamical radii root extract and 10.5 U ml−1 CS-abf and 10.5 U ml−1 CS-bgal sup-

www.elsevier.com/locate/nbt S81 BIOCATALYSIS New Biotechnology · Volume 31S · July 2014 from, on average, from 218 to 330 nm for 1:5 and 1:10 cross-linked can hydroxylate 1,8-cineole. Compared to the well characterised trypsin, repectively. P450cin from Citrobacter braakii, the S. yanoikuyae enzymes have The physical properties of the enzyme species were studied limited amino acid sequence identity, appear to have differing by electrophoresis, gel filtration, circular dichroism, nanoprticle stereo- and regioselectivity and based on testing completed so tracking analysis and MALDI-TOF mass spectrometry. The stabil- far the preferred substrate is 1,8-cineole. Current investigations ity of these enzyme species was studied by activity assays, urea involve mining the S. yanoikuyae genome for the natural electron denaturation, diffferential scanning calorimetry and autolysis. transport partners to optimise the hydroxylation process. The modified trypsin showed increased resistance against ther- http://dx.doi.org/10.1016/j.nbt.2014.05.1799 mal inactivation and autolysis, better storage stability but stability against urea inactivation was unchanged. The proteolytic activity against azocasein improved for the cross-linked trypsins but PA-14 was slightly reduced for D-glucosamine conjugated trypsin. The species were found to become basophilic upon conjugation and Human flavin monooxygenase 2: Heterologous expres- cross-linking. D-glucosamine conjugated trypsin was found to be sion in E. coli and API modification slightly structurally altered which and as consequence displayed Margit Winkler 1,∗,1 , Thorsten Bachler 2,2, Martina Geier 2,2, Steven P. 1.2 times higher catalytic efficiency (kcat/Km) than native trypsin Hanlon 3,3, Matthias Kittelmann 4, Anton Glieder 2,2, Stephan Lütz 4, against the substrate L-BAPNA. Beat Wirz 3,3 http://dx.doi.org/10.1016/j.nbt.2014.05.1798 1 ACIB GmbH c/o Institute of Molecular Biotechnology, Graz University of Tech- nology 2 ACIB GmbH PA-13 3 F. Hoffmann-La Roche Ltd 4 Novartis Pharma AG 1,8-cineole-hydroxylating cytochrome P450s from Sphin- gobium yanoikuyae Abstract The flavin monooxygenase (FMO) isoenzyme 2 is Birgit Unterweger 1,∗ , David J. Midgley 2, Paul Greenfield 3, Dieter M. known as the “pulmonary” FMO because it is expressed predomi- Bulach 4, Dena Lyras 5, Priscilla Johanesen 5, Geoffrey J. Dumsday 6 nantly in the human lung. FMO2*1 is the active FMO2 allele found in Africans and Hispanics [1]. The enzyme is membrane associ- 1 Department of Microbiology, Monash University, Clayton, VIC 3800, Australia ated and oxidizes xenobiotics with soft nucleophiles such as sulfur and CSIRO Future Manufacturing Flagship, Clayton, VIC 3168, Australia 2 CSIRO Animal, Food and Health Sciences, North Ryde, NSW 1670, Australia and nitrogen at the expense of NADPH and oxygen. The aim of 3 CSIRO Computational Informatics, North Ryde, NSW 1670, Australia the current study was to generate simple and efficient whole cell 4 Victorian Bioinformatics Consortium, Monash University, Clayton, VIC 3800, biocatalysts for the preparation of FMO2 drug metabolites on the Australia multi-milligram scale. Recently, we demonstrated the possibility 5 Department of Microbiology, Monash University, Clayton, VIC 3800, Australia to express human FMOs in E. coli, however, the expression level 6 CSIRO Materials Science and Engineering, Clayton, VIC 3168, Australia of the FMO2*1 isoform was very low in comparison to FMO3 and FMO5 [2]. This prompted us to investigate truncated variants of In Australia, extensive Eucalyptus plantations have been estab- FMO2*1 and to compare their performance in whole cell biotrans- lished to prevent dry land salinity and novel applications for formations of different active pharmaceutical ingredients such as this renewable resource are being sought. One approach is to e.g. ethionamide–an antitubercular drug [3] add value to the leaf oil from these plantations via the oxyfunc- Key-words: Biocatalysis, flavin monooxygenase, drug metabo- tionalisation of its chemically unreactive component 1,8-cineole. lites, oxidation, membrane protein. Hydroxyl-groups create starting points for further modification and/or incorporation into more complex molecules such as poly- References mers. Biooxidation of 1,8-cineole promises a more sustainable [1].(a) Dolphin C, et al. J Biol Chem 1998;273(46):30599–607; route to hydroxylated intermediates with greater stereospecificity (b) Krüger SK, et al. Drug Metabol Disposit 2004;32(12):1337–40. compared to conventional chemistry. The availability of biocat- [2].Hanlon SP, et al. Chem Commun 2012;48(48):6001. alysts known to catalyse the oxidation of 1,8-cineole, however, [3].Francois AA, et al. Drug Metabol Disposit 2009;37(1):178–86. is limited. To expand the range of biocatalysts, several microbes http://dx.doi.org/10.1016/j.nbt.2014.05.1800 capable of hydroxylating 1,8-cineole were isolated including a Sphingobium yanoikuyae strain. Genome sequencing revealed the presence of multiple genes encoding P450s and potential electron transport partners. To confirm the identity of genes encoding 1,8-cineole-hydroxylating P450s, proteins exhibiting the characteristic spectroscopic shift upon addition of 1,8-cineole were purified from S. yanoikuyae cultivated in the presence of 1,8- cineole. In addition to this the genes were cloned and the gene products heterologously expressed in Escherichia coli and puri- 1 ACIB GmbH, Petersgasse 14/III, 8010 Graz, Austria fied from clarified cell lysates. Spectroscopic characterisation and 2 F. Hoffmann-La Roche Ltd., 4070 Basel, Switzerland whole-cell biotransformation have demonstrated that the proteins 3 Novartis Pharma AG, 4070 Basel, Switzerland

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PA-15 from a reaction mixture is often difficult. The gram-negative bacteria Zymomonas mobilis and Zymobacter palmae are promising host Co-factor regeneration at the cell surface–upgrading the organisms for industrial applications due to their high ethanol pro- biotechnological potential of whole cell biocatalysts ductivity and resistance towards rough reaction conditions. Until Jan Schüürmann ∗ , Joachim Jose now, no functioning surface display system for these organisms has been reported. For this reason, we tested if the autotransporter Westfälische Wilhelms Universität Münster secretion pathway can be utilized in Z. mobilis and Z. palmae. Here we present the surface display of two industrially relevant Whole cell biocatalysts offer significant advantages compared enzymes, namely Burkholderia gladioli esterase EstA and Bacillus to purified enzymes, specifically cheap and efficient production subtilis endoglucanase Cel5A, and demonstrate that the enzymes combined with obsolete purification. However, there are limi- retain their activity on the cell surface of both organisms. This tations to the applicability of whole cells. Cross reactions with work represents the first step towards using Z. mobilis and Z. palmae endogenous enzymes and membrane impermeability of substrates as expression platforms for surface display applications. and products might complicate their use. Autodisplay presents enzymes on the surface of E. coli cells elim- http://dx.doi.org/10.1016/j.nbt.2014.05.1802 inating mass transfer problems and possible side reactions. The technique replaces the passenger domain of a native autotransporter protein by a peptide or protein of choice. However, a major PA-17 challenge for some enzymes to be used in biotechnological appli- Combined Surface Display of Cytochrome P450 1A2 and cations is the regeneration of co-factors, which are too expensive its Reductase on Escherichia coli to be added stoichiometrically. Here, we report the development of a cell surface based NADPH regeneration system. To circum- Paul Quehl ∗ , Joachim Jose vent laborious protein purification and keep the advantages of University of Münster surface displayed catalysts we used the Autodisplay technology to present various dehydrogenases on the cell surface of E. coli. Human cytochrome P450 monooxygenases (CYPs) play a Surface display was confirmed by protease accessibility tests and prominent role in drug metabolism as these enzymes are involved FACS analysis. NADPH production was measured photometrically in the breakdown of literally every drug. They catalyze a variety of at 340 nm. We first concentrated our efforts on an engineered for- oxidations of a broad range of substrates and have a major impact mate dehydrogenase. The enzyme showed similar specific activity on bioavailability and drug-drug interactions. CYPs obtain the compared to the soluble enzyme, but the efficiency of the system electrons from the NADPH-dependent cytochrome P450 reduc- was limited by the number of enzymes on the bacterial surface and tase (CPR). However, recombinant expression of both enzymes in high amounts of whole-cell catalysts were needed for effective pro- bacteria is often not feasible and they exhibit poor stability after duction of NADPH. Thus, we are currently exploring alternative purification. Moreover, the membrane associated proteins CPR and enzymes with higher specific activities for surface display and cell CYPs require membrane surroundings for activity. CYPs alone can associated co-factor regeneration. be displayed functionally active on the surface of Escherichia coli http://dx.doi.org/10.1016/j.nbt.2014.05.1801 with externally added CPR. Here, we tested the co-expression of CYP1A2 and its NADPH dependent Cytochrome P450 reductase (CPR) on the E.coli outer membrane. Surface display is facilitated PA-16 by usage of the autotransporter secretion pathway for which the enzyme of interest is combined with an N-terminal signal peptide, Surface display of enzymes on Zymomonas mobilis and a C-terminal linker and a beta-barrel domain. Surface presenta- Zymobacter palmae using the autotransporter secretion tion was confirmed by FACS analysis and protease accessibility pathway tests. Both CYP1A2 and CPR bind their cofactors and the sur- Iasson E.P. Tozakidis 1,∗ , Annika Meyers 1, Tatjana Brossette 2, face displayed NADPH-dependent cytochrome P450 reductase is Joachim Jose 1 able to react with Cytochrome C. The functional interaction of CYP1A2 with the CPR is currently under investigation by testing 1 Institute of Pharmaceutical and Medicinal Chemistry, Westfälische Wilhelms- the enzymatic activity towards the substrates 7-ethoxyresorufin Universität Münster 2 Autodisplay Biotech GmbH and phenacetin. http://dx.doi.org/10.1016/j.nbt.2014.05.1803 Displaying enzymes on a microbial cell surface has the potential to significantly reduce the costs of large scale biocatalytic conversion processes. In contrast to intracellularly expressed enzymes, surface displayed enzymes do not have to be purified prior to their use. Instead, the host cells can directly be employed in a reaction, and neither substrates nor products need to cross a membrane barrier. Additionally, whole cells can easily be harvested and used in multiple process cycles, whereas the recovery of free enzymes

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PA-18 PA-19

Biochemical and Structural Characterization of a Functional Characterization of Putative UDP-Glucose Thermophilc L-Arabinose Isomerase from Geobacillus 4-Epimerase (TM0509) from the Hyperthermophilic kaustophilus Eubacterium Thermotoga maritima

Dong-Woo Lee 1,∗ , Yong-Jik Lee 1, Jin Myung Choi 2, Sun-Mi Shin 1, Sun-Mi Shin 1,∗ , Jin Myung Choi 2, Yong-Jik Lee 1, Sang-Jae Lee 3, Sang-Jae Lee 3, Han-Seung Lee 3, Sang Jun Lee 4, Sung Haeng Lee 2 Sang Jun Lee 4, Sung Haeng Lee 2, Dong-Woo Lee 5 1 Kyungpook National University 1 Kyungpook National University 2 Chosun University 2 Chosun University 3 Silla University 3 Silla University 4 KRIBB 4 Korea Research Institute of Bioscience and Biotechnology 5 Kyungpook National Univ Thermophilic L-Arabinose isomerase (AI), that catalyzes the interconversion of L-arabinose to L-ribulose, can also isomerize UDP-glucose 4-epimerase (GalE; EC 5.1.3.2) catalyzes the D-galactose to D-tagatose as a natural sugar substitute, which interconversion of UDP-glucose (UDP-Glc) and UDP-galactose is of commercial interest in the food and healthcare indus- (UDP-Gal), which is a pivotal step in the Leloir pathway for galac- tries.Recently, biochemical and mutational studies revealed that tose metabolism. Although GalEs are widely distributed in Bacteria unlike mesophilic AIs, thermophilic AIs showed the distinct and Eukaryotes, there is little information on hyperthermophilic metal dependence for their catalytic activity and thermostabi- GalE. Herein we cloned and overexpressed the TM0509 gene lity at elevated temperatures. However, it still remains unclear encoding a putative GalE from Thermotoga maritima (TMGalE) as how mesophilic and thermophilic AIs showed different substrate a fusion protein containing an N-terminal hexa-histidine sequence preferences and metal requirements at molecular levels. Herein in Escherichia coli. This gene encodes a 309-amino acid protein we characterized a thermophilic AI from Geobacillus kaustophilus with a calculated molecular weight of 34899 and a theoretical (GKAI) and presented the first crystal structures of the apo and holo pI of 5.72.The recombinant protein was purified to homogene- 2+ forms of GKAI by X-ray crystallography to 2.40 and 2.30 A,˚ respec- ity by heat precipitation, Ni affinity chromatography followed tively. We also determined the crystal structure of holo enzyme by size-exclusion chromatography. The native enzyme was esti- bound to L-arabitol as a substrate analog at 2.25 A˚ resolution.In mated to be a homodimer with a molecular mass of 70 kDa. The combination with biochemical and site-directed mutagenesis stud- recombinant TMGalE could reversibly catalyze the epimerization + ies, the structures identified the structural elements of metal of UDP-Gal and UDP-Glc in the presence of NAD at elevated binding and substrate recognition.In comparison with the crystal temperatures. The apparent optimal temperature and pH for epi- ◦ structures of Escherichia coli AI (ECAI) as a mesophilic counterpart, merization activity were 85 C and pH 7.0, respectively. In order the GKAI structures revealed quite conserved structural features to further characterize TMGalE at molecular levels, we determined for substrate and metal binding, except forsubtle interactions of a not only the crystal structure of TMGalE at 1.9 A˚ resolution, but few polar residues with water molecules near the substrate bind- also the co-crystal structure of TMGalE bound to UDP-glucose at ing region.Our comparative analysis proposes a metal-mediated 2.0 A˚ resolution. These biochemical and structural data showed substrate binding model for the isomerization reaction at ele- that TM0509 is an UDP-galactose 4-epimerase involved in galac- vated temperatures, providing a versatile strategy to engineer the tose metabolism, which is the first detailed characterization of a promiscuity of substrate specificity for sugar isomerases as well thermostable GalE from hyperthermophilic bacterium. as thermostability for mechanistic studies and industrial applica- http://dx.doi.org/10.1016/j.nbt.2014.05.1805 tions. http://dx.doi.org/10.1016/j.nbt.2014.05.1804 PA-20

Microscale tools for evaluating the biological and process options of alkane biooxidations

Johannes Kolmar 1,∗ , Frank Baganz 1, Philip Engel 2 1 University College London 2 Evonik Industries AG

The direct ␻-oxyfunctionalisation of aliphatic alkanes in a regio- and chemoselective manner remains difﬁcult to perform by industrial organic chemistry. Monooxygenases such as the AlkB enzyme complex from Pseudomonas putida efﬁciently catalyse these readily available substrates to primary fatty alcohols and acids under mild conditions. These are of considerable interest as potential intermediates in the chemical, pharmaceutical and cosmetics industry.

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The ability to rapidly screen biotransformation reactions for atenolol and propranolol. These alterations in enzyme properties characterisation and optimisation is of major importance in pro- by chemical modification should be due to changes in the struc- cess development and biocatalyst selection. Studies at lab scale ture of the active form of CALB. Therefore, solid phase chemical are time consuming and labour intensive with low experimental modification of immobilized lipases may become a powerful tool throughput. The feasibility of a parallel microwell platform for in the design of lipase libraries with very different properties. two-liquid phase whole-cell bioconversions has previously been shown for longer chain alkane substrates [1]. However, excessive References evaporation has limited the use of the microscale approach for [1].Rodrigues RC, Ortiz C, Berenguer-Murcia A, Torres R, Fernández- volatile substrates. Lafuente R. Modifying enzyme activity and selectivity by immobi- This study developed a microwell system for use with highly lization. RSC 2013;42:6290–307. volatile n-alkane substrates. Particular attention was paid to mate- [2].Davis BG. Chemical modification of biocatalysts. Curr Opin Bio- rial compatibility, evaporation and oxygen transfer. It was shown technol 2003;14:379–86. that the use of 24 deep square microwell plates machined from http://dx.doi.org/10.1016/j.nbt.2014.05.1807 a fluoropolymer alleviates problems of organic phase permeation and absorption. In combination with a new sealing approach to avoid substrate evaporation, the reproducibility of results was PA-22 increased. However, a comparison with unsealed plates revealed that, despite similar cell growth the bioconversion productivity Newly engineered diol dehydrogenase from Clostridium was lower in sealed plates. The effect of sealing the plates on oxy- butyricum as promising agent in cell-free biosystems for gen transfer will be discussed. biomanufacturing ∗ Reference Marta Jankowska , Włodzimierz Grajek, Agnieszka Olejnik- Schmidt, Marcin Schmidt [1].Grant C, Pinto A, Lui H, Woodley J, Baganz F. Biotechnol Bioeng 2012;109(9):2179–89. Poznan´ Uniwersity of Life Sciences http://dx.doi.org/10.1016/j.nbt.2014.05.1806 Economical and environmentally friendly production of biochemicals and biotechnology-based polymers is a critical goal of modern biotechnology. The use of enzymes as catalysts for chem- PA-21 ical transformations has emerged as more ecological synthesis. Chemical modification of lipase B from Candida Generally, naturally occurring enzymes do not have appropriate antarctica for improving biochemical properties of features necessary for their use in chemical industry. The devel- activity, stability and selectivity opment in protein engineering allows optimization of particular enzyme traits which make them more suitable to chemical process. Rodrigo Torres Sáez 1,∗ Claudia Ortiz López 2 Oveimar Barbosa 3 , , , 1,3-Propanediol is a key chemical bulk for the synthesis Roberto Fernández-Lafuente 4 of polytrimethylene terephthalate with desired properties for 1 School of Chemistry. Universidad Industrial de Santander large volume markets. 1,3-Propanediol dehydrogenase (PDOR, 2 School of Bacteriology and Clinical Laboratory. Universidad Industrial de EC 1.1.1.202) encoded in Clostridium butyricum by dhaT gene is Santander, Colombia involved in the conversion of glycerol into 1,3-propanediol. 3 School of Chemistry. Universidad Industrial de Santander, Colombia 4 Department of Biocatalysis. ICP-CSIC, Spain The study aimed to improve enzyme activity by directed evolution and rational design methods to generate a more efficient Chemical modification of enzymes can be used to modulate 1,3-propanediol production. enzyme properties by means of modification of protein surface or Error-prone PCR, employed to re-engineer PDOR resulted in key residues from the enzyme structure [1,2]. In this work, it was enzyme variants with higher enzymatic activity. Variants with carried out chemical modifications of Candida antarctica lipase B highest activity showed twice higher oxidative activity and twelve (CALB) preparations immobilized on octyl-agarose, BrCN-agarose times higher reductive activity, respectively. Subsequently three- and Eupergit-C supports using different chemical compounds, dimensional structure of PDOR was predicted by homology e.g. ethylenediamine (EDA), succinic anhydride (SA) and 2,4,6- modeling with Lactaldehyde reductase from Escherichia coli as tem- trinitrobenzensulfonic acid (TNBS). These modifications of the plate. Location of the mutations, important for activity and their enzyme surface caused changes in physical properties such as potential impact on the structure and function of the enzyme were charge (isoelectric point) or hydrophobicity (solubility), and specified. proved to be practical methods to enhance the biocatalyst per- Results of study indicate mutations that might influence the formance (stability, activity and enantio-selectivity) when the effectiveness of cofactor binding to protein surface as well as on enzyme preparations were submitted to different ranges of pH (4-9) the enzymatic activity of PDOR. and temperature (25-70 ◦C) and organic co-solvents such as ace- This work was part of POIG 01.01.02-00-074/09 project co- tonitrile or tetrahydrofuran at 50% (v/v). These immobilized and funded by The European Union through The European Regional chemically modified CALB preparations displayed high enantiose- Development Fund within the framework of the Innovative Econ- lectivity during the kinetic resolution of (R/S)-methyl mandelate omy Operational Programme 2007-2013. in aqueous solution and esterification of beta-blocker drugs such as http://dx.doi.org/10.1016/j.nbt.2014.05.1808

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PA-23 are being studied with an aim to optimise biotransformation processes, and to observe the effect of increased biofilm production Sequence and function relationship of Escherichia coli on biotransformations. flavin mononucleotide binding fluorescent protein References Byung-Kwan Cho ∗ , Hyeonseok Shin [1].Tsoligkas AN, Winn M, Bowen J, Overton TW, Simmons MJH, Korea Advanced Institute of Science and Technology Goss RJM. Engineering Biofilms for Biocatalysis. ChemBioChem 2011;12:1391–5. [2].Tsoligkas AN, Bowen J, Winn M, Goss RJM, Overton TW, Simmons Flavin mononucleotide (FMN)-binding fluorescent proteins MJH. Characterisation of spin coated engineered Escherichia coli can provide in vivo reporter system, without oxygen. Here, biofilms using atomic force microscopy. Colloids and Surfaces B: Bioin- we present the functional landscape of individual amino acid terfaces 2012;89:152–60. sequence of Escherichia coli FMN-binding fluorescent protein (EcF- [3].Perni S, Hackett L, Goss R, Simmons M, Overton T. Optimisation bFP). We used random mutagenesis to generate the mutant of engineered Escherichia coli biofilms for enzymatic biosynthesis of l-halotryptophans. AMB Express 2013;3:66. libraries, which were screened by function loss or retained. The function and sequence relationship of the mutants were analyzed http://dx.doi.org/10.1016/j.nbt.2014.05.1810 in single high-throughput sequencing, resulting in 329 tolerant mutations and 259 sensitive mutations that show retained fluorescence or loss of fluorescence respectively. In addition, the PA-25 enrichment of tolerant or sensitive mutations in each amino acid residues were weighed to find functionally important residues n Library sequencing strategies for comparative analysis EcFbFP. The mutation enrichment analysis show that the positions of stress resistance mechanisms in Escherichia coli strains critical to the function of EcFbFP lies among the FMN binding Rebecca Lennen ∗ , Ida Bonde, Anna Koza, Markus Herrgård pocket, turns and loops of the protein where dynamic confor- Novo Nordisk Foundation Center for Biosustainability, Technical University of mational changes occur and the Glu56-Lys97 salt bridge which Denmark is critical to structural stability of EcFbFP. Collectively, the mutational scanning results provide a functional landscape of each Transposon insertion sequencing (Tn-Seq) has recently amino acid of EcFbFP. emerged as a powerful next-generation sequencing method This work was supported by Intelligent Synthetic Biology Cen- that enables querying the contributions of all genes in a bacte- ter of Global Frontier Project (2011-0031957, 2011-0031962). rial genome toward the fitness of a growing organism. In this http://dx.doi.org/10.1016/j.nbt.2014.05.1809 method, transposon insertion mutant libraries are constructed and subjected to growth selections. Following selection, the locations of all insertions in the population are counted and can PA-24 be compared between a control and a target condition, enabling the identification of genes that are both conditionally essential Analysis and Optimisation of the Physiology of Engi- and conditionally detrimental. We have exploited Tn-Seq to neered Biofilms for Biotransformations probe the basis for the large variations in osmotic and acetate James Thomas Leech 1,∗ , Isaac Vizcaino-Caston 1, Tania Barberi 2, stress tolerance of different laboratory strains of Escherichia coli Rebecca Goss 2, Mark Simmons 1, Tim Overton 1 (K-12 MG1655, BL21(DE3), W, and Crooks). Little is currently known to explain the source of this variation and to enable 1 University of Birmingham 2 University of St Andrews rational engineering to impart stress tolerance. Tn-Seq revealed many differences and similarities in resistance mechanisms at the genetic level across strains, allowing correlations to be made with Engineered biofilms formed from reaction-competent recombi- growth phenotypes. Cross-strain comparisons of conditionally nant Escherichia coli provide a useful platform for biotransforma- essential genes and their relative essentiality also suggest a large tion reactions [1,2]. Biofilms form in response to chemical and degree of variation in metabolic flux distributions and regulation physical stresses such as organic solvents and shear, and thus of gene expression between strains. A number of direct targets are able to resist conditions encountered in flow reactor bio- for metabolic engineering of stress resistance via loss-of-function transformation reactions far better than planktonic cultures [3]. mutations were also discovered, and we show that deletion of a Improving attachment and maturation of engineered biofilms is selection of these genes results in improved growth under the paramount to the optimisation of their use in biotransformations. original selection condition. Through the use of fluorescent reporter genes, flow cytometry and confocal laser scanning microscopy, the physiology and compo- http://dx.doi.org/10.1016/j.nbt.2014.05.1811 sition of the biofilm has been studied, in addition to the spatial and temporal expression of biofilm constituents such as curli, poly-N-acetlyglucosamine (PGA/PNAG) and colanic acid. Molec- ular biology techniques have been used to bypass normal biofilm signals, creating increased biofilm formation. Furthermore, the physiological effects of biotransformation reactions on the biofilm

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PA-26 enzymes were investigated and the biocatalytic method for the stereoselective synthesis of a-quaternary a-amino acids was devel- Microorganisms respond in different ways to oscillations oped. in large-scale bioreactors: Conclusions from scale-down Acknowledgements: The research leading to these results approaches has received funding from the Innovative Medicines Initiative ◦ Peter Neubauer 1,∗ , Anja Lemoine 2, Sergej Trippel 2, Eva Brand 2, Joint Undertaking under grant agreement n 115360, resources of Robert Spann 2, Dennis Runge 2, Ping Lu 2, Basant El Kady 2, Chris- which are composed of financial contribution from the Euro- tian Reitz 2, Stefan Junne 2 pean Union’s Seventh Framework Programme (FP7/2007-2013) and EFPIA companies’ in kind contribution. 1 TU Berlin, Chair of Bioprocess Engineering 2 TU Berlin References [1].(a) Steinreiber J, Fesko K, Reisinger C, Schurmann M, van Assema F, Gradients of various growth related parameters are observed Wolberg M, Mink D, Griengl H. Tetrahedron 2007;63:918–26; in large scale bioreactors by the limited mass transfer and are (b) Steinreiber J, Fesko K, Mayer C, Reisinger C, Schürmann M, enhanced by the application of the substrate limited fed-batch Griengl H. Tetrahedron 2007;63:8088–93. [2].Fesko K, Uhl M, Steinreiber J, Gruber K, Griengl H. Angew Chem technology. Consequently, cells are exposed to oscillations, which 2010;122:125–8. Angew. Chem. Int. Ed. 2010, 49, 121–124. cause a specific physiological adaptation. Our studies with different scale-down approaches indicate that http://dx.doi.org/10.1016/j.nbt.2014.05.1813 microorganisms adopt with different strategies to oscillations, which is closely connected to their response to oxygen limitation. While Escherichia coli, similar to Saccharomyces cerevisiae reacts with PA-28 increased glycolytic fluxes, Bacillus subtilisdecreases the maximum The effect of salt-preconditioning Torulaspora del- glucose uptake capacity. In contrast, Corynebacterium glutamicum brueckii cells on fermentation performance shows a high robustness to oscillations. In all cases, the oscillations influenced the pools of various Stilianos Logothetis 1,∗ , Fotini Drosou 1, Arhodoula Hatzilazarou 1, amino acids, which are closely connected to the central carbon Panagiotis Tataridis 1, Anastasios Kannelis 2, Elias Nerantzis 1, metabolism. When we connected the two-compartment scale- Graeme Walker 2 down bioreactor with the rapid sampling unit Bioscope to use 1 TEI of Athens Department of Enology and Spirit Technology 13 C-labelling for a study of the metabolic fluxes in E. coli,we 2 Abertay University of Dundee observed that the preferred route towards the synthesis of branch- chain amino acids after a pulse depends on the history of growth Abstract This paper concerns research into the influence of conditions. salt on physiology of the yeast, Torulaspora delbrouekii. Specifically, The presented methodology, which also includes other novel the work focused on how NaCl affected the growth, viability and analytical instruments, provides a bridge between systems biol- fermentation performance of this yeast in laboratory-scale experi- ogy for the investigation of the cellular regulatory networks under ments. One of the main findings of the research presented involved industrially relevant conditions. the influence of salt “preconditioning” of yeasts which repre- http://dx.doi.org/10.1016/j.nbt.2014.05.1812 sents a method of pre-culturing cells in the presence of salt in an attempt to improve subsequent fermentation performance. Such an approach resulted in preconditioned T. delbruekii yeasts having PA-27 an improved capability to ferment high-sugar containing media (up to 30% w/v of glucose) with increased cell viability and with Exploring the new threonine aldolases with broad donor elevated levels of produced ethanol. Salt-preconditioning most specificity likely influenced the stress-tolerance of yeasts by inducing the ∗ synthesis of key metabolites such as trehalose and glycerol which Kateryna Lypetska (Fesko) , Gernot Strohmeier, Rolf Breinbauer act to improve cells’ ability to withstand osmostress and ethanol Graz University of Technology toxicity. Overall, this research has demonstrated that a relatively simple method designed to physiologically adapt yeast cells–by Threonine aldolases have great biotechnological potential, as salt-preconditioning–can have distinct advantages for alcohol fer- they catalyze the formation of unnatural amino acids with high mentation processes. enantioselectivity. The enzymes have been efficiently applied for http://dx.doi.org/10.1016/j.nbt.2014.05.1814 the aldol condensation of an aldehyde and glycine to produce L- and D-␤-hydroxy-␣-amino acids.[1] Aldolases are tolerant towards acceptor aldehyde, but they are quite rigid for the amino acid donor. In our previous work we have isolated two natural threonine aldolases, which were able to accept alanine and serine as donor.[2] Here we present the identification and characterization of a range of L- and D-threonine aldolases with broad donor specificity. The kinetic properties and the substrate specificity of new

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PA-29 The two isolated Thermobifida cutinases are very similar but had different kinetic parameters on soluble substrates. We determined Protein engineering of arylmalonate decarboxylase vari- the structure of both enzymes. Structural analysis revealed surface ants with promiscuous racemising activity regions of Thc Cut1 and Thc Cut2, which differ in electrostatic Robert Kourist ∗ , Sarah Gaßmeyer, Nadine Hülsemann, Robin Dorau and in hydrophobic properties. Modeling studies suggest that these regions interact with PET during turnover which may explain Ruhr-Universität Bochum the differences in hydrolysis efficiencies which were observed for the two enzymes. Enzymatic racemization allows the smooth interconversion of stereocenters under very mild reaction conditions. Racemases find http://dx.doi.org/10.1016/j.nbt.2014.05.1816 frequent applications in deracemization and dynamic kinetic reso- lutions [1]. Arylmalonate decarboxylase (AMDase) from Alcaligenes bronchosepticus [2] has high structural similarity to cofactor-free PA-31 amino acid racemases. The racemase-like catalytic machinery of Characterisation of a recombinant patchoulol syn- mutant G74 C conveys it a unique activity in the racemisation thase for the biocatalytic production of high valuable of pharmacologically relevant derivates of 2-phenylpropionic acid sesquiterpenes (profenes), which makes AMDase G74 C an interesting object for the mechanistic investigation of cofactor-independent racemases. Thore Frister 1,∗ , Steffen Hartwig 2, Katharina Schnatz 2, Thomas While the racemase has high activity towards small ary- Scheper 2, Sascha Beutel 2 laliphatic acids, larger substrates such as ibuprofen are converted 1 Leibniz University Hannover, Institute of Technical Chemistry much slower. Objective of this study is the extension of the sub- 2 Leibniz University Hannover strate range by rational design, either by creating space in the binding pocket or by stabilizing the substrate by the introduction Sesquiterpenes are a structurally diverse class of secondary of hydrophobic interactions. metabolites, which are mainly produced by various plants. Due to their special odor sesquiterpenes are widely used as fragrance References compounds in exclusive perfumes, scented household goods and [1].Felfer U, Goriup M, Koegl ME, et al. Adv Synth Catal 2005:951–61. as naturally flavor additives in food. These days the majority of [2].Kourist R, Miyauchi Y, Uemura D, Miyamoto K. Chem Eur J sesquiterpene production is still based on isolation techniques 2011:557–63. such as solvent extraction and steam distillation. Recent biotech- http://dx.doi.org/10.1016/j.nbt.2014.05.1815 nological approaches for the production of sesquiterpenes require time and cost-extensive pathway engineering. We hereby present a biocatalytic method for the production PA-30 of high valuable sesquiterpenes such as patchoulol and germa- crene A by converting synthetic farnesyl diphosphate (FPP) with Enzymatic Hydrolysis of PET: Structural Diversity and a recombinant patchoulol synthase (PTS). [1] For the production Kinetic Properties of Cutinases from Thermobifida of the PTS we have established a bioprocess combined with a reli-

,∗ able purification strategy. The product spectrum, substrate scope Altijana Hromic 1 , Doris Ribitsch 2, Andrzej Lyskowski 1, Georg and important parameters concerning the kinetic properties and Steinkellner 1, Helmut Schwab 3, Georg Gübitz 2, Karl Gruber 1 enzyme stability have been studied. FPP is produced in a short 1 ACIB GmbH c.o. IMB Graz and fast synthesis starting from chemically derived, cheap farnesol 2 ACIB GmbH using an optimized method according to Keller et al. [2] The bio- 3 ACIB GmbH c.o. Institute of Molecular Biotechnology, Graz University of Tech- nology conversion of FPP was performed in different multi-phase model reactor in order to increase sesquiterpene yield and maintain an Poly(ethyleneterephthalate) (PET) is one of the most widely easy product recovery. used polymers worldwide. Its use ranges from different medi- References cal and thermoforming applications to fibers for textiles. It is [1].Deguerry F, Pastore L, Wu S, Clark A, Chappell J, Schalk M. Archives highly hydrophobic which makes this polymer difficult to be func- of Biochemistry and Biophysics 2006;454:123–36. tionalized. Therefore, industrial applications often involve surface [2].Keller R, Thompson R. Journal of Chromatography A 1993;645:161–7. activation prior to final treatment. http://dx.doi.org/10.1016/j.nbt.2014.05.1817 Plasma or aggressive chemistry methods are energy consuming or environmentally harmful and in some cases they lead to a decrease in polymer weight or strength. The exchange of unpleasant chemical by enzymatic processes would avoid these problems. Cutinases from Thermobifida cellulosilytica DSM44535 (Thc Cut1 and Thc Cut2) hydrolyze PET. Their ability to hydrolyze the polymer was compared with other enzymes hydrolyzing natural polyesters including the PHA depolymerase from Pseudomonas fluorescens and two other cutinases from Thermobifida fusca KW3.

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PA-32 activity. Streptomyces roseochromogenes could be used as microbial whole cell catalyser for the biotechnological transformation of Nuclear Magnetic Resonance Spectroscopy: An Alter- hydrocortisone to 16a-hydroxy hydrocortisone by using fermen- native Fast Tool for Quantitative Analysis of the tation technologies. Previous studies demonstrated the possibility Solvent-free Ethanolysis of Coconut Oil Using Fungal to obtain 0.508 ± 0.01 g·L−1 of 16␣-OH-HC in 2-L pulsed batch Resting Cells fermentations at 30 ◦C and pH 7, by using a malt extract- and Ramon Canela Garayoa 1,∗ , Edinson Yara-Varón 2, Mercè Balcells 2, yeast extract-based medium. In this study the influence on the Mercè Torres 2, Jordi Eras 2 conversion ratio and the by-product formation of growing conditions, like pH and temperature, was investigated in both shake 1 The University of Lleida-DBA Center flask experiments and 2-L batch fermentations. Once determined 2 The University of Lleida the optimal conditions for both bacterial growth and steroid conversion,different feeding strategies and substrate addition profiles Ethyl fatty esters (EFE) from coconut oil have broad applications were exploredin pulsed batch fermentations, and a 16␣-OH-HC in the flavouring and fragrance industries. A solvent-free synthesis maximum production of 0.630 ± 0.02 g·L−1 was reached. Fed- of EFE using fungal resting cells was conducted. A reliable and fast batch experiments, carried out scaling-up the process in a 15-L analytical method was needed to optimize the biocatalytic process. fermentor, allowed to reach a maximum of 0.804 ± 0.05 g·L−1 The method had to allow the quantification of the starting material of 16␣-OH-HC, while further investigations will be performed to (TAG), the various intermediates (monoacylglycerols–MAG- and drive the process on industrial scales. diacylglycerols–DAG-) and the EFE. The analysis of acylglycerols and EFE has been carried out by http://dx.doi.org/10.1016/j.nbt.2014.05.1819 employing analytical techniques such as high performance liquid chromatography (HPLC), and gas–liquid chromatography (GLC). However, these methods are time-consuming. Recently, nuclear PA-34 magnetic resonance spectroscopy (NMR) has been proposed in the preparation of biodiesel and DAG as an alternative analytical tool Immobilization of cells and enzymes to PVA gel [1,2]. The usefulness of NMR has been increasingly recognized for Martin Rebros 1,∗ , Michal Rosenberg 1, Radek Stloukal 2 its non-invasiveness and rapidity to detect a wide range of com- 1 Slovak University of Technology pounds that can be detected in a single measurement (spectrum), 2 LentiKats whereas little or no need for sample pre-treatment is required. In the present study, a single NMR experiment was used as a fast Compared to conventional free cell and enzyme processes and effective method for studying the progress of the biocatalytic immobilized one offers several important advantages such as: the synthesis of EFE. A new algorithm was developed to determine the possibility of repetitive use of immobilized cells and enzymes, the contents of TAG, DAG, MAG, EFE and free fatty acids in the crude improvement of enzyme stability, the reduction of non-productive samples resulting from various experimental conditions. Results cell growth phases, faster reaction rates at increased cell density were in accordance with those resulting from GLC. and higher yields. A veryeffective and useful matrix for entrapment immobilization is polyvinyl alcohol (PVA). The gelation of PVA References hydrogel is based on partial drying at room temperature and there- [1].Hatzakis E, Agiomyrgianaki A, Kostidis S, Dais P. J Am Oil Chem Soc fore is very gentle to both types of biocatalysts. Due to cell growth 2011;88:1695–708. within the immobilizates the volumetric productivities may grad- [2].Prakash P, Aulakh SS. J Basic Microbiol 2011;51:607–13. ually increase and reached higher values compared to free cell http://dx.doi.org/10.1016/j.nbt.2014.05.1818 processes. By immobilization enzymes improved their stability. These phenomena were successfully applied to various whole-cell and enzyme biocatalysts. PA-33 Acknowledgement: This work was done during implementation of the project Development of Competence center for Enhancing hydrocortisone transformation to research and development in molecular medicine, ITMS code 16a-hydroxy hydrocortisone by Streptomyces roseochro- 26240220071, supported by the Research and Development Oper- mogenes ational Program funded by ERDF. Paola Diana ∗ , Odile Francesca Restaino, Mariacarmela Marseglia, http://dx.doi.org/10.1016/j.nbt.2014.05.1820 Maria Giovanna Borzacchiello, Chiara Schiraldi Second University of Naples

In the last years the biotechnological production of steroids by microbial transformations has became a common practice and a reliable method to produce structural tailored-cut molecules also in large scales. Hydrocortisone is a pharmaceutical active molecule generally used as drug; its hydrolylated forms, like 16a-hydroxy hydrocortisone, have higher and more efﬁcient anti-inﬂammatory

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PA-35 bility, the folding of the expressed protein took place also in the periplasm in addition to cytosolic protein production by cloning Screening of Lipase Production Among Different cut16606 gene downstream of the pelB signal peptide. The het- о Microorganisms erologous expression was induced with IPTG in 16 C for 20 h Duygu Elif Yılmaz 1,∗ , H. Tansel Yalçın2, Nihat Alpagu Sayar 1 of incubation. The recombinant cutinase was purified from the cytosol or culture supernatant using immobilized-metal affinity 1 Marmara University chromatography (IMAC), resulting in a monomeric protein of ca. 2 Ege University 23 kDa that is optimally active at 40 ◦C. FoCut5a cutinase was tested for its potential use in sur- The design and manifecture of single enantiomers of chiral face modification of PET fabrics, with the intention to increase intermediates have been attracting attention in the pharmaceu- their hydrophilicity and to improve their properties. Towards tical industry during the last decades. Single enantiomers that this direction, the enzymatic hydrolysis of two model sub- are potential biocatalysts due to their higher degree of enantio- strates bis(benzoyloxyethyl)terephthalate (1) and commercial selectivity and regioselectivity are usually prepared by chemical bis(2-hydroxyethyl)terephthalate (2) was attempted, in order to catalysis. evaluate the capability of the recombinant cutinase in PET modifi- L-amino acid esters are promising intermediates for various cation. FoCut5a succeeded in hydrolyzing both models, releasing active drug components and their catalytic biosythesis through bis(2-hydroxyethyl)terephthalate and benzoic acid from substrate transesterification reactions could be a rational alternative way. 1 and a bis(2-hydroxyethyl)terephthalate derivative in case of sub- Besides, they can be effectively producted by using lipase from strate 2. Experiments are in progress for the surface modification various microorganisms as a biocatalyst. The selection of the most and functionalization of PET fibers. appropriate organism among different strains can be a valuable starting point for this study. Various isolates from various potantial http://dx.doi.org/10.1016/j.nbt.2014.05.1822 microorganisms which differ significantly from each other, may exhibit different catalytic effectiveness. The microbial isolates for this study are maintained by monthly PA-37 transfers on MGYP agar slants and stored at + 5◦C. For the production stage, microorgansims are transferred to MGYP preculture Synthesis of biological active compounds using carbohy- medium. After overnight incubation, this culture is inoculated to drate esterases as biocatalysts production medium including different types of oil. The culture is Paul Christakopoulos 1,∗ , Io Antonopoulou 1, Evangelos Topakas 2 incubated in appropriate conditions. Samples are taken at different 1 Luleå University of Technology time intervals to determine the growth and lipase activity. 2 National Technical University of Athens The enzyme activity is shown by p-nitrophenyl palmitate (pNPP) as substrate. P-nitrophenol is obtained from pNPP and Various fungal and bacterial carbohydrate esterases represent absorbance is measured spectrophotometrically against an enzyme appealing biocatalysts that have the ability not only to deconstruct free sample. One unit of lipase activity (U) is defined as the amount plant biomass but also to modify compounds with a potential use ␮ of enzyme which liberates 1 mol p-nitrophenol/min under assay in food, cosmetic and pharmaceutical industries. Feruloyl esterases conditions. Protein is measured by the Coomassie Blue G-250 bind- (FAEs, E.C. 3.1.1.73) have been proved promising candidates for ing method using bovine serum albümin as standard. the enzymatic synthesis of antioxidants allowing more flexible http://dx.doi.org/10.1016/j.nbt.2014.05.1821 process configurations. Among the advantages they provide are use of lower temperatures (50-60 ◦C) comparing to the coun- о terpart chemical process (150 C), one step production of one PA-36 product instead of mixtures and no need of by-product and catalyst residues removal in order to produce clean and high quality A cutinase from Fusarium oxysporum with potential for substances. Glucuronoyl esterase (GE) synthetic ability needs to be PET surface modification explored towards the production of alkyl branched glucuronic acid Evangelos Topakas ∗ , Efstratios Nikolaivits, Maria Kanelli, Paul derivatives which are non-ionic surfactants and have good surface Christakopoulos properties, including biodegradability. In addition, due to their tastelessness, non skin-irritation and non toxicity, these bioactive National Technical University of Athens compounds find diverse uses in the cosmetic and pharmaceutical industries. Cutinases are small extracellular serine hydrolases whose natu- Aim of this work is the development of competitive and ral function is the hydrolysis of the polyester cutin. Their ability to eco-friendly bioconversions based on transesterification reactions hydrolyze a wide range of substrates (from low molecular weight catalyzed by FAEs and GEs, for the production of molecules esters to high molecular weight polymers) makes cutinases very with antioxidant activity, such as phenolic fatty and sugar esters. useful biocatalysts for various applications. In the present study, The synthesis of four biological active compounds (prenyl fer- a cutinase (FoCut5a) from the ascomycete fungus Fusarium oxys- ulate, prenyl caffeate, 5-O-(trans-feruloyl)-arabinofuranose, and porum was functionally overexpressed in Escherichia coli BL21, glyceryl ferulate) was evaluated using recombinant FAEs from harboring pET22b(+)-cut16606. In order to improve enzyme sta- Myceliopthora thermophila and Fusarium oxysporum, while the

S90 www.elsevier.com/locate/nbt New Biotechnology · Volume 31S · July 2014 BIOCATALYSIS synthesis of benzyl D-glucuronate and prenyl-D-glucuronate trial process. Compared to chemical catalysts, the limited rate was evaluated using recombinant GEs from M. thermophila. and low throughputs are amongst the challenges which need All reactions were carried out in ternary systems of n- to be addressed for broader application of biocatalysis. And also hexane/alcohol/water forming surfactantless microemulsions. biocatalyst and process development require expensive and time http://dx.doi.org/10.1016/j.nbt.2014.05.1823 consuming experimentation. Mathematical modeling and simulation tools will be used as solution for process development duration problems. The use of PA-38 model based techniques will facilitate the reduction of unnecessary experimentation accelerate optimisation and automation of Biodegradation of esfenvalerate by bacteria from Brazil- processes, resulting in a reduction in cost and time. ian biome mangrove Enzyme-catalyzed esterification has gained increasing attention in many applications, due to the significance of the derived prod- André Luiz Meleiro Porto 1,∗ , Willian G. Birolli 2, Eloá B. Meira 2, ucts. More specifically, the lipase-catalyzed esterification reactions Nitschke Marcia 2, Luciene P.C.Romão 3 have attracted research interest during recent years, due to an 1 Institute of Chemistry of São Carlos - University of São Paulo increased use of organic esters in biotechnology and the chemical 2 Instituto de Química de São Carlos, Universidade de São Paulo industry. 3 Departamento de Química, Universidade Federal de Sergipe Selective substrate and desired product have also significant value for scientific field. The final products may be new molecules Esfenvalerate is a widely used pyrethroid insecticide. This with different characteristics and properties. work aimed the biodegradation of the commercial formulation We aim to focus on kinetic and mathematical modeling of a of esfenvalerate (EsfCom) by environmental bacteria isolate from lipase catalyzed esterification reaction as a model system. Esterifi- Brazilian biome mangrove, especially from turfa soil. The turfa cation will be carried out with various substrates such as different soil is a material of plant origin, partially decomposed, found amino acids and carbohydrates. in layers, usually in swampy areas and mountains. The four The kinetic model will be developed using Matlab program- bacterial strains (P5CBNB, P5MNB, P8CNB, R5RaNB) assessed ming environment with extra possible use of Mathematica in this work were able to grow in a solid and liquid culture software. Depending on the type of process designed for the ester- medium (nutrient agar) in presence of esfenvalerate (100 mg.L−1). ification reaction a process model will be developed using the The insecticide and its main metabolites [3-phenoxybenzoic acid kinetic model as a basis. (PBAc), 3-phenoxybenzaldehyde (PBAld), and 2-(4-chlorophenyl)- 3-methylbutyric acid (ClAc)] were quantified. It was observed that http://dx.doi.org/10.1016/j.nbt.2014.05.1825 all the evaluated strains promoted the esfenvalerate biodegradation after 14 days.The residual esfenvalerate ranged between 103.0 mg.L−1 (similar to method recovery) and 41.6 mg.L−1, while PA-40 the PBAc formation was 8.1-1.2 mg.L−1, ClAc was 10.6-0.0 mg.L−1. Gluconobacter oxydans used to production of natu- PBAld was not detected in any of the biodegradation experiments. ral aroma - 2-phenylacetic acid in immobilized system The EsfCom biodegradation promoted the accumulation of PBAc (LentiKats form) and ClAc, which are considered toxic compounds. The formed PBAld was completely transformated by strains and it was the main Monika Vidová 1,∗ , Ivana Slezáková 2, Martin Rebrosˇ 2, L’udmila metabolite in the abiotic degradation. It was observed that some Kristofíkováˇ 2, Michal Rosenberg 2 strains were efficient in esfenvalerate biodegradation and might be 1 Institute of Biotechnology and Food Science, Slovak University of Technology used in bioremediation. A biodegradation pathway for esfenvaler- 2 Institute of Biotechnology and Food Science, Faculty of Chemical and Food ate by bacteria isolated from turfa soil was proposed. Technology, Slovak University of Technology Acknowledgements: FAPESP, CNPq, CAPES, USP-SGA http://dx.doi.org/10.1016/j.nbt.2014.05.1824 System of generation of natural aromas using G. oxydans in interesting in many cases. Acetic acid bacteria G. oxydans specifically oxidates in unresting condition 2-phenylethanol PEA into PA-39 phenylacetic acid PA on membrane surface, so product is easily remove to reaction mixture which significantly facilitates the Lipase Catalyzed Esterification Reactions–A Kinetic separation. Both PEA and PA have aromatic properties and in Model biotransformation step with whole cells biocatalysts change kind of flavor (from rose to honey like) and physical properties also. Ceyda Kula ∗ , Nihat Alpagu Sayar For highlighting is the fact that biomass in fermentation step is Marmara University Bioengineering Department obtaied by using renewalbe source, cheap waste product - glycerol. Immobilization of G. oxydans into biocompatible polyvinyl Biocatalysis has attracted important interest in chemistry alcohol gel in form of LentiKats may increase the benefits of prepar- and engineering fields. Substrate specificity, enantioselectivity, ing natural flavorings repeated using of biocatalysts cells. The regiospecificity, chemoselectivity and soft reaction conditions are work presents comparison of repeated bioconversion with free and important advantages for enzyme-based synthesis in an indus- immobilized cells, benefits of immobilized system and measured

www.elsevier.com/locate/nbt S91 BIOCATALYSIS New Biotechnology · Volume 31S · July 2014 technologically important factors - the effect of pH, oxygen, differ- pretreatment of lignocellulosic waste and hydrolysis. In this con- ent concentrations of the substrate and also scale-up experiments text, hydrolysis must be successful, inexpensive, and have low in controlled fermentation unit. energy requirements with lower fermentation time and less by- Keywords: G. oxydans, 2-phenylethanol, 2-phenylacetic acid, products. In hydrolysis, it is very difficult to obtain higher yield immobilization, LentiKats, glycerol of fermentable sugars due to lignin. Sugars obtained with enzy- Acknowledgment: This study was supported by the grant matic hydrolysis can be fermented easily because of the relative of Agency for Research and Development of the Slovak Republic absence of by-products and avoidance of undesirable operating ITMS APVV-0302-10 and by the grant of VEGA - Scientific Grant conditions. Pycnoporus sanguineus is a white rot fungus with high Agency of the Ministry of Education, Science, Research and Sport lignocellulolytic potential. of the Slovak Republic VEGA 1/0229/12. Aim: Production of enzyme mixture (laccase and cellulase) http://dx.doi.org/10.1016/j.nbt.2014.05.1826 from P. sanguineus DMSZ 3024 by the hydrolysis of hazelnut husk is aimed in this study. Methods: Both laccases that catalyze the oxidation of lignin PA-41 related compounds and cellulases that hydrolyse cellulose to obtain smaller ␤-glucose oligomers and ␤-D-glucose were produced Enzyme mixture production from Pycnoporus sanguin- from P. sanguineus by the hydrolysis of hazelnut husk. Enzyme eus DMSZ 3024 using a lignocellulosic waste, Hazelnut mixture and hazelnut husk concentration for hydrolysis condi- husk: A case study for laccase and cellulase tions were optimized at different ratios and reaction times. All enzyme activities were measured. Sugar and total sugar content Orkun Pinar 1,∗ , Bas¸akKoç han 2, Nihat Alpagu Sayar 2, Kübra Karaos- were determined by the phenol-sulfuric acid and DNS method. manoglu˘ 2, Dilek Kazan 2 Results and Conclusion: An appropriate method for hydrol- 1 Marmara University, Bioengineering Department ysis of hazelnut husk with enzyme mixture using, P. sanguineus was 2 Marmara University obtained to overcome hydrolysis problems. Acknowledgement: This work was supported by the Turkish Introduction: Hazelnut husk is a promising important lig- Scientific and Technical Research Council (113M053). nocellulosic material for fermentable sugar production. According http://dx.doi.org/10.1016/j.nbt.2014.05.1827 to Fiskobirlik, 774,000 to 1,032,000 tons of hazelnut husk are left in fields as waste or incinerated. In different methods, cellulose and hemicellulose are converted into fermentable sugars by the

S92 www.elsevier.com/locate/nbt New Biotechnology · Volume 31S · July 2014 BIOFUELS, BIOCHEMICALS AND BIOENERGY

Biofuels, biochemicals and bioenergy is to obtain a variety with high productivity and resistant to low temperatures specific to continental winters as can occur in Roma- PB-01 nia. This variety may be seeded as autumn culture under minimal tilling conditions. Minimization of Bacterial Contamination with High As starting material have been used a local camelina cultivar Solid Loading during Ethanol Production from Lignocel- resistant to low temperatures and an international variety GP202 lulosic Materials with high productivity and oil content. It has been taken into Mofoluwake Ishola 1,∗ , Tomas Brandberg 2, Mohammad account classical approach, the use of immature embryo rescue Taherzadeh 2 technique. The first hybrid generation was obtained by castra- tion and pollination; the immature embryos were cultivated in 1 Swedish Centre for Resource Recovery, University of Borås MS medium w/o hormones; new plantlets were cultivated till full 2 University of Borås maturity under greenhouses conditions. The obtained seeds have been used in open field for other randomized hybridization. Abstract Ethanol is the most important renewable fuel in the The new hybrid registered the following characteristics: the transportation sector. Its production from lignocellulosic mate- plant lengths and the branching level are non-significantly dif- rials, commonly referred to as second generation ethanol, is ferent compared to the parental lines (ANOVA tests); in terms of considered more attractive than production from starch and sugar productivity, GP202 variety kept its top position, while the new crops. Bacterial contamination by lactic acid-producing bacteria is hybrid has proven top position for oil content (2.13% higher in still a major problem during ethanol production processes. Bacteria total fat). Regarding the winter resistance, the hybrid has registered compete with the yeast by consuming the sugars and the nutrients 10% more survived plants in open land. As a remark, during the required by the yeast for efficient ethanol production. This often experiments GP202 showed the highest sensibility to the mildew causes substantial economic losses at industrial fermentations. attack. The further aim of the work is to fix the new characters in In this study, without any sterilization of the substrate, simul- order to homologate the new variety. taneous saccharification and fermentation (SSF) was performed using cellulase Cellic® Ctec2 enzyme for hydrolysis and Baker’s http://dx.doi.org/10.1016/j.nbt.2014.05.1829 yeast, Saccharomyces cerevisiae, was used as the fermenting organism with different loads of suspended solids - 8%, 10% and 12%. With 8% and 10% SS, there was a significant contamination, which PB-03 caused consumption of both hexoses pentose sugars in the fer- Advanced Biomass Value: Microalgae biomass as a new mentation medium, this resulted in lactic acid concentrations of source of sustainable aviation biofuels and lubricant 43 g/L and 36 g/L from 10% SS and 8% SS respectively. In con- production trast, only 2.9 g/L lactic acid was observed with 12% SS. An ethanol concentration of 47 g/L was produced from high solid loading of Felix Bracharz ∗ , Jan Lorenzen, Farah Qoura 12% SS while just 26 g/L and 23 g/L were produced from 10% and TUM, Industrial Biocatalysis 8% SS respectively. Our results show that SSF with 12% SS has an increased concentration of inhibitors, particularly acetic acid The aviation industry grows 5% p.a. and by 2020 has to comply which selectively inhibited the bacterial growth without affecting with strict governmental emission standards that mandate a 20% the metabolic activities of the yeast during the fermentation CO2 reduction compared to the emission levels measured at 2005. Keywords Bacterial contamination; Lignocellulosic ethanol; Together with the eminent end of fossil resources these drivers Saccharomyces cerevisiae, lactic acid. force the development of sustainable aviation fuel alternatives http://dx.doi.org/10.1016/j.nbt.2014.05.1828 that are carbon neutral and in compliance fuel standard regulations such as Jet A. In this study a new, mass-and energy efficient algae biorefinery concept for the integrated production of aviation

PB-02 fuels, industrial lubricants and CO2 adsorbing building materials has been developed. The process chain is based on production of Breeding low temperature resistant Camelina sativa for fast growing microalgae biomass containing up to 20%w/w lipids. biofuel production Subsequently, algae lipids are separated from the biomass frac- Florentina Matei 1,∗ , Florentina Sauca 2, Paul Dobre 3, Stefana tion and converted to high performance, high value lubricants Jurcoane 2 by targeted functionalisation using a cascade of optimized biocatalytic processes. The biomass residue is enzymatically hydrolysed 1 University of Agronomical Sciences and Veterinary Medicine Bucharest, Fac- ulty of Biotechnologies and used as a fermentation substrate for oleaginous yeast strains. 2 Centre of Microbial Biotechnology Biotehgen, Bucharest, Romania Since these fast growing, oleaginous yeasts can accumulate up to 3 University of Agronomical Sciences and Veterinary Medicine Bucharest, Fac- 80% w/w lipids, they are the ideal biomass base for the produc- ulty of Agriculture, Bucharest, Romania tion of drop-in aviation fuels. Conversion of wet yeast biomass is accomplished using a streamlined thermochemical process that In the European effort to produce sustainable aviation fuel our features optimized heterogenous catalysts. In this process carbon team is involved in a FP7 project (ITAKA) in breeding activities rich coke is a residue of the thermochemical biomass processing. of Camelina sativa L. as main feedstock. One of our purposes it This residue stream is used as a settling modiﬁer in the production

www.elsevier.com/locate/nbt S93 BIOFUELS, BIOCHEMICALS AND BIOENERGY New Biotechnology · Volume 31S · July 2014 of CO2 adsorbing building materials. The integrated biorefinery PB-05 process does not produce any waste streams and adds value to every process intermediate. The development of biodiesel production using lipase from mutant and selected yeast http://dx.doi.org/10.1016/j.nbt.2014.05.1830 Aree Rittiboon ∗ , Wannisa Pansuk, Marisa Jatupornpiput King Mongkot’ s Institute of Technology, Ladkrabang PB-04 The aim of our research was to select and characterise yeast Enzymatic production of biodiesel that avoids glycerol isolates from soil and other materials produced in the oil palm as byproduct, by using immobilized Rhizopus Oryzae industry. Of the strains isolated from palm kernels after com- lipase pression, strain SLP27 produced the highest lipase activity (0.28 Carlos Luna 1,∗ , Cristobal Verdugo 2, Enrique D. Sancho 3, Diego unit/ml). Strain SLP27 was mutated sequentially with ultraviolet Luna 4, Juan Calero 4, Alejandro Posadillo 5, Felipa M. Bautista 4, light for 38 seconds. The resulting strain, UV79, was mutated with Antonio A. Romero 4 ethyl methane sulfonate for 58 minutes to produce strain EM107, 1 University of Cordoba which was then mutated with gamma rays at 2 kGy to yield strain 2 Crystallographic Studies Laboratory, Andalusian Institute of Earth Sciences, GAM47. The maximum lipase activities of these strains were 0.30, CSIC 0.36 and 0.70 unit/ml, respectively. Lipase from GAM47 could be 3 Department of Microbiology, University of Cordoba used as a catalyst in the transesterification reaction for biodiesel 4 Department of Organic Chemistry, University of Cordoba production. This selected strain was identified to species level by 5 Seneca Green Catalyst S.L analysis of the D1/D2 domain of 26S ribosomal RNA sequence as Candida orthopsilosis. Immobilized Rhizopus oryzae lipase (ROL) was used as biocatalyst on different supports in the selective transesterification http://dx.doi.org/10.1016/j.nbt.2014.05.1832 reaction of sunflower oil with ethanol to generate a new second generation biodiesel. As ROL it was applied a low cost powdered enzyme preparation from Biocon®-Spain (BIOLIPASE-R), PB-06 a multipurpose additive used in food industry. In this respect, Production of 3-hydroxybutyrate from waste biomass by it was carried out a study to optimize the support used and metabolically engineered Escherichia coli the immobilization process, as well as the best pH conditions in each case. It has been also evaluated the physical adsorp- Johan Jarmander 1,∗ , Mónica Guevara 1, Mariel Perez Zabaleta 1, tion of this enzyme on a demineralised sepiolite as well as the Gustav Sjöberg 1, Jaroslav Belotserkovsky 1, Jorge Quillaguamán 2, 1 covalent immobilization of this lipase on amorphous AlPO4 sup- Gen Larsson port by using two different linkers (p-hydroxybenzaldehyde and 1 Industrial biotechnology, School of biotechnology, KTH benzylamine-terephthalic aldehyde, respectively).On the other 2 Center of biotechnology, Faculty of science and technology, San Simón Uni- ® hand, the resulting new biofuel, already patented as ECODIESEL versity is composed by a mixture of fatty acid ethyl esters and monoacylglycerols (FAEE/MG) blended in a 2/1 molar relation. This novel There is a vast interest in establishing biorefineries that rely biofuel, which present the advantage of integrating glycerol as on microbial conversion of biomass for production of fuels and monoacylglycerols (MG) into biofuels composition, exhibits sim- value-added compounds. Especially waste biomasses, such a lig- ilar physicochemical properties than the conventional biodiesel nocellulose waste from e.g. agriculture, and municipal food waste and it is produced through a process which minimizes waste are, due to their low cost, attractive substrates for production generation and maximizes efficiency. Finally, one of these immo- of biochemicals. Escherichia coli is one of the most commonly bilization processes of lipases not only was effective with vegetal used organisms for production of recombinant products since it oils, it has been even successfully carried out the transesterifica- is well documented, easily manipulated and fast growing in sim- tion reaction with animal fat from butchery wastes, using this ple media. In this work we aim to use E. coli for production of the biocatalyst covalently immobilized with p-hydroxybenzaldehyde, chiral compound 3-hydroxybutyrate from waste biomass. This was achieving a viable and functional biofuel from low quality raw done by introducing part of the pathway to poly-hydroxybutyrate materials as animal waste. from the halophilic bacterium Halomonas boliviensis into E. coli. http://dx.doi.org/10.1016/j.nbt.2014.05.1831 3-hydroxybutyrate is an industrially important compound as it can be utilized as a drug, as well as in synthesis of various co-polymers. By employing flux modelling and metabolic engineering, the objective is to identify how the internal fluxes and concentrations of metabolites and co-factors can be shifted towards an increased productivity of 3-hydroxybutyrate. This knowledge is also applied in the process design, where limiting one or several elements in the cultivation medium

S94 www.elsevier.com/locate/nbt New Biotechnology · Volume 31S · July 2014 BIOFUELS, BIOCHEMICALS AND BIOENERGY can have the desired effect of increased carbon ﬂux towards PB-08 3-hydroxybutyrate. The usage of carrot pomace as a feedstock for bioethanol http://dx.doi.org/10.1016/j.nbt.2014.05.1833 production

Ekin Demiray ∗ , Sevgi Ertugrul Karatay, Gönül Dönmez, Sedat Dönmez PB-07 Ankara University

Achievement of a biofuel-like biodiesel by regioselective Keywords: Bioethanol; Saccharomyces cerevisiae transesterification of sunflower oil with mucor miehei lipase In today’s world there is urgent need for alternative energy Juan Calero 1,∗ , Juan Calero 2, Diego Luna 2, Enrique D. Sancho 3, sources due to rapid depletion of the fossil fuels. Renewable sources Carlos Luna 2, Cristóbal Verdugo 4, Alejandro Posadillo 5, Felipa M. are good candidates instead of fossil fuels because of their environ- Bautista 2, Antonio A. Romero 2 mentally friendly and less toxic properties. It has been estimated 1 University of Cordoba that bioethanol will be the most widely used renewable source in 2 Department of Organic Chemistry, University of Córdoba the near future. The usage of agricultural wastes for bioethanol 3 Department of Microbiology, University of Córdoba production has some advantages such as lower production costs. 4 Crystallographic Studies Laboratory, Andalusian Institute of Earth Sciences, Therefore in this study we investigated the potential of carrot CSIC pomaces as a feedstock for bioethanol production by using Saccha- 5 Seneca Green Catalyst S.L romyces cerevisiae. For obtaining fermentable sugars carrot pomaces were hydrolysed in 1.5% H SO (v/v). The yeast growth, initial and In previous researches, we have developed a biofuel that avoid 2 4 consumed sugar concentrations were monitored periodically. The the production of glycerol as by-product. This biofuel is similar bioethanol concentration was determinated with gas chromotog- to the conventional biodiesel, being in the same way applicable raphy. The microbial growth media containing carrot pomace as to diesel engines. Thus, glycerol is kept as monoglyceride (MG), a carbon source and different nitrogen sources were prepared to together to two fatty acid ethyl esters (FAEE) molecules. In this increase the bioethanol production. respect, this biofuel is obtained by a partial ethanolysis of sun- It has been observed that yeast cells used 35.4 g/L sugar in flower oil with Mucor Miehei lipase as biocatalyst. the medium containing 0.5 g/L KH PO and1 g/L (NH ) SO .As Results obtained by using M Miehei lipase have shown that 2 4 4 2 4 a result the media that were prepared with carrot pomace sug- this lipase is an efficient biocatalyst in the 1,3-selective enzy- ars supported the growth of Saccharomyces cerevisiae. These results matic ethanolysis reaction of triglycerides. Thus, reactions were show that carrot pomaces are suitable feedstocks for bioethanol performed to determine the optimal conditions, such as amount production. of lipase, volume of NaOH 10 N aqueous solution, temperature and oil/ethanol molar ratio. It was always used 12 mL of sunflower oil, http://dx.doi.org/10.1016/j.nbt.2014.05.1835 in a 25 mL round bottom flask, with a conventional magnetic stirrer at 300 rpm, during 2 h. Finally, a study of reuses is also carried out. PB-09 The optimized conditions obtained were 3.5 mL of absolute Lactic acid production from lignocellulosic hydrolysates ethanol (oil/ethanol molar ratio 1/6), 37.5 ␮l of NaOH 10 N solu- ◦ under non-sterilized conditions using Bacillus coagulans tion, temperature of 30 C and 15 mg of M Miehei lipase. Operating IPE22 under these experimental conditions, eighteen successive reactions were efficiently carried out after recovering the lipases by Yinhua Wan ∗ , Yuming Zhang, Xiangrong Chen, Benkun Qi, Yi Su centrifugation. Institute of Process Engineering, Chinese Academy of Sciences

Acknowledgements A thermophilic lactic acid (LA) producer was isolated and identiﬁed as Bacillus coagulans strain IPE22. The strain showed Grants from the Spanish Ministry of Economy and Com- remarkable capability to ferment pentose, hexose and cel- petitiveness (Project ENE 2011-27017), Spanish Ministry of lobiose, and was also resistant to inhibitors from lignocellulosic Education and Science (Projects CTQ2010-18126 and CTQ2011- hydrolysates. Based on the strain’s promising features, it was used 28954-C02-02), FEDER funds and Junta de Andalucía FQM 0191, to produce lactic acid (LA) from mixed sugar and wheat straw PO8-RMN-03515 and P11-TEP-7723. hydrolysates under non-sterilized conditions. In order to eliminate http://dx.doi.org/10.1016/j.nbt.2014.05.1834 the sequential utilization of mixed sugar and feedback inhibition during batch fermentation, membrane integrated repeated batch fermentation (MIRB) was used to improve LA productivity. With MIRB, a high cell density was obtained and the simultaneous fermentation of glucose, xylose and arabinose was successfully realized. The separation of LA from broth by membrane in batch fermentation also decreased feedback inhibition. MIRB was carried

www.elsevier.com/locate/nbt S95 BIOFUELS, BIOCHEMICALS AND BIOENERGY New Biotechnology · Volume 31S · July 2014 out for 5 cycles repeated culture using wheat straw hydrolysates from renewable resources. Although microbial production of diesel (29.72 g/L glucose, 24.69 g/L xylose and 5.14 g/L arabinose) as car- has been reported, production of another much demanded trans- bon source, a 2.33-folds increase of LA productivity (2.35 g/L/h) port fuel, gasoline, has not yet been demonstrated. Here we report was obtained as compared with conventional batch fermentation. the development of platform Escherichia coli strains that are capa- http://dx.doi.org/10.1016/j.nbt.2014.05.1836 ble of producing short chain alkanes (gasoline). The b-oxidation pathway was blocked by deleting the fadE gene to prevent the degradation of fatty acyl-CoAs generated in vivo and the activity PB-10 of 3-oxoacyl-ACP synthase (FabH) was enhanced to promote the initiation of fatty acid biosynthesis by deleting the fadR gene. A Solid state fungal fermentation as a biological pre- modified thioesterase was employed to convert short chain fatty treatment strategy to convert lignocellulosic raw mate- acyl-ACPs to the corresponding FFAs, which were consequently rial into fermentable sugars converted to short chain alkanes by the sequential reactions of fatty acyl-CoA synthetase, fatty acyl-CoA reductase and fatty alde- Chenyu Du ∗ , Nattha Pensupa, Siobhan Knight, Jwan Abdullah hyde decarbonylase. The final engineered strain produced up to The University of Nottingham 580.8 mg l−1 of SCAs consisting of nonane (327.8 mg l−1), dodecane (136.5 mg l−1), tridecane (64.8 mg l−1), 2-methyl-dodecane Pre-treatment is one of the key challenges in the conver- (42.8 mg l−1) and tetradecane (8.9 mg l−1) together with small sion of lignocellulosic raw materials into bioethanol. Various amounts of other hydrocarbons. pre-treatment strategies have been developed, such as dilute acid [This work was supported by the Advanced Biomass Research pre-treatment, alkali pre-treatment, steam explosion and ammo- and Development Center of Korea (ABC-2010-0029799) through nia explosion pre-treatment. These methods are energy intensive the Global Frontier Research Program of the Ministry of Sci- as these are normally operated at high temperature, high presence, ICT and Future Planning (MSIP) through the National sure or high chemical loading rate. Inhibitor formation during Research Foundation (NRF). Systems metabolic engineering work the pre-treatment is another concern in these methods, which was supported by the Technology Development Program to Solve significantly impacts the efficiency of the following fermentation Climate Changes on Systems Metabolic Engineering for Biorefiner- processes. ies (NRF-2012-C1AAA001-2012M1A2A2026556) by MSIP through In the University of Nottingham, we developed a solid-state NRF]. fungal fermentation-based pre-treatment strategy to convert ligno- http://dx.doi.org/10.1016/j.nbt.2014.05.1838 cellulosic raw materials into a fermentable hydrolysate. Aspergillus niger was firstly cultured on biomass for production of cellulolytic enzymes and then the biomass was hydrolyzed by the enzyme PB-12 solution into a fermentable hydrolysate. Various lignocellulosic raw materials have been tried, including wheat straw, willow, mis- Statistical optimization of critical parameters for alka- canthus, palm oil tree branches, napier, sago extract and municipal line treatments of canola agricultural residue by solid waste. In solid-state fermentations of most of these raw advanced regression model materials, around 4-10 U/g cellulase activity could be obtained Seung Wook Kim 1,∗ Hah Young Yoo 1 Da Un Jung 1 Sung Bong after 5 days’ culture. The acid or alkali modification of biomass , , , Kim 1 Ja Hyun Lee 1 Chulhwan Park 2 at mild condition improved the cellulase production to over 10 , , U/g. The addition of yeast extract (0.5% w/v) and minerals sig- 1 Korea University 2 nificantly improved the cellulase production, for examples, to Kwangwoon University 24 U/g in wheat straw, 22 U/g in napier. The fungal culture filtrate from the solid-stage fermentation showed higher cellulolytic In this study, canola agricultural residue from Honam area hydrolysis ability then the commercial cellulase Ctec2 at the same of Korea was pretreated by alkaline reagents (ammonium and enzyme loading rate. Moreover, no inhibitor was detected in the sodium hydroxide) in order to enhance the enzyme accessibil- hydrolysate examined. ity, and the treatment conditions were optimized by statistical method. Generally, most researches only focused on the enhance- http://dx.doi.org/10.1016/j.nbt.2014.05.1837 ment of enzymatic digestibility in the process but if not mentioned about solid recovery then the overall sugar yield can be reduced. Therefore, a novel experimental response (biomass to glucose con- PB-11 version; BtG) which modified from solid recovery and enzymatic Metabolic Engineering of E. coli for the production of digestibility is applied toregression analysis, in current study. As a alkanes result, the optimal conditions were carried out by BtG model and the sugar yield was improved compared with the previous work. ∗ Yong Jun Choi , Sang Yup Lee In optimal conditions, the predicted solid recovery, enzymatic Korea Advanced Institute of Science and Technology digestibility and BtG were 75.8%, 78.3% and 25.7% at ammonium hydroxide treatment and 49.2%, 86.4% and 24.3% at sodium Our increasing concerns on limited fossil fuels and global envi- hydroxide treatment, respectively. The experimental results show ronmental problems are urging us to develop sustainable biofuels

S96 www.elsevier.com/locate/nbt New Biotechnology · Volume 31S · July 2014 BIOFUELS, BIOCHEMICALS AND BIOENERGY numerically over 90% in general. Finally, under the overall mass of the red fluorescence appeared to change following the affinities balance, glucose yield was about 3 fold increased by the treatments. between the LZs, as observed by fluorescence imaging and flow http://dx.doi.org/10.1016/j.nbt.2014.05.1839 cytometry. Currently, we are applying the proposed method to construct an in vivo matrix to entrap different enzymes in E. coli cytosole while maintaining their catalytic activities. In addition, PB-13 easy detection of localization to IBs provides a unique platform for the engineering and analyses of protein-protein interactions Biogas production from 3 strains of Napier grass in E. coli.

Pramote Sirirote 1,2,∗ , Farida Promma 2, Dusanee Thanaboripat 2 http://dx.doi.org/10.1016/j.nbt.2014.05.1841 1 King Monkut’Institute of Technology Ladkrabang, Biogas production from 3 strains of Napier grass, (Pennisetum purpureum) 2 Department of Biology, Faculty of Science, King Mongkut’s Institute of Tech- PB-15 nology Ladkrabang, Bangkok, Thailand Direct biocatalytic conversion of methane-to-methanol Keywords: Biogas; Napier grass; Batch anaerobic digestion using methane and ammonia-oxidizing bacteria Eun Yeol Lee ∗ , In Yeub Hwang Abstract The aim of this research was to study the potentials Kyung Hee University of biogas production from 3 strains of Napier grass (Pennisetum purpureum); i.e, King grass, Napier Pakchong1 and Alafal. Batch Production of methanol from methane is the first step for anaerobic digestion was performed on five ratios of different grass achieving methane-based production of a wide range of chemicals. and inoculum volume at 1:1, 1:2, 1:3, 2:1 and 3:1, and performed Methanol is a precursor to various chemicals and a liquid fuel that on working volume of 5 L digestors for 45 days at room temper- can be blended with gasoline. In this study, we employed methane ature (29-34 ◦C). The results showed that the highest cumulative and ammonia-oxidizing bacteria to partially oxidize methane biogas production was 22.45, 26.25 and 24.29 L at the ratios of 1:3, to methanol. To prevent methanol catabolism, thus allowing 1:2 and 1:2 for King grass, Napier Pakchong1 and Alafal, respec- methanol accumulation in the medium, various inhibitors for tively. Initial pH was 6 - 6.5 and biogas yield were 0.37, 0.53 and methanol dehydrogenase were used. Further, sodium formate 0.47 L biogas/g VS. The efficiencies for the COD removal were was supplied as reducing power regeneration because methane 82.8, 76.9 and 85.0% at the best grass inoculum ratios, respec- moonoxygenase-catalyzed oxidation of methane to methanol was tively.Therefore, Napier Pakchong1 and Alafalat grass to inoculum limited by reducing equivalents supply. ratio of 1:2 were selected for further experimentation.The digestion was performed on working volume of1 L and adjusted initial Acknowledgment pH to 7.1 for 36 day. The resulst showed that the cumulative biogas production were 2.46 and 6.97 L for Napier Pakchong1 and This work was supported by the New & Renewable Energy of Alafal, respectively. Therefore, the results indicated that Alafal has the Korea Insitute of Energy Technology Evaluation and Planning higher potential for biogas production than King grass and Napier (KETEP) grant funded by the Korea government Ministry of Knowl- Pakchong1. edge Economy (No. G031595311). http://dx.doi.org/10.1016/j.nbt.2014.05.1840 http://dx.doi.org/10.1016/j.nbt.2014.05.1842

PB-14 PB-16 Controlled localization of functionally active enzymes to Encapsulation of Candida rugosa lipase in chitosan inclusion bodies using leucine zippers beads as biocatalyst for biodiesel production via non- Seung-Goo Lee ∗ , Haseong Kim, Bong-Hyun Sung alcohol route

Korea Research Institute of Bioscience and Biotechnology Heri Hermansyah ∗ , Merisa Bestari Faiz, Intan Sipangkar, Rita Arbianti Inclusion bodies (IBs) are typically non-functional particles Department of Chemical Engineering, Universitas Indonesia, Depok 16424, of aggregated proteins. However, some proteins in fusion with Indonesia amyloid-like peptides, viral coat proteins, and cellulose binding domains (CBDs) generate IB particles retaining the original Lipase-catalyzed biodiesel production offers many advantages functions in cells. Here, we attempted to generate CBD IBs display- such as high level of selectivity and it has good ability to catalyze ing functional leucine zipper proteins (LZs) as bait for localizing organic reactions in aqueous or non-aqueous media. Unfortu- cytosolic proteins in E. coli. When a red ﬂuorescent protein was nately, lipase is expensive and it cannot be reused because it is tested as a target protein, microscopic observations showed that dissolved in reaction media. To overcome the problem, in this the IBs red-ﬂuoresced strongly. When different LZ pairs with KDs research, we immobilized Candida rugosa lipase (CRL) in chitosan of 20–1,000 mM were tested as the bait and prey, the localization beads through encapsulation method. The optimum condition of

www.elsevier.com/locate/nbt S97 BIOFUELS, BIOCHEMICALS AND BIOENERGY New Biotechnology · Volume 31S · July 2014 enzyme to support mass ratio, immobilization time and cross- the overall ethanol production efficiency and energy yield would linking agent concentration were investigated in the range of increase. 1:4 to 1:8, 50 to 150 minutes and 0.6% to 3%w/v, respectively. http://dx.doi.org/10.1016/j.nbt.2014.05.1844 Enzyme concentration was measured through spectrophotometry method. The highest enzyme loading was 97.24% when the operating conditions were 1:6 mass ratio of lipase to chitosan, PB-18 120 minutes immobilization time and 0.6%w/v of cross-linking agent concentration. The immobilized enzyme then was used as Effect of hydraulic retention time on the performance of biocatalyst in biodiesel production. Non-alcohol route was used a novel tubular MFC fed with petroleum hydrocarbons as a route for biodiesel production because it can prevent enzyme Oluwaseun Adelaja ∗ , Godfrey Kyazze, Taj Keshavarz deactivation which is commonly found in conventional route. The operating condition for biodiesel production process was University of Westminster 37◦ C, 4%w/w biocatalyst and 1:12 mole ratio of used cooking oil (as triglyceride source) to methyl acetate (as acyl acceptor). The Pollution of groundwater by petroleum hydrocarbons is a biodiesel, fatty acid methyl ester (FAME), concentration was deter- serious threat to human health as the hydrocarbons are toxic, mined using High Liquid Performance Chromatography (HPLC). mutagenic and carcinogenic. Microbial fuel cells (MFCs) could be Through 50 hours batch production process, 94.06% biodiesel employed in the treatment of these recalcitrant pollutants with yield was achieved. Whereas through continuous process in a concomitant bioelectricity generation. For practical application packed bed reactor sized 11 mm ID and 150 mm length with the MFCs would have to be effective, efficient and robust. 1ml/hour flow rate, 93.67% biodiesel yield was achieved. This study investigated the performance of a novel tubular Keywords: Biodiesel, immobilization, biocatalyst MFC, operated in a continuous mode at different hydraulic retention times, HRT, in the range 2.5 to 10 days at room temperature. http://dx.doi.org/10.1016/j.nbt.2014.05.1843 Bromate was employed as the catholyte and the inoculum was an adapted anaerobic microbial consortium. A mixture of ben- zene and phenanthrene was used as the substrate. Total chemical PB-17 oxygen demand removal efficiencies and peak power densities 2 Enhancing energy yield through saccharification of decreased from 74 to 57% and 3.4 to 1.1mW/m respectively sorghum bagasse and second generation bio-ethanol pro- when HRT was decreased from 10 to 2.5 days.The removal duction efficiencies were higher than 90% for both phenanthrene and ben- zene.Bromate removal efficiencies increased from 52.5-78.6% as Teodor Vintila ∗ Adrian Trulea Daniela Vintila˘ Georgeta Pop Iosif , , , , HRT was raised from 2.5-10d. Gergen Kornel Kovacs , The outcome of this study suggests the application of MFCs University of Agricultural Science Timisoara in the simultaneous removal of petroleum hydrocarbons and bromates (with concomitant power production) in anoxic envi- Sweet sorghum is an important source of sugar that can be ronments, especially deep groundwater reservoir. MFC technology utilized for ethanol production. Sorghum bagasse resulted after could possibly be a substitute for the more expensive conventional sweet juice extraction can be further processed to obtain more technologies such as permeable reactive barrier (PRB) and elec- energy as lignocellulosic ethanol. In our study, we used six com- troremediation which are currently employed in remediation of mercial products consisting of biomass degrading enzymes to hydrocarbon pollutants in subsurface environments. hydrolyze cellulose from pretreated sorghum bagasse and obtain http://dx.doi.org/10.1016/j.nbt.2014.05.1845 fermentable sugars. Tests containing combinations of steam- alkaline, mechanical pretreated bagasse and different enzymes cocktails were conducted. The results indicated the combination PB-19 of steam-alkaline pretreatment and NS22086 cellulase complex (Novozymes) as the most efficient. Next, these conditions were An integrated lignocellulose-based bioprocessing for the applied to hydrolyze three types of Sorghum bicolor bagasse (Sugar production of a generic microbial feedstock Graze, Jumbo and Fundulea FT132). The hydrolysis rates obtained Chen-Wei Chang ∗ , Colin Webb in the three sorghum types are between 32% and 40%. Concentra- tion of total sugars and glucose released in hydrolysis buffer was University of Manchester monitored. The conversion process continued with fermentation of hydrolyzates by Saccharomyces cerevisiae in 500 ml fermenters Conversion of ligno-cellulosic materials to sugars through a equipped with NIR sensors (BlueSens) to evaluate in real time the purely biological treatment is a key to sustainable chemicals pro- ethanol and CO2 concentration. Ethanol concentrations between duction. Sequential solid-state fermentation of sugarcane bagasse 1.65 g·ml−1 and 1.96 g·ml−1 were obtained in fermentation media with soybean hull and enzyme hydrolysis using Trichoderma lon- of the hydrolyzed bagasse from three sorghum varieties, represent- gibrachiatum was performed. Microorganism incubation through ing ethanol yields between 330 g·g−1 and 392 g·g−1 reported at DM solid-state fermentation resulted in process, not only producing bagasse. Applying biotechnology developed in this study in combi- an abundant enzyme complex, but also degrading the recalcitrant nation with current sugar extraction method applied in sorghum, structure of the ligno-cellulosic materials.

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The process includes three stages: (1) Simultaneous decon- PB-21 struction of recalcitrant lignocellulosic materials and cellulolytic enzyme production through filamentous fugal bioconversion Studies on the role of different culture conditions on the usingTrichoderma longibrachiatum; (2) generic fermentation feed- growth and biochemical composition of marine microal- stock production through enzymatic hydrolysis of fermented solid gae Nannochloropsis sp associated with fungi autolysis; (3) subsequent fermentation by Maria Savvidou ∗ , Fragiskos Kolisis native Saccharomyces cerevisiaefor ethanol production. This process National Technical University of Athens provides cost-competitive and environmental friendly alternative to produce chemical and biofuel from lignocellulosic wastes. The climate change, the rising fossil-fuel prices and overall http://dx.doi.org/10.1016/j.nbt.2014.05.1846 the societal concerns have raised attention to the sustainable production of advanced biofuels, as well as high added value bioproducts such as fatty acids (␥-linolenic, eicosapentaenoic, PB-20 docosahexaenoic acids, etc.) pigments (carotenoids, ficobilins), vitamins and metabolites. A synthetic biology approach towards improved cellu- The rapid growth rates, the accumulation of large quan- lolytic activity of Clostridium acetobutylicum ATCC 824 tity of lipid and other compounds of interest and the ability Katalin Kovacs ∗ , Benjamin Willson, Nigel Minton to be cultivated in seawater or brackish water on non-arable University of Nottingham land make algae an exciting addition to the sustainable fuel portfolio. Microalgae are sunlight-driven cell factories that use Several Clostridia species employ large, self-assembled multi- photosynthesis to efficiently and quickly convert CO2 and other enzyme complexes, or cellulosomes, for efficient decomposition of greenhouse gasses into potential biofuels, foods, animal feeds the plant cell wall cellulosic polysaccharides cellulose and hemicel- and bioactive compounds, the cultivation conditions and the lulose. Clostridium acetobutylicum, the best studied of the butanol role of nutrient media characteristicsare still under investigation. producing Clostridia, produces small amounts of a non-functional Amongst various microorganisms, Nannochloropsis sp., an oleagi- cellulosome and is therefore unable to grow on cellulose. In our nous eukaryotic marine alga, is well appreciated in aquaculture due studies, we employed synthetic biology approaches to create sta- to its nutritional value and the ability to produce valuable chem- ble C. acetobutylicum strains with the potential to utilise various ical compounds, such as pigments, proteins and polyunsaturated cellulosic substrates by genome integration of synthetic celluloso- fatty acids. mal subunits derived from various cellulosic degrading bacterial The aim of the present study is to quantify some biochemi- species (Clostridia and other species) as well as integrating non- cal compounds (lipids, chlorophyll a, carotenoids, proteins, total cellulosomal cellulolytic enzymes (bacterial and other sources). cell biomass) in cultures of Nannochloropsis sp. grown in dif- We use standardised synthetic parts (optimized DNA sequences) in ferent culture conditions. The microorganism is cultivated in BioBrick2 format to assemble a range of synthetic genes encoding photobioreactors containing sterilized seawater enriched with f/2 cellulosomal scaffoldin proteins, glycoside hydrolases (GHs) and medium nutrients under standard illumination conditions. The synthetic cellulosomal operons. All synthetic genes and operons effect of parameters such light intensity, pH, sodium bicarbonate are integrated into the C. acetobutylicum genome using the recently supplementation and sodium nitrate concentration on growth and developed allele-coupled exchange (ACE) technology. Heterolo- biochemical composition of Nannochloropsis sp. are extensively gous protein expression levels of the synthetic genes and the studied. self-assembly of the mini-cellulosomes are assayed by Western http://dx.doi.org/10.1016/j.nbt.2014.05.1848 blot, native PAGE and enzyme activity. We have demonstrated the successful expression, secretion, self-assembly and activity of the mini-cellulosomes produced by recombinant C. acetobutylicum PB-22 strains, providing a platform for the construction of novel strains with finely tuned cellulolytic properties Effect of different components of media prepared with sugar beet hydrolysate on cell growth and ethanol pro- http://dx.doi.org/10.1016/j.nbt.2014.05.1847 duction

Meltem Yesilcimen Akbas ∗ , Sar Taner Gebze Institute of Technology

Ethanol is an alcohol made from the fermentation of the carbohydrate or sugar fraction in biomass materials. Sugar beet composition makes it a potential and attractive raw material for the production of the second generation bioethanol. The aim of this research was to assess the usefulness of dilute enzymatic hydrolysis of sugar beet molasses and the different media components for enhancement of cell growth and ethanol production of

www.elsevier.com/locate/nbt S99 BIOFUELS, BIOCHEMICALS AND BIOENERGY New Biotechnology · Volume 31S · July 2014 ethanologic E. coli strain (FBR5). Sugar beet molasses was hydrol- Such characteristics have increased the interest in these molecules ysed with a dilute sulfuric acid at room temperature overnight however, their large scale production is currently more expen- to convert sucrose to glucose and fructose, followed by auto- sive comparatively to synthetics. The use of cellulosic material claving. The enhancement of growth and ethanol production of can improve economics of biosurfactant production and also strain FBR5 were investigated in a variety of growth media compo- contribute to reducing greenhouse gas emissions. This work inves- nents including nutrients (peptone and yeast extract) or thiamin tigates the production of surfactin by Bacillus subtilis using the or/and trace elements. The growth properties of the strain were liquor from sisal (Agave sisalana) pulp hydrolysis as substrate. also evaluated before and after overliming with Ca(OH)2 treat- The liquor deriving from acid and enzymatic hydrolysis of sisal ment. Overliming treatment did not affect the growth and the cellulose was utilized as carbon source in culture media. The ethanol production. The hydrolysate (diluted to 20%; v/v) medium partially-puriﬁed product obtained using acid hydrolysate showed including nutrients, thiamin and trace elements enhanced the cell a surface tension (ST) of 29.8 mN/m, interfacial tension (IT) against biomass and ethanol yield. hexadecane of 5.7 mN/m and a critical micelle concentration http://dx.doi.org/10.1016/j.nbt.2014.05.1849 (CMC) of 1394.0 mg/L whereas when enzymatic hydrolysate was utilized the product showed a ST of 28.7 mN/m, IT of 3.8 mN/m and a CMC of 64.0 mg/L. The enzymatic derived liquor demon- PB-23 strates to be more suitable generating the BS with the best surface active properties. In conclusion, the liquor from sisal cellulose Potential use of different hydrolyzing methods of potato hydrolysis can be explored as an alternative sustainable substrate and corn processing industry wastes for ethanol produc- for biosurfactant production. tion http://dx.doi.org/10.1016/j.nbt.2014.05.1851 Meltem Yesilcimen Akbas ∗ , Fatma Sumer Gebze Institute of Technology PB-25

Bioethanol produced from renewable sources, such as starch Use of hardwood sulphite spent liquor for acclimating or lignocellulosic materials are promising sources of alternative a polyhydroxyalkanoate storage capacity of a mixed energy resources. Potato and corn processing industry waste (PCW) microbial culture is a zero value waste rich in starch and lignocellulosic material.The Diogo Queirós 1 Luísa Seuanes Serafim 1,∗ Simona Rossetti 2 purpose of present research was to investigate the potential use , , of different hydrolyzing methods of PCW. The cell growth and 1 CICECO, Chemistry Department, University of Aveiro 2 ethanol production of ethanologic E. coli strain (FBR5) were com- Water Research Institute, CNR pared. PCW was hydrolyzed with different methods by using different concentrations of dilute acid and/or enzymes such as Polyhydroxyalkanoates (PHAs) are biodegradable and bio- xylanase, cellulase or both enzymes. The sugar contents (glucose, compatible biopolymers that emerge as a possible solution as xylose and arabinose) of different hydrolysates were investi- substitutes of petroleum-based plastics. PHAs can be produced gated by Thin Layer Chromatography. The dilute acid treatment within the Biorefinery concept, in which wastes and by-products methodology yielded the highest levels of glucose, xylose and of numerous industries are used as carbon source. The effectiveness arabinose; 7.6%, 2.7% and 1.8%, respectively. The growth prop- of PHAs production process includes a first stage of selection of a erties and ethanol production of the strain FBR5 were evaluated MMC with a stable PHAs-producing capacity, which determines by using hydrolysate medium including nutrients, thiamin and the success of subsequent PHA accumulation step. This could be trace elements. The experiments resulted in higher cell numbers done resorting to hardwood spent sulphite liquor (HSSL) which and ethanol yield. is a complex feedstock originated from the pulp industry and meets the concept of a lignocellulosic-based biorefinery, due to its http://dx.doi.org/10.1016/j.nbt.2014.05.1850 abundance and affordability, wide variety and good marketing of the bio-based products. To complement the selection of the PHA- storing populations, the evolution of microbial community must PB-24 be evaluated in order to identify the best producers and determine Potential application of liquor from sisal pulp hydroly- the individual relative abundance, allowing for the design of oper- sis as alternative substrate for biosurfactant production ating conditions favoring the most important PHA-accumulating microorganisms. ∗ Marcia Nitschke , Claudia Marin Abadia, Joice Kaschuk, Elisabete In this project, a MMC collected in a wastewater treat- Frollini ment plant was submitted to Aerobic Dynamic Feeding (ADF) University of São Paulo in a Sequencing Batch Reactor (SBR) in order to select PHA- accumulating organisms using HSSL. The reactor has been Microbial-derived surfactants are more environmentally- operating for near 500 days in a steady state condition, produc- friendly, biocompatible and biodegradable in comparison with ing a copolymer, poly(hydroxybutyrate-co-hydroxyvalerate). The conventionally produced detergents from petroleum sources medium, rich in xylose, acetic acid and lignosulphonates, led to moreover, their production supports the concept of biorefinery. a selection of a co-dominant culture between Alphaproteobac-

S100 www.elsevier.com/locate/nbt New Biotechnology · Volume 31S · July 2014 BIOFUELS, BIOCHEMICALS AND BIOENERGY teria and Betaproteobacteria, determined by fluorescent in situ hydrolyzer of agricultural biogas plant, cattle slurry and manure, hybridization. A clone library was constructed and genera like and represents the following genera: Bacillus, Providencia, Ochrobac- Agrobacterium, Rhizobium, Brachimonas, Acidovarax, Flavobacterium, trum. Leadbetterella and Dyadobacter were identified. The main aim of this study was to verify whether the con- http://dx.doi.org/10.1016/j.nbt.2014.05.1852 structed consortium can degrade lignocellulosic material and enhance the production of biogas from maize silage and cattle manure. The enhancement of the methane production by supple- PB-26 mentation with the MCHCA consortium was monitored in batch bioreactors with the use of maize silage as substrate and cattle Bioethanol Production: Adaptation of Scheffersomyces manure as methanogens inoculum. In the first step of the pro- stipitis to Hardwood Spent Sulfite Liquor cess, maize silage (1% d.m.) was pretreated by 5% (v/v)or10% (v/v) MCHCA consortium (with a density ∼108cells/ml) for 72 h AnaMRBXavier 1,∗ , CláudioJRFrazão 1, Susana R Pereira 2, Violeta at 300C, and then pretreated substrate was subjected to anaero- Sanches i Nogué 3, Luísa S Serafim 2, Marie F Gorwa-Grauslund 3 bic digestion with cattle manure (20% v/v) for 21 days at 370C. 1 CICECO, Chemical Department, University of Aveiro, Portugal During the experiment the following parameters were monitored: 2 Chemical Department, University of Aveiro, Portugal cellulolytic activity, pH, level of volatile fatty acids and stability of 3 Division of Applied Microbiology, Department of Chemistry, Lund University the consortium structure by DGGE (denaturing gradient gel electrophoresis) analysis. Methane concentration and volume of gas Biofuels, which consist of fuels produced from biomass, are in the culture was measured after 7, 14 and 21 days. suitable renewable alternatives to conventional motor fuels (e.g. The experimental results showed that constructed MCHCA gasoline, diesel). Bioethanol and biodiesel are the most promising consortium can increase the efficiency of biogas production up biofuels. Biofuels can be generated from various raw materials, like to 20-30%, when used 5% addition of the consortium. lignocellulosic biomass and its derivatives. Hardwood Spent Sulfite Liquor, HSSL, is the side-product of the http://dx.doi.org/10.1016/j.nbt.2014.05.1854 acidic sulfite pulping process, and it is rich in sugars (40–45 g.L−1), mainly the pentose sugar xylose that can be converted to ethanol by yeast Scheffersomyces stipitis. However, HSSL contains toxic PB-28 compounds (e.g. acetic acid and phenolics) that inhibit yeast Production of 1,3-propanediol from glycerol by C. metabolism. butyricum: Optimization of medium composition and The aim of this study was the use of evolutionary engineer- kinetic studies ing to generate a S. stipitis strain with increased tolerance to HSSL inhibitors and maintained xylose conversion rate. Wojciech Białas ∗ , Marta Pikuła, Katarzyna Mroczyk, Włodzimierz A continuous reactor with increasing HSSL concentrations (20- Grajek 60% (V/V)) was operated. The final population, POP, obtained after Poznan University of Life Sciences 382 generations in HSSL, was characterized and compared to the parental strain, PAR. A two-step approach was employed to optimize culture medium By using this approach, improved fermentation performance composition for enhanced production of 1,3-propanediol (1,3- was obtained. POP showed a higher xylose consumption rate PD) by Clostridium butyricum. In the first step, a simple two-level −1 −1 −1 (0.33 g.L .h ) and maximum ethanol concentration (6.93 g.L ) screening experimental design was used to evaluate the influence −1 −1 −1 than PAR (0.10 g.L .h and 1.76 g.L , respectively). of individual components of the medium on 1,3-PD production. http://dx.doi.org/10.1016/j.nbt.2014.05.1853 In the second step, a central composite experimental design and response surface methodology were employed to derive a statistical model describing the impact of yeast extract, potassium phosphate PB-27 dibasic and iron (II) sulfate on the 1,3-PD production. The final titer of 1,3-PD produced by C. butyricum cultured in Microbial Consortium with High Cellulolytic Activity the optimized medium, with the starting glycerol concentration of (MCHCA) for enhanced biogas production 80 g/L, was of 38.8 ± 1.23 g/L, resulting in the process yield of 0.49. Lukasz Drewniak 1,∗ , Krzysztof Poszytek 2, Martyna Ciezkowska 2 The results obtained under the optimal fermentation conditions were used as a starting point for development of a kinetic model 1 Faculty of Biology, University of Warsaw describing the glycerol utilization, 1,3-PD and biomass production 2 Laboratory of Environmental Pollution Analysis, Faculty of Biology, University of Warsaw rates. http://dx.doi.org/10.1016/j.nbt.2014.05.1855 In our previous work we have constructed Microbial Consor- tium with High Cellulolytic Activity (MCHCA). This consortium involves twenty two bacteria strains, which were isolated on minimal salt medium containing carboxymethylcellulose as the sole carbon source. Isolates were derived from: sewage sludge,

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PB-29 to the atmosphere or must be destroyed in a reduction furnace. Biotechnology offers an innovative way to overcome the limita- Lignin utilization by Bacillus sp. associated with the tions and disadvantages of the chemical processes to make diacids. growth enhancement and the molecular weight distri- Yarrowia biocatalyst are able to convert long-chain fatty acids bution change of lignin directly to long-chain diacids through overexpressing ␻-oxidation ,∗ ␤ Gyeongtaek Gong 1 , Han Min Woo 1, Youngsoon Um 1, Tai Hyun pathway and blocking the -oxidation pathway. This study was Park 2 developed and demonstrated key biocatalyst to produce cost- competitive long-chain dodecanedioic acid, DCA12. The focus of 1 Korea Institute of Science and Technology this study was to increase the rate of conversion of glucose into the 2 School of Chemical and Biological Engineering corresponding fatty acid feedstocks through overexpress the gene of fatty acid synthesis, Acetyl-CoA carboxylase (AccD) and Fatty Lignin is one of the major components in lignocellulosic acid synthase (FA-1, FA-2, FB-1), in the yeast biocatalyst (Yarrowia biomass and is the most abundant natural aromatic polymer. To lipolytica). In order to enhance DCA12 content, the Yarrowia- utilize lignocellulosic biomass efficiently, lignin-degradation is an codon acyl-carrier protein thioesterase gene, BTE from Umbellularia important issue; however, lignin is known to be recalcitrant for califoenica, FatB3 from Cocos nucifera were also expressed. Finally, biodegradation. Previously, we isolated Bacillus sp. GWD275 from the RNA inference technology was also used to enhance DCA12 mud flat thorough the culture in kraft lignin agar plate and the production in this research. Azure B decolorization screening method for lignin degradation. In this study, lignin utilization by Bacillus sp. GWD275 was investi- http://dx.doi.org/10.1016/j.nbt.2014.05.1857 gated by observing growth enhancement in the presence of lignin. Also, the molecular weight distribution of lignin in culture media was analyzed by Gel Permeation Chromatography (GPC) during PB-31 the growth of Bacillus sp. GWD275 in the presence of lignin. The growth of GWD275 was compared with Luria-Bertani (LB) and LB Butanol production by metabolically engineered supplemented with lignin (1 g/L). The growth in LB supplemented Clostridium acetobutylicum with in-situ butanol removal with lignin was increased by 1.8-fold compared with LB medium Sang-Hyun Lee 1,∗ , Min-A Kwon 1, Yong-An Shin 1, Kyoung Heon only, implying lignin utilization by Bacillus sp. GWD275. Further- Kim 2 more, when the defined medium with glucose or xylose without 1 GS Caltex Corporation complex nitrogen source was used, the growth of GWD275 was 2 Korea University also enhanced in the presence of lignin (0.5 g/L), possibly by using lignin as a co-substrate for growth. Interestingly, the molec- Clostridium acetobutylicum is industrially important strain for ular weight distribution of lignin in the cultures with the defined butanol production. However, economically feasible production medium was shifted to a lower molecular weight portion, while it of butanol by wild type Clostridium acetobutylicum is still limited was shifted to a higher molecular weight portion in cultures with by butanol toxicity and by-product formation, resulting in low LB medium. The results presenting here would provide an insight butanol yield and productivity. To overcome the cellular toxicity of for efficient use of limited lignocellulosic bio-resources by lignin butanol, we developed fermentation strategy with in-situ butanol utilization using Bacillus sp. GWD275. recovery (ISBR) by addition of butanol selective synthetic resin http://dx.doi.org/10.1016/j.nbt.2014.05.1856 to the bioreactor. Also, we constructed the recombinant strain, which are deletion mutant of acid formation pathways to reduce by-products, acetic acid and butyric acid. Fed batch ISBR fermen- PB-30 tation profiles showed the increse in yield, butanol selectivity and productivity of butanol production. In continuous fermentation Biosynthesis of nylon precursor dodecanedioic acid from with ISBR for 140 h, the recombinant strain showed that the over- fatty acid all yield, butanol selectivity and volumetric producivity were 35%, Liang-Jung Chien ∗ , Szu-Min Yu 78%, and 2.6 g/L/h, respectively, when feeding 200 g/L glucose solution containing 3% corn steep liquor as a nutrient Ming Chi University of Technology Acknowledgement: This work was supported by the Center for Organic Wastes to Energy Business under the Environmen- Aliphatic a,w-dicarboxylic acids (DCA) of the type addressed by tal Technology Development Program funded by the Ministry of this program are used in a wide variety of plastics and other chem- Environment, Korea. ical applications. The DCA12 produced in the largest quantity (>40 MM lb/yr) as a pure chemical intermediate is dodecanedioic http://dx.doi.org/10.1016/j.nbt.2014.05.1858 acid (C12); it is used in polyamides such as nylon 6,12, which is noted for high moisture resistance. The dodecanedioic process is based on non-renewable petrochemical feedstocks. The multi- step conversion process produces unwanted byproducts such as cyclooctadiene and vinyl cyclohexene, which result in yield losses. The nitric acid oxidation step yields NOx, which is either released

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PB-32 PB-33

Production of gaseous biofuels and fine chemicals from The role of adhe in thermophile ethanol production food industrial wastes Tianyong Zheng ∗ , Daniel Olson 1,∗ 2 2 3 Gabor Rakhely , Balazs Balint , Rita Beres , Agnes Kis , Kornel Dartmouth College Kovacs 2, Krisztian Laczi 2, Andrea Nyilasi 4, Andras Fulop 2, Zoltan Bagi 2 Etelka Kovacs 2 Gergely Maroti 5 Katalin Perei 6 , , , Clostridium thermocellum is a thermophilic, gram-positive obli- 1 Institute of Environmental Sciences and Department of Biotechnology, Uni- gate anaerobe that is a candidate organism for converting cellulosic versity of Szeged biomass into ethanol through consolidated bioprocessing. C. ther- 2 Department of Biotechnology, University of Szeged mocellum has one of the highest rates of cellulose utilization 3 Institute of Biophysics, Biological Research Center, Insitute of Environmental Sciences, University of Szeged known, but ethanol production in wild type C. thermocellum was 4 Institute of Biophysics, Biological Research Center reported to be < 30 g/L. This is relatively low comparing to an engi- 5 Institute of Biochemistry, Biological Research Center neered strain of Thermoanaerobacterium saccharolyticum, which has 6 Department of Biotechnology, Institute of Environmental Sciences, University acheived theoretical yields of ethanol. However, because T. sac- of Szeged charolyticum cannot use cellulose as an energy source, we seek to reconstruct the T. saccharolyticum ethanol production pathway in Food industrial activity is accompanied by the emission of C. thermocellum. The bi-functional alcohol/acetaldehyde dehydro- various kinds of organic wastes including protein, fatty and sugar- genase AdhE was shown to be a key enzyme in ethanol production based materials. These substrates have distinct values, some of in many organisms, but this enzyme has never been individu- them could be utilized as animal nutrients but this is often limited ally studied in C. thermocellum or T. saccharolyticum. Therefore, by public sanitation. this study characterized and compared the activity of AdhEs in Protein containing wastes could be used for both biohydrogen different strains of C. thermocellum or T. saccharolyticum, through and biogas production. Keratin was a substrate of a two-stage pro- expression in E. coli and purification. The Km values for substrates cedure for biohydrogen production. Alternatively, various protein and product inhibition were measured, the enzyme specificity for substrates could be converted into biogas via a microbial adapta- co-factors (NADH/NADPH) were also determined. The end goal of tion processes which were monitored by metagenomic approach. this study is to find a better performing AdhE that can be expressed Sugar-based biowastes could also be used for either biohydrogen in C. thermocellum to generate higher yields of ethanol. or biogas production and the yield could be stimulated by addition of either nutrients or properly chosen microbes. Both processes http://dx.doi.org/10.1016/j.nbt.2014.05.1860 were monitored by new generation sequencing based approaches. Fatty acids could be degraded by several microbes. Unctuous wastes were effectively utilized by Rhodococcal strains and the process was monitored by whole cell transcriptome analysis. During fermentation, surfactant molecules were produced which might be utilized in e.g. cosmetics. Food industry and dark fermentation processes produced numerous organic acids which can be utilized either for biohydrogen production or for synthesizing bioplastics. The bioplastic metabolism - also related to CO2 capture - was shown to be in con- nection with hydrogen metabolism in purple phototrophic cells. Thus, the overall process has benefits in recovery of food industrial wastes in valuable products and also in CO2 capture.

Acknowledgments

“The Project is supported by the European Union and co-ﬁnanced by the European Social Fund (grant agreements no. TÁMOP-4.1.1.C-12/1/KONV-2012-0012, TÁMOP-4.1.1.C- 12/1/KONV-2012-0014).” http://dx.doi.org/10.1016/j.nbt.2014.05.1859

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Stem cells, gene therapy, biomarkers and PC-02 diagnostic tools Genotyping of Campylobacter jejuni using a novel Polymerase-Chain-Reaction-Microsphere approach PC-01 Ross Barnard 1,∗ , Fang Liang 2, Lawrence Wong 2, Anna Weis 2, Jil- Lysophosphatidic acid enhanced angiogenic capabil- lian Templeton 3, Patrick Blackall 4 ity of chondrocytes by regulating Gi/NF-kB-dependent 1 angiogenic factor expression The University of Queensland 2 School of Chemistry and Molecular Biosciences, The University of Queensland 3 Hung-Chih Hsu 1,∗ , Pey-Jium Chang 2, Chang-Zern Hong 3 Department of Agriculture Fisheries and Forestry 4 Queensland Alliance for Agriculture and Food Innovation, The University of 1 Chang Gung Memorial Hospital Queensland 2 Graduate Institute of Clinical Medical Sciences, College of Medicine, Chang Gung University 3 Department of Physical Therapy, Hung Kuang University, Taichung, Taiwan Campylobacter is a major cause of foodborne disease, with Campylobacter jejuni contributing more than 90% of reported cases. Lysophosphatidic acid (LPA) regulates myeloid differentiation, For diagnosis and monitoring of transmission, several genotyping osteogenesis, cell proliferation and migration, and inhibits apopto- methods have been developed, including multi-locus sequence sis in chondrocytes. We investigated the effect on the angiogenic typing (MLST) [1]. However, there is still a need for fast, cost capability of human chondrocytes and the underlying mecha- effective methods for routine analysis. We developed a technique nism using human chondrocyte cell line, CHON-001, and human combining allele-specific PCR with a microsphere-flow cytome- vascular endothelial cells (HUVECs). Angiogenic capability was ter system [2]. Seven loci with single-nucleotide polymorphisms determined by capillary tube formation, monolayer permeability, (SNPs) with the highest Simpson’s index of diversity (D) were cell migration and cell proliferation. Angiogenesis protein array selected from an MLST database. With these loci as a target, multi- kit used to evaluate the angiogenic factors secretion. Angiogenin, plex allele-specific PCR was conducted on microspheres in a single insulin-like growth factor-binding protein 1 (IGFBP-1), interleukin reaction and fluorescence signal was detected in a flow cytome- (IL)-8, chonocyte chemoattractant protein-1 (MCP-1), matrix met- ter. The signal on the microspheres indicated which allele specific alloproteinase (MMP)-9 and vascular endothelial growth factor primer was consumed in the PCR. By this means all seven loci (VEGF) mRNA and protein expression were evaluated by EIA could be determined. This approach has a turnaround time of 4 h. and Q-RT-PCR, respectively. LPA receptor (LPAR) expression was Results To date the method has been tested with two strains determined by RT-PCR. Signalling pathways were clarified using of Campylobacter jejuni possessing known SNP patterns and six of inhibitors, Western blot analysis and reporter assays. LPA pro- seven loci have been correctly determined for these strains in a motes the angiogenic capability of CHON-001 cells, resulting in single PCR reaction. enhanced HUVEC capillary tube formation, monolayer permeabil- Conclusion A new allele specific PCR-microsphere-based ity, migration and cell growth. Angiogenin, IGFBP-1, IL-8, MCP-1, method for genotyping Campylobacter jejuni is under development. MMP-9 and VEGF mRNA and protein expression were signifi- This approach will be useful in laboratories utilizing PCR and flow cantly enhanced in LPA-treated chondrocytes. In addition, LPA2, cytometers. 3, 4 and 6 were expressed in CHON-001 cells. Pre-treatment with References the Gi/o type G protein inhibitor, pertussis toxin (PTX) and the [1].Cornelius AJ, et al. Applied and environmental microbiology NF-kB inhibitor, PDTC, significantly inhibited LPA-induced angio- 2010;76:1533–44. genin, IGFBP-1, IL-8, MCP-1, MMP-9 and VEGF expression. PTX [2].Liang F, et al. Anal Biochem 2013;432:23–30. pre-treatment also inhibited LPA-mediated NFkB activation, sug- http://dx.doi.org/10.1016/j.nbt.2014.05.1862 gesting the presence of active Gi/NF-kB signaling in CHON-001 cells. The effect of LPA on inducing chondrocyte angiogenesis may be PC-03 due to increased angiogenesis factor expression via the Gi/NF-kB signaling pathway. Immunocapture and labeling of cd133- and cd54-positive http://dx.doi.org/10.1016/j.nbt.2014.05.1861 cells by using magnetic microspheres coupled with antibody via oriented immobilization

Wen-Chien Lee 1,∗ , Wei-Chih Kuan 1, Daniel Horák 2 1 National Chung Cheng University 2 Institute of Macromolecular Chemistry, Academy of Sciences of the Czech Republic

Magnetic microspheres coupled with cancer biomarkers can be very useful for clinical cancer diagnosis. Cancer stem cells (CSCs) have been identiﬁed as a subpopulation of cells within tumor which are responsible for tumor growth and metastasis. CD133 is considered a marker of CSCs in many malignant

S104 www.elsevier.com/locate/nbt New Biotechnology · Volume 31S · July 2014 STEM CELLS, GENE THERAPY, BIOMARKERS AND DIAGNOSTIC TOOLS tumors. Furthermore, to detect the presence of circulating tumor improvements over standard PCR in terms of minority profile cells (CTSs) in cancer patients, ICAM-1 (intercellular adhesion signal. molecule-1) also known as CD54 is a potential target since CD54 Results and discussion Our preliminary data show that is expressed on endothelial cells in attracting CTCs in blood. COLD PCR principle is not fully transferable from SNP (single CD54 was also found to be highly expressed on cell surface in nucleotide polymorphism) typing to STR typing. hepatocellular carcinoma. In the present work, anti-CD133 and anti-CD54 antibodies were separately immobilized on the amino- Acknowledgements containing magnetic poly(glycidyl methacrylate) (NH2-PGMA) microspheres. Antibody molecules were oxidized on their carbo- This project was partially supported by grants CZ.1.07/2.3.00/ hydrate moieties and bound to the magnetic microspheres via 30.0004, CZ.1.07/2.3.00/30.0041,and CZ.1.05/2.1.00/01.0030. a site-directed (oriented) procedure. Experimental results suggest http://dx.doi.org/10.1016/j.nbt.2014.05.1864 that the orientation immobilization could preserve the antigen recognizable site of antibody for binding with its antigen. Also, results indicate that antibody molecules were evenly fixed on PC-05 the surface of magnetic microspheres. A bound antibody density up to 104.2 mg/g-magnetic microspheres was achieved. The The detection of TNT by the periplasmic ribose binding anti-CD133 antibody-immobilized microspheres were found effec- protein RbsB of Escherichia coli is not yet solved tively for capturing CD133 positive cells from human cancer cell Artur Reimer ∗ , Shantanu Roy, Manupriyam Dubey, Vladimir lines. Flow-cytometric analysis confirmed the immunolabeling of Sentchilo, Jan Roelof van der Meer CD54-positive cell and the enrichment of CD133-expressing cells by using these antibody-bound magnetic microspheres. UNIL http://dx.doi.org/10.1016/j.nbt.2014.05.1863 There exists a wide range of naturally occurring biological sensing systems, which can be combined in biosensors with versatile application possibilities. In addition, it has been proposed that PC-04 the natural range of biological sensing proteins can be expanded Application of CO-amplification at Lower Denaturation through predictions from computational simulation algorithms. temperature (COLD) PCR principle to DNA profiling by A landmark paper in 2003 described the computational simu- STR genotyping lation and subsequent construction of mutant periplasmic ribose binding protein from Escherichia coli to create a biosensor capable Tereza Tichá 1,∗ Jiríˇ Drábek 2 Filip Kokásˇ 1 Karolína Burdová 1 Jana , , , , of detecting TNT (trinitrotoluene). The results from this work sug- Stránská 1 Veronika Holinková 2 Miroslava Rabcanováˇ 2 , , gested that periplasmic binding proteins can be used as universal 1 Palacky´ University, Olomouc scaffolds for new target specificities, which could then be plugged 2 Institute of Molecular and Translational Medicine into a unique single E. coli host cell that carries a hybrid chemo- taxis/osmoregulation cascade by which de novo gene expression Introduction and aims The sensitivity of DNA profiling can be induced. methods based on microsatellite genotyping process has increased Here we describe an independent reconstruction and exami- since the introduction of the first commercial fluorescent mul- nation of the scaffold idea and of the quality of the produced tiplex kit through improvements in PCR (polymerase chain TNT biosensor. The reconstructed E. coli hybrid scaffold is an excel- reaction) or primer chemistry and low-template modifications lent biosensor for ribose in the nM-range, using wild-type RbsB. In of protocol (i.e. increase of cycle number, post-PCR cleaning of contrast, both in vitro and in vivo results suggest that the mutant amplicons, and/or increase of injection time). These changes with RbsB protein is not able to bind TNT, nor able to induce reporter various successes tried to faithfully generate electrophoreogram gene expression in presence of TNT. In order to better understand representing signals from minute amounts of single or mixed bio- the importance of the various amino acid residues in protein fold- logical sample. However, it is sometimes desirable to increase the ing, stability and receptor binding, we substituted each residue of minor profile in the mixture (i.e. to boost a signal of felon over sig- RbsB by alanine and measured gene expression through the hybrid nal of victim). In this project, we tested whether CO-amplification signaling pathway. This information will be implemented in the at Lower Denaturation temperature (COLD) PCR that is used in used simulation algorithm to increase the predictive power of the clinical genetics settings able to improve the detection of single calculations. nucleotide mutant tumour DNA in a surplus of wildtype DNA, http://dx.doi.org/10.1016/j.nbt.2014.05.1865 and if so,whether it is amenable to STR (short tandem repeats) typing. Materials and methods DNA was extracted from cell lines and/or EDTA (ethylenediaminetetraacetic acid) treated blood using QiaGen kits, amplified in mixture or unblended either by homemade fluorescent singleplex (locus DXS101) or by commercial multiplex GenomeLab Human STR Primer Set (Beck- man Coulter, USA) to see if COLD PCR protocol bring any

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PC-06 (PCR). A correlation between recurrence of disease after surgery and analgesics or anesthetics used during or after surgery was also High Performance Computing Based Smart Scan for the found. Analgesia and anesthesia can affect amount of CTC because Identification of Species Based Unique DNA Sequences of their immunomodulatory effects. Kivanc Bilecen 1,∗ , Behnam Rahnama 2, Ender Altiok 2 Methods and patients: Influence of morphine and piritramide was studied in 121 patients with colorectal carcinoma in 1 (1) Okan University, (2) Duzen Laboratories Group our research. Detection of CTC was based on real-time PCR in sam- 2 Okan University ples from peripheral blood and bone marrow with using epithelial genes as markers (carcinoembryonic antigen - CEA and cytokeratin Nucleic acid based tools and techniques such as PCR, RT- 20 - CK20). Afterward, the presence of CTC was evaluated depend- PCR, DNA microarrays and lately DNA-hybridization-on-a-chip ing on the type of analgesia and disease-free and overall survival of devices provide reliable detection and identification of microor- patients. The main objective was to optimize analgesic techniques ganisms. These platforms rely on the presence of target specific after surgery to decrease risk of cancer recurrence. DNA sequences to be known in advance. These sequences, how- Results: One month after surgery, morphine-based analge- ever, can be hard to identify in close species or on the strain level. sia usually induced higher level of CTC. Disease-free survival of In this work we have developed a parallel algorithm to accurately patients was also shorter in case of morphine. specify and classify species and strain specific DNA sequences. Conclusion: Piritramide seems to be better analgesic tech- The parallel implementation of the intelligent search algo- nique than morphine, which negatively influences prognosis of rithm runs on the parallel GPGPU cores on an HPC server. Each patients with colorectal carcinoma after surgery. Kepler K20X computational card provides 2688 fine cores acces- Acknowledgement: This study was supported by sing to 6 GB of DDR5 shared memory. Such massive parallelization grants IGA UP LF 2014 019, CZ.1.05/2.1.00/01.0030 and allows us to compare variable window size of base pairs in an CZ.1.07/2.3.00/30.0004. unmatched performance in comparison with conventional methods. The intelligent scan results bidirectional and circular stream http://dx.doi.org/10.1016/j.nbt.2014.05.1867 search as well as comparing not only the same instance but also similarity scan up to a defined threshold. Well-established CUBLAS library allows comparison of the determinant of sample matrices in PC-08 portion of microseconds rather than sequential scanning of each base pare. Differential Expression of Circulating miRNAs Fol- Our algorithm successfully identified unique markers (70 to lowing Resistance Exercise and Carbohydrate/Protein 120 bp) to differentiate Bacillus cereus and B. subtilis. Locations Supplementation of these markers on the chromosome have also been taken into Foued S. Espindola 1,∗ , Olga Bocanegra 2, Renata Teixeira 2, Maria account. Our second set will include Salmonella group as this Siqueira 2, Matheus Gomes 2, Miguel Diaz 2 group is highly important for the food industry. Our results will 1 Universidade Federal de Uberlândia compare marker selection and validation studies, and also their 2 Institute of Genetics and Biochemistry - Universidade Federal de Uberlândia identification power in PCR and RT-PCR. http://dx.doi.org/10.1016/j.nbt.2014.05.1866 We investigated the levels of expression of 12 circulating miR- NAs (c-miRNAs) involved in cell proliferation, differentiation, angiogenesis, inflammation and glycemic control in response PC-07 to resistance exercise (RE) and dietary supplementation. Twelve subjects performed 10 sets of 10 repetitions with 80% of Influence of analgesia on circulating tumor cells in their respective 1RM followed by either carbohydrate or carbo- patients with colorectal carcinoma hydrate/protein supplementation in a randomized single-blind Hanusˇ Slavík 1,∗ , Emil Berta 2, Josef Srovnal 2, Andrea Prokopova 2, counter-balanced design. Samples of blood were collected before Lenka Radova 2, David Vrana 3, Marian Hajduch 2 RE, 03 and 24 hours afterwards. The relative expression data of all of the genes were analyzed using a two-way analysis of variance 1 Palacky´ University Olomouc with repeated measures. The molecular response in the group that 2 Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacky University, Czech Republic supplemented with protein was more pronounced for c-miRNAs 3 Department of Oncology, Faculty of Medicine and Dentistry, Palacky Univer- involved in the regulation of myogenesis, particularly hsa-miR- sity, Czech Republic 133a and 503. Both treatments revealed a differential expression of the c-miRNAs hsa-miR-126 and 16, which are associated with Introduction: Circulating tumor cells (CTC) can create metas- angiogenesis. We argue that hsa-miR-133a might be associated tases, which are responsible for 90% causes of deaths at patients with satellite cell proliferation, and possibly partially responsible with a cancer. CTC are located in a blood stream or bone marrow for muscle hypertrophy following RE. Further, both the up- and and they are most common cause of cancer recurrence after surgi- down-regulation of hsa-miR-126 and 16, respectively, are likely to cal resection of the primary tumor. Presence of CTC has become reflect neovascularization. These findings support the hypothesis an important prognostic and predictive factor, which is possible that circulating miRNAs bear paracrine-like functions and thus, are to study by molecular methods such as polymerase chain reaction involved in cell-to-cell crosstalk.

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Support: FAPEMIG, CNPq and CAPES principal objectives in diagnostics. Using random M13 phage dis- http://dx.doi.org/10.1016/j.nbt.2014.05.1868 play libraries on the whole U937 cells, we selected a clone, named EIII6, displaying a peptide RKIVHAQTP that preferentially recog- nizes these cells. The human promonocytic cell line U937 is a PC-09 model for leukemia, cancer therapeutics and in vitro hematopoietic cell differentiation. The relationship between fragile sites gene of FHIT in Therefore, we directly labeled the phage clone EIII6 with fluo- type II pneumocytes and idiopathic pulmonary fibrosis rescein isothiocyanate (FITC) for microscopy imaging application. This method allowed to identify fixed U937 cells, while preserving Fei Duan ∗ , Duan Fei, Gu Yu binding affinity of labeled phage clones. Microscopic observations Hebei University showed that U937 cells were fluorescently labeled. In order to further investigate the interaction between U937 Objective: The relationship between fragile sites gene of FHIT cells and EIII6, we utilized Raman Spectroscopy. Spectral peaks in type II pneumocytes and idiopathic pulmonary fibrosis(IPF). observed at about 980, 1008, 1185, 1215, 1320,1340,1455, 1589 Methods: With the techniques of cellular biology, molecular and 1660 cm−1, are commonly assignable to proteins, saccharides biology and immunocytochemisty (ICC), changes in expression of and lipids components of U937. These Raman peaks in U937-EIII6 fragile sites genes, FHIT and WWOX, were investigated to provide complex are shifted in position or reduced in intensity when com- a foundation for gene therapy against IPF. pared to U937 alone, suggesting that phage-probe were selectively Results: 1.WWOX and FHIT were found to be expressed highly localized at cell membrane. Thereafter, we realised networks con- in type II pneumocytes of rat in sham-operated group. Com- sisting of EIII6 phage clone and Ag-Nanoparticles (AgNPs) as signal pared with sham-operated group, no significant difference in the reporters in Surface Enhanced Raman Spectroscopy (SERS) to bet- protein expression of WWOX and FHIT were found in model ter discriminate U937 cells. With this approach, U937-EIII6 Raman group for 7 days (P>0.05). After 14 and 28 days, expression of spectral features become sharper and more intense, confirming the WWOX and FHIT at the protein level was reduced greatly in selective phage-cell interaction. model group (P<0.05). Nevertheless, tetramethylpyrazine signifi- Both methodological approaches, proposed in this work, allows cantly enhanced FHIT and WWOX expression, compared to model to quickly and selectively identify U937 and could be extended to group (P<0.05). the identification of other types of neoplastic cells. 2. After 7 days, no significant difference in the mRNA expres- http://dx.doi.org/10.1016/j.nbt.2014.05.1870 sion of WWOX and FHIT were found between all groups. After 14 days, the expression of WWOX and FHIT at the mRNA level was reduced greatly in model group when compared with sham- PC-11 operated group. Nevertheless, tetramethylpyrazine significantly enhanced mRNA expression of FHIT and WWOX, compared to Glycosphingolipids as specific markers during the differ- model group. Results obtained after 28 days of administration were entiation of mini-pig bone marrow mesenchymal stem similar to observation after 14 days. cells toward neuronal cells Conclusion: We demonstrate that the expression of fragile Young-Kug Choo 1,∗ Malg-Um Lim 1 Dong Hoon Kwak 1 Ju-Hyoung sites gene of FHIT and WWOX is altered during IPF. Tetram- , , , Lee 1 Sung-Youn Heo 1 Ji-Su Kim 2 Sun-Uk Kim 2 Kyu-Tae Chang 2 ethylpyrazine suppresses the alteration in FHIT and WWOX, , , , , suggesting the medication could play a protective role for IPF. 1 Wonkwang University 2 Korea Research Institute of Bioscience and Biotechnology(KRIBB) http://dx.doi.org/10.1016/j.nbt.2014.05.1869

For development of specific markers during the differentiation of mini-pig bone marrow mesenchymal stem cells (mpBMSCs) PC-10 toward neuronal cells, we studied their glycosphingolipid pattern, Phage display as a tool for rapid in vitro cell characteri- with particular attention to gangliosides. mpBMSCs contained zation by fluorescence imaging and Raman spectroscopy GD3 etc as major gangliosides. In order study, their distribution in mpBMSCs and its possible change during the differentia- 1,∗ 1 1 Laura De Plano , Federica Calabrese , Germana Lentini , Marco tion of neuronal cells. When mpBMSCs were cultured under 1 1 2 3 Nicolò , Domenico Franco , Enza Fazio , Sebastiano Trusso , neural differentiation media contained BME/DMSO/BHA, most 4 2 1 Alessandro Allegra , Fortunato Neri , Salvatore Guglielmino of mpBMSCs acquired the distinctive morphological features 1 Department of Biological and Environmental Sciences, University of Messina like neural cells. In differentiated cells, expression of neural 2 Department of Physics and of Earth Sciences, University of Messina markers such as neural precursor marker (nestin), neuronal mark- 3 C.N.R. Chemical-Physical Processes Institute (Messina) ers (␤-tubulin, neurofilament-M) and astrocyte marker (GFAP) 4 Department of General Surgery, Oncology and Pathological Anatomy, Uni- were further demonstrated by revered transcription-PCR, western versity of Messina blotting and immunofluorescence. Specifically, we find that a significant increase in GM1 and GD3 expression was observed during The discovery of new markers for the identification and dis- neural differentiation of mpBMSCs. These results suggest that GM1 crimination of cell types such as neoplastic cells is one of the

www.elsevier.com/locate/nbt S107 STEM CELLS, GENE THERAPY, BIOMARKERS AND DIAGNOSTIC TOOLS New Biotechnology · Volume 31S · July 2014 and GD3 may play a role in the neural differentiation process of Aim: To test and analyse dynamics of biofilm formation in mpBMSCs. clinical strains of S. epidermidis and S. haemolyticus http://dx.doi.org/10.1016/j.nbt.2014.05.1871 Matherials and Methods: The study covered 10 strains of each S. epidermidis and S. haemolyticus. Overnight cultures in Tryp- tic Soy Broth (TSB) were diluted 1:100 in fresh TSB and placed ◦ PC-12 in sterile 24-well plates and incubated in 37 C. After 2 h, 6 h, 12 h, 24 h or 48 h of incubation, the contents of each well were AKT and ERK dependent differentiated Mesenchymal aspirated and the remain biofilm was washed three times with Stem Cells survival promoted by Pulse Electromagnetic 200 ␮l of phosphate-buffered saline. Biofilms were fixed, dried and Field then stained with crystal violet. At the end, 96% ethanol was added to each weel and the optical density (OD) of the stained Jung-Keug Park , Enerelt Urnukhsaikhan, Hyunjin Cho biofilms was measured at wavelength of 600 nm. The measurement Dongguk University was repeated two times and averaged. Fresh uninoculated TSB treated with the same procedure as test samples was used as neg- Researchers have reported that BM-MSC have ability to differ- ative control. Strong biofilm producing Staphylococcus epidermidis entiate into neuron cells in induction media. Also some studies strain RP62A was used as positive control. Study was supported by reported that magnetic field has a role in promoting BM-MSCs dif- K/DSC/001393 grant. ferentiation into neural cells. However from in vitro and in vivo Results and conclusions: Tested strains differed in dynam- studies it is not clear how the methods used induce BMMSCs ics and strength of biofilm formation. S. epidermidis strains were to differentiate into neural cells. Successfully differentiated the stronger biofilm-producers than S. haemolyticus. BM-MSCs would be useful for developing clinical stem cell trans- http://dx.doi.org/10.1016/j.nbt.2014.05.1873 plantation strategies against central nervous system diseases. After differentiation, cells are no longer live, especially after chemical differentiation. We focus on maintaining cellular survival through PC-14 the AKT, ERK pathway. In this work, we show the effect of Pulsed Electromagnetic Field (PEMF) on cell survival and differentiation A simple pre-analytical tool to enrich bacterial DNA: potential of hBM-MSC in vitro. hBM-MSC were cultured with implications for blood sepsis diagnosis application induced medium, and they displayed neuron-like morphology. Ngo Tat Trung , Le Huu Song The cells viability was increased after exposure to 10 mT PEMF Tran Hung Dao University Hospital for 24 h. Differentiation medium treatment combined with PEMF exposition reduced the death cells rate. NeuroD1, NF-L proteins Despite of improved modern medicine, blood sepsis is still are expressed those early stage markers of neuronal differentia- remaining as a high risk of mortality. One barrier to therapeutic tion and p-ERK, p-AKT after induced by PEMF. Our result is shown advancement of the disease is the challenges to accurately iden- that PEMF could play a role in regulating ERK and AKT pathway. tify causative pathogens in a limited time frame. In this content, Also phosphorylation of AKT and ERK promote a survival effect by blood culture is still widely accepted as routine detection approach. inhibiting or regulating the pro-apoptotic, anti-apoptotic proteins However, this conventional method embeds various drawbacks. such as BAX, BAD and Bcl-xL. Furthermore PEMF exposure could Nevertheless, the multiplex PCR based detection art, although is be inducing differentiation of hBM-MSCs. considered to be a complimentary approach, it is still confronted http://dx.doi.org/10.1016/j.nbt.2014.05.1872 by many challenges; of that, the most important factor is the inhibitory effect of human DNA: Imagine that an optimized PCR reaction using DNA extracted by common DNA extraction kits PC-13 can only sense pathogen’s ribosomal 16S pieces if the bacterial load exceeds roughly 500 CFU/ml, whereas typical blood sepsis The dynamics of biofilm formation in clinical strains patients would have bacterial density of 100–10000 CFU/ml blood, of Staphylococcus epidermidis and Staphylococcus therefore in many case, the PCR is unable to detect pathogen’s haemolyticus associated with neonatal infections genetic materials. To circumvent this problem, various techni- Monika Grzebyk ∗ , Anna Piotrowska, Monika Brzychczy-Włoch, cal prompts have been resorted to deplete human DNA prior to Piotr Heczko the PCR reaction. A common DNAse-based bacterial DNA isolation Kit (MolYsis) can acquire an enrichment ratio of 40.000 Jagiellonian University Medical College, Chair of Microbiology hence allowing to detect 50 S. aureus CFU/ml blood, however, its price is hardly to be accepted in lower income community Introduction: Staphylococcus epidermidis and Staphylococcus (about £10/extraction). In the recent study, we introduce a simple haemolyticus are major causes of device-related infections in pre-analytical protocol that combines the use of polar detergent Neonatal Intensive Care Units. The main virulence factor in S. epi- solvent and basic pH to remove 99% human DNA from patients’ dermidis is biofilm formation and S. haemolyticus also form biofilm, specimens hence making our downstream PCR assays’ technical but this process haven’t been well decribed jet. Biofilm formation sensitivity of 10 to100 CFU bacteria/ml blood. is usually tested in 24- or 48-hour cultures, but the dynamics and the early stages of biofilm formation are still unlcear. http://dx.doi.org/10.1016/j.nbt.2014.05.1874

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Biomedical materials PD-02

PD-01 Putative motility-related genes in Gluconacetobacter xylinus. Initial verification of their influence on Bio- Role of clpP and tpi genes in bionanocellulose biosynte- NanoCellulose biosynthesis hesis by Gluconacetobacter xylinus ∗ Katarzyna Kubiak , Bartłomiej Porebski˛ , Marzena Jedrzejczak-˛ Marzena Jedrzejczak-Krzepkowska ∗ , Malgorzata Parniewska, Krzepkowska, Przemysław Rytczak, Karolina Ludwicka, Marek Klaudia Jadczak, Michal Rozanski, Katarzyna Kubiak, Przemys- Kołodziejczyk, Stanisław Bielecki law Rytczak, Karolina Ludwicka, Marek Kolodziejczyk, Stanislaw Institute of Technical Biochemistry, Technical University of Lodz Bielecki Institute of Technical Biochemistry, Lodz University of Technology BioNanoCellulose (BNC) synthesized by Gluconacetobacter xylinus is intensively investigated as biomaterial for medical appli- Among the microorganisms that produce bionanocellulose, cations (best known as wound healing and bone regeneration acetic bacteria of the species Gluconacetobacter xylinus are the most material) and as a drug delivery system [1,2] but also has potential efficient producers. Bacterial cellulose produced by these orga- in various materials preservation (such as skin, paper and textile) nisms have unique properties such as high hydrophilicity, the and in electronics. The best known commercial usage of BNC is lack of cytotoxicity, excellent biocompatibility, is non-allergic, not Nata-de Coco dietary dessert popular in Asia. Scientifically BNC mutagenic nor teratogenic. Due to these properties and possibility gained an attention of tissue engineers, since its features make it a of modification the bionanocellulose has found wide application suitable material for scaffolds production [1,3]. However, in some in the food, paper, textile and chemical industry as well as in applications porosity of BNC is not accurate for mammalian cells medicine, e.g. production of wound dressing. This biopolymer ingrowth (pores diameter < 20 ␮m). One approach to address this has been tested for usefulness as implants of the ears, cartilage issue is genetic modification of G. xylinus in order to obtain strain implants, a mesh for hernia operation, as well as a material for capable to synthesize BNC with desired features. The aim of this tracheal reconstruction [1]. Industrial-scale production of bio- project is to influence the motility of the cells secreting cellulosic nanocellulose is still difficult and not sufficiently cost effective. fibers. These bacteria are susceptible to phenotypic variation, which very After sequencing the G. xylinus genome [4] we have searched often decreases the ability for the biosynthesis of cellulose. Unfor- for genes involved in auxiliary motility mechanism and identi- tunately, current knowledge about the molecular mechanisms of fied two encoding proteins potentially related to it. Subsequently, cellulose biosynthesis and its regulation is insufficient to effec- adequate disruption mutants of G. xylinus ATCC53582 strain tively affect the intensification of biosynthesis (increase efficiency were obtained. Motility test, conducted on semi-solid agar plates, or accelerate the process of production) and change the properties showed less intense colony spreading. The initial analysis of and structure of the obtained cellulose membranes. mutants’ phenotype revealed inhibition and limited BNC pro- The aim of current research is to verify the effect of genes such duction. Furthermore structural changes in BNC membranes were as clpP (encoding ATP-dependent Clp protease proteolytic subunit) investigated. and tpi (encoding triosephosphate isomerase) selected in previous References studies of transcripts differentiating the cells of G. xylinus synthe- [1].Arch Med Sci 2013;9:527. sizing cellulose (Cel + ) from these non producing cellulose (Cel-) [2].J Pharm Sci 2012;102:579. on cellulose biosynthesis. [3].Tissue Eng Part A 2010;15:1091. Reference [4].J Biotechnol 2014;176:18. [1].Kowalska-Ludwicka K, Cala J, Grobelski B, Sygut D, Jesionek- http://dx.doi.org/10.1016/j.nbt.2014.05.1876 Kupnicka D, Kolodziejczyk M, Bielecki S, Pasieka Z. Arch Med Sci 2013;9(3):527–34. http://dx.doi.org/10.1016/j.nbt.2014.05.1875

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Biopharmaceuticals Objective: The aim of the present study was to investigate the effect of P-Protein inhibition, by using P-protein small interfering PE-01 RNA (siRNA), on melanogenesis in Melan-a melanocyte. Methods: si-RNA for p Protein was introduced into Melan-A Functions of natural pigments on gastric ulcer and can- cells. Melanin content, cell viability, PCR and western blot for cer tyrosinase were performed. Hyo-Ihl Chang 1,∗ , Sun-Joong Kim 2, Jee Min Kim 1 Results and Conclusions; It has been observed the both P- protien and mRNA level were significantly lowered by the siRNA 1 Korea University treatment. siRNA of P-Protein also suppressed melanin synthesis 2 UC burkeley without any cytotoxicity in the melan-a melanocyte cells. These results suggest that molecular approaches using siRNA targeting The Natural pigments have many applications in inflammatory, P-protein may provide a novel approach for the control of the and oxidative related damage as well as in cancer chemother- melanogenesis apy. Recently, precise cellular roles of natural pigments, such as modulator of key cellular signaling pathway on variety dis- http://dx.doi.org/10.1016/j.nbt.2014.05.1878 eases, are elucidated. On based on antioxidant, anthocyanins reduced naproxen-induced gastric ulcer. Anthocyanins reduced the level of lipid peroxidation and increased the level of the PE-03 antioxidant enzymes. Anthocyanins increased the expression of Sex hormones regulate gender-dimorphic hepatic fetuin Nuclear factor E2-related factor 2 (Nrf2) which is transactivator expression in rats for cellular defense genes. Interestingly, anthocyanins induced gastrointestinal-glutathione peroxidase expression via Nrf2 that Jong Won Yun ∗ , Sang Woo Kim, Jae Heon Choi, Jung Won Choi bind to regions of antioxidant response element (ARE) in GI-GPX Daegu University promoter. Otherwise, Shikonin, and genipin stimulates production reactive oxygen species (ROS) in gastric cancer cells. They To date, there are limited studies on the sex-specific relation- induced apoptotic cell death in gastric cancer cells in a caspase ship between fetuins (Ft-A and Ft-B) and metabolic diseases. Our dependent manner. They also induced cell cycle arrest at G2/M recent proteomic study has shown that fetuins may play sex- phase via regulation of p21 by early growth response1 (Egr1). dependent roles in obesity and diabetes. In the present study, we The p21 contains promoter region of Egr1 binding motif. Tran- investigated the expression of hepatic fetuins with respect to the sient expression of Egr1 in AGS cells enhanced shikonin and effects of sex hormones both in vivo and in vitro. A sex hormone- genipin-induced p21 promoter activity, whereas suppression of treated rat model was established in order to study the effects of Egr1 expression by small interfering RNA attenuated the ability sex hormones on hepatic fetuin expression. Animal experiments of shikonin and genipin induced p21 promoter activity. Antho- revealed that 17␤-estradiol (E2)- and dihydrotestosterone (DHT)- cyanins improve gastric ulceration through Nrf2 associated with treated rats showed opposite effects in terms of body weight gain antioxidant enzymes, such as GI-Gpx pathways. And, shikonin in both genders. Interestingly, Ft-A and Ft-B were sex-dependently and genipin induced cell damage in AGS cells through the expressed in the livers of rats, responding to different regulatory Egr1/p21 pathways. modes of sex hormone receptors (ER␣,ER␤, and AR). To validate http://dx.doi.org/10.1016/j.nbt.2014.05.1877 in vivo data, rat normal liver cells were treated with E2 or DHT at different concentrations, and similar expression patterns as those in the animal-based experiments were confirmed. We found that PE-02 these changes were mediated via sex hormone receptors using antagonist experiments. The results of the present study indicate Effect of P-Protein Inhibition by si-RNA on Melanogene- that sex hormones induce gender-dimorphic expression of hepatic sis in Melan-A Cells fetuins directly via sex hormone receptors. To the best of our Eunki Kim ∗ , E. Noh knowledge, this is the first approach to address the effects of sex hormones on hepatic fetuin expression as well as possible roles of Inha University fetuins in gender-dimorphic obesity development.

Background; Hyperpigmentation diseases, especially http://dx.doi.org/10.1016/j.nbt.2014.05.1879 melasma and lentigines, are major psychological diseases in most of the societies. The obsession for the clean, fair and healthy skin is one of the basic instincts of every individual. For such diseases, a number of melanogenesis inhibitors have been screened. They were effective but their side effects cause many complications. Pink eye dilution protein (P-protein) is a structural protein in the melanosome that plays a critical role in cellular melanogenesis.

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PE-04 (5 ml/kg). Morphological examination includes ulcer index for hemorrhage and hematoxylin and eosin staining was performed to Immunomodulatory activity of natural polyamines in analyze the severity of ulceration. Gelatin zymography was done murine macrophages to determine the expression of MMP-9 and a number of biolog- Sun Chul Kang ∗ , Anil Kumar Chauhan, Souren Paul, Rekha Jakhar ical and immunological tests were carried out to determine the antioxidant and cytokine levels. Rats challenged with alcohol, Daegu University developed severe injury in gastric mucosa with increased level

of pro-inflammatory mediators like TNF-␣, Prostaglandin E2 and Macrophages are the key players of innate immunity, defend- nitric oxide. Expression of MMP-9 was up-regulated and less pro- ing the body from foreign invaders through phagocytosis. In duction of antioxidant enzymes (SOD and GSH) was documented the present study we investigated the potent role of natural after treatment with alcohol. In thymol pretreated rats, a signifi- polyamines in modulation of macrophage activity by analysing cant decrease in ulcer index, level of pro-inflammatory cytokines all the sequential steps involved during phagocytosis. Initially, and nitric oxide was observed. Expression of MMP-9 was down- we treated splenocytes with three natural polyamines (PUT, SPD regulated and antioxidant enzymes production was increased in and SPM) to test their ability in proliferation of cells and involve- thymol pretreated group. In conclusion, study suggests that thy- ment in mitosis. Thereafter, we detected their role in promoting mol protects gastric mucosa injury from ethanol consumption by membrane fluidity on RAW 264.7 cells, which is essential during down-regulation of MMP-9 and inflammatory cytokines. uptake of particles. Phagolysosome fusion activity of macrophages in the presence of polyamines was then evaluated by measuring http://dx.doi.org/10.1016/j.nbt.2014.05.1881 acid phosphatase through p-nitro phenyl phosphate. Further, we determined the capacity of polyamines in generation of superoxide anion to create respiratory burst inside the macrophages. PE-06 We find decrease in the proliferation of splenocytes in the pres- 7-Hydroxydehydronuciferine induces human melanoma ence of polyamines which proved their potent role as strong A375.S2 autophagy and apoptosis and inhibits metasta- immunomodulator. Uptake capacity of macrophages was observed sis in vitro and in vivo to be enhanced after treatment with polyamines and also increased lysosomal activity of macrophages was detected at a concentra- Hui Min Wang tion dependent manner. Percentage of NBT reduction calculated Kaohsiung Medical University for superoxide anion generation revealed that polyamines poten- tiated this property of macrophages too. We also determined the Melanoma is the deadliest cancer. We identified 7- anti-complementory activity of polyamines by detecting classical hydroxydehydronuciferine (7-HDNF) isolated from the pathway of complement which showed to be effective, suggesting leaves of Nelumbo nucifera Gaertn cv. Rosa-plena to be a its command over unwanted activation of complement. This study bio-active agent against human melanoma A375.S2 cells. 7- presents the potential role of polyamines as an immunostimula- hydroxydehydronuciferine (7-HDNF) was known to induce tory drug, which could be effective to treat various immunological autophagy and apoptosis response mechanisms, and anti- disorders. migratory activity of melanoma in vitro and in vivo. Cell http://dx.doi.org/10.1016/j.nbt.2014.05.1880 proliferation assay was used to test cell viability. Acridine orange (AO) staining and flow analysiswere applied to observe cell morphology. The apoptotic cell death ratio was measured via PE-05 two-dimensional flow cytometry by annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) double stained. Western Thymol protects gastric ulcer induced by alcohol blot was applied to examine protein expressions whereas wound through regulation of matrix metalloprotein 9 activity healing assay was to examine cell activity. Strong anticancer Anil Kumar Chauhan ∗ , Sun Chul Kang effects of 7-HDNF were exhibited in a dose-dependent man- nerand displayed minor cytotoxicities on normal human skin Daegu University cells.7-HDNF induced the formation of intracellular vacuoles and the augmentation of acidic vesicular organelles (AVO). 7-HDNF In gastrointestinal disorders, ulcer is a common disease with increased the cellular arrest in cell cycle at G2/M phase. Cellular multiple etiologies and most of them are observed due to high membrane asymmetry loss was confirmed. Protein expressions consumption of alcohol. Matrix metalloproteinases (MMPs) are were discovered to verify autophagy and apoptosis response a family of zinc-dependent enzymes capable of degradation of mechanisms sharing the associated pathways. 7-HDNF presented extracellular matrix and are key players in various inflamma- the high-quality anti-migratory activity. 7-HDNF inhibited tory diseases and among them MMP-9 is found to play major melanoma tumor growth in mice xenograft model, accompanied role during gastric inflammation. In this study, we aimed to with a decrease of phosphorylation of AKT. We demonstrated the determine the roles of thymol in expression of MMP-9 during mechanism of this compound starting with the formation and ethanol induced gastric ulcer model in vivo. Sprague-Dawley accumulation of AVO leading to autophagy. 7-HDNF caused the rats, pretreated with thymol (10 mg/kg) or normal saline as con- cellular membrane asymmetry loss, triggering the G2/M cell cycle trol were subjected to intragastric administration of 95% ethanol arrest in caspase-dependent apoptosis. 7-HDNF presented high-

www.elsevier.com/locate/nbt S111 BIOPHARMACEUTICALS New Biotechnology · Volume 31S · July 2014 quality anti-migratory bio-functions and inhibited melanoma animal models.[2,3] Therefore, they become promising candidates tumor growth in mice xenograft model. for new drug development. http://dx.doi.org/10.1016/j.nbt.2014.05.1882 The main interest of our group is to modify lupane and oleanane derivatives in order to improve their pharmacological properties and to increase their therapeutic index. Whereas des-E ␤ PE-07 -ketoacid (des-E lupane derivative) is our most active semisyn- thetic triterpenoid (cytotoxic on a set of 30 cell lines including

Oleuropein attenuates visceral adiposity in high fat diet- MDR phenotypes with IC50 0.32 ␮mol/L on K562) causing fast induced obese mice through the modulation of WNT10b- and selective apoptosis, the A-ring modified betulin and betulinic and galanin-mediated signalings acid derivatives that we prepared later have a completely different mechanism of action. In order to obtain another hit compounds, Taesun Park ∗ , Narae Kuem, Sujin Song we synthesized a set of derivatives with various substituents in the Yonsei University position 2 of the skeleton and found them to be highly cytotoxic on T-lymphoblastic leukemia CCRF-CEM cell line. The mechanizm The aim of the present study was to investigate the anti- of action is still being investigated; however, we are able to give obesity effect of oleuropein on high-fat diet (HFD)-induced body some basic assumption about the structure–activity relationships. weight gain and visceral adiposity in mice, and to explore the underlying mechanisms involved. C57BL/6 N mice were fed with the normal diet (ND), high-fat diet (HFD; 40% fat of total References energy) and HFD-supplemented with 0.03% oleuropein (OD) for [1].DzubakP, Hajduch M, Vydra D, Hustova A, Kvasnica M, Biedermann 10 weeks. OD-fed mice significantly reduced HFD-induced body D, Markova L, Urban M. J Sarek Nat Prod Rep 2006;23:394–411. weight gain and visceral adiposity. Oleuropein also significantly [2].Salvador J-A-R. Pentacyclic Triterpenes as Promising Agents in Cancer. reversed the HFD-inducedelevations of adipogenic-related gene Hauppauge NY: Nova Science Publishers, Inc; 2010. expression involved in WNT10b- and galanin-mediated signal- [3].Murph M. Research on Melanoma. InTech; 2011. ingsin adipose tissue of mice. Consistent with in vivo findings, http://dx.doi.org/10.1016/j.nbt.2014.05.1884 oleuropein dose-dependently suppressed lipid accumulation in 3T3-L1 cells during preadipocyte diffentiation. Additionally, exposure of the 3T3-L1 preadipocytes to oleuropein resulted in a PE-09 marked attenuation of the galnon (galanin receptor agonist) or SFRP2 (WNT inhibitor)-induced cellular lipid accumulation. This Intranasal Delivery of Nanoparticles Containing n- study demonstrated that oleuropein reduced body weight gain Butylidenephthalide to Treat Malignant Brain Tumor and visceral adiposity in HFD-fed mice. The protective effect of ,∗ Tzyy-Wen Chiou 1 , Yu-Shuan Chen 1, Yu-han Chiu 1, Yuan-Sheng oleuropein against HFD-induced adiposity in mice appeared to be Li 1, Dean-Kuo Hsieh 2, Horng-Jyh Harn 3 mediated through the upregulation of genes involved in WNT10b- 1 mediated signaling and downregulation of genes involved in Graduate Institute of Biotechnology,National Dong Hwa University, Hualien, Taiwan galanin-mediated signaling cascades. 2 Department of Applied Chemistry, Chaoyang University of Technology, http://dx.doi.org/10.1016/j.nbt.2014.05.1883 Taichung, Taiwan 3 Department of Medicine, China Medical University, Taichung, Taiwan

PE-08 Malignant brain tumor is a highly invasive disease with a very high death rate. The effective treatment method for this disease is Biologically active triterpenoids from birch and still an unmet medical need. Intranasal delivery method is a non- sycamore bark invasive administration route, may bypass the blood brain barrier and reduce the required drug dosage. n-Butylidenephthalide Lucie Borkova 1,∗ , Milan Urban 2, Marian Hajduch 3, Jan Sarek 1 (BP) has previously been shown as a drug candidate for treat- 1 Department of Organic Chemistry, Faculty of Science, Palacky University in ing glioblastoma multiforme (GBM) and temozolomide-resistant Olomouc, Czech Republic GBM. However, its hydrophobic property may limit some of 2 Department of Organic Chemistry, Faculty of Science and Institute of Molec- ular and Translational Medicine, Faculty of Medicine and Dentistry, Palacky the applications. Therefore, in this study, emulsiﬁcation method University in Olomouc, Czech Republic was used to prepare BP-containing nanoparticles in order to 3 Institute of Molecular and Translational Medicine, Faculty of Medicine and provide desirable features. The obtained nanoparticles showed Dentistry, Palacky University in Olomouc, Czech Republic high permeation ability as tested by artiﬁcial cellulose membrane or nasal septum squamous cells. The BP-containing nanoparti- Triterpenes are natural compounds usually occurring in plants cles also exhibited cytotoxic effect in human brain glioblastoma or marine animals and are often used in natural medicine in Asia. multiforme (GBM 8401) with IC50 vaule of 85 ␮g/ml. Moreover, They have various biological activities (e.g. anti-HIV).[1] A large the nanoparticles delivered by aerosol could reduce the tumor number of triterpenes are cytotoxic against wide range of tumor size in transgenic GBM mice model after the treatment for 30 cell lines, their anticancer activity was often observed in preclinical days. Based on these results, it is suggested that the preparation of BP-containing nanoparticles and its application in nasal

S112 www.elsevier.com/locate/nbt New Biotechnology · Volume 31S · July 2014 BIOPHARMACEUTICALS adminstration to treat GBM may have the potential in further PE-11 development for clinical use. Investigation on preventive measures of virus diseases http://dx.doi.org/10.1016/j.nbt.2014.05.1885 Zinaida Klestova 1 , Marina Marchenko 2, Viktor Tashuta 2,∗, Alla Voronina 3 PE-10 1 State scientiﬁc-control institute of biotechnology and strains of microorganisms The chemical synthesis of sulfur analogues of lysophos- 2 the Institute of veterinary medicine pholipids and cyclophospholipids 3 The Institute of pharmacology and toxicology

Przemyslaw Rytczak ∗ , Maria Koziołkiewicz, Andrzej Okruszek Viruses that caused diseases worldwide spread and have pos- Lodz University of Technology, Institute of Technical Biochemistry sibility for mutations. More dangerous viruses attack animal and human organisms, causing economic losses. Many countries are Development of new synthetic methods for the preparation creating biosafety systems for preventing viral infections, for of biologically active phospholipid derivatives is a challenging example, by developing new prophylaxis and antiviral methods. problem of membrane-chemistry and biochemistry today. In fact We investigated the possibility of using some plants and structural and dynamic studies of biomembranes for the establish- dosage forms in anti-viral therapy in sensitive model systems. In ment of structure-activity relationships, phospholipids-proteins the investigation we used: continuous cell culture of versenised interactions and mechanisms of action of phospholipids metab- swine embryonic kidney and BHK-21; test-model virus-member of olizing enzymes require the preparation of a great number of Coronaviridae family; propylenglycol extracts of plants- Aloe vera, phospholipids derivatives as the key step in advancing membrane Camellia sinensis var. ɑssamica, Echinacea purpurea, Húmulus lúpulus. biochemistry. Lysophospholipids have recently become the focus Results of experimental research of cytotoxic and antiviral of special attention since it was discovered that in addition to their action of preparations of a different origin are presented. MTD −5 −4 role in phospholipid metabolism they function as second messen- is10 mkl/ml. Index of CC50 -10 mkl/ml. In experiments on gers, exhibiting a broad range of biological activities in their own cell cultures we obtained the data that showed antiviral activity right. to 1,8 ± 0,04lg TCID50/ml. The chemical synthesis of new sulfur analogues of http://dx.doi.org/10.1016/j.nbt.2014.05.1887 lysophospholipids has been described, including phosphorothioate/phosphorodithioate derivatives of lysophosphatidic acids (LPA), phosphorothioate/phosphorodithioate derivatives PE-12 of cyclic phosphatidic acids (cPA) and phosphorothioate/phosphorodithioate derivatives of lysophosphatidylcholine In vitro assay by bioengineering of new antiviral drugs (LPC). For the preparation of LPA, cPA and LPC derivatives 1,∗ 2 3 both oxathiaphospholane and dithiaphospholane approaches Viktor Tashuta , Zinaida Klestova , Alla Voronina , Shota 4 have been employed. Each lysophospholipid analogue has been Dgebuadze synthesized as a series of five compounds, bearing five different 1 Institute of Veterinary Institute fatty acid residues, both saturated (12:0, 14:0, 16:0, 18:0) and 2 The State scientific control institute of microorganisms strains 3 unsaturated (18:1). The Institute of pharmacology and toxicology 4 Georgian Technical University Acknowledgements: This work was supported by a grant (PBZ-MNiSW-07/I/2007) from the Polish Ministry of Science and The viral diseases are still increasing. There is a wide spectrum Higher Education and by a grant (011/01/B/ST5/06383) from the of tested antiviral compounds, application of which inhibits or National Science Center (NCN-Poland). stops viral activity, but universal antiviral means are absent. http://dx.doi.org/10.1016/j.nbt.2014.05.1886 The investigation was aimed to finding the new approaches of the infection eradication. Methods: test-model viruses–members of Herpesviridae and Coronaviridae family; continuous animal cell cultures:–cell culture of versenised swine embryonic kidney (CCVSEK), BHK-21, Vero, SK-6. For investigation of anti-virus properties we have applied a complex of standard methods. Results of experimental research of cytotoxic and antiviral actions of 10 new substances from indol derivates are presented. We show the data about all examined means that significantly reduced the infection activity of tested virus strains in vitro systems in different ways. We tested different schemes of antivirus actions of tested substances in creation of treatment by viral infection process, as preventive as treatment. But the highest antiviral activity

www.elsevier.com/locate/nbt S113 BIOPHARMACEUTICALS New Biotechnology · Volume 31S · July 2014 among tested substances was shown by decreased viral infection The results were obtained through STCU grant # p450 by ﬁnan- activity to 7,04 ± 0,04 lg TCID50/ml. cial support of KCP. Conclusion: All tested means are suitable in using for clini- http://dx.doi.org/10.1016/j.nbt.2014.05.1888 cal research for testing in eradication virus infections. The results conﬁrm the perspective of tested means for production of new antiviral drugs.

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Bioprocessing and engineering Estimation of the optimal culture conditions needs a continuous analysis concerning biomass data, growth rate, oxygen PF-01 concentration and pH. In the context of this work a multisensory platform (shake ﬂask reader) was evaluated by monitoring

RAMOS: New applications of an established online tech- of three basic cultivation parameters (pH, pO2, and biomass) [1]. nology for shaken vessels The biomass sensor is detecting the cell growth by backscattered Tibor Anderlei 1,∗ , Tim Bürgin 2, Andreas Richter 2, Cedric Bürki 3, light through the flask wall at a wavelength of 625 nm. The mea- Wolf Klöckner 4, Kristina Meier 4 surement is fully non-invasive and the measuring interval can be reduced to a minimum of 7 s. The sensor signal is calibrated against 1 Forum Shaking Technology CDW or OD by application of logarithmic functions. With the 2 Kühner AG 600 3 ExcellGene SA given calibration it is used e. g. for screening procedures in order to 4 AVT Aachen determine the adequate moment of induction. Backscattered light is also sensitive to changes in particle size and opacity. Thus there RAMOS determines the oxygen transfer rate (OTR), the carbon is evidence that inclusion body formation within the cells can be dioxide transfer rate (CTR) and the respiratory quotient (RQ) of monitored online. microbial, plantal and cell cultures online. The respiration rates [1] Ude C., Beutel S., Findeis M., Andrzejewski D., John G. (OTR, CTR) are the most suitable measurable variables to quan- T., Scheper T. (2013): Online biomass monitoring of shake flask tify the physiological state of fermented cultures. RAMOS is the cultures via an optical multisensory platform, Dechema Früh- right tool to meet the PAT initiative of the FDA regarding shaken jahrstagung 2013, Frankfurt am Main, Germany 2013 bioreactors. http://dx.doi.org/10.1016/j.nbt.2014.05.1890 On the one hand RAMOS was made to measure in 250 mL glass shake flasks. Therefore the application field was limited. To overcome this bottle neck Kühner developed three new add-ons: PF-03 - MicrOTR: With this tool the user is able to measure OTR and CTR in microtiter plates. The main application of this tool is to Developing strategies to improve the recovery of find the right fermentations conditions for microtiter plates. a periplasmicaly expressed recombinant protein by - Adapter for disposable flasks: With this new development dis- manipulation of fermentation conditions posable flasks can be directly attached to the RAMOS-System. ,∗ Ioannis Voulgaris 1 , Alex Chatel 1, Gary Finka 2, Mark Uden 2, Mike Therefore the RAMOS system can now easily be used for the Hoare 1 application field cell cultivation. 1 On the other hand the application field was enlarged by apply- University College London 2 GlaxoSmithKline ing the RAMOS technology for testing of plastic components to determine their impact on the cultivation. A significant problem for the clarification of E.coli broths is the All the new features and their advantages will be presented and amount of cytoplasmic DNA which may have been released into results from microbial, plant and mammalian cell cultures will be the extracellular space by cell lysis. This leads to large increases shown. in viscosity which for example can make removal of cell solids http://dx.doi.org/10.1016/j.nbt.2014.05.1889 difficult. E.coli is not a natural protein secretor; hence, a degree of cell lysis is required for the product to be released from the periplasm to the extracellular space. Changes in the relation- PF-02 ship between protein and DNA release favouring protein release should favour the performance of the subsequent separation Online Monitoring of pH, Oxygen and Backscattered stages. Therefore, the balance between product and DNA release Light during Heterologous Protein Production in Dispos- must be carefully monitored and if possible controlled during the able Shake Flasks fermentation. Gernot Thomas John 1,∗ , Christian Ude 2, Thomas Scheper 2, Sascha We present strategies to facilitate a better recovery of an anti- Beutel 2, Michael Findeis 1, Damian Andrzejewski 1 body fragment from recombinant E.coli whilst diminishing the 1 PreSens Precision Sensing GmbH extent of the effect of nucleic acid release. These include by: (i) 2 Institut für Technische Chemie, Leibnitz Universität Hannover the manipulation of the cell growth rate in order to increase the outer cell membrane permeability (ii) the incorporation of Keywords Single-Use shake flask, online backscattered light, reagents within the broth designed to increase the cell membrane shakes flask reader, protein production, inclusion bodies permeability, (iii) the incorporation of reagents within the broth Shake flask cultivation is one of the classical methods to per- designed to remove selectively nucleic acids released during cell form proliferation of cells with low effort and cost. It provides lysis. precultures for the scale up and is suitable for standard screening Using an ultra-scale-down approach we have determined the routines like the evaluation of new media or cultivation condi- maximum level of DNA release into the culture while still allowing tions. satisfactory removal of cell solids by continuous flow pilot scale centrifugation. In this way a strategy may be developed integrating

www.elsevier.com/locate/nbt S115 BIOPROCESSING AND ENGINEERING New Biotechnology · Volume 31S · July 2014 fermentation operation and cell recovery to optimise the recovery cells and minimize the energy consumption in the process. In this of protein product in a well-clarified broth suitable for subsequent work a conceptual design of downstream process using supercriti- processing. cal fluid extraction with carbon dioxide and solvent extraction was http://dx.doi.org/10.1016/j.nbt.2014.05.1891 compared with traditional distillation process for butanol recovery in the broth by simulation. The raw material used in fermentation was glycerol and the main products of the metabolism of PF-04 C. pasteurianum were butanol and 1,3 propanediol. Supercritical extraction with carbon dioxide was applied to remove 1,3 propane- Aqueous Phase Partitioning–from Analytics to Process diol and glycerol then solvent extraction using n-butyl-butyrate was applied for butanol recovery. An integrated process using Jonathan Huddleston ∗ , Rana Hameed, Derek Fisher, Svetlana Igna- single supercritical extraction was also developed. Simulations tova were performed using SuperPro Design®. An economic evaluation Brunel Institute for Bioengineering was carried out to compare these systems. The integrated process using supercritical fluid extraction with carbon dioxide was an The presentation will focus on aspects of our work on the economically attractive scenario, although, butanol has a lower application of Aqueous Phase Partitioning both to the develop- purity than this process followed by solvent extraction with butyl ment of liquid-liquid extraction based approaches to the recovery butyrate. of biopharmaceuticals and to the development of a simple bio- http://dx.doi.org/10.1016/j.nbt.2014.05.1893 process analytical technology. We will consider the recovery of monoclonal antibodies using Counter Current Chromatography and highlight important differences in the dynamic flow regimes PF-06 of different configurations of equipment and their impact on the structural integrity of biomolecules. In addition, consideration will Assessing the volumetric productivity of an ultrafil- be given to some of the different operational regimes available in tration membrane bioreactor during the synthesis of this type of equipment and how these can impact upon overall galacto-oligosaccharides process efficiency. Andres Córdova ∗ Carolina Astudillo Andres Illanes Cecilia Guer- In contrast we will also present some aspects of our work , , , rero to understand the molecular basis of phase partitioning in the development of partitioning based assays having a strong struc- Pontificia Universidad Católica de Valparaíso turally related component. We will show from a theoretical and practical perspective how the technique of Analytical Phase Par- Galacto-oligosaccharides (GOS) are a potent prebiotic which titioning may be applied to the quantitative determination of allows the upgrade of underutilized lactose of the dairy industry. process derived changes and post-translational modifications of However, this industry is somehow reluctant to use enzymes, due biopharmaceutical products. to their high-cost owing to the high-volumes of lactose processing http://dx.doi.org/10.1016/j.nbt.2014.05.1892 that entails. Ultrafiltration membrane bioreactor (UF-MBR) allows the bioconversion of lactose into GOS, removing the reaction products while retaining the enzyme for re-use in a single step. PF-05 The objective of this research was to evaluate the effect of operational variables on volumetric productivity (␲) of GOS synthesis Simulation of butanol production by an integrated fer- when using an UF-MBR. mentation process using supercritical fluid extraction A tubular ceramic-membrane (50 kDa) and retentate recircula- with carbon dioxide tion mode was used. The reacting mixture was a lactose solution ␤ 1,∗ 2 (40%w/w, pH 4.5) containing Aspergillus oryzae -galactosidase Bernadete Delgado , Fernando Luiz Pellegrini Pessoa ◦ ◦ dosed at 50IU/glactose. The temperature (40 Cto60 C), the trans- 1 Federal University of Rio de Janeiro membrane pressure (PT) (2.5 to 4 bar) and cross-flow velocity (CFV) 2 UFRJ (3.5 to 7 m/s) were varied according to a 2k factorial design. All reactions were conducted for 240 min. Petroleum is becoming a scarce resource and alternative bio- Lower conversions were obtained at the higher levels of CFV fuels has been studied as biobutanol. It can be blended directly and PT, and at 40 ◦C.This can be attributed to a greater shear-force with standard oil-based fuels and has the advantage of been pro- on the enzyme, and more compaction of the solute on the mem- duced from glycerol. Nowadays glycerol is an excellent source brane which may adsorb the biocatalyst, decreasing its activity. At of raw material derived from biodiesel production. Clostridium 40 ◦C partial precipitation of lactose and a higher permeate viscos- pasteurianum uses glycerol as the main source of carbon but is ity may explain this situation. An optimized ␲ value of 30.53 (g productivity and butanol concentration is low. The main prob- GOS/L x h) was achieved at 60 ◦C, 3.5 m/s and 2.5 bar, increasing lem related to fermentation for butanol production is its toxicity ␲ by 318% (compared to a conventional batch-synthesis) by using to the microorganism Clostridium. Downstream process for recov- enzyme in two reaction-cycles. ering solvents in the diluted fermentation broth may increase butanol productivity by reducing the toxicity of solvents to the

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Work funded by Grants 1130059 and 11110402 FONDECYT, this study, the influence of solid state fermentation medium com- and CONICYT-PhD-Scholarship, Chile positions containing wheat bran, sugar cane bagasse and lemon http://dx.doi.org/10.1016/j.nbt.2014.05.1894 peel powder as agricultural by-products for pectin lyase production using Rhizomucor pusillus DSM 1331 were accessed. Response surface methodology (RSM) was used for medium optimization, PF-07 fermentation conditions, and the interactions among all the experimental parameters at flask level. Several physico-chemical Continuous photofermentative production of bio- parameters: pH, temperature, moisture content and fermentation hydrogen with Rhodobacter sphaeroides DSM 158 time of the production media were optimizedby using the D- optimal Design. The results show that pectin lyase production was Karsten Helbig ∗ , Rico Hellwig, Felix Krujatz, Thomas Bley, Jost significantly affected by fermentation time and moisture content Weber which stimulates themaximal enzyme production to 100 U/mL TU Dresden and specific activity of 45.24 U/mg at 30 ◦C temperature; 6 days of fermentation and moisture content of 120%. Under these opti- Because of the coming lack of fossil energy sources and to mized conditions, the predicted maximal activity was 107 U/mL. mitigate the global climate change there is a need for an environ- The obtained activity was two times higher than some of the mentally friendly fuel. Hydrogen could be produced from nearly common pectin lyase producers. Additionally, the fermentation omnipresent water and its utilization does not cause the emission process was scaled up from 10 g to 1 kg using a rotating drum type of environmentally harmful pollutants (like CO2). Light is a highly solid-state bioreactor in order to evaluate the difference between available source of energy. Thus, hydrogen production with pho- pectin lyase production in flask and at bioreactor level. In con- totrophic microorganisms has a potential to make a significant clusion, the application of response surface methodology had a contribution to use this energy and produce a renewable fuel. For noteworthy enhancement in pectin lyase production indicating industrial application there is a need to establish a continuous this method was a promising approach for enzyme production process. The aim of this study was to examine conditions for a and cost reduction continuous hydrogen production with purple-non-sulfur bacteria. http://dx.doi.org/10.1016/j.nbt.2014.05.1896 Rhodobacter sphaeroides is able to produce molecular hydrogen with the enzyme nitrogenase. For the study of production conditions a 1-L stirred glas bioreactor was operated as a chemostat and PF-09 equipped with 12 radial installed 50-w-tungsten lamps for light supply. The dilution rate has been varied to determine its influence Advanced clarification of cell culture supernatant by onto the hydrogen production rate. 3MTM EmphazeTM AEX Hybrid Purifier for fast and eco- Throughout the experiments the influence of different dilution nomic bioprocessing of recombinant proteins rates onto hydrogen production and the biomass concentration Michael Maurer 1,∗ Frederik Hoppe 2 Harald Schillinger 3 Melanie has been determined. Dilution rates have been regulated from , , , − − Hutter 2 Renate Kunert 4 Kurt Eyer 3 0.024 to 0.216 h 1. At a dilution rate of 0.12 h 1 the highest , , production rate (152 mL h−1 L−1) and a biomass concentration of 1 FH Campus Wien/School of Bioengineering 2 2.15 g L−1 were observed. Neither higher nor lower dilution rates FH Campus Wien/School of Bioengineering/ACIB 3 3 M Alpine/3 M Purification improved productivity. The higher the dilution rate the smaller 4 University of Applied Life Sciences an Natural Resources Vienna was the concentration of biomass. The shown data show the potential of Rhodobacter sphaeroides for a continuous photo-biological Mammalian cells are the most important expression platform hydrogen production. for the production of biopharmaceutical proteins. More than 50% http://dx.doi.org/10.1016/j.nbt.2014.05.1895 of all registered products are produced in those expression systems according to Ferrer et al. During this work, we used a CHO cell line secreting the fusion protein EPO-Fc for the simulation of a PF-08 biopharmaceutical manufacturing. For pharmaceutical products it is of particular interest to reduce Optimization of medium composition for the novel and eliminate biological contaminates, such as genomic DNA, host pectin lyase producer Rhizomucor pusillus DSM 1331 cell protein, endotoxins and viruses by efficient DSP operations. through response surface methodology On the other hand, biopharmaceutical companies are in a harsh Amira Rizk 1,∗ , Sonja Diercks-Horn 2, Mahmoud Yousef 2, Marcelo economic competition, what generates a strong need for shorter, Fernández-Lahore 2 save and economical processes (reduction of unit operations and the rise of single use systems). 1 Jacobs university Bremen TM TM 2 Downstream Bioprocessing Laboratory, School of Engineering and Science, In this study we evaluated the new 3M Emphaze AEX Jacobs University, Campus Ring 1, D-28759 Bremen, Germany Hybrid Purifier, which combines depth filtration with an all- synthetic construction, substantial chromatographic capability, Pectin lyase is a member of pectinases that plays an impor- and a defined 0.2 ␮m pore size in one single-use cartridge, for clar- tant role in food processing industries (e.g. juice clarification). In ification and purification very early in the manufacturing process.

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Further we compare the performance to a state of the art depth fil- PF-11 ter, 3MTM ZetaPlusTM EXT, and show the scalability from lab scale (25 cm2) to pilot scale (340cm2). Whisky by-products: a valuable source of protein and Data on a dramatic reduction of gDNA and a significant reduc- potential applications in aquaculture TM TM tion of HCP by the new 3M Emphaze AEX Hybrid Purifier will Jane White ∗ , Julio Traub, Dawn Maskell, Paul Hughes, Alan Harper, be presented. The advantage of early removal of biological con- Nik Willoughby taminates and the protection of chromatographic material will be Heriot-Watt University discussed. Ferrer-Miralles N., Domingo-Espín J., Corchero J., Vázquez E., Scotland is famous for its whisky, a tradition stretching back Villaverde A. Microbial factories for recombinant pharmaceuticals. over 500 years. There are over 100 malt distilleries in Scotland with Microbial Cell Factories 2009, 8:17 the capacity to produce 280 million litres of pure alcohol annu- http://dx.doi.org/10.1016/j.nbt.2014.05.1897 ally. This results in the generation of by-products, one of which is pot ale, the liquid residue remaining after the first distillation stage. At least 8 litres of pot ale is produced with every litre of PF-10 alcohol. It contains over 30% protein on a dry matter basis, originating from yeast and barley residues and its main value is as pot Adhesion of anaerobic microbial beer spoilers to stain- ale syrup, an animal feed produced by evaporation. Evaporation is less steel energy intensive, the high temperatures have a deleterious effect Gita Prochazkova ∗ , Milan Bittner, Martina Brozova, Tomas Branyik on protein quality, all components in addition to the protein are Institute of Chemical Technology Prague concentrated and it is only an option for larger distilleries. New markets for pot ale could be realised if cost-effective methods to To reduce the public health threat posed by food/beverage separate the protein components were available. These proteins pathogens or spoilers many sources of contamination have to be are a good match with the protein requirements of salmon feed considered. Increased focus is necessary in the case of anaerobic and may offer a local, sustainable and secure supply of protein for microorganisms. In the case of breweries, the highest risks are asso- Scottish salmon. The work presented here demonstrates the devel- ciated with the genus Pectinatus, but also the genus Megasphaera opment of novel processes for the energy-efficient extraction and must not be neglected. Due to their ability to form or to be a part recovery of valuable proteins. The composition of pot ale, meth- of microbial biofilms that are present on the surfaces of pipelines, ods for producing novel by-products and potential applications in floors, machinery etc. these microorganisms pose a continuous aquaculture are discussed. threat to beer contamination. http://dx.doi.org/10.1016/j.nbt.2014.05.1899 The focus of the presented work is help localize conditions/materials prone to colonization by anaerobic bacteria and thus to prevent undesirable biofilm formation in breweries. The PF-12 work can be divided into three parts: (i) characterization of the physicochemical properties (contact angles and zeta potentials) Pilot-scale biotrickling filter for hydrogen sulfide of two anaerobic bacteria (Pectinatus frisingensis and Megasphaera removal from biogas under continuous discharge flow cerevisiae) and of typical construction material’s surfaces (stainless Martín Ramírez ∗ , Fernando Almenglo, José Manuel Gómez, steel), (ii) prediction of the cell adhesion to stainless steel accord- Domingo Cantero ing to thermodynamic, classical and extended colloidal models, University of Cadiz and (iii) comparison of model predictions with experimental data from real adhesion tests carried out in test tubes with model sur- Biogas is a valuable renewable energy source. However, the aver- faces (stainless steel microparticles) under model conditions. The age H S concentration (0.1-2%) into the biogas stream limits many (in)consistency between model predictions and adhesion exper- 2 applications. Biotrickling filters (BTFs) have been studied under iments can help identify the crucial interactions for microbial aerobic and anoxic (nitrate like electron acceptor) conditions. In adhesion. the conventional mode operation, the liquid phase is recirculated http://dx.doi.org/10.1016/j.nbt.2014.05.1898 from bottom to the top of the column and a portion volume or small split of the flow is discharge to help to remove the oxidation products (sulfate and elemental sulfur). In this study, a pilot-scale BTF (diameter column 0.5 m, bed height 0.85 m, packing material: open-pore polyurethane foam) was feed with real biogas and treated water (TW) from a wastewater treatment plant (WWTP). The aim of this work was study the performance under discharge flow equal to the recirculation flow. Moreover, the mass transfer coefficient was determined. Nitrate concentrate solution was mixed with TW stream at the top of the BTF. The trickling flow rate was set to 1.7 m3 h−1, biogas

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flow rate were of 1, 2 and 3 Nm3 h−1 and nitrate mass flow rate PF-14 2– −1 were of 20 and 70 g N-NO3 h . The elimination capacities (EC) A comparative study of chromatographic matrices for were equal under both nitrate mass flow rate. The critical EC (H2S removal efficiency (RE) of 99%) was 37.9 gS m−3h−1 and the max- affinity chromatography of the diphtheria toxin variant imum EC was 88.4 gS m−3h−1. However, the nitrate consumption Cross-Reacting Material 197 was superior at the highest nitrate mass flow rate, probably due to Alessandra Stefan ∗ , Mattia Boiani, Luca Longanesi, Alejandro the higher gradient concentration and therefore the higher mass Hochkoeppler transfer to the biofilm. University of Bologna http://dx.doi.org/10.1016/j.nbt.2014.05.1900 Cross-Reacting Material 197 (CRM197) is a variant of the diphtheria toxin characterised by a single mutation, a glycine-glutamic PF-13 acid substitution at position 52 [1]. This mutation reduces its toxicity but maintains the same inflammatory and immunostim- Chromatographic Separation of Mono-PEGylated Teri- ulant properties. The conventional industrial-scale production of paratide Isomers CRM197 is performed using cultures of Corynebacterium diphthe- Wen-Yih Chen 1,∗ , Ching-Wei Tasi 1, Wei-Hung Kao 1, Li-Chiao riae, but recently we have proposed an alternative process for its Chang 2, Ruoh-Chyu Ruaan 1 over-expression in Escherichia coli [2]. Accordingly, the recombi- 1 National Central University nant protein, bearing a short artificial histidine tag, was purified 2 Scinopharm Inc., Taiwan by a metal chelating affinity chromatography (IMAC), performed either under denaturing and native conditions. Peptide drugs covalently conjugated with polyethylene gly- In order to investigate possible non-specific interactions col (PEG) polymers can effectively prevent protease digestion and between CRM197 and the matrix employed for the IMAC, we prolong the circulation half-life. Chromatographic purification of compared three types of polymers: a matrix consisting of agarose PEGylated peptide drugs is essentially critical in pharmaceutical beads (GE Healthcare Life Sciences), a macroporous silica matrix industry, especially for the positional PEGylated peptide isomers. (Machery-Nagel) and the Profinity IMAC resin, based on UNO- In this study, we aimed to interpret the separation mechanisms sphere beads (Bio-Rad). The agarose-based matrix showed the of mono-PEGylated Teriparatide isomers by reversed-phase chro- higher non-specific sorption of the CRM197 protein, leading to matography (RPC) and further to provide guidance for a better a low final recovery. On the contrary, the Profinity IMAC resin chromatographic separation of PEGylated isomers. Two mono- proved the maximum yield, likely due to a low retention of the pro- PEGylated Teriparatide isomers, N-terminal and Lys13 PEGylated tein in the column via non-specific interactions with the matrix. Teriparatide, were synthesized through the succinimidyl ester Moreover, the Profinity resin was found to be suitable for both the functionalized methoxy PEG (5 KDa) by controlling the pH of purification and the refolding of CRM197 and, accordingly, the PEGylated reaction buffer. Both two PEGylated Teriparatide iso- recovered protein featured enzymatic activity. mers exhibit high protease stability against trypsin, but their structural helicity decrease dramatically. However, two PEGylated References Teriparatide isomers are co-eluted by acetonitrile/H2O containing [1].Uchida T, Pappenheimer Jr AM, Greany R. J Biol Chem of 0.1% (v/v) trifluoroacetic acid. To separate these isomers, two 1973;248:3838–44. approaches were carried out. One is to tune the pH value of mobile [2].Stefan A, Conti M, Rubboli D, Ravagli L, Presta E, Hochkoeppler A. J phase. The results showed that two positional isomers are grad- Biotechnol 2011;156:245–52. ually separated as the pH value increased from 2.0 to 9.0. The http://dx.doi.org/10.1016/j.nbt.2014.05.1902 other is to alter the eluent composition. Based on the solubility parameters theory (thermodynamics theory), we changed the eluent composition from acetonitrile/H2O to tetrahydrofuran/H2O, PF-15 resulting in baseline separation of PEGylated peptide isomers. Both A two-step one-pot bioprocess for production of 11␣- approaches revealed that the difference of polarity term (Ddp) between peptide isomers plays an important role for baseline sep- hydroxyandrost-4-ene-3,17-dione from phytosterol aration. Consequently, we suggested that the tuning of mobile Dmitry Dovbnya 1 , Vyacheslav Kollerov 2, Sergey Khomutov 1, phase composition based on solubility parameters theory makes Danila Malov 1, Marina Donova 2,∗ it possible to achieve the separation of PEGylated isomers in chro- 1 G.K. Skryabin’s Institute of Biochemistry and Physiology of Microorganisms matographic operation. RAS http://dx.doi.org/10.1016/j.nbt.2014.05.1901 2 Pharmins Limited

11␣-Hydroxyandrost-4-ene-3,17-dione (11␣-HAD) is a primary adrenal steroid in mammalians and the key precursor in the syntheses of halogenated corticoids (pharmaceutically valuable analogs of natural corticosteroids). Conventional routes for its obtaining are based on chemical synthesis, or microbial hydrox-

www.elsevier.com/locate/nbt S119 BIOPROCESSING AND ENGINEERING New Biotechnology · Volume 31S · July 2014 ylation of androst-4-ene-3,17-dione (AD). AD in turn is produced ogy with affinity chromatography to efficiently purify the pDNA primarily with microbial biotransformation of natural sterols by results in a powerful tool for industrial manufacturing. some actinobacteria. References The aim of this work was to develop a bioprocess for obtaining of 11␣-hydroxyandrost-4-ene-3,17-dione from phytosterol. [1].Nunes JC, et al. Journal of Membrane Science 2012;415-416:24–35. [2].Sousa A, et al. Journal of Separation Science 2009;32:1665–72. Specific biochemical activities of two microbial strains were used as a basis for the two-stage bioprocess. On the first stage phy- http://dx.doi.org/10.1016/j.nbt.2014.05.1904 tosterol was converted to AD by Mycobacterium sp. NRRL 3805B. The conditions of biotransformation were optimized to get approx. 70% molar yield of AD from 12 g/l of the substrate. On the second PF-17 stage AD accumulated in the biotransformation broth was regio- Establishment of Efficient Microalgal Harvesting Tech- and stereo-specifically hydroxylated into C-11-alpha position by nique by the Concomitant Application of Red or Blue the mycelial fungus Aspergillus ochraceus VKM F-830Y. Cultivation Light Wavelength with Chitosan conditions for preparation of the fungal biocatalyst with higher specific activity and mode of the biocatalyst application were opti- Dae Geun Kim 1,∗ , Yoon-E. Choi 2 mized. 1 Department of Bioprocess Engineering, Chonbuk National University Both biotransformations were carried out in a single laboratory- 2 LED Agri-bio Fusion Technology Research Center, Chonbuk National Univer- scale bioreactor thus allowing exclude AD isolation and sity purification procedures. The two-stage bioprocess provided 65- 68% molar yield of 11␣HAD from phytosterol for 65-72 h. The Microalgae are considered to be one of the most promising product was separated and purified to 95% by step-wise crystalliza- feedstocks for biodiesel, due to their rapid growth and high lipid tion and re-crystallizations from a system of polar organic solvents. content. However, microalgal harvesting is one of indispensable For our knowledge, microbial production of 11␣-HAD from step claiming almost 20-30% of total biomass production cost. Chi- phytosterol was not so far reported. tosan is a natural, non-toxic, polycationic polymer with multiple http://dx.doi.org/10.1016/j.nbt.2014.05.1903 applications in pharmaceuticals, food, agricultural, and chemical industries. There are multiple lines of reports that chitosan can also be a promising alternative flocculants for microalgal harvesting. In PF-16 our previous study, light-emitting diodes(LEDs) especially red and blue color were demonstrated to govern the specific microalgal Plasmid DNA purification by integrating membrane cell biology. Blue light illumination led to significantly increased technology with arginine affinity chromatography cell size, whereas red light resulted in small-sized cell with active divisions. Based on that, in this study, we attempted to establish a João Queiroz ∗ , Catherine Nunes, Ângela Sousa, José Nunes, novel harvesting strategy of microalgal biomass by the concomi- António Morão, Fani Sousa tant applications of both blue light illumination and chitosan. We University of Beira Interior successfully proved that microalgal cells cultivated under blue light settled more rapidly than those of biomass cultivated under red The implementation of clarification and purification processes light illumination. Next, the combinationary effects of different to isolate the supercoiled (sc) plasmid isoform at industrial scale light wavelengths and chitosan concentration were thoroughly becomes crucial. In the present study, membrane filtration tech- tested. The data suggested that the optimal harvest condition nology was performed to isolate and clarify the sc plasmid DNA under the blue light with chitosan were significantly deviated from (pDNA) from lysates. Microfiltration process was implemented to those under the red light with chitosan. Our strategy, based on eliminate the suspended solids and to perform a diafiltration of the the illumination of blue light in conjunction with chitosan, will solution, followed by an ultrafiltration technique to concentrate contribute to setting up the future biomass harvest process using the plasmid and to remove the different types of RNA [1]. Finally, microalgae. a suitable chromatographic strategy is essential to remove residual http://dx.doi.org/10.1016/j.nbt.2014.05.1905 impurities and to obtain the sc pDNA as a highly pure product. Affinity chromatography with amino acids as ligands, such as arginine, has been employed for this objective due to its high PF-18 selectivity for the sc isoform and also because of the mild elution conditions required to achieve its purification [2]. Thereby, the Optimization of an industrial primary protein recovery sample resultant from the ultrafiltration process was applied in the process by Design of Experiment arginine chromatographic matrix, to attain an adequate strategy ∗ for sc pDNA purification. The nature of the arginine support and Tanja Buch , Ian Marison the use of moderate salt concentrations render this operation more Dublin City University economically sustainable and viable to be used in large scale systems. The separation of sc isoform was proved by electrophoretic The primary recovery of proteins at the end of a fermentation and HPLC analysis. Overall, the integration of membrane technol- process can be considered as a keystone for the overall protein loss during purification. Recombinant proteins are widely produced in

S120 www.elsevier.com/locate/nbt New Biotechnology · Volume 31S · July 2014 BIOPROCESSING AND ENGINEERING batch and fed-batch fermentations at industrial scale since the PF-20 early 1980’s using Escherichia coli or other microbial cells. The main challenge remains in the isolation and recovery of the pro- Sustainable Manufacture of Industrially Relevant Plat- tein from the fermentation broth at the end of the fermentation, form Chemicals Using Microbial Bioprocesses which leads to increase loss in the protein of interest, especially at Laura Jeffrey 1,∗ , Alison Arnold 2, Brian McNeil 1, Linda Harvey 1 manufacturing scale. 1 University of Strathclyde The primary protein recovery process of an industrial E.coli fer- 2 Ingenza Ltd mentation at large scale was characterised based on the protein concentration and protein mass balances were set up to iden- As petroleum stocks decrease on a global scale, it is essen- tify critical process parameters. A loss of more than 80% of the tial that industry decreases its dependency on oil and petroleum recombinant protein was observed occurring at two main criti- based materials in favour of more sustainable resources. Biologi- cal process steps (CPS). A major problem at the critical process cally produced chemicals could provide a sustainable route for the steps was identified to be the solubility levels of the recombinant manufacture of high value monomers. Such biological processes protein. The protein solubility at the CPS was optimized using should have high productivities, use simple media, be operable Design of Experiment (DoE) with a central composite face-centred at large scale, produce a final process fluid with a high concen- design. Statistical analysis was applied to determine significant tration of suitable bio-product which can easily be recovered and factor interactions and to identify optimal process conditions. easily integrated into further chemical conversions. At present, no The optimization of the solubility of the recombinant protein such system is in place within the industrial biotechnology sector. led to a four-fold improvement in the recombinant protein recov- This presents the unique opportunity to develop a novel process ery and an enhancement of the overall process yield. wherein amino acids can be converted into high value chemicals. http://dx.doi.org/10.1016/j.nbt.2014.05.1906 Corynebacterium glutamicum, a Gram positive, non-sporulating bacterium was chosen as a model organism. Since its discovery it has become an industrial workhorse in the production of amino PF-19 acids, especially L-glutamate. For this process to be industrially competitive, efficiency is paramount. Therefore, production of Production of ethanol and biomass from thin stillage L-glutamate was examined in batch processes using standard L- using edible Neurospora intermedia glutamate induction conditions including; biotin limitation, heat Jorge Ferreira ∗ , Patrik R. Lennartsson, Mohammad J. Taherzadeh induction and ethambutol addition. As biotin limitation exhibited a 10 fold yield increase during batch conditions compared to University of Borås other chosen methods a fed-batch process was developed where balancing carbon and nitrogen was crucial. Strain screening under Thin stillage is a prime candidate for improvement of optimal production conditions was employed to increase produc- the industrial ethanol process. Production of biogas, cell oil, tivity further. Once a sufficient titre was achieved, downstream eicosapentaenoic acid and biomass for animal feed has been inves- processing on the culture broth could be examined, converting tigated using thin stillage. In this work, the edible ascomycete this relatively low value amino acid into a high value, desirable fungus Neurospora intermedia was investigated for production of platform chemical for industrial purposes. ethanol and biomass from mostly wheat-derived thin stillage. Aeration rate influenced the production of ethanol and biomass http://dx.doi.org/10.1016/j.nbt.2014.05.1908 during cultivation in a 26 L capacity airlift reactor; the highest amount of ethanol (3.2 g/L) was obtained at lower aeration rate of 0.5 vvm, while the highest amount of biomass (9.2 g/L) was PF-21 obtained at 2 vvm. The reactor was also used as a bubble col- Overproduction of glutathione by recombinant umn. Similar amounts of ethanol (3.5 g/L) and biomass (5.0 g/L) Escherichia coli expressing bifunctional glutathione were obtained. N. intermedia was also investigated in continuous − synthetase mode in the bubble column; dilution rates up to 0.2 h 1 could − be used without cell wash-out. At dilution rate of 0.1 h 1, 5 g/L Zhimin Li of ethanol and 4 g/L of biomass containing 50% protein were East China University of Science and Technology obtained. The solid content in the thin stillage was reduced by 18%. The inclusion of this process using N. intermedia can lead to Glutathione is an important bioactive substance being applied a 5.5% improvement on ethanol production considering a facility in pharmaceutical and food industries widely. Traditionally, the producing 200,000 m3 ethanol/year. The produced ethanol can be production of glutathione was conducted in yeast system. In sent to the beginning of the process and follow the main stream the present study, a recombinant Escherichia coli strain express- towards the distillation column. The high value biomass can be ing the gene gshF coding for bifunctional glutathione synthetase used for animal feed e.g. fish feed, while the reduction of solids can was used as a producer and the fed-batch cultivation was investi- have positive effects on energy savings and water recirculation. gated. During this process, glucose was the sole carbon and energy http://dx.doi.org/10.1016/j.nbt.2014.05.1907 source. Without extra addition of three amino acids, 0.79 g/l of glutathione was obtained and the productivity was 56 mg/l/h. With

www.elsevier.com/locate/nbt S121 BIOPROCESSING AND ENGINEERING New Biotechnology · Volume 31S · July 2014 the addition of 75 mM glutamic acid, cysteine and glycine, 11.3 g/l PF-23 glutathione was formed with a productivity of 628 mg/l/h, which were 13 and 10 times respectively higher than those in the absence The use of enzymes in the coating industry– of amino acids addition. environmentally friendly strategies for curing and hydrolysing coatings http://dx.doi.org/10.1016/j.nbt.2014.05.1909 Katrin Greimel 1,∗ , Veronika Perz 1, Karolina Haernvall 1, Enrique Herrero Acero 1, Georg Guebitz 2 PF-22 1 acib Gmbh 2 University of Natural Resources and Life Sciences, Institute of Environmental Isolation of microorganisms for biosurfactant produc- Biotechnology tion

Franco Liporace 1 , Carla Quevedo 1,∗, Ana María Giulietti 2, Juan The use of enzymes in industrial processes has become more Olivera 1 and more common. For the coating industry, it could be envisaged that enzymes will replace siccatives, paint removers or acticides. 1 Facultad Regional Delta-UTN-Argentina We investigated the potential use of laccases as siccatives for 2 Facultad de Farmacia y Bioquímica-UBA-Argentina coatings containing alkyd resins as well as the potential use of hydrolases as paint removers. Microorganisms with surfactant producing ability were iso- Alkyd resins are binders for coating formulations containing lated from hydrocarbon-contaminated soil and water in order to unsaturated fatty acids. During the hardening process the fatty carry out processes for biosurfactant production.The samples were acids are cross-linked. Cobalt complexes are the most widely used obtained from a petroleum distillery (RHASA) located at Cam- siccative systems, but because they are suspected to be carcino- pana, (Argentina). Isolation was performed by enrichment cultures genic, they need to be replaced. The potential of a laccase from in a mineral salt medium (MSM) containing 4,5% of a mixture Trametes hirsuta in combination with two mediators was evaluated of three different hydrocarbons (HC) or polluted lagoon water regarding its capability to crosslink the unsaturated fatty acids [1]. (AgLag) as both carbon and energy source. The strains isolated were The drying reaction was evaluated using different methods, like cultured in 250 ml erlenmeyer flasks containing 25 ml of MSM FTIR spectroscopy, oxygen measurements, GC chromatography supplemented with HC (pH 7.00) and incubated at 25 ◦C ± 2 ◦Cin and drying time recorder measurements. orbital shaker at 150 rpm for 7 days. The biosurfactant producing The removal of coatings is usually performed using harsh chem- ability of isolated microorganisms was estimated by emulsifica- icals or mechanical strength. Also in this case the development of tion capacity, surface tension measurement using Du Nouy ring environmentally friendlier methods will sooner or later be of great tensiometer, oil spreading techniques, drop collapse method and importance. We investigated the possibility of using enzymes for haemolytic activity. Only six of the isolated strains were able to the hydrolysis of polyester based coatings [2]. Two enzymes - a reduce the growth media surface tension in more than 40%. One lipase from Thermomyces lanuginosus and a cutinase from Humicola of them, Ag.A.1HC strain, was cultured in a stirred tank bioreac- insolens–showed different activities in hydrolysing two different tor (NewBrunswick BioFlo115) containing 2500 ml MSM medium model substrates for polyester based coatings. with a mixture of HC, operating at 200 rpm and 25 ◦C ± 2 ◦C. Biomass concentration and surface tension were evaluated during References the process. A decrease in the surface tension value of the cell- [1].Greimel, et al. Green Chemistry 2013. free supernatant was observed. The initial and final values were [2].Greimel, et al. Reactive and Functional Polymers 2013. 46,85mN/m and 34,90mN/m, respectively. The highest cell concentration was found between day 5 and 7 of culture. According http://dx.doi.org/10.1016/j.nbt.2014.05.1911 to these results the Ag.A.1 HC strain, isolated from hydrocarbon- contaminated water would have a potential use in both production of biosurfactant and bioremediation processes of environments PF-24 contaminated with hydrocarbons. Hollow fibre membranes as a high mass transfer gas dif- http://dx.doi.org/10.1016/j.nbt.2014.05.1910 fusing system for microbial CO fermentation

Muhammad Yasin ∗ , Shinyoung Park, Yeseul Jeong, In Seop Chang Gwangju Institute of Science and Technology (GIST)

Recent research on the biological conversion of carbon monox-

ide (CO) and hydrogen (H2) into multi-carbon compounds have revealed that syngas fermentation is one of the potential alternatives for the production of more sustainable fuels and chemicals. However, poor mass transfer of the sparingly soluble gaseous

substrates (CO and H2) and low cell density in the fermentation media are the big hurdles in the commercialization of the technology. These issues can be resolved by using the membrane based

S122 www.elsevier.com/locate/nbt New Biotechnology · Volume 31S · July 2014 BIOPROCESSING AND ENGINEERING reactors (MBR) instead of most widely employed stirred tank reac- that can be obtained in characterizing microbial populations using tors (STR) and less common bubble column reactors (BCR). The multiple molecular methods. purpose of this study was to develop a simple lab scale hollow fiber http://dx.doi.org/10.1016/j.nbt.2014.05.1913 membrane bioreactor (HFMBR) for addressing the issues of mass transfer and kinetic limitations in syngas fermentation. The first phase of the research has been completed and a hydrophobic PVDF PF-26 (Polyvinylidene fluoride) membrane has been successfully utilized to achieve high mass transfer. The performance of the system has Online estimation of metabolic state in industrial-scale been examined by measuring the gas-liquid volumetric mass trans- bioreactors through the dissolved oxygen response to fer coefficient (kLa). Pressure and membrane surface area have been feed rate perturbations used as two controllable factors to achieve high mass transfer. 1,∗ 2 3 −1 Ola Johnsson , Jonas Andersson , Gunnar Lidén , Tore We have found a kLa of 135.72 h under 13.6psi transmem- Hägglund 1 brane pressure at AS/VL (membrane surface area/working volume −1 −1 1 of the liquid) = 0.27 cm . High kLa of 155.16 h was achieved by Lund University, Department of Automatic Control 2 −1 Novozymes A/S increasing AS/VL to 0.62 cm under lower transmembrane pres- 3 Lund University, Department of Chemical Engineering sure of 5.4psi. http://dx.doi.org/10.1016/j.nbt.2014.05.1912 Overflow metabolism is a significant problem in many industrial bioprocesses, which can lead to decreased productivity as well as total process failure. Avoiding excessive overflow metabolism PF-25 while maintaining a high feed rate and hence productivity is therefore highly desirable. This is complicated by difficulties in Evaluation of dynamic microbial communities in a modelling and online sensing in industrial processes using com- styrene-degrading biotrickling filter using 16S rDNA plex media. tag pyrosequencing and denaturing gradient gel elec- The current study employs a method for online estimation trophoresis of the metabolic state in relation to overflow metabolism, based Kevin Portune ∗ , María Carmen Pérez, Francisco Javier Álvarez- on the response to sinusoidal perturbations in the feed rate. This Hornos, Carmen Gabaldón method requires only measurement of dissolved oxygen, for which University of Valencia robust and precise measurement devices are universally available in industrial bioprocesses. The method is based on directly study- Accurately characterizing microbial communities within biore- ing the effects of the saturation in oxidative metabolism causing actors undergoing dynamic operating conditions is an essential overflow, meaning that it allows estimation of metabolic state first step towards understanding the relationship between micro- regardless of which substrates are used. bial community structure and bioreactor performance. A detailed The study includes a number of experiments in industrial assessment of the changes in microbial populations within a production-scale fermentations (>100 m3), using an industrial styrene-degrading biotrickling filter was carried out using samples Bacillus licheniformis strain and a complex starting medium. Firstly, collected at multiple time points ranging from 21 to 155 days of these have shown that for feed rate perturbations in a certain biotrickling filter operation. Examination of microbial populations frequency interval fermentor mixing dynamics can be approxi- was conducted by 16S rDNA tag pyrosequencing and denaturing mated by a simple model and that the perturbations do not have a gradient gel electrophoresis (DGGE). Validation of pyrosequencing negative impact on growth and productivity. Secondly, they have results was performed by quantitative polymerase chain reaction shown that the method’s estimation of the current metabolic state (qPCR) in order to examine the relative changes in percentages of is consistent with offline measurements of main substrate and by- selected taxonomic groups. Pyrosequencing results revealed a pre- product. The method therefore provides a useful measure of the dominance of bacteria assigned to the phylum Proteobacteria for metabolic state, which can be used for both online diagnosis and all sampling time points in the bioreactor. Relative fluctuations closed-loop control in industrial fermentations. in percentages of total bacterial sequences assigned to selected http://dx.doi.org/10.1016/j.nbt.2014.05.1914 taxonomic groups detected by pyrosequencing during biotrickling filter operation were confirmed by qPCR. Pyrosequencing revealed substantial changes in the community structure between sampling time points, with observed differences in microbial diversity indices and operational taxonomic units (OTUs) among certain samples. DGGE further revealed shifts in the dominant microbial species during changes in biotrickling filter operational parameters. The application of several different molecular tools to examine changes within microbial populations from bioreactors allows a more detailed view of the community structure as compared to using only one molecular method. This study high- lights both the complementary as well as contrasting information

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PF-27 for the small-scale cultivation under defined conditions could be developed so far. Improved scale down of industrial fermentations by Rapid prototyping allows the design and manufacturing of fine- application of oxygen enrichment on lab scale structured reactor components. Here, we present the first rapid Rogier Meulenberg ∗ , Rogier Meulenberg, Jeroen Van Santen, Erik manufactured cultivation system for phototrophic microorgan- Van der Lucht, Wouter Van Winden isms with a working volume of 12 milliliters equipped with organic light emitting diodes (OLED) as lighting source and an optical DSM Biotechnology Center process monitoring sensor system. OLED light technology is characterized by a very homogenous Lab scale improvement programs of industrial fermentations light distribution (area light source) and low self-heating. Impor- require a good scale down of the process. Only then it is possible tant process parameters of microalgal cultivation like pH, pO2 or to test process adaptations under representative conditions and, pCO2 can be monitored online by integrated optical sensor spots. moreover, scale up of the improved process will be faster and more Moreover, cell-specific parameters likeoptical density and fluores- successful. cence signals (e.g. marker proteins or chlorophyll fluorescence) are In the absence of headspace overpressure (in case of glass detected online by optical readout approaches (LED-excitation and fermenters) and negligible hydrostatic pressure, oxygen transfer spectral sensitive photodiodes). capacity in lab scale fermenters is often lower than on industrial Thus, this reactor-setup represents an ideal screening and pro- scale. Therefore, process intensity needs to be decreased for proper cess optimization tool for photo-biotechnological processes. scale down. For such a scale down approach it is crucial that the limiting factor on industrial scale is the same as on lab scale. If http://dx.doi.org/10.1016/j.nbt.2014.05.1916 not, production strain physiology may be different and represen- tativeness of the lab scale process is lost. However, in some cases it turned out to be difficult to confirm that the same limiting factor PF-29 was maintained after process intensity scaling. Bacterial growth modeling for rapid fermentation pro- In contrast to this, lab scale oxygen transfer capacity can also cess development using a parallel mini-bioreactor system be increased to industrial levels by application of oxygen enrichment of the sparged air. With this approach, the saturating oxygen Aarron Erbas 2,∗ , Tony Allman 1, Frank Baganz 2 solubility on lab scale can be increased to similar levels as on indus- 1 Infors AG trial scale at overpressure. In some cases this prevents the need for 2 UCL decreasing process intensity for a proper downscale. Examples of different scale down approaches will be discussed. In order to establish a generic framework for the rapid develop- http://dx.doi.org/10.1016/j.nbt.2014.05.1915 ment and optimisation of scalable fermentation processes a novel methodology will be explored which integrates small scale fermentations with model-based experimental design and predictive PF-28 control strategies. In the first instance mathematical models will be developed to predict microbial growth kinetics. Most commonly Rapid prototyping meets bioreactor–a novel small-scale the first order kinetics logistic and Gompertz models are compara- organic light emitting diode based photobioreactor with tively used to assess the model fit to empirical data. Optical density integrated sensor systems measurements may provide a quick off-line analysis of the growth ,∗ curve of microbial populations, as compared to cell plate counts Felix Krujatz 1 , Karsten Fehse 2, Matthias Jahnel 2, Thomas Bley 1, or dry weights that require more time. Here we propose a mod- Jost Weber 1 ified four parameter logistical model for batch growth of E. coli 1 TU Dresden w3110 in a minimal medium. The mean square error (MSE) was 2 Fraunhofer COMEDD used to measure the fit of the empirical model to the experimental data that were obtained using a quadruple parallel mini-bioreactor Microalgae represent a promising raw material for various system (Multifors). The average optical density after 23 hours was industries due to their wide range of valuable ingredients. By pho- 4.46 + /- 0.09 (n = 4) demonstrating excellent reproducibility. The tosynthetic processes carbon dioxid is fixed under the influence best MSE for the model was MSE = 0.034 showing specifically a of light and converted into products like proteins, fats, oils or pig- good fit to the log and stationary phases of bacterial growth but ments. Despite the great potential of algae research only 150 of the also enabling fitting to the lag phase with a best MSE = 0.0028. Such estimated 400,000 algae strains are used for industrial applications. models have the potential to predict typical microbial cell growth State of the art of microalgal cultivation is the illumination and thereby aiding the development of advanced fermentation with inorganic semiconductor elements known as light emitting control strategies. diodes (LEDs). However, this technology has disadvantages like an inhomogeneous ligth distribution (point light source), a strong http://dx.doi.org/10.1016/j.nbt.2014.05.1917 self-heating and thus the need to integrate cooling elements. Because of these limitations no phototrophic screening system

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PF-30 with size separation techniques (gel filtration or ultrafiltration). Bearing in mind the importance of biotechnological produc- A novel lab-scale two-stage reactor for biogas production tion of glycosaminoglycans the aim of this research was to through the use of efficient and stable microbial consor- develop new methods for purifying the capsular polysaccharide of tia Escherichia coli K4 (chondroitin-like) in order to obtain a pyrogen- Martyna Ciezkowska 1,∗ , Krzysztof Poszytek 1, Otton Roubinek 2, free product with high recovery yields and purity grade. This Jacek Palige 2, Aleksandra Sklodowska 1, Lukasz Drewniak 1 product may than be suitable for preclinical in vitro evaluation of potential biological activity. During fermentation these bacte- 1 Laboratory of Environmental Pollution Analysis, Faculty of Biology, University ria release into the culture media, together with the K4 capsular of Warsaw 2 Institute of Nuclear Chemistry and Technology polysaccharide, the lipopolysaccharide molecules that result the main contaminant during the downstream process. The removal The anaerobic digestion is very fragile and sensitive process, of the lipopolysaccharides is difficult because of their amphiphilic that requires to maintain the balance among different microbial nature and because of structural similarities between its oligosac- populations. To achieve stable biogas production different source charidic portion and the capsular polysaccharide chain. Strategies of microorganism were tested and a variety and numerous techni- based on activated charcoal either in powder form and/or packed cal solutions were developed. disks where compared to newly developed solvent phase separa- The main aim of this work was verification and optimization tion processes. of a novel anaerobic digesters for laboratory studies of two-phase In both case pyrogen free chondroitin of 30 KDa, with a purity biogas production with the use of efficient and stable microbial >98% was recovered. consortia. The developed biogas production system consists of two http://dx.doi.org/10.1016/j.nbt.2014.05.1919 reactors, one dedicated to hydrolysis process (2L tank operated in 30 ◦C) and second designed to fermentation (25L tank operated ◦ in 37 C). Both reactors use hydraulic agitation and operate in a PF-32 quasi-continuous mode. The optimization of biogas production with the constructed Single-use bioreactors for microbial application reactors was carried out with the use of stable microbial consortia Nico Oosterhuis 1,∗ , Stefan Junne 2, Peter Neubauer 2 isolated from fermentation tank of biogas plant and was conducted 1 CELLution Biotech in four phase until the stabilization of methane production. For 2 Chair of Bioprocess Engineering, Institute of Biotechnology, Technische Uni- each phase the bioreactors were supplied with maize silage from 1 versität Berlin, Germany to 5% DM increasing during process. During methane fermentation the following parameters were analyzed: CH4 content; biogas Nowadays single-use bioreactors are fully accepted in the bio- yield, concentration of VFAs and COD, and microbial community pharmaceutical industry. Reactors up to 2000 L working volume structure and activity changes. are commonly used. However, these bioreactors are limited in The conducted analyses showed that the microbial consortia terms of mass-transfer and mixing capabilities and therefore have effective and stable production of biogas after 20 days of pro- only suited for application in mammalian cell culture. Single-use cesses and constructed reactors are perfect for the analyses of each processing offers same benefits for microbial processes as for mam- step (hydrolysis/acetogenesis and methanogenesis) of two phase malian processes. As microbial expression systems are widely use biogas production process. in the biopharma industry, there is a strong need for single-use http://dx.doi.org/10.1016/j.nbt.2014.05.1918 bioreactors applicable for microbial processes as well. The CELL-tainer® technology, based on a 2-dimensional rock- ing motion is available now with a working volume from 0,15–25 L PF-31 in one and the same bag as well as from 10 – 150 L in one bag size. −1 kLa values of 300 hr and above have been reported for both sizes Methods for removing endotoxin contaminants from of reactors. Recently achieved culture data of E.coli and of a Rho- biotechnological chondroitin dutorula yeast show that the CELL-tainer® single-use bioreactor is Mariacarmela Marseglia ∗ , Paola Diana, Alberto Alfano, Katia Della comparable to stirred fermenters and thus suitable for microbial Corte, Rosaria Di Nuzzo, Chiara Schiraldi cultivations. Application in both the seed train as well as cultivation system for small batches in GMP-production is possible seconda università degli studi di Napoli now.

Endotoxins released from the cellular membranes of gram nega- http://dx.doi.org/10.1016/j.nbt.2014.05.1920 tive bacteria during their growth constitute dangerous pyrogens to be removed in case of biotechnological production of pharmaceuticals, expecially in case of parenteral drugs. Polysaccharide-based pharmas have wide biomedical applications but also these products have to be endotoxin free to become suitable for speciﬁc uses. Puriﬁcation of mannans, for example, was performed

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PF-33 cell debris, and suspended materials on the stationary phase. We have employed extended Derjaguin– Landau–Verwey–Overbeek Butanol production by fermentation of Clostridium ace- (xDLVO) theory, based on colloid theory principles, as an effec- tobutylicum: Solventogenic kinetics tive tool in predicting these unfavourable interactions. We have Alessandra Procentese 1 , Francesca Raganati 1,∗, Giuseppe Olivieri 1, adapted capillary rise method to characterize the surface thermo- Maria Elena Russo 2, Piero Salatino 1, Antonio Marzocchella 1 dynamics of EBA adsorbents, and have used streaming potential measurements to describe surface electrostatics. Both these meth- 1 Dipartimento di Ingegneria Chimica, dei Materiali e della Produzione Indus- ods now avoid mechano-chemical alterations in surface properties triali - Università degli Studi di Napoli Federico II 2 Istituto di Ricerche sulla Combustione, Consiglio Nazionale delle Ricerche associated with the bead disruption, which was required for earlier characterization methods. The corresponding xDLVO interactions The awareness of fossil supply depletion and the impact of energies calculated were used to generate a minimum adsorbent- petroleum fuel emissions have increased the research for alterna- biomass interaction energy surface as a function of pH and ionic tive fuel sources. Particular relevant are the fuels produced from strength, which led to the predictive optimization of EBA process renewable resources such as rapid growth biomasses. Among bio- conditions in terms of solution chemistry. Consequently, product fuels, butanol is particularly valuable because it possesses many recovery was shown to increase without compromising on purity favourable physical properties [1]. (99%). Acetone–butanol–ethanol (ABE) fermentation from renewable http://dx.doi.org/10.1016/j.nbt.2014.05.1922 resources is one of the biotechnological routes to butanol production. The potential of cheese-whey as feedstock for butanol production has been pointed out by several authors (e.g. [2]). PF-35 The traditional batch fermentation process for butanol production suffers from two major issues: i) low butanol specific High throughput systems and mathematical models for productivity, which means large fermentors and long fermen- prediction of protein renaturation processes tation periods; ii) severe product inhibition, which limits the Bernhard Mißbichler 1,∗ , Cornelia Walther 2, Sabrina Mayer 3, butanol concentration and increases the industrial cost for solvent Dorota Antos 4, Alois Jungbauer 3, Astrid Dürauer 3 recovery. Continuous operation mode provides several advantages 1 ACIB GmbH over batch processes. Increased productivity was also achieved by 2 University of Natural Resources and Life Sciences Vienna, Department of increasing cell concentration by cell recycling or immobilization Biotechnology, Muthgasse 18, 1190 Vienna, Austria [2]. 3 1ACIB Austrian Centre of Industrial Biotechnology, Muthgasse 18, 1190 The objective of this study was to characterize a continuous Vienna, Austria 4 butanol production system by means of Clostridium acetobutylicum Rzeszow University of Technology, Chemical and Process Engineering Depart- ment, a. Powstancow warszawy 6, 35-959 Rzeszow, Poland adopting lactose as the sole carbon source. A cell recycling system (CSTR equipped with a microfiltration unit) has been adopted. Continuous cultures were carried out under a wide interval of Heterologous recombinant expression of biopharmaceutical operating conditions (dilution rate and recycle flux) in order products in E. coli often leads to high density protein aggregates to characterize the fermentation process under solventogenesis in inclusion bodies. In contrast to empirical strategies for process phases. development we developed high throughput screening methods for inclusion body solubilization and subsequent refolding on ␮- References scale. Thereof parameters were extracted to predict this crucial process steps in a laboratory scale stirred tank reactor controlled [1].Cascone. Chem Eng Prog 2008 Aug:S4–9. [2].Raganati, et al. Bioresour Technol 2013;138:259–65. by inline monitoring. Mathematical modelling using solubility curves and dissolu- http://dx.doi.org/10.1016/j.nbt.2014.05.1921 tions kinetics was carried out. The solubilization process could be described by a homogeneous layer model with a high order of reaction which enables evaluation of the process on a numerical level. PF-34 Hence, solubilization conditions and process parameters can be Evaluation & optimization of yield, productivity, and related to the rate and yield of the solubilization. Approximation sustainability of the EBA process; A surface energetics of the resistances potentially affecting the IB solubilization showed approach that the process is controlled predominantly by pore diffusion. Maintaining the homogeneity of the IB suspension is sufficient for ∗ Prasad Babu Kakarla , Roy D’Souza, Marcelo Fernández Lahore efficient solubilization, and further power input will not improve Jacobs University Bremen the process. The HTS on ␮-scale can predict solubilization in STR over a range of 500 and can be used to determine optimal solubi- Expanded bed adsorption (EBA) chromatography is an lization conditions for laboratory and industrial scale. advanced integrated unit operation for the downstream processing Furthermore, kinetics of the refolding process determined in of biomolecules. Biomass-adsorbent interactions not only lead to different scales – from static vial over beaker stirred by magnetic unstable hydrodynamics in these systems, but also to the elutria- stirrer to laboratory scale STR were equivalent keeping different tion of adsorbent beads, and the deposition of intact cell particles, scale-up criteria such as phase number and Reynolds number

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within a certain range. The established models and prediction tion which enables evaluation of the process on a numerical level. enable the engineering based and thus material and cost efficient Hence, solubilization conditions and process parameters can be process development of IB solubilization and refolding steps. related to the rate and yield of the solubilization. Approximation http://dx.doi.org/10.1016/j.nbt.2014.05.1923 of the resistances potentially affecting the IB solubilization showed that the process is controlled predominantly by pore diffusion. Maintaining the homogeneity of the IB suspension is sufficient for PF-36 efficient solubilization, and further power input will not improve the process. The HTS on ␮-scale can predict solubilization in STR Gamma ray-mediated functionalization of monolithic over a range of 500 and can be used to determine optimal solubi- cryogels for macro-biomolecule purification lization conditions for laboratory and industrial scale. Furthermore, kinetics of the refolding process determined in Naveenkumar Singh 1,∗ , Roy N. Dsouza 1, Mariano Grasselli 2, different scales – from static vial over beaker stirred by magnetic Marcelo Fernández-Lahore 1 stirrer to laboratory scale STR were equivalent keeping different 1 Jacobs University Bremen gGmbH scale-up criteria such as phase number and Reynolds number 2 Universidad Nacional de Quilmes, Bernal, Argentina within a certain range. The established models and prediction enable the engineering based and thus material and cost efficient Recently introduced megaporous cryogels are relatively new process development of IB solubilization and refolding steps. and have not been fully exploited for the downstream processing http://dx.doi.org/10.1016/j.nbt.2014.05.1925 of macro-biomolecules. Weak anion-exchange functionality was incorporated by gamma irradiation-induced grafting. The total ionic capacity obtained for DEAE-functionalized cryogel was 0.59 PF-38 meq/g. The resulting weak anion-exchange cryogels showed a pore ␮ size distribution of up to 100 m with dynamic binding capaci- Precipitation: A powerful tool for continuous purifica- ± ties of ca. 27 3 mg/mL. The adsorbents presented in here showed tion of monoclonal antibodies better column efficiency when compared to packed-bed adsor- 1,∗ 1 2 bents. The dynamic binding capacities of monolithic cryogels were Ralf Sommer , Anne Tscheliessnig , Henk Schulz , Bernhard 2 3 found to be independent of the applied flow rate. Scanning elec- Helk , Alois Jungbauer tron microscopy confirmed that the porous nature of the cryogels 1 University of Natural Resources and Life Sciences Vienna/Department of remained unaffected by the irradiation-grafting procedure. Due to Biotechnology 2 the large pores, high permeability, disposability, and cost-efficient Novartis Pharma AG 3 University of Natural Resources and Life Sciences Vienna nature, megaporous cryogels are an attractive alternative for the purification of macro-biomolecules like mAbs, mRNA, pDNA and viruses. Currently, recombinant biopharmaceutical protein production operates discontinuous, in batch processes. Till 2012, the num- http://dx.doi.org/10.1016/j.nbt.2014.05.1924 ber of approved mAbs increased to 30. These trends require a more economical mAb production processes which only can be achieved with a change from batch to continuous production. PF-37 Most promising way for improvement of mAb production is the implementation of novel continuous downstream processes. High throughput systems and mathematical models for For mAb purification a combination of caprylic acid (CA) and prediction of protein renaturation processes polyethylene glycol (PEG) precipitation was developed. Target of ,∗ Bernhard Mißbichler 1 , Cornelia Walther 2, Sabrina Mayer 1, novel continuous precipitation purification was to reach the high- Dorota Antos 3, Alois Jungbauer 1, Astrid Dürauer 1 est possible purity values. Caprylic acid precipitation reduced both 1 ACIB GmbH HMWI and HCP contents. For IgG capturing a PEG precipitation 2 BOKU was performed, which further reduces HCP content. Adequate 3 Rzeszow University of Technology yield and purity of the final product showed that CA/PEG precipitation was competitive with affinity chromatography. Heterologous recombinant expression of biopharmaceutical Furthermore, additional combination of precipitation methods

products in E. coli often leads to high density protein aggregates such as of CA, PEG, CaCl2, and cold ethanol precipitation were in inclusion bodies. In contrast to empirical strategies for process tested to determine whether biopharmaceutical purity could be development we developed high throughput screening methods achieved with a continuous performable process. The most promis- for inclusion body solubilization and subsequent refolding on ␮- ing combination provided a low HCP content (300 ppm), without scale. Thereof parameters were extracted to predict this crucial aggregates, and a yield of approximately 70%. That purity is nearly process steps in a laboratory scale stirred tank reactor controlled suitable for a biopharmaceutical agent and the solitary use of pre- by inline monitoring. cipitation methods lead to a continuous performable process. Mathematical modelling using solubility curves and dissolu- http://dx.doi.org/10.1016/j.nbt.2014.05.1926 tions kinetics was carried out.The solubilization process could be described by a homogeneous layer model with a high order of reac-

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PF-39 For this, inoculants were prepared in Erlenmeyer flasks, grown in ‘shaker’ for 7 days, with temperatures of 30 ◦C, 6 Klux illumi- Development of a rapid and low-cost tool for biophar- nance and agitation of 150 rpm, with an initial concentration of maceutical formulation screening 50 mg L−1 in standard mineral medium. After that, the microor- Amanda Quigley 1,∗ , Stuart Hassard 1, Dan Bracewell 2 ganism was separated by filtration. The permeate was adsorbed on an ion exchange column using the resin IRA 402 (Rohm and 1 deltaDOT Ltd Haas), washed with distilled water and eluted with a NaCl gradi- 2 University College London ent of 0.5 to 5.0% (w/v). The content of phycocyanin, chlorophyll, beta-carotene and amino acid present in the solution were evalu- One of the most significant challenges in protein formulation ated by spectrophotometry UV. As a result, it was observed that the is the tendency of proteins to aggregate. Aggregation can have greatest concentration values were obtained for elution with 5% severe implications for the safety and effectiveness of biophar- NaCl. It was possible to obtain high concentrations of amino acids maceuticals therefore methods to predict solution conditions that 13 times, chlorophyll 16 times, phycocyanin 5 times and beta- limit aggregation would be of great industrial value. However, very carotene in 4 times at the exit of the column. Therefore, according little progress has been made in this area in recent years and cur- to the experimental conditions under which this work was per- rent formulation screening methodologies are lengthy, outdated formed, the ion exchange showed promise in the extraction of the and empirical and often incompatible with high protein concen- compounds analyzed in Arthrospira (Spirulina) platensis cultivation. trations. This has generated great interest in the measurement of the http://dx.doi.org/10.1016/j.nbt.2014.05.1928 weak protein-protein interactions (quantified by the osmotic second virial coefficient, B22) involved in protein aggregation as a method for rapidly predicting protein stability. Measurement PF-41 of protein-protein interactions also has a potential method for predicting other factors of importance to formulation such as sol- Development of microfluidic system for screening and ubility and viscosity. reaction optimization for lipase This experimental study reports on the development of a Hsiang-Yu Wang ∗ , Chong-Yi Ho novel screening platform to measure B . The advantages of this 22 National Cheng Kung University methodology over other techniques include significantly reduced experimental time and consumption of materials including valu- This study presents microfluidic platforms for the rapid able protein. In addition to increased resolution of peak shape screening of lipase and the corresponding optimization of lipid due to deltaDOT’s multipixel detection and algorithms, providing hydrolysis reaction. The conventional screening methods of lipase important extra information on protein stability. involve time-consuming processes and high-end instrument, mak- B data is presented on a formulation screen of varying buffer 22 ing the analysis not accessible. The proposed microfluidic systems type, pH and NaCl concentration for lysozyme as a model pro- utilize microdroplet (either dynamic or stationary) to enable rapid tein, as well as 2 industrial monoclonal antibodies. This data is analysis of the hydrolysis reaction and use instrument which is compared to that obtained via benchmark techniques for stability readily accessible in common laboratories. Due to the merit of screening; size exclusion chromatography (SEC) and dynamic light high surface area per volume (SAV) ratio of microdroplet, which scattering (DLS). This work establishes the decisive importance of can be as high as 2.5 × 106 m−1 in this study, the lipid hydroly- B as a predictor of protein aggregation. 22 sis reaction occurring on the interface of droplet is extremely fast. http://dx.doi.org/10.1016/j.nbt.2014.05.1927 The analysis for hydrolysis of soybean oil by the Burkhoderia sp. lipase can be accomplished within 5 min. Reaction temperature and pH can be easily adjusted via hydrodynamic control for reac- PF-40 tion optimization. In the dynamic microdroplet system, droplets containing lipase and fluorogenic reagents that react with glyc- Extraction of compounds from the cultivation of erol are generated at T-junction and flow through a serpentine Arthrospira (Spirulina) platensis using ion exchange microchannel to allow lipase to react with the continuous phase Fernando Lisboa 1,∗ , Luana Agassi 2, Evelin Brandão 2, Marlei (soybean oil). The retention time of the droplet inside the device Barbosa 2, Mônica Okura 2, Lúcia Pelizer 2 can be adjusted from less than 1 sec to 10 min; therefore, this system is suitable for analyzing the initial rate to obtain the maximum 1 Instituto Federal de Educação, Ciência e Tecnologia do Triângulo Mineiro - Campus Uberlândia - Brazil/Universidade Federal do Triângulo Mineiro - Brazil reaction rate and Michaelis constant. The stationary droplet sys- 2 Universidade Federal do Triângulo Mineiro - Brazil tem traps the droplet in an indentation for observation as long as 30 days; therefore, it is applied on long-term examinations such Arthrospira (Spirulina) platensis is known because of its high as screening of microorganisms producing lipases. concentration of compounds, so separating them is necessary to http://dx.doi.org/10.1016/j.nbt.2014.05.1929 promote their use, increasing the viability of the process. In this context, the aim of this study was to analyze the extraction of phycocyanin, chlorophyll, amino acids and beta carotene in the liquid phase of this cyanobacterium’s cultivation using ion exchange.

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2 PF-42 (%RECbot) in bottom phase (r = 0.93). Similar result was obtained when the variable was partition coefficient (K). For the parti- From small synthetic ligands to small protein scaffolds: tion coefficient of lipase and recovery lipase (%RECbot) in bottom Affinity reagents for biologics purification phase, the influence of pH and temperature was negative and the Ana Roque 1,∗ , Ana Pina 1, Cláudia Fernandes 1, Ricardo Branco 1, influence of concentration of TX-114 was positive. The majority Ana Dias 1, Iris Batalha 1, Olga Iranzo 2, Christopher Lowe 3 of lipase partition was in the micellar-rich phase (bottom phase) (K = 7.7 and 4.7; 93.8 and 73.5% recovery in bottom phase and 1 FCT-UNL purification factor PF = 1.2 and 1.97, respectively). The greater 2 University of Marseille 3 University of Cambridge extraction of lipase was in the bottom phase, this result was probably influenced by the hydrophobic residues present on lipases. The Over the past 40 years monoclonal antibodies and derived micellar-rich phase showed to be very clear and transparent. Thus, structures became the standard binding proteins representing this micellar ATPMS presents an alternative to the conventional powerful tools in biotechnology and biomedicine, namely on pro- purification/extraction of lipase from L. scottii L117. tein purification, biocatalysis, diagnostic imaging and targeted http://dx.doi.org/10.1016/j.nbt.2014.05.1931 therapy. Other protein binding scaffolds, with the robustness and versatility required, are recently being explored. We employed biological and chemical combinatorial libraries supported by com- PF-44 putational design tools to develop robust peptidomimetics based on different scaffold molecules. The scaffold molecules ranged Risk assessment of feed additives of microbial origin in from small synthetic ligands based on the triazine and Ugi reac- the European Union ␤ tions, to peptide-based -hairpin engineered scaffolds and larger Jaime Aguilera ∗ , Montserrat Anguita, Rosella Brozzi, Jaume natural scaffolds. We studied the potential of these scaffold affin- Galobart, Paola Manini, Jordi Tarrés-Call, Claudia Roncancio Pena˜ ity reagents to find binding partners against recombinant proteins (de novo designed fusion proteins), phosphorylated peptides and European Food Safety Authority, Italy viral particles. These affinity reagents were employed as ligands for the purification of the target biomolecules using standard affinity Microorganisms can be used in animal nutrition as probiotics chromatography and magnetic fishing methodologies. The per- or as silage agents, and are the source of other feed additives like formance of the new affinity adsorbents was benchmarked with enzymes, amino acids and vitamins. In the European Union, all currently available purification and enrichment methodologies, feed additives need, by law, to undergo a risk assessment, which is showing high-performance and re-usability potential at low costs. conducted by the European Food Safety Authority. A proper characterisation of the microorganism is fundamental for its safety http://dx.doi.org/10.1016/j.nbt.2014.05.1930 assessment. This includes an unequivocal identification of the species, consideration of its pathogenic or toxigenic potential and the presence of resistance to antimicrobials, the genetic basis of PF-43 which may need to be established. Genetically modified strains require particular attention, including a full molecular characteri- Optimization of lipase extraction/recovery produced by sation of the modification. If the microorganism is the source of an the psychrotrophic yeast Leucosporidium scottii L117 additive which is purified after fermentation, it is necessary to test using aqueous two-phase micellar systems whether the final product is free from the production organism ,∗ Alysson Duarte 1 , André Lopes 2, João Molino 2, Adalberto and from its DNA if it encodes antimicrobial resistances. Pessoa 2, Lara Sette 3 The characterisation of the microorganism determines the 1 UNICAMP nature and extent of further tests to be done to establish the safety 2 USP of the product. EFSA grants a qualified presumption of safety (QPS) 3 UNESP status to certain species having well documented safety. Strains fulfilling the requirements for QPS are considered safe for target This study aims to evaluate the lipase extraction by micellar species, consumers and the environment without the need of fur- system using nonionic detergent Triton X-114 (TX-114)/McIlvaine ther testing. buffer systems. Additionally, the parameters affecting the extrac- EFSA has published a complete set of guidance stating the data tion/recovery of lipase by ATPMS were optimized by Central needed for the safety evaluation. The conclusions of the assess- Composite Design (CCD23). The micellar system extraction was ments are published in form of scientific opinions, and allow the ◦ ◦ prepared with TX-114 at 25 C and 28 C, both with 2.0% TX- European regulatory authorities to make scientifically sound deci- 114/McIlvaine buffer systems (pH 7.0). The variables optimized sions regarding market authorisations. were pH (3.5–7.5), temperature (19–31 ◦C) and TX-114 (2–14% http://dx.doi.org/10.1016/j.nbt.2014.05.1932 (%w/w). The volume of the top and bottom phases were recovered (with sterile disposable syringes) and evaluated. The lipase activity was measured using the synthetic substrate p-nitrophenyl palmitate (p-NPP). The three variables were statistically significant (p < 0.05) when the response variable was of lipase recovery

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Cell factories acids (L-tryptophan, L-tyrosine and L-phenylalanine) are strictly controlled on transcriptional and kinetic levels and therefore are PG-01 difficult to manipulate. We engineered S. cerevisiae for increased production of aro- Fine-tuning of defined media for chemical production by matic compounds by eliminating degradation, up-regulating the lactic acid bacteria key enzyme encoding genes, and removing feed-back inhibition Kadri Aller ∗ , Kaarel Adamberg, Veronica Timarova, Andrus Seiman, in the pathway. In order to test the strain performance we over- Raivo Vilu expressed heterologous pathway for coumaric acid production. We obtained 4-fold higher concentrations of coumaric acid in the The Competence Center of Food and Fermentation Technologies engineered strain compared with the reference strain. Overexpress- ion of the enzymes prephenate dehydrogenase and transketolase The lactic acid bacterium Lactococcus lactis has emerged as had a negative effect on production of coumaric acid. an effective cell factory for recombinant protein production and In summary, we developed a S. cerevisiae platform strain suitable secretion [1] and for the synthesis of various biochemicals [2]. In for production of aromatic amino acids-derived compounds. order to produce chemicals of interest, the growth physiology of the expression host has to be well characterized [3]. Chemically http://dx.doi.org/10.1016/j.nbt.2014.05.1934 defined media (CDM) are required for studying the metabolism of cells as well as producing recombinant proteins [3]. However, if the medium contains an excess of nutrients, analytical measurements PG-03 are rendered imprecise and the interpretation of metabolic data is Homologous and heterologous expression of dehydroge- aggravated. nases and oxidoreductases of Ralstonia eutropha H16 We have previously elucidated the growth requirements of L. ∗ lactis IL1403 and developed several CDMs where the utilization of Steffen Gruber , Petra Koefinger, Helmut Schwab each amino acid is larger than the measurement error (5%). Now Graz University of Technology we compare the growth of IL1403 on 3 of those media in continuous cultivation experiments and show the differences in metabolic Ralstonia eutropha is a Gram-negative, strictly respiratory fac- behavior. Naturally, decrease in the concentrations of components ultative chemolithoautotrophic bacterium which can use H2 and leads to lower maximum specific growth rate values. Nevertheless, CO2 as sole sources of energy and carbon in the absence of organic biomass yield and productivity were higher and energy spilling substrates. It has attracted great interest for its ability to degrade was lower in media with reduced concentrations of nutrients, e.g. a large list of chloroaromatic compounds and chemically related the metabolism of IL1403 is more efficient in minimalized media. pollutants. Furthermore it was already applied for the production Thus, the importance of fine-tuning a growth medium for a specific of biodegradable polymer polyhydroxyalkanoates on an industrial strain and process cannot be underestimated. scale. R. eutropha serves as a model organism for the mechanisms involved in the control of autotrophic carbon dioxide fixation, References hydrogen oxidation and denitrification. [1].Morello, et al. J Mol Microbiol Biotechnol 2008;14(1–3):48–58. In our project we are interested in establishing specialized R. [2].Gaspar, et al. Biotechnol Adv 2013;31(6):764–88. eutropha based cell factories by genetic engineering. The particular [3].Marreddy, et al. PLoS One 2010;5(4):e10317. interest is constructing cells efficiently performing oxidoreductase http://dx.doi.org/10.1016/j.nbt.2014.05.1933 reactions by overexpression of homologous and/or heterologous enzymes. One of the main types of oxidoreductase reactions is per- PG-02 formed by dehydrogenases, particularly alcohol dehydrogenases, which have a wide range of possible biotechnological applications. Development of a yeast cell factory for production of Biotransformations involving the interconversion of alcohols, aromatic products aldehydes and ketones have great potential for the commercial Angelica Rodriguez Prado 1,∗ , Kanchana Kildegaard 1, Mingji Li 1, production of pure optically active compounds and also for other Irina Borodina 1, Jens Nielsen 2 processes such as the treatment of industrial effluents. The genome of R. eutropha H16 contains a remarkable diver- 1 Novo Nordisk Foundation Center for Biosustainability, Technical University of sity of oxidoreducteases. A selection of alcohol dehydrogenases as Denmark 2 Department of Chemical and Biological Engineering, Chalmers University of well as short chain dehydrogenases of R. eutropha H16 was cloned Technology and expressed in native versions in Escherichia coli. Their activity was analyzed by NAD/NADH dependent enzyme activity assays There is much interest in aromatic chemicals in the chemical with different substrates. Currently we are working on the homol- industry as these can be used for production of dyes, anti-oxidants, ogous expression of these enzymes in R. eutropha H16 and their nutraceuticals and food ingredients. Yeast is a widely used cell functional analysis. factory and it is particularly well suited for production of aro- http://dx.doi.org/10.1016/j.nbt.2014.05.1935 matic chemicals via complex biosynthetic routes involving P450 enzymes. In Saccharomyces cerevisiae the fluxes towards aromatic

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PG-04 that PK activity significantly influenced epothilone production. In addition, the expression of pyk sce4540 and putative oxidase Applicability of a mechanosensitive channel in gene (OX) in S. cellulosum 2161 treated with vitamin K3 was Corynebacterium glutamicum as a versatile exporter remarkably inhibited, while the response of pyk sce5197 was not Ken-ichi Hashimoto ∗ , Tomoyuki Konichi, Isamu Yabe, Tsuyoshi sensitive. These results indicate that PK is one of the key enzymes Nakamatsu, Hisashi Kawasaki in epothilone biosynthesis across the metabolic network in S. cellulosum 2161. It might also play an important role in the biosynthesis Tokyo Denki University of epothilones. Keywords: Epothilones, pyruvate kinase, inhibitor, Sorangium Corynebacterium glutamicum is used worldwide in the industrial cellulosum 2161, qRT-PCR fermentative production of glutamic acid. In 2007, the involvement of the NCgl1221 gene, which encodes http://dx.doi.org/10.1016/j.nbt.2014.05.1937 a homolog of the mechanosensitive channel of small conductance protein, in glutamic acid overproduction was reported [1]. We used electrophysiological methods to obtain direct evidence PG-06 of the excretion of glutamic acid through the NCgl1221 protein Engineering of the UDP-precursor biosynthesis pathway channel. We showed that the NCgl1221 protein functions as a to enhance the production of capsular polysaccharide in mechanosensitive channel [2], and we found direct evidence of E. coli K4 glutamic acid excretion through this channel by passive diffusion [3]. Furthermore we found that aspartic acid, phenyl propionic Elisabetta Carlino ∗ , Chiara Schiraldi, Ottavia Argenzio, Ileana Dello acid and lysine were also transported across the cytoplasmic mem- Iacono, Donatella Cimini brane via NCgl1221 by passive diffusion. We further evaluated Seconda Università degli Studi di Napoli the possibility of the effective application of this mechanosensitive channel as a versatile exporter, for the creation of microbial Chondroitin sulphate is a linear polysaccharide chain made of cell factories for the production of valuable chemicals other than alternating units of glucuronic acid and N-acetyl-galactosamine, glutamate. If successful, this would be a substantial development modified by sulfation in various positions depending on both the in exporter engineering (ExE). A gain-of-function mutation in animal source and tissue of origin [1]. Besides its established use in NCgl1221 was expressed in a phenylalanine producer (Escherichia the treatment of osteoarthritis, potential applications as an anti- coli AJ12741) and an inosine producer (Escherichia coli FADR add inflammatory drug and surprising antiviral properties have been edd AJ13473). These strains displayed remarkably high productiv- suggested. ity compared with a control strain. These data suggest that utilizing E. coli K4 produces a capsule that belongs to the class of the mechanosensitive channel as a versatile exporter could be an GAG-like polymers; in fact the molecule can be described as a effective method for improving fermentation yield. chondroitin backbone decorated with fructose side branches on References the GlcA residues. The synthesis of the necessary sugar precursors for chain elon- [1].Nakamura J, et al. Appl Environ Microbiol 2007;73:4491–8. gation is performed by the cells through two different metabolic [2].Hashimoto K, et al. Biosci Biotechnol Biochem 2010;74:2546–9. [3].Hashimoto K, et al. Biosci Biotechnol Biochem 2012;76:1422–4. pathways. The first starts from glucose 6-phosphate and ends with the production of UDP-GlcA. The second one starts from fructose http://dx.doi.org/10.1016/j.nbt.2014.05.1936 6-phosphate and ends with the synthesis of UDP-GalNAc. In the present work two E. coli K4 recombinant strains were engineered to improve the productive yields of K4 capsular PG-05 polysaccharide (CPS) by overexpressing genes involved in the Research on the Relationship between Pyruvate Kinase biosynthesis of one of the UDP-sugar precursor. The E. coli wild and the Biosynthesis of Epothilones in Sorangium cellu- type strain 05:K4:H4 and EcK4r3 strain (previously obtained in losum 2161 our laboratories) were both transformed with the construct. In order to evaluate the production of K4 CPS by each strain, ∗ Xinli Liu , Lin Zhao, Xiaona Wang shakeflask and microbioreactor experiments were performed. Qilu University of Technology Pathway modifications were also analysed to correlate the productive yields of K4 CPS to gene overexpressions. Pyruvate kinase (PK) catalyzes the formation of pyruvate and Reference ATP from phosphoenolpyruvate and ADP, and it is a key enzyme [1].Cimini D, et al. Homologous overexpression of RfaH in E. coli K4 of glycolysis. Pyruvate has been one of most important substrates improves the production of chondroitin-like capsular polysaccha- for the biosynthesis of epothilones. There are two coding genes ride. Microb Cell Fact 2013;12:46, 2013 May 9. of PK in Sorangium cellulosum 2161: pyk sce5197 and pyk sce4540. http://dx.doi.org/10.1016/j.nbt.2014.05.1938 In this paper, we analyzed the relationship between PK activity, epothilone production, and the gene expression quantity of pyk sce5197 and pyk sce4540 in S. cellulosum 2161 incubated with

vitamin K3, which is a speciﬁc inhibitor of PK. Results showed

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PG-07 marine yeasts. These samples were enriched using a nutrient rich media with antibiotics for three cycles as the first step. In the The isolation of novel marine yeasts; a new procedure second step, single colonies with different morphologies were Abdelrahman Saleh Zaky ∗ , Chenyu Du isolated on plates of agar medium lacking antibiotic after 24–48 h of incubation at 30 ◦C. These isolates were further confirmed to University of Nottingham be pure cultures of yeast by streaking them on agar plates and checking their purity and morphology under a microscope. In The recent research on marine yeasts highlighted that they are this study, 14 samples from different marine habitats in Egypt, UK able to produce many bioactive substances including enzymes, and USA were used for the isolation. 122 isolates were obtained amino acids, killer toxins, and vitamin C with many potential and confirmed to be pure yeast cultures. Our 3-step isolation applications in food, pharmaceutical, fuel, cosmetics and chem- method for marine yeast has demonstrated high level of efficiency ical industries [1]. However, the employment of marine yeast in comparing with two reference methods. research and industry is limited due to the lack of suitable isolation methods. Current methods suffer from fungus interference Reference and/or low number of yeast isolates. In this report, a novel 3-step [1].Zaky, et al. Marine Yeast Isolation and Industrial Application FEMS Yeast isolation method has been developed. Samples were taken from Research 2014 (accepted). seawater, sea sand or seaweed which could potentially contain http://dx.doi.org/10.1016/j.nbt.2014.05.1939

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Environmental biotechnology bility. One important problem of MBR technology is membrane fouling. In this study, Bdellovibrio activity was analysed for reduc- PH-01 ing membrane fouling caused by sludge bacteria, and potential of B. bacteriovirus as a biological cleaning method for MBR sys- Formate oxidation-driven calcium carbonate precipita- tems was investigated. For this aim, nineteen bacteria were isolated tion by Methylocystis parvus OBBP for a concomitant from wastewater sludge and characterized by 16SrRNA analysis. building material surface protection and atmospheric Bacteria were mostly found to be a member of Gammaproteobacte- methane removal ria, Betaproteobacteria and Actinobacteria families. B. bacteriovirus Giovanni Ganendra 1,∗ , Giovanni Ganendra 2, Adrian Ho 3, Nico showed high lysis activity on Aeromonas, Proteus, Alcaligenes Boon 2 and Bordatella species. B. bacteriovirus activity was also tested on membranes plugged after filtration of wastewater. Pore size of the 1 Ugent membranes was found to be important for B. bacteriovirus activity. 2 Ghent university 3 Netherlands Institute of Ecology Flux through Poly(ether)sulphone (PES) membrane with a pore size of 0.05 micrometer was improved after cleaning by predator Microbially Induced Carbonate Precipitation (MICP) is the basis bacteria. B. bacteriovirus successfully degraded the biofilm formed for several biotechnological applications in the construction sec- by sludge bacteria on the membrane surface. tor e.g., concrete surface protection. The typically used urea based MICP poses several disadvantages, such as ammonia release to the Acknowledgement air. Therefore, an alternative MICP for the application on building materials should be investigated. In this study, MICP was driven by We thank Scientific and Technological Research Council of formate oxidation by Methylocystis parvus OBBP, a methanotrophic Turkey for supporting this study (Project no: 112Y156). bacteria. http://dx.doi.org/10.1016/j.nbt.2014.05.1941 Up to 91.4 ± 1.6% of the initial calcium was precipitated in the methane amended cultures, but only a maximum of 35.1 ± 11.9% when methane was not added. Because the bacteria could only uti- PH-03 lize methane but not formate for growth, a higher culture density and subsequently a higher calcium removal was exhibited by the Comparison of inoculums in the removal of 2- bacteria when methane was added. The methane oxidation rate butoxyethanol from air emissions by biotrickling −1 filter: Performance and microbial monitoring (MOR) of the bacteria decreased from 3.15 ± 0.32 ␮gCH4 (ml h) ± ␮ −1 ,∗ when formate was not added to 0.52 0.01 g CH4 (ml h) when Maria del Carmen Perez 1 , Francisco Javier Alvarez-Hornos 1, −1 2.88 g L of formate was added (i.e., the maximum formate addi- Daniel Dobslaw 2, Karl-H. Engesser 2, Carmen Gabaldon 1 −1 tion). An optimum 0.67 ± 0.03 g CaCO3 g Ca(CHOOH)2 calcium 1 University of Valencia carbonate precipitate yield was obtained when 109 cells ml−1 and 2 University of Stuttgart 2.5gL−1 of calcium formate was used. Compared to the currently used biogenic urea degradation as 2-butoxyethanol is one of the most used glycol ether in indus- the basis for MICP, several advantages are presented here: the pre- trial activities and the treatment of air 2-butoxyethanol-emissions vention of ammonia release to the air and nitric acid production become necessary. Biotechnologies are potential treatment tech- and the atmospheric methane removal. nologies due to their low operational costs. The use of two http://dx.doi.org/10.1016/j.nbt.2014.05.1940 inoculums in the treatment of 2-butoxyethanol by biotrickling filters (BTFs) packed with polyurethane-foam was studied. A pure culture of Pseudomonas putida, previously adapted to 2- PH-02 butoxyethanol, was used as inocula in a BTF operated in the University of Stuttgart. Fresh activated sludge from a munici- Activity of Bdellovibrio on Sludge bacteria and its poten- pal waste water treatment plant was used as inocula in a BTF tial use for cleaning of Membrane Bioreactors operated in the University of Valencia. An empty bed residence 3 Melek Özkan ∗ , MerveAkayÇ elik, Pınar Karagöz, Hilal Yılmaz, ¸C isel time of 12.5 s and inlet concentrations of 400 and 800 mg/Nm ¸S engezer were applied. After 40 days of operation at 400 mg/Nm3, the BTF inoculated with Pseudomonas putida reached removal efficiencies Gebze Institute of Technology, Environmental Engineering Department, 41400 ∼ Gebze Kocaeli, Turkey (REs) 80%, whereas the BTF inoculated with activated sludge presented REs ∼ 60%. At 800 mg/Nm3, the BTF inoculated with ∼ Bdlelovibrio bacteriovirus is a gram negative predator bacterium Pseudomonas putida reached REs 60%. Microbial community was feeding on other gram negative bacteria. Its activity on biofilms monitored in both BTFs by using denaturing gradient gel elec- formed by different bacterial species especially pathogenic ones trophoresis analysis (DGGE) with subsequent 16S sequencing and has been investigated in recent years. One of the recent tech- plating methods using 2-butoxyethanol as sole carbon source. nologies for wastewater treatment is Membrane bioreactors(MBRs) Acknowledgements which have high effluent capacity and good disinfection capa- The research leading to these results has received funding from the People Programme (Marie Curie Actions) of the European

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Union’s Seventh Framework Programme FP7/2007-2013/under theticum, Brevibacillus laterosporus and Pantoea ananatis) were REA grant agreement n◦ 284949. Financial support from Ministerio isolated from an agricultural soil located on Chihuahua state, de Ciencia e Innovación (CTM2010-15031/TECNO) and General- Mexico. The most intense effects were observed on C. indotheticum itat Valenciana (PROMETEO/2013/053), is also acknowledged. Ma and on B. laterosporus, e.g., on C. indotheticum superﬁcial damages Carmen Pérez acknowledges her FPU contract (cavities and pits) were observed on its membrane, and on B. lat- http://dx.doi.org/10.1016/j.nbt.2014.05.1942 erosporus severe oxidative stress and the presence of Cu in the outer cell membrane were detected. References PH-04 [1].Gajjar P, Pettee B, Britt DW, Huang W, Johnson WP, Anderson AJ. Antimicrobial activities of commercial nanoparticles against an Diversity of fungi present in the Sossego copper mine in environmental soil microbe, Pseudomonas putida KT2440. Journal of Pará State, Brazil Biological Engineering 2009;3(9). [2].FentK. Ecotoxicology of engineered nanoparticles. F.F.H. Berlin: Springer- Luciana Jandelli Gimenes ∗ Bruno Karolski Claudio Nascimento , , , Verlag; 2010. Elen Perpetuo, Ingrid Avanzi, Louise Gracioso, Marcela Baltazar, [3].DasM, S.K.H., An SSA, Yi DK. Review on gold nanoparticles and their Marcela Veiga, Tatiana Reis, Benedito Correa applications. Toxicol Environ HealthSci 2011;3(4):193–205. Universidade de São Paulo http://dx.doi.org/10.1016/j.nbt.2014.05.1944

Industrial growth, which results from technological development and activities considered essential to human life, has caused PH-06 serious environmental problems. For some time, fungi have been used in organic waste bioremediation. Recently, a good alterna- The use of dairy processing waste as a media for growth tive in the bioremediation of heavy metals has been discovered. of recombinant microorganisms This work aims to isolate, identify and characterize fungi and Michael Ryan ∗ , Gary Walsh evaluates mechanisms and strategies for remediating areas con- University of Limerick taminated by copper. Soil and water samples have been collected from Sossego Mine (Pará State, Brazil) and have been grown on A laboratory-based study was undertaken to assess the potential PDA culture medium, incubated at 28 ◦C for 5 days. After this of whey waste as a media for the growth of recombinant Escherichia period, the isolates were identiﬁed by morphologic and molecular coli, which remain a preferred choice for process-scale manufacture methods. Since the project started, 49 fungi were isolated, and vari- of many recombinant proteins. ous species among 12 different genders were identiﬁed: Aspergillus, Growth characteristics of a recombinant strain of Escherichia Aureobasidium, Bionectria, Eupenicillium, Eurotium, Fusarium, Lenti- coli (MC1061) was assessed on 2 whey-based media, and com- nus, Microspheropsis, Nigrospora, Penicillium, Purpureocillium and pared to growth on standard LB (Luria broth) media, known to Rhizopus. This work has great importance due to the low cost of achieve high cell densities. Whey-based media were: whey only repair systems compared to conventional ones. It also allows a (WM), and whey mixed with LB media (9:1 ratio; W:LB). Media better use of copper wastes and, consequently, a better mining pH was adjusted to 7 prior to autoclaving. economic return. Moreover, the main advantage of this technol- All experiments entailed media inoculation (100 ml) with ogy is further reduction of environmental impact by the mining 1.0 ml of the recombinant strain (grown in Luria-Bertani broth [LB] activity. ◦ to OD600 of 1.5) at 37 C in a shaking incubator (250 rpm). Growth http://dx.doi.org/10.1016/j.nbt.2014.05.1943 kinetics and maximum biomass yield was followed by absorbance at 600 nm and dry cell weight determination, respectively.

E. coli growth rates (A600, 6 h cultures, n = 3) PH-05 were: WM (0.257 ± 0.0448) < W/LB (0.348 ± 0.0246) + LB (1.178 ± 0.0872). Dry cell weights attained after 24 hrs were: CuO nanoparticles toxicity against bacteria strains iso- WM (4.73 mg ± 0.001387) < W/LB (19.8 mg ± 0.00593) + LB lated from agricultural soil (31.9 mg ± 0.00699). Sandra I. Concha-Guerrero a , Elcia M.S. Brito b, Hilda A. Pinón-˜ Un-supplemented whey waste is a poor media for E. coli growth. Castillo a, M. Antonia Luna-Velasco a, Erasmo Orrantia-Borunda a,∗ However, optimally nutrient supplemented whey may yet prove a a Centro de Investigación en Materiales Avanzados, SC, Chihuahua, México viable and inexpensive media for E. coli fermentation to high cell b Universidad de Guanajuato, Guanajuato, México densities, converting a potentially waste product into a valuable commodity. The intensiﬁed use of nanoparticles by human society brings This work is funded by the Irish EPA under the Science, Tech- out the risk of exposure to these particles. In fact, some kinds nology, Research & Innovation for the Environment (STRIVE) of nanoparticles were found to effects on the organisms or on Programme 2007–2013. 2012-WRM-MS-9 their cells [1–3]. In the present work, we studied the interaction http://dx.doi.org/10.1016/j.nbt.2014.05.1945 and effects produced by oxide copper nanoparticles (CuONP) to native bacterial strains. Selected strains (Chryseobacterium indo-

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PH-07 tion without cultivation of microorganisms or further purification of crude extracts. This study suggests that crude plant extracts Effects on Tomato Growth and Soil Bacterial Community of Canavalia ensiformis have the potential to be used in place of by Application of Arthrobacter woluwensis ED Immobi- purified forms of the enzyme during remediation of heavy metal lized in Alginate Beads contaminated soil. Thus, we report the molecular characterization Hong-Gyu Song ∗ , Seung-Tak Kwon of the microbial diversity and composition change of the mine impacted soil or waste ore taken from the site during bioremedi- Kangwon National University ation processes are presented. To evaluate the stability of DGGE patterns in remediated mine soils in comparison with before or For the promotion of plant growth and increase of persistence after plant extract treatment, a number of soil samples with time of plant growth promoting rhizobacteria (PGPR) in rhizpsphere, interval in the mine impacted soil were compared using PCR- tomato growth was examined after application of PGPR Arthrobac- DGGE of 16S rDNA. ter woluwensis ED immobilized in alginate bead. When tomato seedlings were treated with A. woluwensis ED of 1 × 106 cells g http://dx.doi.org/10.1016/j.nbt.2014.05.1947 soil−1 and incubated for 30 days in a plant growth chamber, shoot and root length, fresh weight and dry weight of the grown tomato plants treated with the suspended inoculants significantly PH-09 increased by 36.2, 59, 51.1 and 37.5%, respectively compared Investigation of organic wastes from Mediterranean to the uninoculated control. The treatment of the immobilized plants to produce biogas by anaerobic digestion bacteria increased those by 42, 67.4, 62.5 and 60.4%, respectively compared to the uninoculated control. Therefore, the enhance- Camille Menard ∗ , Anais Fantoni, Pascale Bradesi, Eric Leoni, ment of tomato growth by the treatment of the immobilized Dominique Cancellieri bacteria was higher than those by the suspended inoculants. The Universite de Corse effects of the inoculation on soil bacterial community and the fate of the inoculated bacteria were monitored by DGGE analysis. The The University of Corsica is contributing to the research of DNA band intensity of A. woluwensis ED in the tomato rhizosphere both efficiency and integration of renewable energy in the main treated with the suspended inoculants continuously decreased electrical grid. Since 2013, a scientific program concerning the after inoculation, but the band intensity in the tomato rhizosphere valorization of biomass energy has been developed to investigate soils treated with the immobilized inoculants showed the max- the methane potential through the anaerobic digestion process imum at 1 week after inoculation and the decreasing rate was of lignocellulosic resources. In Corsica, due to the clearing brush less than that of the suspended inoculants, which indicated the policy to prevent forest fires, cellulosic wastes are generated. longer maintenance of the immobilized bacteria at rhizosphere. Besides, Corsica is an important producer of essential oil from Therefore, encapsulation of PGPR in alginate beads may be more Mediterranean species thanks to a process generating an important effective than liquid inoculant for the plant growth promotion amount of dried vegetation as waste every year. and survival of PGPR at plant rhizosphere. The aim of this preliminary study on biomass as renewable http://dx.doi.org/10.1016/j.nbt.2014.05.1946 energy was to characterize and to select the most appropriate substrates for the anaerobic digestion process. Fiber contents and Biochemical Methane Potential (BMP) were performed on five PH-08 substrates. Heather, rockrose and strawberry tree were chosen as representative of forest fuels. Water distillation residues of laurel Bioremediation of heavy metal contaminated soil using and immortelle were considered as natural resource wastes. plant extract as biomaterial On this work, we focus on the three species which had the In-Hyun Nam ∗ , Chul-Min Chon, Jae-Gon Kim best BMP. Rockrose, dry residue of immortelle and strawberry tree 3 produced 139, 124 and 87 Nm CH4 per grams of volatile solids, Korea Institute of Geoscience and Mineral Resources (KIGAM) respectively. The ratio holocellulose: lignin, thanks to the Van Soest method of fiber determination, was determined for those An indigenous plant extract was used to produce calcite from species: 3.8, 3.4 and 1.7. This parameter is a key factor to take Canavalia ensiformis as effective biomaterial, and its ability to into account for the correlation of the BMP with the composi- form under stable conditions was compared to that of purified tional characteristics. Further work will be performed on these urease. X-ray diffraction and scanning electron microscopy were chosen substrates to optimize the anaerobic digestion process in employed to elucidate the mechanism of calcite formation from 15 L-reactors. the crude plant extracts. The results revealed that urease in the plant extracts catalyzed the hydrolysis of urea in liquid state cul- http://dx.doi.org/10.1016/j.nbt.2014.05.1948 tures and decreased heavy metal amounts in the contaminated soil. The heavy metal amounts were decreased in the leachate from the treated mine soil; 55.4% of Pb, 35.6% of Cu, 33.6% of Mn, 32.0% of As, and 25.6% of Fe, respectively. The procedure described herein is a simple and beneficial method of calcite biomineraliza-

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PH-10 can decolorize RBBR more efficiently in continous mode than in batch mode. Biomolecules for Environmental Application: Direct Use Acknowledgements: of Biorecovered Precious Metal Catalyst from Artificial The research wassupported by the following projects: OPVK Wastewater for Chlorinated Environmental Pollutant CZ 1.07/2.3.00/30.0019, FP7-KBBE-2012-6-singlestage no. 312100 Degradation BIOCLEAN, CZ.1.05/2.100/03.0100 (IET) and CR National Feasi- Ian Sofian Yunus ∗ , Shen-Long Tsai bility Programme I no.LO1208. National Taiwan University of Science and Technology http://dx.doi.org/10.1016/j.nbt.2014.05.1950

In the last decades, precious metals have been extensively studied for their potential use as catalysts for chlorinated pol- PH-12 lutant degradations. These pollutants are resulted from various Composting of creosote-impregnated wood via com- industrial processes, groundwater, pharmaceuticals wastewater posting with green wastes: Ecotoxicity and microbial treatment plant (WWTP) effluents, and many other sources. In community dynamics during polycyclic aromatic hydro- this study, a new recovery method of precious metal catalyst for carbons degradation process dechlorination of environmental pollutant was examined. The precious metal catalyst was produced by reduction of its ionic state, Stefano Covino ∗ , Zdena Kresinováˇ , Monika Cvanˇ carováˇ , Alena which was recovered from artificial wastewater, using biomolecules Filipová, Tomasˇ Cajthaml based adsorbent. This study demonstrates that the newly discov- Institute of Microbiology AS CR, v.v.i ered biorecovery concept of precious metal catalyst from artifical wastewater using biomolecules based adsorbent and its direct use Composting has been shown to be a suitable bioremediation as catalyst for chorinated pollutant degradation are both feasible method for the clean-up of polluted matrices. In our pilot-scale and applicable. test, creosote-impregnated wood with an overall polycyclic aro- http://dx.doi.org/10.1016/j.nbt.2014.05.1949 matic hydrocarbons (PAH) contamination of 26498 mg kg-1 was the target material and two different substrates were used as bulking agents, namely grass cuttings and pre-treated broiler litter. PH-11 Incubation took place in 400 l static composters over a period of 240 days (40 days active composting followed by 200 days Degradation of recalcitrant polymers and synthetic dyes maturation). The effectiveness of the two composting processes by microrganisms isolated from polluted soils was comparatively evaluated throughout the whole incubation Ashutosh Kumar Verma 1,∗ , Dornakova Veronika 2, Hana Kotulova 2, period by means of contaminant degradation analyses and tox- Katerina Malachova 2, Cenek Novotny 3 icological testing whereas shifts in microbial community structure were assessed via phosholipid fatty acid analysis (PLFA) and 454- 1 University of Ostrava pyrosequencing. The grass substrate promoted an almost complete 2 Ostrava University 3 Academy of Sciences of the Czech Republic removal (i.e. 97%) of the total PAH content in creosote wood, while overall PAH depletion using broiler litter as bulking agent was 81% An attempt was made to isolate microorganisms which may of the original concentration. The acute toxicity test towards the have potential to degrade polymer plastics and recalcitrant luminescent bacterium Vibrio fischeri and the phytotoxicity test organopollutants. The prescreening was carried out on agar plates based on germinability of barley seeds (Hordeum vulgare L.) showed containing 100 ppm of a model dye, Azure B, whose decolorization that the PAH degradation process was accompanied by a significant was considered to be a tool indicating an enhanced degrada- drop in toxicity. Regardless of the treatment typology, PLFA pro- tion capacity towards recalcitrant polymers and xenobiotics. The filing highlighted an increased incidence of Gram- bacteria and compost, sludge and polymer-polluted soils had been used for iso- fungi in the crucial phases of PAH dissipation. Morevoer, fungi lation. More than 50 monocultures including bacteria, yeast and appeared to be dominant also in the maturation phase, especially fungi belonging to various genera showed efficient decolorization when broiler litter was the substrate. Pyrosequencing analyses of of Azure B. Identification of the isolates was carried out using ribo- 16S rRNA and ITS gene sequences for bacteria and fungi respec- somal molecular markers. A few isolates had been used for the tively, are in progress. degradation of virgin and pre-treated plastic polymers with limited http://dx.doi.org/10.1016/j.nbt.2014.05.1951 success. Culture-independent approach was applied to assess the diversity of microbial communities in polymer-polluted soil(s). A fungal isolate affiliated with Trametes sp. had been further used for the decolorization of an anthraquinone dye, Remazol Brilliant Blue R (RBBR) at a concentration of 50 ppm. Our aim was to evaluate the efficiency and behaviour of the fungus under the conditions of rotating biological contactor reactor in both batch and continuous mode. The results indicated that the selected fungal species

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PH-13 PH-14

Selection and characterization of indigenous Characterization of sulphate reducing bacteria commu- hydrocarbon-degrading bacteria from tourist ports nities in sediments from tourist ports in the Mediter- in the Mediterranean Sea Basin ranean Sea Basin

Enrica Bullita 1 , Claudio Ruggeri 1, Simona Sergi 1, Laura Serreli 1, Claudio Ruggeri 1,∗ , Paolo La Colla 2, Enrica Bullita 2, Simona Sergi 2, Giovannimatteo Erby 2, Alessio Nieddu 2, Alessandra Carucci 2, Elena Francesco Vitali 3, Giorgio Mastromei 3, Elena Tamburini 2 1,∗ Tamburini 1 University of Cagliari 2 1 University of Cagliari - Department of Biomedical Sciences University of Cagliari - Department of Biomedical Sciences 3 2 University of Cagliari - Department of Civil-Environmental Engineering and University of Florence - Department of Biology Architecture Ports receive pollution from land, ships and port facilities. Pollution by petroleum hydrocarbons is one of the major envi- Furthermore, tourist ports are subject to seasonal anthropogenic ronmental problems in ports and it is mainly associated with impacts. Hydrocarbon contamination associated with port activ- ship/boat traffic and related facilities. Ports are not closed systems ities poses major concerns for human health and coastal and their pollution may impact adjacent coastal areas. Hydrocar- ecosystems. bon degraders and particularly the obligate hydrocarbonoclastic This study was carried out within MAPMED, a multidisciplinary bacteria carry out a fundamental and global activity in biological project aimed to improve the environmental sustainability of removal of hydrocarbons in marine habitats. tourist ports in the Mediterranean Sea with regard to monitoring This study was carried out within MAPMED, a multidisciplinary and reduction of hydrocarbon pollution. project aimed to improve the environmental sustainability of Three tourist ports were selected as case study sites: Cagliari tourist ports in the Mediterranean Sea with regard to monitoring (Italy), El Kantaoui (Tunisia), and Heraklion (Greece). In each port, and reduction of hydrocarbon pollution. sampling stations were located in different sectors according to Three tourist ports were selected as case study sites: Cagliari their different uses. Three sampling campaigns were carried out in (Italy), El Kantaoui (Tunisia), and Heraklion (Greece). The degra- winter, spring at the beginning of tourist season and late summer at dation potential of the autochthonous bacterial communities was the end of tourist season. Samples of surface and anoxic sediments evaluated enumerating heterotrophs and hydrocarbon degraders were collected at different stations. by MPN in the surface seawater. Heterotrophs were significantly The dsrAB gene was chosen as genetic marker to specifi- more abundant in seawater from Cagliari port as compared to cally characterise sulphate reducing bacteria (SRB). It codes for El Kantaoui and Heraklion ports. On the contrary, higher viable the dissimilatory sulphite reductase catalysing the last step in titles of diesel- and phenanthrene-degraders were found in sea- the sulphate reduction pathway. Hydrocarbon degradation under water from El Kantaoui port compared to the other two areas. sulphate-reducing conditions is an important process in marine Hydrocarbon-degrading bacteria were isolated and characterized sediments in anoxic environments. T-RFLP analysis of dsrAB gene regarding their phylogenetic position and catabolic abilities. The was employed to elucidate the community structure of SRB. This hydrocarbon degradation activities were evaluated by GC-MS in work provides a spatial comparison of SRB at two scales, different aerobic batch reactors on diesel as carbon source. The majority port sectors within each port area and different port areas across of degraders from Cagliari were assigned to Pseudomonas whereas the Mediterranean Sea, as well as a temporal comparison among strains from El Kantaoui and Heraklion were assigned to Alcanivo- different seasons. rax and Marinobacter. The selection of the most appropriate methodologies for the The selection of the most appropriate methodologies for the eco-efficient remediation of petroleum-hydrocarbon contamina- eco-efficient remediation of petroleum-hydrocarbon contamination of selected sites is currently in progress. tion of selected sites is currently in progress. http://dx.doi.org/10.1016/j.nbt.2014.05.1953 http://dx.doi.org/10.1016/j.nbt.2014.05.1952

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PH-15 tional expression of laccases in yeast was detected as an ability to convert ABTS (2,2-azino-bis(3-ethylbenzothiazoline-6-sulphonic Experimental design in the degradation of pyrene by acid) to a green product. The level of expression and the com- marine-derived fungi Chaunopycnis alba CBMAI 1346 position of the expression media were optimized. All recombinant and Xylaria sp. CBMAI 1464 laccases were produced as secreted proteins due to their native N- Maria Vasconcelos 1,∗ , Rafaella Bonugli-Santos 1, Marili Rodrigues 2, terminal signal sequences, and thus they were easily isolated from Sinésio Boaventura 2, Lara Sette 3 the medium by ion-exchange and gel chromatography. The highest specific activity was found for the laccase from Trametes trogii, 1 Center of Chemical, Biology and Agricultural Research which, moreover, was the only laccase showing the ability of dye 2 Organic Chemical and Pharmaceutical Division 3 São Paulo State University decolorization. This work was supported by TA CR grant TA0101 1461. Marine-derived filamentous fungi are considered strategic for References biotechnological applications, including bioremediation of envi- [1].Riva S. Trends in Biotechnology 2006;24(5):219–26. ronmental pollutants under saline conditions. Chaunopycnis alba [2].Bulter T, et al. Applied and Environmental Microbiology CBMAI 1346 and Xylaria sp. CBMAI 1464 isolated from marine 2003;69(2):987–95. sponges were subjected to experimental design in order to opti- [3].Cassland P, Jonsson LJ. Applied Microbiology and Biotechnology mize their ability to degrade pyrene. At first a Plackett-Burman (PB) 1999;52(3):393–400. [4].Colao MC, et al. Microbial Cell Factories 2006;5(31). model was used, with nine variables, providing 20 assays in total. The matrix was analyzed statistically using STATISTICA 7.0. After http://dx.doi.org/10.1016/j.nbt.2014.05.1955 7 days C. alba CBMAI 1346 and Xylaria sp. CBMAI 1464 reached 79.39% and 46.00% of pyrene degradation, respectively. For the fungus C. alba CBMAI 1346, salinity, concentration of malt extract, PH-17 peptone and yeast extract and the presence of MnSO4 presented a positive effect on the pyrene degradation, being significant at Expression analysis of APX and CAT genes in eggplants 90% (p < 0.1). For Xylaria sp. CBMAI 1464, pH, salinity, concen- subjected to Cu + 2 and Zn + 2 heavy metals tration of malt extract, peptone and yeast extract and inoculum ˙Ilker Büyük , Semra Soydam Aydın, Demet Cansaran Duman, Sumer presented a positive effect on the pyrene degradation, however, Aras ∗ none of them was significant at 90% (p < 0.1). In the second PB Ankara University composed by 5 variables (total of 16 assays) the fungus C. alba CBMAI 1346 reached 58.28% of pyrene degradation. The variables Stress could be called as any change in unfavorable growing riboflavin and bibasic potassium phosphate (KH2PO4) presented a conditions that disrupts homeostasis in plants and can lead to negative effect on the process, suggesting that the inducers might lower yields and possible crop failure. Biotic and abiotic stress fac- work as inhibitors. C. alba CBMAI 1346 was subjected to frac- tors are the sources of environmental stress in plants and heavy tional factorial (FF) using four variables, reaching 94.13% of pyrene metal toxicity is one of the global environmental problems. To degradation. Based on these results a central composite rotational overcome negative effects of heavy metal contaminations on plant design (CCRD) was performed. However, a higher percentage of health, evaluation of the genes responsible for stress tolerance is degradation was not obtained and the result from the FF was vali- the first stage of producing stress-tolerant plants. dated. For this purpose, Solanum melongena L. plants were exposed var- http://dx.doi.org/10.1016/j.nbt.2014.05.1954 ious concentrations of Cu+2 and Zn+2 heavy metals (blank, 80 ␮M, 160 ␮M, 320 ␮M, 640 ␮M, 1280 ␮M) for 24 h. Gene expression analysis of catalase (CAT) and ascorbate peroxidase (APX) genes PH-16 were performed using Light Cycler Nano Real-Time PCR instrument with cDNAs which were synthesized from mRNAs isolated Heterologous expression of three laccases from different from the leaf tissues of eggplant samples. Results indicated that all origin in Saccharomyces cerevisiae and their use for envi- concentrations of Cu+2 and Zn+2 triggered the expression changes ronmental applications of the CAT and APX genes. Almost all concentrations of both metal Klara Richterova 1,∗ , Zuzana Antosova 1, Jiri Dostal 2, Iva Pichova 2, contaminations led to increases in mRNA levels of CAT and APX Hana Sychrova 1 genes and this increment pointed to the importance of these genes 1 Institute of Physiology, Academy of Sciences of the Czech Republic v.v.i for stress defence in eggplant. The overexpression and silencing of +2 +2 2 Department of Biochemistry, Institute of Organic Chemistry and Biochemistry, the APX and CAT genes in eggplants under Cu and Zn stresses Academy of Sciences of the Czech Republic v.v.i will be identified in further studies. http://dx.doi.org/10.1016/j.nbt.2014.05.1956 Laccases are “eco-friendly” oxidoreductases with a wide range of biotechnological applications [1]. Three laccase genes were cloned into the S. cerevisiae expression vectors under various constitutive promoters. The genes originated from Myceliophthora thermophila [2], Trametes versicolor [3], and Trametes trogii [4]. Func-

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PH-18 a diffusion barrier to fluxes of reactants and products. We will present Raman and FTIR microspectroscopic evidence toward the Oxidation of low density polyethylene by a laccase- identification of secondary minerals formed during chalcopyrite mediator system passivation in the presence of iron and sulphur-oxidizing bacteria Guillermo Huerta ∗ , Marcela Ayala isolated from the mines of HCM. Potassium jarosite is the initial product followed by the formation of ammonia-jarosite. Covellite Instituto de Biotecnología, Universidad Nacional Autónoma de México and elemental sulphur are detected in the passivation layer. The mechanism of passivation and testing strategies to minimize it will Synthetic polymers such as low density polyethylene (LDPE) be presented. represent a big ecological problem in todays environment. Scien- tists have explored and proposed a variety of possible solutions to http://dx.doi.org/10.1016/j.nbt.2014.05.1958 alleviate this problem [1]. Biological degradation is an eco-friendly process in plastic waste management, first, the polymer must suffer physical and chemical changes to facilitate the mineralization or PH-20 degradation process by microorganism [2]. Such changes become Characterization of a bacterial collection isolated from possible when the plastics are oxidized by abiotic factors or by a extreme environment with potentiality of use in catalysts. biotechnology industry Laccases are oxidoreductases catalyzing the oxidation of organic substrates. Laccases have been described as efficient cata- Jose Manuel Gomez 1,∗ , Arelys Diaz 2, Jeannette Marrero 2, Gema lysts for many applications, from paper bleaching, decolourization Cabrera 1, Orquidea Coto 2 of textile effluents, biosensors and bioremediation [3]. In this work 1 University of Cadiz we investigated the use of a laccase and mediators, to modify LDPE 2 University of Havana films. We observed changes in chemical composition of enzyme- treated LDPE films, mostly the presence of carbonyl groups in Heavy metal pollution is still a worldwide problem. The metal- − the infrared region of 1720 cm 1; along with these oxidation sig- lic elements can not be degraded but only could be transformed nals, there was a loss of surface hydrophobicity (measured by from an oxidation state or organic compound to another. There- contact angle) and changes in mechanical properties, such as rela- fore, bioremediation is a attractive option to be applied for tive elongation. The enzyme-treated LDPE films were incubated in cleaning up the environment and recovery of soils. Microbiote the presence of selected microorganisms in order to evaluate their isolated from extreme environmemts is considered as a source potential biodegradability. of enzymes and active metabolites which could be used in the Acknowledgments. Authors acknowledge the financial sup- biotechnology industry. In this work bioactive compounds and port of Conacyt 179241. remotion capacity were evaluated in seven strains ofSerratia mar- cencensisolated from nickel lateritic ores located in Moa, Cuba. References 100% of the strains produced extracellular enzymes including DNases, proteases, lipases, lecitinases and caseine hydrolases as [1].Panda AK, Singh RK, Mishra DK. Renewable and Sustainable Energy Reviews 2010;14:233–48. well as were able to solubilize inorganic phosphorus. The strains [2].Bonhomme S, Cuer A, Delort AM, Lemaire J, Sancelme M, Scott G. were classified as highly resistant strains based on the minimal Polymer Degradation and Stability 2003;81:441–52. inhibitory concentration (MIC) of Ni(II) (>25 mM) and Co(II) [3].Rodriguez-Cuoto S, Toca-Herrera JL. Biotechnology Advances (>12 mM). Biomasses of the strainsS. marcescensC-1, 16 and 19 2006;24:500–13. showed removal capacity Ni(II) and Co(II) from solution until http://dx.doi.org/10.1016/j.nbt.2014.05.1957 values higher than 100 ppm in two hours of biomass - metal contact. Removal capacity of strain 16 in bioreactor reached values of 36% of Co(II) and higher than 50% of Ni(II), Cu(II) and Zn(II) PH-19 from multimetallic solutions. The results presented here suggest the potentiality of the use of theses strains in the medical and Probing the formation of secondary minerals in the biotechnology industry. bioleaching reactions of chalcopyrite by Raman and FTIR microspectroscopy http://dx.doi.org/10.1016/j.nbt.2014.05.1959

Constantinos Varotsis 1,∗ , Chrystaleni Giangou 1, Sotiris Papadatos 1, Elena Xenofontos 1, Ioannis Vyrides 1, Giorgos Manos 2, Nicolas Messios 2, Constantinos Xydas 2 1 Cyprus University of Technology 2 Hellenic Copper Mines

Chalcopyrite passivation reduces the yields from leaching and bioleaching. Despite the great efforts the problem has not been successfully resolved. Passivation involves the formation of a layer of secondary minerals on chalcopyrite surface which becomes

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PH-21 PNL-MK25. The genome of P. putida PNL-MK25 strain has been sequenced and annotated, coupling with biological assays relating Harnessing microbial communities in tropical peatlands to plant growth promoting properties. as resources for novel biocatalysts for plant biomass Results indicated that the estimated genome size of the bac- deconstruction terium is around 5.8Mb, encoding 5313 putative open reading Nicole Chua 1,∗ , PuiYiYung1, Shivshankar Umashankar 1, I. Made frames. Whole genome sequence comparison between strain Sudiana 2, Sanjay Swarup 1 MK25 to other sequenced genomes placed it closest to Pseudomonas fluorescens Pf01, sharing ∼82% of ORFs. Substrate utilization 1 National University of Singapore revealed that the strain is capable of utilizing a range of car- 2 LIPI boxylic acids, esters and fatty acids. It is also resistant to several classes of known antibiotics. Plant growth promoting assays have Large amounts of plant biomass are generated annually, usually revealed that strain MK25 promotes lateral root formation in from factory and municipal wastes, forest- and agricultural- model plant Arabidopsis. Genes potentially responsible for plant residues. Plant-derived lignocellulosics represents a major source of growth promotion, for example the production of antimicro- renewable organic matter as raw material for fuel, macromolecules bial compounds and auxin, has been found and/or functionally and aromatics. However, lack of efficient technologies to con- confirmed. vert these “waste” into useful product resulted a great deal of them being burnt for heat generation, or merely to reduce the http://dx.doi.org/10.1016/j.nbt.2014.05.1961 volume of biomass. Common rate limiting steps during biological degradation of biomass include accumulation of microbial growth inhibitors during treatment process, and the lack of diverse PH-23 enzymes available for degradation of the heterogenic lignin- and lignin-bounded chains. ’Biodesalination’: a synthetic biology approach for the Microbial enzymes are well known for their vast functional use of photosynthetic bacteria in water treatment diversity, which have increasing recognitions from industries. We Annegret Honsbein 1,∗ , Mary Ann Madsen 2, Jaime M. Amezaga 3, investigated the functional capabilities of the microbial communi- Catherine A. Biggs 4, Tom Bond 5, Catherine J. Gandy 3, Esther ties in tropical peatlands for their ability to degrade lignocellulosic Karunakaran 4, Linda Lawton 6, Konstantinos Minas 6, Michael R. compounds. Using both culturing and culture-independent tech- Templeton 5, Anna Amtmann 2 niques, enzymes capable of lignocellolytic activities were screened. 1 University of Glasgow To date, we have found microbial isolates capable of hydrolyz- 2 Institute of Molecular, Cell and Systems Biology, University of Glasgow ing hemicelluloses such as xylan and arabinoxylan. The same 3 School of Civil Engineering and Geosciences, Newcastle University isolates are also capable of growth in high concentration of growth 4 Department of Chemical and Biological Engineering, University of Sheffield 5 inhibitors such as syringaldehyde, hydroquinone and furfural. Department of Civil and Environmental Engineering, Imperial College London 6 Institute for Innovation, Design and Sustainability, Robert Gordon University, Culture-independent, sequence-guided metagenomic approaches Aberdeen have revealed the microbial communities in tropical peatlands contain several classes of multi-copper oxidases (e.g. laccases), dye- Shortage of freshwater is a serious global problem, and expected decolorizing peroxidases, and feruloyl esterases. Sequence analysis to become even more urgent over the next decades. Many of revealed these enzymes are distinct from existing enzymes in the the driest regions worldwide are close to the sea, but irrigation database. of fields with seawater–even if diluted–leads to the build-up of http://dx.doi.org/10.1016/j.nbt.2014.05.1960 salt levels in the soil that are toxic to all common food crops (http://www.unwater.org). Current desalination technologies such as membrane-based reverse osmosis, are successfully used in PH-22 large-scale desalination plants, however, they are expensive and energy inefficient [1]. Our multi-disciplinary team of biologists Functional and genomic analysis of the plant growth and engineers from 5 UK universities is working on an innova- promoting bacterium I tive desalination technology based on biological processes [2]. Pui Yi Maria Yung ∗ , Wei Ling Ng, Yong Jian Lee, Shao Bing Johanan The “Biodesalination” strategy envisions the use of photosynthetic Aow, Boon Kiat Lennon Lim, Nicole Chua, Shivshankar Umashankar, cyanobacteria modified with light-driven ion transport proteins to Sanjay Swarup function as ion exchangers that selectively remove sodium chloride from seawater. This process would harness solar energy to National University of Singapore provide a more cost effective and energetically sustainable desalination process. Plant growth promoting Pseudomonas species are of great agricultural interest, especially in food crops. Using biological agent References to enhance plant growth is both environmentally friendly (reduce [1].Plappally AK, Lienhard JH. Costs for water supply, treatment, end- use of chemicals and fertilizers) and economical in a long term. use and reclamation. Desalination and Water Treatment 2013;51: We studied both the physiological and genomic properties of 200–32. our model plant growth promoting bacterium Pseudomonas putida [2].Amezaga JM, Amtmann A, Biggs CA, Bond T, Gandy CJ, Hons- bein A, Karunakaran E, Lawton L, Madsen MA, Minas K, Templeton

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MR. ’Biodesalination’: a case study for applications of photo- the modern biorefineries [1]. Lignin derived compounds released synthetic bacteria in water treatment. Plant Physiology 2014;164: during lignocellulose processing can be used as redox mediators 1661–76. of laccases or as bioactive precursors for the enzymatic synthesis http://dx.doi.org/10.1016/j.nbt.2014.05.1962 of natural products. However, the industrial implementation of fungal laccases is hampered by the lack of powerful expression systems and the need for active and stable enzymes under the harsh PH-24 operational conditions. Recently we developed fungal laccases of high-redox poten- Characterisation of Metal Transport Proteins for provid- tial functionally expressed in S. cerevisiae by directed evolution ing metal stress tolerance in green microalgae [2,3]. Using these platforms as the starting points and ad-hoc Aniefon Ibuot ∗ , Andrew Dean, Jon Pittman high-throughput screening methods [4,5] we developed a more robust laccase with improved catalytic activity towards phenolic University of Manchester compounds under preferred pH conditions, which represents a step forward to aid the conversion of lignocellulosic biomass into Characterisation of Metal Transport Proteins for providing chemicals, materials and biofuels. metal stress tolerance in green microalgae This work has been funded by the Spanish Project BIO2010- The Cation Diffusion Facilitator (CDF) family is known for pro- 19697. viding significant tolerance in metals such as Mn2+ and Zn2+. These proteins are well characterised in organisms such as human, yeast References and plants but not in unicellular green algae. There are five CDF genes in Chlamydomonas reinhardtii. MTP1 to MTP4 were cloned [1].Canas˜ AI, Camarero S. Biotechnol Adv 2010;28:694–705. and functionally characterised by yeast heterologous expression [2].Maté D, García-Burgos C, García-Ruiz E, Ballesteros A, Camarero S, Alcalde M. Chem Biol 2010;17:1030–41. using a Zn2+ and Co2+ sensitive mutant yeast strain zrc1cot1 and a [3].Camarero S, Pardo I, Canas˜ AI, Molina P, Record E, Martínez AT, 2+ Mn sensitive yeast strain pmr1. The MTP-expressing yeast strains Martínez MJ, Alcalde M. Appl Environ Microbiol 2012;78:1370–84. were screened for suppression of metal sensitivity to ascertain the [4].Pardo I, Chanaga X, Vicente AI, Alcalde M, Camarero S. BMC Bio- transport function each MTP protein. MTP1 was able to strongly technol 2013;13:90. rescue the Zn2+ and Co2+ sensitivity of the zrc1cot1 strain, while [5].Pardo I, Vicente AI, Alcalde M, Camarero S. Biotechnol Bioeng 2012;109:2978–86. MTP3 could weakly mediate Zn2+ and Co2+ growth. MTP2 and MTP4 appeared to have no Zn2+ or Co2+ transport activity. MTP2 http://dx.doi.org/10.1016/j.nbt.2014.05.1964 to MTP4 but not MTP1 could strongly rescue the Mn2+ sensitivity of the pmr1 strain. This clearly confirms the metal transport functions of the MTP family in Chlamydomonas. Further studies are PH-26 over-expressing these MTP proteins in Chlamydomonas to deter- Investigation of polyvinylchloride biodegradation by mine whether enhanced metal uptake and tolerance of specific microbial consortia enriched from digested sludges metals can be achieved. This research could increase our understanding of how yeast and algae could be utilized as a potential Fabio Fava ∗ , Noura Raddadi, Lucia Giacomucci, Nadia Lotti environmentally sustainable tool for the remediation and decon- University of Bologna tamination of metal-polluted wastewater. Keywords: Metal transport proteins, metals, yeast, Chlamy- During last decades, production of synthetic plastics has domonas reinhardtii increased dramatically to reach approximately 280 million tonnes http://dx.doi.org/10.1016/j.nbt.2014.05.1963 in 2011. The accumulation of plastic waste in the environment is raising concerns about its effects both on human and the environment. In EU, about 40% of plastic waste is currently disposed of in PH-25 landfills, where partially undergoes photodegradation, producing microplastics which can absorb toxins and toxic chemicals and Engineering high-redox potential laccases in the lab to together with plasticizers enter the marine environment and thus aid biomass conversion into chemicals, materials and the food chain, where they exert toxic effects. Furthermore, colo- biofuels nization of plastics by sessile organisms may permit transport of Susana Camarero 1,∗ , Ana I. Vicente 1, Miguel Alcalde 2, Isabel alien species in the ocean environment and may threaten marine Pardo 1 biodiversity. Therefore it is necessary to find new eco-friendly techniques for safe handling and degradation of plastic wastes. 1 Centro de Investigaciones Biológicas, CSIC 2 Instituto de Catálisis y Petroleoquímica, CSIC In this work, ten microbial communities enriched from waste plastics from digested sludges were screened for their capability of Laccases catalyze the oxidation of a variety of aromatic com- degrading non-pretreated films of polyvynil chloride (PVC) and pounds without any other requirement than oxygen from air. In polypropylene (PP). After six months of anaerobic incubation in previous studies we have highlighted the biotechnological poten- the presence of the plastic films as main carbon source, growth tial of fungal laccases to improve the utilization of plant biomass in of microbial community was recorded in all enriched consortia. Thermogravimetric analysis (TGA) performed on PP and PVC films

www.elsevier.com/locate/nbt S141 ENVIRONMENTAL BIOTECHNOLOGY New Biotechnology · Volume 31S · July 2014 showed biodegradation of only PVC plastic film by 5 communities. PH-28 Further analyses, including ATR-FTIR and SEM analyses to analyze surface film modifications and GPC for the evaluation of the reduc- Analysis of the effectiveness of a cereal milling by tion of the polymer molecular weight on biodegraded PVC films product monocomponent medium for the low cost pro- are ongoing. duction of Bacillus thuringiensis Acknowledgments. The support of the FP7 EU project BIO- Gihane Rahbany 1,∗ , Dominique Salameh 1, Cedric Brandam 2, CLEAN (GA-312100) is acknowledged. Roger Lteif 1 http://dx.doi.org/10.1016/j.nbt.2014.05.1965 1 Université Saint-Joseph Liban 2 Université de Toulouse; INPT, UPS; Laboratoire de Génie Chimique France

PH-27 Bacillus thuringiensis is a facultative anaerobe, gram positive, spore forming bacterium. The biotechnological importance Electrical conductivity in granular biomass. Standard- of this bacterium resides in its ability to produce, during sporu- ization and evaluation lation, crystal proteins known as ␦-endotoxins which express Diego Andres Suarez Zuluaga ∗ , Sebastian Canizales, Annemiek ter specific insecticidal activity. At industrial scale, the culture media Heijne, Jan Weijma, Cees J.N. Buisman represents an important part of B. thuringiensis based biopes- ticides production cost. According to the literature, different Wageningen University agro-industrial residues and byproducts were used as sources of proteins in order to reduce the cost of B. thuringiensis culture Biologically produced oxidation-reduction reactions in anaero- medium, but carbohydrates (glucose, starch or molasses) and/or bic granules require electron transfer between bacteria and archaea mineral sources were added. cells. Anaerobic types of biomass perform this electron transfer by In this work, a cereal milling by-product (CMB) as a mono- mean of different mechanisms. Understanding how this process component medium was investigated and compared to synthetic occurs would help in the optimization and development of pro- mediums in terms of ␦-endotoxin yield and productivity in sub- cesses for anaerobic wastewater treatment. So far, electron transfer merged fermentation of different strains of B. thuringiensis. The via outer-surface c-type cytochromes, long-range electron trans- CMB was shown efficient to be used as a complete substrate (source fer via nanowires, electron flow through a conductive biofilm of proteins, carbohydrates and minerals) for B. thuringiensis matrix containing cytochromes and soluble electron shuttles are production. The optimal CMB ratio in the culture medium was the mechanisms proposed for explaining such process. A simple found to be 6% in shake flasks experiments. The consumption method, based on the one developed by Morita et al. (2011) [1], for of the CMB sugars by the bacteria was analyzed. Production of the measurement and analysis of electrical conductive of biomass the bio-insecticide in lab-bioreactor in controlled conditions was granules is assessed in this study. By means of electrochemical equally performed to give basic elements for extrapolation in interface equipment and a probe consistent of two electrodes sep- industrial conditions. arated by a non-conductive gap, voltages were applied to granular biomass and current was measured. Different physical and micro- http://dx.doi.org/10.1016/j.nbt.2014.05.1967 biological factors that could affect the measurement were studied and their influence determined. A methodology was standardized and voltage vs. current graphs were generated. From them, PH-29 conductance (and conductivity) was calculated. Biomass granules NaCl addition for increased polyhydroxyalkanoate pro- originated on Emmtec (sulphate reducing sludge) and Eerbeek duction by Cupriavidus necator (methanogenic sludge) were used for the standardization of the parameters of the tests and presenting conductance up to 625.86 Pearl Passanha ∗ , Gopal Kedia, Richard Dinsdale, Alan Guwy, San- and 46.94 nS respectively. By relating the measured conductance dra Esteves with the origin, composition and specific activity of each gran- University of South Wales ule a further understanding of the mechanisms used to transfer electrons was obtained. The effect of five different NaCl concentrations, namely 3.5, Reference 6.5, 9, 12 and 15 g/l NaCl on PHA productivity using Cupriavidus [1].Morita M, et al. mBio 2011;2(4):1–6. necator has been investigated alongside a control (no added NaCl) when acetic acid was used as the sole carbon source. A dielectric http://dx.doi.org/10.1016/j.nbt.2014.05.1966 spectroscopy probe was used to measure real-time PHA accumulation online in conjunction with the chemical offline analysis of PHA. The highest PHA production was obtained with the addition of 9 g/l NaCl, which yielded 30% higher PHA than the control. Increasing the addition of NaCl to 15 g/l was found however to inhibit the production of PHA. NaCl addition can be used as a simple, low cost, sustainable, non toxic and non reactive external stress strategy for increasing PHA productivity when compared

S142 www.elsevier.com/locate/nbt New Biotechnology · Volume 31S · July 2014 ENVIRONMENTAL BIOTECHNOLOGY to previous increased temperature and other types of chemical phenanthrene as sole carbon and energy source. The bacterial stress such as ethanol, hydrogen peroxide or metal stress. The isolates were grouped and classified using ISSR-PCR technique order of PHA accumulation for the above salt concentrations were through four different primers. The best phenanthrene degrader 9 g/l > 6.5 g/l > 3.5 g/l > control > 12 g/l > 15 g/l NaCl, which clearly was chosen and identified using 16S rRNA gene sequencing as indicates that addition of NaCl, which up to 9 g/l enhanced PHA Lysinibacillus fusiformis EHTH1 and was able to degrade up to production. 150 mg/l phenanthrene. The bacterial isolate was able to degrade http://dx.doi.org/10.1016/j.nbt.2014.05.1968 another single, double and triple ring containing hydrocarbons as sole carbon and energy source. The strain was able to produce rhamnolipid biosurfactant which hasn’t been detected for this PH-30 genus before. The 800 bp amplified fragment of RhlA gene was successfully cloned into TOPO vector with subsequent transfor- Enhancement of treatment of poly aromatic hydro- mation into E. coli (Top10 strain). The results showed successful carbon contaminated water using new biosurfactant cloning and transformation and showed the ability of the cloned producing marine bacterium strain to degrade phenanthrene at higher concentrations as sole carbon and energy source. Tarek Taha 1,∗ , ELsayed Hafez 1, Hesham Mahdy 2, Saad Alamri 3 http://dx.doi.org/10.1016/j.nbt.2014.05.1969 1 City for Scientific Research and Technology Applications 2 Alazhar University 3 King Khalid University

Poly Aromatic Hydrocarbons (PAH) are well known for their slight solubility in water which considered one of the most obstacles that face the bacterial isolates to degrade hydrocarbons. Our study succeed to isolate nine bacterial isolates able to consume

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General biotechnology glutamicum acquired an ability to grow under stress of 10 mM H2O2. In the transcriptome results of the adapted strain, a PI-01 total of 1,180 genes (38.6% of the genome) were up-regulated more than 2-fold, and 126 genes (4.1% of the genome) were G-protein alpha subunit gene, CGA1, is involved in the down-regulated less than 2-fold under oxidative stress condition resistance against heat and osmotic stress in Chlamy- compared with those of wild-type (WT) strain under non-stress domonas reinhardtii condition. Especially the genes encoding enzymes in the ␤- Changsu Lee 1,∗ , Yoon-E Choi 2 ketoadipate pathway (pca genes) were up-regulated more than 3-fold. To develop an artificial oxidative stress-resistant strain, the 1 Department of Bioprocess Engineering, Chonbuk National University pca gene clusters in the ␤-ketoadipate pathway were expressed 2 LED Agri-bio Fusion Technology Research Center, Chonbuk National Univer- sity, 79 Gobong-ro, Iksan-si, Jeollabuk-do in the WT strain. WT strain was unable to grow under 2 mM H2O2, while the strains expressing pca gene clusters restored The function of heterotrimeric GTPase (G-protein) has been growth. The expressions of pca gene clusters also enabled the characterized in eukaryotic cells. G-proteins are composed of three WT strain to increase its resistance against various oxidative subunits, the alpha (␣), beta (␤), gamma (␥) subunits. The gen- stressors including H2O2. The oxidative stress resistance of those eral GTPase activity of G␣ induces the hydrolysis of the bound strain was correlated to the reactive oxygen species (ROS)- GTP, thereby playing pivotal roles in relaying the signals. How- scavenging activity of the cytosol. These findings would be useful ever, microalgal G-proteins including G-protein alpha subunit to develop an oxidative stress-resistant synthetic strain for indus- have remained largely unknown. In this study, we characterized trial applications. a gene, CGA1, encoding G-protein alpha subunit in Chlamy- [This study was financially supported by the Korean Ministry domonas reinhardtii. Independent knock-down mutants of CGA1 of Science, ICT & Future Planning (Intelligent Synthetic Biology were generated via the RNA interference (RNAi) approach. CGA1 Center of Global Frontier Project 2012M3A6A8054887)] expressions consistently reduced significantly in all of the CGA1 http://dx.doi.org/10.1016/j.nbt.2014.05.1971 mutants (cga1). Interestingly, all of the cga1 displayed higher survival rate at 35 ◦C, compared to wild type. In addition, all of the cga1 has an enhanced survival rate at high osmotic stress, PI-03 compared to that of the wild type. To further understand mechanisms of CGA1-mediated resistance, the extent of reactive oxygen Symbiosis Mechanisms of Lactic Acid Bacteria and Yeasts species (ROS), the expressions of several putative downstream in Inner Mongolian Traditional Fermentative Milk Prod- genes including heat shock proteins(HSPs) and MAP kinases were ucts thoroughly analyzed. Our data indicated that CGA1 is associated Yinfeng He ∗ , Jianjun Tian, Minmin Liu, Bin Yan with the regulation of resistance against heat or osmotic stress Inner Mongolia University of Agriculture in C. reinhardtii by affecting multiple downstream genes. Since a G-protein alpha subunit is highly conserved across microal- The majority of the traditional dairy products in Inner gal species, our results will shed light on future biotechnological Mongolia, China, are co-fermented with lactic acid bacteria and applications of microalgae under the extreme environmental con- yeasts. So far, forty-seven strains of lactic acid bacteria and nine- ditions. teen strains of yeast have been isolated and identified from http://dx.doi.org/10.1016/j.nbt.2014.05.1970 koumisses, ripened creams, and yogurts. A systematic study was performed to understand the co-culture properties and co- fermentation characteristics between them. The results show: (1) PI-02 The lactic acid bacteria and the yeasts found in the traditional dairy products in Inner Mongolia have symbiotic effects that can Adaptive laboratory evolution study of Corynebacterium increase active strain numbers and enhance acid productivity; glutamicum resistant to oxidative stress and their appli- (2) The metabolites of the yeasts can act as pH buffer in the cation to develop an artificial oxidative stress-resistant broth of the lactic acid bacteria, reduce acid inhibition, enhance strain the growth of the strains, and increase titration acidity. In the Joo-Young Lee 1,∗ , Jiyoon Seo 1, Eung-Soo Kim 2, Heung-Shick Lee 3, meanwhile, the metabolites of the lactic acid bacteria can also Pil Kim 1 promote the growth of the yeasts, increase titration acidity, and 1 The Catholic University of Korea decrease the pH value to an optimum growth range; (3) When 2 Inha University the lactic acid bacteria and the yeasts are co-cultured in skimmed 3 Korea University milks, both small molecular acidic substances, such as the lactic acid and propionic acid, and flavor components that can form In this study, we performed adaptive laboratory evolution aromatic substances such as ethyl acetate can be produced eas- (ALE) study of Corynebacterium gluiamicum in chemostat for ily; and (4) The metabolites produced from the yeasts after a 1,900 hours with gradual increase of oxidative stress-intensity culture time of 60 hours are the best for the lactic acid bacte- to understand the oxidative stress response and develop C. glu- ria, and the optimal amount of the metabolites in the culture tamicum as a platform microbe. Through ALE experiment, C.

S144 www.elsevier.com/locate/nbt New Biotechnology · Volume 31S · July 2014 GENERAL BIOTECHNOLOGY medium is 40 wt%. Details of this study will be discussed in the of this study was to optimize the production of lipase by a strain presentation. isolated from a slaughterhouse effluent, identified as Pseudomonas http://dx.doi.org/10.1016/j.nbt.2014.05.1972 sp, using the CLG as a carbon source. The lipase activity at 233,44 U/mL was obtained by growing the microorganism on minimum medium containing 1% (w/v) of CLG, after 24 hours. The highest PI-04 enzymatic activity (400,22 U/mL) was observed when 2% (w/v) of CLG was used. The optimization of the temperature levels, pH Construction, expression and renaturation of and agitation rate, by Box-Benhken design, resulted in the maxi- ◦ UBI::human insulin analog single chains mum enzyme activity of 517,00 U/mL, obtained at 32 C, pH 9.0 and 200 rpm. These data showed the possibility of using the CLG Diana Mikiewicz ∗ , Anna Wojtowicz-Krawiec, Natalia Lukasiewicz, as substrate for lipase production by Pseudomonas sp and can con- Iwona Sokolowska, Agata Jagiello, Piotr Borowicz, Grazyna tribute in the reduction of production cost of lipases in industrial Plucienniczak, Andrzej Plucienniczak scale, and increase the market value of soybean oil byproducts. Institute Biotechnology and Antibiotics Financial support: CAPES/PNPD http://dx.doi.org/10.1016/j.nbt.2014.05.1974 Insulin is a hormone secreted by the pancreas in response to high blood sugar levels that induces hypoglycemia. Insulin regulates the body’s use of glucose and the levels of glucose in the PI-06 blood. Insulin and its various derivatives are used in large amounts in treatments of diabetes. Production of anti-cancer compound by microbial pro- The aim of this work was using a deubiquitinating protease cess analogue - UBP1 to obtain purified single chain A and B of human Changhyun Roh insulin analog in E. coli. We constructed fusion genes - Ub::ins and protease analogue - UBP1 in one vector together. The region that KAERI codes for the recombinant fusion gene is under control deoP1P2 promoter. The protease analogue - UBP1 gene which is under the In this study, we elucidated that the production of anti-cancer control of pms promoter(WO05066344 A2) was discovered and agent from microbial process for regio-specific hydroxylation of described in our Institute. The recombinant human insulin chain resveratrol. Among the strains examined, a strain showed high genes present downstream of ubiquitin gene was used as a signal regio-specific hydroxylation activity to produce Piceatannol. In a protein. Ubiquitin is composed of 76 amino-acid residues with a 5 L (w.v. 3 L) jar fermentation, the wild type Streptomyces sp.in total molecular mass of 8.6 kDa. This protein is an element of the batch system produced 205 mg of Piceatannol (i.e., 60% yields) universal protein modification in eukaryotes called ubiquitinat- from 342 mg of resveratrol in 20 h. This biotransformation result ion, a phenomenon which does not occur in bacteria. In spite of demonstrates that regio-specific hydroxylated resveratrol exhibit- that, it has been shown that proteins fused to ubiquitin undergo ing more potent anti-cancer activity could be produced on a large greater expression in E coli and are easier to purify and renaturate scale using microbial biotransformation. Using biotransformed than nonhybrid foreign proteins. piceatannol, the in vitro anti-cancer study was performed against We obtained a high level of expression of fusion proteins human cancer cell line (HeLa) and MTT assay was used to analyze Ub::ins analog A chain and Ub::ins analog B chain in E. coli. After the cell growth inhibition. The results showed that the biotrans- cleavage of the ubiquitin by UBP1 analog, good yields of thepuri- formed piceatannol possessed a significant anticancer activity. fied insulin chains were produced. This is first report elucidating production of anti-cancer com- http://dx.doi.org/10.1016/j.nbt.2014.05.1973 pound using microbial process for regio-specific hydroxylation of resveratrol. This study has significant scope that a biotransformation result provides one initiative example such that regio-specific PI-05 hydroxylated compound from resveratrol showing more potent biological activity could be produced in large scale using micro- The crude lecithin gum as a substrate for lipase produc- bial biotransformation. Thus, microbial processes become a good tion by Pseudomonas sp model system to develop an industrial biotransformation system to produce drugs for treasure islands. Maria Antonia Celligoi 1,∗ , Cristiani Baldo 1, Marcos Oliveira 1, Lilian Baggio 1, Fabiana Gasparin 1, Marcelo Melo 1, Dionisio Borsato 2 http://dx.doi.org/10.1016/j.nbt.2014.05.1975 1 State University of Londrina - Department of Biochemistry and Biotechnology 2 State University of Londrina - Department of Chemistry

The reﬁning of soybean oil involves the degumming process which results in byproducts with variety of quality grades. The crude lecithin gum (CLG) contains a mixture of phospholipids and oil, and may be submitted to a series of solvent extraction and precipitation processes to produce the commercial lecithin. The aim

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PI-07 Staph), an edible herb, in the forms of oil and powder on the growth and aflatoxin production of Aspergillus flavus IMI 242684 Induced differential metaproteomics: identification of in maize. Lemongrass oil at concentrations of 1, 2, 3, 4 and 5% cellulases in a methanogenic microbial community at and lemongrass powder at 1, 2, 3, 4, 5, 10, 20, 30, 40 and 50% thermophilic conditions were applied in maize with aflatoxin producing fungus for 7 days Jutta Speda 1,∗ , Bengt-Harald Jonsson 1, Martin Karlsson 2 at ambient temperature. The results show that lemongrass oil at all concentrations inhibited fungal growth whereas lemongrass pow- 1 Linköping University der at 40 and 50% could inhibit fungal growth. For the ability to 2 Rational Enzyme Mining AB inhibit aflatoxin production, it was found that lemongrass powder at 10-30% and at 40-50% could inhibit aflatoxin production for 3 The identification of novel enzymes for use in industrial and 7 days, respectively. biotechnology is an important goal in enzyme discovery. Most industrially relevant enzymes to date have been isolated from pure http://dx.doi.org/10.1016/j.nbt.2014.05.1977 cultured microorganisms. For future discovery of novel enzymes this is however a major bottleneck since it is well established that only a small fraction of all microorganisms can be obtained in PI-09 pure cultures. The possibility to identify enzymes directly from complete microbial communities would therefore give access to a Bioconversion of Ginsenosides from Red Ginseng Extract huge number of novel enzyme candidates. Using Candida allociferrii JNO301 Isolated from Meju Metaproteomics has hitherto mainly been used to understand Sulhee Lee 1,∗ , Yong-Hun Lee 1, Jung-Min Park 2, Dong-Hoon Bai 3, ecosystem functions. We have instead used the dynamics of pro- Young-Seo Park 1 teomics to develop a method based on “induced differential 1 Gachon University metaproteomics”, by which a desired enzyme activity is induced 2 Korean Culture Center of Microorganisms in a full microbial population and compared to a non-induced ref- 3 Dankook University erence of the very same population. In a first example the goal was to induce, select and identify cellulases from a thermophilic Red ginseng (Panax ginseng), a Korean traditional medicinal methanogenic community. plant, contains a variety of ginsenosides as major functional com- Out of several hundred detectable proteins in a 2D-DIGE ponents. It is necessary to remove sugar moiety from the major experiment, 24 proteins could be identified as at least two-fold ginsenosides to make them aglycone form due to their low absorp- up-regulated upon induction. For some proteins spots, the cel- tion rate into the intestine. To screen the microorganisms which lulolytic activity was further validated by activity staining using show the bioconversion activity on the ginsenosides from red gin- 2D-zymography. Mass spectrometry analysis revealed that 21 out seng, total 50 yeast strains were isolated from Korean traditional of the 24 up-regulated proteins are cellulases or associated to meju (a starter made with soybean and wheat flour for the fermen- cellulolytic activity giving a remarkable hit-rate of 88%. This tation of soybean paste). Twenty strains which form the black zone demonstrates the high efficiency and precision of the method, by around the colony on the esculin-YM agar plate were screened which a much wider span of the microbial world can be scanned first, and among them 5 strains which have high β-glucosidase for novel and targeted enzymes. activity on p-nitrophenyl-β-D-glucopyranoside as a substrate were http://dx.doi.org/10.1016/j.nbt.2014.05.1976 then selected. Strain JNO301 was finally chosen as a bioconversion strain in this study on the basis of high bioconversion activity against red ginseng extract by TLC analysis. The selected biocon- PI-08 version strain was identified as Candida allociferrii JNO301 by the nucleotide sequence analysis of 18S rRNA gene. The optimum Effect of lemongrass oil and powder on growth and temperature and pH for the cell growth were 20-30 and pH 5- aflatoxin production by Aspergillus flavus IMI242648 in 8, respectively. By the analysis of TLC, it was confirmed that C. maize allociferrii JNO301 converted ginsenoside Rb1 into Rd and then ,∗ into F2, Rb2 into compound O, Rc into compound Mc1, Rf into Dusanee Thanaboripat 1 , Yaowapa Suvathi 2, Manatsanun Rh1. Quantitative analysis using HPLC showed that bioconversion Kurdhom 1, Suchada Rakphung 1 of red ginseng extract resulted in the increase in the concentration 1 King Mongkut’s Institute of Technology Ladkrabang of Rd, F2, compound O, compound Mc1, and Rh1 with 2.73-, 3.32-, 2 Government Pharmaceutical Organization 33.87-, 16-, 5.48-fold, respectively.

Aflatoxin is one of the most important mycotoxins producing http://dx.doi.org/10.1016/j.nbt.2014.05.1978 in nature and poses health hazard to human and animals. Control of aflatoxin producing fungi and aflatoxin production in various commodities by various means have been investigated. Natural plant extracts may provide an alternative way to prevent food or feed from fungal contamination. The objective of this study is to compare the effect of lemongrass (Cymbopogon citratus (DC.)

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PI-10 was noticed between the extraction with isopropanol and acetone. Samples extracted with ethanol had higher antioxidant activity Construction of a genetically encoded binary counting than the others and exhibited the rice fragrance. module in Escherichia coli Keywords: Gamma-oryzanol, Antioxidant activity; Rice bran Jia Zhao 1,∗ , Sean Colloms 2, Susan Rosser 3 oil; conventional solvent extraction 1 Institute of Molecular, Cell and Systems Biology, University of Glasgow http://dx.doi.org/10.1016/j.nbt.2014.05.1980 2 University of Glasgow 3 University of Edinburgh PI-12 Binary systems have been applied extensively in computer science because they allow large numbers to be encoded. Introducing Isolation and characterization of antibiotic producing a similar counting system into living cells will be a fundamen- Micromonospora from Thai Jasmine rice tal step towards building cellular computers. One way to do this Chitti Thawai is to encode information in the DNA sequence using site-specific King Mongkut’s Institute of Technology Ladkrabang recombination to invert the orientation of a DNA segment, making the information heritable and easily detectable. Serine phage integrases catalyse recombination between specific DNA sites, attP and Thirty eight actimonycete strains were isolated from tissue of attB, producing sites attL and attR which are no longer substrates root and stem of Thai jasmine rice (Oryza sativa). These strains for integrases alone. However, in the presence of a recombination were grouped using phenotypic, chemotypic and genotypic char- directionality factor (RDF) this directionality is reversed so that acteristics into 5 groups. Phylogenetic position, chemotaxonomic attL and attR recombine to recreate attP and attB. If two att sites analyses including some phenotypic characterisation revealed that are placed in inverted repeat, recombination flips the orientation the representative strains in each group belonged to the members of the intervening sequence between two possible states, which of the genus Micromonospora. The fermentation broths of these can represent a single binary digit (0 or 1) heritably stored in the isolates were extracted with ethyl acetate and were tested for anti- DNA. Based on this principle, a toggle switch has been constructed microbial activity. The results showed that more than 60% of in E.coli. By first expressing the integrase, and then the integrase Micromonospora strains inhibited the growth of pathogenic agents and the RDF together, its state can be changed between 0 and 1 of leaf blight disease (Xanthomonas oryzae) and blast disease (Pyric- under the control of different signals. This switch will be developed ularia grisea). Based on these results, we conclude that endophytic to a binary counting module by placing the RDF under the control Micromonospora strains are great and should represent an excellent of the sequence state and using a single chemical signal to control source for discovery of the bioactive compounds. the expression of the integrase. By linking N units together, each Keywords: Actinomycetes, Micromonospora, Antibiotic, using a different phage integrase, a binary counter can be made to Blight and Blast disease. help cells count to 2N-1. http://dx.doi.org/10.1016/j.nbt.2014.05.1981 http://dx.doi.org/10.1016/j.nbt.2014.05.1979

PI-13 PI-11 Androgen’s effect on MCP-1 and monocyte attraction to Evaluation of gamma-oryzanol in Thai rice bran oil prostate cancer cells extracted by conventional solvent extraction method Eun-Kyung Kim ∗ , Eun-Ju Choi and its antioxidant activities Konkuk University Sujitra Sukonthamut ∗ , Chitti Thawai, Duangkamol Ruen-ngam King Mongkut’s Institute of Technology Ladkrabang Trigger of inflammatory monocytes to the tumor site is mediated by monocyte chemoattractant protein-1 (MCP-1) through Rice bran oil was extracted by solvent-assisted extraction with binding to its receptor. We assumed that androgen could hexane, ethylacetate, acetone, isopropanol and ethanol using a modulate MCP-1 expression in hormone-responsive prostate solvent-to-rice bran ratio of 4:1 (w/w). The experiments were done cancer cells and then promote recruitment of monocytes. Dihy- in triplicate at room temperature (30 ◦C) with a total extraction drotestosterone (DHT) stimulated a time-dependent (0–72 hours) time of 2 hours/sample. The oil components were separated by and concentration-dependent (0–1 nmol/L) increase in MCP- reverse-phase HPLC and quantified with a fluorescence detector. 1 mRNA levels in androgen-responsive human prostate cancer The radical scavenging capability of the oil was tested with DPPH cells (LNCaP). This increase in MCP-1 mRNA corresponded with and was expressed as ␮mol Trolox Equivalent Antioxidant Activity. increased secretion of MCP-1 protein. The effect of DHT was Among these solvents, ethanol was the best solvent for the extrac- mediated through an androgen receptor (AR)-dependent pathway tion of gamma-oryzanol as compared with the others solvents for as small inhibitor RNA (siRNA) against AR negated the induc- conventional solvent extraction while isopropanol was better for tion of MCP-1. Although DHT also induced TWIST1 mRNA, an oil yield extraction at room temperatures. No difference in oil yield epithelial–mesenchymal transition (EMT)–related factor, and pur- ported inducer of MCP-1, blocking its expression with siRNA

www.elsevier.com/locate/nbt S147 GENERAL BIOTECHNOLOGY New Biotechnology · Volume 31S · July 2014 did not inhibit DHT induction of MCP-1 mRNA. Moreover, con- used CotE, CotG, CotY spore coat protein as anchoring motives ditioned media from androgen-treated cells promoted human for the successful display of beta-galactosidase, spreptavidin, and monocyte THP-1 cell migration and this effect was inhibited by other interesting target protein with biotechnological application antibody against MCP-1. These results indicate that androgen may such as laccase for the environmental usages. Surface localization regulate MCP-1 and promote inflammatory microenvironment in using flow cytometry and enzymatic activity on the Bacillus sub- prostate cancer. tilis spore was examined for the successful confirmation of surface http://dx.doi.org/10.1016/j.nbt.2014.05.1982 localization of target proteins. Further usage and other application will be discussed. http://dx.doi.org/10.1016/j.nbt.2014.05.1984 PI-14

Suppression of dust mite extract and 2,4- PI-16 dinitrochlorobenzene-induced atopic dermatitis by the water extract of DA-9601 Production of 3-hydroxybutyrate by E. coli: Application of Nitrogen and Phosphorous limitation to steer fluxes Choi Eun-Ju ∗ , Kim Eun-Kyung to product formation Konkuk University Monica Guevara 1,∗ , Johan Jarmander 1, Mariel Perez-Zabaleta 1, Jorge Quillaguamán 2 Gen Larsson 1 DA-9601 is a novel anti-peptic formulation prepared from the , ethanol extracts of Artemisia asiatica possessing anti-oxidative, 1 Industrial biotechnology, School of biotechnology, KTH, Stockholm 2 anti-allergic and anti-inflammatory activities. However, their Center of Biotechnology, Faculty of Science and Technology, San Simón Uni- versity, Cochabamba Bolivia effect on atopic dermatitis (AD) has not been studied yet. In this study, we report that topical application of DA-9601 suppressed house dust mite extract (Dermatophagoides farinae extract, DFE) and Polyhydroxyalcanoates are polyesters produced in large 2, 4-dinitrochlorobenzene (CDNB)-induced AD-like skin lesions amounts by different microorganisms, but not by E.coli. Here we in BALB/c mice model. We established atopic dermatitis model expressed the bacterial polyhydroxybutarate (PHB) pathway of in BALB/c mice by repeated local exposure of DFE/CDNB to the Halomonas boliviensis in E. coli using phbA and phbB genes to ears. Repeated alternative treatment of DFE/CDNB caused AD- produce the monomer 3-HB. Acetyl coenzyme A (AcCoA) is the like lesions. DA-9601 reduced AD-like skin lesions based on ear intermediate of the carbon metabolism and precursor of the PHB thickness and histopathological analysis, and serum IgE levels. DA- pathway. The objective is to design a process to produce 3-HB in 9601 inhibited mast cell infiltration into the ear and elevation of E.coli with high productivity which is a first step to further produce serum histamine in AD model. In addition, DA-9601 suppressed different quality polyesters. DFE/CDNB-induced expression of IL-4, IL-13, IL-31, and TNF-␣ in Production of the wild type microorganisms takes place under the ears. Taken together, our results showed that topical applica- nutrient deficient conditions and excess of a carbon source. The tion of DA-9601 exerts beneficial effects in animal model of AD, hypothesis is that directing the flux to the desired product (3-HB) suggesting that DA-9601 might be a candidate for the treatment by cultivations with controlled feed of nitrogen or phosphorous of AD. will increase the flux to the precursor (AcCoA) and minimize the formation of byproducts http://dx.doi.org/10.1016/j.nbt.2014.05.1983 This will be done by: Fed-Batch cultivations with an excess of glucose and nitrogen limitation. Excess glucose will give a surplus of NADH that will PI-15 inhibit citrate synthase and the AcCoA produced will instead be used for 3-HB production Biotechnological application of Bacillus subtilis spore Fed-Batch cultivations with an excess of glucose and phospho- display system rous limitation. Excess glucose will give a surplus of NADH that will June Hyung Kim inhibit citrate synthase and the AcCoA produced will instead be Dong-A University used for 3-HB production. Also the limitation of phosphorous acti- vates the methylglyoxal pathway also leading to the more energy Bacterial surface display finds its important biotechnological favorable production of AcCoA than central glycolisis. application in the fields of screening tools of evolved enzyme, http://dx.doi.org/10.1016/j.nbt.2014.05.1985 bioremediation, whole cell bioconversion and tool for live vaccine production. For the functional bacterial surface display of active enzyme of multimeric form, which is generally impossible due to molecular assembly of the monomer subunit subsequent to the secretion of displayed target protein outside the cell, a new surface display system based on Bacillus subtilis spore is developed. Here, we tried to develop a new bacterial surface display format for the efficient expression of multi-subunit enzyme. We

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PI-18 ting (FACS),offering an advanced uHTS screening platform (∼108 events/day) for the discovery and reengineering of industrially Development of a flow cytometer-based in vitro compart- attractive enzymes [1].uHTS offers a qualitative differentiation mentalization screening platform for directed protein between positive or negative enzymatic activity, enabling the evolution enrichment of active variants by screening thousands of events per Georgette Wirtz ∗ , Christian Pitzler, Ljubica Vojcic, Ronny Martinez, second. In IVC, single cellulase gene variants are transcribed and Ulrich Schwaneberg translated within a double emulsion compartment and upon translation, a fluorogenic substrate is converted by active variants and Institute of Biotechnology, RWTH Aachen sorted using FACS [2,3]. IVC systems enable linking phenotype and genotype, which is a key requirement in directed evolution experi- We describe the development of an ultra high throughput ments. Additionally, IVC allows the expression of larger libraries (> screening (uHTS) flow cytometer based in vitro compartmental- 1010) by overcoming challenges such as loss of gene diversity due to ization (IVC) screening platformfor directed enzyme evolution. low cloning and transformation efficiency and protein production The system combines IVC (water-oil-water emulsion) and the avoiding technical limitations (e.g. inclusion bodies, toxicity). IVC detection of enzyme activity by fluorescence-activated cell sor- also dramatically reduces the time required for screening enzyme ting (FACS),offering an advanced uHTS screening platform (∼108 libraries, allowing a greater number of directed evolution rounds events/day) for the discovery and reengineering of industrially per campaign, increasing the chance of generating and identifying attractive enzymes [1].uHTS offers a qualitative differentiation improved enzyme variants. between positive or negative enzymatic activity, enabling the enrichment of active variants by screening thousands of events per References second. In IVC, single cellulase gene variants are transcribed and [1].RuffAJ, Dennig A, Wirtz G, Blanusa M, Schwaneberg U. ACS Catalysis translated within a double emulsion compartment and upon trans- 2012;2:2724–8. lation, a fluorogenic substrate is converted by active variants and [2].MastrobattistaE, Taly V, Chanudet E, Treacy P, Kelly BT, Griffiths AD. sorted using FACS [2,3]. IVC systems enable linking phenotype and Chem Biol 2005;12:1291–300. genotype, which is a key requirement in directed evolution experi- [3].Tu R, Martinez R, Prodanovic R, Klein M, Schwaneberg U. J Biomol ments. Additionally, IVC allows the expression of larger libraries (> Screen 2011;16:285–94. 1010) by overcoming challenges such as loss of gene diversity due to http://dx.doi.org/10.1016/j.nbt.2014.05.1987 low cloning and transformation efficiency and protein production avoiding technical limitations (e.g. inclusion bodies, toxicity). IVC also dramatically reduces the time required for screening enzyme PI-19 libraries, allowing a greater number of directed evolution rounds per campaign, increasing the chance of generating and identifying A fluorescent polymer shell-based enzyme screening plat- improved enzyme variants. form for hydrolases using flow cytometry Christian Pitzler 1,∗ , Georgette Wirtz 1, Ljubica Vojcic 1, Stephanie References Hiltl 2, Alexander Böker 2, Ronny Martinez 1, Ulrich Schwaneberg 1 [1].RuffAJ, Dennig A, Wirtz G, Blanusa M, Schwaneberg U. ACS Catalysis 1 RWTH Aachen University, department of biotechnology 2012;2:2724–8. 2 DWI-Leibniz Institut für Interaktive Materialien [2].MastrobattistaE, Taly V, Chanudet E, Treacy P, Kelly BT, Griffiths AD. Chem Biol 2005;12:1291–300. [3].Tu R, Martinez R, Prodanovic R, Klein M, Schwaneberg U. J Biomol A novel whole-cell enzyme screening platform based on a Screen 2011;16:285–94. coupled reaction of glucose-oxidase and a hydrolase (Yersinia mol- laretii phytase, YmPh) was developed and validated in a directed http://dx.doi.org/10.1016/j.nbt.2014.05.1986 phytase evolution experiment. The coupled reaction produces hydroxyl radicals through Fenton’s reaction (hydrogen peroxide and Fe2+) which initiate poly(ethyleneglycol) diacrylate-based PI-18 hydrogel polymerization, including the fluorescent Polyfluor-570- Development of a flow cytometer-based in vitro compart- acrylate. As consequence, fluorescent hydrogel is formed around mentalization screening platform for directed protein E. coli cells that express active YmPh variants. Formation of the evolution fluorescent hydrogel was confirmed by confocal microscopy. In addition, scanning force microscopy was used to visualize the Georgette Wirtz ∗ Christian Pitzler Ljubica Vojcic Ronny Martinez , , , , hydrogel on the cell surface, revealing a distinct structure enclos- Ulrich Schwaneberg ing the cells. Labeled cells were analyzed and sorted by flow Institute of Biotechnology, RWTH Aachen cytometry with a throughput of 1.8 × 107 events/h. Further sorting of mixed populations with a defined content of active/inactive We describe the development of an ultra high throughput cells yielded an enrichment factor of up to 5. Finally, the fluo- screening (uHTS) flow cytometer based in vitro compartmental- rescent polymer shell technology was validated by analyzing an ization (IVC) screening platformfor directed enzyme evolution. error-prone PCR mutant library (4.8 mutations/gene). Screening The system combines IVC (water-oil-water emulsion) and the of 107 events by flow cytometry yielded variant M1 with 97 detection of enzyme activity by fluorescence-activated cell sor- U/mg increased specific activity compared to YmPh wild type

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(315 U/mg). This screening platform is a promising approach for medium containing 93.8 g × l−1 of reducing sugars, or glucose screening mutant and metagenome libraries for novel or improved medium containing 120 g × l−1 of this saccharide. Biomass, reduc- hydrolases, by using a variety of glucose-derived substrates. ing sugars and lactic acid concentrations were estimated during http://dx.doi.org/10.1016/j.nbt.2014.05.1988 fermentation process. In the first 24 h of fermentation, specific production rate of lactic acid was 5.18 g × l−1 × h−1 for juice based medium and 4.77 g × l−1 × h−1 for glucose medium. Biomass con- × −1 PI-20 centration at the stationary phase reached value of 3.9 g l irrespectively of used medium. Specific substrate uptake rate was − − − − Rye as substrate for L-lactic acid production 3.98 g × l 1 × h 1 for juice and 2.80 g × l 1 × h 1 for glucose medium. Reducing sugars were completely utilized after 31 h of Piotr Walczak ∗ , Anna Rygala, Katarzyna Dybka, Patrycja process in juice medium. In glucose based medium after 72 h of Pietraszek, Agata Czyzowska, Anna Otlewska, Elzbieta Oltuszak- fermentation 29.5 g × l−1 of sugar was left unused. Final concentra- Walczak tion of L-lactic acid after 31 h of fermentation reached value of 92 Faculty of Biotechnology and Food Sciences g × l−1 for juice and 62 g × l−1 for the glucose medium respectively. The optical purity of produced L-lactate was 99.13%. The work The aim of work was L-lactic acid (LA) fermentation process was supported by the POIG 01.01.02-10-123/09 Project partially using enzymatic rye hydrolyzates as an inexpensive carbon source financed by the European Union within the European Regional and nutrients supporting growth of lactic acid bacteria. Rye grits Development Fund, Grants for Innovation. or rye flour was enzymatically hydrolyzed in the low temperature http://dx.doi.org/10.1016/j.nbt.2014.05.1990 process (90 - 50 ◦C) with thermostable ␣–amylase Aquazym ATL, glucoamylase Spritase GA 14400L and malt prepared from naked oat Avena nuda as a source of pullulanase. Obtained hydrolyzates PI-22 were diluted to the glucose concentration of about 120 g/l, supple- + + 2+ 2+ 3− mented with mineral salts (K ,Na ,Mg ,Mn ,PO4 ) and yeast A synthetic biology approach to characterise cyanobac- extract (3 g/l) and used as the fermentation medium. L-lactic acid terial promoters fermentation processes were carried out in BioFlo 415 bioreactor ∗ (5 l working volume, 42 ◦C, 44 h) with Lactobacillus rhamnosus as Mary Ann Madsen , Anna Amtmann producing organism. pH was controlled at 5,5 by automatic addi- University of Glasgow tion of 12.5% ammonia water. At the end of fermentation (44 h), − − 109.2 g × l 1 of glucose was utilized and 88.5 g × l 1 of lactic acid Cyanobacteria are a phylum of bacteria with the ability to containing 97.5% of L-isomer was produced. Dry mass of bacterial perform oxygenic photosynthesis using minimal nutrient require- × −1 cells reached value of 2.06 g l and average productivity was 2 ments: mainly sunlight, water and CO2. Cyanobacteria thus − − g × l 1 × h 1. It was proved that rye may replace corn and be com- provide a cost-effective and energetically sustainable chassis for petitive substrate for microbial production of L-lactic acid in rye biotechnological applications. Cyanobacteria are relatively new to belt countries with cold climate. The work was supported by the the field of biotechnology, however, and the ‘tools’ available to POIG 01.01.02-10-123/09 Project partially financed by the Euro- genetically modify them are currently very limited. We therefore pean Union within the European Regional Development Fund, describe an approach to identifying promoters activated in high Grants for Innovation. density cultures and characterization using a synthetic biology http://dx.doi.org/10.1016/j.nbt.2014.05.1989 approach. http://dx.doi.org/10.1016/j.nbt.2014.05.1991

PI-21 PI-23 Dynamics of calcium L-lactate fermentation by Lacto- bacillus rhamnosus in sugar beet thick juice and glucose Differential Adaptive Fermentation of E. coli K-12 and based media B Strains in the Absence of adhE Gene Under Anaerobic Condition Elzbieta Oltuszak-Walczak ∗ , Piotr Walczak, Agata Czyzowska, Anna Rygala, Patrycja Pietraszek, Katarzyna Dybka, Anna Otlewska SLee1,∗ , Hyun Ju Kim 1, Haeyoung Jeong 1, Dong-Woo Lee 2 Faculty of Biotechnology and Food Sciences 1 Korea Research Institute of Bioscience and Biotechnology 2 Kyungpook National University Production of pure L-lactate stereoisomer from renewable carbon sources is required for the manufacturing of green Alcohol dehydrogenase (AdhE) plays a key role in the main- biodegradable tactic polymer poly-L-lactide (PLLA).The aim of tenance of redox balance in E. coli under anaerobic condition. work was evaluation of process dynamics of calcium L-lactate fer- However, in the absence of AdhE, oxidized redox cofactors such mentation using sugar beet thick juice or glucose as substrates. as NAD+ could not be efﬁciently recycled in cells. Therefore, Fermentation processes with industrial strain of Lactobacillus rham- highly-reduced status could be alternatively oxidized by lactate nosus were carried out at 42 ◦C for 48-72 h in the juice based dehydrogenase in E. coli adhE mutant cells. Indeed, under anoxic

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condition E. coli K-12 adhE mutant cells rapidly adapted to the accompanied by aberrant chromosomal alignment. Remarkably, stressed condition and grew with lactate fermentation, whereas the mitotic defects caused by ADD1 depletion were rescued by E. coli BL21(DE3) adhE mutant cells begun very slowly adapt- re-expression of ADD1 but not of an ADD1 mutant defective in ing to anaerobic growth. It was observed that the recycling of Myo10 binding. Together, our ﬁndings unveil a novel function NAD+ was very poor in E. coli BL21(DE3) adhE mutant cells during for ADD1 in mitotic spindle assembly through its interaction with anaerobic culture, which caused severe redox stress and in turn Myo10. decreased cellular viability. In contrast, K-12 adhE mutant cells http://dx.doi.org/10.1016/j.nbt.2014.05.1994 maintained cellular redox balance (NAD+/NADH ratio) and viability under anoxic condition. This difference allows K-12 and B cells to undergo differential adaptations to anaerobic stress. PI-26 http://dx.doi.org/10.1016/j.nbt.2014.05.1992 Degredable polymers of D-lactate from renewables

Ozlem Osmanagaoglu 1,∗ , Haluk Hamamcı 2, Onlu Harun 3 PI-24 1 Ankara University 2 Middle East Technical University Department of Food Engineering Dihydroxyacetone-catalyzed phosphorylation 3 Ankara University Faculty of Sciences Department of Biology Roland Wohlgemuth 1,∗ , Dominik Gauss 1, Israel Sanchez-Moreno 2, Eduardo García-Junceda 2 The production of biodegradable plastics is of increasing inter- 1 Sigma-Aldrich est because of environmental concerns. For the same reasons the 2 CSIC utilization of renewable materials for the production of such materials is also being supported. Lactic acid polymers are among the Biocatalytic phosphorylations have been shown to provide biodegradable materials that are being produced and whose pro- a number of advantages over chemical phosphorylations like duction capacity is increasing. Lactic acid has two chiral forms, increased selectivity and high step economy [1,2]. D (-) and L (+); both of which can be produced by fermentation. The use of recombinant dihydroxyacetone kinase from Cit- These chiral forms, when polymerized may have distinct proper- robacter freundii in biocatalytic asymmetric phosphorylation has ties; so both are important from the materials science point of been analyzed by direct quantitative 31P-NMR analysis of the view. kinetics. The phosphoenolpyruvate/pyruvatekinase-system was In this work we attempted to produce D (-) lactic acid from thereby selected for the regeneration of the cofactor ATP. This renewable materials like sunﬂower seed hulls or wood shavings system looks promising for further exploration. by using Lactobacillus delbrueckii subsp. bulgaricus strains isolated from different home-made yogurts. The strains were tested for their References chiral lactate production and the better producers were chosen and [1].D. Gauss, B. Schönenberger, R. Wohlgemuth, Carbohydrate subjected to fermentation tests. By several methods of neutraliza- Research, in press (2014). tion, N-source supplementation and Na-Acetate addition so far we [2].Matsumi R, Hellriegel C, Schönenberger B, Milesi T, van der Oost J, could reach a level of 80 g/L D (-) lactate production. Wohlgemuth R. RSC Advances 2014;4(25):12989–94. http://dx.doi.org/10.1016/j.nbt.2014.05.1995 http://dx.doi.org/10.1016/j.nbt.2014.05.1993

PI-27 PI-25 The Effect of Oral Administration of Pediococcus pen- Adducin-1 is essential for mitotic spindle assembly tosaceus OZF through its interaction with myosin-X Harun Onlu 1,∗ , Zhannatgul Sakyp 2, Fadime Kıran 2, Harun Onlu 2, Hong-Chen Chen ∗ , Po-Chao Chan ˙Ilker Büyük 2, Sümer Aras 2, Özlem Osmanagao˘ glu˘ 2 National Chung Hsing University 1 Ankara University Faculty of Science Department of Biology 2 Ankara University Mitotic spindles are microtubule-based structures, but increasing evidence indicates that F-actin and F-actin-based motors are The intestinal effects of Pediococcus pentosaceus OZF, a promis- components of these structures. Adducin-1 (ADD1) is an actin- ing probiotic strain and its encapsulated form was evaluated binding protein that has been shown to play important roles in in TNBS model of rat colitis. To increase the viability of OZF the stabilization of the membrane cortical cytoskeleton and cell- strain under gastrointestinal conditions, encapsulation was car- cell adhesions. In this study, we show that ADD1 associates with ried out by whey proteins and calcium alginate. Female Wistar mitotic spindles and is crucial for proper spindle assembly and rats (n = 6) were treated daily with oral administration of OZF mitotic progression. Phosphorylation of ADD1 at Ser12 and Ser355 strain (7 log cfu/ml) and its encapsulated form for 14 days, start- by cyclin-dependent kinase 1 enables ADD1 to bind to myosin-X ing one week before the treatment of TNBS. The body weight, (Myo10) and therefore to associate with mitotic spindles. ADD1 water and food intake and the appearance of feces were recorded depletion resulted in distorted, elongated, and multipolar spindles,

www.elsevier.com/locate/nbt S151 GENERAL BIOTECHNOLOGY New Biotechnology · Volume 31S · July 2014 daily throughout the study. One week after induction of colitis, PI-29 all animals was killed and colonic damage was evaluated both histologically and biochemically including the determination of Characterization of Pichia pastoris Golgi and plasma glutathione, superoxide-dismutase, malondialdehyde (MDA) and membrane catalase contents. MDA level, as a marker of oxidative stress was Andreas Grutsch 1,∗ , Karlheinz Grillitsch 1, Pablo Tarazona 2, Erich significantly increased in the colitis group. However, OZF admin- Leitner 3, Ivo Feussner 2, Günther Daum 4 istration markedly decreased the MDA contents in colonic tissue. 1 Austrian Centre of Industrial Biotechnology (ACIB) In addition, pro-inflammatory cytokines (IL-1beta and IL-6) in 2 Department for Plant Biochemistry, University of Göttingen serum and colonic tissue were assessed by Real-Time PCR and a sig- 3 Institute of Analytical Chemistry and Food Chemistry, TU Graz nificant reduction was detected when compared to TNBS control 4 Institute of Biochemistry,TU Graz/Austrian Centre of Industrial Biotechnology animals. In conclusion, P. pentosaceus OZF could be a beneficial (ACIB) adjuvant/agent in the treatment of inflammatory bowel diseases due to its intestinal anti-inflammatory activity. Pichia pastoris has a prominent status regarding expression of recombinant proteins and is worldwide used as a highly efficient http://dx.doi.org/10.1016/j.nbt.2014.05.1996 cell system for the production of heterologous proteins. Despite the importance of Pichia pastoris in biotechnology, little information is available about the cell biology of this microorganism. This PI-28 tempted us to intensify our studies on Pichia pastoris organelles Application of confocal microscopy methods for esti- with special emphasis on subcellular fractions involved in the mation of lipid and proteins dynamics in thylakoid classical secretory pathway. The Golgi harbors many processes of membranes post-translational protein modifications and is a major branching point within the secretory pathway. The plasma membrane is Marketa Husakova 1,∗ Karolina Ditrychova 2 , important for protein secretion as it is the last barrier on the way 1 University of Palackiana in Olomouc, Faculty of Science of polypeptides to be externalized. We have established protocols 2 Charles University in Prague to isolate Pichia pastoris Golgi and plasma membrane at sufficient yield and high purity. Lipid profiling of the isolated membranes The mobility of photosynthetic proteins is an important factor highlighted characteristics of the specific organelles. Whereas the for light-energy conversion in photosynthesis. It has an important phospholipid patterns were organelle specific, the fatty acid com- role in regulation of photosynthesis and in protein transport after position was similar in all membranes. By contrast to evidence its synthesis or repair. Mobility of photosynthetic proteins outside from Saccharomyces cerevisiae, a sterol gradient along the secre- the thylakoid membrane is less understood. Cyanobacterial phyco- tory route was not found in Pichia pastoris. Sphingolipids of Pichia bilisomes attached to the thylakoid membrane can move relatively pastoris showed a clear organelle dependent distribution pattern. fast. We can measure specific feature of photosynthetic proteins Ceramides and hexosyl-ceramides were predominantly found in mobility in vivo using microscopic methods such as FRAP (Fluo- Golgi fractions, whereas inositol containing phosphorylceramides rescence Recovery After Photobleaching) which allows to observe were almost exclusively enriched in plasma membrane fractions. autofluorescence. In our study we used procaryotic photosynthetic Similarities of Pichia pastoris and Saccharomyces cerevisiae became cyanobacteria Synechococcus PCC 7942 and Synechocystis PCC 6803 evident, although certain differences have to be kept in mind. as model organisms. To measure mobility on membranes we Comparison of the two yeasts is important for understanding dif- used confocal microscopy and FRAP method as mentioned. FRAP ferent properties and designing new strategies to improve Pichia method is based on laser technology, thus we used stronger laser to pastoris strains for industrial applications. bleach spots on the membrane and observed recovery of fluores- http://dx.doi.org/10.1016/j.nbt.2014.05.1998 cence caused by mobility of proteins in membrane. We used three different sizes of bleaching spots to compare rate of proteins mobility. We also studied mobility of lipids, therefore we used fluorescent PI-30 dye BODYPI because lipids are not autofluorescent whereas phy- cobilisomes are. Main question of this study was, whether is FRAP Somatic embryogenesis from cultured zygotic explants limited by diffusion or phycobilisome binding to the photosystem. of some olive cultivars (Olea europaea L.) http://dx.doi.org/10.1016/j.nbt.2014.05.1997 ,∗ Sara Oulbi 1 , Ibrahim Toufik 2, Ilham Belkoura 3 1 National School of Agriculture, Meknes, Morocco 2 Department of Biology, Faculty of Sciences, University Moulay Ismail, Meknès, Morocco 3 Department of basic sciences, National school of Agriculture, Meknes, Morocco

Mature zygotic embryos taken from three olive cultivars (Dahbia cv, Moroccan Picholine (PM) cv and Arbequine cv) were used to test the embryogenic capacity of their juvenile

S152 www.elsevier.com/locate/nbt New Biotechnology · Volume 31S · July 2014 GENERAL BIOTECHNOLOGY explants (cotyledons and radicles). These explants were cul- PI-32 tured in Olive Medium (OM) (Rugini, 1984) supplemented with following combinations of auxin and cytokinin: AIB and 2iP RNA-Seq analysis of the degradation of haloacetate by (6-dimethylallylaminopurine), AIB (Indole-3-butyric acid) + TDZ Burkholderia caribensis MBA4 (Thidiazuron) and AIB + Zeatin for 3 weeks; they were then trans- Jimmy Tsang ∗ , Yanling Pan, Nan Zheng ferred on OM free hormone. Calluses formation began during the School of Biological Sciences, The University of Hong Kong first subculture in the three media; in term of the 3 weeks of culture in the first medium, the highest rates of callus were obtained Burkholderia caribensis strain MBA4 was isolated for its abil- when medium supplemented with 2iP for Arbequine cotyledons ity to utilize monobromoacetate as carbon and energy source. (100%) and Moroccan Picholine radicles (81.5%) and TDZ for This bacterium produced an inducible haloacid dehalogenase that radicles of Dahbia (92%). However, somatic embryogenesis was transforms monohaloacetate to glycolate and to glyoxylate by gly- observed on Dahbia and Arbequine cotyledons and radicles culti- colate oxidase. Genomic analysis of the bacterium showed that it vars during the second week of culture on OM free hormone when contains three glycolate oxidases: ETY79679-81, ETY80271-3 and calluses were formed on AIB + 2iP, whereas Picholine explants ETY84258-60. Transcriptomic analysis showed that ETY79679-81 induced nodular calluses without somatic expression. Maturation was expressed constitutively to a reads per kilobase transcript of somatic embryos was observed when OM medium was supple- per million reads (RPKM) value of around 100 no matter the mented with 1 g of activated charcoal; indeed, globular somatic substrate was pyruvate, glycolate or chloroacetate. ETY80271-3 embryos were transformed to cotyledonary embryos (16.7% and gave values of 7 in pyruvate-, 867 in chloroacetate- and 1260 in 7.8% for Dahbia and Arbequine respectively). On attempt to ger- glycolate-grown cells. ETY84258-60 gave values of 20 in pyruvate- mination, somatic embryos were cultured on various media in a , 1880 in chloroacetate- and 178 in glycolate-grown cells. A 16/8 photoperiod. Neoformation of buds was observed on somatic malate synthase G gene, ETY84261, was found downstream of cotyledons (37.5%) whereas rooting of somatic embryos was more ETY84258-60. Apparently, ETY80271-3 converted glycolate to gly- difficult to obtain. oxylate and through the glycerate pathway to pyruvate. When http://dx.doi.org/10.1016/j.nbt.2014.05.1999 MBA4 was grown on chloroacetate, gene products of ETY84258- 61 were mainly used and malate was generated. Putative regulator GlcC genes, ETY80275 and ETY84257, can be found upstream of PI-31 ETY80271-3 and ETY84258-60, respectively. The expression profiles of these glycolate oxidases suggested that both GlcC were Effects of 4 essential oils on growth of afaltoxin produc- activated by glycolate and chloroacetate with ETY80275 more ing fungi responsive to glycolate and ETY84257 more reactive towards Sittichai Chareonsettasilp 1,∗ , Dusanee Thanaboripat 1, Chanita chloroacetate. While the relative transcript levels of ETY80275 Sarutipaisan 1, Chutima Puangtong 1, Phurin Chatpongsatorn 1, were rather stable, expression of ETY84257 was enhanced in Yaowapa Suvati 2 glycolate- and even more in chloroacetate-grown cells. The charac- 1 King Mongkut’s Institute of Technology Ladkrabang terization of the degradation of haloacetate by B. caribensis MBA4 2 Government Pharmaceutical Organization is made possible with the use of RNA-seq analysis. http://dx.doi.org/10.1016/j.nbt.2014.05.2001 There have been records that many essential oils shown antimicrobial properties. Some essentials oils can inhibit the growth of aflatoxin producing fungi and aflatoxin production. In this study PI-33 we compare the ability of 4 plant essential oils. i.e. ginger oil, anise star oil, cajuput oil and cinnamon oil for controlling aflatoxin pro- Analysis of the physiology of an Aspergillus nidulans ducing fungi. The oils at concentrations of 0, 0.5, 1, 2, 3, 4 and 5% mutant lacking the aoxA (cyanide-resistant alternative were tested against Aspergillus flavus IMI 242684 and A. parasiti- oxidase encoding) gene cus IMI 102566 on Potato Dextrose Agar (PDA). The fungi were ∗ Erzsébet Fekete , Ákos P. Molnár, Mojtaba Asadollahi, Szabina cultured and incubated at 30 C for 7 days. The results show that Balázsi, Erzsébet Sándor, Levente Karaffa anise star oil at all concentrations had the most inhibitory effect on both Aspergillus flavus IMI 242684 and A. parasiticus IMI 102566 University of Debrecen with significant difference followed by cinnamon oil and cajuput oils. Ginger oil had the least inhibition effect. One of the most characteristic features of fungal mitochondria is the presence of an additional terminal oxidase, the alterna- http://dx.doi.org/10.1016/j.nbt.2014.05.2000 tive oxidase, the activity of which is insensitive to inhibitors of cytochrome c oxidase and the bc1 complex. Responsible for this activity in the model fungus Aspergillus nidulans is the cyanide- resistant enzyme alternative oxidase, a quinol oxidase localized in the inner mitochondrial membrane encoded by the nuclear gene aoxA.

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Fungal alternative oxidase was reported to be involved in a could be used in the development of harmful heavy metal bio- variety of stress-related cellular responses. Typically, speciﬁc activ- sensor. ity of the alternative oxidase as well as the level of transcription http://dx.doi.org/10.1016/j.nbt.2014.05.2003 of the encoding gene increased upon stress events. In this study we aimed for a quantitative characterization of the physiological consequences of the deletion of the aoxA gene in A. nidulans in PI-35 comparison with the wild-type reference strain. Growth properties on various carbon sources at different concentrations were com- High cell density cultivation of the chemolithoau- pared. Furthermore, osmotic, heat and stress tolerance of the two totrophic bacterium Nitrosomonas europaea in a dialysis strains were analysed. membrane bioreactor

Levente Karaffa 1,∗ , Tibor Török 2, Antal Kökényesi 1, István Kolláth 1, Acknowledgement Erzsébet Sándor 1, Zoltán Németh 1, Noémi Lipták 1, Erzsébet Fekete 1

The research was supported by the EU and co-financed 1 University of Debrecen 2 by the European Social Fund under the project ENVIKUT TEVA Pharmaceutical Ltd., Safety and Environmental Department, Debrecen, Hungary (TÁMOP-4.2.2.A-11/1/KONV-2012-0043), and also by the Hungar- ian Scientific Research Fund (OTKA Grant K1006600 to Dr. Erzsébet Nitrosomonas europaea is a chemolithoautotrophic nitrifier Fekete). Gram-negative bacterium that gains all of its energy for growth http://dx.doi.org/10.1016/j.nbt.2014.05.2002 from the oxidation of ammonia into nitrite ions. A major pre- requisite to produce a high cell density N. europaea culture is to ensure that inhibitory metabolic by-products remain at minimal PI-34 concentrations. The principal inhibitory metabolite being continuously formed during N. europaea fermentation is nitrite. As a Insights into diversity and specificity of heavy metal consequence, N. europaea cannot grow into high cell density under resistance and efflux systems in Bacillus oceanisediminis conventional batch conditions. 2691 A suitable method presented in this study to overcome this Hyun Ju Kim 1,∗ , Yong-Jik Lee 2, Haeyoung Jeong 3, Dong-Woo Lee 2, problem is the application of a single-vessel dialysis membrane Sang Jun Lee 4 bioreactor system. Fermentations were performed in a reactor 1 Biosystems & Bioengineering Program, University of Science and Technology with 2 L total/1.5 L useful and 6 L total/5.5 L useful volumes of (UST) medium in the inner and outer chambers, respectively. Growth 2 School of Applied Biosciences, Kyungpook National University, Daegu, of N. europaea was monitored via cell density determinations and Republic of Korea by the measurement of nitrite formation. Metabolic activity was 3 Korean Bioinformation Center, Korea Research Institute of Bioscience and also visualized by Acridine Orange staining. Maximal cell den- Technology (KRIBB), Daejeon, Republic of Korea 4 Korea Research Institute Bioscience & Biotechnology sity and maximal calculated specific growth rate were over five times of the value achieved under conventional batch conditions. We previously determined the genome sequence of aerobic, We concluded that dialysis fermentors are suitable tools to over- endospore-forming, Gram-positive Bacillus oceanisediminis 2691 come growth limitations observed in standard batch cultures of N. that was isolated from marine sediment of the South Korean europaea. coast (Lee YJ et al., 2012 J Bacteriol.). Many genes encoding heavy metal resistance and efflux systems were found in the Acknowledgement genome. Genes encoding putative cadmium efflux pumps, arsenic efflux pumps, a chromate transporter, and lead-, cadmium-, zinc-, The research was supported by the EU and co-financed and mercury-transporting ATPases were found. Putative resis- by the European Social Fund under the project ENVIKUT tance proteins of copper, cobalt-zinc cadmium, and tellurium (TÁMOP-4.2.2.A-11/1/KONV-2012-0043), and also by the Hungar- were also identified. Apparently, transcriptions of those genes ian Scientific Research Fund (OTKA Grant K1006600 to Dr. Erzsébet are controlled by CadC homologous metal responsive repres- Fekete). sors. The transcription profiles of CadC-controlled genes were http://dx.doi.org/10.1016/j.nbt.2014.05.2004 monitored in the presence of various heavy metals in B. oceanisediminis 2691. Furthermore, six cadC promoter-operator-structural genes were transcriptionally fused with egfp gene in E. coli.A variety of heavy metals were treated and specific fluorescence intensities were measured. Taken together, the results showed that CadC proteins specifically respond to heavy metals and may play separate roles in heavy metal resistances, which have been evolved in the heavy metal abundant marine sediment milieu. In addition, CadC-controlled transcriptional modules

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PI-36 Despite current trends and benefits in increased use and production of contents in new formats (e.g. video, interactive), and use Offer, demand, and needs in training and education: a of new tools and technologies (e.g. e-learning, b-learning), train- study focusing on microbial culture collections within ing within MIRRI still has an overwhelming dominance of classic the MIRRI Consortium content types and delivery methodologies. Also, we identified a André Antunes 1 , Veerle Piessens 2, Nelson Lima 1, The MIRRI Con- much wider untapped market for education and training within sortium our customer base, and we estimate a spike in demand of training from culture collections, particularly from the profit sector. 1 Micoteca da Universidade do Minho, Centre of Biological Engineering, Uni- Additional efforts are clearly necessary in adjusting our offer, versity of Minho, Braga, Portugal 2 BCCM/LMG Bacteria Collection, Faculty of Sciences, Ghent University, B-9000 adapting contents and content delivery and focusing on cost- Ghent, Belgium efficiency and proper advertising to increase visibility, and better serve the needs of our customers. The bioeconomy is fueled by Biological Resource Centers, Reference which play a vital role in harnessing and preserving the world’s [1].OECD. Underpinning the future of life sciences and biotechnology. OECD, biodiversity [1]. MIRRI (the Microbial Resource Research Infra- Biological Resource Centres; 2001. p. 1–66. structure: www.mirri.org) is an EU-project involving a total of 33 partners, aiming to provide facilitated access to microbial http://dx.doi.org/10.1016/j.nbt.2014.05.2005 resources, associated data and expertise, and promote knowledge transfer and foster innovation. One crucial step is to properly define our stakeholder community and identify their current and future needs, and match them to our offer. In order to achieve these goals, MIRRI surveyed both its partners, and current and potential users of microbial resources and services. Here we present and analyze some of the results of this survey, focusing on training and education.

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Glycobiology tion, without the need for sample labeling. Here, we employed SPR to analyze carbohydrate-protein interactions, particularly PJ-01 GM1-related carbohydrate-Vibrio cholera toxin interactions. The interaction between cholera toxin subunits A (ctxA) and B (ctxB) Cloning and characterization of novel β-glucosidases was similar to general ligand-receptor interactions. After the direct from Aspergillus and their functional expression in immobilization of thiol-containing GM1 pentasaccharide on a methylotrophic yeast Pichia pastoris gold surface, the GM1-ctxB interaction kinetics were evaluated, Richard Auta ∗ , Paul Hooley, Iza Radecka and they showed a similar degree of kinetics as reported in previous reports. We found that ctxA had a high affinity for the GM1- University of Wolverhampton ctxAB complex, although its equilibrium dissociation constant was 10-times lower than that of GM1-ctxB binding. Comparative The production of fermentable sugars from lignocellulosic analyses for GM1-related carbohydrates-ctxAB interactions were material has attracted interest in the optimization of conditions also conducted to determine the kinetic values of several GM1 related to conversion of cellulose to glucose for biofuel production. analogues with different structures, although their kinetic values β Aspergillus strains are known as efficient producers of -glucosidase were one-order of magnitude lower than those of the GM1-ctxAB which is a rate limiting factor during enzymatic hydrolysis of cellu- interaction. The kinetic analysis results for the interactions of β lose.A bioinformatics based approach to characterize -glucosidase GM1 analogues and ctxAB indicated that the sialic acid thumb encoding enzymes in the genus Aspergillus is described and the is important for recognition, and the terminal galactose and N- application of bioinformatics in the selection and expression of acetylgalactosamine finger are required to stabilize the GM1-ctxAB target genes is explained. Five Pichia clones (carrying A. nidu- interaction. Taken together, our results indicate that the direct lans AN2227.2, AN2612.2, AN0712.2, AN1551.2 and AN1804.2 immobilization of carbohydrate in an SPR-based analytical system in pPICZ vectors) that exhibit satisfactory levels of expression of can be used to evaluate the structural contribution of carbohydrate β recombinant -glucosidase were obtained from the Fungal Genet- moieties in carbohydrate-protein interactions, as well as provide ics Stock Centre (FGSC). A study to compare their hydrolytic valuable information that can be used to understand the interac- activities and relate these to their growth profiles using different tions. media was carried out. The characteristics of these enzymes and the use of P pastoris in the expression of these proteins are discussed http://dx.doi.org/10.1016/j.nbt.2014.05.2007 Keywords ␤-glucosidase; Aspergillus; Bioinformatics; Hydrolysis http://dx.doi.org/10.1016/j.nbt.2014.05.2006

PJ-02

Kinetic evaluation of carbohydrate-protein interaction using SPR

Jeong Hyun Seo 1,∗ , Chang Sup Kim 2, Hyung Joon Cha 2 1 Yeungnam university 2 POSTECH, Korea

Surface plasmon resonance (SPR) can provide kinetic information about an interaction, and it can also be used to rapidly monitor dynamic processes, such as adsorption and degrada-

S156 www.elsevier.com/locate/nbt New Biotechnology · Volume 31S · July 2014 GREENHOUSE GASES AND GLOBAL WARMING

Greenhouse gases and global warming PK-02

PK-01 Emerging Biotechnologies For Landfill Greenhouse Gases’ Efficiency And Development Of Useful Web CO2 gas conversion to oxaloacetate by Escherichia coli Drawing Utilities For Public Health Protection harboring codon-optimized carbonic anhydrase and ∗ Tilemachos Koliopoulos phosphoenolpyruvate carboxylase derived from marine bacteria Director Telegeco - Scient. Colaborator Technological Educational Institute of Athens - Soohyun Park 1,∗ , Soohye Hong 1, Sangwoo Kim 1, Seung Pil Pack 2, Jinwon Lee 1 This paper analyses the effects of different waste manage- 1 Sogang university ment landfill biotechnology techniques influencing on produced 2 Korea University greenhouse emissions, leachate emissions, acids and landfill mass biodegradation stages. The biodegradation of Mid Auchencarroch

Increased emission of CO2 is a major problem which causes experimental landfill project is studied in four different cells. The global warming throughout the world. Interest in bio-refinery pro- variations of the examining emissions are analysed in order to cess has increased remarkably because microbial conversion of develop an efficient project management for the right operational

CO2 to chemicals has large number of industrial applications, measures for food safety in areas adjacent to landfill boundaries and also it can alleviate the increasing residual CO2. Carbonic and public health protection. An analysis is made for emerging anhydrase (CA) is an enzyme, which plays a role in carbon seques- landfill biotechnology designs and modern spatial monitoring tration process. And, phosphoenolpyruvate carboxylase (PEPC) is systems are developed utilizing properly web drawing utilities a biocatalyst which converts phosphoenolpyruvate to oxaloac- and associated information technologies (IT’s). Useful emerging etate. Oxaloacetate can be converted in to various valuable C4 landfill biotechnologies and simulation models are presented for chemicals (succinate, malate etc.) in TCA cycle. First, novel CA produced landfill greenhouse gases making useful conclusions. and PEPC genes were screened and found in marine bacteria by Keywords basic local alignment using the Escherichia coli PEPC amino acid landfill biotechnology; greenhouse gases; landfill emissions; (AA) sequence as a query. The CA gene from Hahella chejuensis experimental landfill design; public health protection; water and PEPC gene from Photobacterium profundum SS9 were selected. resources; global warming; climate change; food security; emerg- The codons of the heterologous CA and PEPC genes were syn- ing technologies. thesized using codon-optimization technique. Codon-optimized http://dx.doi.org/10.1016/j.nbt.2014.05.2009 genes were cloned into pETDuet1 and transformed in E. coli BL21(DE3). Due to the SDS-PAGE results, it was confirmed that the codon-optimized genes were expressed as soluble protein forms in E. coli. The specific activity value of the codon-optimized enzymes were relatively high compared to the previously known CA and PEPC activities (codon-optimized CA (HC-aCA) = 478 ± 63 WAU/mg protein in the presence of Zn2+, codon-optimized PEPC

(OPPP) = 80.3 U/mg protein). In this study, a CO2 gas converting platform technology based on the co-expression system of CA and PEPC is developed and the potential of CO2 gas conversion platform technology is shown. http://dx.doi.org/10.1016/j.nbt.2014.05.2008

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High value plant products reached 75% and the clearance rate was above 60%. The solubility of the hydrolysate was as high as 93%. Emulsifying property and PL-01 the emulsifying stability decreased in the pH range from 3.0 to 5.0, increasing with increased pH. Compared with the measured glob- Effect of pH on chromium and nickel biosorption by ulin, the foamability of hydrolysate was significantly increased, Litchi chinensis seeds in single and binary metal systems lowest at pH 5.0. The viscosity of hydrolysate was reduced during Liliana Morales Barrera ∗ , Liliana Morales-Barrera, Imelda Guerrero- the whole process. Coronilla, Víctor Zambrano-Pérez, Jessica Reyes-Ledezma, Griselda http://dx.doi.org/10.1016/j.nbt.2014.05.2011 Chávez-Camarillo, Eliseo Cristiani-Urbina National Polytechnic Institute PL-03 The extensive industrial use of hexavalent chromium [Cr(VI)] Combinatorial optimization of synthetic operons for the and divalent nickel [Ni(II)], as well as their improper disposal have microbial production of monolignols in Escherichia coli led to heavy metal contamination of water [1]. Biosorption is an ∗ efficient and low-cost alternative for removing toxic metals from Philana van Summeren-Wesenhagen , Raphael Voges, Stephan aqueous solutions. The purpose of this work was to evaluate the Noack, Michael Bott, Jan Marienhagen effect of pH on chromium and Ni(II) removal by Litchi chinensis Forschungszentrum Jülich seeds (LCS) in single and binary metal systems. LCS was able to remove chromium and Ni(II) from mono- The monolignol p-coumaryl alcohol is an important precursor metal solutions, and its removal capacity depended on the pH. of lignans and key building block of the plant polymer lignin, LCS exhibited the highest removal capacity of Cr(VI) (86.79 mg/g), which is widely recognized as cheap source of aromatic com- total chromium (64.74 mg/g) and Ni(II) (20.0 mg/g) at pH values pounds. However, due to its complex and irregular structure the of 1.0, 2.0 and 7.5, respectively. uitilization of lignin is technically challenging. In contrast, micro- At a pH value of 1, a difference between Cr(VI) (86.79 mg/g) and bial production of p-coumaryl alcohol and other monolignols total chromium removal capacity (34.4 mg/g) was observed and represents a promising alternative. Recently, the first synthetic this was probably due to the fact that part of the Cr(VI) initially pathway for the production of p-coumaryl alcohol from L-tyrosine present in the solution was reduced to trivalent chromium [Cr(III)] in E. coli was published [1]. Here we introduce a fast and robust by LCS. method to optimize the product titers of this four gene pathway, In the bimetal systems, Ni(II) did not have any effect on based on the Phosphorothioate based Ligase-Independent Gene Cloning chromium removal at any pH value. Ni(II) biosorption capacity (PLICing) method [2]. An operon library was generated in which was not affected by the presence of chromium in acidic solutions; the translation efficiency of every gene was systematically varied in contrast, at neutral pH values the capacity improved signif- by different spacings between the ribosomal binding site and the icantly. These results suggest that chromium and Ni(II) do not START-codon. Screening of this library yielded mutants producing compete between them for the same LCS binding sites. up to 55 mg/L p-coumaryl alcohol, 7 times more compared to the starting strain under similar cultivation conditions. Reference References [1].Park D, Yun YS, Yim KH, Park JM. Effect of Ni(II) on the reduction [1].Jansen F, Gillessen B, Mueller F, Commandeur U, Fischer of Cr(VI) by Ecklonia biomass. Bioresource Technol 2006;97:1592–8. R, Kreuzaler F. Metabolic engineering for p-coumaryl alcohol http://dx.doi.org/10.1016/j.nbt.2014.05.2010 production in Escherichia coli by introducing an artificial phenyl- propanoid pathway. Biotechnology and Applied Biochemistry 2014, doi: 10.1002/bab.1222. (published ahead of print). [2].Blanusa M, Schenk A, Sadeghi H, Marienhagen J, Schwaneberg U. PL-02 Phosphorothioate based Ligase-Independent Gene Cloning - A method for cloning of mutant libraries in directed evolution experiments. Preparation and Functional Characteristics of Peptide Anal Biochem 2010;406:141–6. from Naked Oat Globulin by Protease Hydrolysis http://dx.doi.org/10.1016/j.nbt.2014.05.2012 Meili Zhang ∗ , Rui Lin Inner Mongolia Agricultural University

Globulin of naked oat was prepared by the Osborne methods and hydrolyzed by alkaline protease. The degree of protein hydrolysis and clearance rate of hydroxyl free radicals were used to identify the best enzyme hydrolysis process. Solubility, emulsifying property, viscosity and foamability of hydrolysate were tested. The result showed that the optimum conditions were as follows: dose of enzyme, 10000U/g; concentration of substrate, 5%; the temperature, 60◦C; and pH 9.0. After 3 h, the degree of hydrolysis

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PL-04 It could be shown that the fermentation of tofu with L. lactis is an efﬁcient and cost-effective alternative. In vivo and in vitro cultures of Lavandula angustifolia References Mill. for essential oils production

∗ [1].Pongtharangkul T, Demirci A. Appl Microbiol Biotechnol: Pana Lohasupthawee , Prangmas Srisurat 2004;65:268–72. King Mongkut’s Institute of Technology Ladkrabang [2].International Organization for Standardization ISO/TS 27106 Geneva Switzerland 2009. The aim of this research was to extract the essential oils of http://dx.doi.org/10.1016/j.nbt.2014.05.2014 lavender (Lavandula angustifolia Mill.) from callus culture, multiple shoots culture and hydroponic culture. The chemical composition of each extract was analyzed by gas chromatography and PL-06 mass spectrometry (GC-MS). In vitro culture of lavenders, multiple shoots were obtained in culture medium fortified with Murashige Fermentation-like incubations of Theobroma cacao L. - and Skoog (MS) nutrients and 1.5 mg/l benzyladenine which pro- Good quality in shorter time duced 12 shoots per explant. Callus was achieved by using leaf Claudia Bahmann ∗ , Tumforde Thomas, Lieberei Reinhard explants and showed best growth in MS medium supplemented University of Hamburg with 0.5 mg/l 2,4-dicholorophenoxyacetic acid. In vivo culture, hydroponic lavenders were achieved by using deep flow tech- In the course of the post-harvest treatment of cocoa seeds (fer- nique system. The leaves and roots of hydroponic lavenders were mentation) the pulp is microbially degraded. During fermentation extracted for essential oils separately. The GC-MS results showed first ethanol, then acetic acid is produced in combination with a that the leaves of hydroponic lavenders and in vitro shoots demon- temperature increase. The conditions in the fermentation mass strated the components of lavender essential oils whereas callus entail the acidification of the cotyledon tissue and an extensive and roots of hydroponic lavenders showed no essential oils pro- proteolysis of storage proteins. The latter is carried out by endoge- duction. nous proteases that are activated by acidic conditions. The cleavage http://dx.doi.org/10.1016/j.nbt.2014.05.2013 activity of the proteases as well as the conservative amino acid sequences of storage proteins provide a defined pattern of flavor precursors of cocoa. In the study at hand seeds have been incu- PL-05 bated under conditions analog to those of fermentation, because of the ability to control different parameters separately. Media with Fermentative Nisin Production in Tofu for its Preserva- different pH-values, all in the acid range, and different organic tion acids have been used. The basic effects of these factors on the seed Nicole Illas ∗ , Raphael Cziskus, Caterina Hünniger, Myriam Bello, with regard to the proteolysis are examined. In order to imitate the Xuan Zhu, Markus Fischer, Bernward Bisping temperature conditions of fermentation, incubations are carried out under different temperature sequences. University of Hamburg In the course of the incubation process comparatively higher amounts of the amino acids and phenolic compounds examined Tofu has a high nutritional content and serves as stable food in are achieved already after three days. Comparable values in the eastern Asia. In developing countries pasteurization is not afford- fermentation procedures are achieved not until after six days. This able because tofu production takes place mostly in small factories. illustrates the efficiency of the incubation procedure. In this study, A cost-effective and natural preservation of tofu may be thenisin an endogenic bioconversion has been proven to take place in production in tofu cubes (2*2 cm) submerged in water by fer- cocoa seeds in the course of incubations. Furthermore, the striven mentation with Lactococcus lactis ssp. lactis DSM 20729. Optimal accumulation of characteristic chocolate aroma precursors can be fermentation was conducted by adding 3.9% soy peptone. Further- influenced by processing seeds under defined external conditions. more it could be shown that there is a non-uniform distribution of nisin in tofu cubes. The nisin concentration decreases from http://dx.doi.org/10.1016/j.nbt.2014.05.2015 the surface to the interior of tofu but it was sufficient to extend the shelf-life of tofu. Two methods for the detection of nisin in fermented tofu were tested. The comparison of an inhibition PL-07 test (modified [1]), and LC-ESI-MS/MS method (based on ISO/TS 27106:2009 [2]) illustrates that the detection limit of the inhibition Glucanocellulosic biomass: learning from marine test was significantly lower. Concentrations of 0.19 mg/kg nisin in biomass to optimize terrestrial biomass conversion tofu and 0.06 mg/L in the supernatant could be determined. The Christian Voigt ∗ , Claudia Zwikowics detection limit for nisin using LC-ESI-MS/MS was 0.34 mg/kg in University of Hamburg tofu. Matrix calibration of the liquid substrate could not be carried out by LC-ESI-MS/MS, because the background noise of the The recalcitrance of the plant cell wall is one of the obsta- matrix was too high. cles in improving biomass conversion. We followed a strategy to enrich biomass with a polymer that is easily degradable and would

www.elsevier.com/locate/nbt S159 HIGH VALUE PLANT PRODUCTS New Biotechnology · Volume 31S · July 2014 not impair the physiology of the plant. In marine biomass from Applying the optimized processing for glucan-enriched biomass the brown algae Fucus vesiculosus, the polymer (1,3)-β-glucan is a on M. giganteus leaf biomass, we increased bioethanol production major biomass component showing these characteristics and is by 13% compared to non-adapted conversion and fermentation also present in terrestrial plants. We used this glucan-enriched, strategies. To further improve bioethanol production with this marine biomass to optimize hexose release and bioethanol produc- adapted processing, we overexpressed a (1,3)-β-glucan synthase tion. The addition of a bacterial (1,3)-β-glucanase to a commercial from Arabidopsis thaliana in M. giganteus, which further increased enzyme cocktail as well as the usage of an optimized Saccharomyces (1,3)-β-glucan content in leaf biomass to 8.5% and improved cerevisiae strain that we engineered for fermenting glucan-enriched bioethanol production by 20%. Our results suggest that generation biomass resulted in a 50% increase in bioethanol production. To of glucan-enriched biomass via synthetic biology approaches com- test the optimized processing of glucan-enriched biomass on ter- bined with optimized processing for glucanocellulosic bioethanol restrial biomass, we screened for plants with a high (1,3)-β-glucan production is a promising alternative in increasing efﬁciency of content and identiﬁed leaves from the energy crop Miscanthus x biomass conversion. β giganteus with an exceptionally high (1,3)- -glucan content of 5%. http://dx.doi.org/10.1016/j.nbt.2014.05.2016

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Metabolic engineering kinase of C. acetobutylicum encoded by the bukII gene instead of butyrate kinase I encoded by the buk gene was employed. Further- PM-01 more, metabolic pathways were engineered to further enhance the NADH-driving force. Batch fermentation of the metabolically engi- Sex-Dimorphic Expression Of Caveolin 1 Is Linked To neered C. acetobutylicum strain at pH 6.0 resulted in the production Obesity Development In Rats of 32.5 g/L of butyric acid with a butyric-to-acetic acid ratio (BA/AA Jae Heon Choi ∗ , Rajib Mukherjee, Sang Woo Kim, Jong Won Yun ratio) of 31.3 g/g from 83.3 g/L of glucose. These results suggested that the buk gene knockout was essential to get a high butyric acid Daegu University selectivity to acetic acid in C. acetobutylicum. [This work was supported by the Technology Development Pro- Recently, it has been reported that CAV1 is an important target gram to Solve Climate Changes on Systems Metabolic Engineering protein in sex hormone-dependent regulation of various metabolic for Biorefineries from the Ministry of Science, ICT and Future Plan- pathways, particularly in cancer and diabetes. To clarify distinct ning (MSIP) through the National Research Foundation (NRF) of roles of CAV1 in sex-dependent obesity development, we inves- Korea (NRF-2012-C1AAA001-2012M1A2A2026556).] tigated the effects of high fat diet and sex steroid hormones on CAV1 expression in adipose tissues of male and female rats. Results http://dx.doi.org/10.1016/j.nbt.2014.05.2018 of animal experiments revealed that estrogen (17-β-estradiol, E2) and androgen (dihydrotestosterone, DHT) had opposite effects on body weight gain as well as on the regulation of CAV1, hor- PM-03 mone sensitive lipase (HSL) and uncoupling protein 1 (UCP1) The parologues pyruvate kinases in Streptomyces coeli- in adipose tissues. Furthermore, sex hormone receptors and aro- color have distinct roles in growth and antibiotic matase were differentially expressed in a sex-dependent manner in production response to E2 and DHT treatments. In vivo data were confirmed ,∗ using 3T3-L1 and HIB1B cell lines, where Cav1 knock down stim- Jana Hiltner 1 , Pablo Cruz-Morales 2, Lorena Fernandez-Martinez 3, ulated lipogenesis but suppressed sex hormone receptor signaling Hrovje Petkovic 4, Iain S. Hunter 1, Francisco Barona-Gomez 2, Paul A. proteins. Most importantly, co-immunoprecipitation enabled the Hoskisson 1 identification of previously unrecognized CAV1-interacting mito- 1 University of Strathclyde chondrial or lipid oxidative pathway proteins in adipose tissues. 2 Langebio Cinvestav Taken together, current data showed that CAV1 may play impor- 3 John Innes Centre 4 tant preventive role in the development of obesity, with more Acies Bio Ltd prominent effects in females, and proved to be an important target protein for the hormonal regulation of adipose tissue metabolism Streptomyces species are prolific producers of antibiotics, nev- by manipulating sex hormone receptors and mitochondrial oxida- ertheless analysis of complete genomes still shows that there tive pathways. Therefore, we can report, for the first time, the are many biosynthetic clusters present that are silent under lab- molecular mechanism underlying the effects of sex steroid hor- oratory cultivation conditions. The current increase in clinical mones in the sex-dimorphic regulation of CAV1. antibiotic resistance requires the discovery of new antibiotics, but also a greater understanding of antibiotic production for indus- http://dx.doi.org/10.1016/j.nbt.2014.05.2017 trial exploitation. Our interest is in studying the transition of primary metabolites into secondary metabolism. We focus on the Phosphoenolpyruvate-Pyruvate-Oxaloacetate (PEP-PYR-OAA) PM-02 node of central carbon metabolism using Streptomyces coelicolor as Metabolic engineering of Clostridium acetobutylicum for a model and have identified pyruvate kinase influences antibi- highly selective butyric acid production otic production. The genome encodes two parologue pyruvate kinase genes - SCO2014 (pyk1) and SCO5423 (pyk2). Phenotypic ∗ Yu-Sin Jang , Sang Yup Lee analysis of the two mutants revealed differences in their physi- Korean Advanced Institute of Science and Technology (KAIST) ological role, pyk2 exhibits altered growth on glucose, whereas pyk1 shows a difference in antibiotic production. We used cross- Butyric acid, a saturated four-carbon carboxylic acid, has been species complementation experiments with E.coli pykF, pykA widely used in chemical, food, pharmaceutical, and animal feed and pykApykF and complemented these with pyk1 and pyk2 industries. The well-known clostridial native butyric acid produc- from S. coelicolor on different media to clarify their physiologi- ers include C. butyricum, C. thermobutyricum, C. tyrobutyricum, C. cal roles. Furthermore Pyk1 and Pyk2 were overexpressed in E. coli acetobutylicum and C. pasteurianum. A typical characteristic of such for a detailed characterisation of their biochemical properties. Our butyric acid-producing Clostridium is coproduction of both butyric data show that gene parologues in primary metabolism have dis- and acetic acids. Increasing the butyric acid selectivity important tinct physiological roles in Streptomyces that impact significantly for economical butyric acid production has been rather difficult in on growth and the production of antibiotics, which could be used clostridia due to their complex metabolic pathways. In this work, for industrial strain improvement in the biotechnology industry. C. acetobutylicum was metabolically engineered for highly selec- http://dx.doi.org/10.1016/j.nbt.2014.05.2019 tive butyric acid production. For this purpose, the second butyrate

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PM-04 introduced davB and davA genes from Pseudomonas putida ATCC 12633 together with the endogenous gabT and gabD genes in Re-distribution of carbon flux toward 2,3-butanediol C. glutamicum. DavBA function as metabolic “gap” filling for production in Klebsiella by metabolic engineering glutaric acid production in engineered C. glutamicum since the Borim Kim ∗ , Soojin Lee, Daun Jeong, Jeongmo Yang, Jinwon Lee non-engineered strain does not degrade L-lysine. This engineered strain cultured in a laboratory-scale bioreactor produced 24.9 g/L Sogang university of 5-aminovaleric acid and 11.9 g/L of glutaric acid in 144.5 h using glucose as a sole carbon source. The second synthetic path- As shown in previous studies, Klebsiella pneumoniae showed way comprises the codon-optimized davB and davA genes from P. high potential as a 2,3-butanediol (2,3-BDO) producer, and exhib- putida ATCC 12633 and the codon-optimized davT and davD genes ited great productivity. However, the accumulation of substrates from P.putida KT2440. The engineered C. glutamicum harboring the other than 2,3-BDO such as; lactate, ethanol, and acetate, in the second synthetic pathway produced 23.7 g/L 5-aminovaleric acid log-phase of cell growth remained as an obstacle for efficient large and 10.6 g/L glutaric acid in 150.5 h. These results suggest that C5 scale 2,3-BDO production. Hereby, in order to re-distribute the platform chemicals can be efficiently produced by metabolically substrate-directed carbon flux to 2,3-BDO production metabolic engineered C. glutamicum. This study also presents a strategy for engineering was done. Incorporation of the gene deletion method assembling and establishing synthetic pathways in C. glutamicum (deleting competitive NADH consuming pathway by ldhA gene for the production of chemicals, which will be useful for designing deletion) and gene over-expression method (re-directing carbon- other strains for the bio-based production of chemicals. flux toward 2,3-BDO biosynthesis by budA gene over-expression) were conducted for efficient utilization of glucose conversion to http://dx.doi.org/10.1016/j.nbt.2014.05.2021 2,3-BDO under slightly acidic condition(pH 5.5). The engineered strain SGSB105 showed a 40% increased 2,3-BDO production from glucose, compared to the wild-type strain SGSB100. Also the PM-06 closely related genes in the 2,3-BDO biosynthesis pathway were Functional Expression and Characterization of Codon observed at the gene transcription level by cultivating mutant Optimized Proteorhodopsin in Escherichia coli strains under unify culture conditions. As a result, the gene expression levels of the budB, budA, and budC genes showed a 10% Yong-Jik Lee 1,∗ , Sang Jun Lee 2, Dong-Woo Lee 1 increased transcription level at the log-phase of the cell growth, 1 Kyungpook National Univ compared to the SGSB100. Also the 2,3-BDO gene transcription 2 Korea Research Institute of Bioscience and Biotechnology levels of SGSB105 was maintained at a high level during the log- and stationary-cell growth phase. By incorporating the gene dele- Proteorhodopsin (pR) as an integral membrane light-harvesting tion and over-expression method, the carbon flux was re-directed proton pump generates proton motive force across the cellular to a valuable biochemical producing process, and also by combin- membrane. A wide range of pRs have been studied on the eco- ing the batch culture data with the gene transcription data it shows logical distribution and function, but the expression level of pR an insight for improving the 2,3-BDO biosynthesis metabolic net- and its physiological role in nonphotosynthetic host strains still work for industrial application. remain unclear. Here, we chemically synthesized the SAR 86 gene http://dx.doi.org/10.1016/j.nbt.2014.05.2020 with codon optimization (co SAR86) and expressed the pR gene in Escherichia coli as a fusion protein containing a C-terminal hex- ahistidine sequence. The recombinant enzyme was purified to 2+ PM-05 homogeneity by solubilization of E. coli membrane, Ni affinity chromatography followed by gel filtration chromatography. Metabolic engineering of Corynebacteruim glutamicum Comparison of thephysicochemical properties of both membrane- for production of 5-aminovaleric acid and glutaric acid embedded and purified co SAR86 in the presence and absence of as C5 platform chemicals all-trans retinals established that these enzymes expressed in E. coli ,∗ were integrated properly. To investigate whether the expressed JaeHoShin1 , Si Jae Park 2, Sang Yup Lee 1 co SAR86 can enhance the cellular energy production of host cells, 1 Korea advanced institute of science and technology we compared the growth phenotypes of co SAR86 expressing E. coli 2 Myongji University strain and the wild-type strain under various conditions. Here, we report successful production and initial characterization of a The amino acid, L-lysine can be naturally degraded via multiple functional co SAR86 that supports extra energy production for the conduits including the cadaverine and 5-aminovaleric acid path- growth of E. coli cells under certain growth conditions, which may ways. The degradative intermediates, cadaverine, 5-aminovaleric facilitate the exploitation of pR for commercial biotechnological acid and glutaric acid are C5 platform chemicals that can be used applications. for bio-polyamide production. Here we report the development of Corynebacterium glutamicum strains overproducing 5-aminovaleric http://dx.doi.org/10.1016/j.nbt.2014.05.2022 acid and glutaric acid by metabolic engineering of a classical L-lysine producer and introducing novel synthetic pathways. The first synthetic novel pathway consists of the heterologously

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PM-07 available for this purpose, and use several examples to demonstrate the application of protein engineering for metabolic engineering Generation of Oxalic Acid Hyperproducers by Overex- in amino acid overproduction. pressing the Oxaloacetate Hydrolase Gene in Aspergillus http://dx.doi.org/10.1016/j.nbt.2014.05.2024 niger

Keiichi Kobayashi ∗ , Shotaro Watanabe, Kohtaro Kirimura Department of Applied Chemistry, Faculty of Science and Engineering, Waseda PM-09 University Diffusion in crowded cytoplasm-like environment

The filamentous fungus Aspergillus niger is worldwide used Svyatoslav Kondrat ∗ , Olav Zimmermann, Eric von Lieres in the industrial production of citric acid. On the other hand, Forschungszentrum Jülich under specific cultivation conditions, A. niger accumulates oxalic acid. Oxalic acid is one of the valuable chemicals used as a We perform Brownian dynamics simulations to study short- chelator, detergent, or tanning agent, and industrially produced and long-time diffusion of macromolecules in crowded environ- by the chemical method, but not microbial one. In this study, ment of biological cells. We confirm that the diffusion slows down to generate oxalic acid hyperproducers by metabolic engineer- with increasing volume fraction, and focus on its dependence ing, we constructed transformants overexpressing the gene oahA on macromolecular composition of cell’s cytoplasm. The effect of encoding oxaloacetate hydrolase (OAH; EC 3.7.1.1) in citric acid- composition on different types of diffusion, and its importance for producing A. niger WU-2223L [1] as a host. The mRNA level of modelling metabolic pathways will be discussed. oahA and specific activity of OAH in strain EOAH-1, a representative oahA-overexpressing transformant, were higher than those http://dx.doi.org/10.1016/j.nbt.2014.05.2025 in WU-2223L. [2] To examine the potentiality of oxalic acid production, EOAH-1 was cultivated in OAP30 medium containing PM-10 30 g/L glucose as a carbon source, (NH4)2SO4 as a nitrogen source, and 2-[N-morpholino] ethanesulfonic acid (MES) as a buffering Application of a controllable degron strategy for substance, and produced 28.9 g/L oxalic acid during 12 days of metabolic engineering cultivation. Moreover, by the use of NaNO3 and K2HPO4-KH2PO4 ∗ buffer instead of (NH4)2SO4 and MES in OAP30 medium, EOAH- Christoph Knuf , Jérôme Maury, Simo Jacobsen, Jochen Forster 1 produced 35.8 g/L oxalic acid during 9 days of cultivation. The Novo Nordisk Foundation Center for Biosustainability, DTU yield of oxalic acid for EOAH-1 reached 79.6% of the maximum theoretical yield. Therefore, we succeeded in generating oxalic acid In numerous cases of metabolic engineering, metabolite pools hyperproducers by overexpressing a single gene, i.e., oahA in citric have to be increased in order to obtain flux into heterologous path- acid-producing A. niger. ways. A simple tool for this would be the deletion of genes that References would practically lead to a block of the natural pathway, so that the carbon can flow into the heterologous pathway. Unfortunately [1].Kobayashi K, et al. Biosci Biotechnol Biochem 2013;77:1492–8. [2].K. Kobayashi, et al., J. Ind. Microbiol. Biotechnol., in press (2014). some deletions are lethal, as end products of pathways are needed for cellular growth. One example of such a pathway is the meval- http://dx.doi.org/10.1016/j.nbt.2014.05.2023 onate pathway in S. cerevisiae with ergosterol as one of the most important end products. A great number of bioactive compounds, like various terpenoids, can be produced from intermediates of this PM-08 pathway. Different strategies have been applied in order to down-regulate Protein engineering for strain engineering the expression of enzymes involved in the mevalonate pathway. ∗ Ping Zheng , Jibin Sun All these strategies work on the transcriptional level. This leads Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences to a delay of the actual regulation, as the existing enzyme will still be active. We present a strategy for down-regulation that acts Engineering microbial strains to overproduce chemicals repre- on the protein level and which can therefore be controlled in a sents a promising topic in the field of industrial biotechnology. The more precise manner than the hitherto reported strategies. As a production performance and even the production portfolio can be case study we show the action of the degron strategy for control- well reconfigured by the advanced metabolic engineering technol- ling the pools of intermediates of the mevalonate pathway around ogy. Successful examples can be found in the area of antibiotics, 2,3-oxidosqualene, which is the precursor for triterpenoids. Many amino acids, organic acid, biofuels, biopolymers, and recombinant triterpenoids are pharmaceutically relevant compounds which plant-originated drugs. The gene expression by overexpression or nowadays need to be extracted from plant material through an disruption is often used tools. Further fine-tuning the activity of intricate and resource consuming process. the enzymes in particular by structure-based approach is very pow- http://dx.doi.org/10.1016/j.nbt.2014.05.2026 erful for global optimization of the metabolic pathway but not well-recognized in the literature. Here we discuss the toolboxes

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PM-11 for the rational design of bFMP. This strategy can be applied to various enzymes for the identiﬁcation of the potential target sites for Efﬁcient GABA production system development via the rational design, independent on the three-dimensional struc- introduction of synthetic protein scaffold ture of target protein. ∗ Soonho Hong http://dx.doi.org/10.1016/j.nbt.2014.05.2028 University of Ulsan

Gamma-aminobutyric acid (GABA) is a precursor of one of PM-13 the most promising heat-resistant biopolymers, Nylon-4, and can Statistical Optimization for Simultaneous Production of be produced by the decarboxylation of monosodium glutamate PLA Degrading and Raw Starch Degrading Enzymes by (MSG). In this study, a synthetic protein scaffold was applied Thermophilic Filamentous Bacterium, Laceyella sacchari to improve the GABA conversion in engineered Escherichia coli. LP175 Using Agricultural Crops as Substrates Scaffolds were constructed by assembling a single protein–protein interaction domain SH3 to the glutamate decarboxylase (GadA Vichien Kitpreechavanich ∗ , Thanasak Lomthong, Srisuda Han- and GadB) and attaching a cognate peptide ligand to the gluta- phakphoom mate/GABA antiporter (GadC) at the N-terminus, C-terminus, and Kasetsart University the 233rd amino acid residue. When GadA and GadC were co- overexpressed via the C-terminus scaffold, a GABA concentration Optimization of medium using low cost agricultural products of 5.65 g/L was obtained from 10 g/L MSG, which corresponds to a by statistical mixture design for simultaneous production of PLA GABA yield of 93%. A significant increase of the GABA productiv- degrading and raw starch degrading enzymes by thermophilic fila- ity was also observed where the GABA productivity increased 2.5 mentous bacterium Laceyella sacchari LP175 was investigated to fold in the early culture period due to the introduction of the syn- increase biodegradation of polylactide/starch blend bioplastics. thetic protein scaffold. The GABA pathway efficiency and GABA Total 5 g of different amounts of between cassava chip, soy bean productivity were enhanced by the introduction of the scaffold meal and corncob were used as substrates in 0.035% PLA basal between glutamate decarboxylase and glutamate/GABA antiporter. medium with 7 experiment runs with triplicate in shaking flask This work was supported by a grant from the Next-Generation at 50 ◦C. The highest enzymes productions were obtained from BioGreen 21 Program (SSAC, grant number: PJ00954904) by RDA, the mixed substrates of cassava chip and soy bean meal in ratio and Basic Science Research Program by the MEST (2011-0022392). 1:1 at 24 h cultivation. Cassava starch as the additional carbon http://dx.doi.org/10.1016/j.nbt.2014.05.2027 source was found to be the best for both enzymes productions. The response surface methodology with central composite design was used for enhanced both enzymes production consisted of PLA PM-12 powder and cassava starch in the mixture of cassava chip and soy bean meal as the basal medium. The maximum predicted activity Relationship between amino acid properties and cat- of PLA degrading enzyme was 69.7 U/mL with 92.1 U/mL RSDE alytic function in bacterial flavin-containing monooxy- (raw starch digesting enzyme) activity from the basal medium genase using deep mutational scanning approach consisted of 0.52 g/L PLA powder and 3.34 g/L cassava starch, while Namil Lee ∗ , Jongoh Shin, Byung-Kwan Cho the maximum predicted activity of RSDE was 92.9 U/mL with 69.2 U/mL PLA degrading activity was obtained from the basal medium KAIST consisted of 0.52 g/L PLA powder and 3.04 g/L cassava starch. The validation results of both enzymes were 68.8 and 86.1U/mL PLA Flavin-containing monooxygenases (FMOs) are the promising degrading and RSDE activities, respectively. enzymes that catalyze oxidation reactions of a wide array of substrates, including indole compounds. Thus, the engineered FMOs http://dx.doi.org/10.1016/j.nbt.2014.05.2029 with broad substrate specificity, enhanced catalytic efficiency (kcat/Km), and increased thermal stability have great potential to produce value-added bio-chemicals. However, their rational design for obtaining desired changes in the functional parameters is hampered by the lack of information about functions of amino acid residues of the enzymes. In order to understand a relationship between the amino acid residues and function of the bacterial flavin-containing monooxygenase (bFMO), we performed a saturated mutagenesis that all amino acid residues of the enzyme were replaced by other amino acids, followed by deep sequencing. We classified amino acid residues of the enzyme into function-retained and function-loss subgroups, which are subsequently interpreted based on the three-dimensional structure. The functional relationship between amino acid residues and catalytic function was used

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PM-14 colysis pathway in the industrially important and natural glutamate producer Corynebacterium glutamicum. To this end, Study of the role of Escherichia coli central metabolism a double deletion mutant devoid of the genes for phos- pathways related genes in the synthesis of hydrogen and phorylating NAD+ and NADP+-dependent glyceraldehyde-3- ethanol by using glycerol as carbon source phosphate dehydrogenase gapA and gapB was constructed. Non- + Jorge Bolivar 1,∗ , Antonio Valle 2, Gema Cabrera 3, Domingo phosphorylating NADP -dependent glyceraldehyde-3-phosphate Cantero 3 dehydrogenase from Clostridium acetobutylicum (encoded by gapNCac) irreversibly oxidizes glyceraldehyde-3-phosphate (GAP) 1 University of Cadiz to 3-phosphoglycerate (3-PG) in an NADP+-dependent man- 2 University of Cadiz-Department of Biomedicine, Biotechnology and Public Health-Biochemistry and Molecular Biology; Chemical Engineering and Food ner without addition of Pi, thus, bypassing ATP generation Technology via phosphoglyceratekinase. The resulting recombinant strain C. 3 University of Cadiz-Department of Chemical Engineering and Food Technol- glutamicumΔgapAΔgapB (pEKEx3-gapNCac) was expected to oxi- ogy dize glucose to pyruvate without net ATP yield whereas 2 mol of NADPH are formed. However, this strain did not grow in Earth’s climate warming as a result of anthropogenic emis- glucose minimal medium. Upon prolonged incubation suppres- sions of greenhouse gases, particularly carbon dioxide (CO2) from sor mutants could be isolated. Further analysis of the suppressor fossil fuel combustion, has provoked an urgent need to develop mutants by genome sequencing analysis revealed a SNP shared by clean and renewable energy sources. Bioenergy has emerged as an the four suppressor mutants. The SNP in the ndh gene (cg1656) alternative source of fuel, which includes biodiesel, hydrogen and encoding the non-proton pumping NADH dehydrogenase caused bioethanol. However, the biodiesel industry currently generates a an amino acid exchange. This mutation increased NADPH oxida- huge amount of glycerol as a by-product in such a magnitude that tion by NDH and, thus, provides a unique solution to re-oxidize it has become an environmental problem. An achievable solution NADPH generated in the engineered ATP-neutral glycolytic path- to this problem is the use of waste glycerol as the main carbon way in C. glutamicum. source for microbial transformation to hydrogen and ethanol. This http://dx.doi.org/10.1016/j.nbt.2014.05.2031 is an environmentally safe process that may lead to the production of renewable energy resources, which could contribute to the reduction of the CO2 emissions. Escherichia coli is a very promis- PM-16 ing alternative for glycerol utilization and it has the advantage that is commonly used for metabolic engineering in many biotechno- Metabolic Engineering of Saccharomyces cerevisiae for logical applications. In this work we study how the blockage of Isoprenoid Production some enzymes by using E. coli single knock out strains affect to Stefan Tippmann ∗ Sakda Khoomrung Verena Siewers Jens the H2 and ethanol productions as well as glycerol consumption , , , when the cells grew in a glycerol-based medium. Due to the role Nielsen that the central carbon metabolism plays in the hydrogen and Chalmers University of Technology ethanol synthesis, several mutants of these important pathways have been analysed. We describe here several novel mutant back- This project attempts to establish a yeast cell factory for the grounds that could be useful in order to enhance the ethanol and production of isoprenoids, which were attributed a key function H2 productions in E. coli. in the search for alternative transportation fuels. For this purpose, http://dx.doi.org/10.1016/j.nbt.2014.05.2030 Saccharomyces cerevisiae was chosen as a host organism, whereas the main focus is set on sesquiterpenes such as farnesene, which can be used as diesel alternative in its hydrogenated form far- PM-15 nesane. In order to enable for efficient production of farnesene, two central aspects are being addressed, i.e. metabolic engineer- Metabolic Engineering an ATP-neutral EMP pathway in ing for enhanced synthesis and analytical method development C. glutamicum: adaptive point mutation in NADH dehy- for accurate quantification of intra- and extracellular metabolites drogenase restores growth from two-liquid phase fermentations. In the first part, an exist- ,∗ ing platform optimized for sesquiterpene production was recently Gajendar Komati Reddy 1 , Steffen N Lindner 2, Volker F Wendisch 1 used for the integration of farnesene synthase genes from differ- 1 Chair of Genetics of Prokaryotes, Faculty of Biology and CeBiTec, University of ent plant sources to enable the one-step conversion from farnesyl Bielefeld pyrophosphate to farnesene. As a result, maximal titers of ∼1 g/L 2 Chair of Genetics of Prokaryotes, Faculty of Biology University of Bielefeld were attained in a comparative evaluation in fed-batch cultivations with exponential feeding. Enhanced synthesis, however, will not Microorganisms produce ATP by substrate level phos- only involve heterologous expression of these enzymes, but it will phorylation in Embden–Meyerhof–Parnas pathway and/or by also include further engineering of the endogenous mevalonate electron transport phosphorylation. By bypassing substrate- pathway as well as the integration of different ‘omics’ analysis to level phosphorylation via phosphorylating NAD+-dependent support the cycle of metabolic engineering. glyceraldehyde-3-phosphate dehydrogenase(s) and phosphoglyceratekinase, we engineered a strain with ATP-neutral gly- http://dx.doi.org/10.1016/j.nbt.2014.05.2032

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PM-17 PM-18

Identification of a new putative regulatory protein Identification of engineering targets for improving involved in morphological differentiation and eryth- putrescine production by Corynebacterium glutamicum romycin production in Saccharopolyspora erythraea by Anh Do Quynh Nguyen 1,∗ , Jens Schneider 2, Volker Wendisch 3 omics approaches 1 Cebitec & Genetics of Prokaryotes, University of Bielefeld 1,∗ 2 2 Hrvoje Petkovic , Vasilka Magdevska , Benjamin Kirm , Miha 2 Evonic Industry Tome 2, Marinka Horvat 2, Katarina Karnicarˇ 2, Robert Vidmar 3, 3 Chair of Genetics of Prokaryotes, University of Bielefeld Spelaˇ Baebler 4, Polona Jamnik 5, Stefanˇ Fujs 2, Jaka Horvat 2, Marko Fonovicˇ 3, Boris Turk 3, Kristina Gruden 4, Gregor Kosec 2 Corynebacterium glutamicum shows great potential for the pro- 1 University of Ljubljana, Biotechnical Faculty duction of the polyamide monomer putrescine. Previously, we 2 Acies Bio d.o.o have constructed the putrescine producing strain PUT21 by dele- 3 Institut Jozefˇ Stefanˇ tion of argF, the gene for ornithine transcarbamoylase (OTC), and 4 Nacionalni Institut za Biologijo argR, encoding the L-arginine repressor combined with heterol- 5 Biotechnical Faculty, University of Ljubljana ogous expression of the Escherichia coli gene for the L-ornithine decarboxylase SpeC and low-level argF expression from a plasmid Erythromycin is a medically important antibiotic, biosynthe- addiction plasmid system. sized by the actinomycete Saccharopolyspora erythraea. A significant Acetylputrescine was detected as by-product in fermentations increase in erythromycin yields has been achieved, compared to with PUT21 (39% of putrescine formed). Mutation analysis of 18 the wild type strain over decades of intensive strain improvement. (putative) acetyltransferase genes revealed cg1722 to be responsi- Considering the annual world production and commercial impor- ble for putrescine acetylation. Subsequently, PUT21cg1722 was tance of erythromycin and its semi-synthetic derivatives, current shown not only to produce acteylputrescine, but also 54% more yields remain relatively low. Therefore, there is a clear commercial putrescine than PUT21. incentive to further improve erythromycin production technol- To improve provision of L-glutamate as precursor, the activity ogy. Genes encoding erythromycin biosynthesis are organized in of 2-oxoglutarate dehydrogenase complex (ODHC) was reduced a gene cluster, spanning over 60 kbp of DNA. However, improving by promoter exchange, which resulted in 27% higher putrescine the understanding of regulatory elements involved in erythromy- production than PUT21. Similarly, replacing the translational start cin biosynthesis in S. erythraea remains a challenging task because codon of proB encoding the first enzyme of L-proline biosynthesis no regulatory genes are present inside the erythromycin gene from ATG to TTG resulted in 58% higher putrescine production cluster. The difficulty of identifying key regulatory genes, crucial than the parental strain. for improvement of erythromycin yield, is reflected by the fact A transcriptome analysis revealed increased expression of the that among about 7000 predicted ORFs in S. erythraea genome genes cgmR and cgmA during putrescine production. Overproduc- 15.5% are putative regulatory genes. To address this issue, we tion of the exporter CgmA in PUT21 improved putrescine yield by have carried out a comprehensive comparative omics approach, 48% and productivity by 64%. Transcirional fusions and microar- comparing genome, transcriprome and proteome of erythromy- ray analysis confirmed increased cgmA expression in the absence cin high-producing ABE1441 and WT S. erythraea strains during of the transcriptional repressor CgmR. Binding of CgmR to cgmO the bioprocess closely resembling industrial large-scale production DNA was confirmed and shown to be counteracted by the putative process. Among others, we have identified a new putative regula- inducers putrescine and cadaverine. The current work focusses on tory gene, profoundly overexpressed in the industrial strain, which combining all identified targets to improve putrescine production simultaneously influences sporulation during the life cycle of this in one strain. actinomycete and importantly, significantly affects erythromycin yield of the WT and high-producing strains. Importantly, we have http://dx.doi.org/10.1016/j.nbt.2014.05.2034 shown that “omics” approaches are valuable tools for identification of industrially relevant genes/pathways. http://dx.doi.org/10.1016/j.nbt.2014.05.2033

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Metabolic modeling CD133-expressing liver cancer cells and exo-metabolic profiles of 60 cancer cell lines were integrated with a human generic PN-01 metabolic model, Recon 2, to generate CD133+/--specificliver cancer metabolic models; these two models were employed to simulate Effective Fast Filtration Method for Metabolomics Stud- their metabolic states. In particular, we paid attention to: 1) ies of Mammalian Cells metabolites differently consumed or secreted by each cell, 2) Vinoth Shanmukam 1,∗ , Juan-Antonio Hernandez-Bort 2, metabolic pathways whose overall fluxes appear to be different by Michael Hanscho 2, Christian Leitner 3, Stephan Hann 3, Gunda more than 1.5-fold in the two cancer cells, and 3) potential roles Köllensperger 3, Denise Sonntag 4, Christine Heel 5, Nicole Borth 3 of microRNA on the metabolic states of liver cancer stem cells. Experimental validation of the prediction results from the inte- 1 Austrian Centre of Industrial Biotechnology grative systems metabolic analysis conducted herein will further 2 Austrian Centre of Industrial Biotechnology, Graz, Austria 3 University of Natural Resources and Life Sciences, Vienna, Austria contribute to elucidating metabolic aspects of cancer cell resis- 4 Biocrates Life Sciences AG, Innsbruck, Tirol, Austria tances. 5 Sandoz GmbH, Schaftenau, Austria http://dx.doi.org/10.1016/j.nbt.2014.05.2036

During the last two decades, the advent of metabolomics significantly increased the investigation of cellular processes. Detection, PN-03 quantification and determination of intracellular metabolites highly rely on rapid inhibition of metabolic activity. Thus a quick Design and flux modelling for recombinant production and effective quenching process is essential to corroborate in vivo of 3-Hydroxybutyrate in Escherichia coli conditions. To address this challenge the conventional centrifuga- Mariel Perez-Zabaleta 1,∗ , Johan Jarmander 1, Mónica Guevara 1, tion method is replaced by a modified fast filtration protocol which Jorge Quillaguamán 2, Gen Larsson 1 uses commercially available components. This optimized protocol fulfills the requirement of quenching by avoiding cell leakage and 1 KTH 2 contamination with extracellular metabolites, by preserving cell UMSS membrane integrity and by rapid inhibition of enzymatic activity without metabolite degradation. An added advantage of this Poly (3-hydroxybutyrate) (PHB) is accumulated intracellularly method is that the whole quenching process can be performed in by microorganisms, usually under nutrient deficient conditions less than 15 seconds. and excess of carbon source, is known to possess plastic proper- To understand growth behavior, viability and metabolic activ- ties, biodegradable and biocompatible. 3-hydroxybutyrate (3HB) ity, two batch fermentations were run with protein-free suspension is the monomer of PHB. It is believe that increasing the produc- cultures of CHO-K1 cells grown in medium containing 8 mM glu- tion of 3HB consequently can achieve high concentrations of PHB, tamine, and the same cell line adapted to growth in glutamine free because this monomer can be polymerized outside the cell by dif- medium. Samples were quenched with 13 C labelled internal stan- ferent methods. dards and stored at -80 ◦C. Metabolite extractions were performed H. boliviensis, a native strain of Laguna Colorada, Bolivia, is with 80% cold methanol and samples analyzed by a targeted known for produce large amounts of PHB. The PHB is synthe- approach using LC/MS (HILIC and Atlantis) for the identification sized by the successive action of b-ketoacyl-CoA thiolase (phbA), of metabolites. A comparison of a wide range of metabolites of the acetoacetyl-CoA reductase (phbB) and PHB polymerase (phbC). The two fermentation setups will be presented. genes phbA and phbB of the phbCAB operon from H. boliviensis were introduced into E. coli and the production of 3HB was http://dx.doi.org/10.1016/j.nbt.2014.05.2035 achieved. The phbC gene was not cloned in E. coli because it has unspecified thioesterases that can remove the CoA part of 3HB- CoA and excrete 3HB to the medium. PN-02 Previous studies found that H. boliviensis posses 7 different Identification of metabolic characteristics of liver can- genes involved in the formation acetoacetyl-CoA (phbA) and 3 cer stem cells by integrative systems analysis genes able to produce 3HB-CoA (phbB). We want to determine the effect of the different combinations of these genes on the yield of ∗ Jae Yong Ryu , Hyun Uk Kim, Sang Yup Lee 3HB attained by the recombinant E. coli. Korea Advanced Institute of Science and Technology Wild type E. coli does not have the capacity to synthesize 3HB- CoA but grows fast, at a higher temperature than H. boliviensis and Liver cancer stem cells are known to be responsible for cancer it is easy to clone because is one best understood microorganisms. recurrence, metastasis, and various types of resistances. Espe- Recombinant E. coli metabolism will be modeled in order to obtain cially, understanding mechanisms of their resistance to several a high productivity of 3HB and co-polymers. cancer treatments are critical in combating cancers. To this end, http://dx.doi.org/10.1016/j.nbt.2014.05.2037 integrative systems analysis involving constraint-based modeling and simulation was conducted to better understand metabolic characteristics of liver cancer stem cells and potential cues for their anticancer treatment resistances. Transcriptomic profiles of

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PN-04 the stability of china’s rural economy. Due to the reduction of cultivated area, damages by pests and diseases, food safety issues, the Cotton Breeding Research Progress in China ratio of grain to cotton prices, and other factors, the planting area Wuwei Ye ∗ of cotton in china cannot be increased substantially. How to coor- dinately develop the national cotton production to achieve high Institute of cotton research, CAAS, China cotton quality, and production efficiency is the major and urgent issue for the healthy development of china’s cotton industry. In Cotton production plays a significant role to the economy of response to this issue, the overall situation of China’s cotton pro- china, because china is one of the largest cotton producers world- duction, the cotton molecular assisted breeding progress in China, wide. In addition to these, china is also the largest consumer and the development directions of China’s cotton industry will be country of cotton. Therefore, a healthy and stable development introduced. trend of cotton production is important to promote the efficiency of china’s agriculture, the incomes of Chinese farmers, as well as http://dx.doi.org/10.1016/j.nbt.2014.05.2038

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Metagenomic applications and enriched for several months on lignocellulosic materials, such as wheat straw, poplar and Miscanthus. The most active cul-

PP-01 tures, as evidenced by CO2 production, were selected for further analysis. 16S sequence data showed that significant shifts in the The impact of the compostable packaging material microbial community composition occurred with time. Unexpect- poly(lactic) acid on fungal communities in compost edly, important differences were noticed in submerged cultures Mehlika Karamanlioglu ∗ , Geoff Robson compared to solid medium setups. Bacteriodetes and Actinobacteria prevailed in the solid medium, whereas Firmicutes and Proteobacte- The University of Manchester ria were the dominating phyla in the submerged culture. In parallel, metagenomic DNA was extracted from two of The compostable biopolymer, polylactic acid (PLA), is mostly the enrichments and sequenced. Analysis of Carbohydrate active derived from a renewable source, starch, and increasingly being enzymes (CAZymes) revealed that members of glycoside hydro- used as an alternative to conventional plastics for short shelf- lase (GH) families GH6, GH9 and GH48 (cellulases) as well as life products, disposable bags and packaging materials as it families GH27, GH29 and GH36 (xyloglucan side chain digestion) decomposes at elevated temperatures during composting. Abiotic had accumulated after enrichment. Families of CAZymes for hemi- hydrolysis in the presence of water leads to a progressive decrease celluloses and pectin breakdown, especially GH28, GH30, GH53, in the polymer molecular weight and ultimately the release of GH106 and polysaccharide lyase (PL) families PL1, PL4 and PL22 lactic acid. Despite the increase in the amount of PLA entering were also enriched. Thus, the difference in taxonomical composi- composting systems, few studies have examined the potential tion between submerged and solid medium was also mirrored in impact of PLA degradation on the compost microbial community. the CAZyme family composition. Thermophilic fungi play an import role in the composting process and in this study, the impact of PLA hydrolysis on the compost http://dx.doi.org/10.1016/j.nbt.2014.05.2040 fungal community was examined by incubating PLA films and PLA granules in compost at different concentrations, 0-50% (w/w), by terminal restriction fragment length polymorphism (TRFLP) PP-03 and 454-pyrosequencing. When PLA was incubated at 50 ◦C, a Identification and functional characterization of Est16, discernible PLA disintegration occurred and TRFLP revealed that an esterase isolated from a metagenomic library of the fungal community profile initially changed but shifted back microbe consortium specialized in diesel oil degradation toward the initial compost community profile over time when ,∗ PLA concentration was less than 50% (w/w) in compost. However, Mariana Rangel Pereira 1 , Gustavo Fernando Mercaldi 1, Thaís Car- when PLA was at a concentration of 50% (w/w), fungal commu- valho Maester 2, Andrea Balan Fernandes 3, Eliana G. de Macedo nity profile did not shift back toward to the initial profile and Lemos 2 the fungal diversity decreased due to a marked acidification of the 1 Laboratório Nacional de Biociências compost. 454-pyrosequencing revealed that the presence of PLA 2 UNESP enriched the thermophilic fungus, Thermomyces sp. in the compost 3 USP population over time. Lipolytic enzymes have been attracting global market atten- http://dx.doi.org/10.1016/j.nbt.2014.05.2039 tion because they show enormous biotechnological potential such as production of detergents, processing of leather, production of cosmetics, perfumes and biodiesel. To search for novel PP-02 lipolytic enzymes, a DNA metagenomic library was constructed Exploring biomass degrading communities for lignocel- from a microbe consortium isolated from oil contaminated soil lulolytic activities at Ribeirão Preto, Brazil. After functional screenings using try- butirin, one clone referred to as Est16, was used for further Senta Blanquet 1 Agnès Hébert 1 Franç oise Fayolle-Guichard 1 , , , analysis. The sequence analysis revealed that Est16 shares 87% Pedro Coutinho 2 Bernard Henrissat 2 , with lipases/esterases (ADM63076.1) from uncultured bacterium 1 IFP Energies nouvelles in the database. After an amino acid sequence alignment of the 2 AFMB CNRS/Université Aix-Marseille UMR6098 Est16 with 34 sequences of members of the eight lipolytic families demonstrated that Est16 is a new member of family V. The catalytic Present schemes for bioethanol production from lignocellu- triad (Ser, Asp and His), is highly conserved and the serine is located losic biomass, and especially the enzymatic hydrolysis step, are in the conserved motif GXSMGG. The est16 gene was cloned into currently still too costly. An interesting alternative is the “con- the pET28a vector and expressed as a N-terminal fused His6tag solidated bioprocessing” production scheme (CBP), which uses a protein in Escherichia coli BL21(DE3) cells. The recombinant pro- single organism to catalyse both biomass hydrolysis and fermen- tein was purified as active soluble form and used for activity assays. tation of the liberated sugars, and which therefore represents an Est16 showed wide range of substrates and highest catalytic effi- important potential for cost reduction. The aim of the present ciency against p-nitrophenyl valerate (C5), optimum activities at project was therefore to identify new microorganisms and enzymes mesophilic temperature ranges and the optimum pH of 9.0. In par- employable in such a process. Compost samples were collected ticular, Est16 showed an increase in reaction rate using up to 5%

www.elsevier.com/locate/nbt S169 METAGENOMIC APPLICATIONS New Biotechnology · Volume 31S · July 2014 of DMSO, suggesting tolerance to the presence of organic solvents. PP-05 Here, we demonstrated that metagenomic approach can be used as a DNA source to expand the lipolytic enzymes diversity and Discovery of thermostable hydrolytic enzymes of indus- our results indicate that Est16 has potential for use in industrial trial interest by metagenomic screening process. Dimitra Zarafeta 1,∗ , Georgios Skretas 2, Fragiskos N Kolisis 3 http://dx.doi.org/10.1016/j.nbt.2014.05.2041 1 National Technical University of Athens 2 Institute of Biology, Medicinal Chemistry & Biotechnology, National Hellenic Research Foundation, Athens, Greece 3 PP-04 Laboratory of Biotechnology, School of Chemical Engineering, National Tech- nical University of Athens, Athens, Greece ANASTASIA a versatile web platform for metagenomic analysis Enzymes are biocatalysts used in a wide range of industrial applications and provide a “green” alternative to chemical con- Efthymios Ladoukakis 1 Eleftherios Pilalis 2 Aristotelis , , versions. Hydrolases are a class of enzymes that exhibit high Chatziioannou 2 Fragiskos Kolisis 1 , selectivity and potential synthetic ability when used in non- 1 National Technical University of Athens conventional media. These characteristics render this subcategory 2 National Hellenic Research Foundation of enzymes very appealing to the industry, especially for the production of fine chemicals and pharmaceuticals. Despite their In this work, ANASTASIA (Automated Nucleotide Aminoacid advantages, a very limited amount of hydrolases is currently Sequences Translational plAtform for Systemic Interpretation and being used in biotechnological applications, as many industrially Analysis) web repository is presented. ANASTASIA enables auto- relevant processes require high temperatures where conven- mated massive metagenomic sequence assembly, analysis and tional biocatalysts perform poorly. For such processes, thermo- interpretation tasks under the same workflow. It does so by provid- or hyperthermostable enzymes are required. Thermophilic orga- ing a rich suite of computational tools integrated within numerous nisms remain until today a largely unexplored source of such algorithmic pipelines implementing and integrating versatile data enzymes since the vast majority of those organisms (>99%) can- processing tasks for (meta)genomic sequencing, assembly and pro- not be cultured using standard laboratory techniques. To address tein sequence datasets. The modules of these pipelines incorporate some of these issues, the international consortium HotZyme established bioinformatic algorithms like HMMER and BLAST as was formed with the aim of applying systematic metagenomic well as Python and Perl scripts, which perform annotation and screening approaches in order to identify novel hydrolases from classification tasks in synergy with stand-alone programs, while hot environments. Within the framework of this project, metage- being integrated in an all-in-one inclusive solution exploiting the nomic libraries were constructed from samples collected from Galaxy workflow engine. ANASTASIA supports the labor-free de- diverse high-temperature ecosystems around the world (Russia, novo creation and operation of workflows able to handle the Iceland, Italy, China, New Zealand, Japan and U.S.A). These analytical challenge of large datasets (e.g. from metagenomic libraries were then analyzed using bioinformatic tools and high- experiments) and store the generated annotation results auto- throughput functional screens in Escherichia coli cells to identify matically into a MySQL database running in the background. In open reading frames with desired hydrolytic activities and stabil- addition it promotes workflows and large datasets sharing through ity at elevated temperatures. These approaches have led to the its transparent shell which a user-friendly graphical interface. The discovery of novel hydrolases of biotechnological interest. The current configuration of the ANASTASIA installation comprises a characterization of these novel enzymes will be presented and cluster of 36 CPU cores, 256 GB RAM and a fast storage machine their potential use for the synthesis of fine chemicals will be with 32 TB capacity provided by the University of Copenhagen. discussed. ANASTASIA represents the bioinformatics core of the FP7 project http://dx.doi.org/10.1016/j.nbt.2014.05.2043 HotZyme which targets to the exhaustive analysis of metagenomes in thermal springs, with the scope of tracing proteins with interesting enzymatic properties which will be indispensable in a wide PP-06 range of biotechnological applications; from paper pulp bleaching to production of biofuel from biomass. Functional metagenomic and proteomic characteriza- http://dx.doi.org/10.1016/j.nbt.2014.05.2042 tion of soil microbial community associated with decomposing reeds

Gaetano Perrotta ∗ , Linda Bianco, Fabrizio Carbone, Loretta Daddiego, Paolo Facella, Loredana Lopez ENEA

Recent demands for the production of biofuels from lignocellulose biomass led to an increased interest in soil microbial communities. Ligninolytic microbes have developed a unique strategy to handle lignin degradation based on

S170 www.elsevier.com/locate/nbt New Biotechnology · Volume 31S · July 2014 METAGENOMIC APPLICATIONS a pletora of synergistically acting enzymes. Besides, specific In parallel, decomposing leaves and stems of Arundo donax biomasses can be characteristically affected by these networks and Phragmites australis were collected and used for the isolation of collaborative enzymes, deriving from typical soil microbial of cultivatable fungi. Molecular characterization were performed community. on the isolated fungi, leading to the identification of 7 fungal In order to unravel the microbial environment of plant lit- species. Among them, we isolated a white-rot fungus, belonging ter, we collected soil samples from sites characterized by the to the Polyporales order, showing outstanding metabolic activi- presence of different decomposing plants, Arundo donax and ties in ligno-cellulose degradation. A proteomic characterization Phragmites australis, DNA extracted from soil was used for of this fungal secretome is currently under investigation. meta-genomic analyses using Roche 454 platform. A total of The integration of the data coming from these analyses is 617,604 high quality reads have been collected, corresponding expected to point out a number of microorganisms, genes and to more than 2000 microorganisms. Among them, a bacterial proteins likely involved into lignocellulose degradation pathways consortium potentially involved into biomass deconstruction that could be used to improve biomass deconstruction in industrial was identified. A large number of bacteria belonging to this applications. consortium are enriched in genes coding for ligno-cellulolitic http://dx.doi.org/10.1016/j.nbt.2014.05.2044 enzymes.

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Nanobiotechnology PQ-02

PQ-01 A pH-responsive high density lipoprotein-like nanoparticle of epothilone B The use of chimeric virus-like particles with inserted tar- ,∗ get peptides for tailored antibody production Woo-Jong Lee 1 , Ji-Chun Lee 2, Byoung-Jae Kong 2, Jonghyeok Shin 2, Sung-Gun Kim 3, Heekyung An 1, Chi-Heung Cho 1, Dae- Aurelija Zvirbliene ∗, Indre Kucinskaite-Kodze, Alma Gedvilaite Hyuk Kweon 2 Vilnius University, Lithuania 1 Korea Institute of Industrial Technology, South Korea Protein engineering provides an opportunity to generate 2 Sungkyunkwan University, South Korea new immunogens with desired features. Viral structural proteins 3 Youngdong University, South Korea with their intrinsic capacity to self-assemble to highly-organized Epothilone B (EpoB) is a paclitaxel (PTX)-like microtubule- virus-like particles (VLPs) have been shown to possess high stabilizing agent; it induces mitotic arrest and apoptosis in cells. immunogenicity and were exploited as potential vaccines. We EpoB is a promising anti-tumor agent, and is thought to have have demonstrated that major capsid protein VP1 derivatives of the potential to overcome well-known PTX resistance. However, hamster polyomavirus (HaPyV) harboring foreign sequences at cer- EpoB is barely soluble in water, and is fatal to normal cells due tain surface-exposed regions allowed the formation of chimeric to extremely potent cytotoxicity. To reduce the unwanted cyto- VLPs. The chimeric VLPs meet the requirements for a strong toxicity of EpoB to normal cells, a reconstituted high-density immunogen being able to activate both B cells recognizing the lipoprotein (rHDL) of EpoB (EpoB-rHDL) was generated using surface-located epitopes and T-helper cells providing the necessary apolipoprotein A-I (apoA-I). The EpoB-rHDL, as well as PTX-rHDL signals for Ig class switching and affinity maturation. Moreover, (a HDL-like nanoparticle of paclitaxel), was indeed mild (non- the immunogenicity of inserted peptides is enhanced due to toxic) to certain cell lines such as MCF7, MDA-MB-231, and the repetitive multimeric structure of chimeric VLPs. We have SK-OV-3, while free EpoB and PTX were very toxic to these same exploited the VLP approach for generation of monoclonal antibod- cells. In contrast, the EpoB-rHDL and PTX-rHDL were very effec- ies against short non-immunogenic peptides of human proteins tive in killing the Caco-2 and ZR-75-1 while free drugs were less as well as difficult-to-express antigens such as viral glycoproteins. toxic to these cells. The susceptibility of cell lines to rHDLs was The chimeric HaPyV-VP1 VLPs have been shown to induce in mice dependent on the expression of scavenger receptor class B type strong insert-specific B- and T-cell responses. The generated anti- I (SR-BI), indicating that EpoB-rHDL selectively and efficiently bodies were reactive with native full-length target proteins thus kills only SR-BI-overexpressing cells. Furthermore, the EpoB-rHDL demonstrating the surface localization and proper folding of the released EpoB only at acidic pH, which may facilitate the escape of inserted sequences. drugs from acidic endosome. Thus, EpoB-rHDL shown in this study enables safe and targeted delivery of the potent EpoB to cancer cells http://dx.doi.org/10.1016/j.nbt.2014.05.886 in SR-BI-dependent manner.

http://dx.doi.org/10.1016/j.nbt.2014.05.887

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PQ-03 PQ-05

A nanovesicle-based olfactory biosensor and its applica- Biosynthesis of single nanoparticles using various metal tion to disease diagnosis and grain quality assessment binding proteins

Tai Hyun Park ∗, Jong Hyun Lim, Jung Ho Ahn Yoojin Choi ∗, Sang Yup Lee, Doh Chang Lee

Seoul National University, South Korea Korea Advanced Institute of Science and Technology, South Korea

We integrated the olfactory system to carbon nanotube plat- Recently nanotechnology has attracted attention worldwide forms for biosensing applications. Human olfactory receptor because of the interesting physicochemical properties of these par- (OR)-containing nanovesicles were produced from human embry- ticles. However, most nanoparticles are chemically synthesized onic kidney (HEK)-293 cells. The nanovesicles, which generate and involve the use of expensive catalysts for reactions at high olfactory signals through a cAMP pathway, were integrated into temperature and pressure. The environmental issues related to single-walled carbon nanotubes field-effect transistors (SWNT- the synthesis of nanoparticles have motivated research toward FETs). The nanovesicles and SWNT-FETs play roles in perceiving greener methods that utilize microorganisms such as bacteria, specific odorants, and in amplifying cellular signals, respectively. yeast, and fungi for their ability to reduce metal ions. We syn- In particular, this system can be used for the diagnosis of disease thesized various single nanoparticles using metal binding proteins such as lung cancer and also for the real-time monitoring of fungal on recombinant Escherichia coli (E. coli). The morphology and contamination in grain. Specific olfactory receptors recognizing size of the synthesized nanoparticles was observed by low to the chemical biomarkers were first selected through screening a high resolution transmission electron microscopy (TEM) at 200 kV library of human olfactory receptors. The nanovesicle-integrated and energy-dispersive X-ray (EDX) spectra. Finally, we suggest a device was able to detect a lung cancer biomarker (heptanal) and a possible mechanism of the biosynthesis process that might pro- specific compound generated from contaminated grain (1-octen-3- vide a guide for conditions required for the synthesis of various ol) with excellent sensitivity and selectivity, similar to the original nanoparticles. olfactory system. http://dx.doi.org/10.1016/j.nbt.2014.05.890 http://dx.doi.org/10.1016/j.nbt.2014.05.888

PQ-06 PQ-04 Electro-triggered, spatioselective, quantitative gene Biological synthesis of silver nanoparticles using plant delivery into a single cell nucleus by Au nanowire leaf extracts and their speciﬁc antimicrobial activity nanoinjector

Beom Soo Kim ∗, Bipinchandra Salunke, Shailesh Sawant, Bassam Seung Min Yoo ∗, Sang Yup Lee Alkotaini KAIST, South Korea Chungbuk National University, South Korea Delivery of bioactive materials into a cell is highly impor- Several plant leaf extracts (Kalopanax, Magnolia, Persimmon, tant in the study of cell biology and medical treatments. Pine, Ginkgo, Platanus, etc.) were used for extracellular syn- Ideal nanoinjectors should be able to deliver biomaterials with thesis of silver nanoparticles. Stable silver nanoparticles were high spatial resolution while causing minimum cell damage. formed by treating aqueous solution of AgNO3 with the plant leaf We developed a Au nanowire (NW) nanoinjector that has the extracts as reducing agent. The synthesized silver nanoparticles thinnest diameter among the DNA delivering devices as well were characterized by UV-vis spectroscopy, FT-IR, inductively cou- as optimum mechanical properties, minimizing cell damage. pled plasma spectrometry, energy dispersive X-ray spectroscopy, Well-defined single-crystalline Au surface and high electric con- X-ray photoelectron spectroscopy, high-resolution transmission ductivity of a Au NW nanoinjector allow precisely timed and electron microscopy, etc. Antimicrobial susceptibility tests of sil- efficient electrochemical release of DNA molecules attached ver nanoparticle treatments revealed variability in sensitivity of on a Au NW surface. Both linear DNA and plasmid DNA Bacillus cereus and Saccharophagus degradans. Minimum inhibitory were delivered separately, and showed successful expression. concentration (MIC) values of the silver nanoparticles for B. cereus The Au NW nanoinjector would find important biomedical and S. degradans were found to be 30 ␮g/mL and 10 ␮g/mL, respec- applications in the fields such as gene therapy, DNA vaccina- tively. The mixed culture of B. cereus and S. degradans treated with tion, targeted drug delivery, and probe/control of cell signaling silver nanoparticles at 10 ␮g/mL after 24 h showed presence of only events [1]. B. cereus colonies. This study suggests that plant leaf extract syn- Acknowledgements: This work was supported by the thesized silver nanoparticles can selectively inhibit growth of the Technology Development Program to Solve Climate Changes Gram negative S. degradans and retain the Gram positive B. cereus on Systems Metabolic Engineering for Biorefineries (NRF-2012- at MIC values of S. degradans. C1AAA001-2012M1A2A2026556) of the Ministry of Education, Science and Technology (MEST) through the National Research http://dx.doi.org/10.1016/j.nbt.2014.05.889 Foundation of Korea.

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Reference proteoliposome was increased to withstand up to ∼9 atm and water [1].Yoo SM, Kang M, Kang T, Kim DM, Lee SY, Kim B. Nano Lett puriﬁcation was demonstrated using our device. 2013;13(6):2431–5. http://dx.doi.org/10.1016/j.nbt.2014.05.893 http://dx.doi.org/10.1016/j.nbt.2014.05.891 PQ-09 PQ-07 A comparative study of the effectiveness of ␤-glucosidase Nano-pattern integrated biomimetic system for wound immobilized on CNT-nanoparticles and Ca-alginate healing assay beads ∗ Sun Min Kim ∗, Insu Lee, Galahm Park, Tae-Joon Jeon Ahmad Jameel , Labiba Mahmud, Faridah Yusof

Inha University, South Korea International Islamic University Malaysia, Malaysia

Wound healing process of damaged skin involves various steps Enzymes are extensively used in various industrial, biomedical of cellular behavior and complex combinations of signaling path- and biopharmaceutical applications. However, enzymes in their way. In this research, we simply fabricated nano-patterned surfaces free form are unstable and expensive besides being characteris- with biocompatible PDMS (Polydimethylsiloxane) polymer and tically susceptible to inhibition by high product concentrations integrated a patterned surface with a microfluidic system which and are highly sensitive to pH and temperature changes. Immo- can mimic wound healing rad processes. To form wound damage bilization technology offers solutions to these challenges besides to 3T3 fibroblast cell layer cultured on the surface, we gener- enhancing operational stability, longevity and ease of separa- ated layered flows of cell culture media and trypsin/EDTA in a tion. ␤-Glucosidase has been widely employed as model enzyme microchannel. We monitored cell migration on the pattern dur- for enzymatic studies. Ca-alginate beads provide a gentle envi- ing wound healing process and found that the patterned surface ronment for immobilization, but have certain limitations such guided the migration of cells as well as the intercellular cytoskele- as low stability, high porosity and limitations in biocompat- ton structure. The results demonstrate that cellular behavior can ibility. Carbon nanotubes (CNTs) on the other hand have be controlled for wound healing by mechanical stimuli. We expect excellent mechanical, thermal and electrical properties, as well that the developed 2D skin model can be integrated with various as dimensional and chemical compatibility with biomolecules types of surface and used as a standard assay platform for wound like DNA and enzymes, suitable for biosensor design. Here, healing research. ␤-glucosidase was immobilized in Ca-alginate gel and multi- walled carbon nanotubes (MWCNT) using standard techniques http://dx.doi.org/10.1016/j.nbt.2014.05.892 and their activity was compared with that of free enzyme. The activity was found highest (12.53 U/mL) for the free enzyme and lowest (9.768 U/mL) for the immobilized Ca-alginate. The PQ-08 activity of immobilized MWCNT (12.20 U/mL) was close to the Water purification through cross-linked proteolipo- free enzyme activity. The enzyme reaction was found to fol- somes using a preconcentrator low Michaelis–Menten kinetics. The Michaelis constants, Km and Vmax, determined using Langmuir linearized plots are, respectively, 1,∗ 2 2 2 Tae-Joon Jeon , Hyunil Ryu , Huisoo Jang , Insu Lee ,Ga 0.09048 ␮mol/mL and 0.00989 ␮mol/mL min for immobilized 2 2 Lahm Park , Sun Min Kim Ca-alginate; and 0.0985 ␮mol/mL and 0.01237 ␮mol/mL min for 1 Inha University/Biological Engineering, South Korea immobilized MWCNT. The corresponding values for the free ␮ ␮ 2 Inha University, South Korea enzyme are 0.0854 mol/mL and 0.01263 mol/mL min. Thus, the MWCNT appears to be a promising support material for enzyme Aquaporin is the most efficient filter in nature due to its high immobilization. water selectivity and permeability. However, the manufactural difficulties of efficient and large scaled aquaporin embedded mem- http://dx.doi.org/10.1016/j.nbt.2014.05.894 brane preclude its industrial applications. It is mainly attributed to the fragility of a lipid bilayer or other biomimetic membrane. We have created robust membranes by cross-linking liposomes with linkers. In addition, we concentrated liposomes effectively using a microfluidic preconcentrator which has nanochannels formed by the electrical breakdown of a polydimethylsiloxane (PDMS) membrane at a high electrical bias with no nano-lithography. Proteoliposomes were continuously concentrated at the target position by applying an electric field through the junction of micro- and nanochannels. Amine-terminated proteoliposomes embedded with aquaporin were conjugated to the surface of the PDMS device via EDC/NHS reaction. As a result, the durability of

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PQ-10 PQ-11

Impacts of carbon nanotubes on biochemical reactions: Iron-based nano-particles for Lipolase immobilization insight into interaction between carbon nanotubes and and stabilization DNA polymerase enzyme Surabhi Mehra ∗, Shamsher S. Kanwar Ebru Uysal 1,∗, Meral Yuce 2, Hasan Kurt 1 Himachal Pradesh University, Shimla, India 1 Faculty of Engineering and Natural Sciences, Sabanci University, In our study, a commercial Lipolase 100L (Novozymes, Banga- Istanbul, Turkey lore, India) was covalently immobilized on silane coated modified 2 Nanotechnology Research and Application Center, Sabanci Uni- magnetic nano-particles. These magnetic (Fe O or ␥-Fe O ) NPs versity, Istanbul, Turkey 3 4 2 3 were in the size range of 25–30 nm and their surface modifica- Recently, the Polymerase Chain Reaction technique has begun tion was carried out by coating with Tetra Ethoxy Silane (TEOS) to benefit from nanotechnology. In this paper, effects of carbon by sol–gel reaction and these silane-coated magnetic NPs were nanotubes in the Polymerase Chain Reaction were investigated then used for immobilization. Thereafter, Lipolase immobilized by Electrophoresis, Circular Dichroism Spectrometry and Dynamic nano-particles [30 mg] were separated by magnetic decantation Light Scattering Techniques. and suspended in 6 ml of phosphate buffered saline (pH 7.4). The unique ability to amplify low copy number DNA within These Lipolase-bound NPs were kept in a homogeneous form minutes has made in vitro Polymerase Chain Reaction (PCR) one [20 ␮l] and were assessed [∼2 mg = 118.29 U of enzyme in ml] of the most essential techniques in modern biology. In order to for enzymatic activity, stability and reusability using 3 ml reac- harness this technique to its full potential, certain obstacles, such tion system containing p-nitrophenyl palmitate as a substrate as nonspecific by-products, low yield, and complexity of GC rich in 0.05 M Tris buffer pH 8.2. NPs-immobilized Lipolase showed and long genomic DNA amplification need to be surmounted. enhanced activity (59.2 U/mg NPs) and stability. Effect of C- Nanomaterial-assisted PCR, so-called nanoPCR, is a new area in chain length/substrate specificity of NPs-bound Lipolase was also biotechnology that introduces nanostructured materials into PCR assessed and found maximum towards p-nitrophenyl palmitate reaction to obtain improved results. [139.1 U/ml] followed by p-nitrophenyl myristate [87.8 U/ml], Nanomaterials have unique physical and chemical properties, p-nitrophenyl caprylate [77.7 U/ml] and p-nitrophenyl laureate such as high thermal conductivity, stability and high surface to [75.3 U/ml]. The temperature, pH and reusability measurements volume ratios. The effects of nanomaterials in PCR depend on showed that Lipolase immobilized on NPs were capable of work- their size, shape, concentration, heat conductivity, electron trans- ing at broader pH and higher temperature ranges and was reusable fer properties and surface modifications. up to several cycles. Carbon nanotubes are predicted to bind major PCR components, such as primers, template and polymerase enzyme, via http://dx.doi.org/10.1016/j.nbt.2014.05.896 specific or non-specific interactions. In this paper, we demonstrate the interaction of carbon nanotubes with wild type DNA polymerase enzyme, and the effect of this interaction in PCR for the first time. According to the results, chiral properties of the wild type DNA polymerase enzyme has changed after incubation with amine functionalized multiwall carbon nanotubes, which confirms direct interaction between the enzyme and tubes. Furthermore, this interaction has been found to be temperature dependent via dynamic light scattering spectroscopy. http://dx.doi.org/10.1016/j.nbt.2014.05.895

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Natural and synthetic polymers 0.75 ng/mL of analogues were detected. The product extracted and isolated in its cyanide form had the similar UV spectrum as stan- PR-01 dard cyanocobalamin and as cobalamin produced by Lactobacillus A mathematical model for polyhydroxybutyrate pro- reuteri DSM 20016. No cobalamin was detected in the fermenta- duction by a wild type Bacillus megaterium using raw tion broth containing 1% acetate, and less cobalamin was obtained glycerol from biodiesel industry as sole carbon source when acetate started to be consumed.

1,∗ 2 Paalo Andrea Moreno Yanez˜ , Débora Jung Luvizetto Faccin , http://dx.doi.org/10.1016/j.nbt.2014.05.898 Nilo Sérgio Medeiros Cardozo 2, Humberto Escalante 1, Marianny Y. Combariza 1, Carolina Guzmán 1 PR-03 1 Universidad Industrial de Santander, Colombia 2 Federal University of Rio Grande do Sul, Brazil Ultrasonic-assisted production of active polysaccharides from Crassostrea hongkongensis Polyhydroxybutyrate (PHB) is a biodegradable, biocompat- ,∗ ible and thermoplastic biopolymer, synthesized naturally as Bingna Cai 1 , Jianyu Pan 2, Huili Sun 3 cytoplasmic inclusions by various genera of Gram-positive and 1 No. 164, Xingangxi Rd, Haizhu District, P.O. Box 510301, China Gram-negative bacteria. PHB shares similar properties with 2 South China Sea Institute of Oceanology, Chinese Academy of polypropylene, and could potentially replace it, but its industrial Sciences, Guangzhou, China production is limited by its high cost and low productivity. We 3 Chinese Academy of Sciences, Guangzhou, China have previously reported [1] optimized growth conditions for a wild type Bacillus megaterium with the ability to produce PHB from The beneficial effects of oyster extract against various disor- raw glycerol byproduct of a Colombian biodiesel industry. Aiming ders and diseases induced by oxidative stress have aroused great for a better understanding of the PHB biosynthetic process with concern. Oyster polysaccharides, as immune nutrients, were con- the referred carbon source and wild type Bacillus megaterium,in ducted to provide nutrients for cell metabolism and prevent the the present work we present a mathematical model that allows us side effects such as immunotoxicity and gastrointestinal toxic- to describe the kinetics of microbial growth, substrate consump- ity caused by chemoradiotherapy. Ultrasonic-assisted enzymolysis tion, product formation and also to simulate different cultivation was studied to increase the active polysaccharide yield and purity strategies. In this model, microbial growth and product formation from Crassostrea hongkongensis, showing that it is more efficient were described by the Monod and the Luedeking–Piret equations, than ultrasonic extraction or enzymatic hydrolysis alone. On respectively. Model implementation and parameter estimation the basis of Box-Behnken design and ridge analysis, the opti- were carried out in the EMSO process simulator. Key model param- mum conditions were obtained as ultrasonic treatment time of ◦ eters were estimated from experimental data obtained in batch 24 min, power of 876 W, temperature of 49 C and material-solvent cultivation using a submerged bioreactor. Experimentally, under ratio of 1:6 (w/v). Furthermore, polysaccharide fraction (CHP), optimized bioreactor conditions, a maximum PHB concentration which was obtained by ultrasonic pretreatment and then alcalase ◦ of 0.96 g/L was reached at 12 h. The model provided excellent hydrolysis at the conditions: 3000 U/g, 55 C, pH 8.0 for 4 h, exhib- fitting of the experimental data previously obtained, providing ited obvious scavenging effect on DPPH and hydroxyl radical correlation coefficients around 0.9. (98.48 ± 0.55% and 99.20 ± 0.12%, respectively) and linoleic acid peroxidation inhibition effect (85.48 ± 0.65%) at concentration Reference of 5.0 mg/mL. Then the CHP was separated into three fractions [1].Moreno P, Yanez˜ C, Tarazona N, Cardozo NSM, Escalante H, Com- by graded ethanol precipitation. The 30–60% ethanol precipita- bariza MY, Guzmán C. Statistical optimization of PHB production tion fraction (C30–60%) from CHP showed the highest activities, by a wild type Bacillus megaterium using raw glycerol as sole carbon including promoting RAW 264.7 murine macrophage, tlympho- source. Int J Biol Macromol 2014 (submitted for publication). cyte and IEC-6 cells proliferation (the highest cell proliferation rate was 137.10% at 0.0391 mg/mL, 160.48% at 0.0781 mg/mL http://dx.doi.org/10.1016/j.nbt.2014.05.897 and 153.70% at 0.0195 mg/mL, respectively). The activities of CHP might attribute to its uronic acid, sulfate composition and molec- PR-02 ular weight. These results reveal the potential application of CHP in nutraceutical for cancer patients. Acetobacter pasteurianus DSM 3509 produces cobalamin

∗ http://dx.doi.org/10.1016/j.nbt.2014.05.899 Clemens Bernhardt , Xuan Zhu, Bernward Bisping

Uni Hamburg, Germany

A strain of acetic acid bacteria used in food applications was found to have the ability to synthesize cobalamin. A preliminary genetic study of the gene of uroporphyrinogen-III synthase and a survival test indicated the ability to synthesize cobalamin. By a modiﬁed microbiological assay based on Lactobacillus delbrueckii spp. lactis DSM 20355, 4.57 ng/mL of real cobalamin and

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PR-04 evaluated in its two potential active forms (LPhaR/sPhaR), which differ for the presence of an extra 16 aminoacids long N-terminal New clues to design cell factories for tailor-made extension. This N-terminal tail seems to play a crucial role in deter- biopolymer production: Bacillus cereus as a source of mining PhaR function, promoting PhaR activity as transcriptional polyhydroxyalkanoates biosynthetic proteins regulator in its longer form (LPhaR), and switching its role to PHA Marco Vastano ∗, Angela Casillo, Maria Michela Cosaro, Giovanni synthase (PhaC) activator/stabilizer in its shorter form (sPhaR). Sannia, Cinzia Pezzella PhaB role was found to be crucial in determining biopolymer composition, inﬂuencing monomer chain length depending on University of Naples Federico II, Italy its substrate speciﬁcity. Polyhydroxyalkanoates (PHAs) are biopolymers, accumulated Functional information obtained from this study represents the as intracellular storage-granules by a variety of bacteria. The mate- groundwork to design new cell factories for low-cost production rial properties and the potential biotechnological use of PHAs of tailor-made PHAs. are strictly dependent on their monomeric composition. In PHAs Reference producing Bacilli, the presence of a peculiar biosynthetic protein [1].Rehm B. Polyester synthases: natural catalysts for plastics. Biochem J cluster, encoded by the operon phaRBC has been revealed [1]. 2003;376:15–33. In this work a functional study of the phaRBC operon from B. cereus has been carried out through its heterologous expression http://dx.doi.org/10.1016/j.nbt.2014.05.900 in Escherichia coli. Deletions mutants in phaR, phaB and phaC have been assayed for PHA accumulation in culture media boosted with a related (fatty acids) carbon source. PhaR function has been

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Antimicrobials, pathogens and disease sonal communication) found 4 proteins that have antifungal effect namely: glucan endo-1,3-beta-glucosidase, chitinase, 2S albumin PS-01 storage protein, nonspeciﬁc lipid-transfer protein. During the fer- Genomics-based discovery of macrolide glycosyltrans- mentation process the seed coat proteins seem to be degraded, ferase from Bacillus sp. probably caused by an increase in temperature and by activity of

∗ proteases produced by microbes during the fermentation. To find Won-Gon Kim , Ji Yeong Park, Hyun Ju Kim out more details about the degradation of proteins during the fer- Korea Research Institute of Bioscience and Biotechnology, South mentation, we are currently analyzing 28 protein bands, which we Korea extracted during the fermentation. Temperature characterization showed that the seed coat protein is not stable at 40 and 50 ◦C for Glycosylation of pharmacologically active secondary metabo- 24 h. lites is an attractive method to improve their biological activity as well as pharmacokinetics. The availability of suitable glycosyl- http://dx.doi.org/10.1016/j.nbt.2014.05.902 transferases for naturally unglycosylated compounds, however, is limited because of substrate specificity. Recently, Bacilli have been used as a source for the isolation of GT enzymes involved in the PS-03 modification of aromatic or bulky substrates. In an effort to dis- Inactivation of microbial biofilms by visible light with cover enzymes for the glycosylation of a macrolide compound, we a porphyrinic photosensitizer identified putative glycosyltransferases by the genomic analysis of Bacillus sp. that produces various glycosylated macrolides. The Sandra Beirão, Sara Fernandes, Joel Coelho, Adelaide Almeida, selected glycosyltransferases were screened, leading to the iden- Maria da Graça Neves, Maria do Amparo Faustino, João Tomé, ∗ tification of a glycosyltransferase that could catalyze the transfer Angela Cunha of a glucose residue from UDP-␣-D-glucose to the macrolide com- University of Aveiro, Portugal pound. Using NMR and MS analysis, we determined the chemical structures of the new products to be a new glucosylated macrolide Biofilms are aggregates of microbial cells imbedded in a which exhibited improved water solubility. These data showed matrix composed essentially by water and extracellular poly- that genomics-based discovery of glycosyltransferase from Bacillus meric substances (EPS). The matrix provides a first line of defense sp. was an effective strategy for the isolation of a suitable glycosyl- against biological attack, environmental stress, and biocide diffu- transferase for naturally unglycosylated compounds. sion, making biofilms a challenge to conventional antimicrobial approaches. http://dx.doi.org/10.1016/j.nbt.2014.05.901 The photodynamic inactivation (PDI) of microorganisms relies on the interaction of a non-toxic photosensitizer, molecular oxygen and light. This study aimed the assessment PS-02 of the photodynamic effect on the matrix of model Pseu- Antifungal protein of seed coat extracts of Theobroma domonas aeruginosa biofilms, using the tetra-cationic porphyrin cacao L. during fermentation derivative tetra-iodide 5,10,15,20-tetrakis(1-methylpyridinium-4- yl)porphyrin (Tetra-Py+-Me) as photosensitizer (PS) and white light 1,∗ 1 2 Fahrurrozi Fahrurrozi , Claudia Bahmann , Nicolas Niemenak , (380–700 nm) at an irradiance of 40 W m−2. The photodynamic 1 1 Reinhard Lieberei , Bernward Bisping inactivation of imbedded cells, in single-species or mixed biofilms 1 University of Hamburg, Germany of Staphylococcus aureus, Pseudomonas aeruginosa and Candida albi- 2 University of Yaounde I, Cameroon cans was also determined. A reduction of 81% in the polysaccharides content of matrix of Seed coat is an important tissue for the regulation of imbibition P.aeruginosa biofilms was observed after treatment with a light dose and maintenance of the integrity of seed, and it is also the first seed of 64.8 J cm−2 and 20 ␮M of Tetra-Py+-Me. The photosensitization barrier encountered by pests and pathogens. Seed cotyledons con- with Tetra-Py+-Me also caused inactivation of cells in all tested tain an array of proteins that may be involved in the protection biofilms. The maximum reduction factors were 3, 7 and 6 logs, for of quiescent seeds against fungi. In a previous study (Fahrurrozi biofilms of P. aeruginosa, S. aureus and C. albicans, respectively. In et al., 2013) we found that the seed coat from Theobroma cacao mixed biofilms, the inactivation of S. aureus was as efficient as in L. seeds contains an antifungal activity. Seed coat extract can single-strain biofilms (7 log reduction in colony counts) but was inhibit growth of fungi (e.g. Aspergillus niger, Penicillium citrinum, less efficient (5 log) for the yeast. Penicillium purpurogenum, Penicillium roquefortii) and yeasts (e.g. Photosensitization with Tetra-Py+-Me caused EPS destruction Candida lipolytica, Candida krusei, Rhodotorula rubra, Rhodotorula and a significant inactivation of cells. PDI may, therefore, be mucilaginosa, Saccharomyces cerevisiae). 25 and 10 mg/mL of seed regarded as a promising approach for biofilm control, even in cases coat extract can inhibit growth of fungi and yeasts respectively. of bacteria-yeast mixed assemblages. By separation of seed coat proteins using SDS-PAGE 17 proteins bands were found. Analysis of the bands using mass spectrometry http://dx.doi.org/10.1016/j.nbt.2014.05.903 showed that 3 proteins have antifungal effect namely: glucanase, chitinase and osmotin (Bahmann, 2014). Further Niemenak (per-

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PS-04 baculovirus using the flashBACÔ system to improve the quantity and quality of EV71 VLPs. The VLP produced by this baculovirus Antibacterial activity of marine Bacillus spp. isolated reached a yield of about 200 mg/L (10-fold improvement) with from mangroves of Saudi Arabian Eastern Province lower amounts of degradation products, putatively thanks to the Abdurahman Hirad 1,∗, Ali Bahkali 2, Chandra Santhappa 2 delayed cell death and reduced cellular protease activity. The purified VLP, which mainly consisted of VP0, VP1 and VP3 proteins, 1 King Saud University, Saudi Arabia resembled the intact EV71 capsid in shape, size and composition. 2 College of Botany and Microbiology, King Saud University, Saudi After injection into Balb/c mice, the VLP elicited promising titers Arabia of binding antibody (212) and neutralization antibody (27) Bacteria that synthesize bioactive substances against known against EV71 in immune serum with broad spectrum to cross- human pathogens were screened from water, sediment, and neutralize the infection of different EV71 subtypes (C2, C4 and decomposing leaf litter samples collected from Tarut Island of east B5 subtypes). In conclusion, the improvement of EV71 VLP pro- Coast of Saudi Arabia. Bacillus aquimaris KSAWD2 was selected duction using the 3rd generation recombinant baculovirus moves as a potential strain that showed considerable bioactivity from the EV71 vaccine development one step further towards commer- amongst 320 isolates. This isolate was identified based on mor- cialization and clinical applications. phological, biochemical and gene sequence of 16sRNA. Culture filtrate of this bacterium showed satisfactory inhibition activity http://dx.doi.org/10.1016/j.nbt.2014.05.905 against the ATCC bacterial reference strains and clinical pathogens obtained from Military Hospital laboratory in Riyadh. The targeted PS-06 pathogens included Salmonella enterica subsp. enterica serovar Typhimurium (ATCC 13311), Shigella sonnei (ATCC 11060), E. coli Quantitative analysis of protein orientation in mem- (ATCC 25922) and Pseudomonas aeruginosa (ATCC 15442), Staphy- brane environments by kinase activity lococcus aureus subsp. aureus (ATCC 6538P, 25923, and 33591), Chunshan Quan ∗, Wen Xiong, Wenzhong Hu, Aili Jiang, Streptococcus pneumoniae, Haemophilus influenzae, Campylobacter Shengdi Fan jejuni and Streptococcus pyogens. Inhibitory activity was tested employing cross streaking, agar well diffusion method, agar over- Dalian Nationalities University, China lay method and assay of growth inhibition in liquid medium. This AgrC is a membrane-embedded histidine kinase in Staphylococ- isolate showed a broad spectrum of bioactivity by recording inhi- cus aureus that is thought to act as a sensor for the recognition bition of growth against more than one pathogen. It was observed of environmental signals and the transduction of signals into that the culture filtrate of Bacillus aquimaris KSAWD2 showed the cytoplasm so as to regulate and control a series of related considerable inhibition against the tested pathogens better than pathogenic gene expressions. However, due to the complexity of commercial antibiotic discs including chloramphenicol, gentam- the cell membrane, it turns to be difficult to study AgrC on bacte- icin, tetracycline, penicillin, neomycin and ampicillin. Results rial cell membrane directly. Many researches try to take advantage indicate a scope for deriving potential bioactive compounds from of proteoliposome which could provide an approximate natural this marine Bacillus aquimaris KSAWD2 against well-known human membrane environment and keep the protein activity to study pathogens. the structure and function of membrane proteins. Given that most membrane proteins have vectorial functions, both functional http://dx.doi.org/10.1016/j.nbt.2014.05.904 studies and applications require effective control over protein orientation within a lipid bilayer. Many studies have been carried PS-05 out to determine membrane protein orientation, however, most of the methods are complicated and time consuming. In this study, Development of recombinant baculovirus for high yield AgrC orientation in liposomes was determined based on thiol- production of enterovirus 71 virus-like particle vaccine reactive reagent labeling and kinase activity which showed that Yu-Chen Hu ∗, Shi-Yeh Lin the method based on kinase activity of AgrC could get an accurate percentage of protein orientation and only costs nearly one-sixth National Tsing Hua University, Taiwan of the time compared with the method based on thiol-reactive Enterovirus 71 (EV71) is the major pathogen responsible for reagent labeling. The results showed that an efficient and rapid hand, foot and mouth disease prevalent in East Asia and can cause method was established to determine the orientation of membrane serious neurological complications and even death. Thus EV71 protein kinase, like AgrC. has posed a tremendous threat to children health. To develop the vaccine, we previously constructed recombinant baculovirus Bac- http://dx.doi.org/10.1016/j.nbt.2014.05.906 P1-3CD for the production of EV71 virus-like particle (VLP) that consists of EV71 capsid proteins. The VLP elicited potent humoral and cellular immune responses in mouse and monkey models. However, the previous generations of baculoviruses resulted in relatively low VLP yield and excessive degradation products upon production. In this study we constructed a new generation of

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PS-07 using CRISPR-Cas9 system was developed. Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/CRISPR-associated The impact of MDCK cell source on the production of (Cas) system are adaptive immune system that silences invading influenza and canine adenovirus nucleic acid by using RNA-guide endonuclease activity in bacte- Paulo Fernandes 1, Rute Castro 2, Tanja Laske 3, Yvonne Genzel 3, ria and archaea. Targeting of single guide RNA (sgRNA) with Cas9 Eric Kremer 4, Paula Alves 2,∗, Ana Coroadinha 2 directs sequence-specific double strands DNA cleavage. Using L- arabinose induction of Cas9 and a constitutive sgRNA cassette, it 1 iBET, Portugal bla is shown that cleavage of targeted double-strands DNA resulted in 2 iBET/ITQB-UNL, Portugal degradation of plasmids containing bla gene. Ampicillin-resistant 3 Max Planck Institute for Dynamics of Complex Technical Sys- cells could be re-sensitized by expressing Cas9/sgRNA , thereby tems, Germany bla reducing the number of resistant cells. The strategy demonstrated 4 Institut de Genetique Moleculaire de Montpellier, France in this study enables the continuous use of old antibiotics, which MDCK cells are widely used for the production of viral-based are already developed and the safety issue was cleared, by reverting vaccines, namely influenza virus. We have shown that MDCK are the resistant cells to sensitive cells. also suitable for canine adenovirus type 2 vectors (CAV-2) manufacturing. Apart of their suitability for virus production, there are http://dx.doi.org/10.1016/j.nbt.2014.05.908 several sources and subclones from the original cell line obtained by Madin and Darby. Furthermore, given the well-documented PS-09 heterogeneity of MDCK cell-line population, understanding the Biosurfactant produced by marine bacteria interacts differences between MDCK cells currently available would help to with diffusible pyoverdine produced by Pseudomonas better select producer cells and develop more reproducible biopro- aeruginosa cesses. In this work, MDCK cell-lines from ECACC and ATCC suppliers M. Alejandro Dinamarca 1,∗, Natalia Romo 2, Claudia Ibacache- were adapted to suspension and compared for the production of Quiroga 3 CAV-2 and Influenza. 1 University of Valparaíso, United States Despite similar cell growth, ATCC cells were more easily 2 Universidad de Valparaíso, Chile adapted to suspension. However, ECACC cells showed to be 3 Centro Nacional de Biotecnología CNB-CSIC/Micromarine better CAV-2 producers, attaining productivities 2-9 fold higher Biotech, Spain than ATCC cells. ECACC cells also showed to be more infected, indicating that cells hold different susceptibilities to infection. Fur- Biosurfactants are surface-active molecules with a wide diversity thermore, the evaluation of infection progression and cell volume of biological functions. Recently, we reported that specific surface- increase due to virus production indicates that virus replication active compounds produced by marine bacteria are able to capture kinetics and cell response to infection was compromised in ATCC diffusible chemical signals from bacteria, affecting the cell-to-cell cells. A more effective antiviral response negatively impacting communication system based on quorum sensing. In this context, CAV-2 propagation in ATCC cells is currently under evaluation. the goal of the present work was to study the interaction between Similarly to CAV-2, Influenza productivities were 2-3 fold higher marine surface-active molecules and pyoverdine, a diffusible with ECACC cells in adherent cultures. siderophore produced by Pseudomonas aeruginosa for the acquisi- To our knowledge, this is the first attempt to characterize MDCK tion of iron from the environment. In human hosts, pyoverdine cell-lines from different suppliers with respect to their perfor- production by this opportunistic pathogen promotes its viru- mance. Our findings show that the cell source/supplier represents a lence and the cooperation with other microorganisms, affecting key issue to be considered when implementing a robust bioprocess. health recovery in patients. In order to determine the interaction between the biosurfactant and pyoverdine, P. aeruginosa was http://dx.doi.org/10.1016/j.nbt.2014.05.907 exposed to different concentrations of the selected marine biosurfactant. At these conditions, pyoverdine presence was measured by fluorescence spectrophotometry (460 nm). Also, the transcrip- PS-08 tional levels of PvdS (transcriptional regulator of the pyoverdine Resensitization of antibiotic-resistant bacteria using synthesis), and PvdQ (periplasmic acylase) were measured by qRT- Clustered Regularly Interspaced Short Palindromic PCR. Results show that when P. aeruginosa was exposed to the Repeats (CRISPR)—–Cas9 system marine surface-active compound at the critical micellar concentration, the fluorescence generated by pyoverdine was reduced in Da-Hyeong Cho ∗, Jonghyeok Shin, Myungseo Park, Younghun 81,97%, respect to the condition without biosurfactant. On the Jung, Byoung-jae Kong, Junghoon In, Dae-Hyuk Kweon other hand, gene expression of pvdS and pvdQ were increased 1.85- Sungkyunkwan University, South Korea fold and 1.0-fold, respectively. Since the assays were performed in non-limiting iron conditions, we propose that the marine surface- Antimicrobial drug development is increasingly lagging behind active compound captures the extracellular pyoverdine, inducing the evolution of antibiotic resistance. As a result, there is pressing or simulating an iron limiting condition in P. aeruginosa. need for new antibacterial therapies that can be readily designed and implemented. In this study, a new antibacterial therapy http://dx.doi.org/10.1016/j.nbt.2014.05.909

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PS-10 The dynamic study of TEM-1 ␤-lactamase by our group has shown that binding of the ␤-lactamase inhibitory protein causes Analysis of 16S rDNA sequences of endophytic bacteria changes in the flexibility of regions away from the ␤-lactam bind- associated with Huanglong disease of citrus plants in ing site. In addition to the previously identified H10 helix, which Guangning County, China forms a lid over an allosteric inhibitor binding site, sequence con- Chongbi Li ∗, Feiyun Gao, Min Zhang, Yugan Hao servation analysis has shown that Trp229 residue of H10 is highly conserved and it has a stacking interaction with Pro226 and Pro251 Center of Biopharmaceutical Engineering in Zhaoqing University residues. Citrus yellow shoot disease is an incurable disease of citrus In order to investigate the importance of Trp229, this residue plants that has long been recognised. It is a serious threat to the has been mutated to alanine. To this end, R-TEM-1 ␤-lactamase development of the local citrus industry. The reports a study of gene of pUC18 was cloned into pET28a(+) with an C-terminal the Chinese citrus industry in Guangning County of Zhaoqing. 6xHis tag. The mutation was performed using QuikChange Endophytes were isolated from the roots and stems. Endogenous Site-Directed Mutagenesis Kit (Agilent Technologies, USA). DNA was extracted and bacterial 16S rDNA was amplified by PCR. Enzyme expression was achieved in Escherichia coli BL21 (DE3) ◦ Sequences were analysed and compared with those isolated from cells at 30 C with 0.1 mM IPTG. Using nickel affinity column chro- bacteria on both healthy and diseased plants. The results revealed matography ␤-lactamase was purified and its activity was measured differences between the endogenous dominant bacteria in phloem with CENTA as the substrate. These measurements have shown tissue of healthy plants and plants with Huanglongbing disease. that W229A mutation causes significant loss of activity, hence it Sphingobacterium multivorum, Acinetobacter junii, and Pseudomonas could be an important residue that enables communication with species similar to those that infect the fish, perch, were more abun- the active site. dant in healthy plants. In contrast, Exiguobacterium acetylicum, the Acknowledgements: This work was supported by TUBITAK plant pathogen Pantoea agglomerans and Acinetobacter baumannii project 113M533. were more abundant in infected plants. The discovery of bacteria associated with found only in citrus plants with Huanglongbing http://dx.doi.org/10.1016/j.nbt.2014.05.911 disease of citrus plants should provide a theoretical and experimental basis for the prevention and control of citrus yellow shoot disease. http://dx.doi.org/10.1016/j.nbt.2014.05.910

PS-11

The importance of a conserved residue of the H10 helix in ␤-lactamase

Fatma Gizem Avcı 1,∗, Elif Özkırımlı Ölmez 2, Berna Sarıyar Akbulut 3

1 Marmara University, Bioengineering Department, Turkey 2 Bogazici University, Turkey 3 Marmara University, Turkey

The most common ␤-lactam resistance mechanism utilized by bacteria is the production of ␤-lactamase enzymes that cleave the amide bond in the ␤-lactam ring rendering the antibiotic inactive. ␤-Lactam antibiotics in combination with ␤-lactamase inhibitors are used to overcome bacterial resistance. Thus design of new inhibitors is a promising area.

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Plant genetic engineering immature embryos of maize were treated at different temperatures for different time durations. Our results showed that the peak GUS PT-01 expression occurred when the embryos were subjected to a tem- ◦ ◦ Identification and characterisation of key genes perature of 42 C for 3 min or at 38 C for 9 min. A 9 percent (%) in involved in fruit ripening of the Chilean strawberry the transformation frequency was achieved in a number of experi- ◦ ∗ ments. Heat shock pretreatment at 38 C for 9 min also resulted in Michael Handford , Analia Espinoza, Milagros Bracamonte, a 7% increase in the “embryo positive callus”. Besides, it was found Aliosha Figueroa, Sara Zapata, Uri Aceituno, Lorena Norambuena that 100 mg/L Casein Hydrolysate (CH) could accelerate the redif- Universidad de Chile, Chile ferentiation of somatic embryos; and 2 mg/L Multi-effect triazole (MET) promoted root elongation, lateral root formation and robust The Chilean strawberry (Fragaria chiloensis L. (Duch.)) is a seedlings to improve the survival rate of the plantlets. Therefore, promising fruit product for Chile. The appeal of these whitish- our protocol can help advance the overall breeding of transgenic pink fruits is associated with their higher sweetness and stronger maize. aroma compared to the red fruits of the commercial strawberry (F. x ananassa). Strawberries are non-climacteric fruits and little is http://dx.doi.org/10.1016/j.nbt.2014.05.913 known about the factors governing their ripening. Nevertheless, the ripening of these fruits is coupled with multiple changes in tex- ture, aroma, sweetness and colour, which are affected by hormonal PT-03 and environmental cues. Therefore, this work aims to generate a A simple, novel and high efficiency transformation greater knowledge of these processes in the Chilean strawberry, for method to introduce foreign DNA into corn plants medi- increasing our understanding of fruit ripening, for use in breed- ated by cell-penetrating peptides ing programs and for maximising the commercial potential of this ∗ product. Zhongyi Wu , Jianhua Wei By contrasting four different developmental and ripening Beijing Agro-Biotechnology Research Center, Beijing Academy of stages of the Chilean strawberry, including small green, large Agriculture and Forestry Sciences, China green, large white and ripe whitish-pink fruit (soft), six suppression subtractive hybridization (SSH) libraries were generated. Of the Genetic engineering breeding and identification of novel genes 1809 differentially expressed cDNAs (ESTs), we identified poten- in maize are very dependent on high efficiency of corn transfor- tial events which could be key in fruit development and ripening, mation. The major deficiencies in current plant transformation including genes which code for proteins putatively involved in systems include but are not limited to the production efficiency of the resistance to oxidative and biotic stress, the perception and the system, transformation variability due to genotype or species signalling of auxins and abscisic acid, vasculature development diversity and explant limitations and a long labor-intensive pro- and in anthocyanin synthesis. The expression patterns obtained cess requiring much skill. In particular, there is a continuing need by quantitative real time PCR generally confirm the data obtained in the field of plant biotechnology to provide more efficient, sim- from the SSH libraries and phenotypic observations; for example ple and low cost transformation methods suitable for high capacity transcripts of a potential anthocyanidin synthase gene peaked in production of economically important plants, particularly elite whitish-pink fruits, consistent with pigment accumulation. Fur- cultivars. Cell-penetrating peptides (CPPs) were discovered to pro- ther findings will be discussed. tect DNA from degradation and deliver a variety of molecules Financial support: Anillo ACT-1110. including DNA into cell. Here, we studied the function of CPPs (Tat2) in improving maize transgenic efficiency through the pollen http://dx.doi.org/10.1016/j.nbt.2014.05.912 tube method. The transformation experiments in Jing 24 and 178 corn inbred lines were conducted under the different conditions of linear DNA concentration, ratio of Tat2, calcium, sucrose, ATP, PT-02 and GTP concentration. Screened with herbicide for T0 seedlings, Improvement of Agrobacterium-mediated transforma- the optimal transformation system was obtained by the media- tion in Hi-II maize (Zea mays L.) by heat-shock treatment tion of Tat2 through the pollen tube transformation method, its with immature embryos transgenic efficiency was up to 1–1.5%. Screened with herbicide

∗ for T1 seedlings, genetic inheritance and segregation of T1 progeny Ya Liu , Jiuran Zhao was revealed under this genetic manipulation, supporting a genetic Beijing Academy of Agricultural and Forestry Science, China ratio 3:1 of T1 progeny was about 16–18% in all transgenic lines. This is a novel transgenic method for corn with high efﬁciency, Improving Agrobacterium-mediated transformation of Hi-II safe, low cost and no limitation of corn genotype. maize (Zea mays L.) would be beneﬁcial for transgenic breeding. Although Hi-II maize is used in various maize transformation http://dx.doi.org/10.1016/j.nbt.2014.05.914 research efforts, improvements in the transformation frequency of this genotype are still needed. In the present study, the immature maize embryos were given heat shock treatment prior to Agrobacterium tumefaciens mediated transformation. The

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PT-04 Nicotiana benthamiana and plum (Prunus domestica L.). Resistance assays showed the transgenic plants were efﬁciently resistant to Impact of non-edible transgenic plant on soil microbial PPV. communities

Kijong Lee ∗, Sung-Dug Oh, Tae-Hun Ryu, Soo-In Sohn, Jong-Bum http://dx.doi.org/10.1016/j.nbt.2014.05.916 Kim

NAAS, South Korea PT-06

Studies on the adverse effects of non-edible transgenic plants Inducible expression of Agrobacterium virulence gene on soil microbial colonies are scarce. Here, we evaluated the effect VirE2 for stringent regulation of T-DNA transfer in of virus-resistant trigonal cactus on nearby soil microbial commu- biosafe plant transient expression systems nities. We collected soil samples at the site of genetically modified ,∗ (GM) and non-GM trigonal cactus cultivation. We collected soil Erna Denkovskiene 1 , Sarunas Paskevicius 2, Stefan Werner 3, samples during the vegetative growth period and the post-harvest Anatoli Giritch 3, Ausra Razanskiene 2 period to provide the data for comparative evaluation. Ecoplate 1 Vilnius University Institute of Biotechnology, UAB Nomads, was used to evaluate the functional diversity of soil microbial Lithuania communities. We found no significant difference between the 2 UAB Nomads, Lithuania GM and non-GM soil samples collected during the vegetative 3 Nomad Bioscience GmbH, Germany growth period. However, the post-harvest soil sample exhibited a marked difference in carbon substrate utilization between the Agrobacterial delivery of viral expression vectors is a com- study group and the control group, but the observed change was fortable solution for the transient production of biomolecules not permanent. Principal component analysis showed that simi- in plants. Valuable proteins are successfully produced in plants lar soil sample groups were not formed based on GM or non-GM in contained facilities, avoiding release of transfecting agrobac- trigonal cactus cultivation, but based on the cultivation period. teria to the environment. However, field applications would be Denaturing gradient gel electrophoresis fingerprinting revealed needed to gain the full economical advantage in the production that virus-resistant trigonal cactus cultivation had negligible effect of lower value and high-volume products, or transient molecu- on soil microbial communities including dominant rhizosphere lar reprogramming of plants to resist stresses or modulate time bacteria, actinomycetes, and fungi. We found no clear evidence of to flower. The release of GM agrobacteria into open field envi- GM trigonal cactus cultivation affecting the functional diversity ronment requires appropriate addressing of biosafety issues. The of soil microbial communities. safety of GM Agrobacterium could be increased by controlling its T-DNA transfer. We constructed chemically regulated T-DNA trans- http://dx.doi.org/10.1016/j.nbt.2014.05.915 fer based on inducible expression of the essential Agrobacterium virulence gene VirE2. Looking for the strongest and stringently regulated promoters in Agrobacterium, we evaluated IPTG inducible PT-05 lac, tac, T7/lac, T5/lac promoters in ␤-galactosidase assays and also Development of genetic resistance to plum pox virus in evaluated hybrid cumic acid inducible promoters lacUV5/CuO, Prunus tac/CuO, T5/CuO and VirE/CuO. For inducible T-DNA transfer tests, Nicotiana benthamiana plants were transfected with a VirE2- Aiming Wang deficient A. tumefaciens strain containing transient expression Agriculture and Agri-Food Canada, Canada vectors harboring inducible VirE2 expression cassettes in vector backbone and a marker GFP gene in their T-DNA region. The Plum pox virus (PPV) is the causal agent of the devastat- efficiency of T-DNA transfer was evaluated by counting GFP expres- ing viral disease known as Sharka in Europe, on many stone sion foci on plant leaves. VirE2 expression regulated by tac/CuO, fruit spp. To engineer genetic resistance against PPV through T5/CuO and VirE/CuO promoters resulted in 50-73% of T-DNA the hairpin-mediated RNA silencing (RNAi) approach, previously transfer efficiency in comparison with the wild type Agrobacterium. two plant transformation vectors, pAWp1 and pAWcp were con- We present efficient and tightly regulated promoters for gene structed by cloning two highly conserved regions of the PPV expression in A. tumefaciens and a very new approach to address genome corresponding to portions of viral RNA coding for P1 and plant transfection biosafety concerns in agrobiotechnology. CP, respectively, into a Ti binary vector under the control of the double Cauliflower mosaic virus 35S promoter as inverted repeats http://dx.doi.org/10.1016/j.nbt.2014.05.917 spanned by an intron from the peach endo-polygalacturonase (endo-PG) genomic DNA. The resulting transgenic plants expressing hairpin RNAs either targeting the viral P1 or CP sequences showed resistance to PPV. Here we report construction of a new construct pAWp1-cp by cloning the P1 and CP hairpin sequences together into the Ti vector and the inserted DNA contains a triple-intron double-hairpin sequence simultaneously targeting the P1 and CP sequence of PPV. This vector was transformed into

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PT-07 for consumers. While conventional breeding programs have been successful in achieving such objectives, directed genetic modi- Quantitative determination of phytase activity and inor- fication can provide additional ways to create new, improved ganic phosphorus of transgenic barley and dihaploid ornamental products. In the last two decades the postharvest per- lines formance of a range of varieties has been successfully improved by Tomas Vlcko 1,∗, Marie Hanakova 2, Jana Vaskova 3, Ludmila genetic modification. In our laboratory we used the etr1-1 mutant Ohnoutkova 3 gene for improvement of display life of several ornamentals, such as kalanchoe, campanula and orchid. 1 Centre of the Region Hana for Biotechnological and Agricultural Although significant progress has been made in developing Research, Czech Republic improved new varieties using tools of plant biotechnology, most of 2 Department of Plant Biology, Faculty of Agronomy, Mendel such improved crops never reach consumers. While scientists are University in Brno, Czech Republic developing the products, they are not being released to the mar- 3 Institute of Experimental Botany AS CR, v. v. i. and Centre of ket because of legal obstacles, including lack of interest or financial the Region Hana for Biotechnological and Agricultural Research, incentive of patent owners in negotiating a license. The result is Faculty of Science, Palacky University Olomouc, Czech Republic that such improved products never reach the market. Possibilities Barley is one of the most prominent crops in the world with for ensuring that remarkable varieties do not become neglected thousands of square kilometers of sown fields. Harvested bar- and forgotten somewhere in an experimental greenhouse or ultra- ley grains are commonly utilized as livestock feeding and in the low freezer must be discussed. brewing industry. Phytic acid (about 70%) is the main storage form of phosphorus in plant seeds. Owing to the acidic nature http://dx.doi.org/10.1016/j.nbt.2014.05.919 of the phytic acid molecule it binds with cations forming an insoluble complex. This is considered the main antinutritional factor for the availability of minerals (calcium, zinc and iron PT-09 principally), because the salts of phytic acid are unfortunately indigestible for monogastric species such as swine, fish and fowl. Subcellular localization of maize cytokinin dehydro- Phosphorus in feces ultimately leads to environmental pollution. genases using heterologous expression in Arabidopsis Phytase is an enzyme which catalyzes the degradation of phy- thaliana Ler cell suspension cultures tate complexes thus providing necessary phosphate and cations Patricie Johnová 1,∗, David Zalabák 1, Ondˇrej Plíhal 1, Olga during germination. This enzyme converts the indigestible form Samajováˇ 2, Petr Galuszka 1 of phosphorus into a fully exploitable form. Transgenic barley line SCLW-GP-PHYA developed using biolistic transformation of 1 Department of Molecular Biology, Palacky´ University in immature embryos exhibits stable expression of the microbial Olomouc, CR Haná, Czech Republic [Aspergillus niger] phytase enzyme. This line was used for a hydrid 2 Department of Cell Biology, Palacky´ University in Olomouc, CR programme, crossing with Czech barley cultivar Azit. The activity Haná, Czech Republic of phytase and content of digestible phosphorus in obtained lines Cytokinins are plant hormones playing a key role during many were analyzed. The original transgenic line was also assessed in plant developmental processes, such as shoot initiation, bud for- brewing trials. mation, apical dominance, delay of leaf senescence, etc. As the Acknowledgement: This work was supported by project OP cytokinins act at the nanomolar concentrations, its homeostasis VaVPI CZ.1.05/3.1.00/14.0327 – New biotechnological products of must be precisely controlled on the tissue as well as cellular and IEB ASCR. subcellular levels. One of the ways to control the cytokinin level is its irreversible degradation. This process is mediated via a class http://dx.doi.org/10.1016/j.nbt.2014.05.918 of flavoproteins, cytokinin dehydrogenases (CKX; EC 1.5.99.12). Plant genomes usually encode for small CKX gene families. So PT-08 far, best characterized is the AtCKX gene family from Arabidop- sis thaliana, comprising seven members. Individual CKX isoforms Genetic modification to enhance breeding for improved exhibit various biochemical features e.g. substrate specificity, dis- postharvest quality of ornamental plants – barriers for tinct spatial and temporal distribution. Interestingly, respective commercial application AtCKX’s differ in their subcellular localization too. While four of Margrethe Serek AtCKX proteins are secreted to the apoplast, two are vacuolar and the last one is cytosolic (Werner et al., 2003; Kowalska et al., 2010). Leibniz University Hannover, Institute for Horticulture Production In contrast to Arabidopsis as the representative dicot, the Systems, Germany information on the CKX subcellular localization in monocots Development of novel varieties with improved postharvest is still not clear. So far, only two out of 13 maize (Zea mays) characteristics is an important goal for the floriculture industry. ZmCKX isoforms were studied in detail. It was shown that Improved varieties can secure higher productivity for growers, ZmCKX1 is apoplastic, while ZmCKX10 is cytosolic (Smehilováˇ increased marketing opportunities, reduced postharvest losses dur- et al., 2009). ing storage and distribution and finally provide better products

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The goal of the presented research is to ﬁnd out the localiza- secretion trichomes of Artemisia leaf surface. To increase the tri- tion of remainder ZmCKX isoforms, to complete the information chome number is one of the efﬁcient ways to increase artemisinin on cytokinin catabolism in maize model. For this purpose, the yield. To investigate the development of multicellular glandular ZmCKX-GFP fusions were prepared, heterologously expressed in trichomes in A. annua, AaMYB1, a R3 MYB family gene was cloned Arabidopsis thaliana Ler cell suspension cultures and studied using and analyzed. Overexpression of AaMYB1 in Arabidopsis led to live cell confocal microscopy. the disappearance of trichome and seed mucilage, enhanced root hair number and reduced anthocyanin contents, showing that http://dx.doi.org/10.1016/j.nbt.2014.05.920 AaMYB1 had a similar function with AtCPC. Overexpression of AaMYB1 in A. annua reduced the artemisinin content as well as the trichome number. On the contrary, the suppression of AaMYB1 in PT-10 A. annua increased the trichome number as well as the artemisinin Regulation of trichome density in Artemisia annua content. This study indicates that AaMYB1 is a negative regulator of multicellular glandular trichome development in A. annua Xiaofen Sun ∗, Fangyuan Zhang and could be useful in engineering of A. annua for increasing Shanghai Jiao Tong University, China artemisinin content.

Artemisia annua is the source of the most potent antimalarial http://dx.doi.org/10.1016/j.nbt.2014.05.921 drug, artemisinin, which is synthesized in multicellular glandular

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Recombinant protein production tance. Insect cells have been shown to be an excellent platform for the production of functional recombinant antibodies [1]. In PU-01 the present study, the production of an antibody Fab fragment Protein extraction by means of electroporation and bac- by transient gene expression in insect cells was investigated. The terial viability of E. coli cDNA fragments encoding the Hc (Fd fragment) and Lc genes

∗ of a mouse anti-bovine ribonuclease A Fab fragment with the Saˇsa Haberl Meglicˇ , Tilen Marolt, Damijan Miklavciˇ cˇ Drosophila BiP signal sequence were cloned separately into the University of Ljubljana, Faculty of Electrical Engineering, Slovenia plasmid vector pIHAneo [2], which contained the Bombyx mori nucleopolyhedrovirus (BmNPV) IE-1 transactivator, the BmNPV Proteins extracted from recombinant bacteria have proved of HR3 enhancer, and the B. mori actin promoter for high-level great value in industry and medicine. Established processes to expression. After co-transfection with the resulting plasmids car- extract proteins from bacteria often include use of mechanical rying the Hc and Lc genes, Trichoplusia ni BTI-TN-5B1-4 (High forces or chemicals, which cause complete disruption of the cell Five) cells were incubated with a serum-free medium in static and release of membrane contaminants (endotoxins). Thus, addi- or shake-flask cultures. Transfection conditions were optimized tional purification steps are needed, which at large scales represents with flow cytometric analysis using the green fluorescence pro- up to 80% of the production costs [1]. tein. Western blot analysis and enzyme-linked immunosorbent Extraction by means of electroporation is a quick, chemical free assay (ELISA) of culture supernatants revealed that transfected and cost efficient release of intracellular components from E. coli cells secreted an Fab fragment with antigen-binding activity. [2]. In order to avoid cell disruption and by that the release of Production of over 30 mg/L of Fab fragment was achieved in membrane contaminants, and to obtain adequate quantity of pro- 6 days. teins, electroporation pulse parameters need to be adjusted. Thus, we studied the influence of pulse strength, duration, number and References repetition frequency on extracted proteins from E. coli and on [1].Yamaji H, et al. Cell Eng 2011;7:53–76. bacterial viability. [2].Yamaji H, et al. Biochem Eng J 2008;41:203. Our results show that by increasing electric field strength, pulse repetition frequency and/or pulse duration, also concentration of http://dx.doi.org/10.1016/j.nbt.2014.05.923 extracted proteins increases (maximum 15.29 ␮g/ml), while E. coli viability decreases (minimum 2.2 – log reduction). The correlation was expected, since more bacteria were destroyed and more PU-03 proteins were released into surrounding media. The best choice RMCE reference sites – A valuable tool for comparing under experimental conditions used and for our strain was a train antibody expression capabilities in CHO cells of eight pulses with 1 ms duration, 1 Hz pulse repetition frequency ∗ and electric field strength of 5 kV/cm. Under these conditions the Patrick Mayrhofer , Alexander Mader, Renate Kunert best ratio between extracted proteins and bacterial viability was University of Natural Resources and Life Sciences, Austria observed. Chinese hamster ovary (CHO) cells represent the major host References for recombinant monoclonal antibody production for biotherapeutic applications. Traditional methods for generation of stable [1].Assenberg R, et al. Curr Opin Struct Biol 2013;23:393. antibody producing cell clones are based on random integra- [2].Haberl S, et al. J Membr Biol 2013;246:861. tion of the gene of interest into unpredictable chromosomal loci. Therefore, much effort is undertaken for tedious and time consum- http://dx.doi.org/10.1016/j.nbt.2014.05.922 ing screening and selection procedures. Additionally, integration events into chromosomal loci with variable transcription rates PU-02 and co-introduction of residual vector sequences triggers epigenetic mechanisms in various extent making prediction of expected Production of an antibody fragment by transient gene expression levels and comparisons between cell lines a tedious expression in insect cells task. Hideki Yamaji ∗, Tomohisa Katsuda, Hirotsugu Hamada, Keita The development of site-directed gene integration methods Mori enables the integration of different transgenes into pre-determined chromosomal positions. Department of Chemical Science and Engineering, Kobe Univer- Here we demonstrate that the application of recombinase sity, Japan mediated cassette exchange (RMCE) provides a valuable tool for Monoclonal antibodies and their fragments have been used introducing a re-targetable gene cassette into a stable and tran- in a variety of diagnostic and therapeutic applications. Novel scriptionally active chromosomal locus. antibody-based biologics are currently selected from a large pool of A RMCE competent cassette was introduced into DUKX-B11 lead candidates. High-throughput production systems for rapidly cells by two rounds of RMCE reactions leading to stable and providing a large number of recombinant antibody molecules homogenous gfp reporter gene expression. This RMCE cassette was of high quality and in sufficient quantity are of major impor- replaced by two different anti-HIV antibody variants leading to cell

S186 www.elsevier.com/locate/nbt New Biotechnology · Volume 31S · July 2014 RECOMBINANT PROTEIN PRODUCTION clones with reproducible expression behavior in T25 roux flasks PU-05 and spinner vessels during batch cultivation. Intracellular prod- Establishment of stable, high producing, recombinant uct formation analyzed by flow cytometry and qPCR indicated a CHO cell lines using Rosa26 Bacterial Artificial Chromo- uniform antibody expression behavior of the established cell lines somes for transgene delivery demonstrating the success of targeted integration by RMCE. The established RMCE host cell line enables the comparison Wolfgang Sommeregger 1,∗, Andreas Gili 2, Katalin Zboray 3, of different cell clones and product specific expression patterns in Thoams Sterovsky 2, Emilio Casanova 3, Renate Kunert 1 future projects based on an identical chromosomal environment. 1 VIBT/DBT/University of Natural Resources and Life Sciences, Vienna, Austria http://dx.doi.org/10.1016/j.nbt.2014.05.924 2 Polymun Scientific Immunbiologische Forschung GmbH, Klosterneuburg, Austria PU-04 3 Ludwig Boltzmann Institute for Cancer Research (LBI-CR), Vienna, Austria Development of a method to quantify genomic rearrangements in Chinese Hamster Ovary cells Chinese Hamster Ovary (CHO) cells are the most frequently used mammalian host for the production of biopharmaceuti- Inmaculada Hernandez Lopez 1,∗, Vaibhav Jadhav 2, Martina cals. The achieved volumetric titers using CHO cell factories have Baumann 1, Norbert Auer 2, Angelika Zotter 1, Nicole Borth 3 increased significantly over the past two decades. However, the 1 Austrian Center of Industrial Biotechnology, Austria establishment of well-producing cell lines remains tedious. Many 2 University of Natural Resources and Life Sciences, Austria parameters like the host cell line, the genetic construct, the cell 3 University of Natural Resources and Life Sciences/Austrian Center culture medium, the applied cultivation strategy and the product of Industrial Biotechnology, Austria by itself are influencing volumetric titers. Bacterial Artificial Chromosomes (BACs) harbouring the Rosa26 Chinese Hamster Ovary (CHO) cells are the preferred host cells locus show improvements concerning transcriptional efficiency for the production of therapeutic proteins and the most commonly when used as shuttle vectors for the delivery of transgenes. The used mammalian expression system due to properties such as an high transcription rate of the Rosa26 BACs enables to use the easy cultivation, fast growth, complex protein folding and human- expression system without gene amplification in auxotrophic and like post-translational modifications. non-auxotrophic CHO hosts. Nevertheless, development of recombinant CHO cell lines is In this work we combined the transcriptional efficiency of the a slow and work intensive process that requires testing of thou- Rosa26 BAC with CHO host cell lines growing to high cell densities sands of clones as these tend to be heterogeneous in behaviour. in order to increase the volumetric product titers. The commonly While this is the source of easy adaption and the occurrence of used CHO hosts CHO-K1, -DUKX-B11, -DG44, and CHO-S were extraordinarily high producers, it also means that frequently the screened for their growth behaviour in batch experiments using most promising clones turn out to be unstable with respect to optimized serum-free cultivation strategies. CHO-K1 and CHO- productivity and phenotype. S performed best and were used for the establishment of stable Despite the high frequency of genomic rearrangements due to clones. The model proteins in this study were two anti–HIV-1 IgG-1 genomic instability, no methods are currently available to rapidly antibodies and the HIV-1 gp140 (CN54) envelope protein. assess large scale chromosomal rearrangements, with the excep- For all products we could show that the combination of cells tion of FISH (fluorescence in situ hybridization) and karyotyping. growing to high cell densities and the transcriptional efficiency of The focus of the present work is to adapt a method for this pur- the Rosa26 BAC system leads to the accumulation of high product pose, Amplified Fragment Length Polymorphism (AFLP), which concentrations. Even for the highly glycosylated HIV-1 envelope allows simple detection and quantification of genomic changes protein we achieved titers above 1 g/L. over time using standard laboratory equipment. The approach, which is already established in cancer research and plant breeding, http://dx.doi.org/10.1016/j.nbt.2014.05.926 was modified for use in CHO cells. It defines an initial pattern of electrophoretic bands that enables detection and quantification of changes over time and the determination of the degree of genomic PU-06 differences between CHO strains and subclones and therefore can Modification of signal peptide for enhanced production also be used for cell line identification and characterization. of recombinant erythropoietin from animal cells http://dx.doi.org/10.1016/j.nbt.2014.05.925 Duk Jae Oh 1,∗, Ji Hye Park 2

1 Sejong University, Department of Bioscience and Biotechnology, South Korea 2 Sejong University, South Korea

Background and novelty: Various secretory proteins are synthesized as precursors with additional N-terminal signal peptide. The signal peptide is cleaved off by signal peptidase once it has

www.elsevier.com/locate/nbt S187 RECOMBINANT PROTEIN PRODUCTION New Biotechnology · Volume 31S · July 2014 served its purpose of targeting the protein to, and importing it into, little or no further increase. Further strain optimisation will be the ER. Recently, recombinant DNA research was used to study sig- required to increase production yield of ergopeptine hydrolase nal peptide and made it possible to show the efficient activity of a ErgA to a level suitable for a technological enzyme production proposed signal peptide by fusing it to another protein. process. Experimental approach: In this study, signal peptide of human erythropoietin was replaced with the signal peptide of http://dx.doi.org/10.1016/j.nbt.2014.05.928 human IL-2, then the hydrophobic region of the signal peptide was modified and its effect on the production of the target protein PU-08 (erythropoietin) was evaluated. The nucleotide sequence of modified signal peptide was transported directly into the upstream of Osmolality as a determining factor in screening for high the 5’end of the human erythropoietin gene by performing one performing clones of human hematopoietic expression round of amplification with pfu DNA polymerase. The gene of tar- systems designed for monoclonal antibody production in get protein with modified signal peptide was transiently expressed chemically defined media in HEK293/CHO cells and quantification of protein secretion was Galina Kaseko ∗, Kim Lou, Derrick Theys, Tohsak Mahaworasilpa evaluated. Results and discussion: As a result, we could observe sig- The Stephen Sanig Research Institute, Australia nificantly increased protein secretion up to 4 fold higher by In cell line development for monoclonal antibody (mAb) pro- modifying the native signal peptide, in particular the hydropho- duction, the screening and selection of a highly productive and bic region. This observation is believed to be applicable to improve stable clone from transfectant population in a limited time frame the productivity of recombinant therapeutic proteins from animal remains a major challenge. The clone performance including cells. product quality, productivity, cell growth and cell metabolic profile is often dependent on cell culture conditions. With industry http://dx.doi.org/10.1016/j.nbt.2014.05.927 being pushed towards chemically defined, non-animal components or low protein media, the selection of the best performing PU-07 clone faces an additional challenge as not all antibody-producing cell lines can achieve high yields in such conditions. In attempt Effect of gene copy number on production yield of to evaluate a possibility of obtaining high performance clones recombinant ergopeptine hydrolase ErgA in Pichia pas- of an in-house generated host cell line derived from human toris hematopoietic cells, the cells were cloned in four either chemi- Julia Panhölzl 1,∗, Julia Lindenberger 1, Patricia Menczik 1, Markus cally defined, low protein or animal component free media. We Aleschko 1, Irene Hahn 2, Heidi Schwartz-Zimmermann 2, Gerd found that a key factor in establishing commercially viable clones Schatzmayr 1, Michaela Thamhesl 1, Wulf-Dieter Moll 1 was culture osmolality. Most cell culture media have an osmolality range 270–330 mOsm/kg. The impact of high osmolality seems 1 BIOMIN Research Center, Austria to be cell line specific with reports of inhibition of cell growth 2 Christian Doppler Laboratory for Mycotoxin Metabolism and and specific productivity and an increase in specific productiv- Center for Analytical Chemistry, Department for Agrobiotechnol- ity balanced with reduction in cell growth to yield an increased ogy (IFA-Tulln), University of Natural Resources and Life Sciences, volumetric productivity. In our experiments, osmolality above Vienna, Austria 300 mOsm/kg had deleterious effects on single cell cultures regard- Our long term goal is to prevent ergot alkaloid poisoning in less of the types of culture media used. Reducing osmolality to farm animals by providing enzymes for use as feed additives to 250–290 mOsm/kg lead to an improved clone performance. It degrade ergot alkaloids in the digestive tract. We isolated an ergot remains to be determined whether these results are specific to alkaloid degrading bacterial strain, Rhodococcus erythropolis MTHt3, our host cell line or have broader implications to other expres- from a soil sample, cloned the genes for ergot alkaloid degradation, sion systems, particularly those based on human hematopoietic and characterised the recombinant enzymes. In the present work, cells. we attempted to optimise the production yield of the recombinant ergopeptine hydrolase ErgA in Pichia pastoris by increasing copy http://dx.doi.org/10.1016/j.nbt.2014.05.929 number of the heterologous gene. We cloned plasmids with one, two, and three cassettes of ergA fused to the secretion signal sequence of Saccharomyces cerevisiae alpha-mating factor under control of the GAP promoter. Plasmids were transformed into Pichia pastorisNRRLY-11430 (CBS 7435), recombinant Pichia strains were cultivated in YPD medium in Erlenmeyer shake flasks, and yield of secreted ErgA was measured by SDS-PAGE analysis and enzyme activity assay based on ergo- tamine hydrolysis and HPLC. We found that two copies of the ergopeptine hydrolase gave higher ErgA yield than one copy, but a third copy ofergA showed

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PU-09 these factors have to be evaluated case-by-case for any new target protein and without any guarantee for improvement. Here, we Development of improved methanol dehydrogenases describe the combination of a randomised library approach in using directed evolution and biological methanol sensor conjunction with yeast surface display (YSD) and cytometric system for the elimination of formaldehyde sorting with the aim of selecting producer clones with enhanced Bong Hyun Sung 1,∗, Ji-Yeun Yi 1, Seung-Goo Lee 1, Sun Chang secretory capacities. In order to link the specific secretion rate to Kim 2, Jung-Hoon Sohn 1 individual cells, the recombinant target protein is fused with a cell wall anchor sequence. Thus, the avidity of the recombinant 1 Korea Research Institute of Bioscience and Biotechnology, South protein on the cell surface reflects the secretory capacity of each Korea cell clone. Using immunofluorescence staining, promising candi- 2 KAIST, South Korea dates can be sorted and enriched in consecutive rounds applying Formaldehyde is an important organic precursor to many fluorescence-activated cell sorting (FACS). Finally, isolated clones materials and chemical compounds. Despite its widespread use, are subjected to DNA sequence analysis and the identified genes are exposure to formaldehyde is a significant consideration for human supertransformed in host cells secreting soluble target protein in health due to its toxicity and volatility. Methanol dehydroge- order to achieve proof-of-principle. nase (MDH) is an NAD+-dependent oxidoreductase that catalyzes formaldehyde to methanol reversibly. Reduction activity of MDH http://dx.doi.org/10.1016/j.nbt.2014.05.931 is noticeable in two aspects of the elimination of toxic formaldehyde and the production of methanol as an energy source. Herein, PU-11 we screened improved mutants of Bacillus methanolicus MDH in Purification of tag-free recombinant fumonisin esterase Esehcichia coli that reduce formaldehyde to methanol effectively FumD from Pichia pastoris culture supernatant for use with directed evolution and biological methanol sensor system. To as calibration standard for enzyme quantification examine the resistance to formaldehyde, E. coli strains expressing ∗ each mutant were cultured in 3 mM formaldehyde, toxic concen- Corinna Kern , Markus Aleschko, Wulf-Dieter Moll, Gerd Schatz- tration for E. coli. In the best mutant, three phenylalanine residues mayr were substituted to leucine, valine and serine respectively and all Biomin Holding GmbH, Austria the three substitutions were required to increase reduction activity for formaldehyde. Interestingly, the mutant which has F213V and Fumonisin esterase FumD is used as feed additive for gastroin- F289L increased the reduction relative to the wild type MDH, on testinal hydrolysis and detoxification of fumonisins, carcinogenic the other hand, the mutant that has F356S reduced the reduction. mycotoxins produced by Fusarium verticillioides, which are nat- Based on these results, mutant MDH eliminates formaldehyde effi- urally contained in maize from warm growing regions, in the ciently, therefore it could help solve environmental problem such gastrointestinal tract of farm animals. The goal of our present as sick house syndrome. work was to provide a preparation of pure, stable and active FumD for use as calibration standard for enzyme quantifica- http://dx.doi.org/10.1016/j.nbt.2014.05.930 tion and characterization. Instead of purifying FumD-6x His via affinity chromatography as before, we attempted purification of PU-10 tag-free FumD from Pichia pastoris fermentation culture supernatant. We compared three different purification protocols: anion Optimizing secretion efficiency of industrially relevant exchange (AIEX) versus cation exchange (CIEX) chromatography recombinant proteins in Pichia pastoris by means of and hydrophobic interaction chromatography (HIC). Analysis was yeast surface display and cytometric sorting performed by SDS-PAGE, immunoblotting and/or gel filtration Lars Toellner 1,∗, Fabian Schneider 1, Brigitte Gasser 2, Diethard (GFC) on Superdex 200 resin. IEX led to higher purity of the Mattanovich 2 FumD fraction, and HIC showed higher yield. We tested protein stability by storing a FumD preparation in different buffers at 1 ACIB GmbH, Austria 4 ◦C and -20 ◦C (including freeze-thaw cycles) for defined time 2 BOKU Vienna/ACIB GmbH, Austria periods. After quantification via ELISA and activity analysis, Tris Pichia pastoris has become a competitive host system for the buffer pH 8 without any additives was selected for FumD storage. production of industrially relevant enzymes and biopharmaceuti- The final FumD preparation was generated by two-step purifi- cal proteins. Strong constitutive and inducible promoter systems cation via AIEX-GFC using Tris as final buffer, and offered the and efficient export signals enable production of authentic soluble same specific enzymatic activity as the starting material, suggest- proteins in the range of grams per litre. Still, similar to other host ing no loss during the purification process. After quantification systems, the expression and secretion of recombinant protein can via several spectrophotometrical methods (BCA-assay, Bradford, be impaired drastically when the secretory capacity of the host is UV-absorption), ELISA and Amino Acid Analysis, the FumD cal- overloaded with the additional burden. Co-expression of proteins ibration standard was available with well-defined concentration which support protein folding, ER transport or other relevant and specific activity. steps in expression and secretion, has been shown to potentially increase the yield of secreted recombinant protein. However, http://dx.doi.org/10.1016/j.nbt.2014.05.932

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PU-12 that sorbitol as co-substrate leads to a higher speciﬁc productivity

of ROL (qROL) whereas glycerol allowed higher volumetric produc- Developing a versatile tool set for heterologous gene tivity of ROL (QROL). Besides, there is a synergic effect of glycerol expression in Pichia pastoris feeding and lowering the temperature on QROL. Controversially, we Helmut Schwab 1,∗, Mudassar Ahmad 2, Melanie Hirz 2, Markus have found that decreasing the cultivation temperature from 30 to 2 2 ◦ Kolmbauer , Ingund Rosales Rodriguez 22 C at constant μ did not result in a qROL increase, suggesting that the increase of specific productivity by decreasing the cultivation 1 ACIB/Graz University of Technology, Austria temperature previously reported elsewhere is actually a result of a 2 Graz University of Technology, Austria decrease in μ rather than temperature itself. This may open a new Pichia pastoris has become an important microbial host for approach to optimise production of heterologous proteins based recombinant protein production. We have chosen as basis the on decreasing the cultivation temperature. strain P. pastoris CBS 7435 (also NRRL Y-11430), which was originally patented for single cell protein production via methanol http://dx.doi.org/10.1016/j.nbt.2014.05.934 utilization, and for which the patent protection has expired. A high quality genome sequence of this strain was recently PU-14 determined [1]. On the one hand proper integration vectors based on different promoters and selection markers have been Hydrophobins class I versus class II: Trichoderma virens newly designed and constructed. For engineering P. pastoris HFB9a and HFB9b (class I) are more effective as enhanc- strains, an efficient vector construct was developed for marker- ing agents in enzymatic PET hydrolysis and surface free genome knock-out or integration of gene. Based thereon, a modulators compared to HFB4 and HFB7 (class II) set of amino acid auxotrophic and protease deficient host strains Agnieszka Przylucka 1,∗, Doris Ribitsch 1, Enrique Herrero-Acero 1, was constructed. In addition, an AOX1 promoter based methanol Georg Gübitz 1, Christian P. Kubicek 1, Irina Druzhinina 2 inducible host system was developed allowing for efficient induction with low amounts of methanol. Moreover, a methanol-free 1 ACIB GmbH, Austria alternative has been developed for induction of the AOX1 2 Technical University of Vienna, Austria promoter. Poly(ethylene terephthalate) (PET) is a thermoplastic polyester Reference with excellent tensile and impact strength, transparency, and [1].Küberl A, Schneider J, Thallinger GG, Anderl I, Wibberg D, Hajek appropriate thermal stability. Because of its high production T, Jaenicke S, Brinkrolf K, Goesmann A, Szczepanowski R, Pühler A, (36 mio tons per year), PET constitutes a significant waste mate- Schwab H, Glieder A, Pichler H. High-quality genome sequence of rial, which – although not representing a direct hazard to the Pichia pastoris CBS7435. J Biotechnol 2011;154(4):312–20. environment - is not readily decomposed in nature. Recently, microbial cutinases were shown to be capable of partial or com- http://dx.doi.org/10.1016/j.nbt.2014.05.933 plete hydrolysis of the synthetic polymer PET, therefore offering new possibilities in the processing and recycling of this highly PU-13 inert material. We have demonstrated that the addition of class II hydrophobins of Trichoderma can enhance the rate of PET hydrol- Temperature effect on recombinant protein production ysis by cutinases. in continuous cultures of methylotrophic yeast Pichia It is currently unknown whether this observed effect is pastoris: a comparative study of sorbitol and glycerol as a property of some specific hydrophobins, and whether the a co-substrate hydrophobins need to bind to the cutinases to enhance ∗ their action. In this work we therefore compared the class II Julio Berrios , Maria-Olga Flores, Alvaro Diaz-Barrera hydrophobins (HFB4 and HFB7) to the Trichoderma-unique non- Pontificia Universidad Catolica de Valparaiso, Chile class II hydrophobins (HFB9a and HFB9b) with respect to their activity as enhancers of PET hydrolysis by cutinase I (CutI) from Pichia pastoris is a methylotrophic yeast that is widely used Thermobifida cellulosilytica and surface modulators. Time scan anal- as an expression system of heterologous proteins. The use of co- ysis over 72 h revealed a hydrophobin-specific interaction with substrates (e.g. glycerol, sorbitol) together with methanol, and ◦ cutinases. Whereas the non-class II hydrophobins HFB9a and culture temperatures below 30 C are among the proposed strate- HFB9b where able to increase the rate of PET degradation by CutI, gies to improve recombinant protein productivity. Since these class II hydrophobins were less active: HFB4 showed an increased approaches normally affect the specific cell growth rate (μ), and it activity on the hydrolysis of PET at lower time points, an inhibition is well known that μ also affect the protein productivity, we have was observed when using HFB7. performed an experimental design based on continuous cultures operating at a steady-state, thus fixing μ by a constant dilution rate − http://dx.doi.org/10.1016/j.nbt.2014.05.935 (D = μ = 0.05 h 1). Two sets of continuous cultures were run using either glycerol or sorbitol as co-substrates, exploring at 22 and 30 ◦C, together with co-substrates and methanol mixtures with 30 and 60% of co-substrate in the mix. The recombinant protein Rhi- zopus oryzae lipase (ROL) was used as model. Our results indicate

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PU-15 ble of the production of high yields of a recombinant protein. The system combines both advantages of prokaryotes – high expres- Strategy for on-column refolding of human recom- sion, simple scale-up, cheap production media–, and advantages binant His-tagged prethrombin-2 produced using the of eukaryotes – ability to perform various post-translation modifi- Escherichia coli-based expression system cations. Michaela Osadska ∗, Martin Safranek,ˇ Hana Bonková,ˇ Ján The hGH gene was integrated into genome of P. pastoris under Krahulec, Stanislav Stuchlík, Ján Turnaˇ the control of a constitutive promoter, in order to produce native hGH into the extracellular media. Then, the genome integration Department of Molecular Biology, Faculty of Natural Sciences, was validated through a PCR and expression in flasks. The SDS- Comenius University in Bratislava, Slovakia PAGE results were confronted with a Western blot analyses. hGH One way to obtain an active human thrombin for pharmaceuti- was then successfully produced in 1L fermenter and partially puri- cal purposes is to prepare recombinant human thrombin with the fied using anion-exchange chromatography. use of an Escherichia coli expression system. Prethrombin-2, the Acknowledgements: This publication is the result of the smallest single chain thrombin precursor, modified with His-tag project implementation: “Production of biologically active agents at N-termini forms inactive intracellular inclusion bodies during based on recombinant proteins” (ITMS 26240220048) supported the expression. Therefore the refolding process to obtain an active by the Research and Development Operational Program funded by conformation is necessary. Here we report two on-column refold- the ERDF. ing methods of His-tagged prethrombin-2. Immobilized metal ion affinity chromatography and hydroxyapatite chromatography for http://dx.doi.org/10.1016/j.nbt.2014.05.937 one-step protein purification and on-column refolding were used. Solubilization of aggregated structures was carried out with vari- PU-17 ous buffers composition under the denaturing conditions, where higher yield of solubilized prehrombin-2 was obtained in the Isolation of novel lignin degrading enzymes and lignin presence of 6.0 M guanidine hydrochloride compared to 8.0 M degradation products from bacteria and fungi urea. After the solubilization step gradual refolding of the protein Fatai Bello ∗, Takanori Furukawa, Louise Horsfall was performed on-column using a linear guanidine gradient from 6.0 M to 0.0 M in the presence of 50 mM Tris–HCl and 0.5 M NaCl University of Edinburgh, United Kingdom in the case of IMAC and 1 mM KH PO if hydroxyapatite chro- 2 4 Lignocellulosic biomass offers an alternative to non-sustainable matography was used. There was one protein peak in the A280 fossil fuels for the production of useful chemical substances and is profile during the refolding process, which means that protein was expected to become one of the key renewable energy resources released during reduction of chaotropic reagent concentration. in the near future. Lignin makes up as much as 30% of ligno- Final yield of refolded protein was therefore in minimal concen- cellulosic biomass and is considered one of the most important tration and on-column refolding requires next optimization. limiting factors in the yield of bioethanol obtained from biomass Acknowledgements: This work is result of project imple- fermentation. Currently, industries regard lignin as a nuisance in mentation: “Production of biologically active agents based on the utilization of cellulose and hemicellulose, which are the target recombinant proteins” (ITMS 26240220048) supported by the starting materials for these industries. However, lignin is a poten- Research and Development Operational Program funded by the tial source of a variety of aromatic chemicals, if a system for its ERDF. controlled degradation can be developed. Several species of fungi and bacteria possess lignin-degrading ability through the produc- http://dx.doi.org/10.1016/j.nbt.2014.05.936 tion of different types of ligninolytic enzymes. This project aims to identify ligninolytic enzyme gene sequences from fungi and bac- PU-16 teria. The genes will then be cloned and expressed in bacterial and fungal hosts. The optimum expression conditions will be deter- Preparation of human growth hormone in Pichia pas- mined and the activity of the resulting enzymes on lignin and toris under a constitutive promoter lignin model compounds will be determined. Structural analysis Diana Hopkova ∗, Kristina Jirickova, Zdenko Levarski, Jan by X-ray crystallography will be performed on the best enzymes Krahulec, Stanislav Stuchlik, Jan Turna from the activity studies. The lignin degradation products will be analysed and methods for quantitative estimation are in develop- Department of Molecular Biology, Faculty of Natural Sciences, ment. The enzymes discovered from this study are intended to Comenius University, Slovakia improve the current lignin enzyme treatments used in industry. The human growth hormone (hGH) is the polypeptide used in modern medicine for a treatment of different diseases such http://dx.doi.org/10.1016/j.nbt.2014.05.938 as growth deficiency in children. Moreover, the marketed pharmaceuticals based on hGH got the approval for the treatment of Turner’s syndrome or cachexia associated with AIDS. Pichia pastoris was chosen as the host organism because of its ability to grow to high cell densities and its strong promoters capa-

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PU-18 thioredoxin (Trx). In this work, we compare the expression level, portion of soluble product, efficiency and yield of Trx-hGH fusion Fusion protein and solubility enhancing strategies for protein using three cultivation methods – shake-flask, batch and heterologous expression of novel plant sesquiterpene fed-batch bioreactor runs. While more than 95% of the produced synthases Trx-hGH was in soluble form when cultivated in shake-flask or Steffen Hartwig ∗, Thore Frister, Thomas Scheper, Sascha Beutel bioreactor in batch mode, this number decreased to 47% when produced in fed-batch mode. We have been able to purify 1.045 g Leibniz University of Hannover, Germany of soluble Trx-hGH from the liter of fed-batch culture and 0.8 g Terpenes constitute the largest class of flavor and fragrance from the batch culture. These numbers represent a significant yield molecules in nature. Important representatives of this class are when compared to other published data and are of potential inter- used widely in various foods, beverages, exclusive perfumes and est to the biotech industry. personal care products. Biocatalytic production of terpenes is a Acknowledgements: This work is the result of the project promising method, but requires the availability of large amounts implementation: “Production of biologically active agents based of soluble and active terpene synthases. Plant terpene synthases on recombinant proteins” (ITMS 26240220048) supported by the (TPS) exhibit large variations in amino acid sequence and protein Research and Development Operational Program funded by the fold. The soluble yield when expressed in E. coli varies substantially ERDF. from enzyme to enzyme. In most cases, TPSs are considered hard to express in a heterologous host organism. We chose cDNAs of syn- http://dx.doi.org/10.1016/j.nbt.2014.05.940 thases, partially synthesized as well as extracted from native plants, which all produce chemically interesting and industrial relevant PU-20 terpene products. We identified a cDNA variant of patchoulol synthase from Pogostemon cablin [1,2]. The active expression level Multiple chromosomal gene integration for production of the enzyme variant was increased substantially by fusion to a of pharmaceutical proteins in S. cerevisiae thioredoxin moiety [3]. Malene Jensen 1,∗, Uffe Mortensen 1, Nina Gunnarsson 2, Tomas A previously not characterized zizaene synthase from Vetiveria Strucko 1, Line Due Baron 2 zizanoides was assembled from artificial DNA strings. Because no soluble expression could be detected by conventional strategies, 1 Technical University of Denmark, Denmark induction using a cold shock promoter as well as fusion to the 2 Novo Nordisk, Denmark highly soluble SUMO protein moiety were evaluated. When studying protein folding and secretion the general con- The presented results enable other researchers in the field to ception is that all cells in a population express an equal amount of decide what expression strategy might be most suitable for new protein. Recent work has shown that expression levels vary greatly TPSs, which have not been expressed in E. coli. in cell populations which express proteins on plasmids. Hence a yeast expression platform has been developed at the Department References of Systems Biology, DTU. The platform offers the opportunity to [1].Croteau, et al. Arch Biochem Biophys 1987;256:56–68. express genes on the chromosome in 1 to 10 copies. A comparison [2].Deguerry, et al. Arch Biochem Biophys 2006;454:123–36. between the expression of CFP and RFP by the platform and by [3].Hartwig, et al. Prot Expr Purif 2014;97:61–71. plasmids reveals the problems of plasmid expression. FACS analyses of two cell populations, expressing CFP and RFP on the separate http://dx.doi.org/10.1016/j.nbt.2014.05.939 plasmids or expressing CFP and RFP using the yeast expression platform shows expression varies greatly in a cell population based PU-19 on plasmid expression compared to the yeast expression platform. When expressed on plasmids a few cells are high performers Expression of soluble recombinant human growth hor- on both proteins but the largest fraction of cells is actually not mone in Escherichia coli cultures using shake-flask, batch expressing either of the proteins. The yeast expression platform is and fed-batch cultivation developed to facilitate stable expression of integrated genes. The ∗ integration sites are separated by essential genes which ensure that Jan Turna , Zdenko Levarski, Jan Krahulec, Kristina Jirickova, the integrated genes are not lost by recombination. An amplifica- Diana Hopkova, Stanislav Stuchlik tion method has been developed for the platform which enables Comenius University in Bratislava, Department Mol. Biol., fast integration of genes. Future perspectives involve exploring the Slovakia capabilities of the platform for recombinant protein production including performance and stability studies. Human growth hormone (hGH) was one of the first recombinant proteins approved for the treatment of human growth http://dx.doi.org/10.1016/j.nbt.2014.05.941 disorders. Its small size (191 amino acids), possession of only 2 disulphide bonds and absence of posttranslational modifications make Escherichia coli the host of choice for its production on any scale. We have developed an efficient E. coli based expression system for the production of high levels of soluble hGH fused to

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PU-21 cessing economics) but is challenging due to the formation of inclusion bodies, leading to inactive enzymes. Engineering EK Biosynthesis, purification and biointeraction of human cat for periplasmic secretion generated a basal level of soluble and SCOMT with Parkinson’s disease inhibitors active enzyme. Directed evolution was then applied to screen for Luis Passarinha ∗, Filipa Correia, Augusto Pedro, Diana Oliveira, variants with improved total activities attained during expres- João Queiroz sion. The generated library of variants contained mutations across the following gene sequences: EK , secretion signal peptide, pro- CICS-UBI: Health Sciences Research Centre – Universidade da Beira cat moter, lac operator, and ribosomal-binding site. The solubility, Interior, Portugal stability, and activity of the resultant EKcat variants were examined Catechol-O-methyltransferase (COMT) is an important target and hypotheses given based on sequence, structural, and bioin protein engineering due to its role in human neurological dis- physical findings. Furthermore, variants were also shown to adapt orders such as Parkinson’s and Alzheimer’s disease [1]. Therefore, differently when the fermentation temperature was raised, provid- is relevant to study new COMT inhibitors since it will lead to an ing another dimension for investigating the complex dynamics improvement in Parkinson’s therapy. So, it is necessary a biosyn- between expression and secretion. In addition to improving EKcat thesis process to produce and stabilize the human SCOMT and expression and activity in E. coli, the study presents insights into subsequently, a suitable purification strategy to obtain pure frac- engineering proteins for high functional expression and periplas- tions for protein-inhibitors interaction studies. The biosynthesis mic secretion. process was performed based on pPICZ A and Pichia pastoris as a host cell where hSCOMT was synthesized with a hexahisti- http://dx.doi.org/10.1016/j.nbt.2014.05.943 dine tag. Moderate to high expression values were obtained for defined media at 24 h, 30 ◦C and 250 rpm. Since COMT loses PU-23 rapidly its activity during isolation and storage [2], stabilization siRNA-mediated engineering of DNA recombination for assays were performed in order to improve the main isolation improved transgene integration and expression step by Immobilized-Metal Affinity Chromatography (IMAC) and further storage. Through a neuronal network we studied the pro- Kaja Kostyrko ∗, Nicolas Mermod tein stability in the presence of potential compounds, varying University of Lausanne, Switzerland the time and temperature at which the protein is stored. The best input was selected and applied. Then, to obtain a purified Achieving high levels of stable heterologous gene expression fraction for inhibition trials, IMAC was applied and highly puri- in eukaryotic cells is limited in part by the efficiency of trans- fied hSCOMT fractions were obtained. Finally, biophysical studies gene integration into the genome. This likely relies on one of were performed by microcalorimetry in the presence of specific the cellular DNA repair pathways – non-homologous end-joining inhibitors in order to assess kinetic profiles for human SCOMT. (NHEJ), homologous recombination (HR), or on less well charac- Acknowledgements: A.Q. Pedro acknowledges a doctoral terized microhomology-mediated end-joining (MMEJ) pathways. fellowship (SFRH/BD/81222/2011) from Fundação para a Ciên- However, the exact molecular basis of this process is still not fully cia eTecnologia. This work was partially funded by FEDER understood. funds through Programa Operacional Factores de Competitividade In the effort to identify the transgene genomic integra- – COMPETE:FCOMP-01-0124-FEDER-027563 with the project tion mechanism we silenced important DNA recombination EXPL/BBB478/BQB/0960/2012. repair components in Chinese hamster ovary (CHO) cells using small interfering RNA (siRNA), and analyzed the integration http://dx.doi.org/10.1016/j.nbt.2014.05.942 and expression of transgene-containing plasmid DNA in these cells. In addition, we used vectors bearing matrix attachment regions (MARs), genetic elements commonly used to increase and PU-22 stabilize recombinant gene expression, which were recently pro- Directed evolution to remove expression and secre- posed to also play a role in DNA recombination. tion bottlenecks of heterologous enterokinase in We find that knock-down of the NHEJ pathway components Escherichia coli does not affect transgene integration or expression, while silencing the HR pathway increases both. We also show that the use of a MAR Weiluo Lee ∗, Paul Dalby element stimulates increased transgene integration and expres- University College London, Biochemical Engineering, United sion. However, in the absence of the HR pathway, MAR induces Kingdom an even higher expression, without further increasing integration. Instead, we observe an increased expression per individual gene Bovine enterokinase catalytic subunit (EK ) has significant cat copy. Therefore, we propose that HR knock-down, as well as the industrial potential for selective modification of therapeutic MAR element, stimulate an alternative repair pathway, most likely proteins. The enzyme is typically expressed heterologously in MMEJ, which has a beneficial influence on transgene integration eukaryotic cell strains such as Pichia pastoris to ensure proper and resulting expression. This knowledge should help engineer glycosylation and folding into its soluble active form. EK produc- cat cells for improved recombinant protein production. tion in E. coli offers several advantages (i.e. shorter fermentation times, undemanding growth conditions, and favourable pro- http://dx.doi.org/10.1016/j.nbt.2014.05.944

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PU-24 peroxidases and laccases, which act cooperatively to degrade lignin macromolecules. MAR elements and transposons for improved transgene In this research project, we aim to develop a synthetic biolog- integration and expression ical platform to optimize the degradation process and products Déborah Ley 1,∗, Valérie Le Fourn 2, Pierre-Alain Girod 2, Alexandre of lignin disruption. To this end, several nucleotide sequences Regamey 2, Nicolas Mermod 1 encoding a putative lignin-degrading enzyme were identified from GeneBank at the National Center for Biotechnology Informa- 1 Institute of Biotechnology, University of Lausanne, Switzerland tion (NCBI) based on its putative domain structure and sequence 2 Selexis SA, Geneva, Switzerland similarity to reported lignolytic enzymes. Codon usage of the iden- A critical step for protein production in biotechnology or tified genes was optimized for Pichia pastoris and Saccharomyces expression of a therapeutic gene in gene therapy is the ability cerevisiae to enable high-level protein expression, and heterolo- to achieve consistent gene expression into the target cells. This gous production of the optimized genes was investigated in the implies transgene integration in a locus which allows its efficient two different yeast expression platforms. Furthermore, enzymatic and sustained expression. Among the wide variety of non-viral vec- properties of the expressed proteins were characterized in order tors used to deliver DNA, transposon systems have the advantage to select lignolytic enzymes with the desired properties for the to provide increased frequencies of single-copy integration into future development of synthetic microbes for controlled lignin the host genome. However, as with other methods of transgene degradation. delivery, the use of transposon-based systems are not immune to the risks of silencing and insertional mutagenesis. These unwanted http://dx.doi.org/10.1016/j.nbt.2014.05.946 events may be limited by the use of genetic elements, called matrix attachment region (MAR). Transgenes associated with MARs have PU-26 been shown to have a higher and more stable expression and are less prone to silencing. Here, we assessed the effect a piggyBac (PB) Metabolic model-based prediction of engineering targets transposon containing the MAR-1-68 in CHO cells. Using this com- for increased production of heterologous proteins bination, it was possible to obtain an enriched transgene-positive Justyna Nocon 1,∗, Matthias G. Steiger 2, Martin Pfeffer 3, population harbouring few integrations sufficient for high trans- Seung Bum Sohn 4, Tae Yong Kim 4, Hannes Rußmayer 5, Ste- gene expression, without the need of selection pressure. As a proof fan Pflügl 3, Christina Haberhauer-Troyer 6, Karin Ortmayr 6, of feasibility for biotechnology applications, we demonstrated the Gunda Köllensperger 6, Brigitte Gasser 2, Sang Yup Lee 7, Diethard ability of the system to obtain significant amounts of therapeutics Mattanovich 5 proteins from unselected cell populations 2–3 weeks after transfection. Since antibiotic-enforced selection protocols often result 1 University of Natural Resources and Life Sciences, Austria in a higher integrated copy number and mosaic expression pat- 2 Austrian Centre of Industrial Biotechnology, Vienna, Austria terns, this strategy could benefit many applications in which low 3 Department of Biotechnology, University of Natural Resources integrated copy number and antibiotic-free conditions are desired. and Life Sciences Vienna, Austria 4 Bioinformatics Research Center, Korea Advanced Institute of Sci- http://dx.doi.org/10.1016/j.nbt.2014.05.945 ence and Technology (KAIST), Daejeon, South Korea 5 Centre of Industrial Biotechnology, Vienna, Austria 6 Department of Chemistry, University of Natural Resources and PU-25 Life Sciences Vienna, Austria Exploration and characterization of novel lignin degrad- 7 Korea Advanced Institute of Science and Technology (KAIST), ing enzymes for a synthetic biology platform to aid Daejeon, South Korea enzymatic lignin disruption The production of recombinant proteins can be enhanced Takanori Furukawa 1,∗, Aaron McKerracher 2, Franck Escalettes 2, by influencing transcription, optimizing codon usage, increasing Reuben Carr 2, Loise Horsfall 1 protein folding and secretion. Overproduction of heterologous proteins, however, also directly affects the primary metabolism 1 The University of Edinburgh, United Kingdom of the host organism. The incorporation of recombinant protein 2 Ingenza Ltd, United Kingdom production into the genome scale metabolic model of the yeast Lignin is the most abundant aromatic polymer on earth Pichia pastoris, allowed the simulation of the effects of overpro- derived from photosynthesis and considered to be potential duction. Gene targets for deletion or overexpression for enhanced feedstock for the production of renewable aromatic chemicals. productivity were predicted. Overexpression targets were local- However, due to its structural nature, it exhibits high resistance ized in the pentose phosphate pathway and the TCA cycle, while towards chemical and biological degradation, and this is a major knockout targets were found in several branch points of glycoly- obstacle for achieving efficient conversion of lignin into value- sis. Five out of 9 tested targets led to an enhanced production of added products. Many microorganisms including both fungi cytosolic human copper/zinc superoxide dismutase (hSOD). Ben- and bacteria have been reported to produce a wide variety of eficial mutations were mainly related to reduction of the NADP/H oxidative enzymes that are involved in the degradation of lignin. pool and the deletion of fermentative pathways. Overexpression These include lignin peroxidases, manganese peroxidases, versatile of the hSOD gene itself had a strong impact on intracellular fluxes,

S194 www.elsevier.com/locate/nbt New Biotechnology · Volume 31S · July 2014 RECOMBINANT PROTEIN PRODUCTION which were predicted with high accuracy by the model. Genome PU-28 scale metabolic modeling is shown to predict overexpression and Generation and application of tools for increasing deletion mutants which enhance recombinant protein production periplasmic soluble product yields of recombinant ther- with high accuracy. apeutic proteins in Escherichia coli http://dx.doi.org/10.1016/j.nbt.2014.05.947 Alexander Büttner 1,∗, Simon Stammen 1, Mark Dürkop 1, Artur Schuller 1, Lina Heistinger 2

PU-27 1 Boehringer Ingelheim RCV GmbH & Co KG, Austria 2 University of Natural Resources and Life Sciences Vienna, Austria The production of recombinant cationic a-helical antimicrobial peptides in plant cells induces the for- Soluble production of properly folded target proteins in mation of protein bodies derived from the endoplasmic Escherichia coli can avoid cumbersome protein refolding steps reticulum when compared to inclusion body (IB) production processes. Due to several advantages over the cytoplasm, the periplasmic space of Cristina Ruiz Ramirez 1,∗, Nuri Company 2, Anna Nadal 2, Jose- E. coli is the compartment of choice for production of soluble and Luis La Paz 3, Sílvia Martinez 3, Stefan Rasche 4, Stefan Schillberg 4, correctly folded disulfide-bond containing proteins. Maria Pla 2 To address bottlenecks that can prevent efficient production, 1 Institute for Food and Agricultural Technology (INTEA), Univer- secretion and folding, a toolbox consisting of different host cell sity of Girona, Spain modifications, expression cassettes and process control strate- 2 Institute for Food and Agricultural Technology (INTEA), Spain gies was created and tested for applicability. Cell engineering 3 Center for Research in Agricultural Genomics (CRAG), Spain approaches comprised, e.g., different bacterial strains, genomic 4 Fraunhofer Institute for Molecular Biology and Applied Ecology integration of the target gene expression cassette, its organiza- (IME), Aachen, Germany tion within the integration locus as well as the co-synthesis of helper factors, which improve secretion or folding of the target Synthetic linear antimicrobial peptides with cationic a-helical proteins. A range of inducible and constitutive promoter variants structures, such as BP100, are valuable as novel therapeutics and were tested concerning their applicability to control helper fac- preservatives. However, they tend to be toxic when expressed at tor gene co-overexpression. Different fermentation processes with high levels as recombinant peptides in plants, and they can be diffi- regard to temperature, feeding and induction mode and time were cult to detect and isolate from complex plant tissues because they applied. are strongly cationic and display low extinction coefficient and For several therapeutically relevant proteins of interest, we extremely limited immunogenicity. We therefore expressed BP100 showed that application of one or a combination of several of with a C-terminal tag which preserved its antimicrobial activity the developed methods increased the overall yield of the desired and demonstrated significant accumulation in plant cells. We used product. Additionally, the ratio of recombinant protein was shifted a fluorescent tag to trace BP100 following transiently expression in from insoluble (cytoplasmic and/or periplasmic aggregates) to sol- Nicotiana benthamiana leaves and showed that it accumulated in uble and correctly folded product in several cases. large vesicles derived from the endoplasmic reticulum (ER) along with typical ER luminal proteins. Interestingly, the formation of http://dx.doi.org/10.1016/j.nbt.2014.05.949 these vesicles was induced by BP100. Similar vesicles formed in stably transformed Arabidopsis thaliana seedlings, but the recombinant peptide was toxic to the host during latter developmental PU-29 stages. This was avoided by selecting active BP100 derivatives based Enhanced periplasmic production of therapeutic pro- on their low haemolytic activity even though the selected peptides teins in Escherichia coli remained toxic to plant cells when applied exogenously at high doses. Using this strategy, we generated transgenic rice lines pro- Simon Stammen 1,∗, Alexander Buettner 1, Lina Heistinger 2, Mark ducing active BP100 derivatives with a yield of up to 0.5% total Duerkop 1, Arthur Schuller 1, Franz Kollmann 1 soluble protein. 1 Boehringer Ingelheim RCV GmbH, Austria 2 University of Natural Resources and Life Sciences, Austria http://dx.doi.org/10.1016/j.nbt.2014.05.948 For manufacture of therapeutic proteins that do not require translational modification (e.g. glycosylation) for their biological activity, the production in Escherichia coli is still the host system of choice. Its well-known biology, the ability to grow on inexpensive media, the availability of genetic tools for host cell modifications and last but not least its long history as pharmaceutical work host are only the most prominent benefits. However, pharmaceutical proteins became more and more complex and therefore the requirements on the expression systems grew. Many modern, biologically active proteins contain multiple disulphide bridges,

www.elsevier.com/locate/nbt S195 RECOMBINANT PROTEIN PRODUCTION New Biotechnology · Volume 31S · July 2014 what makes classical production routes (e.g. production as inclu- cellulase master regulator xyr1, suggesting interplay between these sions bodies and subsequent in vitro refolding) more and more factors. challenging. Even though recent advances in folding technology (e.g. high pressure refolding) enable refolding in commercially rel- http://dx.doi.org/10.1016/j.nbt.2014.05.951 evant yields for many target proteins, Boehringer Ingelheim (BI) also significantly increased its efforts in production of properly PU-31 folded target proteins in the E. coli periplasm. We present several approaches to increase the amount of Effects of K87 mutations in the alpha-crystallin domain soluble therapeutic protein within the periplasmic space of of Tpv sHSP 14.3 on oligomerization and in vitro chaper- the bacterial cell. Application of BI’s expression system, which one activity makes use of a genomically integrated product gene signif- ∗ icantly increased the titre of properly folded target protein Semra Kocabiyik , Ilir Sharej when compared to one of the typically used plasmid-based Middle East Technical University, Turkey expression systems. Furthermore, the secretion and refolding machinery was supported by co-overproduction of different Small heat shock proteins (sHSPs) act as molecular chaperones helper proteins and chaperones to enhance cellular productivity. by binding denaturing proteins and protecting them from aggre- The fermentation process was optimized with regard to pro- gation before refolding. Their level is up-regulated in particular cess variables to enhance the yield of properly folded target events, such as stress and pathological conditions. ACD is the sig- protein. nature of the sHSP monomer which is flanked by N-terminal arm of variable sequence and C-terminus with a conserved I/L-X-I/L ␤ http://dx.doi.org/10.1016/j.nbt.2014.05.950 motif. Seven or eight -strands in ACD arranged into antiparallel sheets constituting ␤-sandwich which mediates dimerization. The sHSPs form reversible homo- or hetero-oligomers indicating the PU-30 dynamic plasticity of the quartenary structure that modulate the recognition of substrate. Mutations in the ACD occur in positions Identification of a novel master regulator of cellu- that would have impact on this dynamic behavior. One of the lase and hemicellulase production in Trichoderma reesei well studied mutations is R120G in human CRYAB that causes using genome-wide approach congenital cataract and desmin related myopathy. At the dimer ∗ Mari Häkkinen, Mari Valkonen , Ann Westerholm-Parvinen, interface R120 makes two interface ion pairs with D109. The Nina Aro, Marika Vitikainen, Merja Penttilä, Markku Saloheimo, aim of this study is to determine if the topological equivalent Tiina Pakula of this residue has the same impact on oligomeric structure of non-metazoan sHSPs. We investigated the effect of substitutions VTT Technical Research Centre of Finland, Finland at K87 position of Tpv HSP14.3 (an archaeal sHSP) on oligomer- Trichoderma reesei (anamorph Hypocrea jecorina) is an efficient ization and chaperone activity. The K87R and R87I mutations producer of enzymes degrading lignocellulosic biomass. The cel- resulted in increased chaperone activity when citrate synthase lulases and hemicellulases produced by the fungus are widely was used as the client. Increased substrate binding capacity due to employed in industry, and this production system has a central role R87I mutation can be explained by increased hydrophobicity. The in biorefinery applications. Various environmental and metabolic oligomere size of all mutant sHSPs increased at the temperature factors together with the physiological state of the cell affect the from 25 to 60 ◦C. The notable increase was observed at 25 ◦C for enzyme production in T. reesei. In previous studies, both positively the K87I mutant particles. and negatively acting regulatory factors for cellulase and hemicellulase genes have been characterised in T. reesei. http://dx.doi.org/10.1016/j.nbt.2014.05.952 In this study, an expression microarray data on T. reesei cultivated in the presence of different carbon sources was analysed PU-32 in order to identify additional regulatory genes for cellulase and hemicellulase production. In total, 28 putative regulatory Development of a “toolbox” approach for recombinant factors were chosen to be over-expressed in T. reesei based on the protein production fact that they were induced by lignocellulosic substrates. In the 1,∗ 2 1 primary screening, over-expression of seven of these factors led Tania Selas Castineiras˜ , Steven G. Williams , Jeff A. Cole ,Tim 1 2 to increased production of cellulases and/or xylanases. Among W. Overton , Anthony Hitchcock these factors is a novel master regulator of the cellulose and hemi- 1 University of Birmingham, United Kingdom cellulose genes designated ace3. Its over-expression increased 2 Cobra Biologics Ltd, United Kingdom cellulase production 2 to 4-fold and also enhanced hemicellulase The high demand of recombinant proteins produced in micro- production. Its deletion abolished cellulase production totally, bial hosts, which accounts for more than 30% of the biological decreased hemicellulase production, and in the ace3 deletion products in the market, have reinforced the necessity of the strains the major cellulase gene transcripts were at extremely development of more efficient processes for the production of low levels. Interestingly, the modifications of ace3 also affected recombinant proteins. Higher production efficiencies and lower the mRNA levels of the previously identified hemicellulase and cost have become essential pre-requisites for a commercially viable

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process. Optimisation of fermentation conditions to decrease cell PU-34 stress has been shown to favour the accumulation of soluble and Genetic engineering of Saccharomyces cerevisiae for effi- active recombinant proteins [1]. cient IgG-assembly and secretion We have shown that shake-flask studies are an appropriate tool for the optimisation of fermentation conditions, reducing the Essi Koskela ∗, Alexander Frey time and cost involved in process development. This approach Aalto University, Finland was used for the production of tumour necrosis factor ␣ (TNF␣) in E. coli using the arabinose-inducible T7 expression system. A IgG-antibodies are complex molecules that require multiple ele- − volumetric yield of 3.82 g L 1 of TNF␣ was achieved by fermen- ments to assemble efficiently, including folding factors and an tation, 92% being soluble and active. The proven success of this oxidative environment. The growing demand for biosimilars and approach can be now applied to a broader range of recombinant alternative treatments makes the production process of human proteins. antibodies an intriguing area of research. Although simple micro- We will discuss the selection of adequate expression systems bial expression platforms such as Saccharomyces cerevisiae are not and approaches for the optimisation of cultivation conditions as intrinsically suited for IgG-production, the tools to genetically key factors for the production of recombinant proteins. The main modify the yeast cells for this purpose are versatile. goal of this project is to test and integrate fermentation conditions To improve the secretion of produced protein, we focus on allowing the design of platforms following a “toolbox approach” modifying ER luminal environment and protein folding process. for protein production. One important approach is increasing the size of ER, which can Reference be achieved with a single gene knock-out [1]. The extra space is supplemented with additional folding factors important in [1].Sevastsyanovich Y, Alfasi S, Overton T, et al. Exploitation of GFP IgG-tetramer assembly including molecular chaperones, PDI and fusion proteins and stress avoidance as a generic strategy for the production of high-quality recombinant proteins. FEMS Microbiol Lett PPIases. These factors are tested in different amounts and combi- 2009;299:86–94. nations and the strains producing the highest yields are identified with high-throughput screening. http://dx.doi.org/10.1016/j.nbt.2014.05.953 Genetic engineering requires a substantial amount of labor- intensive cloning work. To optimize this aspect, we recently discovered a new approach to molecular cloning, which outcom- PU-33 petes many of the existing cloning methods in simplicity and In vivo reconstitution of membrane protein by caveolin1 affordability. This novel protocol is presented along with pre- co-expression liminary results of genetic modifications on IgG-secretion. With suitable high-throughput methods in hand, the microbial plat- ∗ Jonghyeok Shin , Paul Heo, Joon-Bum Park, Myungseo Park, form for IgG-production is optimized at the level of the whole Younghun Jung, Da-Hyeong Cho, Byoung-jae Kong, Junghoon system. In, Jichun Lee, Dae-Hyuk Kweon

Sungkyunkwan University/Bioengineering Department, South Reference Korea [1].Schuck S, a Prinz W, Thorn KS, Voss C, Walter P. Membrane expan- Caveolae is a membrane-budding structure which exists in sion alleviates endoplasmic reticulum stress independently of the unfolded protein response. J Cell Biol 2009;187:525–36. many animal vertebrate cells. One of the important functions of caveolae is to form membrane curvature and endocytic vesi- http://dx.doi.org/10.1016/j.nbt.2014.05.955 cle. Recently, It was shown that caveolae could be formed in Escherichia coli by expressing caveolin-1. The heterologous caveolae may host other membrane proteins overexpressed inside the PU-35 cell. We utilized this system for construction of proteo-liposome in Escherichia coli. SNARE proteins (Syntaxin1a, SNAP25, VAMP2) Study on the domain function of Listeria monocytogenes were introduced to prove our in vivo reconstitution system. Here, p60 protein we show that the purified heterologous caveolae indeed contain Minliang Guo ∗, Hao Gu, Qian Xu, Jinrong Zuo the co-expressed membrane protein and the membrane proteins were facing outward. The size of the purified caveolae with mem- College of Bioscience and Biotechnology, Yangzhou University, brane protein reconstituted were measured by dynamic light China scattering. The presence of VAMP2 & Syntaxin1a on this proteo- Listeria monocytogenes p60 protein is an autolysin that can endosome was confirmed by Western blot analysis. Furthermore, hydrolyze the peptidoglycans of bacterial cell walls. L. monocy- membrane proteins (VAMP2 & Syntaxin1) embedded in caveolae togenes p60 protein was required for L. monocytogenes virulence. retained the ability to form SNARE complex. Our study proposes Besides the importance of p60 protein in bacterial pathogenesis, an in vivo membrane protein reconstitution system. p60 protein can also be developed as a new proteinaceous antimicrobial if its activity to hydrolyze the peptidoglycans can be greatly http://dx.doi.org/10.1016/j.nbt.2014.05.954 improved. It contains two independent structural domains, N- terminal LysM domain and C-terminal NlpC/P60 domain, which

www.elsevier.com/locate/nbt S197 RECOMBINANT PROTEIN PRODUCTION New Biotechnology · Volume 31S · July 2014 can be separated at the amino acid residue 270. However, the active PU-37 functions of these two domains in p60 protein and their influences Extracellular transaminases for biocatalysis on the substrate recognition and catalytic activity of p60 protein are unknown. Here we identified both of the functional hot spot Katrin Weinhandl 1,∗, Margit Winkler 1, Anton Glieder 1, Andrea and the mutational hot spot amino acid residues in these two struc- Camattari 2 tural domains by means of the amino acid sequence alignment of 1 Austrian Center of Industrial Biotechnology (ACIB), Austria different p60 variants, including two p60 variants screened in our 2 TU Graz, Austria lab. The functional hot spot and the mutational hot spot amino acid residues were substituted to alanine (A) by using site-directed Branched chain aminotransferase (BCAT, EC 2.6.1.42) of mutation to construct p60 variants. These p60 variants in combi- Escherichia coli is an intracellular protein and an interesting tool nation with some truncated p60 proteins were used to unveil the for the production of chiral amines or amino acids. Secretion of molecular mechanism of substrate recognition and catalysis of p60 BCAT to the culture supernatant was the method of choice to facili- protein in the domain level. Results confirmed that the N-terminal tate industrial enzyme applications and downstream processing by LysM domain in p60 protein could bind to the bacterial cell wall whole cell applications while counteracting limited cell permeabil- tightly, whereas the C-terminal NlpC/P60 domain showed slight ity for target substrates. Pichia pastoris was chosen as expression ability to hydrolyze the cell walls. These fundamental studies on host because of its positive characteristics, such as the ability to p60 protein variants will provide strong support for engineering reach high biomass levels as well as the lack of background proteins the p60 protein molecular. in the extracellular environment during expression. Although secretion of intracellular proteins was reported to be http://dx.doi.org/10.1016/j.nbt.2014.05.956 problematic in the past, we were able to secrete BCAT in Pichia pastoris and obtained a maximum activity level in the supernatant of 150 ␮mol/min/mg total protein (L-leucine conversion in a coupled PU-36 enzymatic assay [1]). GlycoDelete technology: shortcutting mammalian cell N- In order to improve the expression level, several approaches glycosylation were investigated: on the one hand we examined different Pichia strains. On the other hand, alternative signal peptides and dif- Francis Santens ∗, Leander Meuris, Morgane Boone, Nico Calle- ferent promoters were evaluated for improved expression and waert secretion of BCAT. In our hands methanol-induced expression lead VIB Ghent University, Belgium to a higher activity in the supernatant, compared to constitutive expression which still allowed satisfying BCAT secretion. Mammalian complex-type N-glycan synthesis is a multi-step process that results in heterogeneous glycosylation of proteins. Reference Heterogeneity in therapeutic glycoproteins causes difficulties for protein purification and process reproducibility and can lead to [1].Weinhandl, et al. Tetrahedron 2012;68(37):7586–90. variable therapeutic efficacy. Here we report engineered mammalian cell lines that have a shortened Golgi N-glycosylation http://dx.doi.org/10.1016/j.nbt.2014.05.958 pathway, which leads to the expression of proteins with small, sialylated trisaccharide N-glycans. This glycoengineering strategy, PU-38 which we call GlycoDelete [1], results in proteins with substantially reduced glycan heterogeneity. To assess the potential of these A method to stably integrate multiple genetic elements GlycoDelete glycans and their influence on glycosylated phar- into Chinese hamster ovary (CHO) cells maceutical proteins, human GM-CSF and an anti-CD20 antibody ,∗ Sabine Vcelar 1 , Martina Baumann 1, Nicole Borth 2 were produced in 293s and 293sGlycoDelete cells. Both proteins were purified and thoroughly analysed. For hGM-CSF we did not 1 ACIB - Austrian Centre of Industrial Biotechnology, Austria see a significant influence of the GlycoDelete sugars on the activ- 2 Department of Biotechnology, University of Natural Resources ity of the protein. GlycoDelete anti-CD20 on the other hand has and Life Sciences, Vienna, Austria a significantly reduced Fc␥R affinity and an increased circulation CHO cells are the preferred host cells for the production times in mice compared to 293S produced anti-CD20. of therapeutic proteins and the most commonly used mam- Reference malian expression system. Advantages such as an easy cultivation, [1].MeurisL, Santens F, Elson G, et al. GlycoDelete engineering of mam- fast growth, complex protein folding and human-like post- malian cells simplifies N-glycosylation of recombinant proteins. Nat translational modifications are in part set of by slow cell line Biotechnol 2014, advance online publication. and process development. These constraints lead to an increased requirement for CHO cell line modification tools. http://dx.doi.org/10.1016/j.nbt.2014.05.957 The present work focuses on the integration of up to four genetic elements into CHO cells. Two different approaches were established. The principle of both systems is the Recombinase- mediated cassette exchange (RMCE). Systems A is engineered on the basis of transfection vectors comprised of different resistance

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markers and two cloning sites. The incorporation of the genetic PU-40 elements of choice is mediated by RMCE at two specific genomic The employment of a heterologous yeast expression sys- locations. The second system established combines the RMCE system for production of VP1-derived virus-like particles tem with the R4 Integrase System to enable integration of up to originated from novel human polyomaviruses four genes at a single specific genomic location. The stable integration of DNA elements of choice at specific Alma Gedvilaite ∗, Milda Norkiene, Rita Lasickieke genomic locations and the testing of candidate genes for phe- Vilnius University Institute of Biotechnology, Lithuania notypic effects without disturbance by clone specific variation is getting feasible with these methods. Virus-like particles (VLPs) resemble their parent virion in structure, immunogenicity, tropism and transduction efficiency, but http://dx.doi.org/10.1016/j.nbt.2014.05.959 do not contain any viral genetic material. They can be used for diagnostic purposes, vaccination and gene therapy. Polyomaviri- dae is a growing family of naked, double-stranded DNA viruses PU-39 that infect birds and mammals. The major capsid protein VP1 of Construction of pH-sensitive Her2 binding antibody all polyomaviruses (PyV) is sufficient for assembly of VLPs and fragment by directed evolution using yeast display represents the major immunogenic protein of PyV. In the last few years, the human polyomavirus (HPyV) family has expanded to 12 Elisabeth Lobner ∗, Michael Traxlmayr, Florian Rüker, Christian members. Serological studies are the primary tool to investigate the Obinger prevalence of various polyomaviruses in human populations. The University of Natural Resources and Life Sciences, Vienna, Austria recombinant VP1 VLPs are particularly valuable for the serological detection of these viruses as many PyV cannot be easily cultured. The half-life of therapeutic antibodies, which are internal- The earlier discovered PyV VP1-derived VLPs were successfully ized together with their antigenic receptor, can be increased by produced using different eukaryotic and prokaryotic expression decreasing their affinity at acidic endosomal conditions. Here, a systems including yeast. Here, we report that the galactose- directed evolution protocol was developed for construction of pH- inducible yeast S. cerevisiae expression system is efficient for dependent binding sites by using yeast display. The C-terminal high-level production and self assembly of VP1 derived from new structural loops of an antigen binding crystallizable fragment of HPyV: KIPyV, WUPyV, Merkel cell PyV, HPyV6 and HPyV7. The immunoglobulin G1 (Fcab) [1] have been engineered for reduced formation of empty VP1-derived VLPs was confirmed by cesium binding to the extracellular domain of human epidermal growth chloride ultracentrifugation, agarose gel electrophoresis and elec- factor receptor 2 (Her2-ECD) at pH 6 compared to pH 7.4. A tron microscopy. Yeast-generated VP1 VLPs were free of toxins, library based on a Her2-ECD binding lead Fcab was constructed host cell DNA and proteins. The purified VP1 VLPs originating by parsimonious mutagenesis and displayed on yeast. Alternating from KIPyV, WUPyV, Merkel cell PyV, HPyV6 and HPyV7 were suc- selections for binding at pH 7.4 and non-binding at pH 6.0 were cessfully used for generation of monoclonal antibodies and might performed by FACS probing the binding to the antigen as well as be useful for the generation of new diagnostic tools, antiviral vac- a structurally specific ligand. The three best performing variants cines or gene delivery systems. (P1, P2, P3) were selected. Displayed on yeast they showed clear pH-dependent binding to soluble Her2-ECD (decrease in affinity http://dx.doi.org/10.1016/j.nbt.2014.05.961 at pH 6.0 compared to pH 7.4). Additionally, solubly expressed P1, P2 and P3 exhibited pH-dependent interactions with Her2- positive cells whereas their conformational and thermal stability PU-41 was pH-independent. The interaction of P1, P2 and P3 with the Purification by affinity chromatography of recombi- neonatal Fc receptor remained wild-type like showing the inverse nant L-asparaginase I from Saccharomyces cerevisiae pH-dependence compared to Her2-ECD binding. Interestingly, expressed in Escherichia coli two of the three Fcabs did not contain a single histidine mutation but all of them contained variations next to histidines that Adalberto Pessoa-Jr 1,∗, Gisele Monteiro 1, Joao Santos 2, Johanna already occurred in loops of the lead Fcab. Oses 1, Albert Peixoto 3, Juan Santos 1, Joao Molino 1, Laura 4 2 2 1 Reference Oliveira , Joao Coutinho , Sonia Ventura , Andre Lopes 1 [1].Wozniak-Knopp, et al. Protein Eng Des Sel 2010;23:289–97. University of Sao Paulo, Brazil 2 University of Aveiro, Brazil http://dx.doi.org/10.1016/j.nbt.2014.05.960 3 Universidade Estadual do Sudoeste da Bahia, Brazil 4 University of Campinas, Brazil

L-Asparaginase is known by its capacity to catalyse the hydrolysis of L-asparagine into L-aspartic acid and ammonia. This enzyme has been clinically acceptable as an anti-tumour agent for treatment of acute lymphoblastic leukemia and lymphosarcoma. This biopharmaceutical is produced by fermentation processes and subsequently it needs to be correctly separated from the contaminants

www.elsevier.com/locate/nbt S199 RECOMBINANT PROTEIN PRODUCTION New Biotechnology · Volume 31S · July 2014 from the fermentation broth. Here, Affinity Chromatography is heterologous protein secretion, possibly by cluttering the ER with applied as a novel approach to purify (His)6-tagged recombi- an overload of unfolded proteins. nant L-asparaginase I from Saccharomyces cerevisiae expressed in Escherichia coli. The Affinity Chromatography was performed on a http://dx.doi.org/10.1016/j.nbt.2014.05.963 Fast Protein Liquid Chromatography (FPLC) system using a Ni2+- charged, 5 mL HiTrap IMAC FF. A linear gradient of 0 mM to PU-43 500 mM of imidazole at 5.0 mL min−1 was applied in order to identify the lowest imidazole concentration of the elution buffer that Expression of C-terminal Fab fragment variants: the host extracts the higher amount of L-asparaginase. This concentration determines the best variant range was further used in a step imidazole gradient. Two-step con- Brigitte Gasser 1, Rebecca Goengrich 1,∗, Christoph Kiziak 2, centrations of the initial imidazole concentration (32.0 and 54.4%) Diethard Mattanovich 1 were applied. Polyacrylamide gel electrophoresis combined with silver staining and Nessler activity assay were used to confirm the 1 Department of Biotechnology, BOKU University of Natural presence of the purified L-asparaginase (≈45 kDa). In the gradi- Resources and Life Sciences, Vienna, Austria ent step, the eluted recombinant L-asparaginase showed a high 2 Lonza AG, Visp, Switzerland specific activity of 110.1 ± 0.3IU mg−1. Furthermore, a recovery of Expression of antibody fragments is routinely done in microbial 81.00 ± 0.01% was obtained resulting in a purification factor of 17. host cells such as the bacterium Escherichia coli or the yeast Pichia In this respect, the FPLC process has undoubtedly proved to be an pastoris. In yeast, Fabs are usually secreted to the cell supernatant efficient tool for the purification of this enzyme. after passing the secretory pathway, while Fab expression in E. coli Acknowledgements: This research was supported by is usually done in the periplasm. Different Fab variants have been CAPES, CNPq (301248/2010-9) and FAPESP (2013/08617-7), reported in literature, however, it has not yet been comprehen- Brazil; FEDER funds through the program COMPETE/FCT (Pest- sively assessed which work best in which host. Here we compare C/CTM/LA0011/2013); SFRH/BPD/79263/2011; and Santander. different Fab variants expressed in E. coli and P. pastoris, which resulted in a preference for different variants in each host. http://dx.doi.org/10.1016/j.nbt.2014.05.962

http://dx.doi.org/10.1016/j.nbt.2014.05.964 PU-42

Analysis of modiﬁcations in the ER protein quality PU-44 control system on heterologous protein expression in Cell-free incorporation of unnatural amino acids Saccharomyces cerevisiae for cloning-independent engineering of elastin-like ∗ Jorg de Ruijter , Essi Koskela, Alexander Frey polypeptides

Aalto University, Finland Dong-Myung Kim 1,∗, Su-Jin Oh 2, Young-A Son 3

The yeast Saccharomyces cerevisiae is a widely used host in the 1 Department of Fine Chemical Engineering and Applied Chem- production of therapeutic glycoproteins. However, the expression istry, Chungnam National University, South Korea of high levels of heterologous proteins is known to interfere with 2 Department of Fine Chemical Engineering and Applied Chem- various processes in the cells and can trigger stress responses. istry, Chungnam National University of Korea, South Korea High volume production of heterologous glycoproteins is prone 3 Department of Advanced Organic Materials Engineering, Chung- to overﬂow the ER with unfolded proteins. Glycoproteins that nam National University of Korea, South Korea remain unfolded in the ER for too long are predisposed to be The physicochemical nature of a protein is determined by the targeted for degradation by components of the ER associated degra- collective properties of consisting amino acids and their sequential dation (ERAD) pathway. In this present study, the ERAD pathway order in the protein molecule. Although natural proteins exhibit was modiﬁed by deleting genes encoding various components, a wide array of structure and biological functions with the concise with and without a blocking of the unfolded protein response set of 20 canonical amino acids, the ability to introduce unnat- pathway (UPR). In addition, overexpression of the folding chaper- ural amino acids into protein molecules will further expand the ones KAR2, PDI1, ERO1 and CPR5 was introduced. These proteins diversity, creating protein species that have never been explored. are expected to assist in the quick folding of proteins to decrease In this study, we demonstrate the use of a cell-free protein syn- the blocking of the ER by unfolded proteins. thesis system as a versatile platform for synthesis of engineered The production levels of a human IgG1 molecule of these bio-materials through the incorporation of unnatural amino acids mutant strains were analyzed using ELISA. Since changing the into recombinant proteins. Elastin-like-polypeptides (ELPs) were ER quality control system might lead to a decreased quality of chosen as a model protein to investigate the effects of unnatural proteins, the quality of the produced IgG1 was analyzed by PAGE. amino acid incorporation. Herein, we discuss on the manipulation The results at hand indicate that deletion of early ERAD fac- of the transition temperature of ELPs through cell-free incorpo- tors is detrimental for protein production, as that removal of later ration of proline analogues. Through the replacement of proline stages leads to an increase. For the folding chaperones, control of expression levels is key, as high level expression tend to decrease

S200 www.elsevier.com/locate/nbt New Biotechnology · Volume 31S · July 2014 RECOMBINANT PROTEIN PRODUCTION with a variety of its structural analogues, we were able to tune the PU-46 transition temperature of ELPs. Analysis of amino acid residues in the N-terminal region of the beta-neurotoxin CssII and its speciﬁcity for http://dx.doi.org/10.1016/j.nbt.2014.05.965 voltage-gated Na+ channel subtypes

Rodrigo Ibarra Vega 1,∗, Juana María Jiménez Vargas 2, Rita Restano PU-45 Cassulini 2, Georgina Estrada 3, Lourival D. Possani 2, Gerardo Enhancing recombinant protein production with an Corzo 2 Escherichia coli host strain lacking insertion sequences 1 Instituto de Biotecnología, Universidad Nacional Autónoma de Sun-Chang Kim ∗, Myung Keun Park, Jun Hyoung Lee, Kyung México, UNAM, Mexico Seok Yang 2 Instituto de Biotecnología de la Universidad Nacional Autónoma de México, UNAM, Mexico Korea Advanced Institute of Science and Technology, South Korea 3 Centro de investigación Científica de Yucatán, AC. (CICY), The genomic stability and integrity of host strains are criti- Mérida, Yucatán, México, Mexico cal for the production of recombinant proteins in biotechnology. The scorpion toxin CssII from the scorpion Centruroides suffusus Bacterial genomes contain numerous jumping genetic elements, is a peptide that affects the voltage-gated sodium channel Nav 1.6. the insertion sequences (ISs) that cause a variety of genetic rear- The CeII9 peptide toxin of Centruroides elegans is 86% identical to rangements, resulting in adverse effects such as genome and CssII but selectively affects Nav 1.4. The most significant variation recombinant plasmid instability. To minimize the harmful effects between these two peptide toxins is observed at the N-terminus, of ISs on the expression of recombinant proteins in Escherichia coli, specifically in residues 7 and 8. Therefore, these two residues in we developed an IS-free, minimized E. coli strain (MS56) in CssII were substituted for residues corresponding to the peptide which about 23% of the genome, including all ISs and many CeII9 to evaluate if the CssII variants keep or shift their selectiv- unnecessary genes, was removed. Here, we compared the expres- ity for the Nav1.6 or Nav1.4, respectively. Three CssII variants sion profiles of recombinant proteins such as tumor necrosis were generated by site-directed mutagenesis, named CssII K8H, factor-related apoptosis-inducing ligand (TRAIL) and bone mor- CssII S7N and CssII S7N/K8H. Since the CssII native toxin has an phogenetic protein-2 (BMP2) in MG1655 and MS56. Hopping of amidation at the C-terminal and this amidation is important for ISs (IS1, IS3, or IS5) into the TRAIL and BMP2 genes occurred at binding the Navs, the aforementioned variants were constructed the rate of ∼10−8/gene/h in MG1655 whereas such events were not but including basic charges at the C-terminal (T64R/N66R). These observed in MS56. Even though IS hopping occurred very rarely substitutions improve the affinity of the CssII recombinant toxins (10−8/gene/h), cells containing the IS-inserted TRAIL and BMP2 for the Nav1.6. The synthetic genes were cloned into the pQE30 plasmids became dominant (∼52% of total population) 28 h after vector and expressed in E. coli BL21 strain. The recombinant vari- fermentation due to their growth advantage over cells containing ants had an N-terminal fusion peptide composed of a 6His-tag intact plasmids, significantly reducing recombinant protein pro- and a FXa proteolytic cleavage region. The mutants were puri- duction in batch fermentation. Our findings clearly indicate that fied, folded in vitro, digested with FXa and electrophysiological IS hopping is detrimental to the industrial production of recom- assayed on HEK293 cells stably expressing the Nav1.4 and Nav1.6 binant proteins, emphasizing the importance of the development channels. of IS-free host strains.

http://dx.doi.org/10.1016/j.nbt.2014.05.967 http://dx.doi.org/10.1016/j.nbt.2014.05.966

PU-47

Expression of recombinant formate dehydrogenase in Escherichia coli and its immobilization onto magnetic nano-particles

Stanislav Stuchlik 1,∗, Lukas Hason 2, Zdenko Levarski 2, Lucia Bocanova 2, Kristina Jirickova 2, Diana Hopkova 2, Pavol Kois 3, Jan Turna 2

1 Comenius University in Bratislava, Department of Molecular Biology, Slovakia 2 Comenius University in Bratislava, Faculty of Natural Sciences, Department of Molecular Biology, Slovakia 3 Comenius University in Bratislava, Faculty of Natural Sciences, Department of Organic Chemistry, Slovakia

The enzymatic bioconversion of aromatic compounds in food and cosmetic industry is often accompanied by the limitation in

www.elsevier.com/locate/nbt S201 RECOMBINANT PROTEIN PRODUCTION New Biotechnology · Volume 31S · July 2014 terms of co-factor depletion or its insufficient regeneration. To to simplify the synthesis of selenoproteins and therefore expand address this issue, we have constructed an efficient E. coli based their biotechnological and biopharmaceutical utilization. expression system capable of producing functional yeast formate dehydrogenase (FDH), an enzyme used for the regeneration of http://dx.doi.org/10.1016/j.nbt.2014.05.969 oxidized NAD+, during conversion of trans-2-hexenal to trans- 2-hexenol catalyzed by NAD-dependent alcohol dehydrogenase PU-49 (ADH). To further improve this reaction, we have immobilized FDH onto magnetic nano-particles and tested the ability of pro- Develop new tools for investigation of albumin transduced enzyme to improve activity of ADH by NAD+/NADH portation conversion. The activity of FDH has been tested by applying var- Dinh Khoi Nguyen Ly 1,∗, Philip Poronnik 2, David Nikolic- ious physical and chemical condition changes. The results show Patterson 3 that although the activity of the immobilized FDH is decreased compared to the free enzyme, its stability is increased signifi- 1 HiRi RMIT Australia cantly allowing prolonged incubations or repeated use of a single 2 University of Sydney Australia batch. 3 Monash Medical Center Acknowledgements: This work was supported by the Slo- Albumin is most abundant protein in blood and plays impor- vak Research and Development Agency grant APVV-0061-11 and tant homeostatic roles in the regulation of oncotic pressure and is also result of the “Production of biologically active agents based as a carrier of many small proteins, peptides and hormones. Albu- on recombinant proteins” (ITMS 26240220048) project implemen- min in the circulation can be covalently modified at surface lysine tation supported by the Research and Development Operational residues and thus the circulation contains a mixture of native Program funded by the ERDF. albumin (i.e. not modified) and modified albumin. A significant amount of albumin is lost from the blood each day as a result of the http://dx.doi.org/10.1016/j.nbt.2014.05.968 filtration occurring in the capillaries of the renal glomeruli. This filtered albumin in the early filtrate is thought to be mostly taken PU-48 up by and degraded in the early renal proximal tubular epithelial cells, while a small proportion is excreted in the final urine. Production of selenoproteins in yeast via genetically This degradation of albumin by proximal tubules operates via the encoded incorporation of a photocaged selenocysteine megalin scavenger receptor and an endocytic pathway. Rasa Rakauskaite ∗, Viktoras Masevicius, Giedre Urbanaviciute, A major gap in our knowledge is whether the megalin-based Audrone Ruksenaite, Saulius Klimasauskas albumin uptake is a non-selective process, or whether megalin has a significantly different affinity for modified versus native Vilnius University, Institute of Biotechnology, Lithuania albumin. Using yeast expression system, we develop radioactive Recent advancements in protein engineering materialized in a native recombinant albumin. The uptake experiment using 14C- number of valuable products for pharmaceutical, industrial, and rHA suggest the uptake pathway of native and modified albumin research applications. One promising way to achieve advanced are different in opossum kidney cell line. The uptake of 14C-rHA is engineered protein products is based on targeted incorporation not megalin dependent. of rare and unnatural amino acids. For instance, selenocysteine (Sec) provides a selenium atom with unique chemical char- http://dx.doi.org/10.1016/j.nbt.2014.05.970 acteristics (higher nucleophilicity, lower pKa, and lower redox potential) not attainable in common proteins. Although Sec is of significant technological importance as a component of both natural proteins and designed biocatalysts, the availability of such proteins is hampered by technical limitations. Here we report a universal method for production of recombinant selenoproteins in Saccharomyces cerevisiae cells via genetically encoded SECIS-independent incorporation of a photocaged unnatural amino acid, 4,5-dimethoxy-2-nitrobenzyl selenocysteine (DMNB- Sec). Photodeprotection of the incorporated residue in the target selenoprotein can be performed under mild conditions. The new approach was validated by inserting a Sec residue i) at a permissive position (Tyr39) of enhanced green fluorescent protein (EGFP) and ii) replacing an essential catalytic cysteine (Cys103) of a bacterial DNA cytosine-5 methytransferase, M.HpaII. Photodeprotection of the incorporated DMNB-Sec residue in the model EGFP protein demonstrated light-controlled protein dimerization and efficient Sec-specific covalent labeling in vitro. This method has a potential

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Stress responses PV-02

PV-01 Overview of the biomedical research in the newly established Laboratory of Genome Integrity at the Palacky Preliminary analysis of DNA isolated from flax grown University, Olomouc in the radio-contaminated Chernobyl area for six gen- ∗ erations suggests the stability of fatty acid desaturase Zuzana Loubalová , Kamila Jahodíková, Martin Liptay genes Institute of Molecular and Translational Medicine Palacky Univer- Veronika Lancíková 1,∗, Jana Ziarovskáˇ 2, Maksym Danchenko 3, sity Olomouc, Czech Republic Valentyna Berezhna 4, Milan Bezoˇ 2, Katarína Raznáˇ 2, Namik The Laboratory of Genome Integrity (LGI) is part of the recently Rashydov 4, Martin Hajduch 5 opened Institute of Molecular and Translational Medicine belong- 1 Institute of Plant Genetics and Biotechnology of Slovak Academy ing to the Palacky University in Olomouc, Czech Republic. The of Sciences, Slovakia institute was built with the aim to conduct cutting edge research 2 Department of Genetics and Plant Breeding, Faculty of Agrobiol- in the field of drug design and discovery. The collaborative net- ogy and Food Resources, Slovak University of Agriculture, Slovakia work at the institute covers all crucial phases of the drug design 3 Institute of Virology, Slovak Academy of Sciences, Institute of Cell process including search for novel drug targets. Biology and Genetic Engineering, National Academy of Sciences The research in the LGI is focused mainly on various mechanis- of Ukraine, Slovakia tic aspects of the DNA damage response and DNA repair pathways. 4 Institute of Cell Biology and Genetic Engineering, National Both pathways play a key role in the onset and progress of cancer Academy of Sciences of Ukraine, Slovakia and thus represent ideal targets for novel anti-cancer drugs. 5 Institute of Plant Genetics and Biotechnology, Slovak Academy In addition, we have been trying to understand how elevated of Sciences, Institute of Virology, Slovak Academy of Sciences, Slo- replication stress in rapidly growing pre-cancerous and cancerous vakia cells affects the progression of the disease. We would also like to understand in detail how a cell copes with the replication stress at Plants have the ability to survive and reproduce under changed the molecular level. environmental conditions which were induced by biotic and Our next long term goal is to set up specific DDR response based abiotic stress. The area of the Chernobyl nuclear accident is a high throughput screens capable of assessing effect of different very interesting example of plant adaptation to abiotic stress in compounds and environment on DNA integrity in human cells. the form of increased radiation. Two experimental fields were Apart from basic research, we have been also developing novel established in the Chernobyl region – radio-contaminated and research and diagnostic tools, such as devices for more efficient non-radioactive. Flax (Linum usitatissimum L., variety Kyivskyi) was chromosome spreading and non-laborious mammalian cell syn- cultivated in these experimental fields since 2007, and during the chronization. 2012 the sixth generation of seeds was collected. The aim of the presented study was to analyze fatty acid desaturase (FAD) genes in http://dx.doi.org/10.1016/j.nbt.2014.05.972 the sixth generation of flax, specifically FAD3A and FAD3B, using the restriction fragment length polymorphism technique. In a biosynthetic pathway of fatty acids, these genes perform an impor- PV-03 tant function because they encode conversion of linoleic acid to Acanthopanax sessiliflorus stem confers increased resis- ␣-linolenic acid. This study analyzed three parts of FAD3A gene tance to environmental stresses and lifespan extension with following length of the nucleotide sequences 1201, 1500, in Caenorhabditis elegans 1924 base pairs. Also, FAD3B gene was divided into three parts with length of the nucleotide sequences 1246, 1330, 1279 base Sang-Kyu Park, Jin-Kook Park ∗, Chul-Kyu Kim, Sang-Ki Kong, A- pairs. For restriction analysis four different restriction enzymes Reum Yu, Mi-Young Lee were applied – MnlI for restriction digestion of the first part of Soonchunhyang University, South Korea FAD3A and FAD3B genes, NlaIII for the second and third part of FAD3A gene, AciI for the second and Hpy188I for the third part of Acanthopanax sessiliflorus is a native Korean plant and used as FAD3B gene. Based on these preliminary data, no polymorphism traditional medicine or an ingredient in many Korean foods. The of FAD genes in sixth flax generation was detected. However, the free radical theory of aging suggests that cellular oxidative stress stability of FAD genes will be further verified by more detailed caused by free radicals is the main cause of aging. Free radicals analyses. can be removed by cellular anti-oxidants. Here, we examined the anti-oxidant activity of Acanthopanax sessiliflorus extract and its http://dx.doi.org/10.1016/j.nbt.2014.05.971 lifespan-extending effect in Caenorhabditis elegans. Oxidative DNA damage was reduced and survival under oxidative-stress conditions was significantly enhanced by Acanthopanax sessiliflorus stem extract. In addition, Acanthopanax sessiliflorus stem increased resistance to other environmental stresses, including heat shock and ultraviolet irradiation. Treatment with Acanthopanax sessiliflorus stem extract significantly extended both mean and maximum

www.elsevier.com/locate/nbt S203 STRESS RESPONSES New Biotechnology · Volume 31S · July 2014 lifespan in C. elegans. However, fertility was not affected by PV-05 Acanthopanax sessiliflorus stem. In conclusion, Acanthopanax ses- Determination of reliable house keeping gene(s) for siliflorus stem had strong anti-oxidant activity and conferred a qPCR in maize under different boron dosages longevity phenotype without reduced reproduction in C. elegans, which provides conclusive evidence to support the free radical Hasan Can 1,∗, Mehmet Hamurcu 1, tijen Demiral 2, Anamika theory of aging. Pandey 1, Mohd Kamran Khan 1, Seyit Ali Kayıs 1, Zuhal Zeynep Avsaroglu 1, Nimet Can 2, Sait Gezgin 1, Erdogan Esref Hakki 1 http://dx.doi.org/10.1016/j.nbt.2014.05.973 1 Selcuk University, Turkey 2 Harran University, Turkey PV-04 Maize is one of the major cereal crops in the world. In Turkey Transcriptome characteristic of echiuran worm Urechis a production of 5.9 million tons was recorded in the year 2013 unicinctus exposed to sulfide by digital gene expression and became third most important crop of the country. Production analysis of maize has recently increased in Central and South Anato- lian regions of Turkey. However, boron toxicity has emerged as Zhifeng Zhang ∗, Litao Zhang, Xiaolong Liu a major crisis of the Central Anatolian Soil affecting the maize Ocean University of China, China yield and product quality. For solving this important issue, different approaches, including boron-tolerant variety development Sulfide is a well-known toxicant. However, some organisms and use, are implemented. Understanding the mechanism of can tolerate and utilize sulfide. In this study, Urechis unicinctus, boron tolerance in plants is an active research area. Different inhabiting coastal sediment and tolerating high concentration sul- genes are known to be involved in this mechanism. Tracking fide, was used to examine its transcriptional profile in response properly the expression patherns of these genes requires normal- to 50 ␮M sulfide for 24 h by digital gene expression analysis. A ization of the expressions using constantly expressed reference total of approximately 16 million cDNA tags were sequenced and genes. The main aim of our research was to determine reli- 3,909,160 and 4,057,279 clean tags were obtained in the control able house keeping genes for qPCR in maize under the effect and 24 h libraries. Compared with the 0 h library, 1181 tag-mapped of various boron concentrations. Initially, we focussed on the genes were detected as differentially expressed genes (DEGs) in the determination of boron tolerant commercial hybrid varieties. 24 h library. The DEGs were further subjected to GO and path- Further, experiments have been made for the determination way analysis. More than 80% pathways were rarely reported to of the gene/genes with constant expression level among the 6 be related to sulfide stress before. Three key physiological actions candidates reference genes namely, S18, alpha-tubulin, globu- induced by sulfide involving in energy metabolism, DNA dam- lin S, GAPDH, ubiquitin, beta2 tubulin. Results were computed age response and inflammation were discussed in details. To our using efficient software programs like geNorm and NormFinder. best knowledge, this study is the first transcriptome-wide effort to Two genes, S18 and GAPDH, were found to be the most reveal the transcriptional response to sulfide stress by digital gene consistently expressed genes under implemented boron stress expression analysis and the identified DEGs can serve as a basis for conditions. further understanding of sulfide roles in organisms [1,2]. Acknowledgement: This study was financially supported by Acknowledgement: This work was supported by the Natural The Selcuk University Scientific Research Projects Funding Unit Science Foundation of China (31372506). (Project No: 13401089). References [1].Tamizhselvi R, et al. Preprotachykinin-A gene deletion regulates http://dx.doi.org/10.1016/j.nbt.2014.05.975 hydrogen sulfide-induced toll-like receptor 4 signaling pathway in cerulein-treated pancreatic acinar cells. Pancreas 2011;40:444–52. [2].Janssens S, Tinel A. The PIDDosome, DNA-damage-induced apopto- PV-06 sis and beyond. Cell Death Differentiation 2012;19:13–20. Gene expression profiling of Thermoplasma volcanium under extreme stress conditions http://dx.doi.org/10.1016/j.nbt.2014.05.974 Sema Zabcı ∗, Semra Kocabiyik

Middle East Technical University, Turkey

Identifying the stress response mechanisms in Archaea might have potential applications considering the industrial importance of their enzymes. In this study, as an extension of our research on anti-stress mechanisms, we have carried out genome wide transcriptome analysis of Thermoplasma volcanium (grows optimally at pH 2.7 and 60 ◦C) under extreme stress (i.e., heat- shock ◦ at 68 C, hydrogen peroxide, 0.03 mM H2O2 and pH stress at pH 5.0) using Agilent Custom Gene Expression Microarray. The data was analyzed by using GeneSpring Ver 12.6 Software.

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With a pH shift from 2.7 to 5.0 most of the gene expression Acknowledgement: Supported by the Intelligent Synthetic was repressed: of 47 genes differentially expressed by ≥2-fold, Biology Global Frontier Program, and the Next-Generation 66% were up-regulated and 34% were down-regulated. Among the BioGreen 21 Program. notably induced genes, some code for membrane proteins, sugar and amino acid transporters, cation transporter and antiporters http://dx.doi.org/10.1016/j.nbt.2014.05.977 which are crucial for maintaining cellular pH homeostasis. Oxida- tive stress mostly resulted in decreased gene expression; out of 452 PV-08 genes expressions of which changed ≥2 fold, 75% were down regulated. Up regulation of ferrodoxins, oxidoreductases, thioredoxin, The role of S-nitrosoglutathione reductase in defence Fe-S oxidoreductases, aldo/keto reductases and dehydrogenases as response of tobacco plants and cells to elicitins a response to H2O2 may be important for maintaining intracellular Tereza Jendriˇsáková 1,∗, Pavla Moricová 1, Martina Zeleznáˇ 1, redox potentials and detoxifications. Temperature up-shift favored Lenka Luhová 1, Jan Lochman 2, TomáˇsKaˇsparovsky´ 2, Marek the expression of heat-shock proteins (including GroEL, sHSP, Dna Petˇrivalsky´ 1 K, DnaJ, GrpE) as well as genes related to carbohydrate metabolism (e.g., sugar permease). 1 Palacky University Olomouc, Czech Republic 2 Masaryk University Brno, Czech Republic http://dx.doi.org/10.1016/j.nbt.2014.05.976 Nitric oxide (NO) is an important signalling molecule which participates in the plant immune responses. S-nitrosoglutathione PV-07 reductase (GSNOR) is known as key enzyme in NO metabolism through cysteine S-nitrosylation, the post-translational modifica- Stress responsive global regulatory mechanisms in the tion mediated by reactive nitrogen species (RNS). GSNOR belongs methylotrophic yeast Hansenula polymorpha to the family of alcohol dehydrogenases class III (ADH3; EC Ohsuk Kwon 1,∗, Eun Hye Kim 1, Doo-Byoung Oh 1, Hyun Ah 1.1.1.1). GSNOR is highly specific for the S-nitrosoglutathione sub- Kang 2 strate and was demonstrated to indirectly control the total level of protein S-nitrosothiols. 1 Korea Research Institute of Bioscience and Biotechnology, South Elicitins, extracellular proteins secreted by oomycete pathogens Korea of Pythium and Phytophthora spp., represent effective elicitors 2 Chung-Ang University, South Korea that trigger defence responses in plant-pathogen interactions. The thermotolerant methylotrophic yeast Hansenula polymor- The aim of this work was to study a possible GSNOR role in pha has been regarded as an attractive model organism for defence response of tobacco plants and cell culture to selected fundamental studies of methanol metabolism, peroxisome biogen- elicitins. We tested cryptogein and its mutated forms V84F and esis and function, nitrate assimilation, and protein glycosylation. L41F with different ability to trigger the formation of reactive oxy- H. polymorpha has become one of the promising hosts for the gen species (ROS) and cell necrosis. Oligandrin, another elicitin production of recombinant proteins on an industrial scale owing from Pythium oligandrum, does not elicit hypersensitive reaction to the availabilities of strong inducible promoters and a multi- in plant tissue. The production of RNS and ROS was determined copy integration system for target protein expression cassettes after elicitin application using specific fluorescent probes. Cryp- into the genome. In addition, recently H. polymorpha is gaining togein and V84F mutant triggered significantly increased ROS increasing interest for its several peculiar physiological charac- level, which correlated with decreased cell viability. On the con- teristics, such as resistance to heavy metals and oxidative stress, trary, little effect of oligandrin and mutant L41F on growth was and thermotolerance since these properties are advantageous for observed, whereas higher NO production was induced. Increased various biotechnological applications. However, only a limited activity and expression of GSNOR were detected after application amount of information is available for the global regulatory of cryptogein and V84F mutant. The results bring new insight mechanisms for stress response in this organism. In the present into the involvement of ROS and RNS in plant response to elic- study, we investigated the roles of representative signaling and itors and possible regulatory role of GSNOR in control of protein regulatory proteins to understand the regulatory mechanisms S-nitrosylation. governing the osmotic and oxidative stress responses of H. polymorpha. The hybrid histidine sensor kinases Sln1 and Nik1, http://dx.doi.org/10.1016/j.nbt.2014.05.978 histidine-containing phosphotransfer protein Ypd1, response regulator proteins Skn7 and Ssk1, high osmolarity glycerol pathway regulator Hog1, and oxidative stress response regulator Yap1 were functionally characterized by mutant construction, growth phenotype comparison, in vitro protein phosphorylation, and comparative transcriptome analysis. Obtained results indicate that the Skn7/NiK1-Ypd1-Skn7/Ssk2 two-component signal transduction pathway plays a critical role in oxidative, osmotic, and cell wall stress responses in H. polymorpha.

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PV-09 YtfE has a crucial role in repairing iron-sulphur centres damaged by nitrosative stress conditions [1–3]. This poster will show that a Sensitivity of selected root hair mutants of Arabidopsis major role of YtfE is to release nitric oxide that has become bound thaliana to abiotic stress to proteins such as aconitase and fumarase, thereby restoring their Lenka Vaˇskebová 1,∗, Barbora Satnáˇ 1, Miroslav Oveckaˇ 2, Jozef activity under conditions of nitrosative stress. ˇ 2 Samaj References 1 Palacky University in Olomouc, Czech Republic [1].Justino MC, Almeida CC, Goncalves VL, Teixeira M, Saraiva LM. 2 Centre of the Region Haná, Olomouc, Czech Republic Escherichia coli YtfE is a di-iron protein with an important function in assembly of iron-sulphur clusters. FEMS Microbial Lett Plant growth requires the continuous uptake of solutes and 2006;257:278–84. minerals from the rhizosphere by the root as well as mainte- [2].Justino MC, Almeida CC, Teixeira M, Saraiva LM. Escherichia coli di- nance of ion homeostasis and avoidance of intracellular ion Iron YtfE protein is necessary for the repair of stress-damaged iron- sulphur clusters. J Biol Chem 2007;282:10352–9. toxicity. Plants also possess effective molecular, physiological and [3].Overton TW, Justino MC, Li Y, Baptista JM, Melo AMP, Cole developmental mechanisms to minimize harmful effects of exter- JA, Saraiva LM. Widespread distribution in pathogenic bacteria of nal abiotic stress factors. Root hairs are tubular extensions of di-iron proteins that repair oxidative and nitrosative damage to iron- root epidermal cells, and they are formed by specialized tip sulphur centers. J Bacteriol 2008;190:2004–13. growth. Since root hairs effectively extend surface area of the root, they could play signiﬁcant role in plant sensitivity to abi- http://dx.doi.org/10.1016/j.nbt.2014.05.980 otic stresses originating from the soil. Arabidopsis thaliana root hair mutants such as rhd2(root hair defective 2, lacking the activ- PV-11 ity of the NADPH oxidase AtRBOH C, Takeda et al., 2008) and tip1 (tip growth defective 1, inactive in S-acyl transferase, Hems- Nitrosative stress response in Escherichia coli ley et al., 2005) are defective in the tip growth. This suggests Jing Wang ∗, Claire Vine, Jeff Cole their possible involvement in root stress sensitivity. We studied effects of abiotic stresses on root growth and root hair devel- University of Birmingham, United Kingdom opment in certain stages of stress perception in these mutants. Bacteria encounter various stress as the living environment In general, root growth of rhd2-1 mutant was well compara- changes. For instance, in anaerobic growth conditions when nitrite ble to Col-0 wild type, while root growth of tip1-1 mutant was or nitrate is used as electron acceptor, nitric oxide (NO) is generated weaker. Upon application of diverse abiotic stresses, rhd2-1 mutant during the reduction process. Up to present, three NO reduc- showed similar sensitivity as Col-0 and all lines showed a compa- tases, Nrf, Nor and Hmp have been reported in the bacterium rable sensitivity to salt and oxidative stress. Stress responses were Escherichia coli. A recent microarray study revealed several genes tested also in Arabidopsis plants stably overexpressing mitogen- that are highly up-regulated during nitrosative stress, one of which activated protein kinase kinase SIMKK from Medicago sativa. These was hcp, a gene that encodes a hybrid cluster protein (HCP) with results contribute to better elucidation of root hairs as integral a unique structure. parts of the root developmental program in changing environ- We tested two E. coli strains, one lacking all the above men- ment. tioned NO reductases and one with a further deletion of hcp. The hcp mutation decreased nitrosative stress tolerance of E. coli.A http://dx.doi.org/10.1016/j.nbt.2014.05.979 second gene, hcr, is co-transcribed with and located in the same operon as hcp. We detected a positive protein interaction between PV-10 HCR and HCP in vivo using the bacterial two-hybrid system. This supports the hypothesis that HCP might function as another NO Sources and repair of nitrosative stress in Escherichia coli detoxifying enzyme, with HCR being a reductase of HCP. We sub- Basema Balasiny ∗, Claire Vine, Jeff Cole jected the two strains to NO stress and observed that in our control

strain NO was reduced to N2O, probably via a reaction catalysed University of Birmingham, United Kingdom by HCP. Escherichia coli is a Gram negative, facultatively anaerobe. Inside Our data demonstrated that HCP is essential for the reduction the mammalian gut, E. coli utilises various alternative electron of low concentrations of NO to N2O. acceptors to respire efﬁciently when oxygen is unavailable. To survive, it must defend itself against reactive nitrogen species http://dx.doi.org/10.1016/j.nbt.2014.05.981 (RNS) generated as products of its own metabolism, or the innate immune response of the host. Under anaerobic conditions, nitrate reductase NarGHI generates nitric oxide (NO) as a side product during reduction of nitrate via nitrite to ammonia. However, some NO is formed from nitrite even by mutants that lack NarGHI. Nitric oxide is an extremely toxic gas that damages metal centres of proteins, especially iron-sulphur proteins such as aconitase and fumarase. Several studies have reported that the di-iron protein

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PV-12 tolerance in monocot. Here, we report a new member of the chitinase family 19, designated as LcChi2, isolated from Chinese Molecular markers based approach for genetic improve- wildrye (Leymus Chinensis). Sequence analysis demonstrated that ment of tea (Camellia sinensis) LcChi2 belonged to chitinase class II subgroup containing two Rahul Kumar 1,∗, Himanshu Sharma 2, Sanatsujat Singh 2, Rakesh conserved domains, i.e. Cys49Gly71 and Val161Met171. In compar- Kumar Sud 2, Arvind Gulati 2, Paramvir Singh Ahuja 2, Ram Kumar ison with wild-type tobacco, the transgenic tobacco (Nicotiana Sharma 2 tabacum) overexpressing LcChi2 showed increased tolerance to fungal and bacterial pathogens stresses. Semi-quantitative RT-PCR 1 DAV University, India analysis indicated that the expression of LcChi2 in Chinese wildrye 2 CSIR-IHBT, Palampur, HP, India was up-regulated with the treatment of 400 mM NaCl, 100 mM

Tea, due to multiple health benefits with various flavors and Na2CO3 and 20% PEG, so was the activity of LcChi2 product antioxidant properties is the most consumed beverage across the coincidently in similar treatments. Transgenic yeast (Saccharomyces globe and becomes an important agro-based revenue source for cerevisiae) overexpressing LcChi2 exhibited an enhanced toler- many countries in the world including India, which ranks first ance to 1.6 M NaCl, 10 mM Na2CO3, 1.5 M sorbitol, and 10 mM in black tea production. Genetic improvement studies, however, ZnSO4 stresses respectively. Interestingly, LcChi2-overexpressed have been limited in tea due to non-availability of sequence transgenic tobacco presented increased tolerance to 200 mM NaCl, based co-dominant microsatellite markers. Novel microsatellite 3mMNa2CO3 and 500 mM sorbitol stresses during germination markers were identified through construction and sequencing of and to 600 mM NaCl stress during seedling development. This sug- enriched genomic clones and mining of public expressed sequence gested that LcChi2 is likely an interesting candidate gene to be data in tea. These markers along with other dominant DNA used in transgenic breeding to improve broad-spectrum tolerance markers like AFLP and RAPD were utilized for genetic mapping in crop. Current results added information to plant chitinase fam- using pseudo-test cross population comprising of 212 individuals ily in abiotic stress tolerance over its existing functions in biotic derived through crossing between parental clones SA6 (resistant stress tolerance. to blister blight disease) and Asha (susceptible). Of one thou- sand microsatellite markers identified, 576 recorded successful http://dx.doi.org/10.1016/j.nbt.2014.05.983 amplification in selected tea accessions including parental lines. Informative 80 SSR markers along with 102 AFLP and 160 RAPD were used for construction of linkage map in tea. Following a pseudo test cross approach, in total, 400 markers loci (292 AFLP, 81 RAPD, 27 SSR) were found to be segregating in a test cross ratio and 127 of these could be linked to 17 linkage groups covering a total length of 1659 cM. Novel SSRs enriched the limited reper- toire of co-dominant markers in tea and will be further utilized in saturating the linkage map and subsequently in QTL analysis and MAS in tea. http://dx.doi.org/10.1016/j.nbt.2014.05.982

PV-13

A novel chitinase gene homologue from Chinese wildrye enhances tolerance to biotic and abiotic stress in transgenic tobacco

Dongyun Hao 1, Xiangguo Liu 2,∗, Ying Yu 3, Yang Liu 2, Yinning Qu 3, Shuo Yang 3, Siping Han 2, Shudan Feng 4

1 Institute of Agricultural Biotechnology, India 2 Jilin Academy of Agricultural Sciences (JAAS), India 3 Jilin University, India 4 Harbin Normal University, India

Plant chitinase is a glycosyl hydrolase that is believed to be responsible exclusively for pathogens stress. However, there has been no report suggesting its function in relation to salinity stress

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Synthetic Biology chain with an enantiomer sn-glycerol-1-phosphate backbone. This unique structure is believed to be vital for the adaptation of these PW-01 organisms to extreme conditions and the lipid divide is considered Design of the novel catalytic units by key motif-directed significant in a split of prokaryotes into archaea and bacteria. The domain recombination aim of this project is to functionally introduce the archaeal ether lipid biosynthetic pathway into E. coli to examine the properties 1,∗ 2 3 3 Yan Feng , Xiaoli Zhou , Yuan Xie , Guangyu Yang of mixed membrane lipids, to study the archaeal lipid biosynthetic 1 State Key Lab of Microbial Metabolism, Shanghai Jiao Tong Uni- pathway and to understand evolutionary aspects associated with versity, China the lipid divide. 2 Jilin University, China Using a synthetic biology approach, different modules of the 3 Shanghai Jiao Tong University, China pathway were designed for the synthesis of the isoprenoid chain and CDP archaeol, the precursor for polar head group attachment. Diverse functional domains in proteins provide a resource The latter ether-lipid is produced by an uncharacterized enzyme, for designing novel biocatalysts. Recombination of more distant whose gene has not yet been identified in Archaea. Several in vivo sequences in evolution offer greater opportunity for function and in vitro assays were performed to access the enzymatic activities leaps, but also introduces more disruptions in the chimeras. Here, and the hypothetical gene encoding for CDP-archaeol synthase we report two lipase-like chimeras engineered from a mesophilic was expressed in E. coli and validated. lipase and a hyperthermophilic peptidase/esterase with only 14% A membrane integrated precursor of the archaeal lipid biosyn- or 21% amino acid identity. Recombination was at the conserved thetic pathway was produced in E. coli and formation of CDP key motif regions which are part of the hard cores of the proteins archaeol can be reconstituted in vitro. instead of the flexible loops between protein functional domains. The resulting chimeric lipases retained the desirable preference http://dx.doi.org/10.1016/j.nbt.2014.05.985 for long-chain acyl ester substrates of their mesophilic parents, with pNP-C14 as the best substrate. Meanwhile, compared to the mesophilic parent, the chimeras exhibited more than 100- fold increased thermostability at 50 ◦C and the optimum catalytic temperature improved 40 ◦C. These results suggest that the key motif-directed recombination (KMDR) approach promises for the efficient construction of robust hybrid enzymes. http://dx.doi.org/10.1016/j.nbt.2014.05.984

PW-02

Ether-lipid membrane engineering of Escherichia coli

Antonella Caforio ∗, Samta Jain, Arnold Driessen

University of Groningen, The Netherlands

The membrane lipid composition of archaea differs from bacteria and eukarya in having an ether-linked, isoprenoid hydrocarbon

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Use of organic wastes composition, rich in sugars such as cellulose, hemicellulose and pectin, they can be easily assimilated by microorganisms. PX-01 Our research group has demonstrated that high levels of Integral exploitation of olive tree pruning in the paper hydrolytic enzymes are produced by SSF of Aspergillus awamori on industry a mixture of grape pomace and orange peels. Furthermore, these enzymes could be safely added to food preparations for this fungus 1,∗ 1 2 Luis Jiménez , Alejandro Rodríguez , Juan Domínguez , Anto- is considered a GRAS (Generally Recognized as Safe) microorgan- 2 2 2 nio Rosal , Gustavo Cordero-Bueso , Eva Valero ism. However, higher enzyme activities could be achieved by using 1 University of Córdoba, Spain the same methodology but growing different fungi. In fact, it 2 University Pablo de Olavide, Spain would be interesting to be able to produce these enzymes from different agroindustrial residues but in the same conditions, as they This work seeks to obtain valuable products through an integral could be processed in the same manufacturing facilities. More- exploitation of lignocellulosic residues generated by agriculture, over, if possible and considering seasoning crops as raw material, it such as olive tree prunings. These sorts of residues are studied as would be profitable for having the production plant operating for alternative sources for lignocellulosic raw material in the paper a longer time. Thus, the aim of this work is to evaluate the produc- industry and the black liquors generated could be used for several tion of hydrolytic enzymes by SSF of Botryotinia fuckeliana grown purposes, including the production of bioethanol. in several agroindustrial residues with different composition in the To reach these goals, a central composite factorial design was same conditions of fermentation. used to study the influence of operational variables [temperature ◦ (155, 170 and 185 C), cooking time (40, 65 and 90 min) and soda http://dx.doi.org/10.1016/j.nbt.2014.05.987 concentration (10, 14 and 18%)], on pulps and paper sheets properties obtained from olive tree prunings, likewise black liquors generated in these pulping processes were set up to enable both PX-03 yeast strains (Saccharomyces cerevisiae CECT 1170 and Pichia stipitis Liquid chromatography/mass spectrometry based iden- CECT 1922) to be capable of doing an alcoholic fermentation at tification, cloning and characterization of thermostable 30 and 26 ◦C respectively, 5.5 pH, and under 150 rpm shaking. bacterial enzymes useful for the production of long- The results were similar to those obtained with other agricul- chain oligosaccharides from agro-industrial wastes tural residues which are alternative sources for the paper industry. ∗ The best values of the physicochemical properties of cellulose Nomeda Kuisiene , Raimonda Petkauskaite, Dangiras Lukosius, pulps, such as Kappa number (31.98) or viscosity (716.11 mL/g) Andrius Jasilionis and the paper sheets, such as tensile index (608 m) or tear index Vilnius University, Lithuania (1.655 mNm2/g), were reached operating to high values of soda concentration and temperature and slight cooking times. How- Oligosaccharides are important ingredients of the functional ever, the black liquors of this study were not an optimal medium foods. The development of novel and highly functional oligosac- for the bioethanol production. It might be due to the important charides with physiological properties is now continuing. Over presence of inhibitors in the fermentation processes, such as 5- the past few years, long-chain oligosaccharides have evoked a hydroxymethyl-furfural. great interest. Such oligosaccharides are absorbed to a much lower degree and persist longer in the colon than the shorter ones. Con- http://dx.doi.org/10.1016/j.nbt.2014.05.986 sequently, beneficial effect of the long-chain oligosaccharides is greater. Thermophilic microbial enzymes have potential to be used for the production of the long-chain oligosaccharides. PX-02 We report identification of thermostable bacterial enzymes Utilization of agroindustrial residues for hydrolytic involved in the degradation of starch and pectin using liq- enzymes production uid chromatography/mass spectrometry (LC/MS) based analysis.

∗ Zymographic analysis was combined with LC/MS. A range of Ana Belen Diaz, Ana Blandino , Ignacio de Ory, Ricardo Martin, polysaccharide degradation associated enzymes was identified Maria Jose Munoz, Ildefonso Caro using LC/MS of zymographic samples. The obtained peptides University of Cadiz, Spain were used for the construction of primers in order to clone selected enzymes. Recombinant proteins were expressed, purified Agricultural and agroindustrial residues are the most abun- and characterized. The length of oligosaccharides produced by dant resources on earth, which have significantly increased as a recombinant enzymes was determined. In order to determine con- result of industrialization. In recent years there has been a grow- ditions to monitor the chain length of obtainable oligosaccharides, ing trend towards efficient utilization and value-addition of these we also tested activity of recombinant enzymes at suboptimal residues. Biotechnological processes, especially solid state fermen- pH and temperature values. The potential of recombinant ther- tation (SSF), have contributed enormously to such reutilization as mophilic bacterial enzymes in the degradation of agro-industrial it can convert these agro-feedstocks into a wide variety of valuable wastes was also evaluated. It was shown, that long-chain oligosac- chemical products. Agroindustrial residues are generally consid- charides can be produced from starch and pectin (including that ered the best substrate for the solid state processes. Given their

www.elsevier.com/locate/nbt S209 USE OF ORGANIC WASTES New Biotechnology · Volume 31S · July 2014 from apple pomace) using recombinant thermostable bacterial acid producing bacterium and the filamentous fungus Aspergul- enzymes. lus niger. The latter was introduced into a typical Mediterranean Acknowledgement: The authors are thankful to the Lithua- degraded soil as a biotechnological product containing partially- nian Science Council for the financial support (project No. solubilized phosphate (hydroxyapatite of animal bone origin, SVE-08/2011). HABO), mineralized organic matter and mycelial mass. The lactic acid bacterium was entrapped in alginate beads and further inoc- http://dx.doi.org/10.1016/j.nbt.2014.05.988 ulated in the soil, before transplanting plant seedlings (Lavender spica), to serve as a slow-release microbial formulation. Culture filtrate of Piriformospora indica was added to the above system PX-04 in the beginning of the experiment and after two weeks. All Solubilization of animal bone char by Yarrowia lipolytica treatments were supplemented with HABO. Results showed a sig- on medium containing glycerol nificant plant growth promotion in treatments with immobilized bacterial cells (IC). Similar effect was observed when organic mat- Nikolay Vassilev 1,∗, Bettina Eichler-Löbermann 2, Vanessa ter was used as an amendment. The highest plant growth was Martos 1, Maria Vassileva 1 however registered in the treatment with both IC and microbially- 1 University of Granada, Spain treated organic matter. This effect was more pronounced in the 2 University of Rostock, Spain presence of filtrate of P. indica. Our results confirmed the beneficial effect of root-colonizing fungi and PGP microorganisms Phosphorus (P) is accepted as an essential element for all liv- on plant growth. P. indica is a mycorrhiza-like root-colonizing ing organisms. P is mainly derived from mined rock phosphate, fungus which can be cultivated in fermentation systems. In our which is a non-renewable resource. It has been suggested that agri- study, the combination between P. indica filtrate, P-solubilizing IC, cultural demand for P will outstrip mineable resources in 50–100 and, on the other hand, organic matter/soluble P product resulted years and economically mineable resources will be depleted before in a highly effective promotion of plant growth in degraded the end of this century. Biotechnology offers a number of sustain- soil as an alternative of chemical-fertilizer-based conventional able solutions that can mitigate these problems by using various scheme. waste materials as a source of P and, on the other hand, their solubilization by selected microorganisms. In this work we present http://dx.doi.org/10.1016/j.nbt.2014.05.990 results on solubilization of animal bone char (HABO) with a high content of phosphate by Yarrowia lipolytica. Free yeast cells were cultivated using different combinations of glycerol and HABO, PX-06 the latter being simultaneously solubilized by the released citric Production of carotenoids, ergosterol and other lipidic acid. Biomass accumulation of 6.1 g/l, citric acid (8.7 g/l) and sol- compounds by red yeasts cultivated on lignocellulose uble phosphate (379 mg/l) concentrations were measured at 50 g/l waste substrates glycerol and 6 g/l HABO after 96 h of shake-flask cultivation. How- ever, the highest percentage of soluble phosphate of the total P Ivana Marova ∗, Andrea Haronikova, Sinisa Petrik, Stanislav of 44.8% was obtained at 30 g/l glycerol and 2 g/l animal bones Obruca, Iveta Kostovova char. Employing the latter combination, a separate experiment Brno University of Technology, Faculty of Chemistry, Czech was carried out in fermenter where the solubilization efficiency Republic was enhanced to 345 mg soluble phosphate/l (61.6% of the total phosphate) after 40 h of cultivation. The main advantage of HABO Carotenogenic yeasts are a diverse group of unrelated organ- as an alternative P source is its abundance and low price. The EU isms (mostly Basidiomycota), that can be found in soil, fresh and produces about 3 million tons of meat and bone residues. marine water, on plants and also in foods. Due to its ubiquitous and world-wide occurrence, these yeasts have been able to assimilate http://dx.doi.org/10.1016/j.nbt.2014.05.989 various carbon sources, such as glucose, xylose, cellobiose, sucrose, glycerol, etc. Therefore, agro-industrial waste materials including lignocellulose materials can be used as cheap substrates. PX-05 Presented work is focused on growth and production activity Plant growth enhancement by biotechnological tools of red yeast strains Rhodotorula, Sporobolomyces and Cystofilobasid- ium cultivated on some lignocellulose wastes: pre-treated whey Maria Vassileva 1,∗, Massimiliano Fenice 2, Antonia Galvez 1, Niko- straw, pine hydrolyzates, SCG and rapeseed waste. The main aim lay Vassilev 1 of the current investigation was to assess the potentialities of red 1 University of Granada, Spain yeasts to transform these waste substrates to high-value products 2 University of Viterbo, Spain as carotenoids (about 1–3 mg/g CDW), ergosterol (2–4 mg/g CDW), coenzyme Q (0.5–1 mg/g CDW), lipids (11–21% of dry mass) and An alternative, more environmentally friendly strategy for fatty acids as well as enriched red yeast biomass. Production prop- plant growth promotion was developed which include various erties of red yeasts were compared between tested strains and types of plant-soil improvers and low-cost P-bearing material. studied also during scale-up process in 5-L laboratory fermentor. Two types of microbial P-solubilizers were employed: a lactic The yields of about 30 g per liter of biomass enriched by 30–50 mg

S210 www.elsevier.com/locate/nbt New Biotechnology · Volume 31S · July 2014 USE OF ORGANIC WASTES of total carotenoids and 60 mg of ergosterol were obtained by the PX-08 most producing strains. Such biomass which is efficiently enriched NOSHAN EU-project (Sustainable Production of Func- for provitamins A, D and CoQ could serve as an additional tional and Safe Feed from Food Waste): molecular natural source of significant nutrition factors in feed and food characterization of selected food waste materials industry. Acknowledgement: This work was supported by project Tullia Tedeschi ∗, Mariangela Bencivenni, Chiara Bottesini, Judith “Materials Research Centre at FCH BUT-Sustainability and Devel- Muller-Maatsh, Federica Meli, Arnaldo Dossena, Stefano Sforza opment, REG LO1211 NPS I MEYS CR”. University of Parma, Italy http://dx.doi.org/10.1016/j.nbt.2014.05.991 Food processing activities produce in Europe large amounts of by-products and waste. Such waste streams are only partially valorized at different value-added levels (spread on land, PX-07 animal feed, composting), whereas the main volumes are man- Fruit residues: low cost substrates for development of aged as waste of environmental concern, with relevant negative new food products effects on the overall sustainability of the food processing industry. Ninna Granucci ∗, Silas G. Villas-Boas The main focus of NOSHAN1 is to investigate the process and Centre for Microbial Innovation, School of Biological Sciences, technologies needed to use food waste for feed production at low The University of Auckland, New Zealand cost, low energy consumption and with maximal valorisation of starting wastes materials. Nutritional value and functionality as In recent years, there has been increasing interest in devel- well as safety and quality issues are investigated and addressed as oping alternative uses for agricultural residues for production of main leading factors for the feed production using food derived higher added-value products. Bioconversion of these substrates (fruit/plant and dairy). According to this not only wastes are char- using microbial fermentation could be an attractive option. There- acterized for their nutritional potential, but suitable technologies fore, the main goal of my project is to optimise a fermentation to stabilize them and convert them into suitable raw materials for process for bioconversion of fruit residues developed at labora- bulk feed are researched. tory scale using apple pomace as model. In our previous work, The topic of this communication is the characterisation, at Candida utilis and Pleurotus ostreatus were employed for bioconver- molecular level, of the different waste streams selected. In par- sion of apple pomace, resulting in a nutritious enriched whitish ticular a detailed composition of the nitrogen fraction has been substrate (flour-like) with attractive walnut/hazelnut aroma that performed. Determination of amino acids was performed by chro- contained high protein and low sugar. We have data demon- matography methods and racemisation was studied by GC–MS. strating that the sequential fermentation of apple pomace with Peptide and protein profiles were determined by LC/MS. C. utilis and P. ostreatus resulted in 400% enrichment of pro- Waste streams turned out to be quite promising to be trans- tein content in the substrate in addition to increased availability formed in feed or to be exploited as a source of feed additives, as of phosphorus, calcium and potassium as compared to non- far as their content in desirable compounds was concerned. fermented substrate. Moreover, the flavor and appearance of the processed substrate was very attractive, which made it ideal http://dx.doi.org/10.1016/j.nbt.2014.05.993 for a human consumption product. Thus, we envisage the following main steps for further research: 1 Assess other fruit residues to be bioconverted; 2 Scale up fermentation; and 3 Nutri- PX-09 tional assessment of fermented product. This same strategy can Production of polyhydroxyalkanoates from anaerobic be used for the bioconversion of different agriculture wastes, digested grape pomace by employing a pure culture of such as residues of other fruit processing and/or dairy indus- Cupriavidus necator tries. Gonzalo Martinez 1,∗, Lorenzo Bertin 1, Joana Domingos 1, Ger- http://dx.doi.org/10.1016/j.nbt.2014.05.992 hart Braunegg 2, Fabio Fava 1

1 University of Bologna, Italy 2 Technical University of Graz, Italy

The present work was dedicated to verify the possibility of replacing the carbon source commonly employed for the biotechnological production of polyhydroxyalkanoates, i.e., glucose or fructose, with grape pomace (GP), which is a solid organic waste of the winery industry. To this aim, a grown culture was fed with the volatile fatty acids (VFAs)-rich efﬂuent obtained by fermenting

1 Project full title: “Sustainable Production of Functional and Safe Feed from Food Waste” Grant agreement no.: 312140.

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GP under acidogenic conditions (GPAcid). GPAcid mainly contained mine the content of galacturonic acid, their composition in POS (g/L) acetic (14.69 ± 0.57), propionic (0.77 ± 0.04), iso-butyric and their prebiotic activity, in order to define structure–function (0.83 ± 0.03), butyric (4.67 ± 0.21) and caproic (0.55 ± 0.02) acids. relationships. Experiments were carried out in 500-mL Erlenmeyer flasks (working volume: 150-mL) at 30 ◦C and 180 rpm. The whole poly- http://dx.doi.org/10.1016/j.nbt.2014.05.995 hydroxyalkanoates production process was separated into two stages, namely: a balanced cell growth phase (by using DSMZ- PX-11 81 mineral medium amended with 5 g/L of fructose) and an accumulation phase, where harvested cells were suspended in Fungal bioconversions of various lignocellulosic by- aNH4 free-DSMZ-81 medium containing 20 or 40% of GPAcid products to edible biomass (v/v). Higher VFAs concentrations were observed to inhibit poly- Georgios Koutrotsios ∗, Konstantinos Mountzouris, Georgios Zer- hydroxyalkanoates accumulation. A control experiment aimed vakis at indirectly evaluate water matrix inhibition was performed by substituting GPAcid with a VFAs water solution, wherein VFAs Agricultural University of Athens, Greece concentrations were the same of GPAcid. First results showed a The world-wide trend for a continuous increase in the amount higher polymer production when employing 40% of GPAcid, with a of food produced leads to the accumulation of huge quantities of final polyhydroxybutyrate content of 60% (cell dry weight bases). plant residues. Controlled solid state fermentation of various agro- Thermo-gravimetric analyses confirmed gas-chromatography ones industrial and forestry residues rich in lignocellulosics were treated and identical results were obtained in the control experiment. by selected strains of Agrocybe cylindracea, Ganoderma lucidum, Heri- To our knowledge, this work represents the first attempt to pro- cium erinaceus and Pleurotus ostreatus (phylum Basidiomycota), the duce polyhydroxyalkanoates with a pure culture of Cupriavidus processes were monitored, and composition of end-products was necator and a fermented GP as alternative carbon source. comparatively evaluated. Olive mill waste (composted or not), olive prunings and grape marc were among the most promis- http://dx.doi.org/10.1016/j.nbt.2014.05.994 ing substrate ingredients for mushroom production since their use contributed at obtaining significantly higher yields than the PX-10 conventional cultivation media. In most cases, mushroom productivity correlated with substrates nitrogen, lignin, hemicelluloses, Composition of pectins from food waste to be used in and residual mycelial content. Spent substrates exhibited high bulk feed and as feed additives reductions in hemicelluloses and cellulose, and low in lignin; Stefania Baldassarre 1,∗, Stefano Sforza 1, Barbara Prandi 1, Marcela these values in conjunction with elevated protein content advo- Santarelli 1, Neha Babbar 1, Kathy Elst 2, Monica Gatti 1, Stefano cated their use in animal feeds. Mushroom proximate analysis Sforza 1 showed correlations of protein and crude fat with substrates nitrogen and lipids, whereas total carbohydrates, crude fiber and ash 1 University of Parma, Italy content demonstrated relatively less variability among strains 2 University of Antwerp, Italy and cultivation media tested. Mushroom nutritional value could Pectins are heterogeneous carbohydrate polymers found in the be suitably enhanced by appropriate selection/modification of cell wall of higher plants; they are formed by a backbone of (1->4)- the growth substrates. Exploitation (incl. detoxification) of waste ␣-D-galacturonic acid units interspersed with rhamnose residues streams could be combined with the generation of value-added linked to neutral sugar side chains. They contribute to primary wall products and provide a viable solution for the sustainable use of functions such as cell strength, cell adhesion, stomatal function such materials. and defense response. Furthermore pectic oligosaccharides (POS) Acknowledgements: This research has been co-financed by have been proposed as a new class of prebiotics able to exert several European Union (ESF) and Greek national funds (NSRF) through health-promoting effects. the project titled “Metagenomics of ligninolytic microorganisms The main objective of this work is the characterization of – Bioconversion of plant by-products into high-added value prod- pectins in vegetal food waste, in order to ensure their suitabil- ucts” (THALIS–UOA–MIS377062). ity as raw material for feed production. Two raw materials were considered: bulk feed prepared from vegetal food waste (rapeseed http://dx.doi.org/10.1016/j.nbt.2014.05.996 and malted barley mixed with dairy waste) and pectins extracted from vegetal waste to be used as feed additive .In the first case, pectic substances are precipitated with ethanol and the total alcohol insoluble residue is used for the determination of total pectins by quantitatively measuring the total uronic acid through a colorimetric assay after acid hydrolysis. In this way it is possible to assess the technological impact of the processing on the composition and the extractability of pectins. In the latter case, samples of pectic oligosaccharides (POS) extracted by enzymatical means, are analyzed in order to deter-

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PX-12 MFCs provide a dual benefit: wastewater treatment and direct elec- tricity generation. For industrial scale-up, the approach in this Influence of cell size on performance of microbial fuel work is through plurality. Multiple units of relatively small scale cells of single chamber using organic solid waste with can be connected electrically and hydraulically to achieve the nec- Amazonian and High Andeans soils from Ecuador essary capacity [2]. Washington Logrono,˜ Celso Recalde, Magdy Echeverria Initially a single chamber open air cathode MFC of 170 ml volume treating spent wash subsequent to anaerobic digestion Escuela Superior Politécnica de Chimborazo, Ecuador treatment of 2 g/l Chemical Oxygen Demand demonstrated an It was the first work done in Ecuador where compared three average voltage of 0.4 V in open circuit and a COD reduction volumes to produce bioelectricity in Microbial Fuel Cells of Single of 70%. Scaling up, two 100 lt units connected in parallel and Chamber, employing organic solid waste as substrate with 50:50 operating in directly diluted spent wash at 26.4 g COD/l.d demon- relationships (vegetables and fruits) to enrich electrogenic bacte- strated 84% COD removal, maximum voltage 0.9 V and current rias, without renovation of microbial fuel and over a testing time 160 mA. Ongoing experiments using this configuration and previ- of 171 days. The study used soils from Kaiptach Achuar Commu- ously anaerobically treated spent wash demonstrate encouraging nity – AMAZON to 1000 m.a.s.l. and the same procedure with the results, bringing practical industrial scale Microbial Fuel Cell treat- Pichan Central Community-ANDES to 4000 m.a.s.l. The samples ment closer. were taken among 20–40 cm depth of natural soils, each treatment References have used polyethylene containers as bioreactors and carbon fiber [1].Mohana S, Acharya K, Madamwar BD. Distillery spent wash: was used as both electrodes with different surface areas. treatment technologies and potential applications. J Hazard Mater The initial physical chemical analysis indicated differences in 2009;163:12–25. parameters like: P,K, CaO, MgO, Rel.C/N, %H; while the initial het- [2].Gálvez A, Greenman J, Ieropoulos I. Landfill leachate treatment erotrophs counted in the Andean soil showed higher amount, but with microbial fuel cells; scale-up through plurality. Bioresour Technol smaller microbiological diversity, in accordance to morphological 2009;(100):5085-509. description done under aerobic conditions. The means comparison test has indicated that there are significant differences among http://dx.doi.org/10.1016/j.nbt.2014.05.998 sizes both studies cases, at the ANDES the best treatment was the PX-14 intermediate, with a mean generation of 317 mV, moreover at the AMAZON was the smallest size, with 270 mV. It was observed that New application of agro-industrial waste to prevent the cell size is a parameter influencing at performance and stability melanosis of Mediterranean pink shrimp of Microbial Fuel Cells in accordance to the ecosystem. ∗ Giorgio Rizza , Giuseppina Rosaria Antonella Alberio, Rosa Palmeri, Aldo Todaro, Giovanni Spagna http://dx.doi.org/10.1016/j.nbt.2014.05.997 University of Catania, Italy

PX-13 Food byproducts represent an environmental and economic problem because peel, pulp, pulp wash, and yellow water are Optimisation of scale-up of microbial fuel cell for sus- difficult to digest. Additionally, citrus byproducts are relatively tainable wastewater treatment with positive net energy resistant to microbial degradation (high COD and BOD5 indexes) generation due to their high content of bioactive compounds with antimi- Ourania Dimou 1,∗, John Andresen 2, Veyacheslav Feodorovich 3, crobial activity, e.g. ascorbic acid, limonoids, and polyphenols. Igor Goryanin 4, Alan Harper 2, David Simpson 5 In addition, agro-industrial wastes are potential sources of bio- phenols that have proven antioxidant, anti-inflammatory and 1 Heriot-Watt University, UK anticancer properties. Aims of this work is to test how natural 2 Department of Chemical Engineering, Heriot-Watt University, extracts obtained from vegetable waste, in particular orange and Edinburgh Campus, EH14 4AS, UK lemon peel, may inhibit the process of melanosis in pink shrimp 3 M Power World, Vavilova str. 5/3, Moscow, Russia species (Parapeneus longirostris). The treatment with orange peel, 4 Informatics Life-Sciences Institute, Edinburgh University, Edin- extracted in hot water, significantly reduced (p < 0.05) the enzy- burgh EH8 9AB, UK matic activity of the PPO at the level of the cephalothorax of 5 Biological Systems Unit, Okinawa Institute of Science and Tech- shrimp. Ethanolic extract of lemon peels was also effective for inhi- nology, Okinawa 1919-1, Japan bition of melanosis. The results were related to the evaluation of From eight to fifteen litres of liquid by-products are generated the polyphenol quality index (QI) that confirmed the efficacy of for every litre of grain whisky produced. ‘Spent Wash’is the main treatment of the orange and lemon peel extracts. The addition of liquid stream. If discharged untreated into the environment it these natural extracts in pink shrimp samples can be considered a might contribute to pollution such as eutrophication [1]. valid alternative to the use of agro-industrial waste. Microbial Fuel Cells (MFCs) are a natural bio-technology solution to the issue working either independently or in conjunction http://dx.doi.org/10.1016/j.nbt.2014.05.999 with established wastewater treatment technologies. Utilising metabolic reactions of electrochemically active microorganisms,

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Veterinary biotechnology the intracelluar levels of ROS and numbers of apoptotic nuclei in catalpol (100 ␮M) treated blastocysts revealed that ROS levels PY-01 of catapol-treated porcine blastocyst were decreased (P < 0.05) and Expression and bioactivity analysis of the expression of the numbers of apoptotic nuclei were reduced by catalpol treat- PEDV COE gene in Pichia pastoris ment in porcine embryos. Moreover, the blastocyst development

∗ and total cell numbers of blastocysts were significantly increased Lumu Li , Fazhi Xu, Qingsong Hu, Xiaoling Ding, Xiongyuan Si in the catalpol treated group relative to the untreated catalpol ␮ Anhui Agricultural University, China group under H2O2 (200 M) induced oxidative stress (P < 0.05). Furthermore, the intracellular levels of ROS in catalpol-treated The purpose of this study was to express the porcine epidemic group were significantly decreased in the untreated catalpol group diarrhoea virus (PEDV) COE protein in Pichia pastoris, and eval- under H2O2 induced oxidative stress (P < 0.05). In conclusion, our uate the neutralizing ability of mice serum after immunization. results suggest that catapol improves the developmental compe- A pair of primers was designed based on the cloned PEDV S tence of porcine embryos via modulation of intracellular levels of nucleotide sequences. Intestinal tissues were collected from piglet ROS and the apoptotic index during the preimplantation stage. suffering from PEDV, and the mRNA of PEDV was extracted by the TRIzol reagent. The PEDV COE gene was amplified by RT- http://dx.doi.org/10.1016/j.nbt.2014.05.1001 PCR. Furthermore, the PEDV COE gene was inserted into pPIC9K, and the recombinant plasmid of pPIC9K-COE was transformed into P. pastoris GS115 by electroporation. High copy recombinant PY-03 strains were screened and then expression was induced by addi- Study on the establishment of the animal model infected tion of methanol. SDS-PAGE and Western blotting were used to by influenza virus in Microtus brandti analyse the immunogenicity of recombinant protein. The neutral- ∗ izing ability of mice serum antibodies by PEDV COE recombinant Defeng Wu was analyzed by virus neutralization test. PEDV COE gene was Fujian Agriculture and Forestry University, China amplified by RT-PCR, and it was 530 bp in length .The PEDV COE protein was expressed in P. pastoris. The MW of the proteins was In this study, the brandti voles were infected by influenza virus about 30 kDa as analyzed by SDS-PAGE, and the concentration was A/WSN/33(H1N1) intranasally, in order to assess the susceptibility 30 mg/L. Western blotting showed that the protein had immuno- of influenza virus on brandti voles. The brandti voles showed high genicity. The virus neutralization test result showed that the mice susceptiblity to infection. The infection susceptibilty was further immunized with the recombinant PEDV COE protein produced evaluated to establish a possible susceptibility index for brandti specific serum antibodies with neutralizing activity, the neutraliza- voles model infected by influenza virus. tion titer reached 1:36. PEDV COE protein was correctly expressed By means of the method of infecting brandti voles, influenza in P. pastoris and the protein had a good biological activity. virus was separated and identified successfully. The result showed that the virus was widely distributed in brandit vole tissues. http://dx.doi.org/10.1016/j.nbt.2014.05.1000 which also indicated that the brandti vole are highly sensitive to influenza virus, and the major target organ was lung. The animal model of brandti voles infected by influenza virus has been PY-02 established successfully by means of some model indexes, such Protective effects of catalpol against hydrogen perox- as clinical symptoms, body weight, mortality, the index of the ide induced oxidative stress on preimplantation porcine separation of influenza virus and so on. embryos http://dx.doi.org/10.1016/j.nbt.2014.05.1002 Deog-Bon Koo ∗, Yong-Hee Lee, Sung-Hun Min, Jin-Woo Kim, Jae- Hyun Ahn, Geon-Yeop Do, Sung-Kyu Chae PY-04 Daegu University, South Korea A Salmo salar diet based on a marine biosurfactant for Catalpol, an iridoid glucoside, isolated from the root of the profilaxis and treatment of Pisciricketsia salmonis Rehmannia glutinosa, possesses a broad range of biological and ,∗ pharmacological activity including anti-tumor, anti-inflammation Claudia Ibacache-Quiroga 1 , M. Alejandro Dinamarca 2, Juan and anti-oxidant by acting as a free radical scavenger. Therefore, Ojeda 2, José Miguel Troncoso 3 in the present study, the effects of catalpol on blastocyst develop- 1 Centro Nacional Biotecnología/Micromarine Biotech, Spain ment and expression levels of reactive oxygen species (ROS) were 2 Universidad de Valparaíso, Chile investigated in preimplantation porcine embryos. After in vitro 3 Ewos, Chile maturation and fertilization, porcine embryos were cultured for 6 days in porcine zygote medium 3 (PZM-3) with catalpol (0, Aquaculture is a fast growing economic activity due to an 100, 200 and 400 ␮M respectively). Blastocyst development was increase of food demand worldwide, which main challenges are not significantly improved in the catalpol treated groups when bacterial infections that causes significant biomass and economic compared the control group. However, subsequent evaluation of losses. Although classic antibiotic therapy was effective against

S214 www.elsevier.com/locate/nbt New Biotechnology · Volume 31S · July 2014 VETERINARY BIOTECHNOLOGY bacterial infections in aquaculture, nowadays these therapies in which Salmo salar was directly exposed to the fish pathogen are unsuccessful due to an increase of antibiotic resistance. P. salmonis. Fish were fed with food supplemented with BS at a We recently reported that a specific marine biosurfactant (BS) final concentration of 400 mg/kg of fish/10 days, during thirty interferes with quorum sensing (QS) cell-to-cell communication days. Fish diet with BS showed a survival rate 15% higher than system, reducing virulence of bacterial fish pathogens. BS do not the control diet (without BS). The immune makers IL-1␤ and IL-8 act modifying bacterial cellular processes, therefore no resistance evaluated by qRT-PCR showed an increase in gills and intestine to BS is developed, making these molecules an alternative to the respect to the control diet. The food containing the BS and serum use of antibiotics in aquaculture. The aim of this study was to incor- samples form treated fish were active for the inhibition of QS and porate the biosurfactant into fish food and to evaluate the effect growth of P. salmonis, demonstrating the presence of the active of the supplemented diet on fish survival to Pisciricketsia salmonis. principle. BS was incorporated by emulsification into fish food. The effect of the supplemented diet was evaluated through a challenge assay, http://dx.doi.org/10.1016/j.nbt.2014.05.1003

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Yeast and filamentous fungi The production of the rose-like aroma compound 2- phenylethanol (2-PE) by Aspergillus niger spp. is used as an PZ-01 example to investigate the effect of microparticle enhanced culti- New source of hyaluronidase – Fistulina hepatica vation MPEC on the production of fungal secondary metabolites. The production of the higher alcohol 2-phenylethanol from L- 1,∗ 1 1 Lenka Bobková , Dzianis Smirnou , Martin Krcmáˇ ˇr , Veronika phenylalanine was up to now mainly investigated in yeasts. 2 2 3 Moravcová , Martina Hermannová , Vladimír Velebny´ However, Aspergillus niger DSM 821 is also capable of synthesizing 1 Contipro Biotech s.r.o, Czech Republic 2-PE. It yields product concentrations of up to 700 mg/L, which is 2 Contipro Pharma s.r.o, Czech Republic twice as much as previously investigated strains. Screening of 16 3 Contipro Group s.r.o, Czech Republic types of particles of different chemical composition showed that the application of MPEC increased the final concentration by a fur- Hyaluronidases are a group of enzymes capable of hyaluronic ther 30%. Experiments for downscaling the technique to 48 well acid (HA) depolymerization. These enzymes can be divided into microtiter plate scale is under way, results we be available by the groups according to the mechanism of HA cleavage or source time of the congress. of origin. Bovine testicular hyaluronidase (hydrolase) and bacterial hyaluronidases (lyases) are the main ones used in cosmetic http://dx.doi.org/10.1016/j.nbt.2014.05.1005 or medicinal applications nowadays. Fungi as a new source of hyaluronidases were not authentically described until recently [1]. PZ-03 This work was focused on novel hyaluronidase production by the Basidiomycete Fistulina hepatica. The enzyme activity was The cellulase induction system in Trichoderma reesei detected in culture medium. Characterization of the enzyme was strains remains well conserved despite several genera- performed after chromatographic purification. Products of HA tions of random mutagenesis and screening cleavage were identified by HPLC and MS. The reaction pro- ,∗ Dante Poggi-Parodi 1 , Aurelie Pirayre 1, Thomas Portnoy 1, ceeded in an eliminative manner and unsaturated hyaluronan Hugues Mathis 1, Thiziri Aouam 1, Frédérique Bidard 1, Stéphane oligosaccharides were formed. This type of mechanism was so Le Crom 2, Antoine Margeot 1 far described only for hyaluronan-lyases produced by bacteria. Optimal conditions for HA–oligosaccharide production were pH 1 IFP Energies Nouvelles, France 4 and 20 ◦C. The enzyme was stable for 8 days under these 2 Université Pierre et Marie Curie, Laboratory of Developmental conditions. Biology, CNRS UMR7622, Paris, France

Trichoderma reesei is the main industrial producer of enzymes Reference degrading cellulosic and hemicellulosic biomass because of its high [1].Bakke M, Kamei J, Obata A. Identification, characterization, protein secretion capacity. This outstanding level of cellulases pro- and molecular cloning of a novel hyaluronidase, a member of duction is the product of several rounds of random mutagenesis glycosyl hydrolase family 16, from Penicillium spp. FEBS Lett and screening in the past. 2011;585:115–20. In this study, using a well-studied lineage of improved strains from T. reesei, NG14 and RUT C30, we investigated the links http://dx.doi.org/10.1016/j.nbt.2014.05.1004 between the mutations generated during the mutagenesis screens, that allowed development of these strains, and the transcriptome PZ-02 differences in the first hours of the cellulases induction process. Despite a high number of reported mutations and very different Particles prevent pellets: microparticle enhanced culti- productivities, the transcriptome from both strains were surpris- vation MPEC increases 2-phenylethanol production in ingly similar, with few genes being over or under expressed in Aspergillus niger the higher producer strain. Comparison of transcriptomes of the ∗ Maria M.W. Etschmann , Ina Huth, Jens Schrader, Dirk Holt- mother strain QM6a and RUT C30 before induction of cellulase mann production revealed that most of observed changes between NG 14 and RUT C30 were probably due to basal changes to the cell DECHEMA Research Institute, Germany physiology, and that the cellulase production system was essen- Adding microparticles to fungal shake flask cultivations pre- tially intact. Accordingly, very few mutated genes seem to belong vents pellet formation and leads to finely dispersed and highly to the cellulase induction system, suggesting that there is room for productive mycelia. This addresses a problem occurring in many improvement regarding this system. industrial processes using filamentous fungi in submerged cul- This study shows that systems biology methods can gather tures: formation of pellets. Fungi tend to grow in agglomerates information about cellular systems that cannot be obtained by which can be as large as several centimeters in diameter. The cen- traditional techniques, and that modern strain development ter of the pellets is badly supplied with oxygen and nutrients and programs should include both approaches to construct compet- can consist of essentially dead biomass. As only the biomass in itive strains. the outer shell is active, productivity of these processes can be low. Techniques to increase productivity are therefore in high http://dx.doi.org/10.1016/j.nbt.2014.05.1006 demand.

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PZ-04 isolated from different E. purpurascens strains, including epicoc- caene, a broad-range fungicide, discovered and patented by our Characterization of the relevant genes and development group. It is remarkable that the biology of this fungal species of salt-tolerant yeast strains by transposon mutagenesis has not been much studied, and we have very little knowledge Hyun-Soo Kim ∗, Hye-Min Kang on its genetic and phenotypic diversity. Only recently the genus Epicoccum was placed in the newly described family Didymel- Department of Food Science and Industry, Jungwon University, laceae, and great intraspecific diversity of E. purpurascens has been South Korea reported. We determined the genetic and metabolite profiles of Kimchi is a traditional, fermented Korean food prepared with different E. purpurascens strains isolated in New Zealand, and we different vegetables, spices, and ingredients and is an important have found that metabolite profile was closely associated with the dietary source of vitamins, minerals, and other nutrients. The ability of each strain to produce biologically active compounds, waste brine from manufacturing process of kimchi is released which did not match their similarities based on their genetic pro- and eventually results in serious environmental pollution. Sac- file. We have now de novo sequenced and assembled the draft charomyces cerevisiae strains tolerant to salt stress are important genome of our working E. purpurascens strain. Further annotation for the production of single cell protein by using kimchi waste of the genome and specific mining for secondary metabolite gene brine and removal of potential pollutants from the waste water. clusters will strongly facilitate biotechnological use of E. purpuras- In this study, approximately 3000 transformants of the mTn3- cens. mutagenized genome library were selected as leucine prototrophs and replica-plated to both YPD agar and YPD agar containing 10% http://dx.doi.org/10.1016/j.nbt.2014.05.1008 NaCl. Three strains (Tn 1–3) tolerant to up to 10% NaCl were isolated by screening a transposon-mediated mutant library. Three PZ-06 transposon mutants showed higher ethanol production and grew faster than control strain when cultured in rich media contain- The effect of carbon sources on upstream regulatory ing 5%, 7.5%, and 10% NaCl respectively. The determination of regions controlling the expression of the Candida utilis transposon insertion sites and Northern blot analysis identified maltase gene putative genes and revealed simultaneous down-regulations and Ján Krahulec ∗, Hanka Bonková,ˇ Veronika Liˇsková, Michaela disruptions of the genes indicating that salt tolerance can be con- Osadská, Stanislav Stuchlík, Ján Turnaˇ ferred. The genes identified in this study may provide a basis for the application in developing industrial yeast strains. Faculty of Natural Sciences, Comenius University in Bratislava, Slovakia http://dx.doi.org/10.1016/j.nbt.2014.05.1007 In the last few years it has been demonstrated that the industrially important yeast system Candida utilis represents a promising PZ-05 expression host, generating relatively high levels of recombinant proteins. Nevertheless, basic knowledge of its gene structure Epicoccum purpurascens (Didymellaceae, Ascomycota)as and regulation of gene expression, which is needed to allow a non-model source of novel bioactive compounds: more extensive use of this organism, is lacking. The current biotechnological perspectives study presents preliminary characterization of the carbon source- Mikhail Fokin 1,∗, Bevan Weir 2, Ting-Li Han 3, Neethu Arun 3, dependent expression of the C. utilis maltase gene in order to Lydia Yamaguchi 4, Massuo Jorge Kato 4, Silas Villas-Boas 3 compare its regulation. Our results showed that C. utilis maltase is able to hydrolyze cellobiose and soluble starch, and its affinity 1 University of Auckland, New Zealand for cellobiose is even three times higher than for maltose. To fur- 2 Landcare Research, New Zealand ther investigate the function of maltase and the use of the maltase 3 Centre for Microbial Innovation, School of Biological Sciences, promoter for the heterologous protein expression, we successfully The University of Auckland, New Zealand applied the Cre-loxP system to acquire a null mutant strain with 4 Chemistry Institute, University of São Paulo, Brazil disrupted maltase gene and promoter in the polyploid yeast C. Filamentous fungi have been widely used as a source of valuable utilis. The first step was to introduce a mutagenesis cassette har- bioactive compounds for medicine, agriculture and white biotech- boring a marker gene between two loxP sites into the target cells. nology. Recently, genomic studies have significantly improved After identification of a partial mutant strain, the same mutagen- our knowledge of the functional background for known com- esis cassette was repeatedly used and the null mutant strain was pounds and revealed a vast amount of novel secondary metabolites selected on higher antibiotic concentration. The last step was to not commonly expressed in the laboratory. Among fungi these introduce a Cre-expression plasmid and screen for marker rescue studies have been focused mostly on a few model species. Epic- strains. occum purpurascens is a widespread member of Dothideomycetes Acknowledgements: This work is result of project imple- class, which consists of over 20 thousands species. E. purpuras- mentation: “Production of biologically active agents based on cens has been isolated from different substrates such as plants, recombinant proteins” (ITMS 26240220048) supported by the soil, and marine invertebrates. Several bioactive compounds were

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Research and Development Operational Program funded by the PZ-08 ERDF. Selection of preservation conditions for Debaryomyces hansenii yeasts killer toxins http://dx.doi.org/10.1016/j.nbt.2014.05.1009 Barbara Zarowska 1, Lukasz Bobak 2, Piotr Regiec 3, Adam Figiel 4, Monika Grzegorczyk 5, Marek Szołtysik 2, Maria Wojtatowicz 5, PZ-07 Xymena Polomska 5,∗ Characterizing the sterol specificity of microbial and 1 Wroclaw University of Environmental and Life Sciences, Wro- mammalian acyltransferases claw, Poland Holly Stolterfoht 1,∗, Barbara Petschacher 1, Katharina Littringer 1, 2 The Department of Animal Technology and Quality Manage- Martin Lehmann 2, Hans-Peter Hohmann 2, Harald Pichler 3 ment, Wroclaw University of Environmental and Life Sciences, Wroclaw, Poland 1 ACIB GmbH, Austria 3 The Department of Food Storage and Technology, Wroclaw Uni- 2 DSM Nutritional Products Ltd, Austria versity of Environmental and Life Sciences, Wroclaw, Poland 3 Institute of Molecular Biotechnology, TU Graz, Austria 4 The Institute of Agricultural Engineering, Wroclaw University of Sterols are essential lipid components of eukaryotic membranes Environmental and Life Sciences, Wroclaw, Poland influencing membrane fluidity, membrane permeability as well 5 The Department of Biotechnology and Food Microbiology, Wro- as the activity of membrane-bound proteins. In case of excessive claw University of Environmental and Life Sciences, Wroclaw, sterol formation, sterols are stored as sterol esters in lipid parti- Poland cles as energy reservoir or stock of membrane building blocks. Killer toxins of Debaryomyces hansenii yeast species are proteins The free sterol 3-OH group is acylated by the integral membrane with an ecological regulation function: they are active against proteins acyl coenzyme A: sterol O-acyltransferases (ACATs). This other yeast species and filamentous fungi. The study investigated storage mechanism is conserved from yeasts to plants and mam- the possibility of protein concentration by ultrafiltration or evap- mals, although the main membrane sterols differ between the oration under vacuum, and then drying by means of spray drying kingdoms of life. In yeasts, ergosterol is formed while mammalian and lyophilization. membranes contain mainly cholesterol. Ultrafiltration on a membrane with 18 kDa cut-off point has For the two S. cerevisiae acyltransferases, differences in sterol proved to be an effective method for killer protein concentration specificity have been shown for Are2p preferring ergosterol and of the tested strains. The use of membranes with a higher cut-off Are1p esterifying also ergosterol precursors [1]. Here, we compare resulted in the activity decrease of the killer protein in the obtained in a cross-species study the sterol specificities of ACATs. Therefore, concentrates, and at the same time, relatively high activity in fil- we expressed endogenous acyltransferases Are1 and Are2, and sev- trates. Comparably good results, as in the case of the ultrafiltration eral heterologous microbial and mammalian acyltransferases, in with membrane of 18 kDa cut-off, were obtained using the vacuum a S. cerevisiae are1are2 knockout strain. ACAT substrate specifici- evaporator. Spray drying resulted in the loss of activity of lethal ties were determined in vitro by activity assays using microsomal preparations. The smallest losses of 15% were observed in prepa- preparations, radioactively labeled oleoyl-CoA and ergosterol- and rations dried at 100 ◦C of inlet air. The best method to stabilize cholesterol-like sterol substrates. the killer toxin was freeze-drying. Immediately after lyophilization Reference and after three months of storage killer toxins exhibited activity [1].Zweytick D, Leitner E, Kohlwein SD, Yu Chunjiang, Rothblatt J, similar to that before preservation. Daum G. Contribution of Are1p and Are2p to steryl ester synthesis in Acknowledgements: This work was supported by research the yeast Saccharomyces cerevisiae. Eur J Biochem 2000;267:1075–82. grant POIG.01.03.01-02-080/12, co-financed by the European Union from the European Regional Development Found. http://dx.doi.org/10.1016/j.nbt.2014.05.1010 http://dx.doi.org/10.1016/j.nbt.2014.05.1011

PZ-09

Evaluation of Geotrichum candidum deacidiﬁcation potential for yoghurt keeping quality

Imran Muhammad, Saima Riaz, Nawaz Farah ∗

Department of Microbiology, Quaid-i-Azam University, Islamabad, Pakistan

Lactic acid bacteria have received a lot of research attention due to their diverse technological and metabolic characteristics in fermented dairy products. The present study was designed to study the impact of microbial population on maintaining the quality of

S218 www.elsevier.com/locate/nbt New Biotechnology · Volume 31S · July 2014 YEAST AND FILAMENTOUS FUNGI fermented milk. Post fermentation acidification is a serious con- molecules of different size. Those plasmids replicate independently cern in dairy industry owing to its potential detrimental effects from cell nucleus due to the encoding of their own DNA poly- on yogurt safety and quality. In order to rectify post acidification merases together with the terminal protein being a very important problem in yogurt, the deacidifying ability of G. candidum was uti- element of replication initiating complex. lized. G. candidum and starter culture when used in combination In this work seven new D. hansenii plasmid systems (four dou- were found to have positive influence on the stability and qual- ble and three triple) were sequenced by NGS and the sequences ity of the yogurt. Growth parameters such as pH, acidity, syneresis, of genes encoding DNA polymerases were compared to each other total solid content, sensory evaluation and viscosity were analysed. and to previously known plasmid sequences from D. hansenii and Results have shown that G. candidum remarkably maintained pH to other yeast species. All genes of D. hansenii DNA polymerases 3.75 from 48 until 196 h as compared to control yogurt. Similarly shared very high homology with corresponding plasmids (the solid contents were higher and reduced syneresis was recorded for smallest or the largest plasmids from the systems) with identity yogurt with G. candidum. on the level of 95–96%. Also high homology, about 72% of iden- In the light of above findings it could be inferred that this mix tity, was observed with genes of plasmids from other yeast species culture starter could be a better option for industry for controlling like Kluyveromyces lactis, Babjeviella inositovora, MIllerozyma acaciae, the post fermentation acidification problem in fermented dairy Schwanniomyces etchellsii. Within one plasmid system only resid- products thus contributing towards safety and stability of final ual similarity in analyzed genes was observed; a few percent of product. identity. Acknowledgements: This work was financially supported by http://dx.doi.org/10.1016/j.nbt.2014.05.1012 Polish Ministry of Science and Higher Education. ProjectNN312 258138.

PZ-10 http://dx.doi.org/10.1016/j.nbt.2014.05.1013 Genes encoding DNA polymerases on linear dsDNA plasmids of Debaryomyces hansenii yeasts share very high homology

Xymena Połomska 1,∗, Cecile Neuveglise 2, Zbigniew Lazar 3, Bar- bara Zarowska 4

1 The Department of Biotechnology and Food Microbiology, Wro- claw University of Environmental and Life Sciences, France 2 INRA, MICALIS, AgroParisTech, Thiverval-Grignon, France 3 Wrocław University of Environmental and Life Sciences; INRA, MICALIS, AgroParisTech, Thiverval-Grignon, France 4 Department of Biotechnology and Food Microbiology, Wrocław University of Environmental and Life Sciences, Wrocław, Poland

Debaryomyces hansenii yeasts can keep natural plasmids in their cytoplasm occurring in systems with two or three linear dsDNA