DESIGNEKSEMPEL RAPPORTER institutniveau centerniveau

Perostrud eugait amconsenia Volor seniamet conulputpat dolenia AnnualAuismod Report della ipsurmy 2008

CenterCenter forfor MicrobialBiologisk BiotechnologySekvensanalyse Institut for Systembiologi Department of Systems Biology ANNUAL REPORT 2008 CENTER FOR MICROBIAL BIOTECHNOLOGY 

I tilfælde af at rapporter produceres lokalt, kan overskrifter tilprintes på fortrykte forsider. Her benyt- tes skriften Arial. 39 DTU VISUEL IDENTITET DESIGNRETNINGSLINIER 2.1 Center for Microbial Biotechnology Department of Systems Biology Technical University of Denmark Building 221 / 227 DK-2800 Kgs. Lyngby Denmark

Phone: +45 4525 2600 Fax: +45 4588 4148 CMB is an Engineering Center of Excellence funded [email protected] by the Danish Research Agency. It is a collaboration between an acknowledged research manager, www.cmb.dtu.dk his/her institute and university, and the Research Agency. An Engineering Center of Excellence is Articles written by: Chris Tachibana a research institute of first-class quality with Edited by: CMB tradition for cooperation with industry. Contents

Executive summary 5

Communication and cooperation between mould species permits better growth for all 6

A project on DNA double-strand break repair forms a bridge between basic research and industrial applications 8

Identifying Disease Biomarkers: A Networker's Approach 10

Colourful filaments yield powerful antibiotics 12

Complementary competencies in a project to improve citric acid production 14

Highlights 2008 16

Faculty 18

Organisation 19

Publications 2008 20

Staff 30

ANNUAL REPORT 2008 CENTER FOR MICROBIAL BIOTECHNOLOGY 

Executive summary

CMB in 2008 and onwards expertise on Saccharomyces yeast; an increased emphasis When I signed my contract with DTU to replace Jens on secondary metabolites and protein production, Nielsen and become the Director of Center for Microbial while maintaining a strong expertise on primary Biotechnology (CMB) by the start of 2008, I knew that metabolism; and an increased emphasis on regulation I took responsibility for something extremely valuable. and compartmentalisation, while maintaining a strong Having collaborated with Jens Nielsen on several projects expertise on stoichiometric modelling. and having been on the Scientific Advisory Board for several years, I knew CMB well. Among Jens Nielsen’s The IVC support from FTP is part of a larger, long-term many talents are to see quality in people, to see fruitful strategy, reaching beyond 2013. CMB will in 2009-2013 synergy between disciplines, and to use these insights continue to collaborate with many partners and to obtain to build a well-functioning organisational structure. financing from many sources. The specific goals of such I therefore knew I was taking responsibility for a staff activities are distinct from those supported by the IVC unique in technological skills and creativity, working well grant, but the activities will be highly synergistic with the together in a good atmosphere. latter, and will be instrumental for each research group for keeping a state-of-the-art level of technology. Examples It is with joy that I look back at the development of CMB are the set-up of cultivation of mammalian cells for the in 2008. Our first big challenge in the past year was to production of pharmaceutical proteins and activities in prepare an application to the Danish Research Council food safety. In relation to the food area it is important for Technology and Production Sciences (FTP) for a to mention that the International Advisory Board of the second 5-year (2009 through 2013) period of support in Centre for Advanced Food Studies, a centre that supports the form of an ingeniørvidenskabeligt center (IVC). We part of our activities, in October visited DTU Systems submitted the application in March, the international Biology, including CMB. We were praised in a number scientific evaluation panel liked it, and FTP granted of respects, including the “very high international level” us more than DKK 20 million after a co-financing by of our research. DTU of more than DKK 26 million came in place. We are convinced that the positive decision by FTP was stimulated by strong support from the Danish biotech industry, including generous financial support of projects related to the IVC; one company thus contributed with several PhD stipends.

The scientific substance of the continued IVC defines the scientific core of CMB. Despite the inclusion of several new elements, the mission of CMB remains unchanged. We focus on the development of cell factories with a strong emphasis on yeast and other fungi, and our main strategy is to possess a broad range of complementing expertise that will secure optimal state-of-the-art approaches to analysis, modelling and engineering. The in-house expertise includes analytical biotechnology, cell biology, data management, downstream processing, It is with great enthusiasm that I look at the future of CMB. fermentation physiology, genetics, informatics, I enjoy my interactions with my wonderful colleagues, mathematical modelling, microbial biodiversity, who do all the work, and I see challenges being met with molecular biology and natural product chemistry, their high spirit, creativity and capability. and it is obviously supplemented by out-of-house collaboration, both within and outside the mentioned disciplines. In short, the defining strength of CMB is its multidisciplinarity.

We use bio- and chemodiversity and new knowledge within metabolic regulation and subcellular localisation Morten Kielland-Brandt to develop cell factories for industrially relevant products such as flavours, antibiotics, fuels, natural colorants, Director of CMB organic acids and special proteins. New trends in the coming years will thus consist of an increased emphasis on filamentous fungi, while maintaining a strong

ANNUAL REPORT 2008 CENTER FOR MICROBIAL BIOTECHNOLOGY  Communication and coopera- tion between mould species permits better growth for all

CMB Associate Professor Birgitte Andersen investigates how chemical communication between fungal species may allow moulds to cooperate and thrive on water-damaged surfaces like wallpaper, wood and fabric.

By Chris Tachibana

Step one: keep out the water. This is the key to preventing fungal contaminants even more important. Although there mould from growing on your windowsills, walls and are hundreds of thousands of fungal species, Andersen and furniture, says Associate Professor Birgitte Andersen. A colleagues have found that only a limited number grow decade ago she studied fungal growth on building materials indoors. Only 10-15 different types, such as Penicillium, as part of the Moulds in Buildings project, a collaboration of Aspergillus, Chaetomium, Stachybotrys and the three that doctors, mycologists, and builders that looked at the health Andersen is currently concentrating on, Cladosporium, impact of mould in water-damaged schools, and came up Alternaria and Ulocladium, are regularly found on building with ways to renovate mouldy buildings. Andersen has materials. continued work on building moulds, with funding from the Villum Kann Rasmussen Fund. She knows that step one is, in Some of these are the same taxonomic groups that attack fact, impossible to fully implement, because of the humidity food, so Andersen, with the help of undergraduate we generate in our homes when we cook, bathe and wash researchers and a technician, has used knowledge about our clothes. Leaks and majors floods, like after Hurricane food-spoiling fungi as the background for some hypotheses. Katrina in New Orleans, can lead to extensive fungal For example, food-spoilage moulds are known to specialize, growth. So step two is figuring out how to inhibit fungal with particular species and strains adapting to growth in growth, and this requires knowing exactly what nutrients conditions like high salt or high protein. It appears that fungi use on indoor surfaces and what their vulnerabilities building moulds do the same, specializing to grow on wet are. Why not just live with the fungi? Microbial growth wallpaper, wood or insulation material. Andersen has also causes deterioration and discolouration when it breaks found, however, that the building moulds tend to grow down building material. Some people are allergic to in consortia, or cooperative groups. In samples collected airborne mould spores, and in high concentrations, they as part of the earlier project, Andersen found that two or can irritate the respiratory system. Some moulds produce three different species are usually found growing together. mycotoxins that can cause illness, but toxin production is No single species dominates over the others, and all seem to highly species-specific, making precise identification of grow more robustly than when each species is grown alone Ulocladium (black) growing uninhibited together with Trichoderma (bluish green) Inoculated at the same time in the centre of a PCA plate.

Already established Aspergillus (3 orange cultures) “allows” Trichoderma ( centre) to germinate and grow. in pure culture. In addition, some unexpected patterns a consortium, if and how they compete, and the small are seen, such as the appearance of Stachybotrys, which molecule communication or collaboration that occurs does not produce cellulase enzymes, on high-cellulose between them. Together with the Fungal Biodiversity material. Association with cellulase-producing fungi, like Centre (CBS) in the Netherlands they have already used Ulocladium, allows its growth. Other results suggest that the molecular analysis to definitively classify the moulds production of secondary metabolites influences the types in some consortia, and have conducted preliminary of fungi in a consortium, with some compounds acting as metabolite analysis. They have found that different mycotoxin defences for the entire consortium and others fungal species growing together on a Petri dish are not as deterrents for "outsider" fungi. These findings have as competitive as one might expect, as long as they are led to the three main hypotheses of Andersen's current grown on a medium that does not favour one or the other. project: that mould on building materials is a consortium of Rather than one species taking over the culture, if the collaborating fungal species; that each produces compounds medium is poor for all of them and all are slightly stressed, such as enzymes or antibacterials that aid the consortium; they appear to equalize and possibly even collaborate, and that members of the consortium communicate through consistent with the hypotheses. This system could be a compounds that stimulate or restrict other fungi. model for events that lead to fungal consortia "in edificio", on building materials that are acceptable, albeit poor The project is currently a basic research enterprise that growth media for several different collaborative fungi. uses species collected in the earlier study, grown on In the future, Andersen will use HPLC-based metabolite wallpaper samples prepared in the lab. The goals are to mapping to identify the secondary metabolites that serve complete the identification of the fungi and characterize as communicating molecules, attractants, mycotoxins, their individual limits to growth and metabolite anti-fungals and anti-bacterials in the consortia, possibly production. Then, working with the fungal species in leading to targets for controlling indoor mould growth. pairs and groups of three, Andersen and her colleagues For now, though, she advises we all try to follow step one will investigate which species are capable of creating and keep the humidity down and the water out.

ANNUAL REPORT 2008 CENTER FOR MICROBIAL BIOTECHNOLOGY  A project on DNA double- strand break repair forms a bridge between basic research and industrial applications Anyone who has ever worked in a lab knows the feeling. How universal are double-strand At the start of a project, progress is excruciatingly slow. Protocols must be optimized. Techniques must be break repair mechanisms? Why perfected. Then, just as the project deadline approaches do mutations in the related proc- and the funding ends, everything begins to work and ess of homologous recombination major breakthroughs are just one experiment away. Jakob Blæsbjerg Nielsen, who finished his Ph.D project make Aspergillus turn red? Post- with Associate Professor Uffe Mortensen and Assistant doctoral fellow Jakob Blæsbjerg Professor Michael Lynge Nielsen in 2008, experienced this common research phenomenon, so he considers Nielsen's answers to these ques- himself lucky to be continuing his project at CMB as tions may be useful in developing a post-doctoral fellow. CMB is fortunate to keep Dr. Blæsbjerg Nielsen's experience at the Centre, since his industrial Aspergillus strains. work on Aspergillus has implications for the industrial use of this fungus as a cellular factory, as well as possibly By Chris Tachibana addressing its impact on humans as an invasive pathogen and a cause of food spoilage, and a pest that damages building materials and crop plants.

The process of double-strand break repair, or restoring DNA after both strands have been severed, is one that all cells must address. The mechanisms have been extensively studied in unicellular yeast and in multicellular mammals, but Nielsen says, "One or a few model systems cannot tell the whole story. A link – for example from filamentous fungi, is needed between simple and complex organisms." Aspergillus is the ideal, simple, multicellular organism to act as such a link. Uncovering the details of its double-strand break repair mechanism will identify those features that are unique to certain organisms and highlight those that are global. Some double-strand break repair components may be universal, but adapted to different roles in different cell types. In addition to learning about basic DNA repair mechanisms, Nielsen's This makes the project of even greater relevance for project has practical implications. The processes by which the Aspergillus groups at CMB. A project in Dr. Anna cells attempt to paste together exposed DNA ends, such Eliasson Lantz's lab, which is also described in this as homologous recombination, non-homologous end- Annual Report, specifically studies the pathways joining (NHEJ) and alternative end-joining of the broken involved in producing polyketides, many of which are DNA molecules, are also important for transformation the basis of pharmaceuticals like anti-bacterials, and of Aspergillus, that is, introducing genes for producing cholesterol-lowering statins. genetically engineered strains. During his Ph.D research, Nielsen found that strains defective in NHEJ were much The collaborative environment is one of the advantages more efficient at gene targeting, or directing introduced of working at CMB that Nielsen noticed, as a student genes to specific chromosomal locations. He is now and now as a post-doc. "We have an exceptional working on finding out why NHEJ-deficient mutants working environment and great colleagues," he says. still occasionally insert a gene at random chromosomal He cites the dynamic atmosphere of having students locations. Knowing the components of the "back-up" and staff of many nationalities and backgrounds and pathway that causes the random insertions could lead to fields of expertise as contributing to the productivity ways to reduce this side effect, and allow an assessment at CMB. Mostly though, he says the advantage of of the overall rate of mutations, which must be kept low working at CMB is being part of a team of highly in industrial strains. skilled labs with expertise over the entire pipeline of generating cell factories– from drug discovery, to Jakob Blæsbjerg Nielsen's project has recently become, the molecular biology and metabolic engineering of literally, more colourful. He and co-workers discovered strains, to chemical analysis and downstream handling, that strains with mutations in genes involved in all applying a systems biology approach. homologous recombination produce a red secondary metabolite. The pigment is visible in the colonies and is secreted into the medium, and the intensity of the red colour seems to correspond to the severity of the mutation. Strains that are the most defective in recombination appear to be the most red. The hypothesis is that the pigment is produced in response to the replicative stress induced by the inability to carry out DNA repair because of homologous recombination deficiencies. The pigment might have defensive or protective properties and is probably a polyketide.

ANNUAL REPORT 2008 CENTER FOR MICROBIAL BIOTECHNOLOGY  Identifying Disease Biomarkers: A Networker's Approach

not treated. They need a fast, reliable and non-invasive Assistant Professor Kiran Patil diagnostic test, and they need a way to distinguish between different types of type 2 diabetes. For example, in Western and his students are analysing countries, type 2 diabetes is often associated with obesity, existing transcriptome and while in Asian countries, it is not. In the absence of reliable markers to distinguish between forms of type 2 diabetes, metabolome data in silico, to find little can be known about differences in their molecular indicators for type 2 diabetes. mechanisms or consequences. Research on both diagnostic and distinguishing tests for type 2 diabetes would benefit By Chris Tachibana greatly from the identification of biomarkers, or small molecules that act as indicators of the disease state and can be used to follow its progression. The diabetes work in Kiran Patil is a networker. At CMB, he develops algorithms the Patil lab is aimed at identifying these biomarkers. The to analyze biological networks and their interactions. To hypothesis guiding the project is that the cellular objective find experienced personnel for his lab, he tapped into his in regulating metabolic genes is maintaining homeostasis. professional network. He used connections in Portugal Rather than monitoring all possible small molecule to recruit student Simão Soares, who brings expertise in changes at once, the cell relies on altering key metabolic informatics engineering. The lab now has a connection fluxes, with all others following, as a way to maintain to Lithuania with Aleksej Zelezniak, who was recently balance. The lab is looking for those key metabolites that awarded a prestigious Novo Nordisk-Novozymes Master's are the main triggers of change. student scholarship. The team is working with collaborators Dr. Mary Elizabeth Patti at the Joslin Diabetes Centre In 2005 and 2008, the lab published papers describing in Boston, and Allan Ertmann Karlsen, from the Steno Reporter Algorithms, a system that can be applied to Diabetes Centre in Denmark, to untangle the complex genomic, transcriptomic, proteomic and metabolic interconnections between the cell's biochemical pathways, data. The system uses information in public databases the metabolome of small molecules that flow through the to make hypotheses about physiological and biological pathways, and the transcriptome of the cell's expressed constraints that can be imposed on the vast amounts of genes. data generated by global analysis. The method distills all possible interactions between pathways, gene expression Their system for studying these network connections is type patterns and metabolites, down to only the most likely 2 diabetes, a disease of both insulin resistance and impaired and the most informative ones. Taking advantage of the insulin secretion that is linked to changes in metabolism, microbial metabolomics expertise at CMB, the Reporter gene expression and the molecular composition of the Algorithms system was first validated using established cell. Type 2 diabetes currently affects millions of people data on the yeast glucose repression transcriptome. worldwide and its incidence is increasing. In spite of its Now, the methods can be applied to higher organisms. global importance, the causes, consequences and potential This has been demonstrated in plants, but Patil and his cures for type 2 diabetes are still being investigated. To students have extrapolated their method to an even more make progress on these fronts, researchers and physicians complex system: the human diabetes transcriptome. needBy Chris comprehensive Tachibana information on what happens during Their initial analysis identified the TCA, oxidative the development of diabetes, and what happens if it is phosphorylation and fatty acid metabolism pathways as containing likely candidates for diabetes biomarkers. analysis on only those metabolites, out of the thousands Promoter analysis of genes involved in the flux of these of possibilities, that are the most likely to show a strong biomarker candidates led to the identification of the difference between the diabetic and non-diabetic state. transcription factors CREB and USF1 as possible global In the meantime, the Patil lab continues to improve regulators in type 2 diabetes, a finding that could be and extend the Reporter Algorithms, and apply them useful in identifying new drug targets. in making discoveries to guide basic research. An important and surprising finding is the importance of Their next step was to apply the Reporter Algorithms certain highly connected metabolites like NAD+. This to the available data on the human metabolome, and key metabolite occupies a node that connects the TCA, combine those results with the transcriptome data oxidative phosphorylation and fatty acid pathways, which analysis. The results have led to powerful predictions appear to be crucial to metabolic flux in type 2 diabetes, for candidate diabetes biomarkers. Information on and its importance helps explain the multiple effects of the candidates will be given to collaborators who have the disease. In a similar way, the researchers in the Patil expertise in metabolomic analysis by mass spectroscopy, lab also sit at a node, one that connects mathematical who will use human samples to test the hypotheses modelling with the interpretation and application of generated by the Patil lab. The collaborators will use basic research, and the clinical development of diabetes the information on candidate biomarkers to focus their tests and treatments.

ANNUAL REPORT 2008 CENTER FOR MICROBIAL BIOTECHNOLOGY 11 Colourful Ph.D student Sujata Sohoni filaments yield studies Streptomyces coelicolor powerful to create a superhost for polyketide antibiotic production. antibiotics By Chris Tachibana The widely prescribed cholesterol-lowering drug and quantitative metabolic engineering. The facilities lovastatin, the powerful anti-cancer agent doxorubicin, at CMB mean that Sohoni, in addition to occasionally the common antibiotic erythromycin and the last-resort working with ActinoGEN collaborators, can do all her anti-fungal drug amphotericin B all have something strain construction and data collection in-house. The in common, besides their use as life-saving medicines. goal of the ActinoGEN project is to combat increasing Their shared polyketide chemical structure is what trends in antibiotic resistance with new antibiotics from interests student Sujata Sohoni and her supervisors CMB Streptomyces, which is the original source for two-thirds Associate Professor Anna Eliasson Lantz and Professor of currently used antibiotics. The project also aims to Ivan Mijakovic, former of CMB and now at INRA/CNRS, create generic superhost strains that can be used for Thiverval-Grignon, France. The model organism they commercial production of the new antibiotics. Before the work with, the filamentous bacterium Streptomyces ultimate goal can be reached, however, some background coelicolor A3(2), excels at producing polyketides. In fact, research is required. The genome sequence ofStreptomyces , this bacterium is known for the dark violet polyketide published in 2002, revealed approximately twenty pigment it makes, which is used in easy assays for growth antibiotic-producing gene clusters, of which only three and general polyketide production. Polyketide production were previously known. This points to a potential treasure is why the Actinomycetes family, which contains the of new antibiotics, but also indicates the fundamental need genus Streptomyces, is the subject of the European Union to understand the regulation of the different gene clusters, ActinoGEN project, which funds the work of Sohoni and in order to control and manipulate their expression. Lantz. CMB is one of 14 collaborating institutions on the €9 million project, and was chosen for its strength in Sohoni's project investigates the regulation of a specific fermentation technology, and its expertise in flux analysis polyketide antibiotic, actinorhodin, which is naturally produced by S. coelicolor, as a model for polyketide antibiotic production. Sohoni cloned regulation in Streptomyces in general. Since most antibiotic the gene for the second phosphatase, clusters have similar promoter structures, the information and purified the protein fromEscherichia gained about the actinorhodin gene cluster can be applied coli to demonstrate its enzyme activity. to other antibiotic-producing genes. A positive regulator Overexpression of this gene in S. coelicolor severely affected controls the gene cluster for actinorhodin synthesis. the amount of actinorhodin produced, and also caused The gene encoding this regulator, actII orf4, is itself in global effects, like an increase in growth and substrate the middle of the gene cluster, with its own regulatory uptake rates, and a delay in actinorhodin synthesis. These region. Sohoni employed an innovation that had not been types of global effects, along with interactions between previously used in Streptomyces. She generated a synthetic regulatory cascades that ultimately affect the expression promoter library in a technique developed by DTU of key genes in polyketide synthesis, are exactly what the Professor Peter R. Jensen1 to remove the normal regulation ActinoGEN project is looking for, to build its knowledge of actII orf4, and screened for a promoter that gives levels about Streptomyces polyketide synthesis and eventually of actII orf4 that lead to maximal actinorhodin synthesis. develop superhost strains. The library was made by inserting randomly generated, synthetic oligonucleotides into the region between the The ActinoGEN project's investment in the Lantz lab -35 and -10 sequences upstream of the actII orf4 gene, appears to be paying off for the EU. Sujata Sohoni and which is hypothesized to be the binding site for RNA Dr. Bapat, the post-doc who developed the assay Sohoni Polymerase and its regulators. Sohoni then used a high- uses, both came from the Indian Institute of Technology throughput, microtitre-based assay developed by post- in Mumbai, specifically to be trained in the Lantz lab. Dr. doctoral fellow Prashant Bapat, to screen the promoter Bapat has remained in Denmark, taking his expertise library. Two hundred candidate promoters were chosen to a permanent job at Novozymes. Although Sohoni for further analysis, not all of which expressed actII orf4 returns to India every winter for "a dose of sunshine", at maximal levels. Sohoni and Lantz note that "more isn't she is willing to stay in the EU and even in Denmark, if always better", and the most strongly expressing promoters the right industry job or post-doc comes along when she inhibit growth, so optimal expression will need to be graduates in 2010. determined empirically. The preliminary data from the screening looks promising. Actinorhodin, which usually 1Hammer et al. 2006, Trends Biotechnol 24:53 is produced only after growth has ceased, was produced concomitantly with growth in the recombinant strains, and one candidate displayed a several-fold increase in polyketide production.

Another aspect of the project was to find general regulatory mechanisms, for example among the kinases and phosphatases revealed by the S. coelicolor genome sequence. Collaborating with an ActinoGEN lab in the United Kingdom that examined the kinases, the Lantz lab looked at the phosphatases. One tyrosine phosphatase, in particular, was known to increase actinorhodin production when overexpressed, and Sohoni confirmed this in her experiments. However, deletion of the phosphatase gene has no phenotype, suggesting the existence of a second, redundant phosphatase that might affect polyketide Streptomyces coelicolor

ANNUAL REPORT 2008 CENTER FOR MICROBIAL BIOTECHNOLOGY 13 Complementary competencies in a project to improve citric acid production

producing A. niger. In addition, PNNL has the luxury CMB researcher Jette Thykær of generous governmental funding for its projects, but lacks hands to do the work, and Thykær welcomed travels to the United States to the opportunity to spend a few months doing only collaborate on increasing citric bench work. Collectively, the goal of the collaboration project between CMB and PNNL, titled "Aspergillus acid production from Aspergillus, niger proteomics – a systems biology approach for and learns about Halloween and cell factory design", was to gain knowledge about the fundamental process of acid production, so that the Superbowl in the process. ultimately, optimal commercial strains can be designed. Financial support for the project came from the Danish By Chris Tachibana Research Council.

To most people, Aspergillus niger is the mould on the A. niger produces acid as a defence, to inhibit the growth sandwich left in the work refrigerator last week. But of nearby microbes, and give A. niger a competitive A. niger is also a factory, and it has two branches. advantage. In addition to citric acid, the strain produces One is represented by the strain of A. niger that is other compounds, like oxalic acid and gluconic acid, used for industrial protein production, for example, depending on the conditions. For both practical and the amylase enzymes that are used commercially to basic research reasons, Thykær and her colleagues make high-glucose and high-fructose syrups. Another wanted to know what regulates the production of strain is specialized for production of acid, particularly different acids under different conditions. Knowing the citric acid, which is widely used as a preservative regulatory map that triggers production of a particular and flavour additive in the food industry. CMB type of acid is the first step toward manipulating it. researchers have expertise in the protein-producing strain, and were involved in the project to sequence To obtain some baseline information, Thykær and its genome. Researchers at Pacific Northwest National collaborators in Scott Baker's lab at PNNL initiated a Laboratories (PNNL), which is funded by the United study of the manganese switch A. niger growth and acid States Department of Energy, worked on the sequence production. In the absence of manganese, the strain of the acid-producing strain, so when both sequences produces citric acid. When manganese is added, the were available, CMB and PNNL collaborated on a strain changes morphology, becoming more filamentous, large-scale genome comparison. Taking advantage of and stops producing citric acid. With CMB's expertise this productive collaborative channel, and knowing that in fermentation providing the starting materials, the discussing results with colleagues over the phone or by CMB and PNNL labs began an extensive transcriptomic email cannot compare to sitting together at a table, with and proteomic analysis of the two growth and acid- the raw data and a pencil and paper, Associate Professor producing states. Working at PNNL gave Thykær access Jette Thykær travelled with her family to work at PNNL to their particularly effective proteome analysis system, for six months in 2008. Her knowledge on A. niger, which performs statistical analysis on small amounts along with CMB's noted competencies in fermentation of isolated proteins, identified by mass spectrometry, technology, microbial physiology, metabolic mapping thereby bypassing the gel electrophoresis step of and other systems biology applications, complemented other proteomic methods. In the PNNL technique, PNNL's expertise in proteomics and the acid- all cell proteins are collected in an extract, cleaved proteolytically and subjected directly to mass spec. The experimental design is more straightforward and less error-prone than other proteomic techniques, and the statistical analysis allows for quantitation.

The project is now in the data analysis phase. In a continuation of the manganese switch research, student Lars Poulsen carried out a Master's project focusing on identifying potential key regulators of acid production. As part of his thesis work, Lars also travelled to PNNL, where he was involved in constructing strains that will be used to test these regulatory candidates, and the trip allowed him to experience work and life in another lab and another culture. Lars just turned in his Master's thesis, and will continue to work on A. niger fermentation while applying for PhD opportunities.

In addition to the practical laboratory experience and the wealth of data generated in only a few months of collaboration, Thykær published two articles with the Baker lab, one identifying essential metabolic pathways in A. fumigatus using a metabolic flux model developed by CMB post-doctoral researcher Mikael Rørdam Andersen, and a commentary on the need to continuing fungal genome sequencing in the future. Thykær emphasizes the additional benefits she and CMB gained through the collaboration. "Doing the work in another lab allows you to look at your own system from a different perspective and see things you wouldn't see if you didn't go and experience it through work with a collaborator," she says. Thykær also welcomed the opportunity to introduce her family to a different culture, noting that her children loved dressing up and collecting candy on Halloween, and her family even enjoyed the over-the-top experience of watching the championship game of American football, at a Superbowl party. That's definitely an experience with collaborators that you can't get by phone or email.

ANNUAL REPORT 2008 CENTER FOR MICROBIAL BIOTECHNOLOGY 15 C M B A n n u a l R e p o r t Highlights 2008

Ph.D student David Kold honoured at Bioche- mical Engineering Symposium David Kold received top honours and €500 for his poster “Optimization of mass transfer in fed-batch Aspergillus oryzae fermentations using a rotation jet head mixing system”, presented in September at ESBES 7 (7th European Symposium on Biochemical Engineering Science) in Faro, Portugal.

Aspergillus cretensis

CMB recognized for excellence in mycotoxin research CMB has recieved grants to study population CMB has been recognized as one of the top insti- heterogeneity tutions in mycotoxin research, in a report compiled Associate Professor Anna Eliasson Lantz is coordi- by ScienceWatch, which monitors performance in nating a 4-year EU Eranet grant to study hetero- basic research. Measured by the total number of geneity in microbial populations during industrial mycotoxin papers, or by the number of times that fermentation. Furthermore, she is taking part in a papers from an institution were cited, CMB was complementary Danish FøSu project on the same to- fifth in the survey, largely on the strength of As- pic. With partners at Belgian and Dutch universities, sociate Professor Kristian Fog Nielsen placing third and in Danish industry and academia, Dr. Lantz's in the category of citations per paper. group will map the heterogeneity that can occur du- ring growth in large fermentation tanks, and deter- mine the heterogeneity level that gives an optimal "Engineering Centre of Excellence" funded balance between consistency and adaptability. with DKK 20 million CMB received a 20 million kroner continuation of its grant from The Danish Agency for Science, Technology and Innovation, to fund five additio- nal years as an “Engineering Centre of Excellen- ce”. The funding renewal allows continued work in the core research area around methods to im- prove yeast and other fungi as cell factories for industrially relevant products such as antibiotics, fuels, natural colorants, organic acids and proteins of pharmaceutical interest. Highlights 2008 Samson. Rob by aethiopicum Penicillium and cretensis Aspergillus of Photos

Penicillium aethiopicum

Researchers show anticancer properties of a drug from filamentous fungi The anticancer effects of griseofulvin, isolated from filamentous fungi, were discovered by Asso- ciate Professor Thomas Ostenfeld Larsen of CMB, in collaboration with Associate Professor Mads H. Clausen, DTU Department of Chemistry, and Pro- fessor Dr. Alwin Krämer, University of Heidelberg. Poster award leads to California conference The natural product and its synthetic derivatives trip for Ph.D student Lasse Pedersen target a feature of cell division that is unique to Lasse Pedersen won the competition at the DTU cancer cells, so they have the potential to elimi- Society for Biological Engineering (SBE) Symposi- nate many side effects associated with current um of Biotechnology Research for his poster “Gen- chemotherapy. ome wide analysis of Aspergillus niger metabolism during industrial fed-batch fermentations”. The prize was a trip to SBE’s International Conference on Biomolecular Engineering in Santa Barbara, Ca- Two new professors lifornia, which Lasse attended in January 2009. CMB promoted two new professors in 2008. Asso- ciate Professor Michael Lynge Nielsen and Assistant Professor Kaisa Karhumaa. Dr. Nielsen will continue work from his post-doc at DTU on secondary meta- bolite biosynthesis, with the goal of contributing to personalized medical treatment. Dr. Karhumaa, origi- nally from Finland, has a PhD from Lund University and was a post-doctoral fellow at the Carlsberg La- boratory before she came to DTU. She will work on yeast nutrient sensing, with applications in increa- sing the efficiency of industrial microbial growth.

Post-doc Mikael Rørdam Andersen is DTU Sy- stems Biology Researcher of the Year 2008 Mikael Rørdam Andersen was named “Researcher of the Year 2008” and received DKK 10.000 for his modelling of metabolism in the filamentous fungi Aspergillus niger. Professor Søren Molin cal- led his work “a pioneering achievement in systems biology of fungi, which has important implications for our understanding of the biology of these in- dustrially relevant microorganisms”.

ANNUAL REPORT 2008 CENTER FOR MICROBIAL BIOTECHNOLOGY 17 Faculty

Associate Professor Professor Associate Professor Assistant Professor Professor Birgitte Jens Chr. Timothy Kaisa Morten C. Andersen Frisvad Hobley Karhumaa Kielland-Brandt

Associate Professor Associate Professor Associate Professor Associate Professor Associate Professor Anna Thomas Uffe Kristian Michael Eliasson Lantz Ostenfeld Larsen Hasbro Mortensen Fog Nielsen Lynge Nielsen

Associate Professor Assistant Professor Associate Professor Professor (Docent) Gianni Kiran Ib Ulf Panagiotou Patil Søndergaard Thrane Organization

The Organization of CMB has changed to a more flat structure. The three research clusters that previously accommodated all faculty have been replaced by currently six research groups. Each group leader delineates the strategy of the research group in accordance with the overall mission and vision of CMB as defined by the Center Director. The management group is selected by the Center Director amongst faculty.

The Scientific Advisory Board comprises five in- Steering committee provides strategic and mana- ternationally recognised researchers in fields covering the gerial advice and approves significant decisions, e.g. the majority of the research activities at CMB. The advisory approval of new long-term scientific positions. The board board meets in connection with CMB’s annual meeting, meets with the Center director and Head of administra- and assists in the evaluation of research activities. tion twice a year, and once a year writes a brief statement about the center for the Danish Research Agency. The Professor Axel Brakhage, committee consisted in 2008 of the following persons: University of Hannover, D Professor Klaus Bock, Professor Bärbel Hahn-Hägerdal, Chairman of the Danish National Research Foundation Lund University, SE Chief Technology Officer Leif Kjærgaard, Professor Carsten Christophersen, Danisco A/S University of Copenhagen, DK Professor Søren Molin, Professor Jack Pronk, Technical University of Denmark Technical University of Delft, NL Executive vice president Poul Rasmussen, Professor Peter Roepstorff, LEO Pharma A/S University of Southern Denmark, DK Dean of Research Kristian Stubkjær, Technical University of Denmark

Department of Systems Biology

Steering committee Center Director Management Group

Service functions Secretariat

Research Groups Scientific Advisory Board

ANNUAL REPORT 2008 CENTER FOR MICROBIAL BIOTECHNOLOGY 19 Publications 2008

Andersen, B., Dongo, A., Pryor, B. M. (2008) Secondary Borodina, I., Siebring, J., Zhang, J., Smith, C. P., van metabolite profiling ofAlternaria dauci, A. porri, A. solani, Kuelen, G., Dijkhuizen, L., Nielsen, J. (2008) Antibiotic and A. tomatophila. Mycological Research 112: 241-250. overproduction in Streptomyces coelicolor A3 (2) mediated by phosphofructokinase deletion. Journal of Biological Andersen, B., Hollensted, M. (2008) Metabolite Chemistry 283: 25186- 25199. production by differentUlocladium species. International Journal of Food Microbiology 126: 172-179. Bruun, S. W., Søndergaard, I., Jacobsen, S. (2008) Using near infrared spectroscopy for the study of protein Andersen, M. R., Nielsen, M. L., Nielsen, J. (2008) structures during gluten hydration. NIR News 19: 4-6. Metabolic model integration of the bibliome, genome, metabolome and reactome of Aspergillus niger. Molecular Bruun, S. W., Søndergaard, I., Jacobsen, S. (2008) Systems Biology 4: art. 178. Using near infrared spectroscopy for the study of salt perturbation of gluten structures. NIR News 19: 11-13. Andersen, M. R., Vongsangnak, W., Panagiotou, G., Salazar, M. P., Lehmann, L. O., Nielsen, J. (2008) Degenkolb, T., Dieckmann, R., Nielsen, K. F., A trispecies Aspergillus microarray: Comparative Gräfenhan, T., Theis, C., Zafari, D., Chaverri, P., transcriptomics of three Aspergillus species. Proceedings Ismaiel, A., Brückner, H., von Döhren, H., Thrane, of the National Academy of Sciences of the USA 105: U., Petrini, O., Samuels, G. J. (2008) The Trichoderma 4387-4392. brevicompactum clade: a separate lineage with new species, new peptaibiotics, and mycotoxins. Mycological Andersen, N. Johansen, K. S., Michelsen, M. L., Stenby, Progress 7: 177-219. E. H., Krogh, K. B. R. M., Olsson, L. (2008) Hydrolysis of cellulose using mono-component enzymes shows Degenkolb, T., von Döhren, H., Nielsen, K. F., Samuels, synergy during hydrolysis of phosphoric acid swollen G. J., Brückner, H. (2008) Recent advances and future cellulose (PASC), but competition on avicel. Enzyme and prospects in peptaibiotics, hydrophobin, and mycotoxin Microbial Technology 42: 362-370. research, and their importance for chemotaxonomy of Trichoderma and Hypocrea. Chemistry & Biodiversity Arpanei, A., Heebøll-Nielsen, A., Hübbuch, J., Thomas, 5: 671-680. O. R. T., Hobley, T. J. (2008) Critical evaluation and comparison of fluid distribution systems for industrial Faber, C., Hobley, T. J., Mollerup, J., Thomas, O. R. T., scale expanded bed adsorption chromatography columns. Kaasgaard, S. G. (2008) Factors affecting the solubility Journal of Chromatography 1198-1199: 131-139. of Bacillus halmapalus alpha-amylase. Chemical Engineering and Processing 47: 1007-1017. Arpanaei, A., Mathiasen, N., Hobley, T. J. (2008) DNA binding during expanded bed adsorption and Frisvad, J. C., Andersen, B., Thrane, U. (2008) The use factors affecting adsorbent aggregation. Journal of of secondary metabolite profiling in fungal taxonomy. Chromatography A1203: 198-206. Mycological Research 112: 231-240.

Asadollahi, M., Maury, J., Møller, K., Nielsen, K. F., Geng, H., Bruhn, J. B., Nielsen, K. F., Gram, L., Schalk, M., Clark, A., Nielsen, J. (2008) Production of Belas, R. (2008) Genetic dissection of tropodithietic plant sesquiterpenes in Saccharomyces cerevisiae: Effect acid biosynthesis by marine roseobacters. Applied and of ERG9 repression on sesquiterpene biosynthesis. Environmental Microbiology 74: 1535-1545. Biotechnology and Bioengineering 99: 666-677. Greenhill, A., Blaney, B., Shipton, W., Frisvad, J. C., Baker, S. E., Thykær, J., Adney, W. S., Brettin, T. S., Pue, A., Warner, J. (2008) Mycotoxins and toxigenic Brockman, F. J., D’haeseleer, P., Martinez, A. D., Miller, fungi in sago starch from Papua New Guinea. Letters in R., M., Rokhsar, D. S., Schadt, C. W., Torok, T., Tuskan, Applied Microbiology 47: 342-347. G., Bennett, J., Berka, R. M., Briggs, S. P., Heitman, J., Taylor, J., Turgeon, B. G., Werner-Washburne, M., Grove, H., Jørgensen, B., Jessen, F., Søndergaard, I., Himmel, M. E. (2008) Fungal genome sequencing and Jacobsen, S., Hollung, K., Indahl, U., Færgestad, E. M. bioenergy. Fungal Biology Reviews 22: 1-5. (2008) Combination of statistical approaches for analysis of 2-DE data gives complementary results. Journal of Bjarnsholt, T., Kirketerp-Moller, K., Jensen, P. O., Madsen, Proteome Research 7: 5119-5124. K.G., Phipps, R., K., Krogfelt, K., Hoiby, N., Givskov, M. C. (2008) Why chronic wounds will not heal: A novel Herrgård, M. J., Swainston, N., Dobson, P., Dunn, W. hypothesis. Wound Repair and Regeneration 16: 2-10. B., Arga, K. Y., Arvas, M., Blüthgen, N., Borger, S., Costenoble, R., Heinemann, M., Hucka, M., Le Novère, de Jongh, W., Nielsen, J. (2008) Enhanced citrate N., Li, P., Liebermeister, W., Mo, M. L., Oliveira, A. P., production through gene insertion in Aspergillus niger. Petranovic, D., Pettifer, S., Simeonidis, E., Smallbone, Metabolic Engineering 10: 87-96. K., Spasić, I., Weichart, D., Brent, R., Broomhead, D. S.,Westerhoff, H. V., Kirdar, B., Penttilä, M., Klipp, E., Kale, S. P., Milde, N., Trapp, M., Frisvad, J. C., Keller, N. Palsson, B. Ø., Sauer, U., Oliver, S. G., Mendes, P., Nielsen, P. (2008) Requirement of LaeA for secondary metabolism J., Kell, D. B. (2008) A consensus yeast metabolic network and sclerotial production by Aspergillus flavos. Fungal reconstruction obtained from a community approach to Genetics and Biology 45: 1422-1429. systems biology. Nature Biotechnology 26: 1155-1160. Kouskoumvekaki, I., Yang, Z., Jonsdottir, S. O., Olsson, Højer-Pedersen, J., Smedsgaard, J., Nielsen, J. (2008) L., Panagiotou, G. (2008) Identification of biomarkers for The yeast metabolome addressed by electrospray genotyping Aspergilli using non-linear methods for clustering ionization mass spectrometry: Initiation of a mass and classification. BMC Bioinformatics9: art. 59. spectral library and its application for metabolic footprinting by direct infusion mass spectrometry. Krogh, A. M., Beck, V., Christensen, L. H., Henriksen, Metabolomics 4: 393-405. C. M., Møller, K., Olsson, L. (2008) Adaptation of Saccharomyces cerevisiae expressing a heterologous Hong, S.-B., Shin, H.-D., Hong, J., Frisvad, J. C., protein. Journal of Biotechnology 137: 28-33. Nielsen, P. V., Varga, J., Samson, R. A. (2008) New taxa of Neosartorya and Aspergillus in Aspergillus section Krska, R., Berthiller, F., Schuhmacher, R., Nielsen, K. F., Fumigati. Antonie van Leeuwenhoek 93: 87-98. Crews, C. (2008) Determination of ergot alkaloids: purity and stability assessment of standards and optimization Jers, C., Soufi, B., Grangeasse, C., Deutscher, J., of extraction conditions for cereal samples. Journal of Mijakovic, I. (2008) Phosphoproteomics in bacteria: AOAC International 91: 1363-1371. Towards a systemic understanding of bacterial phosphorylation networks. Expert Review of Proteomics Lai, X., Zheng, Y., Jacobsen, S., Larsen, J. N., Ipsen, H., 5: 619-627. Løwenstein, H., Søndergaard, I. (2008) Determination of adsorbed protein concentration in aluminium de Jongh, W., Bro, C., Østergaard, S., Regenberg, B., hydroxide suspensions by near-infrared transmittance Nielsen, J. (2008) The roles of galacitol, galactose-1- spectroscopy. Applied Spectroscopy 62: 784-790. phosphate, and phosphoglucomutase in galactose-induced toxicity in Saccharomyces cerevisiae. Biotechnology and Lubzens, E., Hadas, O., Sukenik, A., Cerda, J., Bioengineering 101: 317-326. Hohmann, S., Worland, R., Clark, M. S., Reinhardt, R.,

ANNUAL REPORT 2008 CENTER FOR MICROBIAL BIOTECHNOLOGY 21 Nielsen, K. F. (2008) Dormancy of cells and organisms - Noonim, P., Mahakarnchanakul, W., Varga, J., Frisvad, Strategies for survival and preservation - Sleeping Beauty. J. C., Samson, R. A. (2008) Two new species of Aspergillus Comparative Biochemistry and Physiology A-Molecular section Nigri from Thai coffee beans. International & Integrative Physiology 151: S34-S35. journal of Systematic and Evolutionary Microbiology 58: 1727-1734. Macek, B., Gnad, F., Soufi, B., Kumar, C., Olsen, J. V. Mijakovic, I., Mann, M. (2008) Phosphoproteome Olivares-Illana, V., Meyer, P., Bechet, E., Gueguen- analysis of E-coli reveals evolutionary conservation of Chaignon, V., Soulat, D., Lazereg-Riquier, S., Mijakovic, bacterial Ser/Thr/Tyr phosphorylation. Molecular & I., Deutscher, J., Cozzone, A. J., Laprevote, O., Morera, Cellular Proteomics 7: 299-307. S., Grangeasse, C., Nessler, S. (2008) Structural basis for the regulation mechanism of the tyrosine kinase Mapari, S. S., Hansen, M. A. E., Meyer, A. S. Thrane, U. CapB from Staphylococcus aureus. PLoS Biology 6: (2008) Computerized screening for novel producers of 1321-1331. Monascus-like food pigments in Penicillium species. Journal of Agricultural and Food Chemistry 56: 9981-9989. Oliveira, A. P., Patil, K. R., Nielsen, J. (2008) Architecture of transcriptional regulatory circuits is knitted over the Mapari, S. S., Meyer, A. S., Thrane, U.(2008) Evaluation topology of bio-molecular interaction networks. BMC of Epicoccum nigrum for growth, morphology and Systems Biology 2: art. 17. production of natural colorants in liquid media and on a solid rice medium. Biotechnology Letters 30: Panagiotou, G., Andersen, M. R., Grotkjær, T., 2183-2190. Regueira, T. U. B., Hofmann, G., Nielsen, J., Olsson, L. (2008) Systems analysis unfolds the relationship between Mapelli., V., Olsson, L., Nielsen., J. (2008) Metabolic the phosphoketolase pathway and growth in Aspergillus footprinting in microbiology: methods and applications nidulans. PLoS ONE 3: e3847. in functional genomics and biotechnology. Trends in Biotechnology 26: 490-497. Panagiotou, G., Pachidou, F., Petroutsos, D., Olsson, L., Christakopoulos, P. (2008) Fermentation characteristics Maury, J., Asadollahi, M., Møller, K., Schalk, M., Clark, of Fusarium oxysporum grown on acetate. Bioresource A., Formenti, L. R., Nielsen, J. (2008) Reconstruction of a Technology 99: 7397-7401. bacterial isoprenoid biosynthetic pathway in Saccharomyces cerevisiae. FEBS Letters 582: 4032-4038. Panagiotou, G., Papadakis, M. A., Topakas, E., Olsson, L., Christakopoulos, P. (2008) Identification of NADH Nielsen, J., Jewett, M. C. (2008) Impact of systems kinase activity in filamentous fungi and structural biology on metabolic engineering of Saccharomyces modeling of the novel enzyme from Fusarium oxysporum. cerevisiae. FEMS Yeast Research 8: 122-131. Process Biochemistry 43: 1114-1120.

Nielsen, J. B., Nielsen, M. L., Mortensen, U. H. (2008) Perrone, G., Varga, J., Susca, A., Frisvad, J. C., Stea, Transient disruption of non-homologous end-joining G., Kocsube, S., Tóth, B., Kozakiewicz, Z., Samson, facilitates targeted genome manipulations in the R. A. (2008) Aspergillus uvarum sp. nov., an uniseriate filamentous fungusAspergillus nidulans. Fungal Genetics black Aspergillus species isolated from grapes in Europe. and Biology 45: 165-170. International journal of Systematic and Evolutionary Microbiology 58: 1032-1039. Nookaew, I., Jewett, M. C, Meechai, A., Thammarongtham, C., Laoteng, K., Cheevadhanarak, S., Nielsen, J., Pildain, M. B., Frisvad, J. C., Vaamonde, G., Cabral, Bhumiratana, S. (2008) The genome-scale metabolic D., Varga, J., Samson, R. A. (2008) Two novel aflatoxin- model iIN800 of Saccharomyces cerevisiae and its producing Aspergillus species from Argentinean peanuts. validation: a scaffold to query lipid metabolism. BMC International Journal of Systematic and Evolutionary Systems Biology 2: art. 71. Microbiology 58: 725-735.

Noonim, P., Mahakarnchanakul, W., Nielsen, K. Pizarro, F. J., Jewett, M. C., Nielsen, J., Agosin, F., Frisvad, J. C., Samson, R. A. (2008) Isolation, E. (2008)Growth temperature exerts a differential identification and toxigenic potential of ochratoxin A- physiological and transcriptional response in laboratory producing Aspergillus species from coffee beans grown in and wine strains of Saccharomyces cerevisiae. Applied and two regions of Thailand. International Journal of Food Environmental Microbiology 74: 6358-6368. Microbiology 128: 197-202. Plate, I., Albertsen, L., Lisby, M., Hallwyl, S. C. L., Feng, Storm, I. M. L. D., Rasmussen, P. H., Strobel, B. W., Q., Seong, C., Rothstein, R., Sung, P., Mortensen, U. Hansen, H. C. B. (2008) Ergot alkaloids in rye flour H. (2008) Rad52 multimerization is important for its determined by solidphase cation-exchange and high- nuclear localization in Saccharomyces cerevisiae. DNA pressure liquid chromatography with fluorescence detection. Repair 7: 56-66. Food Additives and Contaminants 25: 338-346.

Plate, I., Hallwyl, S. C. L., Shi, I., Krejci, L. Müller, Storm, I. M. L. D., Sørensen, J. L., Rasmussen, R. R., C., Albertsen, L., Sung, P., Mortensen, U. H. (2008) Nielsen, K. F., Thrane, U. (2008) Mycotoxins in silage. Interaction with RPA is necessary for Rad52 repair center. Stewart Postharvest Solutions 4: 1-12. Journal of Biological Chemistry 283: 29077-29085. Sørensen, J. L., Nielsen, K. F., Rasmussen, P. H., Thrane, Porsby, C. H., Nielsen, K. F., Gram, L. (2008) Phaeobacter U. (2008) Development of a LC-MS/MS method for and ruegeria species of the roseobacter clade colonize the analysis of Enniatins and Beauvericin in whole fresh separate niches in a Danish turbot (Scophthalmus maximus) and ensilaged maize. Journal of Agricultural and Food - rearing farm and antagonize Vibrio anguillarum under Chemistry 56: 10439-10443. different growth conditions. Applied and Environmental Microbiology 74: 7356-7364. Sørensen, L. M., Jacobsen, T., Nielsen, P. V., Frisvad, J. C., Koch, A. G. (2008) Mycobiota in the processing areas Poulsen, P., Gaber, R. F., Kielland-Brandt, M. of two different meat products. International Journal of (2008) Hyperand hyporesponsive mutant forms of Food Microbiology 124: 58-64. the Saccharomyces cerevisiae Ssy1 amino acid sensor. Molecular Membrane Biology 25: 164-176 Sørensen, L. M., Nielsen, K. F., Jacobsen, T., Koch, A. . G., Nielsen, P. V., Frisvad, J. C. (2008) Determination of Rank, C., Phipps, R. K., Harris, P., Fristrup, P., Larsen, mycophenolic acid in mest products using mixed mode T. O., Gotfredsen, C. H. (2008) Novofumigatonin, reversed phase-anion exchange clean-up and liquid a New Orthoester Meroterpenoid from Aspergillus chromatographyhigh-resolution mass spectrometry. novofumigatus. Organic Letters 10: 401-404. Journal of Chromatography A 1205: 103-108.

Rocha, M., Maia, P., Mendes, R., Pinto, J. P., Ferreira, Thykær, J., Kildegaard, K. R., Noorman, H., Nielsen, E. C., Nielsen, J., Patil, K. R., Rocha, I. (2008) Natural J. (2008) NADPH-dependent glutamate dehydrogenase computation meta-heuristics for the in silico optimizing in Penicillium chrysogenum is involved in regulation of microbiel strains. BMC Bioinformatics 9: art. 499. of beta-lactam production. Microbiology-sgm 154: 1242-1250. Seong, C., Sehorn, M. G., Plate, I., Shi, I., Song, B., Chi, P., Mortensen, U. H., Sung, P., Krejci, L. (2008) Usaite, R., Nielsen, J., Olsson, L. (2008) Physiological Molecular anatomy of the recombination mediator characterization of glucose repression in the strains with function of Saccharomyces cerevisiae Rad52. Journal of SNF1 and SNF4 genes deleted. Journal of Biotechnology Biological Chemistry 283: 12166-12174. 133: 73-81.

Sin, G., Ödman, P., Petersen, N., Eliasson Lantz, Usaite, R., Wohlschlegel, J., Venable, J. D., Park, S. K., A., Gernaey, K. (2008) Matrix notation for efficient Nielsen, J., Olsson, L., Yates, J. R. (2008) Characterization development of firstprinciples models within PAT of global yeast quantitative proteome data generated from applications: Integrated modeling of antibiotic the wild-type and glucose repression Saccharomyces production with Streptomyces coelicolor. Biotechnology cerevisiae strains: The comparison of two quantitative and Bioengineering 101: 153-171. methods. Journal of Proteome Research 7: 266-275.

Skindersø, M. E., Alhede, M., Phipps, R. K., Yang, Vongsangnak, W., Olsen, P., Hansen, K., Krogsgaard, L., Jensen, P. O., Rasmussen, T. B., Bjarnsholt, T., S., Nielsen, J. (2008) Improved annotation through Tolker-Nielsen, T., Høibo, N., Givskov, M. C. (2008) genome-scale metabolic modeling of Aspergillus oryzae. Effects of antibiotics on quorum sensing inPseudomonas BMC Genomics 9: art. 245. aeruginosa 52: 3648-3663. Wattanachaisaereekul, S., Eliasson Lantz, A., Nielsen, Soufi, B., Jers, C., Hansen, M. E., Petranovic, D., M. L. Nielsen, J. (2008) Production of the polyketide Mijakovic, I. (2008) Insights from site-specific 6-MSA in yeast engineered for increased malonyl-CoA phosphoproteomics in bacteria. Biochimica et Biophysica supply. Metabolic Engineering 10: 246-254. Acta-Proteins and Proteomics 1784: 186-192.

ANNUAL REPORT 2008 CENTER FOR MICROBIAL BIOTECHNOLOGY 23 Zalar, P., Frisvad, J. C., Gunde-Cimerman, N., Varga, J., Samson, R. A. (2008) Four new species of Emericella from the Mediterranean region of Europe. Mycologia 100: 779-795.

Zheng, Y. Lai, X., Bruun, S. W., Ipsen, H., Larsen, J. N., Løwenstein, H., Søndergaard, I., Jacobsen, S. (2008) Determination of moisture content of lyophilized allergen vaccines by NIR spectroscopy. Journal of Pharmaceutical and Biomedical Analysis 46: 592-596.

Books and Book Chapters Frisvad, J. C. (2008) Cold-adapted fungi as a source for valuable metabolites. In: Margesin, R. Schinner, F., Marx, J. C., Gerday, C (Eds.) Psychrophiles: From Biodiversity to Biotechnology. Springer, Berlin, pp. 381-387.

Frisvad, J. C. (2008) Fungi in cold ecosystems. In: Margesin, R. Schinner, F., Marx, J. C., Gerday, C (Eds.) Psychrophiles: From Biodiversity to Biotechnology. Springer, Berlin, pp. 137-156.

Peterson, S.W., Varga, J., Frisvad, J. C., Samson, R. A. (2008) Phylogeny and subgeneric taxonomy of Aspergillus. In: Varga, J. & Samson R. A. (Eds.) Aspergillus in the Genomics Era. Wageningen Academic Publishers, Wageningen, pp. 33-56.

Varga, J., Houbraken, J., Samson, R. A., Frisvad, J. C. (2008) Molecular diversity of Aspergillus and Penicillium on fruits and vegetables. In: Barkai-Golan, R. & Paster, PhD Theses N. (Eds.) Mycotoxins in fruits and vegetables. Academic Andersen, M. R. Systems biology studies of Aspergilli Press, Amsterdam, pp. 205-223. - from sequence to science.

Other Publications Asadollahi, M. A. Establishment of yeast platform for Johansen, M., Vynne, N. G., Nielsen, Larsen, T. O., isoprenoid production. Gram., L. (2008) På fisketur efter nye bioaktive og bakteriehæmmende stoffer - en rapport fra verdenshavene. Højer-Pedersen, J. Metabolome analysis - mass spectrometry Dansk Kemi 89: 12-17. and microbial primary metabolites.

Jørgensen, L. N., Thrane, U., Collinge, D. B., Jørgensen, Krogh, K. Growth and biomass degrading enzymes from H. J. L., Jensen, J. D., Spliid, N. H., Nielsen, G. C., Penicillium. Rasmussen, P. H., Nicolaisen, M., Justesen, A. F., Giese, H., Bach, I. C. (2008) Fusarium på korn skader planter, Meijer, S. Creation of an organic acid production platform husdyr og mennesker. Planteforskning.dk. in Aspergillus niger using a system-level approach.

Schäpper, D., Gernaey, K., Eliasson Lantz, A., Stocks, Nielsen, J. B. Understanding DNA repair in Aspergillus S. (2008) Udvikling af mikrobioreaktorer til kontinuerlig nidulans - paving the way for efficient gene targeting. kultuvering af gær. Dansk Kemi 89: 16-19. Oliveira, A. P Towards enhancing metabolic engineering Seifert, K., Crous, P., Frisvad, J. C. (2008) Correcting the through systems biology. impact factors of taxonomic journals by ACT. Inoculum 59 (3): 4. Usaite, R. The Snf1 protein kinase in the yeastSaccharomyces cerevisiae. Huang, L. Characterization and optimization of ACVS expression and ACV production in Saccharomyces cerevisiae.

Jensen, B. G. Characterisation of human monoclonal antibodies against P. falciparum variable surface antigens involved in pregnancy-associated malaria.

Jensen, M. B. LC-MS analysis of bio-active marine bacteria.

Jessen, K. M. Studying glucose signalling in humanised yeast. Johansen, K. T. Secondary metabolites from Penicillium subgenus Biverticillium.

Johansen, M. Microbial natural products chemistry.

Johnsen, L. G. Profiling of fermented milk.

Johnsson, C. In vivo phosphorylation of new substrates by the B. subtilis tyrosine kinase PtkA.

BSc Theses Anyaogu, D. C. Metabolite production in Cladosporium: Influence of substrate composition and identification of major metabolic products.

Frandsen, H.V. Building forensic screening libraries of drugs by UPLC-TOF.

Høegh, L. H. S. S. Investigation of genetic instability in MSc Theses Aspergillus nidulans. Adolph, M. Characterization of Xanthomonas campestris in a rotating jet head fermenter. Jørgensen, L. M. Investigation of central carbon metabolism in Str. coelicolor. Bengtsson, A. Characterization of the Plasmodium falciparum antigen VAR4 with a view to identify surface Kappelgaard, C. S., Nilsson, J. M. The nanoplatform exposed antibody epitopes. concept.

Blom, L., Hansen, J. B. Differentiation of CD4+ T cells Khodaie, M. Z., Thomsen, M. H. High throughput into various subsets. artificialintelligent characterization of microorganisms through image analysis. Carlsen, S. Engineering Saccharomyces cerevisiae for production of isoprenoids Larsen, S. L. Make yeast taste good: Metabolic engineering of flavour compounds inSaccharomyces cerevisiae. Gronemann, M. A. Metabolic engineering of Saccharomyces cerevisiae for lycopene production. Munir, S. Quantitative assessment of collagen type ΙΙ synthesis in joint diseases. Hansen, J. H. Test of nano-platform concept and fusion strategy with the vanillin pathway as a model system. Nielsen, M. A. Determination of extractables from polymers and elastomers using LC-MS and GC-MS. Hansen, M. M. In vivo localisation of the Bacillus subtilis tyrosine kinase PtkA, its modulator and its putative Olsen, M. R. Fungal consortia in water-damaged buildings substrates using fluorescent protein fusions. - identification, chemotaxonomy & systematics.

ANNUAL REPORT 2008 CENTER FOR MICROBIAL BIOTECHNOLOGY 25 Pedersen, C. D. Physiological dynamics of Saccharomyces cerevisiae during adaptive growth on galactose.

Pedersen, D. M. K. Genetic instability in Aspergillus nidulans.

Oral Presentations Andersen M. R. A comparative study of the metabolic capabilities of Aspergilli. Eurofungbase meeting, Girona, Spain.

Andersen, M. R. Comparative transcriptomics of three Aspergillus species. 5th Asperfest, Edinburgh, Scotland.

Frisvad, J. C. From Denmark to the Netherlands, from Penicillium to Aspergillus. CBS Fungal Biodiversity Center, Utrecht, The Netherlands.

Frisvad, J. C. Penicillium: A large ecomically imporant genus. XII International Congress of Mycology, IUMS, Istanbul, Tyrkey.

Frisvad, J. C. P. expansum metabolomics. University of Copenhagen, EU meeting on fungi in fruit products.

Frisvad, J. C. Invited lecture: Metabolomics of Aspergillus fumigatus. In the congress: Thirs Advances Against Aspergillus. Miami Beach, Florida

Frisvad, J. C. Invited lecture. Chemotaxonomy of Penicillium. In ISM (International Society of Mycotoxicology) Workshop. Bari, Italy mutant libraries in filamentous fungi. Fungal Biology Karhumaa, K., Wu, Bo., Kielland-Brandt, M. Functional and Biotechnology in the Genomic Era, 2008, Sant Feliu analysis of transporter-like sugar sensors in Saccharomyces de Guixols, Spain. cerevisiae. 26th Small Meeting Yeast Transport and Energetics, Braga. Salazar, M. P., Nielsen, M. Lynge, Nielsen, J. Regulatory network analysis of genome - wide transcription data Karhumaa, K., Wu, B., Kielland-Brandt, M. from Aspergillus niger. 9th European Conference on Functional analysis of transporter-like sugar sensors Fungal Genetics, 2008, Edinburgh, Scotland. in Saccharomyces cerevisiae. 12th International Congress on Yeasts, 2008, Kiev. Center Seminar Albertsen, L. Nano-platforms for coordinating enzyme Kold, D., Hellmuth, K., Hobley, T. J. Optimization of mass activities - a new approach for optimizing cell factories. transfer in fed-batch Aspergillus oryzae fermentations Andersen, M. T. Could glucose be connected to the using a rotating jet head mixing system. 7th European development of Alzheimer’s? Symposium on Biochemical Engineering Science, 2008, Faro, Portugal. Kjeldsen, K. Improving a L-lysine producing Corynebacterium glutamicum production strain. Lantz, A. E., Ödman, P., Petersen, N., Olsson, L., Gernay, K. Practical aspects of applying near infrared spectroscopy Kold, D. Optimization of mass transfer in viscous for monitoring of filamentous microorganism fermentations using a rotating jet head mixing system. fermentations. EuPAT3, 2008, Gothenburg, Sweden. Mapari, S. A. S. Chemotaxonomic exploration Nielsen, M. L., Nielsen, J. B., Hansen, B. G., Mortensen, of fungal biodiversity for polyketide natural food U. H. High-thoughput strategies for construction of colorants: Discovery and evaluation of cell factories Basilio, C., Houbraken, J., Samson, R., Frisvad, J. C., Tenreiro, R., Crespo, M. T. B. Cork fungal diversity and characterization. CBS Symposium Fungi and Health, Royal Institute of Science, Letters and Fine Arts (KNAW), Amsterdam, The Netherlands.

Borodina, I., Poulsen, L. K., Larsen, J. L., Søndergaard, I., Nielsen, J. Production of wasp venom allergens in Pichia pastoris. 5th EFB Meeting on Recombinant Protein Production, Alghero, Italy.

Brochado A. R., Hansen J., Møller B. L., Mortensen U. H., Patil K. R. Metabolic engineering for vanillin production in Saccharomyces cerevisiae. 3rd Danish Conference on Biotechnology and Molecular Biology, Vejle, Denmark.

Brochado A. R., Hansen J., Møller B. L., Mortensen U. H., Patil K. R. Metabolic engineering for vanillin production in Saccharomyces cerevisiae. Metabolic Engineering VII: Health and sustainability, Puerto Vallarta, Mexico.

Copetti, M. V., Taniwaki, M., Frisvad, J. C. Fungi on cocoa and their mycotoxin production. CBS Symposium Fungi and Health, Royal Institute of Science, Letters and Fine Arts (KNAW), Amsterdam, The Netherlands.

Formenti, L. R., Maury, J., Asadollahi, M., Nielsen, K. F., Nielsen, J., Kielland-Brandt, M., Schalk, M., Clark, A. Improving sesquiterpene production in Saccharomyces cerevisiae through integration of the MEP pathway. and characterization of pigments Ottow, K. Scaleable Metabolic Engineering VII: Health and sustainability, modification and assembly of biological molecules using Puerto Vallarta, Mexico. high gradient magnetic fishing. Frisvad, J. C., Thrane, Andersen. B., Samson, R. Piddocke, M. Yeast stress during high gravity brewing A. Good chemistry between CBS and CMB. CBS – physiological studies and “-omic” analyses. Symposium Fungi and Health, 2008, Royal Institute of Science, Letters and Fine Arts (KNAW), Amsterdam, Sørensen, J. L. Fungi and their mycotoxins in maize at The Netherlands. harvest. Hansen, B. G., Patil, K. R., Mortensen, U. H. Uracil- Poster Presentations excision based cloning: a fast and efficient method for the Bapat, P. M., Patil, K. R., Thykær, J., Lantz, A. E. creation of DNA-constructs. Metabolic Engineering VII: Development of a robust “scaled-down” cultivation Health and sustainability, Puerto Vallarta, Mexico. platform for Streptomyces via morphology engineering. Natural Products Discovery and Production II: Houbraken, J., Frisvad, J. C., Meijer, M., Samson, R. Celebrating the successes of traditional and novel Culture A. Polyphasic taxonomy of Penicillium citrinum and sources, Whistler, British Colombia, Canada. related species. CBS Symposium Fungi and Health, Royal Institute of Science, Letters and Fine Arts (KNAW), Bapat, P. M., Sohoni, S. V., Lantz, A. E. An Integrated Amsterdam, The Netherlands. “low volume high throughput cultivation platform” for industrial systems biology: Streptomyces coelicolor Jensen, R. H., Nielsen, M. L., Mortensen, U. H. a case study. Metabolic Engineering VII: Health and Development of a synthetic lethality screen to uncover sustainability, Puerto Vallarta, Mexico. key genes in filamentous fungi. Symposium for Biotech Research, Lyngby, Denmark.

ANNUAL REPORT 2008 CENTER FOR MICROBIAL BIOTECHNOLOGY 27 Johansen, M., Nielsen, K. F., Hansen, M. A. E., Lyhne, Metabolic Engineering VII: Health and sustainability, E. K., Larsen, T. O., Vynne, N. G., Melchiorsen, J., Puerto Vallarta, Mexico. Gram, L. Dereplication Strategies for Discovery of Marine Microbial Natural Products. Gordon Research Piddocke, M., Kreisz, S., Heldt-Hansen, H. P., Olsson, Conference on Marine Natural Products, Ventura Beach, L. Can –omics help high gravity brewing? World Brewing California, U.S.A. Congress 2008, Honolulu, Hawaii, USA.

Karhumaa, K., Wu, B., Kielland-Brandt, M. Quantitative Schäpper, D., Gernaey, K., Eliasson Lantz, A., characterization of nutrient sensing by transporter-like Stocks, S., Szita, N. Development of continuous sugar sensors in Saccharomyces cerevisiae. 9th International culture microbioreactors. 7th European Symposium on Conference on Systems Biology, Gothenburg, Sweden. Biochemical Engineering Science, Faro, Portugal.

Lingasamy, P., Nielsen, M. L., Maury, J., Olsson, L., Schroers, H. J., Sørensen, J. L., Thrane, U., Nielsen, Panagiotou, G. Cloning and Functional Characterization K. F., Zerjav, M., Munda, A., Frank, J. High incidence of fungal feruloyl esterase. 3rd Danish Conference on of Fusarium avenaceum (Nectriaceae, Gibberella) and Molecular Biology and Biotechnology, Vejle, Denmark. moniliformin in apples with wet core rot systems. 9th International Congress of Plant Pathology, Torino, Mogensen, J. M., Nielsen, K. F., Frisvad, J. C., Larsen, Italy. T. O., Thrane, U., Varga, J.Fumonisins from Aspergillus niger - a new health risk? CBS Symposium Fungi and Sin, G., Eliasson Lantz, A., Gernaey, K. Evaluating Health, Royal Institute of Science, Letters and Fine Arts prediction uncertainty of fermentation models. 7th (KNAW), Amsterdam, The Netherlands. European Symposium on Biochemical Engineering Science, Faro, Portugal. Mølgaard, L., Hansen, B. G., Mortensen, U. H., Patil, K. R. Heterologous expression of polyketides in fungi Sohoni, S. V., Mijakovic, I., Eliasson Lantz, A. An and optimization by using in silico analysis. Metabolic additional phosphotyrosine protein prosphatase (ptp) is Engineering VII: Health and sustainability, Puerto present in Streptomyces coelicolor A3(2). Natural Products Vallarta, Mexico. Discovery and Production II: Celebrating the successes of traditional and novel Culture sources, Whistler, British Nielsen, J. B., Mapari, S. S., Nielsen, M. L., Larsen, T. O., Colombia, Canada. Frisvad, J. C., Mortensen, U. H. Fungal pigments in stress response - new therapeutics? 3rd Danish Conference on Sørensen, J. L, Aveskamp, M., Hansen, M. A. E., Thrane, Molecular Biology and Biotechnology, Vejle, Denmark. U., Crous, P., Andersen, B. Polyphasic analysis of Phoma section Peyronellaea. CBS Symposium Fungi and Health, Pedersen, L., Hansen, K., Nielsen, K. F., Nielsen, J., Royal Institute of Science, Letters and Fine Arts (KNAW), Thykær, J. Genome wide analysis of Aspergillus niger Amsterdam, The Netherlands. metabolism during industrial fed-batch fermentations. Sørensen, J. L., Phipps, R. K., Nielsen, K. F., Schroers, H. J., Thrane, U. Analysis of Fusarium avenaceum metabolites produced during wet apple core rot. 10th International Fusarium Workshop and Fusarium Genomics Workshop 2008, Alghero, Italy.

van de Vondervoort, P., Andersen, M. R., Baker, S. E., Meijer, M., van Peijn, N., Pel, H., Robus, H., Samson, R., Schaap, P. Insights in short term evolution of Aspergillus niger strains. 5th International Aspergillus meeting, Asperfest, Edinburgh, Scotland.

Wollenweber, B., Jiang, D., Jacobsen, S., Søndergaard, I., Porter, J. Implications of high-temperature and drought events for yield and quality in wheat. International Symposium on Crop Modelling and Decision Support, ISCMDS-2008, Nanjing Agricultural University, China. ANNUAL REPORT 2008 CENTER FOR MICROBIAL BIOTECHNOLOGY 29 Staff

Director Lehmann, Linda Kampp, Thomas, IT and Database manager Morten Kielland-Brandt, Professor Mapari, Sameer Karsbøl, Birgitte, Secretary Meijer, Susan Knoth-Nielsen, Lisette, Technician Faculty Mogensen, Jesper M. Kornholt, Martin E., Lab. trainee Andersen, Birgitte, Associate professor Mølgaard, Louise Krøger, Elisabeth, Lab. manager Frisvad, Jens Christian, Professor Olivares, Roberto Laursen, Jytte V., Communications officer Hansen, Michael E. Assistant professor Oliveira, Ana Paula Lyhne, Ellen K., Technician Hobley, Tim, Associate professor Otero, José Manuel Mortensen, Jette, Technician Karhumaa, Kaisa, Assistant professor Ottow, Kim Mylord, Martin, Lab. assistant Lantz, Anna Eliasson, Associate professor Papini, Marta Svendsen, Lou, Office assistant Larsen, Thomas Ostenfeld, Associate professor Pedersen, Lasse Wass, Anne, Office assistant Mijakovic, Ivan, Associate professor Petersen, Trine L. Winther, Pernille, Secretary Mortensen, Uffe, Associate professor Piddocke, Maya P. Nielsen, Kristian Fog, Associate professor Poulsen, Tine R. Guests Nielsen, Michael L. Assistant professor Rank, Christian Abreu, Lucas, PhD student Panagiotou, Gianni, Assistant professor Rasmussen, Rie R. Basilio, Carmo, PhD student Patil, Kiran, Assistant professor Rueksomtawin, Kanchana Copetti, Marina V., PhD student Thrane, Ulf, Professor Rønnest, Mads H. James, Jemila, PhD student Thykær, Jette, Assistant professor Salazar Pena, Margarita Lages, Nuno, PhD student Søndergaard, Ib, Associate professor Sohoni, Sujata Montagud, Arnau, PhD student Storm, Ida D. Polizzotto, Rachele, PhD student Post docs Sørensen, Jens L. Rebacz, Blanka, PhD student Andersen, Mikael R. Sørensen, Marie Wasielewska, Joanna, PhD student Borodina, Irina Tavares, Sabina Bapat, Prashant M. Usaite, Renata Hansen, Bjarne G. Vongsangnak, Wanwipa Mapelli, Valeria Wattanachaisaereekul, Songsak Nielsen, Jakob B. Zhang, Jie Maury, Trine L. Ödman, Peter Maury, Jerome Pedersen, Mona H. Research assistants Phipps, Richard K. Chumnanpuen, Pramote Quriós, Manuel Hansen, Vesna Siewers, Verena Jose, Dinto Vemuri, Goutham Mogensen, Jesper Papadakis, Emmanouil PhD students Partow, Siavash Albertsen, Line Rattleff, Stig Asadollahi, Mohammad A. Brochado, Ana Rita Technical and administrative staff Brogaard, Katrine Abdellatif, Mohammad, Technician Carlsen, Simon Andersen, Dorthe, Secretary Chen, Xiao Andersen, Taja, Lab. assistant Faustrup, Helene Asueva, Anja A., Lab. assistant Formenti, Luca Bro, Trine, Head of administration Hallwyl, Swee Christiansen, Lene, Technician Johansen, Maria Jakobsen, Hanne, Lab. manager Kjeldsen, Kjeld R. Jakobsen, Simo, Lab. trainee Kold, David Jensen, Anni, Technician Krogh, Astrid M. Johansen, Tina, Lab. manager ANNUAL REPORT 2008 CENTER FOR MICROBIAL BIOTECHNOLOGY 31 www.cmb.dtu.dk

Layout: Suzanne Fog v/ J & R Frydenberg A/S // Tryk: J & R Frydenberg A/S - København N - TlF: 35 81 03 33