Exploring the limitations of isobutanol production by engineered industrial Saccharomyces cerevisiae
strains
Dissertation zur Erlangung des Doktorgrades der Naturwissenschaften
vorgelegt beim Fachbereich Biowissenschaften der Johann Wolfgang Goethe-Universität in Frankfurt am Main
von Wesley Cardoso Generoso aus Birigui (Brazil)
Frankfurt am Main 2016 D30
vom Fachbereich Biowissenschaften der Johann Wolfgang Goethe – Universität als Dissertation angenommen.
Dekanin: Prof. Dr. Meike Piepenbring
Gutachter: Prof. Dr. Eckhard Boles Prof. Dr. Jörg Soppa
Datum der Disputation:
CONTENTS
LIST OF CONTENTS
1. Summary ...... 1
2. Introduction ...... 3
2.1 Industrial biotechnology and metabolic engineering ...... 3
2.1.1 Saccharomyces cerevisiae in metabolic engineering ...... 4
2.2 Use of biomass in industry ...... 5
2.2.1 Xylose assimilation pathway ...... 6
2.3 Next-generation biofuels ...... 8
2.3.1 Butanol isomers ...... 10
2.4 Endogenous isobutanol production ...... 11
2.4.1 Branched-chain amino acids biosynthesis in yeast ...... 11
2.4.2 Ehrlich pathway ...... 15
2.5 Metabolic engineering for isobutanol production ...... 18
2.5.1 Isobutanol production in bacteria ...... 18
2.5.2 Isobutanol production in S. cerevisiae ...... 19
2.6 Objectives of the work ...... 22
3. Materials and methods...... 23
3.1 Buffers and media ...... 23
3.1.1 Buffers ...... 23
3.1.2 Media ...... 24
3.2 Microorganisms ...... 25
3.2.1 Bacterial strains ...... 25
3.2.2 Yeast strains ...... 26
3.3 Plasmids...... 28
3.4 Synthetic DNA ...... 30
3.5 Equipment, enzymes, chemicals and kits...... 30
3.6 Molecular biology methods...... 32
CONTENTS
3.6.1 Plasmid and DNA isolation ...... 32
3.6.2 Yeast RNA isolation ...... 32
3.6.3 Separation of nucleic acids fragments...... 33
3.6.4 Polymerase chain reaction (PCR) ...... 33
3.6.5 Error-prone PCR ...... 34
3.6.6 DNA Restriction ...... 34
3.6.7 Sequencing of DNA ...... 35
3.7 Genetic methods ...... 35
3.7.1 E. coli transformation...... 35
3.7.2 Yeast transformation ...... 36
3.7.3 Plasmid construction ...... 36
3.7.4 Gene deletion via Cre/loxP ...... 38
3.7.5 Gene deletion via CRISPR/Cas9 ...... 38
3.8 Growth based methods and fermentations...... 39
3.8.1 Quantification of cell density ...... 39
3.8.2 Serial dilution spot assay ...... 40
3.8.3 Complementation of valine auxotrophy ...... 40
3.8.4 Aerobic growth cultivations ...... 41
3.8.5 Micro-aerobic fermentations ...... 41
3.8.6 Evolutionary engineering ...... 41
3.9 Protein methods ...... 42
3.9.1 Enzyme assay ...... 42
3.9.2 Pull-down assay ...... 42
3.9.4 Western Blot ...... 43
3.10 Metabolite analysis via HPLC ...... 43
3.11 In silico methods ...... 44
3.11.1 Bioinformatics analysis ...... 44
3.11.2 Plasmids and primers design ...... 44
CONTENTS
3.11.3 Statistical analysis ...... 45
4. Results ...... 46
4.1 Isobutanol biosynthetic pathway incorporation ...... 46
4.1.1 Exchange of the co-factor specificity for Ilv5 ...... 47
4.1.2 Minimization of Ilv6 inhibition ...... 49
4.2 Investigation of limitations in Ilv3 ...... 52
4.2.1 Intensification of cytosolic iron-sulfur cluster assembly ...... 52
4.2.2 Overexpression of heterologous ILV3 orthologous ...... 55
4.3 Bioprospection of dehydratases with Ilv3 activity ...... 57
4.3.1 Site-directed mutagenesis of rspA ...... 59
4.3.2 Randon mutagenesis of RspA ...... 61
4.4 Dihydroxy-isovalerate uptake evaluation ...... 61
4.4.1 Search for DIV export proteins ...... 64
4.5 Substrate channeling between IlvC6E6 and Ilv3 ...... 68
4.5.2 3-methyl-butanol production ...... 73
4.6 Redirection of flux from ethanol to isobutanol production ...... 74
4.6.1 Deletion of alternative pathways ...... 78
5. Discussion ...... 80
5.1 Development of the initial isobutanol producing strain ...... 80
5.2 Investigation of Ilv3 activity limitations ...... 83
5.3 Dihydroxy-isovalerate efflux attenuation for isobutanol production ...... 89
5.4 Pyruvate flux redirection from ethanol to isobutanol production ...... 93
6. Zusammenfassung ...... 97
7. References ...... 102
Appendix A: Abbreviations ...... 113
Appendix B: Primer list ...... 115
Appendix C: Zusammenfassung (Kurz) ...... 126
Appendix D: Acknowledgements ...... 128
CONTENTS
Appendix E: Curriculum vitae ...... 129
SUMMARY
1. SUMMARY
Saccharomyces cerevisiae is a natural producer of isobutanol, which has more advantages as biofuel than ethanol, i.e. superior combustion energy, weaker corrosive action and reduced aqueous miscibility. Isobutanol is produced by the combination of the valine biosynthesis and the Ehrlich pathway. In this work, an industrial strain was employed for isobutanol production, in which the valine pathway was relocated into the cytosol. The valine pathway in yeast has a cofactor imbalance, since the glycolysis produces NADH, while Ilv5 employs NADPH for the reaction. Therefore, the cofactor specificity of the pathway was rebalanced with exchange of Ilv5 by an NADH-consuming mutant, IlvC6E6. Furthermore, Ilv6, which regulates the feed- back inhibition of the valine biosynthesis, was tested to boost isobutanol production; however, none of these Ilv6 alternatives could greatly enhance isobutanol production. Therefore, due to a still low production yield, the bottlenecks of the isobutanol pathway were deeper studied. The major observed bottleneck concerned the conversion of DIV into KIV, since high concentrations of acetoin, 2,3-butandiol and, specially, DIV were observed in the fermentation supernatant, while neither KIV nor isobutyraldehyde were detected. This step is performed by the dihydroxy-acid dehydratase, Ilv3, which needs iron-sulfur clusters for its activity. Therefore, the first approach to circumvent this limitation was to increase the FeS assembly and its transference into the cytoplasm; however, Ilv319 activity was not improvement. Afterwards, Ilv3 alternatives were screened for substitution of Ilv319. Heterologous ILV3 orthologous with possible advantages were investigated, but Ilv319 was still the most promising alternative. Furthermore, sugar- acid enolases were tested as Ilv319 substitutes. These enolases also catalyze the dehydration of the substrate in the same way as Ilv3, but uses Mg2+ as cofactor. One of the employed enolases could complement valine auxotrophy; however, it allowed just a very slow growth of the ilv3 strain and its activity could not be enhanced by mutagenesis studies. Interestingly, we observed that once DIV is secreted out of the cell, it cannot be re-uptaken from the medium and this possibly further aggravates the pathway flux and Ilv319 activity. In order to suppress DIV waste, two strategies were formulated: the deletion of the possible DIV transporter, and the substrate channeling of DIV from IlvC6E6 to Ilv319. In order to find possible DIV export proteins, a transcriptome 1
SUMMARY
analysis of a strain producing high amounts of DIV against a strain producing no detected DIV were compared. Several transporters were found upregulated in the DIV producing strain, but, alone, none of these were responsible for the DIV efflux. For the substrate channeling, an artificial enzymatic net was constructed by the fusion of IlvC6E6 and Ilv319 with synthetic zippers, which have high affinity to each other, and as both enzymes are alone organized as oligomers. The use of this enzymatic net enhanced not only the isobutanol production in about 17%, but also 3-methyl-butanol production yield was 25% increased. Nevertheless, together with bottlenecks arising from Ilv3 activity, the isobutanol production is limited by the ethanol production, which is the main product of S. cerevisiae. Therefore, in order to abolish ethanol production, PDC1 and PDC5 were deleted. Moreover, BDH1 and BDH2 were also deleted to create an NADH-driving force towards isobutanol production. However, the isobutanol yield of this mutant was even lower than that of the strain without the mentioned deletions. As a high production of isobutyric acid was observed, and it could be produced directly from KIV, different KIV decarboxylases and isobutanol dehydrogenases were investigated; but without improvement. Then, alternative pathways were abolished in other to favor isobutanol production, e.g. valine, leucine, isoleucine and panthotenate biosyntheses. Nevertheless, isobutanol yields were still low and the main byproducts were glycerol, acetoin, DIV and isobutyric acid. Despite the outcomes were not enough to enhance isobutanol production up to commercially required yields, these results help in the comprehension of the bottlenecks surrounding the isobutanol production pathway and serve as basis for further studies within the branched-chain amino acids biosynthesis and Ehrlich pathway.
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INTRODUCTION
2. INTRODUCTION
2.1 Industrial biotechnology and metabolic engineering
Biotechnology has changed the development of industrial processes, making them gradually less aggressive and less expensive than chemical techniques. The industrial use of microorganisms has been widely spread, from the food processes to the synthesis of high-value biomolecules. Many are the examples, in which industrial biotechnology has been employed for high demand products, e.g. the citric acid production with Aspergillus niger and monosodium glutamate with Corynebacterium glutamicum (Max et al. 2010; Sano 2009). The main objective of these processes is to employ biotechnology to reduce fossil feedstock and to circumvent purely chemical or extractive processes, in order to ensure sustainability and decrease environmental impacts. However, in many of the cases, the yield of production is low and must be improved to be commercially viable. In this aspect, advances in recombinant DNA techniques have boosted the use of biological systems, fostering the understanding of many metabolic pathways of commercial interest. DNA techniques assist from basic science, in which the function and structure of protein are investigated; up to more complex metabolic engineering, aiming to increase productivity of a homologous pathway, or the incorporation of pathways in heterologous system (Badiru 2014; Stephanopoulos 2007). Some biotechnological processes with genetically modified organisms are simple, consisting just in the overexpression of a single protein, which can be already the desired final product. On the other hand, the production of bulk chemicals can involve several pathway manipulations, such as the deletion of branches of the pathway of interest, modulation of genes expression of enzymes involved in the pathway, protein engineering for an increased enzymatic activity and/or elimination of regulatory elements (Badiru 2014). Insulin was the first licensed product made with recombinant DNA technique (Baeshen et al. 2014). For this, the chain A and chain B of the insulin molecule were overexpressed separately in Escherichia coli and the active insulin was obtained by chemical-induced disulfide bonds formation. Another example is the metabolic engineering of E. coli for production of 1,4-butanediol. Yim et al. (2011) engineered the oxidative tricarboxylic acid cycle (TCA), to produce more succinate, which is further 3
INTRODUCTION converted to 1,4-butanediol. Despite the high efficiency of the E. coli for metabolic engineering, it is not robust enough for industrial processes and its application can be either limited by the culture conditions or by the high effort and energy for product purification.
2.1.1 Saccharomyces cerevisiae in metabolic engineering
Saccharomyces cerevisiae is the most ancient example of microbial usage in large scale processes. This yeast has been adopted by humans due to the ability of consuming sugars and producing carbon dioxide and ethanol, which is why it is a key element for wine and beer manufacture and baking. The simplicity to control and manipulate S. cerevisiae has made it the most studied yeast and, therefore, an important eukaryotic model, even for human research (Denoth Lippuner et al. 2014; Voisset et al. 2014). Besides, S. cerevisiae is robust in processes with high sugar concentration, high osmotic pressure, low pH and anaerobic conditions, and, furthermore, yeast is considered as a GRAS (Generally Recognized as Safe) microorganism worldwide (Hong and Nielsen 2012). These reasons and the strong knowledge in yeast genetics, proteomics and metabolomics, made it an important organism for production of high value compounds (Hong and Nielsen 2012; Nielsen et al. 2013). Pharmaceutical molecules are good example of yeast utilization for production of fine compounds. Yeast has been employed for production of the hepatitis B surface antigen for vaccination since 1984 (McAleer et al. 1984). Another example is the production of the antimalarial drug artemisinin. Paddon et al. (2013) produced the artemisinin precursor in a high yield employing a yeast strain with an engineered mevalonate pathway. One of the largest industrial applications of S. cerevisiae is the ethanol production. After the oil crises in 70’s, world governments created initiatives to encourage the development of large scale ethanol production and utilization as automotive fuel (Goldemberg 2013). Currently, ethanol has been produced worldwide, differing of the sugar source, e.g. sugarcane is employed in Brazil, corn in USA and sugar beet in Europe. The ethanol production from sugar cane is the most advantageous, with a cost of less than 0.25 dollars per liter of the final product (Basso et al. 2011). Yeast has been still investigated for further improvement of ethanol
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INTRODUCTION production from sugar, as for example the development of more robust genotypes and less production of byproducts, such as glycerol and acetate (Gombert and van Maris 2015). Nonetheless, the main focus nowadays is the application of yeast for ethanol production from lignocellulosic raw material, such as sugarcane bagasse, rice and corn straw, and forest biomass.
2.2 Use of biomass in industry
Plant raw material has been deeply explored as feedstock for biotechnological processes, because of its sustainable way of obtainment and lower production cost than synthetic formulations. This material is mainly composed of the plant cell wall and is constituted of 35-50% cellulose, 5-35% lignin and 20-30% hemicellulose (Lynd et al. 2002). Cellulose is a glucose homopolymer, organized by inter-chain hydrogen bonds, forming crystalline fibrils. This structure is highly cohesive and has unique strength and partial insolubility in water, what ensures the plant rigidity (Festucci-Buselli et al. 2007). Lignin is a phenolic heteropolymer, which has an amorphous structure and high hydrophobicity. It provides the recalcitrance of the plant wall and a high resistance to hydrolytic attack (Ragauskas et al. 2014). The hemicellulose is a heteropolymer, primordially of pentoses (xylose and arabinose) with lower amount of hexoses (glucose, mannose and galactose), uronic acids and acetyl groups. This heterogeneity plays an important role in plant structure, since the hemicellulose establish the connection between the cellulose fibers and the lignin (Ferreira-Filho 1994). Industrially, lignocellulosic raw material is often burned in boilers to produce electricity, e.g. the bagasse in the ethanol production in Brazil generate enough energy to run the plant (van Haandel 2005). Nevertheless, this material is still rich in sugar polymers that could be employed as feedstock for nobler industrial processes. As these sugars are not ready for microbial utilization, these polymers should undergo a depolymerization process. Nevertheless, before this depolymerization, a pretreatment is necessary to break down the recalcitrance of the biomass and allow the lignin separation (Arantes and Saddler 2010). The pretreatment can be performed chemically or physically. The chemical pretreatments employ either acids, which promote the autolysis of the hemicellulose, or hydroxides, with the intention to break the esters of the 5
INTRODUCTION hemicellulose, allowing thereby the extraction of the lignin (Chaturvedi and Verma 2013; Zheng et al. 2009). In spite of the simplicity and low-cost, these methods generate fermentation inhibitors. On the other hand, the physical pretreatments are capable of promoting the autolysis of hemicellulose with less fermentation inhibitors than chemical pretreatment (Kaar et al. 1998; Karp et al. 2013). Steam explosion is the most well-known method and consists of a high heating (around 200°C) under high pressure in a few seconds, followed by a rapid decrease in pressure. This disrupts the structure of the polymers and forms acetic acid which hydrolyzes the hemicellulose (Kaar et al. 1998). After the pretreatment, lignin is removed with organic solvents, then the hemicellulose sugar is recovery from the cellulose pulp (Hu et al. 2011). At this point, the pulp with a lower recalcitrance is ready for the biomass depolymerization. This depolymerization of the biomass can be performed chemically or enzymatically. The (termo-)chemical method is carried out with the combination of acids, solvents and high temperature, which destroys the polymers and reduce them to a liquor of monomers or oligomers (Galbe and Zacchi 2002). In spite of the efficiency, the chemical methods generate harmful compounds for fermentative process. For this reason, the enzymatic treatment is the more attractive alternative for biomass depolymerization. This treatment employs the specificity of enzymes to decompose the polymers into monomers without the generation of fermentation inhibitors or reduced, at least (Banerjee et al. 2010). Either by thermo-chemical or enzymatic depolymerization, the final hydrolysates are rich in glucose and xylose, and traces of arabinose. In spite of the harsh characteristics of these hydrolysates, they can be successfully employed as medium for S. cerevisiae (Tesfaw and Assefa 2014). Nonetheless, yeast is not able to assimilate pentoses and, consequently, it reduces the potential of the biomass.
2.2.1 Xylose assimilation pathway
The plant hydrolysates can have high amounts of xylose, depending on the biomass, and the consumption of this xylose further reduces the overall cost of in the production of biofuels. There are several microbial species able to consume xylose naturally and one of the alternatives is to use these microorganisms, e.g. specially yeasts from the genera Candida, Kluyveromyces and Pichia (Canilha et al. 2012; Chandel et al. 2011). However, S. cerevisiae is still the most adapted microorganism
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INTRODUCTION to the severe conditions of the hydrolysates and it is a GRAS. Therefore, this ability to consume xylose has been deeply studied (Weber et al. 2010). Xylulose, instead, can be a substrate for yeast, and the isomerization of xylose into xylulose can be achieved in two manners: via oxi-reduction or direct isomerization (figure 2.1). Two enzymes are necessary for the redox conversion of xylose, the xylose reductase (XR) and the xylitol dehydrogenase (XDH), and the reactions depend on NADPH and NAD+, respectively. The cofactor imbalance created by XR is the first limitation of this pathway, not only because NAD+ is the cofactor required in the next reaction, but also because NADH is the main produced cofactor during glycolysis. Nevertheless, this issue was solved by mutagenesis of XR to exchange cofactor specificity (Krahulec et al. 2012; Lee et al. 2012). Yeast has an endogenous XDH (Xyl2); however, heterologous gene expression promoted higher efficiency (Ho et al. 1998; Kötter and Ciriacy 1993).
Figure 2.1 Potential pathways for xylose assimilation in Saccharomyces cerevisiae. The scheme does not discriminate between reversible and irreversible reactions. Continuous-lines stand for enzymatic steps and dotted-lines stand for metabolite transference.
The other alternative to convert xylose into xylulose is with a xylose isomerase (XI). The direct isomerization is the main form of xylose assimilation in bacteria, and also found in filamentous fungi (Kuyper et al. 2003; Madhavan et al. 7
INTRODUCTION
2009). Despite XI is known since 1953, when it was first characterized in Lactobacillus pentosus extract (Mitsuhashi and Lampen 1953), its overexpression in yeast is still challenging and limited due to xylitol inhibition (Weber et al. 2010). Nevertheless, Brat et al. (2009) characterized a XI from C. phytofermentans, which was fully functional in S. cerevisiae. After xylose isomerization, xylulose is then phosphorylated and undergoes the non-oxidative pentose-phosphate pathway (figure 2.1). The non-oxidative pentose- phosphate pathway converts three molecules of xylulose-5-phosphate into two molecules of fructose-6-phosphate and one glyceraldehyde-3-phosphate, which will enter the glycolysis and become pyruvate. In previous research in our group, the industrial strain EthanolRed (Lesaffre, France) was metabolically engineered to consume pentoses (Dietz, 2013). Succinctly, this work employed XI for the direct isomerization of xylose, as well as the genes involved in arabinose consumption (not explained in this work). Additionally, overexpression cassettes of xylulokinase and the native genes of the pentose- phosphate pathway were also incorporated into this strain. The overexpression cassettes were integrated into the genome, but this strain was not able to consume xylose efficiently (Demeke et al. 2013). Therefore, evolutionary engineering for xylose consumption was carried out, and a resulting strain, named HDY.GUF9, was able to grow in xylose as carbon source. Although HDY.GUF9 was already able to consume xylose, this strain presented irregular fermentative profiles. Thus, additional copies of the xylose and arabinose consumption genes were incorporated into HDY.GUF9 genome and this transformant was employed in a new evolutionary engineering, but for arabinose consumption. The resulting strain, HDY.GUF12, had a satisfactory and stable xylose fermentative performance and, in addition, produced ethanol in a yield of 0.513 grams of product per gram of sugars (g/gsugar) in Arundo hydrolysates (Dietz, 2013). Therefore, this strain would be of great value for production of fine chemicals from plant biomass, such as advanced biofuels.
2.3 Next-generation biofuels
In spite of the positive utilization of ethanol as biofuel, it has some disadvantages compared to gasoline, such as a higher corrosive action to the car 8
INTRODUCTION engine, a higher compression necessity for explosion and 30% less energy than gasoline. Moreover, during the production process, high energy is necessary to separate ethanol from the medium, due to its high hydroscopic characteristic (MacLean and Lave 2003). For this reason, new chemicals with possible microbial production have been investigated as biofuels substitutes. Fatty-acids and its derivatives, as alcohols, methyl-esters and alkanes, are good alternatives as biofuels. Due to their long hydrocarbon chain, these molecules are rich energetically and can be great substitutes to fossil diesel (Petrovič 2015). Yarrowia lipolytica has been intensely studied for fatty-acid production, since this microorganism can accumulate high amount of fatty-acids, consuming crude glycerol as carbon source (Papanikolaou and Aggelis 2009; Vorapreeda et al. 2012). S. cerevisiae does not accumulate high amounts of fatty-acids. Two main steps are critical for the improvement of fatty-acids production in yeast: the acetyl-coenzyme A (acetyl-CoA) production and the activity of the acetyl-CoA carboxylase Acc1 (Li et al. 2014). Both bottlenecks have been researched lately (Kozak et al. 2014; Shi et al. 2014; Vorapreeda et al. 2012). Terpenoids are another class of molecules feasible as biofuels. As fatty- acids, the long hydrocarbon chains of terpenoids ensure a high energetic storage. Moreover, some of these terpenoids, e.g. limonane and farnesane, are lighter than fatty-acids derivatives, which is why they are interesting chemicals for drop-in jet-fuel (Brennan et al. 2012). Dimethylallyl diphosphate and isopentenyl diphosphate are the building blocks of terpenoids and these two molecules are produced via the mevalonate or the 2-methylerythritol 4-phosphate (non-mevalonate) pathways. Although yeast possesses a functional mevalonate pathway, its terpenoid production is naturally very small (Buijs et al. 2013; Siddiqui et al. 2012). One of the bottlenecks, is also the acetyl-CoA synthesis, which is the starting point of the mevalonate pathway. Another limitation is the strong feedback-inhibition of the hydroxymethylglutaryl-CoA reductases (Hmg1 and Hmg2) and squalene synthase (Erg9) activities, which attenuate the flux towards sesquiterpenes biosynthesis as they are produced (Asadollahi et al. 2010). Carlsen et al. (2013) tried to incorporate the bacterial non- mevalonate pathway into yeast, but it was not possible due to the inactivity of the iron- sulfur cluster (FeS) possessing enzymes. The most promising biofuel alternatives are the medium-chain alcohols: the butanol and pentanol isomers. Out of these, the main alcohols with potential to be
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INTRODUCTION employed as biofuels are 1-butanol, isobutanol, 2-methyl-1-butanol (2-MB) and 3- methyl-1-butanol (3-MB).
2.3.1 Butanol isomers
The butanol isomers are the most promising substitutes for gasoline for auto mobile among the new biofuels. If compared to ethanol, they have a higher octane rating, a higher energy density, a lower vapor pressure, a higher flash point and are less hygroscopic. These advantages permit that the butanol isomers can be employed as equal as the current fuels, i.e. using the same existing fuel infrastructure (cars, pipelines and stations), and being produced with the same existing infrastructure for ethanol production (Petrovič 2015). Furthermore, the minor water miscibility of the butanols reduce the cost of production, since less energy is necessary to separate them from the fermentation medium; and they can be mixed with gasoline in higher concentrations than ethanol without necessity of changes in the car engine. Three butanol isomers can be produced with microorganisms: 1-butanol, 2- butanol and isobutanol. 1-butanol is produced by fermentation of sugars with anaerobic bacteria, mostly with Clostridium species. Clostridia produce 1-butanol through the named acetone-butanol-ethanol (ABE) fermentation. Due to the strict anaerobically characteristic, these microorganisms consume glucose rapidly and produce acetate and butyrate, in order to generate enough ATP for growth. Afterwards, the excess of NAD(P)H in the cells causes an oxidative imbalance, so that the produced acetate is converted to ethanol and acetone; and butyrate is converted to 1-butanol (Lutke- Eversloh 2014). This pathway has been employed in S. cerevisiae; although the 1- butanol production is still very small and the limitations unclear (Generoso et al. 2015). Schadeweg and Boles (2016) demonstrated that one of the limitations of this pathway in yeast is not only the acetyl-CoA synthesis, but the CoA itself. The 2-butanol biosynthesis was first characterized in Lactobacillus species. In this pathway, meso-2,3-butanediol, a sub-product of acetoin, is converted to 2- butanone, with a diol dehydratase, and then to 2-butanol, with a secondary alcohol dehydrogenase (Ghiaci et al. 2014a). A recombinant S. cerevisiae strain, overexpressing a B12-dependent diol dehydratase from Lactobacillus reuteri and the secondary alcohol dehydrogenase from Gordonia sp., was able to produce just 4 mg/L of 2-butanol (Ghiaci et al. 2014b). The main bottleneck of this pathway is the
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INTRODUCTION heterologous expression of the diol dehydratase, not only for the requirement of B12, but also because this enzyme is composed of three peptides for the catalytic subunit and two more peptides for the activating subunit. Beyond that, these peptides must be expressed in equimolar amounts. The isobutanol is the most promising butanol isomer for biofuel application. Isobutanol has the same advantages as the other butanols and, additionally, is naturally produced by S. cerevisiae.
2.4 Endogenous isobutanol production
S. cerevisiae is naturally capable of producing branched-chain alcohols via the combination of two pathways: the biosynthesis of branched-chain amino acids (BCAAs) and their degradation. The BCAAs, e.g. isoleucine, leucine and valine, are synthetized de novo in yeast. In this process, pyruvate is used to produce -keto acids, which thence receive an amino group and become the amino acids. Afterwards, in the catabolic process, the amine radical of the BCAAs is removed and the deaminated - keto acids are further converted to fusel alcohols. Isoleucine, leucine and valine generate respectively 2-methyl-1-butanol (2-MB), 3-methyl-1-butanol (3-MB) and isobutanol.
2.4.1 Branched-chain amino acids biosynthesis in yeast
Isoleucine, leucine and valine are part of the essential amino acid in human diet. During the evolutionary track, mammals lost the ability of synthetizing the BCAAs; however, plants, bacteria and fungi can produce them (Valerio et al. 2011). The BCAA producing organisms have the BCAAs biosynthesis (ILV pathway) well-regulated by the end products and pathway intermediates. In agriculture, this pathway is very important, because it has been employed as target of herbicides that inhibit intermediate reactions (Singh and Shaner 1995). Besides, the ILV pathway has been explored in microbiology, not only due to the production of the BCAAs, but also because fine chemicals that can be produced from this pathway, as for example the branched-chain alcohols, branched-chain acids, acetoin and 2,3-butanediol. Corynebacterium species and E. coli are the main microorganisms employed for BCAAs production in industry. Most of the BCAAs produced industrially 11
INTRODUCTION employ strains generated by random mutagenesis; and these mutants, despite the high production efficiency, can demonstrate physiological alteration that hinder their utilization with cheap feedstocks (Park and Lee 2010). For this reason, yeast has been studied for production of BCAAs and their derived molecules. S. cerevisiae has the ILV pathway separated into two cellular compartments: mitochondria and cytosol (figure 2.2). The biosynthesis of valine is the shortest via among the complete ILV pathway and is essentially conducted in mitochondria (figure 2.2). The first reaction is carried out by the acetolactate synthase that carbo-ligates two molecules of pyruvate and originates 2-acetolactate and CO2. This reaction needs thiamine diphosphate as cofactor and is one of the steps responsible for the feedback inhibition of the ILV pathway. The acetolactate synthase reaction is performed by Ilv2 and it is regulated by a regulatory subunit, Ilv6 (Pang and Duggleby 1999). Ilv6 controls the activity of Ilv2 according to the presence of branched- chain amino acids (predominantly valine), i.e. Ilv6 inhibit Ilv2 activity if BCAAs are present in the media and, oppositely, the activity of Ilv2 is enhanced in the absence of the amino acids (Pang and Duggleby 1999). However, the sole catalytic subunit is capable to catalyze the reaction (Duong et al. 2011). Ilv2 is also important because of two sub products of the valine biosynthesis, acetoin and 2,3-butanediol (Bae et al. 2016; Ng et al. 2012). Both compounds are produced from diacetyl, which is product of the spontaneous decarboxylation of 2-acetolactate, in the presence of oxygen. Diacetyl can have two steps of reduction by the diacetyl reductases (Bdh1 and Bdh2), first into acetoin, and then into 2,3-butanediol. The second reaction of the valine biosynthesis is the isomerization of 2- acetolactate to 2,3-dihydroxy-isovalerate (DIV). This reaction is catalyzed by the ketol- acid reductoisomerase (Ilv5), which uses NAPDH and a magnesium ion for the isomerization. Petersen and Holmberg (1986) were the first to characterize ILV5 and they observed a constantly high basal expression. Curiously, Ilv5 was later associated with maintenance of the mitochondrial DNA. The purified Ilv5 was demonstrated to bind dsDNA and ILV5 deletion results in mitochondrial abnormalities, such as irregular growth in non-fermentative sugar and, consequently, petite phenotype (Macierzanka et al. 2008; Zelenaya-Troitskaya et al. 1995).
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INTRODUCTION
Figure 2.2 Schematic illustration of the branched-chain amino acid biosynthetic pathways from glucose in Saccharomyces cerevisiae. The scheme does not discriminate reversible and irreversible reactions, regulatory subunits and transcription factors. Continuous-arrows and dotted-arrows in black stand for enzymatic steps and metabolite transference, respectively. Red arrows represent multiple enzymatic steps and blue line display the mitochondrial double membrane.
The dihydroxy-acid dehydratase (Ilv3) performs the next step in the ILV pathway, the dehydration of DIV to 2-keto-isovalerate (KIV). This enzyme is very particular in the ILV pathway, since it is a member of the ILVD_EDD family that includes dehydratases with FeS. In yeast, there are proteins with FeS in the mitochondria, cytoplasm and nucleus, although the de novo assembly of this cofactor takes place inside of the mitochondria. Moreover, the biogenesis of FeS is a well-regulated process, being limited by different factors (Lill and Mühlenhoff 2006; 2008). The final reaction of the valine biosynthesis is the amination of KIV to valine. Yeast has two BCAA transaminases, Bat1 and Bat2, and they differ in their localization in the cell and their expression profile during growth. Bat1 is the mitochondrial transaminase and is expressed during exponential phase, and that is why is more associated with BCAA biosynthesis. On the other hand, Bat2 is cytosolic and is expressed during stationary phase, and is thereby involved in BCAA catabolism (Colon et al. 2011). Nevertheless, both BCAA transaminases can catalyze the reaction in both directions.
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INTRODUCTION
The enzymes employed for isoleucine biosynthesis are the same as those of the valine biosynthesis, with the difference that the isoleucine production starts from threonine (figure 2.2). Threonine is synthetized from aspartate, which in turn, comes from oxaloacetate. In yeast, oxaloacetate is produced by the carboxylation of pyruvate via Pyc1 and Pyc2 and is an important component of the glyoxylate cycle in the cytosol and TCA cycle in the mitochondria (Zelle et al. 2008). Threonine is synthesized either in cytosol or in mitochondria, but the its deamination to keto-butyrate occurs only inside the mitochondria. Ilv1 is responsible to carry out the threonine deaminase and it is another point of regulation of the ILV pathway, since ILV1 transcription is repressed in the presence of the BCAAs in the medium (Holmberg and Petersen 1988). The further steps of the isoleucine biosynthesis occur in parallel to the valine biosynthesis, in which Ilv2 condensates keto-butyrate and pyruvate, then Ilv5 and Ilv3 catalyze the production of the -keto acid, 2-keto-3-methyl-valerate. Thence, the BCAA transaminases execute the amination of 2-keto-3-methyl-valerate to isoleucine. The leucine biosynthesis is connected with the valine biosynthesis through KIV (figure 2.2). KIV receives an acetyl group from acetyl-CoA, becoming 2-isopropyl- malate. This reaction is performed by the 2-isopropylmalate synthases, Leu4 and Leu9. Leu9 is the sole mitochondrial isoform, whereas Leu4 can differ in its locations in the cell (mitochondria or cytosol). LEU4 has two alternative peptides, consequence of two adjacent AUG start codons, in which the further one outcomes in a truncation of the N-terminally mitochondrial targeting sequence (Beltzer et al. 1988). Leu4 is the major expressed 2-isopropylmalate synthase isoform and contributes with the regulation of ILV pathway via feedback inhibition of leucine (Casalone et al. 2000; Cavalieri et al. 1999). The next step of the leucine biosynthesis is the isomerization of 2- isopropylmalate to 3-isopropylmalate. The isopropylmalate isomerase (Leu1) is another FeS containing enzyme, but natively localized in cytosol (Wallace et al. 2004). Afterwards, 3-isopropylmalate is converted to 2-keto-isocaproate by the isopropylmalate dehydrogenase (Leu2). Leu2 is a bifunctional enzyme, catalyzing the dehydrogenation and the decarboxylation of the substrate, generating thereby one additional NADH molecules and CO2 (Kohlhaw 2003). LEU1 and LEU2 are transcriptionally regulated by Leu3, which is an important transcription factor that regulates the ILV pathway. It was already characterized to interact with LEU1, LEU2, LEU4, ILV2, ILV5 and BAT2 loci (Zhou et al. 1990). Furthermore, Leu3 has an
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INTRODUCTION additional regulatory function, i.e. it has a binding site for 2-isopropylmalate, and in the presence of the intermediate, the complexed-Leu3 acts as a repressor; and in the absence, Leu3 alone stimulates the transcription of the mentioned genes of the ILV pathway (Friden et al. 1989; Friden and Schimmel 1988; Zhou et al. 1990).
2.4.2 Ehrlich pathway
When in excess in the medium or in the absence of ammonium, some amino acids can be used as nitrogen source for the biosynthesis of other amino acids. This catabolic process was first described in 1907 by Felix Ehrlich and was thereby named as Ehrlich pathway (Ehrlich 1907). Ehrlich (1907) observed that S. cerevisiae was able to grow in sole sugar and BCAAs as medium; and, as a consequence, a higher amount of fusel alcohols was observed. Yeast has the ability to use seven amino acids through the Ehrlich pathway, e.g. valine, leucine, isoleucine, phenylalanine, tyrosine, tryptophan and methionine (Hazelwood et al. 2008). The aroma and flavour of the branched-chain alcohols, and their different esters, made the Ehrlich pathway very important for the wine making industries (Lilly et al. 2006). In general, this catabolic pathway is composed of three steps: the transference of the amino group from the amino acid into an acceptor, the decarboxylation of the resulting α-keto acid, and the last step can be either the aldehyde reduction to a fusel alcohol or the oxidation to a fusel acid (figure 2.3). The Ehrlich pathway intermediates and final products are listed in the table 2.1, but just the ones from BCAAs will be further explored in here. The starting point of this pathway is the deamination of the BCAAs, which is performed by the already described transaminases Bat1 and Bat2. These enzymes transfer the amine group from the BCAAs to a 2-oxoglutarate, generating a glutamate. Thence, this amine group is further transferred from the glutamate to the biosynthesis of other amino acids. Most of microorganisms have one or two BCAA transferases and they are equally involved in the biosynthesis and the degradation of amino acids (Bondar et al. 2005; Colon et al. 2011; Yvon et al. 2000). In yeast, Bat2 is believed to be more involved in the BCAA catabolism than Bat1 and also Yoshimoto et al. (2002) demonstrated that an overexpression of Bat2 positively affected isobutanol production. However, enzymatically, both directions of both enzymes are possible and quite similar (Prohl et al. 2000). Differently, plants have a bigger number of BCAA aminotransferases than microorganisms and they differ either in their locations in the
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INTRODUCTION cell or different activities (Binder 2010). One example is the Bcat7 from tomato, which favors the amino acids catabolism more than the biosynthesis (Kochevenko et al. 2012).
Figure 2.3 Schematic illustration of the Ehrlich pathway for catabolism of amino acids in Saccharomyces cerevisiae. The scheme does not discriminate between reversible and irreversible reactions.
Table 2.1. Ehrlich pathway intermediates and final products Amino acid α-keto acid Aldehyde Fusel alcohol Fusel acid Valine 2-keto- Isobutanal Isobutanol Isobutyrate isovalerate Leucine 2-keto- Isoamyl aldehyde 3-methyl- Isovalerate isocaproate butanol Isoleucine 2-keto-3-methyl- Methyl- 2-methyl- Methyl- valerate valeraldehyde butanol valerate Phenylalanine Phenylpyruvate 2- Phenylethanol Phenylacetate Phenylacetaldehyde Tyrosine Hydroxy- Hydroxy- Tyrosol Hydroxy- phenylpyruvate phenylacetaldehyde phenylacetate Tryptophan 3-Indole 3-Indole Tryptophol 3-Indole pyruvate acetaldehyde acetate Methionine Keto-methylthio- Methional Methionol 3-methylthio- butyrate propionate
The second step of the Ehrlich pathway is the decarboxylation of the α-keto acids to the corresponding aldehyde. The irreversibility of the decarboxylation reaction insures the flow of the amino acid catabolism. Five yeast genes were already described to be related to this step: PDC1, PDC5, PDC6, ARO10 and THI3 (Dickinson et al. 1998). The pyruvate decarboxylases (Pdc1, Pdc5 and Pdc6) are very active enzymes
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INTRODUCTION in S. cerevisiae and, therefore, they are the main decarboxylases of the amino acid catabolism (ter Schure et al. 1998). Aro10 was described as a broad-substrate- spectrum -keto acid decarboxylase and ARO10 involvement in the Ehrlich pathway was evidenced by its upregulation upon phenylalanine, leucine or methionine use as sole nitrogen source (Vuralhan et al. 2005). Differently, Thi3 was described not only as a catalytic enzyme, but also as a regulatory protein. Dickinson et al. (1997) reported Thi3 as an important decarboxylase for the 3-MB production. Besides, Thi3 is important for the other decarboxylases, to the extent that it is a sensor for the control of homeostasis and biosynthesis of thiamine in yeast and acts as a transcriptional factor for the activation of the THI genes (Mojzita and Hohmann 2006; Nosaka et al. 2005). The final step of the Ehrlich pathway occurs either towards the reduction or oxidation of the fusel aldehyde. The distinction between reduction or oxidation is related on the growth conditions that yeast is submitted. On one hand, in fermentative processes conducted with high glucose concentration, the high glycolytic flux generates a rapid overload of NADH, which consequently redirect the Ehrlich pathway towards the fusel alcohol production. On the other hand, in glucose-limited growth, the Ehrlich pathway is redirected towards fusel acid production to increase NADH production (Hazelwood et al. 2008). In S. cerevisiae, the alcohol dehydrogenases Adh1-7 are able to reduce the aldehydes to fusel alcohol (Dickinson et al. 2003); however, Adh2, Adh6 and Adh7 are described as the most efficient for production of the branched-chain alcohols (Brat et al. 2012; Kondo et al. 2012; Larroy et al. 2002). Furthermore, Dickinson et al. (2003) demonstrated that 3-MB was still produced from leucine even with several dehydrogenases deletions, which suggested that there are still unknown reductases able to produce the fusel alcohols. On the other hand, not much is known about the oxidation of the branched-chain aldehydes. Yeast has six described aldehyde dehydrogenases, three cytosolic (Ald1, Ald2 and Ald6) and three mitochondrial (Ald3, Ald4 and Ald5). From these, Ida et al. (2015) demonstrated that the deletion of ALD6 improved isobutanol production and, therefore, can be an important aldehyde dehydrogenase for isobutyric acid production.
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INTRODUCTION
2.5 Metabolic engineering for isobutanol production
An isobutanol biosynthetic pathway can be created by the connection of the valine biosynthesis and the Ehrlich pathway. Few microorganisms are naturally able to produce isobutanol, e.g. Lactococcus lactis, S. cerevisiae, Candida sp. (Derrick and Large 1993; Smit et al. 2004). Nevertheless, these organisms produce just very little amounts of isobutanol, and this is far from the maximum theoretical yield, which is
0.41 g isobutanol per g glucose (giso/gglu). For this reason, much of metabolic engineering of the isobutanol biosynthetic pathway had been investigated.
2.5.1 Isobutanol production in bacteria
Several bacterial hosts had been employed for isobutanol production. The most frequent are C. glutamicum and E. coli. Nonetheless, these bacteria require the expression of heterologous enzymes, since the crucial limitation of the natural isobutanol production is the inability to catalyze the decarboxylation of the α-keto acid. C. glutamicum is the main microorganism employed for production of several amino acids in large scale, and among these, L-valine can be produced in a yield higher than 0.3 g/gsucrose (Leuchtenberger et al. 2005). Thus, theoretically, C. glutamicum strains that produce high amount of KIV could be employed for high isobutanol production. In one example, Blombach et al. (2011) employed an C. glutamicum strain with deletion of pyruvate competing pathways and overexpression of the ILV pathway genes, i.e. ilvB, ilvN, ilvC and ilvD, orthologous to the yeast genes ILV2, ILV6, ILV5 and ILV3, respectively. To complete the isobutanol pathway, the keto- acid decarboxylase (kivD) from L. lactis and the endogenous alcohol dehydrogenase (adhA) were also overexpressed in the strain. However, the employed pathway had a cofactor imbalance, since in glycolysis two NADH are produced and IlvC and AdhA need NADPH as cofactors. In order to solve this cofactor imbalance, two transhydrogenases from E. coli (pntA and pntB) were employed. Blombach et al. (2011) thereby achieved an isobutanol production of 77% of the theoretical yield. Currently, the higher isobutanol yields were reported using E. coli as host.
Atsumi et al. (2008) described a isobutanol production of 0.35 giso/gglu (86% of the theoretical yield) with a strain overexpressing the ILV pathway, kivD from L. lactis and ADH2 from S. cerevisiae. In this, the acetolactate synthetase from Bacillus subtilis
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INTRODUCTION
(AlsS) was employed instead of the endogenous IlvB and IlvN holoenzyme, due to a higher affinity for pyruvate than the endogenous enzymes. In this study, the deletion of the pyruvate competing pathways were also crucial for the high isobutanol production. Later on, Bastian et al. (2011) reported 100% efficiency in the isobutanol production by using the same strategy as Atsumi et al. (2008), but with a cofactor-balanced pathway. For this, Bastian et al. (2011) employed NADH-dependent mutants of ilvC from E. coli and adhA from L. lactis, namely, ilvC6E6 and adhARE1, respectively. The ilvC mutant was generated through structure-guided mutagenesis, in which the amino acids responsible for the binding of NADPH were addressed and employed for saturation mutagenesis. IlvC6E6 had the cofactor specificity exchanged for NADH, without loss of the activity. The adhA mutant was generated by the combination of the mutations of two error-prone mutagenesis variants. These variants were discovered via in vitro assay of the libraries clones for an increased dehydrogenase activity with isobutyraldehyde as substrate and NADH as cofactor. The use of this two mutants (IlvC6E6 and AdhARE1) in the isobutanol pathway resulted in 100% of the theoretical yield, under anaerobic conditions (Bastian et al. 2011). Nevertheless, despite the high efficiency of isobutanol production in bacterial hosts, the higher alcohol tolerance of S. cerevisiae than these bacteria boosted the metabolic engineering studies for isobutanol production in yeast (Liu and Qureshi 2009). Moreover, as already mentioned, yeast can be employed in fermentative processes with harsh conditions, which would allow future production of isobutanol with lignocellulosic hydrolysates.
2.5.2 Isobutanol production in S. cerevisiae
While in bacteria the main bottleneck of the natural isobutanol biosynthetic pathway is the decarboxylation and reduction of the keto acid, in yeast the main limitation is the separation of the pathway into two different compartments. After glycolysis, pyruvate has to be transported into the mitochondrial matrix to produce KIV (figure 2.2) and afterwards, KIV or valine is transferred back into the cytoplasm for isobutanol production (figure 2.3). Without alteration of the native compartmentalization of the pathways, Chen et al. (2011) enhanced six-fold the isobutanol production with a strain overexpressing the endogenous ILV genes (ILV2, ILV5 and ILV3) and BAT2. With increased valine anabolism and catabolism, the
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INTRODUCTION
isobutanol yield was 3.86 mgiso/gglu, which represents less than 1% of the theoretical yield. Therefore, two major strategies have been explored to minimize this transmembrane barrier: the relocation of the whole pathway either to the mitochondria or to the cytosol. The first attempt to relocate the whole isobutanol pathway to mitochondrial was reported by Avalos et al. (2013). In this study, the authors compared the isobutanol production in two strains: both overexpressing the endogenous ILV pathway, but differing in the localization of the decarboxylase and alcohol dehydrogenase, i.e. either in the cytosol or mitochondria. The native ARO10 and adhARE1 from L. lactis were employed for the downstream pathway, and to redirect them to the mitochondria, a mitochondrial targeting signal (MTS) was incorporated at their N-terminal. The complete mitochondrial pathway resulted in 65% more isobutanol than the split pathway and a final isobutanol yield of 6.40 mgiso/gglu. Later on, in order to further optimize this mitochondrial-based isobutanol pathway, Yuan and Ching (2014) developed a strain with integration of the mitochondrial pathway genes into yeast genome. Aside the native ILV pathway, they employed ARO10 and ADH7 with the same MTS described by Avalos et al. (2013). Their strategy aimed in balance the pathway gene expression levels using a δ-integration system for arbitrary incorporation of different copy number for each gene. The most productive clone achieved 15 mgiso/gglu and had more copies of ILV5 and ADH7. In spite of the positive outcome of relocating the isobutanol pathway into the mitochondria, in both studies, no direct comparison was included with the whole pathway in the cytosol, under the same conditions. The use of mitochondria, or other cell compartments, can be an interesting tool to enhance productivity, since it contributes to limit intermediates diffusion, abolish competing reactions or toxicity of the intermediates, and concentrate the enzymes and their substrates, with favors the reaction rates. Nevertheless, specifically for the isobutanol biosynthetic pathway, the relocation into the mitochondria can be unsuitable. First, yeast reduces the number of mitochondria either in anaerobic processes or upon high sugar concentration, what is the case for most of the industrial processes (Watson et al. 1970). Moreover, pyruvate is the starting substrate of the ILV pathway, and yeast has a high pyruvate decarboxylase activity when cultivated with fermentable carbon-sources, i.e. during isobutanol production from glucose or xylose, pyruvate would earlier converted to acetaldehyde than it is transferred into the
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INTRODUCTION mitochondria (Pronk et al. 1996). For this reason, the cytosolic redirection of the ILV pathway has been the most investigated option. In previous work of our group, Brat et al. (2012) developed the first fully cytosolic pathway for isobutanol production in yeast. In order to maintain the ILV enzymes in the cytoplasm, Brat et al. (2012) investigated various N-terminally truncated mutants of ILV2, ILV5 and ILV3. The variants Ilv2N54, Ilv5N48 and Ilv3N19 were found exclusively in the cytoplasm and, together, they were able to complement the valine auxotrophy of an ILV-deleted strain (ilv2, ilv5 and ilv3). To achieve isobutanol production, the truncated ILV genes were codon optimized and overexpressed, as well as ARO10 and ADH2. However, the deletion of ilv2 and the cultivation in medium lacking valine were crucial for isobutanol production (Brat et al. 2012). In the end, after additional evolutionary engineering for growth in minimal medium, Brat et al. (2012) reached 14.18 mgiso/gglu. Following the same strategy, Matsuda et al. (2013) employed a combination of both isobutanol pathways, the cytosolic and the native mitochondrial. In this study, not only the N-terminally truncated mutants of ILV2, ILV5 and ILV3 were overexpressed, but also the native ILV2. Additionally, kivD from L. lactis and ADH6 were employed, and with the purpose of circumventing the Ilv5 and Adh6 cofactor imbalance, Matsuda et al. (2013) constructed a futile cycle to convert NADH into NADPH. In this transhydrogenase-like shunt, pyruvate is cyclically converted to oxaloacetate, then to malate and back to pyruvate by the pyruvate carboxylase Pyc2, malate dehydrogenase Mdh2, and a cytosolic-mutant of the malic enzyme Mae1, respectively. As Mdh2 consumes NADH and Mae1 produces NADPH, both cofactor could be interchanged in the cytoplasm. In this approach, Matsuda et al. (2013) achieved a yield of 16 mgiso/gglu, with additional disruption of the pyruvate dehydrogenase complex (lpd1). Nevertheless, ethanol is the major product during isobutanol production with yeast, either using the mitochondrial or the cytosolic pathway localization. Therefore, recently, Milne et al. (2016) reported the first attempt to abolish ethanol production in a isobutanol producing strain. In this strain, the three pyruvate decarboxylases were deleted (pdc1, pdc5 and pdc6), and a completely bacterial ILV pathway was overexpressed. For the Ehrlich pathway, just a keto acid decarboxylase (kdcA) from L. lactis was employed, i.e. no alcohol dehydrogenase. However, the resulting yield was
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INTRODUCTION
just 7.4 mgiso/gglu, which is lower than the observed for the strain without deletion of the pyruvate decarboxylases (Brat et al. 2012; Ida et al. 2015; Matsuda et al. 2013).
2.6 Objectives of the work
In spite of all these advances within the isobutanol production with yeast, it is still very inefficient and less than 4% of the theoretical yield was already reached. Moreover, just laboratorial strains were employed for isobutanol production up to date and their application in industry can be limited by their cultivation conditions. Therefore, the first objective of this work was to establish the pathway for isobutanol production in a xylose-consuming industrial strain. For this, the employed strain was HDY.GUF12, which was previously engineered for pentose consumption (see topic 2.2.1). This strain derives from the industrial strain EthanolRed (Lesaffre, France), which is very tolerant to harsh conditions, such as low pH, high sugar concentration and highly concentrated medium, and shows an excellent fermentation capacity and low byproducts formation (Demeke et al. 2013; Mukhtar et al. 2010). The employed strategy for the isobutanol production was the same adopted by Brat et al. (2012), i.e. the solely cytosolic isobutanol biosynthetic pathway. And afterwards, the limitations of the isobutanol production were investigated, as well as possible solutions for these bottlenecks.
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MATERIALS AND METHODS
3. MATERIALS AND METHODS
3.1 Buffers and media
3.1.1 Buffers
The composition of the buffers employed in this work are listed in table 3.1.
The buffers were prepared with double-distilled water (ddH2O).
Table 3.1: List of buffers used in this work.
BUFFERS COMPONENTS
50 mM Tris-HCl (pH 7.5), 15% (v/v) glycerol, 2% (w/v) SDS, 2x protein loading dye 0.6% (v/v) 2-mercaptoethanol, 0.04% (w/v) bromophenol blue
6x DNA loading dye TAE, 10% (w/v) glycerol, 0.04% bromophenol blue
Blocking buffer TBS-T, 5% Skimmed milk powder
Extraction buffer for affinity 100 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1x protease chromatography inhibitor cocktail
Extraction buffer for 50 mM Tris-HCl (pH 7.5), 5 mM MgCl enzyme assays 2
FCC solution 5% (v/v) glycerol, 10% (v/v) dimethyl sulfoxide
Elution buffer 500 mM imidazole, 100 mM Tris-HCl (pH 7.5), 150 mM NaCl
Isothermal reaction (ISO) 100 mM Tris-HCl (pH 7.5), 5% PEG-8000, 10 mM MgCl, buffer 10 mM dithiothreitol, 1 mM dNTP, 5 mM NAD+
PAGE running buffer 25 mM Tris, 0.192M glycine
TAE 40 mM Tris, 40 mM acetic acid, 2 mM EDTA
50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.3% (w/v) Tween- TBS-T 20
36% (w/v) PEG-3350, 100 mM lithium acetate, 270 μg/ml Transformation mix Salmon Sperm DNA
Washing buffer for affinity 100 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.3% (w/v) Tween chromatography 20
100 mM sodium phosphate buffer (pH 7.4), 1 M sorbitol, 25 Zymolyase solution U/mL zymolyase
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MATERIALS AND METHODS
3.1.2 Media
The composition of the media for the cultivation of E. coli and S. cerevisiae are listed in table 3.2. The media were sterilized by autoclavation and the carbon sources and antibiotics, if necessary, were added after the media reached less then 60C. The carbon sources used in this work have the following abbreviations in the medium name: glucose/dextrose (D), glycerol (G) and ethanol (E). The carbon source concentrations were 20 g/L for general cultivation (solid or liquid) and 40 g/L for the micro-aerobic fermentation. For the preparation of solid media (agar plates), 19 g/L agar-agar were added to the media before autoclavation.
Table 3.2: List of media employed in this work.
MEDIA COMPONENTS REFERENCE
10 g/L tryptone, 5 g/L yeast extract, (Sambrook and Russell Luria-Bertani (LB) 5 g/L NaCl. pH 7.5 with NaOH 2001) 1.7 g/L yeast nitrogen base w/o amino acids and ammonium sulfate, (Sherman 1991) Synthetic complete (SC) 5 g/L (NH ) SO , 1x amino acids 4 2 4 (modified) solution (table 3.3) and corresponding carbon source. pH 6.3 with KOH
5 g/L (NH4)2SO4, 0.5 g/L MgSO4, 2.7 g/L KH PO , vitamin and trace Synthetic fermentative 2 4 element solution as Verduyn et al. (Verduyn et al. 1992) (SF) (1992), and corresponding carbon source. pH 5.0 with KOH. 1.7 g/L yeast nitrogen base w/o amino acids and ammonium sulfate, Synthetic minimum (SM) 5 g/L (NH4)2SO4, 20 mM KH2PO4 and (Sherman 1991) corresponding carbon source. pH 6.3 with KOH 10 g/L yeast extract, 20 g/L Yeast extract peptone bacteriological peptone and (Sherman 1991) (YP) corresponding carbon source
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MATERIALS AND METHODS
Table 3.3: Amino acids solution employed in the SC media. SUBSTANCE CONCENTRATION [mM] Adenine 0.083 L-Histidine 0.124 L-Arginine 0.220 L-Isoleucine 0.439 L-Leucine 0.439 L-Lysine 0.439 L-Methionine 0.257 L-Phenylalanine 0.291 L-Threonine 0.484 L-Tryptophan 0.094 L-Tyrosine 0.079 L-Valine* 0.492 Uracil 0.171 * If mentioned, L-valine was omitted from the medium (-V).
3.2 Microorganisms
3.2.1 Bacterial strains
The bacterial strains used in this work are listed in the table 3.4. E. coli strains were cultivated in LB medium at 37C, with addition of 100 μg/mL ampicillin for cultivation of strains carrying plasmids containing amp resistance marker. The other strains were cultivated in YPD at 30C. For cryopreservation of the bacterial strains, containing plasmid or not, 500 μL of the bacterial culture at stationary phase were mixed with 250 μL 50% glycerol, and afterwards stored at -80C.
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MATERIALS AND METHODS
Table 3.4: Bacterial strains employed in this work.
STRAIN SOURCE
Agrobacterium tumefaciens GV3101 Eckhard Boles Group
Clostridium acetobutylicum Eckhard Boles Group
Corynebacterium glutamicum ATCC13032 Eckhard Boles Group
Enterococcus casseliflavus ATCC12755 Eckhard Boles Group
Escherichia coli DH10B Thermo Fisher Scientific, USA
Escherichia coli NEB10 New England Biolabs (NEB), England
Lactococcus lactis MG1614 Eckhard Boles Group
Xanthomonas campestris pv. campestris Eckhard Boles Group
3.2.2 Yeast strains
The yeast strains obtained from other works and companies are listed in the table 3.5. The strains generated in this work are listed in the table 3.6. The yeast strains were recovered from the cryopreservation in YPD plates, and these plates were kept at 4C for no longer than two months. The strains transformed with plasmids were also kept at 4C for no longer than two months. For cryopreservation of the yeast strains, 500 μL 50% glycerol was mixed to 500 μL of the culture at stationary phase, and afterwards stored at -80C. The strains were stored without any plasmid. The employed antibiotics for selection of yeast transformants were G418 (200 μg/mL), hygromycin B (200 μg/mL) or nourseothricin (100 μg/mL), which correspond to the selection markers for kanMX, hphNT1 and natMX, respectively.
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MATERIALS AND METHODS
Table 3.5: Yeast strains obtained for this work. STRAIN GENOTYPE SOURCE CEN.PK113-7D MATa; MAL2-8c; SUC2 Euroscarf, Germany CEN.PK2-1C MATa; leu2-3,112; ura3-52; trp1-289; his3-Δ1; Euroscarf, Germany MAL2-8c; SUC2 ISOY12 CEN.PK2-1C; ilv5::loxP (Brat et al. 2012) EthanolRed Industrial strain Lesaffre, France HDY.GUF9 EthanolRed; ∆gal2::opt.xylA, opt.araB, opt.araD, (Dietz 2013) opt.araA; ∆pyk2::opt.araA, opt.araD, opt.araB, opt.araT, opt.TAL2, opt.TKL2, HXT7, RKI1, RPE1, TKL1, TAL1, opt.XKS1, opt.xylA; unknown mutations for growth with xylose HDY.ISO2 HDY.GUF9; Δilv2::opt.ILV2D54, opt.ILV5D49, (Dietz 2013) opt.ILV3D19; Δpdc1::ARO10, ADH2, udhA; unknown mutations for growth in minimal media HDY.GUF12 HDY.GUF9; hxt2::xylA, HXT9, araA; unknown (Dietz 2013) mutations for growth with arabinose
Table 3.6: Yeast strains created in this work. STRAIN BACKGROUND MODIFICATIONS WGY9 CEN.PK113-7D ilv3::loxP-hphNT1-loxP WGY19 CEN.PK113-7D ilv3::loxP-hphNT1-loxP WGY.CISO1 CEN.PK113-7D ilv2::- WGY.CISO1tpo2.3 WGY.CISO1 tpo2::-; tpo3::- WGY.CISO1hxt9.11.12 WGY.CISO1 hxt9::-; hxt11::-; hxt12::- WGY.CISO1yro2mrh1 WGY.CISO1 yro2::-; mrh1::- WGY.CISO1pdr12 WGY.CISO1 pdr12::- WGY.CISO1hxt5 WGY.CISO1 hxt5::- WGY.CISO1mch5 WGY.CISO1 mch5::- WGY.CISO1pdr5.15 WGY.CISO1 pdr5::-; pdr15::- WGY.CISO1qdr1.2 WGY.CISO1 qdr1::-; qdr2::- WGY.CISO1azr1 WGY.CISO1 azr1::- WGY.CISO1flr1 WGY.CISO1 flr1::- WGY.CISO1aqr1qdr1.2 WGY.CISO1 aqr1::-; qdr1::-; qdr2::- WGY.CISO1arr3 WGY.CISO1 arr3::- WGY.GISO1 HDY.GUF12 ilv2::- WGY.GISO1yro2mrh1 WGY.GISO1 yro2::-; mrh1::- WGY.GISO1hxt5 WGY.GISO1 hxt5::- WGY.GISO2.2 WGY.GISO1 pdc1::-; pdc5::-; bdh1-bdh2::ilvHm
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MATERIALS AND METHODS
STRAIN BACKGROUND MODIFICATIONS WGY.GISO2.3 WGY.GISO2.2 leu4::-; leu9::- WGY.GISO2.4 WGY.GISO2.2 bat2::- WGY.GISO2.5 WGY.GISO2.2 ecm31::- WGY.GISO2.6 WGY.GISO2.2 ald6::- WGY.GISO2.7 WGY.GISO2.2 leu1::- WGY.GISO2.35 WGY.GISO2.2 leu4::-; leu9::-; ecm31::-
3.3 Plasmids
The single-copy and multi-copy plasmids used in this work are indicated in the table 3.7 and 3.8, respectively. The plasmids were constructed with Gibson assembly or homologous recombination in yeast (see topic 3.7.3). The vectors were based on the plasmid series for single-copy (pRS41) and multi-copy plasmids (pRS42) from Euroscarf (Germany) with kanMX or natMX markers. Additionally, one plasmid without any yeast origin (pUG6-H) was used for amplification of the loxP-hphNT1-loxP sequence for gene deletions. All plasmids have amp marker for selection.
Table 3.7: Multi-copy (2-micron) plasmids employed in this work.
NAME MARKER OVEREXP. CASSETES / DESCRIPTION
General plasmid for overexpression of genes under the pRS62N natMX control of the constitutive HXT7-1,-392 promotor fragment (HXT7p) and CYC1 terminator (Farwick et al. 2014) pRS62K kanMX As pRS62N, but with the kanMX resistance marker As pRS62N, but with the resistance marker promoter pRS72N natMX exchanged by TDH3p pRS62N-Atm1 natMX pRS62N with wtATM1 pRS62N-Aft1 natMX pRS62N with wtAFT1UP [C291F]
pRS62N-Ilv319 natMX pRS62N with ILV319 pRS62N-Ilv5-P1 natMX pRS62N with ILV5P1 [G111S, K115D, A117D]
pRS62N-Ilv548 natMX pRS62N with ILV548 pRS62N-Ilv5-P4 natMX pRS62N with ILV5P4 [G111S, S113D, K115D, A117D] pRS62N-Ilv5-P5 natMX pRS62N with ILV5P5
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MATERIALS AND METHODS
NAME MARKER OVEREXP. CASSETES / DESCRIPTION
pRS62N-IlvC6E6 natMX pRS62N with ilvC6E6 from E. coli
pWG39 kanMX FBA1p-ILV254-PGK1t; HXT7p-ILV319-CYC1t
pRS62N-Ilv6-A natMX pRS62N with ILV6A [N61, C167]
pRS62N-Ilv6-B natMX pRS62N with ILV6B [N61]
pRS62N-Ilv6-D natMX pRS62N with ILV6D [N89, C167]
pRS62N-Ilv6-E natMX pRS62N with ILV6E [N89, C150] pRS62N-IlvHm natMX pRS62N with ilvHm from E. coli pRS62N-Sso_IlvD natMX pRS62N with ilvD from S. solfataricus pRS62N-Cgl_IlvD natMX pRS62N with wtilvD from C. glutamicum pRS62N-Lla_IlvD natMX pRS62N with ilvD from L. lactis pRS62N-Ncr_IlvD2 natMX pRS62N with ilvD2 from N. crassa pRS72N-D8ADB5 natMX pRS72N with wtmanD from E. coli pRS72N-A9CG74 natMX pRS72N with wtatu4196 from A. tumefaciens pRS72N-Q8P3K2 natMX pRS72N with wtxcc4069 from X. campestris pRS72N-A6M2W4 natMX pRS72N with wtcbei_4837 from C. acetobutylicum pRS72N-C9CN91 natMX pRS72N with wtecag_02205 from E. casseliflavus pRS72N-C6CBG9 natMX pRS72N with dd703_0947 from D. dadantii pRS72N-D4GJ14 natMX pRS72N with rspA from P. ananatis pRS72N-A2RAU0 natMX pRS72N with dgdA from A. niger pRS72N-G0FDF9 natMX pRS72N with wtrhmD from E. coli pRS62N-RspA natMX pRS62N with rspA from P. ananatis
pRS62K-Ilv319 kanMX pRS62K with ILV319 pRS72N-Ady2 natMX pRS72N with wtADY2 pRS72N-Jen1 natMX pRS72N with wtJEN1 pRCC-K kanMX ROX3p-cas9-CYC1t; SNR52p-gRNA-SUP4t pRCC-N natMX As pRCC-K, but with the natMX resistance marker OBS: Genes with wt were amplified directly from the microorganism genome. The other genes were amplified from synthetic DNA and had their ORFs codon-optimized.
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MATERIALS AND METHODS
Table 3.8: Single-copy (CEN/ARS) plasmids employed in this work.
NAME MARKER OVEREXP. CASSETES / DESCRIPTION
FBA1p-ILV254-PGK1t; GLK1p-ILV548-PTP3t; HXT7p-ILV319- pWG107 kanMX CYC1t; STI1p-ARO10wt-GUS1t; REH1p-ADH2wt-TUB1t
pWG108 kanMX As pWG107, but ILV548 exchanged by ilvCE6E
pWG109 kanMX As pWG108, but ILV319 exchanged by ilvD2
pWG110 kanMX As pWG107, but ILV548 exchanged by ILV5P4 pWG112 kanMX As pWG108, but ARO10 exchanged by kivD
As pWG108, but ilvC6E6 exchanged by ilvC6E6-Z2-N and ILV319 pWG133 kanMX exchanged by ILV3-Z1-C pWG134 kanMX As pWG108, but ARO10 exchanged by kdcA pWG135 kanMX As pWG133, but ARO10 exchanged by kdcA pWG136 kanMX As pWG108, but ARO10 exchanged by ARO10
pWG139 natMX FBA1p-LEU4D578Ywt-PGK1t; HXT7p-LEU3601wt-CYC1t pWG140 kanMX As pWG135, but ADH2 exchanged by adhARE1 OBS: Genes with wt were amplified directly from the microorganism genome. The other genes were amplified from synthetic DNA and had their ORFs codon-optimized.
3.4 Synthetic DNA
The employed oligonucleotides (primers) were synthesized by Biomers.net LLC., Germany. All oligonucleotides and primers are listed in the appendix B. The targeting sequence of the primers were designed for a Tm of 60C (± 2C) within a minimum length of 18 nucleotides. The majority of the synthetic genes were synthesized by GeneArt (Thermo Fisher Scientific, Germany) using the strings DNA fragments method, preferentially, or standard gene synthesis. Alternatively, synthetic genes were also synthesized as gBlocks by IDT, Belgium.
3.5 Equipment, enzymes, chemicals and kits
The equipment, enzymes, chemicals and kits employed in the work are described in the table 3.9. The materials are organized by the purchased company.
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MATERIALS AND METHODS
Table 3.9: Other materials used in this work.
COMPANY EQUIPAMENT/MATERIAL
Amsbio, UK Zymolyase Bacto yeast extract, Bacto tryptone, Difco yeast nitrogen base (w/o BD Bioscience, USA amino acids and ammonium sulfate) Beckman Coulter, Centrifuge Avanti J-25 with JA-10-Rotor USA Bio-Rad, USA Gene Pulser Electroporator Centrifuges (5415D, 5415R, 5702, 5810R), Thermo Stat plus Eppendorf, Germany Thermoblock GE healthcare, UK Ultrospec 2100 pro Spectrophotometer IKA, Germany VXR basic Vibrax, VX 2E Tube Adaptor Macherey-Nagel, NucleoSpin Extract II Germany G418, Durapore membrane filter (PVDF, hydrophilic, 0.22 µm, Merck, Germany 47 mm) Neolab, Germany Agarose Gel-electrophoresis System Restriction endonucleases, Taq DNA polymerase, Q5 High-Fidelity NEB, UK PCR Kit, Phusion High-Fidelity PCR Kit, 1 kb DNA Ladder, 100 bp DNA Ladder, 10 mM dNTP mix, NEB10 electrocompetent cells Oxoid, UK Bacteriological Peptone QIAGEN, USA RNeasy kit, Ni-NTA Superflow Roth, Germany All other chemicals, but the mentioned in another company SciQuip, UK Sensoquest Thermal Cycler G418, salmon sperm DNA (D1626), PEG-3350, Monoclonal Anti- HA antibody, TRI reagent, Anti-Mouse IgG−Alkaline Phosphatase, Sigma-Aldrich, RNAstable Tube kit, Acetoin, 2,3-butanediol, Sodium DL-2,3- Germany dihydroxyisovalerate, Sodium 2-ketoisovalerate, Methyl 2-hydroxy- 2-methyl-3-oxobutyrate NanoDrop 1000 Spectrophotometer, GeneJET Plasmid Miniprep Kit, 1-Step NBT/BCIP Substrate, Dream Taq polymerase, Dionex Thermo Fisher UltiMate 3000 (Semipreparative Autosamplers, ISO-3100SD Scientific, USA Isocratic Analytical Pump, Solvent Rack, TCC-3000SD Thermostatted Column Compartment), refractive index detector Shodex RI-101, HyperREZ XP Carbohydrate H+ 8 µm column Vilber Lourmat, E-Box VX2 (Agarose-gels), Fusion SL3 Xpress System (Western Germany Blot, Plates)
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MATERIALS AND METHODS
3.6 Molecular biology methods
3.6.1 Plasmid and DNA isolation
The isolation of plasmids (miniprep) from bacteria was performed with the GeneJET Miniprep Kit (Thermo Fisher Scientific, USA) from a 5-mL culture pellet, according to the manufacturer’s recommendations. For the isolation of genomic DNA from bacteria, the GeneJET Miniprep Kit (Thermo Fisher Scientific, USA) was employed as for miniprep, however, the lysis buffer was substituted by 0.5% (w/v) SDS. The yeast miniprep was performed employing the GeneJET Miniprep Kit (Thermo Fisher Scientific, USA), with modifications. For this, the pellet of a 5-mL overnight culture was resuspended in 250 µL resuspension buffer and mixed with 200 L glass beads (Ø 0.5 mm). The cells were vortexed for 10 min at 4C, and the miniprep proceeded as manufacturer’s recommendations. For the isolation of genomic DNA, the same procedure described before was employed, however, the lysis buffer was substituted by 0.5% (w/v) SDS. The purification of PCR and restrictions were performed with the NucleoSpin Extract II kit (Macherey-Nagel, Germany), as manufacturer’s protocol. The integrity and concentration of the isolated plasmids and DNA were determined with the NanoDrop 1000 Spectrophotometer (Thermo Fisher Scientific, USA).
3.6.2 Yeast RNA isolation
For the RNA extraction, a 15-mL fermentation pellet was transferred to a 2-ml cryotubes containing 200 µL glass beads (Ø 0.5 mm) and 1 mL TRI Reagent (Sigma-Aldrich, Germany). The sample was vortexed for 10 min at 4°C, and 200 µL chloroform were mixed to the solution. After 5 min at room temperature (RT), the sample was centrifuged at 12,000 g for 15 minutes at 4°C and the supernatant was further purified with the RNeasy kit (Qiagen, USA), following manufacturer’s instructions, but skipping the genomic DNA removal column. The integrity and quantification of the isolated RNA were assessed via agarose gel electrophoresis and with the NanoDrop 1000 Spectrophotometer (Thermo Fisher Scientific, USA). The
32
MATERIALS AND METHODS
RNA samples were shipped to Phalanx Biotech Group for the microarray analysis using the RNAstable Tube kit (Sigma-Aldrich, Germany), following manufacturer’s protocol.
3.6.3 Separation of nucleic acids fragments
Essentially, DNA and RNA fragments were separated in 1% (w/v) agarose gels, but DNA fragments smaller than 500 bp were separated in 1.5% (w/v) agarose gels. TAE buffer was employed for preparation of agarose gels and as running buffer. The 1 kb DNA Ladder (NEB, UK) and 100 bp DNA Ladder (NEB, UK) were used to determine the size of the DNA fragments. The DNA samples were mixed with 6x DNA loading dye before loading onto the agarose gel. The agarose gel run was performed at 100-150 V until the bromophenol blue line achieved 70% of the gel (typically 30-60 min). The agarose gel was incubated in an ethidium bromide bath per 15 min and the DNA fragments were visualized under UV light.
3.6.4 Polymerase chain reaction (PCR)
DNA amplicons employed for cloning were amplified with Phusion or Q5 polymerases (NEB, UK). The Tm of the primer pairs was determined using the software Clone Manager 9 (Sci-Ed, USA). The PCRs were conducted in a 50-µL reaction containing 1 ng DNA template, 0.5 U polymerase, 0.2 mM dNTPs and 0.5 µM each primer in the supplied reaction buffer. The cycling was performed with a 3-minute initial denaturation at 95°C, followed by 30 cycles of 95°C, 30 s; Tm, 30 s; and 72°C, 30 s per kb. For bacterial colony-PCR, instead of the DNA template, 1 µL of the bacterial culture was employed in the PCR reaction. Colony-PCR was employed for confirmation of the deletions (topics 3.7.4 and 3.7.5), and was performed with zymolyase-treated yeast cells. For this, a small amount of cells was suspended in 5 µL of zymolyase solution and incubated at 37°C per 30 minutes. Afterwards, the mixture was boiled for 3-5 minutes and diluted with
20 µL ddH2O. The PCRs were conducted in a 20-µL reaction containing 2 µL zymolyase-treated yeast, 0.5 U DreamTaq (Thermo Fisher Scientific, USA), 0.3 mM dNTPs and 0.5 µM each primer in the supplied green buffer. The cycling was
33
MATERIALS AND METHODS performed with a 3-min initial denaturation at 95°C, followed by 30 cycles of 95°C, 30 seconds; 53°C, 30 seconds; and 72°C, 2 minutes per kb.
3.6.5 Error-prone PCR
Conventional Taq DNA polymerase (NEB, UK) was employed for the mutagenesis method as described by Schadeweg (2013). Four different dNTP mixes (table 3.9) and four different concentrations of magnesium and manganese (table 3.10) were used to promote mistakes of the polymerase. Therefore, 16 different PCR were carried out. Each PCR was conducted in a 50-µL reaction containing 10 ng of DNA template, 1.5 U Taq DNA polymerases, 0.5 uM each primer, 1x dNTPs mix, magnesium and manganese in the supplied buffer. The cycling conditions was a 3-min initial denaturation at 95°C, followed by 30 cycles of 95°C, 30 seconds; 55°C, 30 seconds; and 72°C, 2 min per kb. The PCRs were analyzed by agarose gel electrophoresis and the positive reactions were combined and purified together.
Table 3.9: Setup of the dNTP mixes used in the epPCR.
10x dNTP dGTP [mM] dCTP [mM] dATP [mM] dTTP [mM] Mix1 2 1 2 1 Mix2 1 2 1 2 Mix3 2 2 1 1 Mix4 1 1 2 2
Table 3.9: Magnesium and manganese concentrations used in the epPCR.
CONDITION MgCl2 [mM] MnCl2 [mM] A 3 0.5 B 5 0.25 C 5 0.5 D 7 0
3.6.6 DNA Restriction
The digestion of DNA with restriction endonucleases (NEB, UK) were carried out with the supplied buffers and following the manufacturer's specifications. For the preparative restrictions, i.e. with DNA for posterior cloning, a 25-µL reaction
34
MATERIALS AND METHODS was set with 2 µg DNA and 10 units of the restriction endonucleases. The reaction was incubated in the appropriate temperature for, at least, three hours. Afterwards, the restriction enzymes were denatured and 4 µL of the reaction were analyzed by agarose gel electrophoresis. For the analytical restriction, i.e. for evaluation of plasmid assembly, a 10-µL reaction was set with 0.5 µg DNA and 5-10 units of restriction endonucleases. The reaction was incubated in the appropriate temperature for, at least, one hour and directly used for agarose gel electrophoresis.
3.6.7 Sequencing of DNA
The sequencing of DNA molecules was carried out by the GATC Biotech Inc., Germany. For this, 40-50 ng/µL DNA and 2.5 µM of the appropriate primer were combined in a 10-µL final solution. This solution was shipped to the company and the data was obtained as ab1 file, which was processed with Clone Manager 9 (Sci-Ed, USA).
3.7 Genetic methods
3.7.1 E. coli transformation
The transformation of E. coli was performed by electroporation. E. coli DH10B was employed for general plasmid propagation and the commercial E. coli NEB10 electrocompetent cells were used for transformation of the mutagenesis libraries. Electrocompetent E. coli DH10B was produced following protocol of Sambrook and Russell (2001). Briefly, E. coli was cultivated in 200 mL LB to early exponential phase [optical density at 600 nm (O.D.) 0.7] and, afterwards, the cells were washed twice with 50 mL ice-cold 10% glycerol. The pellet was resuspend in 2 mL ice-cold 10% glycerol and separated in 50-µL aliquots, which were then frozen in liquid nitrogen and stored at -80C. For the transformation, the DNA (5 µL of yeast miniprep or 2 µL Gibson assembly reaction) was mixed to the thawed competent cell aliquot and the suspension was transferred to a 1-mm electroporation cuvettes. The cuvette was pulsed (1.5 kV, 200 Ω and 25 F) and the cells were then immediately plated on LB agar plates containing ampicillin. 35
MATERIALS AND METHODS
The transformation of the commercial E. coli NEB10 electrocompetent cells (NEB, UK) was performed as manufacturer’s guideline.
3.7.2 Yeast transformation
The yeast transformations were performed by lithium-acetate chemical competence as described by Gietz and Schiestl (2007). Briefly, S. cerevisiae was cultivated in 50 mL YPD (YPEG for the PDC-null mutants) to early exponential phase (laboratorial strains: O.D. 0.6-1.0; industrial strains: O.D. 1.0-2.0) and, afterwards, the cells were washed twice with 25 mL ddH2O. The pellet was resuspend in 0.5 mL FCC solution and separated in 50-L aliquots, which were either used on time or stored at -80C. For the transformation, the thawed cells were pelleted and resuspend in 306 L Transformation Mix. DNA (500 ng of each plasmid or DNA fragment) was mixed to the suspension and the mixture was incubated at 42C for 40 or 60 min, for laboratorial and industrial strains, respectively. Afterwards, the solution was transferred to 5 mL fresh YPD or YPEG and this culture was cultivated for 2-4 hours at 30C. After regeneration, the cells were pelleted and plated on selection medium containing the resistance marker antibiotic.
3.7.3 Plasmid construction
The plasmids of this work were constructed, mainly, with the Gibson assembly method (Gibson et al. 2009). In this technique, DNA fragments with overlapping ends are subjected to an in vitro recombination-like reaction, as schematized in the figure 3.1. In order to obtain the fragments with overlapping ends, the primers used in the PCR reaction were designed with a 5’ overhang, which were designed for a Tm above 55C in a length range of 20 to 50 nucleotides. The plasmid backbones employed in the Gibson assembly were derived from the empty vector linearized by digestion at the multiple cloning site. After PCR or restriction, the DNA fragments were purified and quantified. For the Gibson assembly reaction itself, 25 ng plasmid backbone and three-fold equimolar amounts of each insert were combined in a reaction mixture containing 0.008 U T5 exonuclease, 0.05 U Phusion polymerase and 8 U Taq DNA ligase in ISO buffer. The reaction was incubated for one hour at
36
MATERIALS AND METHODS
50C and 2 L of it were then used for transformation in E. coli. The plasmids were analyzed by restriction analysis and the sequences were verified by DNA sequencing. If a plasmid construction was not possible with Gibson assembly, homologous recombination in yeast was employed as alternative (Oldenburg et al. 1997). For amplification of DNA fragments, either the same primers as that of Gibson assembly, or primers with longer 5’ overhangs (> 40-nt), were employed for the PCR. After purification and quantification of the DNA fragments, 500 ng plasmid backbone and 500 ng of each insert were used for transformation of CEN.PK113-7D. Some yeast colonies were inoculated altogether in 5 mL YPD with appropriate antibiotic and used for plasmid miniprep. Then, E. coli was transformed with 5 L of the yeast miniprep and the plasmids were isolated from single bacterial colonies and analyzed by restriction analysis and sequencing. Either by in vitro or in vivo recombination, the E. coli clones transformed with the correct plasmids were preserved in -80C. The PCRs were conducted using genomic DNA extracted from S. cerevisiae CEN.PK113-7D, E. coli DH10B and the bacteria indicated in the table 3.4; also plasmids of the laboratory and ordered synthetic genes were used as DNA template.
a b
Figure 3.1 Schematization of the plasmid assembly design (a) and Gibson assembly reactions overview (b). A, plasmid backbone is showed in black and inserts are
37
MATERIALS AND METHODS
showed in red, blue and green. B, although the Gibson assembly reactions are displayed in separate, all steps occur in parallel in an isothermal reaction.
3.7.4 Gene deletion via Cre/loxP
The deletion of ILV3 or ILV5, for the construction of the WGY9 and WGY19 strains, was performed with the Cre/loxP recombinase system (Güldener et al., 1996; Steensma et al., 2001). For this, the complete ORF of the gene of interest was exchanged by the loxP-hphNT1-loxP sequence. The deletion cassette was amplified via PCR from pUG6-H with a primer pair containing 50-bp overhangs at the 5’ terminus. The forward primer overhang contained the 50-nt downstream of the initiation codon, and the reverse primer overhang contained the 50-nt upstream of the stop codon. The unpurified PCR (20 µL) was used for yeast transformation and the transformants were verified via colony-PCR with primers targeting to the promoter and terminator of the gene of interest, and primers in the deletion cassette. Despite the possibility of exclusion of the hphNT1 gene, the marker was kept in the strain genomes, since no experiment comparing the knock-out strains and the wild types were performed.
3.7.5 Gene deletion via CRISPR/Cas9
All the other gene knock-outs were carried out with the CRISPR/Cas9 system developed in our group (Generoso et al. 2016). The selection of the protospacers was carried out with the CRISPR gRNA Design tool (DNA 2.0), for the single gene deletions, and manual sequence analysis, for protospacers used in simultaneous genes deletions. The protospacers were 20-nt long and previous an NNG PAM (protospace adjacent motif) sequence. The chosen protospacers were incorporated downstream of the guide-RNA (gRNA) sequence in the pRCC-K or pRCC-N, which also have the codon-optimized cas9 from Streptococcus pyogenesis under control of ROX3 promoter. The whole vector was amplified with primer in which the overhangs were the gene specific targeting sequence, as shown in the figure 3.2. To promote the deletion of a targeted gene, 20 µL of the unpurified PCR reaction were used for transformation of the yeast strain, together with 300 pmol of an 80-bp single-stranded oligonucleotide (Donor), which consisted of 40-nt downstream and 40-nt upstream of the gene of interest. In case of multiple simultaneous deletions,
38
MATERIALS AND METHODS
300 pmol of each Donor were transformed with the PCR reaction. After transformation, the transformants were plated on YPD (YPEG for the PDC-null strains) containing the antibiotic according to the CRISPR/Cas9 vector. The clones were screened via colony- PCR employing primers targeting to the promoter and terminator of the gene of interest. The colony with the correct gene deletion was cured from the CRISPR/Cas9 plasmid and employed for further studies.
Figure 3.2 Schematic illustration of the CRISPR/Cas9 vectors, pRCC-K and pRCC-N. The plasmids carry the cassette for expression of the gRNA and the cas9 from S. pyogenesis. Fw and Rv stand for the primers employed in the PCR for incorporation of the protospacers, displayed in orange [modified from Generoso et al. (2016)].
3.8 Growth based methods and fermentations
3.8.1 Quantification of cell density
The cell density in liquid culture was measured by the optical density at 600 nm (O.D.). The O.D. was considered just up to 0.8 units, and samples outside this range were diluted in ddH2O, as well as the media used as blank. Cuvette of polystyrene were employed and the O.D. was measured using the Ultrospec 2100 Spectrophotometer (GE Healthcare, UK).
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MATERIALS AND METHODS
3.8.2 Serial dilution spot assay
The serial dilution spot assay (drop-test) was carried out for comparison of growth among different S. cerevisiae strains. For this, the yeast strains were grown to exponential phase (O.D. 1.2-2.0) in 10-25 mL YPD with appropriate antibiotics. Afterwards, the cultures were centrifuged (4 min, 4,500 g, RT) and the pellet was washed in sterile ddH2O. The pellets were resuspended in sterile ddH2O for an O.D. 1 0 (10 ). Each of these cell suspensions, were ten-fold diluted with sterile ddH2O in a total of three successive dilutions, i.e. 10-1, 10-2, 10-3. From each dilution, 5 µL were dropped onto the agar plate with the characteristics to be evaluated (e.g. SCD-V). The plate was kept at RT until all the drops were dried, and then incubated at 30°C. After growth, the plates were photographed with the Fusion SL3 Xpress System (Vilber Lourmat, Germany).
3.8.3 Complementation of valine auxotrophy
To evaluate the activity of the ILV alternatives, valine auxotroph strains (ilv5 or ilv3) were transformed with the plasmids with the studied genes. The transformants were first selected in YPD with appropriate antibiotic, and then transferred to SCD-V plates. The plates were incubated at 30C and after growth, the plates were photographed with the Fusion SL3 Xpress System (Vilber Lourmat, Germany). To evaluate KIV and DIV uptake, valine auxotroph strains (ilv2 or ilv3) were transformed with the plasmids containing the studied genes, if applicable. The transformants were first selected in YPD containing appropriate antibiotics, and then transferred to SCEG-V containing 0.5 g/L of KIV or DIV. The plates were incubated at 30C and after growth, the plates were photographed with the Fusion SL3 Xpress System (Vilber Lourmat, Germany). The DIV employed for the plates was produced in situ from the micro-aerobic fermentation of WGY.GISO1 transformed with pWG108. The substances in the supernatant were quantified and DIV, ethanol and glycerol concentrations were adjusted for the required in the medium.
40
MATERIALS AND METHODS
3.8.4 Aerobic growth cultivations
Aerobic cultivations were employed to evaluate the growth profile and production yields of the PDC-null strains. For this, the yeast strains were grown in 25 mL YPEG at 30°C, 180 RPM, until O.D. 2-3. Afterward, the cells were pelleted
(4 min, 4,500 g, RT), washed and resuspended in YPD in order to obtain a final O.D. 1. 15 mL of this cell suspension were transferred to a 50 mL flask and the cultivation was carried out at 30°C, 180 RPM. Samples were collected periodically for O.D. and high performance liquid chromatography (HPLC) analysis.
3.8.5 Micro-aerobic fermentations
For the micro-aerobically fermentations, initially, cells were grown in 50 mL YPD at 30°C, 180 RPM, until O.D. 3-4. Afterward, the cells were pelleted and transferred to 400 mL SMD, SFD or SCD medium at 30°C, 180 RPM, until O.D. 3-5. After the growth, the culture was pelleted at 5,000 g per 15 min, RT, suspended in water and pelleted once more. The pellet was resuspended in 50-100 mL SMD, SFD or SCD in order to have a final O.D. 8. The cultures were transferred to 125 mL flasks with a S-shaped Airlock to access the micro-aerobic conditions. The fermentation was carried out at 30°C in magnetic shaker and samples were collected periodically for HPLC analysis.
3.8.6 Evolutionary engineering
The evolutionary engineering was carried out by sequential batch cultivations. Initially, a pre-inoculum was grown in YPEG and 1 mL of it was transferred into 50 mL YPD. The culture was kept in aerobic conditions and when in stationary phase, 200 µL of this cultures were used as inoculum in 50 mL fresh YPD. This process was repeated for 5 consecutive new cultures, every time the culture reached stationary phase. At the sixth culture, the medium was changed to SMD and, again, there were five consecutive new cultures. At the final stage, some clones were isolated and analyzed.
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MATERIALS AND METHODS
3.9 Protein methods
3.9.1 Enzyme assay
For the analysis of enzymatic activity, first, the soluble protein extract was purified from the culture pellets. The pellet of 50-ml cultures in exponential phase (O.D. 2-4) was washed with the extraction buffer and, afterwards, resuspended in 400 µL extraction buffer and 200 µL glass beads (Ø 0.5 mm). The mixture was vortexed for 10 min, 4°C, and then centrifuged at 16,000 g for 5 min, 4°C. The supernatant (the crude extract) was reserved and used for quantification and the assay. The total protein concentration in the extract was estimated with Roti-Quant (Roth, Germany) using bovine serum albumin as standard. The Ilv5 specific activity was assayed in a 100-µL reaction containing 0.25 mM NADH or NADPH, 0.75 mM 2-acetolactate and 1 to 20 µg of protein crude extract in the same extraction buffer. The mixture, without 2-acetolactate, was pre-incubated for 5 minutes at 30°C and the substrate was used to initiate the conversion. The specific activity was quantified by the NADH and NADPH absorbance at 340 nm. The 2-acetolactate substrate was produced in situ by the saponification of methyl 2- hydroxy-2-methyl-3-oxobutyrate with equimolar amount of sodium hydroxide (2 h, RT).
3.9.2 Pull-down assay
The pull-down assay was performed via affinity chromatography in nickel column. For this, the proteins with putative interaction were co-overexpressed in yeast and the soluble protein extract from the culture pellets were employed for the affinity chromatography. In case, one of the proteins (Ilv3) had a N-terminal poly-histidine (HT) tag, for nickel affinity, and the other protein (IlvC6E6) had a human influenza hemagglutinin (HA) tag at C-terminus, for posterior Western blot analysis. For the affinity chromatography, the crude extracts of cells in exponential phase (O.D. 2-4) were employed. For this, 250-mL culture pellets were first washed with the resuspension buffer and then resuspended in 1 mL resuspension buffer, divided into two 2-mL tubes. To each tube, 200 µL glass beads (Ø 0.5 mm) were added and the sample was vortexed for 10 min at 4°C. The mixtures were centrifuged at
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MATERIALS AND METHODS
16,000 g for 5 min, 4°C, and the supernatant (the crude protein extract) was reserved for the pull-down analysis. The nickel affinity chromatography was performed within 1-mL Ni-NTA Superflow resin (Qiagen, USA). Initially, the resin was pre-equilibrated with 25 mL washing buffer and then the protein crude extract was loaded through the resin. The loaded-resin was washed with 25 mL washing buffer and the proteins were eluted with 2 mL elution buffer (twice 1 mL). All protein fractions were recovered and employed, on time, for Western blot analysis.
3.9.4 Western Blot
Initially, the proteins were separated in denaturizing polyacrylamide gel electrophoresis (SDS-PAGE) with 12% (w/v) acrylamide in PAGE running buffer with 0.1% (w/v) SDS (Sambrook et al. 2001). Before loading on the SDS-PAGE, 100 µL sample were mixed with 50 µL 2x protein loading dye and incubated at 95C for 5 min. The SDS-PAGE was loaded with 10 µL of each sample and 5 µL PageRuler prestained protein ladder, and ran at 120 V until the bromophenol blue line got off of the gel (typically 90 min). For the Western blot (Sambrook et al. 2001), the proteins in the resolution gel were transferred to a PVDF membrane via semi-dry blotting (2V per cm2) for 1.5 h, in PAGE running buffer with 20% (v/v) methanol. Afterwards, the membrane was saturated in blocking buffer for 45 min and incubated in TBS-T buffer with the primary anti-body (anti-HA-tag, 1:2,000) at 4C overnight. The membrane was washed six- times (5 min) with TBS-T buffer and incubated in TBS-T buffer with the secondary anti- body (anti-Mouse IgG−Alkaline Phosphatase, 1:10,000) at RT for 1 h. The Western blot was revealed with 2 mL NBT/BCIP (Thermo Scientifc, USA) until the bands could be visualized (typically 5 min). The membrane was dried overnight and photographed with the Fusion SL3 Xpress System (Vilber Lourmat, Germany).
3.10 Metabolite analysis via HPLC
Before the metabolites quantification in HPLC, 450 µL of the culture supernatant were mixed with 50 µL 50% 5-sulfosalicilic acid. After 5 min incubation on ice, the solution was centrifuged at 16.000 g per 5 min, 4°C. The supernatant was 43
MATERIALS AND METHODS recovered and from this, 10 µL were subjected to HPLC (Dionex Ultimate 3000, Thermo Fisher Scientific, USA) with the ionic exchange column HyperREZ XP Carbohydrate H+ (Thermo Fisher Scientific, USA). The liquid-phase was 5 mM sulfuric acid with 0.6 ml/min flux at 65°C. The metabolites were quantified from standard curves and they were glucose, ethanol, glycerol, acetate, acetoin, 2,3-butanediol, 2,3- dihydroxi-isovalerate, 2-ketoisovalerate, isobutanal and isobutanol. Chromeleon 6.8 (Thermo Fisher Scientific, USA) was employed for the chromatogram manipulations and quantifications.
3.11 In silico methods
3.11.1 Bioinformatics analysis
DNA and protein alignments were conducted using Multalin (Corpet 1988) and the aligment figures were generated with ESPript (Robert and Gouet 2014). The mitochondrial targeting sequences were predicted using SignalP (Petersen et al. 2011) and by sequence comparison with bacterial orthologous. The codon-optimization of gene ORFs were carried out according to the yeast glycolytic codon usage (Wiedemann and Boles 2008) and the codon adaptation index (CAI) were evaluated with the Jcat software (Grote et al. 2005). The protein structures were obtained from the RCSB Protein Database (PDB) and the in silico analysis and manipulations were performed with the Maestro software (Schrödinger, USA).
3.11.2 Plasmids and primers design
The vectors and primers were designed with Clone Manager 9 (Sci-Ed, USA). The plasmids were planned were planned based on the plasmid maps of the series for single-copy (pRS41) and multi-copy plasmids (pRS61) from Euroscarf, Germany. The promoters and terminators were selected according to Tochigi et al. (2010), and the sequence length were stipulated by analysis of transcription factor binding sites indicated in the Saccharomyces Genome Database. The sequences were grounded by the Saccharomyces Genome, UniProt and NCBI databases.
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MATERIALS AND METHODS
The primers were designed for a Tm of 60C ( 2C) with a minimum length of 18 nucleotides. Furthermore, the primers were designed for a C or G at the 3’ end, and a 5’ overhang smaller than 50 nucleotides, for primers used in cloning.
3.11.3 Statistical analysis
Prism 5 (GraphPad, USA) was employed for the statistical analysis and exhibition of the results. The results were presented as means with standard deviation among the replicates. The significance of the results of the independent replicates were analyzed by Student’s T-test, employing 0.05 of significance, between the control and each of the conditions. One-way analysis of variation, with Turkey post-test and employing 0.05 of significance, was the employed method when the control and the conditions were analyzed all against each other (e.g. figure 4.33). The differences were discriminated as statistically significant and not statistically significant, with p<0.05 and p>0.05, respectively. For sake of figure clearness, asterisk (*) was employed either to display means with significant differences or without significant differences, and the option of presentation is described in the figure legend.
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4. RESULTS
4.1 Isobutanol biosynthetic pathway incorporation
In previous research of our laboratory, the strain HDY.GUF9 (see topic 2.2.1) was used for the production of isobutanol. The strain HDY.GUF9 is derived from the industrial strain EthanolRed, which was adapted for xylose consumption (Dietz, 2013). In this strain, initially, both native ILV2 alleles were substituted by cassettes for overexpression of the N-terminally truncated Ilv2, Ilv5 and Ilv3 described by Brat et al. (2012). This strain presented slow growth in media without valine, therefore, the growth was improved by evolutionary engineering using minimal medium. Afterwards, one PDC1 allele was substituted by the cassettes for overexpression of Aro10 and Adh2, and the bacterial UdhA (pyrimidine transhydrogenase) to enable conversion of NADH into NADPH. This strain was named HDY.ISO2 and in anaerobic fermentation using glucose and xylose as carbon sources, isobutanol was produced in significant yields
(9.3 mg/gglu and 13 mg/gxyl); however, a large amount of DIV was found in the culture medium, while KIV and isobutanal could not be detected (Dietz, 2013). Although HDY.ISO2 could already produce significant amounts of isobutanol, this strain exhibited some instabilities regarding cultivation and fermentation profiles, which were also observed in the HDY.GUF9. For this reason, the strain HDY.GUF12, which derives from HDY.GUF9 and exhibits better stability, was employed for production of isobutanol in this work. In our case, the employed strategy was the simple deletion of both ILV2 alleles and expression of the isobutanol pathway genes from a single centromeric plasmid. The HDY.GUF12 with ILV2 deletion was named WGY.GISO1 and the plasmid created (pWG107) contained cassettes for overexpression of Ilv2Δ54, Ilv5Δ48, Ilv3Δ19, Aro10 and Adh2. Nevertheless, even after the construction of WGY.GISO1, HDY.ISO2 was still employed in some of the following experiments. Furthermore, before the any experiment with the strain WGY.GISO1 carrying pWG107, some facts about the pathway were investigated, as described below. The following experiments were performed in order to understand the bottlenecks and to find possible solutions, and the development of technologies for a strain with reduction of the ethanol production (∆pdc1 and ∆pdc5). Also, the
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RESULTS experiments were performed only with glucose, since the strain already exhibits an efficient xylose consumption profile and the bottlenecks are independent of the sugar consumption pathway.
4.1.1 Exchange of the co-factor specificity for Ilv5
A problem in the native pathway for isobutanol production is the cofactor imbalance created by Ilv5, which needs NADPH as a cofactor, whereas NADH is generated during the glycolysis. In E. coli, the cofactor specificity exchange of IlvC was important for achieving 100% of isobutanol production yield (Bastian et al. 2011). Therefore, Ilv5∆48 was employed for mutagenesis, in order to exchange its cofactor specificity for NADH. As Ilv5 has no solved crystal structure, the sequence of the E. coli IlvC6E6, the NADH-dependent mutant characterized by Bastian et al. (2011), was used as template for site-direct mutagenesis. The exact mutated amino acids of IlvC6E6 were not found in Ilv5∆48; therefore, two mutants were generated: Ilv5P1, which contained the mutations G111S, K115D and A117D; and Ilv5P4, with G111S, S113D, K115D and A117D (figure 4.1). Additionally, a Ilv5∆48/IlvC6E6 chimera (named Ilv5P5) was created by the exchange of the NADPH-binding region of Ilv5∆48 by the IlvC6E6 NADH-binding sequence (figure 4.1). IlvC6E6 and Ilv5∆48 were also used as controls. The sequences, all with codon optimization and without signal peptide, were cloned into pRS62N and the recombinant plasmids were used for complementation of the valine auxotrophy of WGY19 (figure 4.2). For the growth tests, together with the plasmid containing Ilv5∆48 (and alternatives), another plasmid (pWG39) was co-transformed into WGY19 for overexpression of Ilv2∆54 and Ilv3∆19, since 2-acetolactate and DIV transport across the mitochondrial membrane seemed to be limited. The spot assay showed a superior performance of IlvC6E6 for growth, while the chimera (Ilv5P5) was practically inactive. Ilv5P1 and Ilv5P4 were able to promote some growth, but, very poorly if compared to the control or IlvC6E6. To further investigate the changes in the cofactor specificity, enzyme assays were conducted to determine the specific activity using NADPH or NADH, and the crude extract of WGY19 transformed with pRS62N containing the Ilv5 substitutes (figure 4.2). IlvC6E6 showed the highest activity with NADH as cofactor, as well as the
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RESULTS highest NADH/NADPH ratio (figure 4.2). Ilv5P4 also had a slight change in the cofactor specificity, but with a significant decrease of the specific activity.
Figure 4.1 Sequence comparison of Iv5 wildtype and mutants from S. cerevisiae with IlvC and IlvC6E6 from E. coli. Dark boxes indicate identity amino acids identical and arrows indicate the mutation sites employed in this work.
Figure 4.2 Growth assay for evaluation of the valine auxotrophy complementation and specific activity of the Ilv5 variants. The serial dilution test was performed with WGY19 transformed with pWG39 and pRS62N containing the Ilv5 variants. The 0 transformants were plated in SCD without valine. 10 stands for O.D.600nm 1. Image registered after five days of incubation. For the enzyme assay, WGY19 was transformed with pRS62N containing the Ilv5 variants and grown in YPD. mU.mg-1 stands for the specific activity in mmol.min-1.mg-1 and SD stands for standard deviation among the three replicates.
Afterwards, fermentative tests were performed to investigate the benefits of Ilv5P4 and IlvC6E6 for the isobutanol production. For this, two plasmids were constructed from pWG107: pWG108 and pWG110, in which ILV548 was exchanged by ilvC6E6 and ILV5P4, respectively. The WGY.GISO1 strain was transformed with pWG107, pWG108 and pWG110 and the transformants were employed for micro-aerobic fermentations. As can be seen in the figure 4.3, isobutanol and DIV were not significantly improved by the use of Ilv5P4 and IlvC6E6, but both variants produced less acetoin and 2,3-butanediol. Acetate and glycerol yields were also reduced, which could be related to the rebalance of the redox relation. Nevertheless, no great improvement
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RESULTS was observed in the isobutanol production itself, as Ilv5 does not seems to be the limiting step in the pathway. IlvC6E6 was therefore employed in some further experiments. Part of these results were generated during supervision of the bachelor thesis of Lisa Eberle (Eberle, 2014).
Figure 4.3 Yields of the micro-aerobic fermentation of WGY.GISO1 overexpressing the genes of isobutanol pathway, varying Ilv5. The fermentations were conducted in SMD with 4% glucose and the yields were calculated at the end of glucose consumption. The presented metabolite bars display the average and error bars display the standard deviation among the three replicates. Asterisk displays mean with significant difference (p<0.05) with the control (Ilv5). 23BD stands for 2,3-butanediol.
4.1.2 Minimization of Ilv6 inhibition
In order to enhance the flux of pyruvate into the isobutanol pathway, Ilv6 was employed to increase Ilv2 activity. However, as the final plan is to have a strain as robust as possible, the simple overexpression of Ilv6 would bring difficulties related to the valine inhibition, if fermentations are carried out with complex media. Thus, different truncated ILV6 mutants and an E. coli truncated ilvH were investigated, aiming to achieve just the enhancement of Ilv2 activity. The truncations for ILV6 were adopted according to sequence analysis and the literature: ∆N1-61, exclusion of signal peptide; ∆N62-89 and ∆C167-310, reduced valine sensibility from IlvH (Slutzker et al. 2011); ∆C150-310, further reduction of valine sensibility of IlvH (Zhao et al. 2013). The truncated ILV6 mutants (listed in the table 4.1) and the minimum activation peptide derived of ilvH (ilvHm) described by Zhao et al. (2013) were cloned in pRS62N (with codon optimized sequences) and evaluated in fermentative experiments.
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Table 4.1 Truncated ILV6 variants. Truncation amino acids are referenced to the wild type. Mutant N-truncation C-truncation Length (aa) ILV6A ∆1-61 ∆167-310 106 ILV6B ∆1-61 - 249 ILV6D ∆1-89 ∆167-310 78 ILV6E ∆1-89 ∆150-310 61
An initial analysis was performed with Ilv6A, Ilv6B and Ilv6D, in order to investigate whether a still high valine sensibility was present. For this, the truncated isoforms were overexpressed in the strain HDY.ISO2 and employed for fermentation in minimal medium. The aim was to observe a block in isobutanol biosynthesis by the natural valine production by the cell, which would reduce the final yield of the intermediates with concomitant increase of ethanol yield. The mutant Ilv6D was only to show significant changes in the yields (figure 4.4), which is why Ilv6A and Ilv6B were excluded of the next experiments. Ilv6E and IlvHm was not employed in this test.
Figure 4.4 Relative yields of the micro-aerobic fermentation of HDY.ISO2-G overexpressing the Ilv6 mutants. The yields were normalized to results of the strain containing the empty pRS62N vector. The fermentations were conducted in SF with 4% glucose and the yields were calculated at the end of glucose consumption. The metabolite bars display the average and error bars display the standard deviation among the three replicates. Asterisk displays mean with significant difference (p<0.05) with the control (Empty). Acn, 23Bd and Iso stand for acetoin, 2,3-butanediol and isobutanol, respectively.
In a second approach, ILV6D, ILV6E and ilvHm plasmids together with pWG107 were used for transformation of WGY.GISO1. The empty pRS62N with pWG107 was used as control. These transformants were analyzed in fermentations with minimal medium (SMD), complex medium (YPD) and minimal medium supplemented with 0.5 mM valine (SMD+V). As can be seen in figure 4.5, Ilv6D could enhance Ilv2 activity in minimal medium, but still demonstrated valine inhibition. Ilv6E demonstrated just a bad influence in Ilv2 in every case. IlvHm showed an interesting result, the isobutanol production was higher with YPD than the control, although the 50
RESULTS sum of the pathway intermediates where not the highest (figure 4.6). However, IlvHm was just employed in the strain without PDC activity (see topic 4.6).
Figure 4.5 Relative yields of the micro-aerobic fermentation with WGY.GISO1 overexpressing the genes of isobutanol pathway and the Ilv6 mutants. Values for ethanol (a) and the sum of acetoin, 2,3-butanediol, DIV and isobutanol (b). The employed media were SMD, SMD with 0.5 mM valine, and YPD, all with 4% glucose. The yields were calculated at the end of glucose consumption and compared to the results of the strain containing the empty pRS62N vector in the fermentation performed with SMD. The metabolite bars display the average and error bars display the standard deviation among the three replicates. Asterisk displays mean without significant difference (p>0.05) with the control (Empty).
Figure 4.6 Relative isobutanol yields (a) and ratio between isobutanol and ethanol (b) of the micro-aerobic fermentation of WGY.GISO1 overexpressing the genes of isobutanol pathway and the Ilv6 mutants. The employed media were SMD, SMD with 0.5 mM valine, and YPD, all with 4% glucose. The yields were calculated at the end of glucose consumption and compared to the results of the strain containing the empty pRS62N vector in the fermentation performed with SMD. The metabolite bars display the average and error bars display the standard deviation among the three replicates. All means demonstrated significant difference (p<0.05) with the control (Empty).
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4.2 Investigation of limitations in Ilv3
As mentioned before, the always large amount of DIV in the culture medium, led us to suggest Ilv3 as the main bottleneck in the metabolic pathway. This enzyme is a member of the IlvD_EDD family, which is known to include proteins containing iron-sulfur cluster (FeS). The synthesis of FeS is very well regulated in the cell and FeS absence in the protein leads to inactivity. Therefore, two stratagies were investigated to enhance Ilv3 activity: fostering of cytosolic iron-sulfur cluster assembly, and use of a heterologous Ilv3 alternative.
4.2.1 Intensification of cytosolic iron-sulfur cluster assembly
FeS biosynthesis has been deeply studied in yeast, but remains still an unclear field (Lill and Mühlenhoff 2006; 2008; Netz et al. 2013). In eukaryotes, it is known that FeS are build inside the mitochondria and then transferred into the cytoplasm to be incorporated into proteins (Sharma et al., 2010). Also, FeS biosynthesis, and the iron homeostasis itself, tend to be very well regulated by the cell, due to the high toxicity of iron in the cytosol (Ihrig et al. 2010; Li and Kaplan 2004). Therefore, in order to increase the availability of FeS to Ilv3, a FeS transporter (Atm1), and a key gene for iron homeostasis (Aft1) were investigated. Atm1 is an important transporter for the assembly of the cytoplasmic FeS, because it is responsible for the transference of FeS from mitochondria into the cytoplasm (Sipos et al. 2002). Thus, we expected that the overexpression of the carrier could increase the number of functional Ilv3∆19. AFT1 encodes a transcription factor that controls the external iron uptake and intracellular iron homeostasis. However, Aft1 has a post-translational regulation, requiring the mutation C291F to stay in the constitutively active form, so called Aft1up (Miao et al. 2011; Yamaguchi-Iwai et al. 1995). Consequently, with the overexpression of the transcription factor, it was expected that the intracellular iron availability would be increased and enhance the FeS assembly. Both genes (ATM1 and AFT1up) were cloned into pRS62N and the plasmids used for transformed of HDY.ISO2. In addition, as a high expression of Aft1up could lead to problems of iron toxicity, the ORF was also cloned under the control of a weaker promoter (GAL2p). The transformants were employed for micro-aerobic fermentations
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RESULTS and, in order to assess some possible performance abnormality related to iron homeostasis, the fermentations were monitored periodically (figures 4.7, 4.8, 4.9 and 4.10). Interestingly, up to 96 hours, the glucose was not completely consumed by the strains overexpressing the studied genes; however, there was alteration of the fermentative pattern. Nevertheless, none of the combinations tested improved isobutanol production. This can be clearly observed in the isobutanol yield obtained with the fermentation tests (figure 4.11), since the control produced more isobutanol than the transformants with the investigated genes. This result demonstrates that if there was a higher amount of FeS in the cytosol, it was not transferred to Ilv3∆19 and did not helped the pathway bottleneck.
Figure 4.7 Micro-aerobic fermentation of HDY.ISO2 with overexpression of Atm1. The fermentations were conducted in SF with 4% glucose. The analyzed metabolites are presented in the legend and their concentrations are displayed on the y-axis.
Figure 4.8 Micro-aerobic fermentation of HDY.ISO2 with overexpression of Aft1up under control of HXT7 promoter. The fermentations were conducted in SF with 4% glucose. The analyzed metabolites are presented in the legend and their concentrations are displayed on the y-axis. 53
RESULTS
Figure 4.9 Micro-aerobic fermentation of HDY.ISO2 with overexpression of Aft1up under control of GAL2 promoter. The fermentations were conducted in SF with 4% glucose. The analyzed metabolites are presented in the legend and their concentrations are displayed on the y-axis.
Figure 4.10 Micro-aerobic fermentation of HDY.ISO2 with empty vector. The fermentations were conducted in SF with 4% glucose. The analyzed metabolites are presented in the legend and their concentrations are displayed on the y-axis.
Figure 4.11 Relative isobutanol yields of the micro-aerobic fermentation of HDY.ISO2 with overexpression of Atm1 and Aft1up. The employed medium was SF with 4% glucose. The yields were calculated at the end of glucose consumption and compared to the results of the strain containing the empty vector. H-AFT1 and G- AFT1 represents AFT1up under control of HXT7 and GAL2 promoters, respectively. Bars display the average and error bars display the standard deviation among the two replicates. Asterisk displays mean without significant difference (p>0.05) with the control (empty vector). 54
RESULTS
4.2.2 Overexpression of heterologous ILV3 orthologous
As the increase of Ilv3∆19 activity was not possible via stimulation of FeS assembly, some interesting heterologous genes orthologous to ILV3 were tested (table 4.2). IlvD from Sulfolobus solfataricus was selected for being characterized as possessing no FeS, or at least a very stable one (Kim and Lee 2006). IlvD from Lactococcus lactis was characterized for possessing [2Fe-2S] iron sulfur cluster (Flit et al. 2010). IlvD from C. glutamicum was chosen because of the industrial use of this microorganism for production of BCAAs. And IlvD2 from Neurospora crassa due to its native cytoplasmic localization (Altmiller and Wagner 1970; Dundon et al. 2011). Therefore, it was anticipated that these enzymes could present advantages compared to the cytosolic relocated Ilv3.
Table 4.2 Heterologous ilvD genes employed as Ilv3 substitutes. Gene Organism NCBI access Sso_IlvD Sulfolobus solfataricus WP_009990927 Lla_IlvD Lactococcus lactis AAB81918 Cgl_IlvD Corynebacterium glutamicum WP_003854128 Ncr_IlvD2 Neurospora crassa XP_963045
For the screening of Ilv3 activity, the valine auxotroph strain WGY9 was employed. The heterologous genes were cloned into pRS62N. The sequences for Sso_IlvD, Lla_IlvD and Ncr_IlvD2 were codon optimized. The empty vector and ILV3∆19 were employed as negative and positive control, respectively. The recombinant plamids were used for WGY9 transformation, in order to investigate the ability of complementing the valine auxotrophy. As can be seen in figure 4.12, only the strains overexpressing Cgl_IlvD and Ncr_IlvD2 could restore growth in media without valine. To further investigate Cgl_IlvD and Ncr_IlvD2, their vectors were transformed into HDY.ISO2, as well as Ilv3∆19 and the empty pRS62N, as positive and negative controls. The transformants were employed for fermentation, but no improvement was observed with the heterologous genes (figure 4.13). Nevertheless, no clear difference between Ilv3∆19 and Ncr_IlvD2 was able to be distinguished; therefore, both genes were further investigated in another background. For this, the plasmids pWG108 and pWG109, which contain the enzymes for the isobutanol biosynthetic pathway varying Ilv3∆19 or Ncr_IlvD2 (respectively), were used for transformation of WGY.GISO1 and the transformants were employed in fermentation 55
RESULTS with minimal medium. As observed in figure 4.14, Ilv3∆19 or Ncr_IlvD2 provided nearly the same DIV and isobutanol yields. For this reason, Ilv3∆19 was still employed in our biosynthetic pathway.
Figure 4.12 Valine auxotrophy complementation test with WGY9 with overexpression of the Ilv3 alternatives. The transformants were grown first in YPD, then shift to SCD without valine. Image registered after three days of incubation. C+ and C- stand for WGY9 overexpressing Ilv3∆19 and with empty vector, respectively. Sso, Lla, Cgl and Ncr represent the specie name of the Ilv3 alternatives.
Figure 4.13 Relative isobutanol yields of the micro-aerobic fermentation of HDY.ISO2 with additional overexpression of the Ilv3 alternatives. The employed medium was SF with 4% glucose. The yields were calculated at the end of glucose consumption and compared to the results of the strain with the empty vector (control). Bars display the average and error bars display the standard deviation among the three replicates. Asterisk displays mean with significant difference (p<0.05) with the control (empty vector).
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Figure 4.14 Yields of the micro-aerobic fermentation of WGY.GISO1 overexpression the genes of isobutanol pathway, varying Ilv3. The fermentations were conducted in SMD with 4% glucose and the yields were calculated at the end of glucose consumption. The presented metabolite bars display the average and error bars display the standard deviation among the three replicates. Experiments showed no significant difference within the metabolite means (p>0.05).
4.3 Bioprospection of dehydratases with Ilv3 activity
As none of the options tested before could enhance isobutanol production, some dehydratases were investigated as possible substitutes for Ilv3. These studied dehydratases are part of a group of the enolases that catalyzes the dehydration of saccharides with a carboxylic group (e.g. mannonate or gluconate). These enzymes act in the alpha-carbon of the substrate and require, instead of FeS, a metallic cation (predominantly Mg2+), for stabilization of the intermediate compound (Wichelecki et al. 2014a; Wichelecki et al. 2014b). Therefore, as the same reaction happens with DIV to generate keto-isovalerate, one of these enolases might substitute Ilv3, and would not present additional limitations, such as the limitations of the FeS biosynthesis. The enolase sequences were selected from literature and databases, and their annotation codes are shown in the table 4.3. The acid-sugar enolases were cloned into pRS72N and used for evaluation of the complementation of the valine auxotrophy transformed with WGY9. Three types of enolases were selected: some have higher activity for one specific substrate; others are promiscuous, acting on more than one substrate; and a third group, which the main substrate has not being characterized, but has sequence similarity. Eight of the sequences are from mesophilic bacteria and one is from Aspergillus niger. The figure 4.15 presents their phylogenetic relationship.
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Table 4.3 Enolases employed as Ilv3 substitutes. Substrate Organism Uniprot Unknown Clostridium acetobutylicum A6M2W4 Unknown Enterococcus casseliflavus C9CN91 D-galactonate/D-mannonate Dickeya dadantii C6CBG9 D-galactonate Pantoea ananatis D4GJ14 D-mannonate Escherichia coli D8ADB5 D-galactonate Aspergillus niger A2RAU0 D-galactonate Agrobacterium tumefaciens A9CG74 L-rhamnonate Escherichia coli G0FDF9 L-fuconate Xanthomonas campestris Q8P3K2
Figure 4.15 Phylogenetic relationship of the enolase amino acid sequences applied in this study. The sequence alignments were performed using ClustalW, and the phylogenetic trees were constructed using neighbor-joining method. The sequence names are displayed as the initials of the specie name followed by the UniProt code.
Among the studied enolases, the D-galactonate dehydratase (RspA) from Pantoea ananatis was the only enzyme able to restore the valine auxotrophy, but only allowing a very slow growth of the WGY9 strain (figure 4.16). With this results, we decided to focus on rspA and employ mutagenesis approaches to enhance its activity towards DIV. The mutagenesis was carried out by site-directed mutagenesis in hotspots of the active site and random mutagenesis.
Figure 4.16 Valine auxotrophy complementation test with WGY9 overexpressing RspA from P. ananatis. After transformation, the cells were directly plated on SCD without valine. Image registered after 45 days of incubation. Empty vector stands for the empty pRS72N. 58
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4.3.1 Site-directed mutagenesis of rspA
RspA has elucidated structure, therefore we analyzed it and looked for interesting amino acids to favor the dehydratase activity towards DIV. RspA structure is submitted in the RSCB protein database under entry 3T6C and was crystalized together with the substrate, D-galactonate, and Mg2+. As can be seem in the figure 4.17, the protein possesses a cavity where the reaction occurs. Analyzing this active site by changing the substrate structure from D-galactonate to DIV (figures 4.18 and 4.19), we observed that the amino acids His225, His235 and Asp329, which are responsible to direct D-galactonate during activity, could interfere in DIV access, because of DIV’s hydrophobic branch. For this reason, these three positions were employed for saturated mutations, since we could not preview which would be the best amino acid choice.
Figure 4.17 Active site cavity and substrate surface representation of RspA from Pantoea ananatis. Gray surface displays active site cavity of the enzyme and blue surface presents D-gluconate surface.
For the mutagenesis, first the rspA ORF was subcloned into pRS62N and, then, the recombinant plasmid was re-amplified with degenerated primers, which were designed with NNK bases substituting the codon triplets for the amino acids His225, His235 and Asp329. The NNK code allows that all amino acid codons can occur in the library. The amplicons containing the mutations were assembled with Gibson assembly and further transformation in E. coli. The pre-assembled plasmids were then transformed into WGY9 and screened for the ability of complement valine auxotrophy in SCD-V medium. The library derived from 17.942 CFU E. coli, which were transformed into WGY9. However, out of 720 CFU screened yeast transformants, no colony was observed in the SCD-V medium. These results were generated during
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RESULTS supervision of Navadurka Navabalasingam in her master thesis (Navabalasingam, 2016).
Figure 4.18 Structural features of the active site of RasA connected with D-gluconate. The four amino acids responsible for the substrate redirection are labelled: Gln43. His225, His325 and Asp329. The substrate is shown in red and light blue.
Figure 4.19 Structural features of the active site of RasA connected with Dihydroxy- isovalerate. The four amino acids responsible for the original substrate redirection are labelled: Gln43. His225, His325 and Asp329. DIV is shown in red and light blue.
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4.3.2 Randon mutagenesis of RspA
In parallel to the site-directed mutagenesis, rspA was used in a random mutagenesis approach. The random mutagenesis methods are interesting protein engineering tools, because they generate mutations through the whole ORF, different combinations and unforeseen point-mutation. However, these methods can easily lead to inactivity of the enzyme or be silent. The method employed was error-prone PCR (epPCR) and it bases on the reduction of the Taq DNA polymerase fidelity by modifications in the reaction composition, i.e. addition of manganese or use of imbalanced dNTP mixes to disturb the polymerase fidelity (Cadwell and Joyce 1992). In our approach, we employed four different concentrations of magnesium and manganese, 4.5 to 7 mM and 0 to 0.5 mM, respectively; and four different dNTP mixes (see topic 3.6.5). Accordingly, the rasA ORF was amplified in the epPCR conditions and subcloned in pRS62N via Gibson assembly and transformated in E. coli. The pre- assembled plasmids were transformed into WGY9 and screened for the ability of restoring valine auxotrophy in SCD-V medium. The epPCR library derived from 18,072 CFU E. coli, which were transformed into the valine auxotroph yeast. However, out of 6,254 CFU yeast transformants, no colony was observed in the SCD-V medium. These results were generated during supervision of Navadurka Navabalasingam in her master thesis (Navabalasingam, 2016).
4.4 Dihydroxy-isovalerate uptake evaluation
As no improvement was obtained with substitution of Ilv3 or enhancement of its activity, other possible reasons that could further impair isobutanol production were explored. One interesting fact observed in the isobutanol production assays was that the high concentration of DIV produced during glucose consumption remained unchanged afterwards. In other words, even if not efficiently, some consumption of DIV should occur after a while, what does not happen. Therefore, the ability of uptake DIV from the media was further investigated. For this, WGY.GISO1 carrying the plasmid pRS62N-ILV3∆19 was plated in SCEG-V supplemented with DIV or KIV. WGY9 and CEN.PK113-7D were employed as negative and positive controls, respectively. As shown in the figure 4.20, WGY.GISO1 could not grow in medium supplemented with
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DIV. Unexpectedly, the KIV uptake was not efficient either, allowing a limited growth of WGY.GISO1 (figure 4.20b).
Figure 4.20 Valine auxotrophy complementation test using DIV (a) and KIV (b). YPD was used as control (c). Images registered after three days of incubation. The following strains were employed: 1, WGY.GISO1 overexpressing Ilv319; 2, WGY9; and 3, CEN.PK113-7D. The transformants were grown first in YPD, then shift to the plates containing DIV or KIV.
Our first attempt to circumvent the DIV waste was to enable DIV re-uptake from the medium. For this, Jen1 and Ady2 were investigated for the transport of DIV. Jen1 is a membrane symporter with ability to transport monocarboxylic acids, e.g. pyruvate, acetate and lactate (Casal et al. 1999; Paiva et al. 2013). Ady2 is also a monocarboxylate permease involved in the transport of acetate, propionate, formate and lactate (Pacheco et al. 2012; Paiva et al. 2004). Therefore, as DIV is also a monocarboxylic acid, both genes could promote the ability of uptake DIV from the medium. For this, both ORFs were cloned into pRS72N, and then they were
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RESULTS transformed into WGY.GISO1, which already was carrying the plasmid pRS62K-ILV3∆19. The transformants were plated in SCEG-V supplemented with DIV or KIV, and YPD for control. WGY.GISO1 transformed with pRS62K-ILV3∆19 and the empty pRS72N was employed as negative control. As can be seem in figure 4.21, neither Jen1 nor Ady2 could promote DIV uptake. Surprisingly, Jen1 improved KIV uptake expressively (figure 4.21b).
Figure 4.21 Valine auxotrophy complementation test using DIV (a) and KIV (b) with WGY.GISO1 overexpressing Jen1 and Ady2. YPD was used as control (c). Images registered after three days of incubation. The following strains were employed: 1, WGY.GISO1 overexpressing Ilv319 and Jen1; 2, WGY.GISO1 overexpressing Ilv319 and Ady2; and 3, WGY.GISO1 overexpressing Ilv319. The transformants were grown first in YPD, then shift to the plates containing DIV or KIV.
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4.4.1 Search for DIV export proteins
Another alternative to avoid DIV exclusion would be the deletion of transporters involved in the secretion of DIV. For this, the development of an efficient and quick deletion method would be crucial, which is why we established a variation of the CRISPR/Cas9 method. CRISPR/Cas9 system is comprised by the Cas9 (Crispr associated) protein, which complexes with specific RNA structures, named guide RNA (gRNA), and then, together, they scan the genome for a specific gRNA sequence. Wherever this target is found, there is a cleavage of the DNA, similarly to an endonuclease. In our technique variation, a single plasmid was created with the cas9 from S. pyogenes under control of a medium strength promoter (ROX3p), and also the gRNA expression cassette (Generoso et al. 2016). To create the gRNA of interest, the whole plasmid was just amplified via PCR with primers containing 20-bp homology, which was exactly the targeting sequence for the gene of interest (see topic 3.7.5). Afterwards, 20 µL of the unpurified PCR was transformed together with 300 pmol of the donor DNA, to enable the genome modification. As donor DNA, we employed an 80-bp long single-stranded primer containing the 40-bp sequence downstream and 40- bp upstream of the gene to be deleted. Within this method we achieved high efficiency of deletion, even in the industrial strain (Generoso et al. 2016). As the discovery of the DIV transporter by trial and error could be long and hard, a transcriptome analysis was carried out with a strain producing high amount of DIV in comparison to the wild-type strain. This means, upregulated proteins related to metabolite transport in the microarray analysis could be targets for reduction of DIV efflux. For the microarray analysis, two strains were compared: WGY.GISO1 transformed with pWG108 (for high DIV production) and HDY.GUF12 transformed with the control plasmid pRS41K (basal DIV production). The transformants were submitted to three independent micro-aerobically fermentation with synthetic minimal medium with 40 g/L glucose and the cells were pelleted when glucose concentration was between 20 and 15 g/L. The metabolites in the supernatant of the fermentations were analyzed for ensuring DIV discrepancy and are displayed in the figure 4.22. A good quality RNA (figure 4.23) was extracted with a combination of the TRI reagent (Sigma-Aldrich, Germany) and the RNeasy kit (Qiagen, USA) (see topic 3.6.2). The RNA labeling and the microarray hybridizations were performed by the company Phalanx Biotech Group (USA), which also possesses specific array for S.
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RESULTS cerevisiae (Yeast OneArray) and offers service for transcriptome analysis. As displayed in the histogram in the figure 4.24, the predominant mRNAs were unchanged in the conditions investigated, and, considering a cut-off of 1.5, 682 genes were differentially expressed: 334 downregulated and 348 upregulated. A large number (more than 40%) of unknown proteins or unknown biological processes were observed among the differentially expressed genes. Furthermore, even under micro-aerobic conditions, the strain with the isobutanol pathway presented a considerable number of upregulared genes related to mitochondria maintenance and mitochondrial ATP synthesis. Remarkably, there were upregulated proteins associated with transport of metabolites and, among them, 13 genes were selected to be further studied for DIV efflux (table 4.5).
Figure 4.22 Yields of the micro-aerobic fermentation of HDY.GUF12 and WGY.GISO1 overexpression the genes of isobutanol pathway. The fermentations were conducted in SMD with 4% glucose and the yields were calculated at the end of glucose consumption. The presented metabolite bars display the average and error bars display the standard deviation among the replicates. Experiments presented significant difference within the metabolites (p<0.05).
Figure 4.23 Total RNA extracted from Saccharomyces cerevisiae. 1% agarose gel, showing in M, GeneRuler 1 kb ladder (Thermo Fisher Scientific, USA); R, total RNA extracted from HeLa (control); 1-3, RNA from HDY.GUF12 with pRS42K; 4- 6, RNA from WGY.GISO1 with pWG108.
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Figure 4.24 Histogram representing the distribution of log2(fold change) for analyzed genes in the microarray analysis. Fold change was calculated for HDY.GUF12 versus WGY.GISO1 with pWG108.
Table 4.5 Upregulated genes studied for DIV efflux. Log2 p Gene Short Description Codeletion 5.46 0.006 TPO2 Polyamine transporter TPO3 Putative hexose transporter and involved in pleiotropic drug HXT9, 3.84 0.012 HXT11 resistance HXT12 3.72 0.003 YRO2 Protein with a putative role in response to acid stress MRH1 2.68 0.001 TPO3 Polyamine transporter of the major facilitator superfamily TPO2 2.34 0.001 PDR12 Plasma membrane weak-acid-inducible ABC transporter - 2.07 0.001 HXT5 Hexose transporter with moderate affinity for glucose - 1.81 0.001 MCH5 Similarity to mammalian monocarboxylate permease - Short-lived membrane ABC transporter, actively exports various 1.76 0.001 PDR5 PDR15 drugs 1.66 0.001 QDR2 Multidrug resistance transporter QDR1 1.65 0.001 AZR1 Azole drugs transporter - 1.61 0.001 FLR1 Multidrug transporter; involved in efflux of drugs - QDR1, 1.59 0.001 AQR1 Short-chain monocarboxylic acids resistance transporter QDR2 1.53 0.001 ARR3 Arsenite transporter -
In order to address which of these upregulated transporters could be related to DIV efflux, they were deleted in the strain WGY.CISO1. WGY.CISO1 is CEN.PK113- 7D strain with deletion of ILV2, and this strain overexpressing the ILV pathway present the same DIV efflux phenomenon observed with WGY.GISO1 (data not shown). The deletions were performed with the CRISPR/Cas9 system and together with the upregulated gene, some paralogs or related genes were also deleted (table 4.5). The knock-out mutants were transformed with the pWG108 and employed in fermentative 66
RESULTS tests with SCD medium and the pathway intermediates were analyzed. As can be seen in the figure 4.25, none of the target genes could cease DIV efflux. Interestingly, the DIV in the supernatant was reduced in the strains with deletion of YRO2/MRH1, QDR2/QDR1, AZR1, FLR1 and ARR3; but, there was not a higher isobutanol production in these cases. Nevertheless, for WGY.CISO1∆yro2∆mrh1 fermentation, DIV concentration in the media was 40% reduced, acetoin and 2,3-butanediol 78.5% increased and isobutanol 22% increased. For the fermentation with WGY.CISO1∆hxt5, the isobutanol yield was more than double, but with the same DIV yield of the control (WGY.CISO1). Drawing on these results, Yro2, Mrh1 and Hxt5 could be involved in DIV efflux, but not as the only transporters.
Figure 4.25 Yields of the micro-aerobic fermentation WGY.CISO1 and, its knock-out mutants, overexpression the genes of isobutanol pathway. The fermentations were conducted in SMD with 4% glucose and the yields were calculated at the end of glucose consumption. Showing in a, acetoin and 2,3- butanediol yields; b, DIV yields; and c, isobutanol yields. The bars display the average and error bars display the standard deviation among the two replicates. Bars with asterisk have significant difference with the control (p<0.05). Acn and Bdo stand for acetoin and 2,3-butanediol, respectively.
In order to further prove Yro2/Mrh1 and Hxt5 effects, both deletions were performed in another background, WGY.GISO1. WGY.GISO1∆yro2∆mrh1, WGY.GISO1∆hxt5 and WGY.GISO1 were transformed with pWG108 and used for fermentation in SMD. The same effect observed with WGY.CISO1∆yro2∆mrh1 and WGY.CISO1∆hxt5 was repeated with WGY.GISO1∆yro2∆mrh1 and WGY.GISO1∆hxt5, respectively (figure 4.26). Nevertheless, for WGY.GISO1∆yro2∆mrh1 fermentation, DIV production was just 10% reduced; acetoin and 2,3-butanediol was 40% increased and isobutanol 10% increased. For WGY.GISO1∆hxt5 fermentation, the isobutanol increase was just 36.5%.
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Figure 4.26 Yields of the micro-aerobic fermentation WGY.GISO1, WGY.GISO1∆yro2∆mrh1 and WGY.GISO1∆htx5 overexpression the genes of isobutanol pathway. The fermentations were conducted in SMD with 4% glucose and the yields were calculated at the end of glucose consumption. The bars display the average and error bars display the standard deviation among the two replicates. Bars with asterisk have no significant difference with the control (p>0.05). Acn and Bdo stand for acetoin and 2,3-butanediol, respectively.
4.5 Substrate channeling between IlvC6E6 and Ilv3
The results presented before demonstrated that the intermediate dihydroxy- isovalerate, once is outside of the cell, cannot undergoes in the pathway anymore. Consequently, even if occasioned by Ilv3 inefficient activity, DIV secretion additionally complicates its activity, and therefore should be avoided to enhance isobutanol production. For this, we sought about two alternatives: searching for the transporters that favor DIV secretion (explained in topic 4.4.1); and to create a substrate channeling of DIV between IlvC6E6 and Ilv319. The substrate channeling was attained with a pair of synthetic zippers (synZIP), which have high affinity for each other (Reinke et al. 2010), creating thereby an enzymatic net with the enzymes IlvC6E6 and Ilv319. One synZIP pair was employed, synZIP1 (Z1) and synZIP2 (Z2). Thus, synZIP1 and synZIP2 were screened in both enzymes, IlvC6E6 and Ilv319, at their N- and C-terminus. As IlvC is organized in tetramers (Tyagi et al., 2005) and Ilv3 in dimers (Velasco et al., 1993), a hetero- polymer would be generate as schematized in the figure 4.27.
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Figure 4.27 Schematization of the enzymatic net between IlvCE6E and Ilv3. Ilv3 are displayed in blue, IlvCE6E are displayed in green and the synZIP interactions are displayed in black.
Initially, Ilv319 with different synZIP combinations was tested for valine auxotroph complementation. For this, ILV3∆19 was cloned into pRS62K with synZIP1 or synZIP2 at N- or C-terminus. These genes were named ILV3-Z1-N, ILV3-Z1-C, ILV3-Z2-N and ILV3-Z2-N. The four recombinant plasmids were transformed into WGY9, then plated directly into SCD-V plates. pRS62K-Ilv3∆19 and the empty pRS62K vector were employed as positive and negative controls, respectively. As can be seen in the figure 4.28, all of the tagged Ilv319 were able to complement the valine auxotrophy. However, Ilv319 with both synZIPs at C-terminus guaranteed better growth compared to the zippers at N-terminus, as far as the colonies of the N-terminally tagged enzymes needed nine days more to grow to the same size as the C-terminally tagged Ilv319. As Ilv3 activity is easily lost once in contact with oxygen, no enzyme assay could be carried out to evaluate the changes in the activity of the tagged enzyme; therefore, both C-terminally tagged Ilv319 were further studied.
Figure 4.28 Valine auxotrophy complementation test with WGY9 overexpressing Ilv3 with synZIPs. After transformation, the cells were directly plated on SCD without valine plates. Images registered after three days of incubation, except for Ilv3-Z1- N and Ilv3-Z2-N, which were incubated for 12 days. A shows Ilv3∆19 with synZIP1 (Z1) or synZIP2 (Z2) at N- and C-terminus; B shows WGY9 with overexpression of Ilv3∆19 and with empty vector.
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Afterwards, all possible IlvC6E6 pair combinations were cotransformed with their correspondent tagged Ilv319. For this, ilvC6E6 was cloned with synZIP1 or synZIP2 at N- or C-terminus into pRS62N. These genes were named ILV5-Z1-N, ILV5- Z1-C, ILV5-Z2-N and ILV5-Z2-N. The ilvC6E6 variations cloned into pRS62N, together with the correspondent tagged ILV3∆19 plasmid, were transformed into ISOY12 (ilv5∆ strain), then tested for the ability to grow in SCD-V medium. The untagged ilvC6E6 and ILV3∆19 were employed as controls. As shown in the figure 4.29, just IlvC6E6 tagged with synZIP2 at N-terminus could satisfactorily complement valine auxotrophy. Furthermore, in a general way, ISOY12 transformed with the plasmids could not grow adequately in synthetic media, which is evidenced by the poor growth when using untagged ilvC6E6 and ILV3∆19. The experiment was also tried with the WGY19 strain, but the growth was worse than that using ISOY12. Specifically, IlvC6E6 was investigated together with the Ilv319, because former research in our group demonstrated that the use of a single synZIP, i.e. without the correspondent partner, can lead to a earlier degradation of the tagged protein (Mignat, 2015).
Figure 4.29 Valine auxotrophy complementation test with ISOY12 overexpressing IlvC6E6 Ilv3 with synZIPs. After transformation, the cells were cultivated in YPD, then plated on SCD without valine. Images registered after seven days of incubation. A shows IlvC6E6 with synZIP1 (Z1) or synZIP2 (Z2) at N- and C- terminus together with Ilv3-Z1-C; B shows IlvC6E6 with synZIP1 (Z1) or synZIP2 (Z2) at N- and C-terminus together with Ilv3-Z2-C.
To investigate changes in the activity of IlvC6E6, an enzyme assay was conducted with the tagged and untagged IlvC6E6. The specific activity was measured during the conversion of acetolactate to DIV using the crude extracts of the strains ISOY12 overexpressing Ilv3∆19-Z1-C and IlvC6E6-Z2-N, or Ilv3∆19-Z1-C and IlvC6E6 for control. The specific activity of the tagged IlvC6E6 was around five times lower than the activity of the control (figure 4.30). As we had no other feasible combination, since
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IlvC6E6-Z2-N was the only active tagged IlvC6E6, the experiments were continued with the combination Ilv3∆19-Z1-C and IlvC6E6-Z2-N though.
Figure 4.30 Relative activity of the IlvC6E6 alternatives. For the enzyme assay, ISOY12 was transformed with ILV3∆19-Z1-C and either ilvC6E6, for control, or ilvC6E6-Z2- N. The specific activities were assayed and compared with the mean of the control. Bars display the average and error bars display the standard deviation among the three biological replicates. The experiments presented significant difference (p<0.05).
In order to confirm whether there is the formation of the enzymatic net, the enzymes were submitted to pull down analysis. Thus, ILV3∆19-Z1-C had a polyhistidine-tag (HT) fused at the N-terminus for binding to the nickel column; and ilvC6E6 and ilvC6E6-Z2-N had a human influenza hemagglutinin tag (HA) fused at the C-terminus for detection in the Western blot. For the pull down, yeast was transformed with two combinations: ILV3∆19-Z1-C-HT and ilvC6E6-Z2-N-HA; and ILV3∆19-Z1-C-HT and ilvC6E6-HA as control. The plasmids were transformed in CEN.PK113-7D and the crude extract of cultures at exponential growth (O.D. 2-4) were used for the Western blot. As observed in the figure 4.31, IlvC6E6 could be detected in the elution fractions when tagged with synZIP2, i.e. IlvC6E6-Z2-N-HA stuck to the column due to the interaction between synZIP2 and synZIP1 of Ilv3∆19-Z1-C-HT. A very faint band was observed in the elution fraction of the experiment with untagged IlvC6E6, which is possibly due to overload of the column (figure 4.31a). Hence we could validate the interaction between both enzymes, Ilv3∆19- Z1-C and IlvC6E6-Z2-N were employed in fermentative tests to evaluate the reduction of DIV efflux. Both tagged genes were cloned substituting their untagged version in the plasmid pWG108, becoming the plasmid pWG133. Also, pWG108 was used as control of the pathway without substrate channeling. The plasmids were used for transformation of WGY.GISO1 and then the transformants were used for micro-aerobic fermentation. The figure 4.32 shows that the use of both zipped enzymes brought benefits to the pathway, i.e. the concentration of DIV in the supernatant was reduced 71
RESULTS and isobutanol slightly increased. Moreover, less ethanol was produced, which suggest that the flux toward the isobutanol production was enhanced, and thereby glycerol production was increased, as Ilv3 is still limiting the pathway. Nevertheless, despite the benefits to isobutanol production, the reduction in the yield of DIV (-6,03 mg/gglu) was not proportional to the enhancement in isobutanol yield (+0,67 mg/gglu), and more 3-MB was produced, which suggests that not all KIV produced was converted into isobutanol. Thus, to better evaluate the channeling effect, another strategy was traced and is presented ahead.
Figure 4.31 Western blot of the pull-down assay with extract from strains with overexpression of Ilv3∆19-Z1-C-HT and IlvC6E6-HA (a) or IlvC6E6-Z2-N-HA (b). For the assay, Cen.PK113-7D was transformed with the plasmids were cultivated in YPD. They display in M, molecular marker PageRuler; 1, crude extract; 2, column flow through; 3, column washing; 4, first elution with 500 mM imidazole; 5, second elution with 500 mM imidazole.
Figure 4.32 Yields of the micro-aerobic fermentation of WGY.GISO1 overexpression the genes of isobutanol pathway, with IlvC6E6 and Ilv3 with and without synZIPs. The fermentations were conducted in SMD with 4% glucose and the yields were calculated at the end of glucose consumption. The presented metabolite bars display the average and error bars display the standard deviation among the replicates. The experiments showed significant difference within the metabolite means (p<0.05).
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4.5.2 3-methyl-butanol production
The connection between IlvC6E6 and Ilv319 brought benefits to the isobutanol pathway, although the channeling effect was not markedly pronounced. Therefore, another approach was drawn to further investigate the channeling effect. As not all KIV produced was converted into isobutanol and a considerably higher amount of 3-MB was produced, the leucine production pathway was stimulated in the strain. 3-methyl-butanol is produced from 2-ketoisocaproate, which is the precursor of leucine (see topic 2.4). Park et al. (2014) demonstrated that, among other alterations, the use of mutant forms of Leu3 and Leu4 together with the isobutanol genes increased 3-MB production. Leu3 is a transcription factor that regulates genes involved in the branched-chain amino acids either upregulating LEU1 and LEU2 expression or acting as a repressor in the absence of 2-isopropylmalate (Friden et al. 1989; Friden and Schimmel 1988). However, the mutant Leu3∆601, which has an internal truncation from the amino acid 173 to 773, maintains constitutive activation the target genes, independently of 2-isopropylmalate (Friden et al. 1989). Leu4 is the first enzyme of leucine biosynthesis and its activity is attenuated in presence of leucine; nonetheless, with the mutation in the asparagine 578 to tryptophan, this inhibition is abolished (Oba et al. 2005). In this way, an additional plasmid was created containing LEU3∆601 and LEU4∆D578Y, and this plasmid (pWG140) was transformed together with the plasmid with the genes for isobutanol production. Additionally, ARO10 was substitute by kdcA from L. lactis in the plasmids pWG108 and pWG133, becoming pWG134 and pWG135. Milne et al. (2015) measured with KdcA a higher decarboxylase activity to keto-isovalerate and keto-isocaproate than Aro10, what could reduce potential byproducts of both keto acids. Thus, the plasmids pWG134 or pWG135 together with pWG140 (or pRS41N for control) were transformed into the strain WGY.GISO1, and the transformants were evaluated in micro-aerobic fermentation with SMD. As can be seen in the figure 4.33, not only 3-MB was higher, but also isobutanol production. Interestingly, the use of KdcA did not influenced isobutanol production, if compared with the plasmids with Aro10 (figure 4.32), what is probably due to Pdc1 and Pdc5 activity. Furthermore, ethanol production was reduced with use of the zipped enzymes, and, as consequence, glycerol was enhanced. Nevertheless, the channeling approach 73
RESULTS could not cease DIV efflux completely, but can be further studied as a powerful tool to enhance isobutanol production.
Figure 4.33 Yields of the micro-aerobic fermentation of WGY.GISO1 overexpressing the genes of isobutanol pathway, with IlvC6E6 and Ilv3 with and without synZIPs, and the mutant version of Leu3 and Leu4. The fermentations were conducted in SMD with 4% glucose and the yields were calculated at the end of glucose consumption. The strains with the leucine pathway genes are presented with “+Leu”, and the other have pRS41N for empty vector control. The presented metabolite bars display the average and error bars display the standard deviation among the replicates. Bars connected by brackets have no significant difference (p>0.05). Ace and 23-Bdo stand for acetoin and 2,3-butanediol, respectively.
4.6 Redirection of flux from ethanol to isobutanol production
Despite all the approaches employed so far, isobutanol production is still under 2% of the theoretical yield, whereas ethanol is still the main product. In order to acchieve higher isobutanol production, a strategy was raised to redirect the pyruvate flux from ethanol production to isobutanol production, in the strain WGY.GISO1. To this end, the ethanol production was abolished with the deletion of the two main pyruvate decarboxylases (∆pdc1 and ∆pdc5). This might create a redox imbalance that challenges the cell to regenerate NADH via the production of isobutanol. This strain, named WGY.GISO2, was transformed with the plasmid pWG108, but it could not grow properly. Therefore, this transformant was employed for evolutionary engineering. First, WGY.GISO2 with pWG108 was grown under aerobic conditions, in YPEG. When the culture reached stationary phase, 1 mL was used to start new culture, also under aerobic conditions, but with YPD instead. This process was continued for five consecutive new cultures, every time it reached stationary phase. Afterwards, the
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RESULTS medium was then changed for SCD and re-inoculed for five cultures again. However, the final evolved strain was not able to produce isobutanol, but high amount of DIV, 2,3-butanediol and isobutyrate (data not shown). This evolutionary engineering approach was tried again under micro-aerobic conditions, either using glucose or xylose as carbon source, but the strain was not able to grow and consume the sugar of the medium. In order to reduce NADH and NADPH consumption for 2,3-butanediol production and force isobutanol production, additionally, BDH1 and BDH2 were deleted in the WGY.GISO2. As BDH1 and BDH2 are sequential in the genome, their loci were exchanged by the cassette for overexpression of IlvHm to decrease pyruvate accumulation (see topic 4.1.2). This strain was named WGY.GISO2.2 and, in absence of oxygen, the NADH should act like a driving force for the isobutanol biosynthesis, as shown by the diagram in figure 4.34.
Figure 4.34 Simplified scheme of metabolic pathways for production of isobutanol Saccharomyces cerevisiae. The scheme comprises the pathway with native and heterologous enzymes employed in this work. Bold arrows represent multiple enzymatic steps and red bars represent blocked alternative pathway.
As Dickinson et al. (1998) suggested that isobutyric acid could be produced directly from KIV and the acid production in the WGY.GISO2 strain could be a result of an inefficient decarboxylase activity. Furthermore, as Pdc1 and Pdc5 are important 75
RESULTS decarboxylases for KIV (ter Schure et al. 1998), and they are absent in WGY.GISO2.2, different keto acid decarboxylases were investigated. ARO10 was exchanged in pWG108 by its codon-optimized version (coARO10), and two, also codon optimized, keto-acid decarboxylases: kivD from Lactococcus lactis subsp. lactis (UniProt: Q684J7), and kdcA from Lactococcus lactis (UniProt: Q6QBS4). The pathway plasmids with kivD (pWG112), kdcA (pWG134) and the codon-optimized ARO10 (pWG136) were used for transformation of WGY.GISO2.2. Firstly, the strains were screened for aerobically and micro-aerobically growth, in minimal (SMD) and complex media (YPD); however, either in SMD or micro-aerobically (SMD and YPD), the growth and glucose consumption were extremely poor (data not shown). For this reason, the transformants were solely evaluated through aerobic cultivation in YPD. The use of both bacterial keto-acid decarboxylases promoted a better growth and glucose consumption of the strain (figure 4.35). As expected, neither ethanol nor 2,3-butanediol was produced. Interestingly, the higher isobutanol yield was achieved with KdcA (1.95 mg/gglu). On the other hand, with Aro10 there was no production of isobutanol, nor even 3-MB (figure 4.36). Surprisingly, the isobutanol yields were lower than the obtained with the strain WGY.GISO1, and isobutyrate was produced instead. Also, the higher was the keto-acid decarboxylase activity, as indicated by Milne et al. (2015), the lower was DIV production (figure 4.26). This supports the suggestion that KIV could be interfering in the pathway. Nonetheless, KdcA was selected for further experiments.
Figure 4.35 Aerobic growth of WGY.GISO2.2 overexpressing the genes of isobutanol pathway, varying the keto-acid decarboxylases. The cultivations were conducted in YPD with 2% glucose. The employed keto-acid decarboxylases are presented in the legend. Glucose concentrations are displayed on the left y-axis and optical density on the right y-axis. Continuous lines stand for glucose concentration and dashed lines stand for O.D.
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Figure 4.36 Yields of the aerobic cultivation of WGY.GISO2.2 overexpressing the genes of isobutanol pathway, varying the keto-acid decarboxylases. The cultivations were conducted in YPD with 2% glucose and the yields were calculated at the end of glucose consumption. The presented metabolite bars display the average and error bars display the standard deviation for the triplicate. All experiments show significant difference within the metabolite means (p<0.05).
The next step was to check whether a possible low activity of Adh2 could be responsible for the high isobutyrate production. adhARE1 is a mutant of adhA from L. lactis, which arose of a random mutagenesis library for increased isobutyraldehyde using NADH as cofactor, and has a catalytic efficiency 40-fold higher than the wild- type (Bastian et al. 2011). Thus, ADH2 was exchanged by the codon-optimized adhARE1 in the plasmid pWG134, becoming pWG140. The plasmids pWG134 and pWG140 were used for transformation of WGY.GISO2.2 and the transformants were investigated through aerobic cultivation with YPD. Nevertheless, independently of the alcohol hydrogenase employed, the isobutanol production was reduced; and isobutyrate further increased with the use of the AdhARE1 (figure 4.37).
Figure 4.37 Aerobic growth (a) and metabolite yields of cultivation of WGY.GISO2.2 overexpression the genes of isobutanol pathway (b), varying Adh2. The cultivations were conducted in YPD with 2% glucose and the yields were calculated at the end of glucose consumption. The presented metabolite bars display the average and error bars display the standard deviation among the replicates (n=3). All experiments show significant difference within the metabolite means (p<0.05).
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4.6.1 Deletion of alternative pathways
Another hypothesis for the poor isobutanol yield could be due to alternative routes from KIV. KIV is an important intermediate for valine, leucine and pantothenate biosynthesis (figure 4.38), and these pathways either include redox steps that could be promoting NADPH production, or their intermediates could be interfering in isobutanol dehydrogenase reaction. Other competitive pathways could be either the biosynthesis of isoleucine, since the same enzymes are used in both pathways, or the production of acetoin, which also depends on NADH. For this reason, different deletion mutants of WGY.GISO2.2 were generated in order to remove or reduce these alternative pathways. Therefore, the chosen genes for deletion were LEU4/LEU9, BAT2, ECM31, ALD6 and ILV1, becoming the strains WGY.GISO2.3, 2.4, 2.5, 2.6 and 2.7, respectively. An additional strain was constructed with the deletion of LEU4/LEU9 and ECM31, which was named WGY.GISO2.35. Ald6 was selected since it is the only cytosolic NADP+-dependent aldehyde dehydrogenase (Saint-Prix et al. 2004), which could thereby play the main role in isobutyrate production in our circumstance. To reduce acetoin production, no plan was set, since BDH1 and BDH2 are already absent in the strain and there is acetoin production still, what suggests that other reductases are able to convert diacetyl.
Figure 4.38 Simplified scheme of metabolic pathways with keto-isovalerate in Saccharomyces cerevisiae. The scheme comprises the pathway with native and heterologous enzymes employed in this work. Bold arrows represent multiple enzymatic steps. Labels in blue represent the group of isoenzymes.
For the analysis, the knock-out strains were transformed with the plasmid pWG134 and the transformants were investigated through aerobic cultivation with 78
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YPD. Unexpectedly, as can be seen in the figure 4.39, none of the deletions improved isobutanol production, but further reduced it instead. Conversely, isobutyrate production was increased. Neither isobutanol nor 3-MB were detected in the supernatant of the strain WGY.GISO2.35.
Figure 4.39 Yields of the aerobic fermentation of the deletion strains overexpressing the genes of isobutanol pathway. The cultivations were conducted in YPD with 2% glucose and the yields were calculated at the end of glucose consumption. WGY.GISO2.2 was used as control. The presented metabolite bars display the average and error bars display the standard deviation among the three replicates. Bars with asterisk have no significant difference (p>0.05) with the control (WGY.GISO2.2).
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5. DISCUSSION
Isobutanol has attracted much attention among the second-generation biofuels. If compared to ethanol, isobutanol has a higher combustion power and a lower hydrophilicity, closely comparable to gasoline. Moreover, isobutanol is an advantageous alternative biofuel to ethanol, because it can be produced by several microorganisms and its biosynthesis requires less enzymatic steps among the next generation biofuels (e.g. butanol and fatty-acid or terpene derivatives). Isobutanol production using bacteria already reached the theoretical yield; however, to employ the same infrastructure of the existing ethanol plants, yeasts are better alternatives for isobutanol production. S. cerevisiae, the most adapted yeast to harsh industrial conditions, is capable of producing isobutanol and tolerates it in substantial amounts. S. cerevisiae produces isobutanol via the combination of the valine biosynthesis and its further degradation, through the Ehrlich pathway. However, yeast produces isobutanol in a very low yield naturally, which is why this metabolic pathway has been focus of optimizations. Therefore, this work describes the investigation of the bottlenecks that limits the isobutanol biosynthetic pathway in yeast. For this, a xylose- adapted industrial strain, named HDY.GUF12 and derived from EthanolRed (Lesaffre, France), was employed for metabolic engineering studies.
5.1 Development of the initial isobutanol producing strain
The employed strategy for establishment of the isobutanol biosynthesis in HDY.GUF12 was the fully cytosolic pathway described by Brat et al. (2012). In this approach, the valine biosynthesis is relocated into the cytosol, in order to create a short-cut between the glycolysis and the Ehrlich pathway, without metabolite transferences across the mitochondrial membranes. In our strategy, the native ILV2 was deleted in the HDY.GUF12 genome in order to eliminate the mitochondrial valine pathway. This strain was named WGY.GISO1. The ILV pathway genes, without MTS, ARO10 and ADH2 were overexpressed from a centromeric plasmid in WGY.GISO1. In a first trial, a 2-micron plasmid was used for the overexpression of the pathway; however, the transformed strain demonstrated irregular growth in plate and a scarce growth in minimal medium (data not shown). Probably, this was result of a high energy demanded to keep the high copy number vector and the high transcriptional rate of the
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DISCUSSION genes, since the plasmid had around 12 kb with five genes under control of strong promoters. Other authors also reported problems related to the use of multicopy or big plasmids, and employing centromeric plasmids or direct integration of the pathway into the genome, these problems could be overcome (de Jong et al. 2015; Krivoruchko et al. 2013; Schadeweg and Boles 2016). For this reason, the 2-micron origin was exchanged by CEN/ARS in the plasmid, and WGY.GISO1 transformed with the single copy vector was able to grow homogeneously. Moreover, differently of the stated by Brat et al. (2012) and Dietz (2013), WGY.GISO1 transformed with the centromeric pathway plasmid could promptly grow in minimal medium, without necessity of evolutionary engineering. Our first concern about the employed pathway was the possible cofactor imbalance generated by ILV548, which could, therefore, limit the isobutanol production. This happens because NADH is the major redox cofactor produced during glycolysis and Ilv5 needs NADPH for the conversion of 2-acetolactate into DIV. Dietz (2013) employed a bacterial transhydrogenase (UdhA) to convert NADH into NADPH. Similarly, Matsuda et al. (2013) also employed a transhydrogenase-like shunt to overcome the cofactor imbalance. However, we opted to modify the cofactor specificity of Ilv5 towards NADH, so that the rapid generation of NADH during the glycolysis could act as a driving force for the isobutanol production. For this, as Ilv5 has no elucidated structure, the sequence of an already described NADH-dependent acetohydroxyacid reductoisomerase mutant from E. coli (IlvC6E6) was employed as example. The mutations in IlvC6E6 were situated on the cofactor binding pocket, such that the amino acids responsible for the interaction with the phosphate group of NADPH were exchanged for either negatively charged amino acids or amino acids with longer side- chain (Bastian et al. 2011). As the same mutated amino acids of IlvC6E6 were not found in Ilv5, the same idea was employed with the amino acids G111, S113, K115 and A117 (figure 4.1); however, only the mutant Ilv5P4 [G111S:S113D:K115D:A117D] presented a satisfactory change in the cofactor preference, but with decrease of its activity (figure 4.2). Interestingly, IlvC6E6 itself was fully active in yeast and exhibited the best kinetics results (figure 4.2). Despite the better NADH preference of IlvC6E6, the isobutanol production was very similar in the fermentative tests, regardless of the employed Ilv5 variant (figure 4.3). Possibly, this is result of the versatile ability of yeast to produce cytosolic NADPH. Yeast has two main sources of cytosolic NADPH: the deviation of the glycolysis to the pentose-phosphate pathway, and the acetate production. In the
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DISCUSSION first case, glucose-6-phosphate is the branch point out of the glycolysis, and along this deviated path, the glucose-6-phosphate dehydrogenase (Zwf1) and the 6- phosphogluconate dehydrogenases (Gnd1 and Gnd2) produce NADPH (Minard and McAlister-Henn 2005). However, the deviation of the glycolysis to the pentose- phosphate pathway leads to an additional decarboxylation step, which reduce the theoretical yield of isobutanol from glucose. The NADPH generation via the acetate production occurs because of Ald6, which is the major cytosolic aldehyde dehydrogenase during glucose cultivations and is NADP+-dependent (Saint-Prix et al. 2004). Nevertheless, in a PDC-null strain (∆pdc1, ∆pdc5 and ∆pdc6), Ald6 activity would be useless, since no acetaldehyde is produced. For this reason and also because of the lower accumulation of the byproducts acetoin, 2,3-butanediol and glycerol (figure 4.3), IlvC6E6 was the selected Ilv5-variant for our pathway. One intriguing point of the solely cytosolic isobutanol pathway is the unfeasibility to produce isobutanol in complex media or media with valine (Brat et al. 2012). The same observation was not emphasized in the other studies with cytosolic isobutanol pathways; however, just media without valine was employed for isobutanol production in these cases (Ida et al. 2015; Matsuda et al. 2013; Milne et al. 2015). Natively, the first reaction of the ILV pathway is catalyzed by Ilv2 in combination of Ilv6, which is one of the most important step of the feedback inhibition of the pathway. Ilv6 either reduces or stimulates Ilv2 activity according to the presence or absence of valine. As a choice to overcome the low isobutanol production in complex medium, Ilv6 was combined into the employed pathway to boost Ilv2 activity. Curiously, the sole overexpression of the cytosolic Ilv2 was enough to enable isobutanol production; however, the activity of the sole Ilv2 is reported to be only 10-15% of the stimulated holoenzyme (Duong et al. 2011; Zhao et al. 2013). Nevertheless, the valine inhibition is not result of the dissociation of the complex, but, when complexed with valine, Ilv6 alters the catalytic site of Ilv2 and the holoenzyme has the affinity massively decreased (Pang and Duggleby 1999; 2001). Therefore, the additional overexpression of Ilv6 would further reduce Ilv2 activity if yeast is cultivated with complex media, which is why different truncated ILV6 mutants were investigated. These truncations had as objective to access a peptide that would only stimulate Ilv2, but have no affinity for valine. Zhao et al. (2013) performed the same study with IlvH, an Ilv6 orthologous from E. coli, and they obtained a small peptide (IlvHm) capable of stimulating Ilv2 from S. cerevisiae without valine inhibition. However, this demonstration was only made in vitro.
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Therefore, besides the truncated Ilv6 mutants, IlvHm was also tested for the enhancement of the isobutanol production, in vivo. The highest increase in isobutanol production was observed in the fermentation performed with the isobutanol producing yeast carrying ILV6D in minimal medium (figure 4.6). IlvHm was not the best trigger of Ilv2 activity (figure 4.5), but its overexpression led to a higher isobutanol production in complex medium than the control (figure 4.6). Moreover, either the use of IlvHm in the pathway or more 2-acetolactate seemed to pull the activity of the other enzymes of the isobutanol pathway or some other unknown protein important for the Ehrlich pathway, since this higher isobutanol production, than the control, was not a reflect of sole Ilv2 increased activity (figures 4.5 and 4.6). Nevertheless, IlvHm was just employed in the pdc1/pdc5 strain, because of the impossibility of growth in minimal media (see topic 4.6).
5.2 Investigation of Ilv3 activity limitations
Nevertheless, neither improvements in Ilv5 nor Ilv2 enhanced isobutanol production with our producing strain. Remarkably, high amounts of DIV were produced in every conducted fermentation, and neither KIV nor isobutyraldehyde were detected in the supernatant. For this reason, Ilv3 could be the major bottleneck of the isobutanol pathway. As already mentioned (see topic 2.4.1), Ilv3 is the enzyme responsible to convert DIV into KIV. Ilv3 needs iron-sulfur cluster (FeS) for the dehydration reaction, and this cofactor must be assembled de novo before incorporated into the apoproteins. In yeast, the beginning of the FeS assembly takes place inside the mitochondria and involves more than 15 proteins for this (Lill 2009). The FeS assembly starts with protein scaffolds, in which their cysteine sulfurs pre-assemble [2Fe-2S] clusters with rough iron, and, afterwards, these [2Fe-2S] groups are transferred to carriers that either deliver the cofactor to the apoproteins or further build [4Fe-4S] before it (Lill 2009; Lill and Mühlenhoff 2006). In spite of the mitochondrial location of the FeS assembly, iron- sulfur proteins are also found in cytosol and nucleus (Sharma et al. 2010). In these cases, it is not the apoproteins that reach the mitochondria to receive the clusters, but FeS that is transferred from the mitochondria into the cytosol. Yeast has a cytosolic iron-sulfur cluster assembly machinery (CIA), which receives [2Fe-2S] from the mitochondria and, in a parallel way to the mitochondria, either deliver the cofactor to
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DISCUSSION apoproteins or further assemble [4Fe-4S] FeS and hence incorporate them into apoproteins (Netz et al. 2014). Two characteristics made mitochondria the best environment for FeS assembly in yeast: the higher iron and the lower oxygen concentration inside mitochondria than other compartments. Due to a toxic effect of high concentration of iron in the cytosol, yeast stores iron excess inside the mitochondria or vacuole (Ihrig et al. 2010; Outten and Albetel 2013). Also, oxygen and oxygen species (e.g. nitric oxide) promote the loss of electrons from the FeS, which generate unstable forms of the clusters and completely inactivate the proteins and leads it to premature degradation (Fitzpatrick and Kim 2015; Imlay 2006). Thus, as Ilv3 is natively mitochondrial and it was relocated into the cytosol, either its activity could have been limited by an inefficient delivery of FeS to the cytoplasmic apoprotein or the FeS of Ilv3 might have a short half-life in the cytosol. In order to examine whether an inefficient incorporation of FeS into Ilv3 was the reason for isobutanol low yield, two genes were employed together with the isobutanol pathway: ATM1 and AFT1. As mentioned before, during the maturation of the cytosolic FeS, [2Fe-2S] clusters are transferred from the mitochondrial machinery into the CIA. The manner in which this transference occur is still unknown, but Kispal et al. (1999) identified an important mitochondrial transporter for the assembly of the cytoplasmic FeS, Atm1. The deletion of ATM1 leads to unload of FeS into cytosolic and nuclear iron-sulfur proteins, to the extent that no influence is observed for mitochondrial proteins (Kispal et al. 1999; Sipos et al. 2002). Therefore, Atm1 is the transporter in charge of transferring [2Fe-2S] into the cytosol. Differently, Aft1 is not involved in the cytosolic FeS assembly itself, but it is an important transcription factor for iron uptake and homeostasis. Aft1 is responsible for the regulation of several genes for iron uptake, including enzymes necessary for the reduction of ferric (Fe3+) into ferrous iron (Fe2+) and transporters involved in the Fe2+ uptake itself (Ihrig et al. 2010; Miao et al. 2011; Yamaguchi-Iwai et al. 1995). Also, Shakoury-Elizeh et al. (2004) associated Aft1 overexpression with the upregulation of genes necessary for the recycle of iron inside of the cell, as from heme groups for example. Therefore, with the overexpression of Atm1 and Aft1, the load of FeS to the cytosolic Ilv3 should be enhanced, which would increase the number of functional Ilv3 and improve isobutanol production. However, it has not happened and both proteins had not helped isobutanol production in our experiments, probably because this extra cytosolic FeS could not
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DISCUSSION reach Ilv319. Moreover, another hypothesis is the fragility of Ilv3 FeS in the cytosolic environment, which could causes a short half-time of Ilv319. Therefore, in order to investigate whether a possible instability of Ilv3 was causing DIV accumulation, different heterologous ILV3 orthologous were studied as substitutes. The Ilv3 alternatives were selected from literature, considering potential advantages that these enzymes could have in place of Ilv319. The ILV3 orthologous from S. solfataricus, L. lactis, C. glutamicum and N. crassa were studied and their choice are explained below. S. solfataricus is a thermophilic and acidophilic archaea, which has a very atypical dihydroxy-acid dehydratase. IlvD from S. solfataricus is a promiscuous enzyme and is associated, besides the ILV pathway, with the conventional Entner- Doudoroff pathway (EDp), in place of the 6-phosphogluconate dehydratase, and also in the non-phosphorylative EDp, dehydrating gluconate and glycerate for production of pyruvate (Carsten et al. 2015; Kim and Lee 2006). Furthermore, IlvD from S. solfataricus exhibits a high stability against oxygen degradation, which suggests that this enzyme either possesses no FeS or a very stable one (Carsten et al. 2015; Kim and Lee 2006). Another employed alternative for Ilv3 was IlvD from L. lactis, which was characterized for possessing [2Fe-2S] (Flit et al. 2010). The advantage of the [2Fe-2S] concerns the amount of FeS necessary for the enzyme, since two [2Fe-2S] are necessary to create one [4Fe-4S] (Sharma et al. 2010). However, two points should be emphasized: the native Ilv3 from S. cerevisiae is speculated to be also composed of [2Fe-2S] (Mühlenhoff et al. 2011); and the majority of cytosolic and nuclear FeS proteins are composed of [4Fe-4S], what suggests a bigger number of FeS delivers for [4Fe-4S] than [2Fe-2S] groups in the cytosol (Netz et al. 2014). As already mentioned, Corynebacteria are the most employed microorganisms for large-scale production of BCAAs. For this reason, IlvD from C. glutamicum was chosen as Ilv3 alternative, since this high yield production of BCAAs with this organism might be result of high activity of the ILV pathway enzymes. And lastly, IlvD2 from N. crassa is the only described dihydroxy-acid dehydratase from eukaryotes, which is natively active in the cytoplasm (Altmiller and Wagner 1970; Dundon et al. 2011). Employing differential centrifugation experiments, Altermiller and Wagner (1970) observed dihydroxy-acid dehydratase activity in mitochondria isolates and also in the cytosolic fraction. Years later, with the sequencing of the whole N. crassa genome, two different sequences were found for
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DISCUSSION the dihydroxy-acid dehydratases (Galagan et al. 2003). Furthermore, both genes are unlikely to be arisen from gene duplication, since ilvD1 and ilvD2 are included in two different chromosomes and their product sequences have not more than 60% identity. Therefore, as N. crassa is not a strict anaerobe and it is evolutionarily close to yeast, IlvD2 could possibly be functional in the cytosol of S. cerevisiae and bring benefits to our isobutanol pathway. Nevertheless, among the four employed heterologous Ilv3 orthologous, IlvD from S. solfataricus and IlvD from L. lactis could not complement the valine auxotrophy of the ilv3 yeast (figure 4.12). Possibly, the environmental conditions of yeast cytosol are not favorable for IlvD from S. solfataricus, since the higher enzyme activity was measured around 80C, and almost no activity was detected under 40C (Kim and Lee 2006). Crude extract of the strain overexpressing IlvD from S. solfataricus was employed for enzyme activity assay conducted as described by Kim and Lee (2006), in the optimal temperature and pH conditions of the enzyme, and no activity was detected, however, we had no positive control to validate the enzyme assay (data not shown). On the other hand, the reasons why IlvD from L. lactis was inactive in our experiments is still unclear. Milne et al. (2016) employed IlvD from L. lactis in their isobutanol pathway, and it was fully functional; however, our employed isoform had some amino acid differences if compared with that one from Milne et al. (2016). Besides, Gevo Inc. and Butamax LLC. filed patents in which IlvD from L. lactis demonstrated high DIV dehydratase activity in yeast (Flit et al. 2010; Urano and Dundon 2012). IlvD from C. glutamicum and IlvD2 from N. crassa were both able to complement the valine auxotrophy of WGY9 (figure 4.12). IlvD from C. glutamicum did not improve isobutanol production, but reduced it instead (figure 4.13). For IlvD2 from N. crassa, no clear discernment from fermentations with Ilv319 was observed, since the use of both enzymes led to similar intermediates and products yields (figure 4.14). This unexpected equal performance of IlvD2 and Ilv319 could be also result of an inefficient loading of FeS into IlvD2, although we expected that the FeS assembly and delivery machinery from N. crassa would be similar to that of S. cerevisiae. Nonetheless, the overexpression and improvement of enzymes possessing FeS in yeast is something very complex and challenging, and an extensive effort and research can be still insufficient for improvement of their activities (Benisch and Boles 2014; Carlsen et al. 2013).
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For this reason, we investigated the use of dehydratases without FeS as substitutes for Ilv3. These studied Ilv3 alternatives are sugar-acid dehydratases (enolases), which are important components of the non-conventional EDps. Beyond the classical EDp, two modified versions of the EDp were described for microorganisms: the semi-phosphorylative and non-phosphorylative EDp (figure 4.1). The major difference among the EDps is the point of phosphorylation of the carbon source. In the conventional EDp, glucose is phosphorylated into glucose-6-phosphate; while in the semi-phosphorylative EDp, glucose is first converted into 2-keto-3-deoxy- gluconate, which is then phosphorylated. And lastly, in the non-phophorylative EDp, glycerate either can be phosphorylated to glycerate-2-phosphate, or dehydrated directly to pyruvate; however, this is rare and just observed in thermophilic archaea (Ahmed et al. 2005; Carsten et al. 2015; Peekhaus and Conway 1998).
Figure 4.1 Schematic illustration of the Entner-Doudoroff pathways. The scheme does not discriminate between reversible and irreversible reactions. The enzyme names are not displayed in the schematization. Black arrows stand for single enzymatic steps and red arrows represent multiple enzymatic steps. The blue contrasts display the remification reaction among the conventional (C EDp), semi-phosphorylative (SP EDp) and non-phosphorylative EDp (NP EDp).
These non-conventional EDps are important for allowing microorganisms to consume a broad spectrum of sugars as carbon sources, e.g. glucose, fructose,
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DISCUSSION galactose, mannose, arabinose, xylose (Ahmed et al. 2005; Peekhaus and Conway 1998). This wide range of substrates for the non-conventional EDps is not only because of several sugar-acid enolases within a microorganism, but also consequence of a promiscuity of the gluconate dehydratase, and other enolases (Groninger-Poe et al. 2014; Wichelecki et al. 2014a; Wichelecki et al. 2014b). Interestingly, these enolases catalyze the dehydratase reaction employing a metallic cation, predominantly Mg2+, for stabilization of the intermediate compound. Furthermore, the reaction occurs in the alpha-carbon of the substrate, as well as Ilv3 converts DIV into KIV (Groninger- Poe et al. 2014). Therefore, because of this promiscuity of the enolases, and also because IlvD from S. solfataricus can act like an enolase and dihydroxy-acid dehydratase, some enolases were screened for the ability to convert DIV into KIV in vivo. Nine enolases were tested for complementation of the valine auxotrophy of WGY9: eight from bacteria and one from A. niger. However, just one of them, RspA from P. ananatis, could restore growth in medium without valine, but very poorly (figure 4.16). For our surprise, the D- galactonate dehydratase from D. dadantii could not promote growth of the ilv3 strain, given that its sequence is very similar to the sequence of RspA from P. ananatis (figure 4.15). Nonetheless, the perspective of using RspA in our isobutanol pathway was so attractive that further investigation was carried out to make it more active in yeast. As the molecular structure of RspA is already elucidated, side-direct mutagenesis was employed to exchange the substrate preference. For this, the natural substrate of RspA, D-galactonate, was exchanged by DIV in silico and this new configuration was analyzed (figures 4.18 and 4.19). Three charged amino acids (H225, H235 and D329), which were important for guiding of the natural substrate, seemed to be obsolete for the binding of DIV to the pocket, since DIV has a partially nonpolar chain. These three amino acids were thereby mutated via saturated mutagenesis, using NNK codon. NNK codons are more versatile than NNN for saturated mutagenesis, because they cover all amino acid codons and exclude TAA and TGA stop codons. However, no improvement of the RspA activity towards DIV was achieved, i.e. no mutant demonstrated better growth in media without valine. Therefore, rspA was also employed for mutagenesis with error-prone PCR, which performs point mutations in random points of the ORF. This technique is often employed for engineering of enzymes, since no knowledge about the structure is necessary (Bastian et al. 2011; Blombach et al. 2011; Slutzker et al. 2011). However,
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DISCUSSION no improvement was obtained with the epPCR library as well. Possibly, this difficulty to obtain an RspA mutant with improved activity towards DIV was due to the small number of mutants screened in WGY9. In other words, for the site-direct mutagenesis, as three amino acids were mutated into 20 possible amino acids, more than eight thousand clones should have been screened, and this number ignores the repetitions of similar amino acid combination. And as epPCR leads to a higher number of detrimental mutations than site-direct mutagenesis, an even higher number of clones must be screened to find the expected phenotype (Liu and Jiang 2015).
5.3 Dihydroxy-isovalerate efflux attenuation for isobutanol production
As no improvement of isobutanol production was achieved with modulation of Ilv3, other possibilities were investigated to increase DIV utilization. Interestingly, an impossibility to reuptake DIV was observed for S. cerevisiae (figure 4.20). Possibly, the inefficient Ilv3 activity drives the DIV exclusion of the cell, and as it cannot be reuptake from the medium, the flow towards isobutanol production is impaired. There are two forms for transport of carboxylic acids trough plasma membrane: undissociated acids diffuse passively through the membrane, while the anionic acid requires transporters (Casal et al. 2016). As the predicted pKa of DIV is 3.8 (Yeast Metabolome Database) and the employed pH for the media in our fermentations were 6.3, DIV must use a transporter to be pumped. Therefore, the first approach to overcome DIV waste was the use of two acid transporters to allow DIV reuptake. For this, Jen1 and Ady2 were investigated. Jen1 is an important transporter for short-chain mono acids, and was already associated with the transport of pyruvate, lactate and acetate (Casal et al. 1999; Paiva et al. 2013). Moreover, McDermott et al. (2010) reported that Jen1 is able to transport selenite, mimicking monocarboxylic acids, with high affinity. However, JEN1 is tightly regulated in presence of glucose, not only at transcriptional level, but its mRNA is quicker degraded during the exponential growth; moreover, Jen1 is removed from the plasma membrane and transferred into the vacuole for degradation in yeast cultivation with fermentable carbon sources (Andrade et al. 2005; Paiva et al. 2002). Interestingly, JEN1 deletion has not abolished yeast capability of uptaking acetate, and, thereby, a second monocarboxylic acid transporter was identified, Ady2 (Paiva et al. 2004). Ady2 is associated with the ability of yeast to transport proprionate, formate, lactate and acetate through the plasma membrane (Pacheco et al. 2012;
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DISCUSSION
Paiva et al. 2004). Likewise for JEN1, ADY2 is also repressed in the presence of glucose at transcriptional level (Abate et al. 2012). Furthermore, Ady2 is mainly overexpressed when carboxylic acids, as acetate, are used as carbon-source (Abate et al. 2012; Paiva et al. 2004). Taken together, Jen1 and Ady2 could be suppressed under the conditions employed for the isobutanol production. Therefore, both transporters were overexpressed in WGY.GISO1, and the transformants was then plated in minimal medium without valine, but supplemented with DIV and KIV. None of the transporters allowed WGY.GISO1 growth from DIV; however, overexpression of Jen1 improved KIV uptake (figure 4.21). Remarkably, this property of Jen1 was not reported in the literature, up-to-date. Since the reuptake of DIV could not be accomplished, another strategy was carried out to reduce DIV waste. This other strategy considered the deletion of possible DIV transporters, which would lead to accumulation the intermediate inside of the cell. In order to identify these transporters, a microarray analysis was conducted with a strain producing high amounts of DIV against a wildtype strain with undetectable DIV concentration in the fermentation supernatant. WGY.GISO1 transformed with the ILV pathway plasmid was employed as the high DIV-producing strain, and HDY.GUF12 was the reference strain (figure 4.22). The microarray results demonstrated that the metabolism of the DIV-producing strain was demanding production of energy, since mitochondrial genes related to ATP production were upregulated in this strain, even during micro-aerobic fermentation. Interestingly, 13 transporters were found upregulated in the transcriptome analysis (table 4.5). The majority of the upregulated transporters in the DIV-producing strain are genes associated to multidrug resistance in yeast (e.g. TPO2, TPO3, HXT11, PDR12, PDR5, QDR2, AQR1, AZR1 and FLR1). These transporters are responsible for secretion of different toxic compounds and thereby protect yeast against them. Regarding their way of action, these efflux pumps are divided into two different groups: the H+-antiporters, in which the drug is secreted while a proton is taken into the cell; and the ATP-binding cassette (ABC) transporters, in which energy is employed to actively secret the compound (Panwar et al. 2008; Sa- Correia and Tenreiro 2002). Although the H+-antiporters do not need ATP for their action, ATP is necessary to secret the imported H+ a posteriori, in order to restore the acid/base equilibrium in the cytosol (Sa-Correia and Tenreiro 2002). Therefore, these facts corroborate with the assumption that DIV has been actively secreted of the cell,
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DISCUSSION instead of passive diffusion; and one or some of these upregulated transporters might be involved in the DIV efflux. Thus, to investigate which of the 13 transporters were indeed associated to DIV efflux, their ORFs and their paralogs, were deleted in the strain WGY.CISO1. Afterwards, the knocked-out strains were transformed with the ILV pathway plasmid and employed in fermentations. Surprisingly, none of the studied transporters alone was responsible for DIV efflux (figure 4.25). Nevertheless, two strains showed interesting results: WGY.CISO1∆yro2∆mrh1 and WGY.CISO1∆hxt5. Yro2 and Mrh1 are two paralogous transporters involved in tolerance to weak acids in yeast. Takabatake et al. (2015) demonstrated that sole and double ∆yro2 and ∆mrh1 mutants are more susceptible to acetate and lactate, which reflected in worse fermentative performances in biomass hydrolysates. Although MRH1 expression is not so responsive to weak acid stress as YRO2, ∆mrh1 mutants are hypersensitive to external acetic acid (Keller et al. 2001; Takabatake et al. 2015). In our case, WGY.CISO1∆yro2∆mrh1 fermentation revealed less DIV in the supernatant and higher acetoin, 2,3-butanediol and isobutanol production (figure 4.25). This could be results of a diminution of DIV efflux and, as far as Ilv3 activity is still limiting the flux towards KIV, Ilv5 converted DIV back into 2-acetolactate. To further evaluate ∆yro2/∆mrh1 effect, both genes were deleted in WGY.GISO1 and this employed for isobutanol production. The YRO2/MRH1 deletion affected the intermediate yields of the isobutanol pathway in the same manner as for WGY.CISO1; however, with less intensity (figure 4.26). Differently to Yro2 and Mrh1, Hxt5 is not related to weak acid or drug stresses, and in our investigation, the ∆hxt5 mutants did not exhibited discrepant yields of the ILV pathway intermediates if compared to the parental strains; however, isobutanol production was increased with HXT5 deletion (figures 4.25 and 4.26). Hxt5 is a hexose transporter with low to moderate affinity to glucose, which is mainly expressed in growth with low glucose concentration or with non-fermentative sugars, and also during the stationary phase of glucose-based growth (Boles and Hollenberg 1997; Diderich et al. 2001). Although no clear phenotype is associated in hxt5 strains, HXT5 transcription is also trigged by different stress conditions, e.g. high temperature, starvation and high osmotic pressure (Buziol et al. 2002; Gasch et al. 2000; Verwaal et al. 2002). Therefore, this elevated mRNA level of HXT5 in the DIV-producing strain might be result of the stress conditions wherein this strain is submitted, e.g. deficit of energy, high glycerol and high acid production. However, the reason why HXT5
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DISCUSSION deletion improved isobutanol production is still unclear. And lastly, either DIV efflux is caused by different transporters or the main DIV transporter was not covered by our strategy. Since the two above-mentioned strategies (DIV uptake or impair of DIV efflux) could not circumvent the waste of DIV, substrate channeling was employed to enhance the conversion of DIV into KIV. The use of substrate channeling has become a strong tool in metabolic engineering. This is a natural cellular mechanism in which pathway enzymes are held in proximity of each other with the purpose of enhancing yield by reducing the diffusion of the intermediates, the secretion, the use in other pathways or the toxicity to the cell (Levskaya et al. 2009; Shiue and Prather 2012). The substrate channeling occurs via the isolation of reactions in a smaller compartment inside the cell, the use of a sole holoenzyme for several reactions or even by organization of enzymes in proximity (Shiue and Prather 2012). As examples: the de novo assembly of FeS is not only compartmentalized inside the mitochondria, but the enzymes responsible for that are connected to each other as a scaffold (Lill and Mühlenhoff 2006; Netz et al. 2014); likewise, in some cellulolytic bacteria, the biomass degrading enzymes are organized into structures named cellulosomes, which are protein scaffolds that connect the hydrolases and the cellulose-binding domains and the bacterial cell wall (Bayer et al. 1998). Applying scaffolds in E. coli metabolic engineering, Dueber et al. (2009) avoided the accumulation and diffusion of 3-hydroxy- 3-methyl-glutaryl-CoA, which in E. coli is a toxic intermediate of the mevalonate pathway. Furthermore, the use of channeling improved the pathway flux by 200% (Dueber et al. 2009). Also, employing a bacterial cellulosome in yeast, Tsai et al. (2013) described a 2-fold higher ethanol production from cellulose if compared with the yeast expressing the free enzymes. In previous work in our group, Mignat (2015) employed substrate channeling between pyruvate kinase and two pyruvate-requiring pathways, lactate and 2,3-butanediol production, in order to attenuate ethanol production. However, channeling has just improved 2,3-butanediol production. In our substrate channeling strategy, a pair of synthetic coiled-coil zippers (synZIPs) were fused to IlvC6E6 and Ilv319. These employed synZIPs were characterized by Reinke et al. (2010) and were demonstrated to have more stable interactions than other protein ligands, not only because of the inexistence of crosstalk, but also due to a strong interaction affinity (Kd < 10nM). Moreover, Thompson et al. (2012) showed that this high affinity is also maintained in yeast cytoplasm. Therefore,
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DISCUSSION as IlvC and Ilv3 naturally form homo-oligomers and the incorporated synZIPs would enforce a hetero-oligomerization between both enzymes, we expected that DIV would be confined inside this enzymatic net and the flux towards isobutanol would be enhanced. Interestingly, despite the decrease of the specific activity of the tagged IlvC6E6 (figure 4.30), the use of both zipped enzymes together improved isobutanol production (figure 4.32). However, the isobutanol production was not enhanced in the same proportion as the DIV reduction, and 3-MB production was improved with the use of the zippers. Therefore, Leu3 and Leu4 mutants were also overexpressed in the strain, in order to consume KIV towards the production of 3-MB as well. With this, the channeling effect between IlvC6E6 and Ilv319 was more evident than just with isobutanol production, i.e. both isobutanol and 3-MB yields were enhanced with the enzymes with zippers (figure 4.33). Surprisingly, the faster consumption of KIV altered the pyruvate flux from the ethanol production into the ILV pathway, evidenced by the overexpression of the leucine pathway genes together with the unzipped IlvC6E6 and Ilv319 (figure 4.33). This suggests that either KIV itself inhibits the ILV pathway or Ilv3 might perform the reversible reaction. Drawing on these results, the substrate channeling was a very interesting approach against DIV waste, although DIV efflux could not be abolished, probably because of a still high number of apoenzyme within the artificial enzymatic net, i.e. many Ilv319 without functional FeS linked to IlvC6E6.
5.4 Pyruvate flux redirection from ethanol to isobutanol production
Despite the metabolic engineering of the isobutanol pathway in E. coli had already reached 100% efficiency, isobutanol production with yeast is still insignificant for biofuel purposes. Although two companies, Gevo Inc. and Butamax LLC., are leading the imminent large-scale commercialization of yeast from isobutanol within the next years (Upton 2015), not much is known scientifically. Another important obstacle for the isobutanol production with yeast, beyond what was already described before, is the high preference for ethanol production, due to the strong influence of the Crabtree effect in S. cerevisiae (Pfeiffer and Morley 2014). Some attempts were investigated to abolish ethanol production, mainly by knocking out the pyruvate decarboxylases: PDC1, PDC5 and PDC6 (Mohamed et al. 2015; Oud et al. 2012; van Maris et al. 2004; Zhang et al. 2015). However, the high glucose sensibility of these PDC-null mutants complicates their use for biotechnological applications.
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In our strategy for minimization of the ethanol production, PDC1 and PDC5 were deleted from WGY.GISO1 genome. Hohmann (1991) showed that the deletion of these two isoforms is enough to reduce pyruvate decarboxylase activity to almost zero. Indeed, no ethanol was detected in the supernatant of the ∆pdc1/∆pdc5 strain (WGY.GISO2) transformed with the isobutanol pathway plasmid. However, this recombinant strain could barely growth in medium with glucose, even after evolutionary engineering attempts to increase growth rate. Moreover, as also observed by Milne et al. (2016), the main byproducts of this strain was glycerol, 2,3-butanediol, DIV and isobutyric acid. The high glycerol and 2,3-butanediol production with WGY.GISO2 was a consequence of the excess of NADH in the strain, considering that isobutanol could not be produced instead. Glycerol biosynthesis derives from glycolysis, more specifically from dihydroxy-acetone-phosphate (DHAP), and requires one NADH per glycerol molecule produced. In this pathway, DHAP is first reduced to glycerol-3- phosphate by the glycerol-3-phosphate dehydrogenases Gpd1 and Gpd2, and then dephosphorylated to glycerol. Yeast produces glycerol as the main source to overcome redox imbalance and, consequently, the double GPD1/GPD2 deletion negatively affects yeast during growth with glucose (Bloem et al. 2016; Hubmann et al. 2011; Knudsen et al. 2015). On the other hand, 2,3-butanediol biosynthesis can be abolished in yeast without growth anomalies (Gonzalez et al. 2000). 2,3-butanediol is produced through two consecutive NADH-dependent reductions from diacetyl, which is a product of the spontaneous decarboxylation of 2-acetolactate. Yeast has two 2,3-butanediol dehydrogenases, Bdh1 and Bdh2. Nevertheless, bdh1/bdh2 mutant (strain WGY.GISO2.2) only abolished 2,3-butanediol production, whereas acetoin was still highly produced (figure 4.36). Interestingly, the strain WGY.GISO2.2 carrying the pathway plasmid preferentially produced isobutyrate, instead of isobutanol. Besides isobutanol, isobutyric acid is also a product of the Ehrlich pathway (figure 2.3), but occasioning one additional NAD+ reduction. However, Dickinson et al. (1998) suggest that isobutyrate can be produced from KIV in a similar way as acetate is produced from pyruvate via the pyruvate dehydrogenase complex. Also, Vuralhan et al. (2005) detected no Aro10 activity in cells cultivated in complex and minimal media with glucose, even if under control of a very strong promoter (TDH3p). Therefore, one hypothesis was that a possible inefficient Aro10 activity was driving the isobutyrate production. To further investigate it, ARO10 was exchanged by other keto-acid
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DISCUSSION decarboxylases in the isobutanol pathway plasmid and the most promising substitute was kdcA from L. lactis (figure 4.36). Among several keto-acid decarboxylases already tested in yeast, KdcA from L. lactis was the one that demonstrated higher activity to KIV (Milne et al. 2015; Stribny et al. 2016). Nevertheless, despite the further force towards NADH oxidation (bdh1/bdh2) and higher KIV decarboxylase activity, isobutyrate yield was still much higher than isobutanol yield. Interestingly, the higher the activity of the employed KIV decarboxylase was, the lower was DIV production (figure 4.36). This suggests that Ilv3 activity can be affected by KIV availability, either by reverting the reaction into DIV or by activity inhibition. Moreover, KIV was never detected in the fermentation supernatant in any of the experiments performed with our isobutanol production strategy, what suggests that KIV was always further consumed by other pathway (or Ilv3 itself). The difficulty in the in vitro activity assay for Ilv3 precludes the further investigation of these hypotheses. As KIV decarboxylation alone was not causing the high isobutyrate production, the next step of the pathway was also examinated. Adh2 was the employed alcohol dehydrogenase for the isobutanol reduction so far and Brat et al. (2012) demonstrated that Adh2 has the highest activity to isobutyraldehyde among the other native alcohol dehydrogenases, employing NADH as cofactor. Albeit Adh2 has no described post transcriptional regulation, for the sake of clarity ADH2 was exchanged by the bacterial mutant adhARE1 (Bastian et al. 2011). Nevertheless, an even lower isobutanol production was achieved with AdhARE1 (figure 4.37). Beyond isobutanol production, KIV is an important intermediate for other pathways. Therefore, we examined whether these competing pathways could be the driving force for the unexpected low isobutanol and high isobutyrate production. KIV is known as precursor of valine, leucine and pantothenate biosynthesis in yeast (figure 4.38). In our case, Bat2 is expected to be the main responsible for valine production, as the isobutanol pathway is conducted in the cytoplasm. Moreover, glutamate can be cyclically restored from oxoglutarate via the glutamate dehydrogenases (Gdh1 and Gdh2), which employs ammonia and mainly NADPH as cofactor (DeLuna et al. 2001). Therefore, for valine production, NADPH would be necessary, and thereby synthetized via isobutyrate production; and the deletion of BAT2 could enhance isobutanol production. In a similar way, Bat2 could be promoting an overproduction of leucine, which also derives from KIV. Although leucine biosynthesis also requires acetyl-CoA, which is expected to be scarce in our case, and generates an additional NADH (figure
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2.2), LEU4 and LEU9 deletions were also investigated. Following the same hypothesis that NADPH necessity was driving isobutyrate production, ECM31 was deleted in the WGY.GISO2.2. Ecm31 (Ketopantoate hydroxymethyltransferase) is the first step of the panthotenate biosynthesis from KIV, but the second enzyme of the pathway, 5- ketogluconate reductase (Pan5), employs NADPH as cofactor for panthotenate production (White et al. 2001). And as a last approach to directly abolish generation of NADPH via isobutyrate production in the cytosol, ALD6 was deleted. S. cerevisiae has three described cytosolic aldehyde dehydrogenases (Ald1, Ald2 and Ald6), although Ald6 is the only NADPH-dependent enzyme (Saint-Prix et al. 2004). Moreover, Ida et al. (2015) demonstrated that the deletion of ALD6 further improved isobutanol production. Therefore, LEU4/LEU9, BAT2, ECM31, ALD6 and LEU4/LEU9/ECM31 deletions were investigated for isobutanol production. Nevertheless, none of these deletions improved isobutanol production, but further increased isobutyrate yield instead (figure 4.26). The last alternative pathway deletion was the isoleucine biosynthesis. The biosynthesis of isoleucine occurs in parallel to valine production, i.e. the same enzymes employed in for isobutanol production could be enhancing isoleucine production as well (figure 2.2). The difference in the substrates for Ilv2 differs both pathways, i.e. for valine production, Ilv2 catalyze the carbo-ligation of two pyruvates, while for isoleucine, Ilv2 substrates are one pyruvate with one ketobutyrate (see topic 2.4.1). Therefore, as ketobutyrate is the product of Ilv1, ILV1 was deleted for elimination of isoleucine biosynthesis. Nevertheless, as the other competing pathways deletions, ILV1 deletion did not improve isobutanol production, but further increased isobutyrate yield. In conclusion, the reason for the lower isobutanol production employing a pdc1/pdc5 strain in comparison to the normal ethanol producing strain is still unclear for us and additional investigations would be necessary to reveal insights behind these unexpected results.
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6. ZUSAMMENFASSUNG
Die Biotechnologie macht industrielle Prozesse schrittweise nachhaltiger und kostengünstiger als klassische chemische Techniken. Das Hauptziel dieser Bemühungen ist es, den Verbrauch von fossilen Rohstoffen zu senken, umweltschädliche chemische Verfahren zu umgehen und diese durch nachhaltigere Prozesse zu ersetzen. Verschiedene Mikroorganismen werden heute für die Produktion von Grundchemikalien verwendet, von denen das Bakterium Escherichia coli und die Hefe Saccharomyces cerevisiae zu den wichtigsten zählen. Insbesondere ist für industrielle Prozesse mit hoher Zuckerkonzentration, hohem osmotischen Druck, geringem pH und anaeroben Bedingungen S. cerevisiae der robusteste Mikroorganismus. Zusätzlich besitzt dieser Organismus weltweit GRAS (generally recognized as safe) Status. Diese Hefe ist bereits ein elementarer Bestandteil in der Produktion von Wein, Bier und Brot. Die größte industrielle Verwendung von S. cerevisiae ist die Produktion von Ethanol. Weltweit wird Ethanol biotechnologisch produziert und die Produktionsweisen unterscheiden sich lediglich in der Zuckerquelle. In Brasilien wird Zuckerrohr, in USA wird Mais und in Europa werden Zuckerrüben und Getreide verwendet. Aktuelle Forschungsanstrengungen konzentrieren sich darauf, Hefe für die Produktion von Ethanol aus lignocellulosischer Biomasse wie Zuckerrohrbagasse, Stroh von Reis und Mais sowie Forstbiomasse einzusetzen. Lignocellulosisches Material besteht zu 35- 50% aus Cellulose, 5-35% aus Lignin und 20-30% aus Hemicellulose. Die Hauptmenge an Zuckern, die nach Vorbehandlung und Depolymerisierung dieser Biomasse gewonnen werden, sind Glucose und Xylose. Trotz der guten Verwertungseigenschaften von S. cerevisiae in lignocellulosischen Hydrolysaten ist S. cerevisiae natürlicherweise nicht in der Lage Xylose zu verstoffwechseln. Daher wurde in dieser Arbeit ein industrieller Hefestamm verwendet, der zuvor für Xyloseverstoffwechselung angepasst wurde, um damit anstelle von Ethanol Isobutanol zu produzieren. Unter den Butanolisomeren gilt Isobutanol als der geeignetste Biokraftstoff, um fossile Kraftstoffe zu ersetzen. Im Vergleich zu Ethanol besitzt Isobutanol eine höhere Oktanzahl, eine höhere Energiedichte, einen geringen Dampfdruck, einen höheren Flammpunkt und ist weniger hygroskopisch. Diese Vorteile ermöglichen es, dass Isobutanol in der vorhandenen, auf fossilen Kraftstoffen basierenden Infrastruktur
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(Autos, Rohrleitungen, Tankstellen) verwendet und in bestehenden Ethanolproduktionsanlagen hergestellt werden kann. S. cerevisiae ist ein natürlicher Produzent von Isobutanol, welches durch die Kombination von Valinbiosynthese und dessen Degradation durch den Ehrlichstoffwechselweg synthetisiert wird. Die Valinbiosynthese findet in den Mitochondrien statt und sie beginnt mit der Ligation von zwei Pyruvatmolekülen zu 2-Acetolactat durch die Acetolactat Synthase (Ilv2). Anschließend reduziert die Acetohydroxysäure Reductoisomerase (Ilv5) durch Verbrauch von NADPH 2-Acetolactat zu 2,3-Dihydroxyisovalerat (DIV). Als nächstes wird DIV durch die Dihydroxysäure Dehydratase (Ilv3) zu 2-Ketoisovalerat (KIV) dehydratisiert. Schließlich wird KIV zu Valin durch verzweigtkettige Aminotransferasen (Bat1 und Bat2) umgewandelt. Die Isobutanolproduktion ist das Ergebnis des Valinabbaus über den Ehrlichstoffwechselweg. Dieser Stoffwechselweg findet im Cytosol statt und ist für die Hefe eine Möglichkeit Stickstoff für die Biosynthese von anderen Aminosäuren in der Abwesenheit von Ammonium bereitzustellen. In diesem Stoffwechselweg wird Valin zurück zu KIV umgesetzt, welches dann zu Isobutyraldehyd decarboxyliert wird. Viele Enzyme wurden bereits für diese Reaktion beschrieben, aber Pdc1, Pdc5 und Aro10 besitzen die höchsten Aktivitäten hierfür. Anschließend wird Isobutyraldehyd durch Alkoholdehydrogenasen (hauptsächlich Adh2) zu Isobutanol reduziert. In dieser Arbeit wurde ein Stoffwechselweg eingesetzt, der vollständig im Cytosol lokalisiert ist. Diese Strategie basiert auf der Arbeit von Brat et al. (2012), die eine Isobutanolausbeute von 14,18 mg/g Glucose mit der Relokalisation von Ilv2, Ilv5 und Ilv3 ins Cytosol erreichten. Hierfür wurde ILV2 aus dem Genom des Stamms HDY.GUF12 deletiert, der auf dem Stamm EthanolRed (Lesaffre) basiert. Für die Etablierung des Isobutanolstoffwechselweges wurde ein single-copy Plasmid mit Kassetten für die Überexpression von N-terminal gekürzten Ilv2Δ54, Ilv5Δ48 und Ilv3Δ19 sowie zusätzlich Aro10 und Adh2 erstellt. Eine Limitation in diesem Stoffwechselweg ist das Ungleichgewicht an Cofaktoren. Ilv5 verwendet NADPH, in der Glykolyse werden jedoch zwei NADH Moleküle gebildet. Das Enzym Ilv5Δ48 wurde durch eine bakterielle NADH- verwendende Mutante IlvC6E6 ausgetauscht, um ein Gleichgewicht an Cofaktoren herzustellen. Die Verwendung von lvC6E6 führte zur Produktion von weniger Nebenprodukten (z.B. Glycerin, Acetat und 2,3-Butandiol), auch wenn keine höheren Isobutanolausbeuten erzielt wurden. Eine weitere Limitation, die in der Arbeit von Brat
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ZUSAMMENFASSUNG et al. (2012) gezeigt wurde, ist, dass Isobutanol in Anwesenheit von Valin nicht produziert wird. Um diese Behinderung zu umgehen, wurde Ilv6 mit dem Isobutanolstoffwechselweg coexprimiert. Ilv6 reguliert die Aktivität von Ilv2, indem es die Aktivität bei Anwesenheit von Valin reduziert oder sie steigert, wenn kein Valin vorhanden ist. Daher wurden verschiedene Ilv6-Mutanten getestet, um die Aktivität von Ilv2 zu steigern und den Rückkoppelungsmechanismus zu unterbinden. Keine der Mutanten konnte jedoch die Isobutanolproduktion verbessern. Trotz der Arbeiten an den Enzymen Ilv5 und Ilv6 konnte keine der eingebrachten Änderungen die Isobutanolproduktion erhöhen. Es wurde jedoch festgestellt, dass die Hauptlimitierung bei der Isobutanolproduktion die Umwandlung von DIV zu KIV ist, da im Fermentationsüberstand stets hohe Konzentrationen von Acetoin, 2,3-Butandiol und insbesondere DIV gemessen wurden, während KIV und Isobutyraldehyd nicht vorlagen. Diese Reaktion wird durch Ilv3 katalysiert, das Eisen- Schwefel-Cluster (FeS) für seine Aktivität benötigt. Der erste Ansatz war deshalb, die FeS Assemblierung und Übertragung ins Cytoplasma mit Aft1 beziehungsweise mit Atm1 zu erhöhen. Aft1 ist ein Transkriptionsfaktor, der bei der Aufnahme und der Selbstregulation von Eisen beteiligt ist. Atm1 ist ein mitochondrieller Transporter, der Eisen-Schwefel-Cluster vom Mitochondrium ins Cytosol transportiert. Die Überexpression dieser Gene steigerte die Aktivität von Ilv3Δ19 jedoch nicht. Die zweite Alternative war es, heterologe Ilv3-Orthologe zu testen. Die Ilv3- Alternativen wurden aus der Literatur nach entsprechenden Vorteilen gegenüber Ilv3Δ19 ausgewählt. Die ausgewählten Alternativen waren die Ilv3-Orthologe von S. solfataricus, L. lactis, C. glutamicum und das cytosolische Ilv3 von N. crassa. Diese Enzyme wurden in einem wachstumsbasierten Komplementierungsassay gescreent, aber nur IlvD von C. glutamicum und IlvD2 von N. crassa konnten eine ilv3-Mutante komplementieren. Nach eingehender Untersuchung blieb jedoch Ilv3Δ19 das vielversprechendste Enzym für den Isobutanolstoffwechselweg. In einem weiteren Ansatz wurden Zuckersäure-Enolasen als Ersatz für Ilv3 getestet. Diese Enolasen katalysieren die Dehydratisierungsreaktion in der gleichen Art und Weise wie Ilv3, benötigen jedoch Mg2+ als Cofaktor. Neun Enolasen wurden in einem Δilv3-Komplementationsassay auf Herstellung einer Valinprototrophie getestet: acht von Bakterien und eine von A. niger. Jedoch konnte nur RspA von P. ananatis Wachstum auf Medium ohne Valin vermitteln; jedoch sehr langsam. Daher wurde dieses Enzym mit Mutagenesemethoden modifiziert, um die Substratspezifität für DIV
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ZUSAMMENFASSUNG zu erhöhen. Site-directed und error-prone PCR wurden als Mutagenesemethoden verwendet, die jedoch zu keiner Verbesserung führten. Neben der niedrigen Aktivität von Ilv3Δ19 konnte eine weitere Limitierung festgestellt werden. Wenn DIV ins Medium sekretiert wurde, konnte es durch die Hefezellen nicht mehr aufgenommen werden, was den Fluss des Stoffwechselweges zusätzlich erschwert. Mit zwei Strategien sollte der Verlust von DIV umgangen werden: die Deletion möglicher DIV-Transporter und Substrate Channeling von IlvC6E6 zu Ilv3Δ19. In einer Transkriptomanalyse wurde ein Stamm, der hohe Mengen an DIV produziert, mit einem Stamm verglichen, der keine messbaren Mengen an DIV herstellt, um dadurch mögliche DIV-Transporter zu identifizieren. Es wurden zahlreiche Transporter im DIV-produzierenden Stamm entdeckt, die hochreguliert waren. Diese Transporter wurden deletiert und die neuen rekombinanten Stämme wurden bezüglich DIV-Export charakterisiert. Keiner dieser Transporter alleine war für den Verlust von DIV ins Medium verantwortlich, jedoch führte die kombinierte Deletion von YRO2 und MRH1 und die einzelne Deletion von HXT5 zu interessanten Ergebnissen. Der Stamm mit der Deletion von YRO2 und MRH1 produzierte weniger DIV und mehr Acetoin sowie 2,3-Butandiol. Dies lässt vermuten, dass der DIV-Verlust reduziert wurde und Ilv3 immer noch den Stoffwechselweg limitiert, da mehr 2- Acetolactat akkumuliert wurde. Der Stamm mit HXT5-Deletion produzierte unveränderte Mengen an Intermediate, aber fast die doppelte Isobutanolausbeute. Hierfür konnte keine klare Erklärung gefunden werden. Bei der zweiten Strategie wurden Substrate Channeling Strategien zwischen IlvC6E6 und Ilv3Δ19 etabliert, um den DIV-Verlust zu verhindern. Hierfür wurde ein künstliches Enzymnetz konstruiert, indem synthetische Zipper an IlvC6E6 und Ilv3Δ19 fusioniert wurden. Diese synthetischen Zipper wurden durch Reinke et al. (2010) charakterisiert und binden untereinander mit hoher Affinität. Da beide Enzyme Oligomere bilden und die Zipper Protein-Interaktionen ermöglichen, würde ein Heteropolymer aus IlvC6E6 und Ilv3Δ19 entstehen. Interessanterweise führte diese Substrate Channeling Strategie zu einer 17% höheren Isobutanolproduktion und zu einer 25% höheren Produktion von 3-Methyl-Butanol mit dem getesteten Industriestamm. Dennoch wurde bei der Isobutanolproduktion mit dem Industriestamm noch nicht einmal 2% der theoretischen Ausbeute an Isobutanol von Glucose erzielt und
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Ethanol blieb das Hauptprodukt in allen durchgeführten Experimenten. Um die Ethanolproduktion zu unterbinden und somit die Isobutanolproduktion zu erhöhen, wurden die Pyruvatdecarboxylase-Gene PDC1 und PDC5 deletiert. Zusätzlich wurden die Butandioldehydrogenase-Gene BDH1 und BDH2 deletiert, um eine NADH- treibende Kraft in Richtung Isobutanolproduktion zu etablieren. Die Isobutanolausbeute dieses Stammes war jedoch geringer als des Stammes ohne PDC-Deletion. Zusätzlich konvergierte der Ehrlichstoffwechselweg in Richtung Isobuttersäureproduktion anstatt der Isobutanolproduktion. Um den Fluss zu ändern, wurden unterschiedliche KIV-Decarboxylasen und Isobutanol Dehydrogenasen getestet, wovon KdcA von L. lactis und nach wie vor Adh2 aus Hefe die besten Ergebnisse erzielten. Trotzdem war die Isobuttersäureproduktion höher als die von Isobutanol. Alternative Stoffwechselwege, in denen Intermediate verloren gehen könnten, wurden durch Deletionen blockiert, um die Isobutanolproduktion zu erhöhen. Zu den untersuchten Stoffwechselwegen gehören die von KIV ableitenden Stoffwechselwege (z.B. Valin-, Leucin, und Panthotenatbiosynthese), Isobuttersäurebiosynthese und Isoleucinproduktion. Daher wurden BAT2, LEU4/LEU9, ECM31, ALD6 und ILV1 im Stamm ohne PDC-Allele deletiert. Keine der Deletionen konnte die Isobutanolproduktion erhöhen, sie verringerte sich stattdessen. In diesen Fällen waren die Hauptnebenprodukte Glycerin, Acetoin, DIV, Isobuttersäure. Ohne eine Steigerung der Dihydroxysäure-Dehydratase Aktivität scheinen die weiteren Optimierungen der Isobutanolproduktion keinen entscheidenden Effekt zu zeigen.
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ABBREVIATIONS
APPENDIX A: ABBREVIATIONS
A. niger Aspergillus niger FeS iron-sulfur cluster B. subtilis Bacillus subtilis C. glutamicum Corynebacterium glutamicum
D. dadantii Dickeya dadantii GRAS generally regarded as safe E. coli Escherichia coli gRNA guide-RNA L. lactis Lactococcus lactis
N. crassa Neurospora crassa P. ananatis Pantoea ananatis S. cerevisiae Saccharomyces cerevisiae h hours S. pyogenes Streptococcus pyogenes HA human influenza hemagglutinin tag S. solfataricus Sulfolobus solfataricus hphNT1 hygromycing resistance cassette HPLC high performance liquid chromatography HT polyhistidine-tag
HXT hexose transporters ø diameter
2-MB 2-methyl-1-butanol 23BDO/BDO 2,3-butanediol 3-MB 3-methyl-1-butanol i.e. id est (that is) Inc. incorporation
ABE acetone-butanol-ethanol ACN acetoin KanMX kanamycin resistance cassette ADH alcohol dehydrogenases KIV 2-ketoisovalerate ADP adenosine-5’-diphosphate kp kilobase pairs ALD aldehyde dehydrogenase ATP adenosine-5’-triphosphate ARS autonomous replication sequence ILV isoleucine-leucine-valine
B12 cobalamin LB lysogeny broth BCAA branched-chain amino acids LiAc lithium acetate bp base pairs LLC. limited liability company BSA bovine serum albumin
min minutes CAI codon adaptation index mRNA messenger RNA cDNA complementary DNA MTS mitochondrial targeting sequence CEN centromere CFU colony-forming unit CIA cytosolic iron-sulfur assembly machinery NAD+ nicotinamide adenine dinucleotide CoA coenzyme A (oxidized form) CRISPR clustered regularly interspaced short NADH nicotinamide adenine dinucleotide palindromic repeats (reduced form) NADP+ nicotinamide adenine dinucleotide phosphate (oxidized form) ddH2O double-distilled water NADPH nicotinamide adenine dinucleotide DIV 2,3-dihydroxyisovalerate phosphate (reduced form) DHAP dihydroxy-acetone-phosphate NatMX nourseothricin resistance cassette dsDNA double-stranded DNA
O.D. optical density at 600nm EDD 6-phospho-D-gluconate dehydratase ORF open reading frame EDp Entner-Doudoroff pathway EDTA ethylenediaminetetraacetic acid e.g. exempli gratia (for example) PAGE polyacrylamide gel electrophoresis epPCR error-prone PCR PEG Polyethylene glycol et al. et alii (and others) PCR polymerase chain reaction PDC pyruvate decarboxylases
113
ABBREVIATIONS
PDB RCSB Protein Data Bank PPP pentose phosphate pathway PVDF polyvinylidene fluoride U unit of enzyme activity UK United Kingdom UniProt universal protein resource database RNA ribonucleic acid USA United States of America UV ultra violet s seconds SC synthetic complex medium XDH xylitol dehydrogenase SDS sodium dodecyl sulfate xg gravitational forces SF synthetic fermentative medium XI xylose isomerase SM synthetic minimum medium XK xylulokinase ssDNA single stranded DNA XR xylose reductase synZIP/Z synthetic coiled-coil zippers
YEP/YP yeast extract peptone medium TAE tris/acetate/EDTA TCA tricarboxylic acid cycle
Amino acid code: Nucleotide code:
1-letter 3-letter Amino acid Code Nucleotide A Ala alanine A adenosine C Cys cysteine T thymine D Asp aspartate C citosine E Glu glutamate G guanine F Phe phenylalanine U uracil G Gly glycine N A, T, C or G H His histidine K T or G I Ile isoleucine K Lys lysine L Leu leucine M Met methionine N Asn asparagine P Pro proline Q Gln glutamine R Arg arginine S Ser serine
T Thr threonine V Val valine W Trp tryptophan Y Tyr tyrosine
114
PRIMER LIST
APPENDIX B: PRIMER LIST
The table B.1 contains the information concerning the primers employed in this work.
Table B.1. Primers employed in the work. Name Sequence (3' - 5') Description c.op- AACAAAGAATAAACACAAAAACAAAAAGTTTTTTTAAT Cloning of ILV3 (mitochondrial) 34 ILV3_Fw TTTAATCAAAAAATGGGTTTGTTGACCAAGG into pRS62 vectors c.op- GGGGGGAGGGCGTGAATGTAAGCGTGACATAACTAA Cloning of ILV3 (mitochondrial) 35 ILV3_Rv TTACATGACTCGAGTTAAGCGTCCAAAACACAACC into pRS62 vectors T-c.op- AACAAAGAATAAACACAAAAACAAAAAGTTTTTTTAAT Cloning of ILV3 (N-truncated) 36 ILV3_Fw TTTAATCAAAAAATGGCTAAGAAGTTGAACAAGTAC into pRS62 vectors Sso-ILV3- AACAAAGAATAAACACAAAAACAAAAAGTTTTTTTAAT Cloning of ilvD (S. solfataricus) 44 Fw TTTAATCAAAAAATGCCAGCTAAGTTGAACTC into pRS62 vectors Sso-ILV3- GGGGGGAGGGCGTGAATGTAAGCGTGACATAACTAA Cloning of ilvD (S. solfataricus) 45 Rv TTACATGACTCGAGTTAAGCTGGTCTGGTAACAG into pRS62 vectors Cloning of AFT1up (mutation M_AFT1_ AGAATAAACACAAAAACAAAAAGTTTTTTTAATTTTAA 61 Cys291Phe) into pRS62 TCAAAAAATGGAAGGCTTCAATCCGG Fw vectors Cloning of AFT1up (mutation M_AFT1_ CATTCCAACAGCTACAATCCCCTTTGATTGTGGTTTA 62 Cys291Phe) into pRS62 ACAAATGAAATAC mut_Fw vectors Cloning of AFT1up (mutation M_AFT1_ GTATTTCATTTGTTAAACCACAATCAAAGGGGATTGT 63 Cys291Phe) into pRS62 AGCTGTTGGAATG mut_Rv vectors Cloning of AFT1up (mutation M_AFT1_ GAGGGCGTGAATGTAAGCGTGACATAACTAATTACAT 64 Cys291Phe) into pRS62 GACTCGAGCTAATCTTCTGGCTTCACATACTTC Rv vectors M_Gal2p GTGTGAAATACCGCACAGATGCGTAAGGAGAAAATA Cloning of AFT1up (GAL2 65 _Fw CCGCATGAGAGAATTAACCAAGACATCAAG promoter) into pRS62 vectors M_Gal2p- AAGTAAACACAAGATTAACATAATAAAAAAAATAATTC Cloning of AFT1up (GAL2 66 AFT1_Fw TTTCATAATGGAAGGCTTCAATCCGG promoter) into pRS62 vectors M_Gal2p Cloning of AFT1up (GAL2 67 TATGAAAGAATTATTTTTTTTATTATGTTAATC _Rv promoter) into pRS62 vectors M_Atm1_ AGAATAAACACAAAAACAAAAAGTTTTTTTAATTTTAA Cloning of ATM1 into pRS62 68 Fw TCAAAAAATGCTGCTTCTTCCAAGATGTC vectors M_Atm1_ GAGGGCGTGAATGTAAGCGTGACATAACTAATTACAT Cloning of ATM1 into pRS62 69 Rv GACTCGAGTCATAGTTCTTGCTGGTCTTTTAGTTC vectors Ilv5_3mut GTGTTAGAAAGGACAGCGCTTCTTGGGACGCTGACA Cloning of ILV5P3 into pRS62 81 _Fw TTGAAGACGGTTGGGTTCC vectors Ilv5_3mut CAACCGTCTTCAATGTCAGCGTCCCAAGAAGCGCTG Cloning of ILV5P3 into pRS62 82 _Rv TCCTTTCTAACACCAATAATAAC vectors Ilv5_4mut GTTAGAAAGGACAGCGCTGATTGGGACGCTGACATT Cloning of ILV5P4 into pRS62 83 _Fw GAAGACGGTTGGGTTCC vectors Ilv5_4mut CCGTCTTCAATGTCAGCGTCCCAATCAGCGCTGTCC Cloning of ILV5P4 into pRS62 84 _Rv TTTCTAACACCAATAATAAC vectors Ilv5mitIlv GGAATCTATTGCTGAAAAGGACGCTGACTGGAGAAA Cloning of ILV5P5 into pRS62 85 C_Fw GGCTACCGAAAACGGTAAGAACTTGTTCACCGTTG vectors TTTTCGGTAGCCTTTCTCCAGTCAGCGTCCTTTTCAG Ilv5mitIlv Cloning of ILV5P5 into pRS62 86 CAATAGATTCCTTTCTCAAAGCGTAAGAAACGTTCAA vectors C_Rv ACCGTTGTCTC CAAAGAATAAACACAAAAACAAAAAGTTTTTTTAATTT Cloning of ILV5 (N-truncated) 87 Ilv5_Fw TAATCAAAAAATGAAGCAAATTAACTTCGGTG into pRS62 vectors GGGGAGGGCGTGAATGTAAGCGTGACATAACTAATT Cloning of ILV5 (N-truncated) 88 Ilv5_Rv ACATGACTCGAGTTATTGGTTTTCTGGTCTCAAC into pRS62 vectors GGGGAGGGCGTGAATGTAAGCGTGACATAACTAATT Ilv5_6Ht_ Cloning of ILV5 (N-truncated) 89 ACATGACTCGAGTTAGTGGTGGTGGTGGTGGTGTTG into pRS62 vectors Rv GTTTTCTGGTCTCAAC 115
PRIMER LIST
Cloning of ilvC6E6 (NADH- IlvCE6E_ AACAAAGAATAAACACAAAAACAAAAAGTTTTTTTAAT consuming mutant, 100 Fw TTTAATCAAAAAATGGCTAACTACTTCAACACCTTG Escherichia coli) into pRS62 vectors Cloning of ilvC6E6 (NADH- IlvCE6E_ GGGGGGAGGGCGTGAATGTAAGCGTGACATAACTAA consuming mutant, 101 Rv TTACATGACTCGAGTTAACCAGCAACAGCAATTCTC Escherichia coli) into pRS62 vectors pFBA1_Il CTTTTTCTTTTGTCATATATAACCATAACCAAGTAATA 103 v3D19_F Build of pWG39 CATATTCAAAATGGCTAAGAAGTTGAACAAG w tPGK1_Ilv GAAAGAGAAAAGAAAAAAATTGATCTATCGATTTCAA 104 Build of pWG40 3D19_Rv TTCAATTCAATTTAAGCGTCCAAAACACAAC Cloning of ilvD (C. Cgl_IlvD_ AGAATAAACACAAAAACAAAAAGTTTTTTTAATTTTAA 105 glutamicum) into pRS62 TCAAAAAATGATCCCACTTCGTTCAAAAG Fw vectors Cloning of ilvD (C. Cgl_IlvD_ GAGGGCGTGAATGTAAGCGTGACATAACTAATTACAT 106 glutamicum) into pRS62 GACTCGAGTTAGTCGACCTGACGGACTG Rv vectors CoLla_Ilv AGAATAAACACAAAAACAAAAAGTTTTTTTAATTTTAA Cloning of ilvD (L. lactis) into 111 D_Fw TCAAAAAATGGAATTCAAGTACAACGGTAAG pRS62 vectors CoLla_Ilv GAGGGCGTGAATGTAAGCGTGACATAACTAATTACAT Cloning of ilvD (L. lactis) into 112 D_Rv GACTCGAGTTACAAATCGGTAACACAACCTTC pRS62 vectors CoNcrILV AGAATAAACACAAAAACAAAAAGTTTTTTTAATTTTAA Cloning of ilvD2 (N. crassa) 113 D2_Fw TCAAAAAATGGCTTCCAACCAAGATAAC into pRS62 vectors CoNcrILV GAGGGCGTGAATGTAAGCGTGACATAACTAATTACAT Cloning of ilvD2 (N. crassa) 114 D2_Rv GACTCGAGTTAGTAAGCATCACCACCC into pRS62 vectors CTAAGAACAAAGAATAAACACAAAAACAAAAAGTTTTT Cloning of ILV6B into pRS62 138 Ilv6_Fw TTAATTTTAATCAAAAAATGTCTATTATTTACGAAACC vectors CCAGCTCC GGGGGAGGGCGTGAATGTAAGCGTGACATAACTAAT Cloning of ILV6B into pRS62 139 Ilv6_Rv TACATGACTCGAGTTAACCTGGTGGCAATTGAGAAAT vectors GTCAAC GGGGGGAGGGCGTGAATGTAAGCGTGACATAACTAA shortIlv6_ Cloning of ILV6A into pRS62 140 TTACATGACTCGAGTTAAATTCTAGCCATAACCAATT vectors Rv CTCT Eco_Fw_ AACACAAAAACAAAAAGTTTTTTTAATTTTAATCAAAA Cloning of D8ADB5 (Enolase) 156 D8ADB5 AGTTAACATGCATCACGACAAGCGATG into pRS62 vectors Eco_Rev GTGAATGTAAGCGTGACATAACTAATTACATGACTCG Cloning of D8ADB5 (Enolase) 157 _D8ADB5 AGGTCGACTTACCAGTTCCACAGCGTG into pRS62 vectors Eco_Fw_ AACACAAAAACAAAAAGTTTTTTTAATTTTAATCAAAA Cloning of Q8FHC7 (Enolase) 159 Q8FHC7 AGTTAACATGCATCACGACAAGCGATG into pRS62 vectors Eco_Rev GTGAATGTAAGCGTGACATAACTAATTACATGACTCG Cloning of Q8FHC7 (Enolase) 160 _Q8FHC7 AGGTCGACTTACCAGTTCCACAGCGTG into pRS62 vectors At_Fw_A AACACAAAAACAAAAAGTTTTTTTAATTTTAATCAAAA Cloning of A9CG74 (Enolase) 162 9CG74 AGTTAACATGAAAATCGATCGCATGCG into pRS62 vectors At_Rev_A GTGAATGTAAGCGTGACATAACTAATTACATGACTCG Cloning of A9CG74 (Enolase) 163 9CG74 AGGTCGACTCAGGCGAAGGCATAAGAAC into pRS62 vectors St_Fw_Q AACACAAAAACAAAAAGTTTTTTTAATTTTAATCAAAA Cloning of Q8ZL58 (Enolase) 165 8ZL58 AGTTAACATGGCTTTAAGCGCGAATTC into pRS62 vectors St_Rev_ GTGAATGTAAGCGTGACATAACTAATTACATGACTCG Cloning of Q8ZL58 (Enolase) 166 Q8ZL58 AGGTCGACTTAAGGGCGTTTGCCAAATTC into pRS62 vectors Ecoli_Fw AACACAAAAACAAAAAGTTTTTTTAATTTTAATCAAAA Cloning of C9QR62 (Enolase) 171 _C9QR62 AGTTAACATGACCCTACCAAAAATTAAACAGGTTCG into pRS62 vectors Ecoli_Rev GTGAATGTAAGCGTGACATAACTAATTACATGACTCG Cloning of C9QR62 (Enolase) 173 _C9QR63 AGGTCGACTTAGTGGTGGCTGTAGGGGCG into pRS62 vectors Xcamp_F AACACAAAAACAAAAAGTTTTTTTAATTTTAATCAAAA Cloning of Q8P3K2 (Enolase) 174 w_Q8P3K AGTTAACATGCGCACCATCATCGCCC into pRS62 vectors 2 Xcamp_R CGTGAATGTAAGCGTGACATAACTAATTACATGACTC Cloning of Q8P3K2 (Enolase) 176 ev_Q8P3 GAGGTCGACTTAGGCCTTCGCCTTGCTGGC into pRS62 vectors K3
116
PRIMER LIST
Dda_C6C AACACAAAAACAAAAAGTTTTTTTAATTTTAATCAAAA Cloning of C6CBG9 (Enolase) 201 BG9_Fw AGTTAACATGTCTAAGTTGAAGATTACCAACG into pRS62 vectors Dda_C6C GTGAATGTAAGCGTGACATAACTAATTACATGACTCG Cloning of C6CBG9 (Enolase) 202 BG9_Rv AGGTCGACTTATGGTCTAGAAGCGGTACC into pRS62 vectors Cac_A6M AACACAAAAACAAAAAGTTTTTTTAATTTTAATCAAAA Cloning of A6M2W4 (Enolase) 203 2W4_Fw AGTTAACATGGAACCAACCATTATTACCG into pRS62 vectors Cac_A6M GTGAATGTAAGCGTGACATAACTAATTACATGACTCG Cloning of A6M2W4 (Enolase) 204 2W4_Rv AGGTCGACTTATGGGGTAACAATGGTACC into pRS62 vectors A1- 215 TACCGGCTTGGCTTCAGTTG ILV2 deletion control PCR Ilv2_pCC A4- 216 TAGCTGGCTCCTGATGTACC ILV2 deletion control PCR Ilv2_pCC GATACTAACGCCGCCATCCAGTGTCGAAAACGCGGT 233 pCC1_Fw Build of CrisprCas plasmid GTGAAATACCGCACAGATG CTTGGTGGTGTTCGTCGTATCTCTTAATCATAGAAGC 234 pCC1_Rv Build of CrisprCas plasmid AGACAATGGAG TGTTGTCTGACATTTTGAGAGTTAACACCGAAATTAC 235 pCC2_Fw Build of CrisprCas plasmid CAAGGCTC ATTCTCTTCATTCTTTCTCTAGAGTTCTTTTGACCCTT 236 pCC2_Rv Build of CrisprCas plasmid TTGGGTG CAAGCCAGAAAACATTGTTATTGAAATGGCTAGAGAA 237 pCC3_Fw Build of CrisprCas plasmid AACCAAACC GTACAAGAAGTTAACGTACTTAGATGGCAAAGCCAAT 238 pCC3_Rv Build of CrisprCas plasmid TCGTTAC AACGGTAGAAAGAGAATGTTGGCTTCTGCTGGTGAA 239 pCC4_Fw Build of CrisprCas plasmid TTGCAAAAGG AAAACGCCAGCAACGCGGCCTTTTTACGGTTCGGCG 240 pCC4_Rv Build of CrisprCas plasmid AATTGGGTAC pCC- 241 backbone GTAAAAAGGCCGCGTTGC Build of CrisprCas plasmid _Fw pCC- 242 backbone CATCTGTGCGGTATTTCACAC Build of CrisprCas plasmid _Rv S-Cas9- CrisprCas Plasmid 243 TCTTCTTGAAGTAGTCTTCC 1_Rv Sequencing S-Cas9- CrisprCas Plasmid 244 TCTGACTACGACGTTGAC 2_Fw Sequencing S-Cas9- CrisprCas Plasmid 245 GGCTATTGTTGACTTGTTG 3_Fw Sequencing pCC4- TGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGC CrisprCas Plasmid 250 new_Rv CAGCAACGCGGGCTAGGGATAACAGGGTAATTC Sequencing TCGGCGTTCGAAACTTCTCCGCAGTGAAAGATAAAT CC_Ilv2- ILV2 targeting seq for 252 GATCAATTGCCACGAGCGGTAGACGTTTTAGAGCTA CrisprCas Fw GAAATAGCAAGTTAAAATAAGGCTAGTCC GGACTAGCCTTATTTTAACTTGCTATTTCTAGCTCTAA CC_Ilv2- ILV2 targeting seq for 253 AACGTCTACCGCTCGTGGCAATTGATCATTTATCTTT CrisprCas Rv CACTGCGGAGAAGTTTCGAACGCCGA CC_PDC GCAGTGGTTTCAGAAATGTGTTTTAGAGCTAGAAATA PDC1 and PDC5 targeting seq 254 15-Fw GCAAGTTAAAATAAGG for CrisprCas CC_PDC ACATTTCTGAAACCACTGCGATCATTTATCTTTCACTG PDC1 and PDC5 targeting seq 255 15-Rv CGGAG for CrisprCas ACTTATTTCACATAATCAATCTCAAAGAGAACAACACA DR_PDC ATACAATAACAAGAAGAACAAAGCTAATTAACATAAA 256 PDC5 Donor (CrisprCas) 5-Fw ACTCATGATTCAACGTTTGTGTATTTTTTTACTTTTGA AGGTTAT ATAACCTTCAAAAGTAAAAAAATACACAAACGTTGAAT DR_PDC CATGAGTTTTATGTTAATTAGCTTTGTTCTTCTTGTTA 257 PDC5 Donor (CrisprCas) 5-Rv TTGTATTGTGTTGTTCTCTTTGAGATTGATTATGTGAA ATAAGT TCTCAATTATTATCTTCTACTCATAACCTCACGCAAAA DR_PDC TAACACAGTCAAATCAATCAAAGCGATTTAATCTCTAA 258 PDC1 Donor (CrisprCas) 1-Fw TTATTAGTTAAAGTTTTATAAGCATTTTTATGTAACGA AAAATA
117
PRIMER LIST
TATTTTTCGTTACATAAAAATGCTTATAAAACTTTAACT DR_PDC AATAATTAGAGATTAAATCGCTTTGATTGATTTGACTG 259 PDC1 Donor (CrisprCas) 1-Rv TGTTATTTTGCGTGAGGTTATGAGTAGAAGATAATAA TTGAGA Cen- GGCGTATCACGAGGCCCTTTCGTCAGGCCTATGAAA 270 Building of pWG109 Ilv2t_Fw GGTGACAAACGCCTAG Ilv2t- CGAAATTGTTCCTACGAGAGGCCTTTGAAAATTGATT 271 Building of pWG109 Hxt7p_Rv CTGTTGTATTTATCTCC Ilv2t- ATAAATACAACAGAATCAATTTTCAAAGGCCTCTCGT 272 Building of pWG109 Hxt7p_Fw AGGAACAATTTCGGGC Hxt7p- TGGTTGGAAGCCATGCGGCCGCTTTTTGATTAAAATT 273 Building of pWG109 IlvD_Rv AAAAAAACTTTTTGTTTTTGTG Hxt7p- AAAAGTTTTTTTAATTTTAATCAAAAAGCGGCCGCATG 274 Building of pWG109 IlvD_Fw GCTTCCAACCAAGATAACAAG IlvD- CATAACTAATTACATGACTCGAGGCGGCCGCTTAGTA 275 Building of pWG109 Cyc1t_Rv AGCATCACCACCCAAATC IlvD- GGGTGGTGATGCTTACTAAGCGGCCGCCTCGAGTCA 276 Building of pWG109 Cyc1t_Fw TGTAATTAGTTATGTCAC Cyc1t- TCAAAGTTACAAAAGGTACTTGAAGGATAGCAAATTA 277 Building of pWG109 Gus1t_Rv AAGCCTTCGAGCG Cyc1t- CGCTCGAAGGCTTTAATTTGCTATCCTTCAAGTACCT 278 Building of pWG109 Gus1t_Fw TTTGTAACTTTG Gus1t- TTAAAAGAAATAAAAAATAGGGATCCGCATCACATAA 279 Building of pWG109 Aro10_Rv GTAATGTATATACATATTTATG Gus1t- ATGTATATACATTACTTATGTGATGCGGATCCCTATTT 280 Building of pWG109 Aro10_Fw TTTATTTCTTTTAAGTGCCGC Aro10- AAACAACCTATACGCAAGAAAGGGATCCATGGCACC 281 Building of pWG109 Sti1p_Rv TGTTACAATTGAAAAG Aro10- TTTCAATTGTAACAGGTGCCATGGATCCCTTTCTTGC 282 Building of pWG109 Sti1p_Fw GTATAGGTTGTTTTAG Sti1p- CCTATCATTATTTACGTAATGACCCAAATTTTCCCCCC 283 Fba1p_R Building of pWG109 GTCATAAGTTC v Sti1p- GAACTTATGACGGGGGGAAAATTTGGGTCATTACGT 284 Fba1p_F Building of pWG109 AAATAATGATAGG w Fba1p- GCTGGTTCTGGCATCCCGGGTTTGAATATGTATTACT 285 Building of pWG109 Ilv2_Rv TGGTTATGGTTATATATG Fba1p- ATAACCATAACCAAGTAATACATATTCAAACCCGGGA 286 Building of pWG109 Ilv2_Fw TGCCAGAACCAGCTCCATC Ilv2- CTATCGATTTCAATTCAATTCAATCCCGGGTTAGTGC 287 PGK1t_R Building of pWG109 TTACCACCGGTTCTC v Ilv2- GAACCGGTGGTAAGCACTAACCCGGGATTGAATTGA 288 PGK1t_F Building of pWG109 ATTGAAATCGATAGATC w Pgk1t- ATTTCACGGATTTCATTAGTAATTGAAATAATATCCTT 289 Building of pWG109 Tub1t_Rv CTCGAAAGCTTTAAC Pgk1t- AAAGCTTTCGAGAAGGATATTATTTCAATTACTAATGA 290 Building of pWG109 Tub1t_Fw AATCCGTGAAATAATC Tub1- GATACGTTGTTGACACTTCTAAATAACCGCGGGGTTC 291 Building of pWG109 Adh2_Rv ACTCTCGCCCCC Tub1- GGGGGCGAGAGTGAACCCCGCGGTTATTTAGAAGT 292 Building of pWG109 Adh2_Fw GTCAACAACGTATC Adh2- CAGGATTTACATTTTGAAAGCCGCGGATGTCTATTCC 293 Building of pWG109 Reh1t_Rv AGAAACTCAAAAAGC Adh2- TTTTTGAGTTTCTGGAATAGACATCCGCGGCTTTCAA 294 Building of pWG109 Reh1t_Fw AATGTAAATCCTGCTG Reh1- CGAAAAACCCGTATGGACATACAGAACTTGGTACATA 295 Building of pWG109 Glk1_Rv CCCTAATTTAC
118
PRIMER LIST
Reh1- GTAAATTAGGGTATGTACCAAGTTCTGTATGTCCATA 296 Building of pWG109 Glk1_Fw CGGGTTTTTCG Glk1p- TGTTGAAGTAGTTAGCCATGGCGCGCCCTTGTGTAT 297 Building of pWG109 IlvC_Rv GATAGAGTTGTATTAGTGG Glk1p- AATACAACTCTATCATACACAAGGGCGCGCCATGGC 298 Building of pWG109 IlvC_Fw TAACTACTTCAACACCTTG IlvC- TCAAATACATTCATATTAGCCTAAGGCGCGCCTTAAC 299 Building of pWG109 Ptp3t_Rv CAGCAACAGCAATTCTCTTC IlvC- ATTGCTGTTGCTGGTTAAGGCGCGCCTTAGGCTAAT 300 Building of pWG109 Ptp3t_Fw ATGAATGTATTTGATCTC Ptp3t- ATGCTTTTGAAATAAATGTTTTTGAAATGCTAGCCTAT 301 Building of pWG109 Ilv2t_Rv TGGGATGAACTAAGCGTAC Ptp3t- TAGTTCATCCCAATAGGCTAGCATTTCAAAAACATTTA 302 Building of pWG109 Ilv2t_Fw TTTCAAAAGCATTTTC Ilv2t- TTTTACGGTTCCTGGCCTTTTGCTGGCTAGCCAGAG 303 Building of pWG109 pBR_Rv CGTTTAGCTGGCTC Ilv2t- GAGCCAGCTAAACGCTCTGGCTAGCCAGCAAAAGGC 304 Building of pWG109 pBR_Fw CAGGAACC Cen- GGCGTTTGTCACCTTTCATAGGCCTGACGAAAGGGC 305 Building of pWG109 Ilv2t_Rv CTCGTGATAC S1- 306 pCenIlv_F GATCGCAGGTGTTTTACTGG Sequencing of pWG109 w S2- 307 pCenIlv_ CAACAAGATTGTATAGTATACCCTTC Sequencing of pWG109 Rv S3- 308 pCenIlv_F CATCAGTGCCCAACTCAG Sequencing of pWG109 w S4- 309 pCenIlv_ TGTCTCAAGTTCAAGGTGTTG Sequencing of pWG109 Rv S5- 310 pCenIlv_F GATGGACAGATTGTCTAACCC Sequencing of pWG109 w Pa_RasA TTTTAATTTTAATCAAAAAGTTAACATGTCTAACTTGTT Cloning of rspA (Enolase) into 323 _Fw CATCACTAACG pRS62 vectors Pa_RasA ACTAATTACATGACTCGAGGTCGACTCATGGTCTCCA Cloning of rspA (Enolase) into 324 _Rv AACAGTACC pRS62 vectors M_Pan- GTTGCACGACGCTNNKGAAAGAATCACTCCAATCAA 329 H225X- Mutation rspA - His225X CGC Fw M_Pan- GGAGTGATTCTTTCMNNAGCGTCGTGCAACAATTCA 330 H225X- Mutation rspA - His225X AC Rv M_Pan- ACTGCTTGGNNKTCTCCAGGTNNKATCTCTCCAATCG Mutation rspA - His325X ; 333 H325X_F GTGTTTGTG Asp329X w M_Pan- AGAGATMNNACCTGGAGAMNNCCAAGCAGTTCTAAC Mutation rspA - His325X ; 334 H325X_R ACCGTTC Asp329X v Glk1- ACTCTATCATACACAAGGGCGCGCCATGAAGCAAAT 335 Ilv5mut_F Building of pWG110 TAACTTCGGTGG w Ilv5mut- CATTCATATTAGCCTAAGGCGCGCCTTATTGGTTTTC 336 Building of pWG110 PTP3_Rv TGGTCTCAACTTTC Fba1p- TTCTTTTGTCATATATAACCATAACCAAGTAATACATA 337 KanMX_F Building of pWG109 TTCAAACCCGGGATGGGTAAGGAAAAGACTCACG w
119
PRIMER LIST
Ilv2- GAGAAAAGAAAAAAATTGATCTATCGATTTCAATTCAA 338 KanMX_R TTCAATCCCGGGTTAGAAAAACTCATCGAGCATCAAA Building of pWG109 v TG AGTTTTTTTAATTTTAATCAAAAAGTTAACATGTCGTC Cloning of JEN1 into pRS62 339 Jen1-Fw GTCAATTACAGATG vectors ACATAACTAATTACATGACTCGAGGTCGACTTAAACG Cloning of JEN1 into pRS62 340 Jen1-Rv GTCTCAATATGCTCC vectors AGTTTTTTTAATTTTAATCAAAAAGTTAACATGTCTGA Cloning of ADY2 into pRS62 341 ADY2-Fw CAAGGAACAAACGA vectors ACATAACTAATTACATGACTCGAGGTCGACTTAAAAG Cloning of ADY2 into pRS62 342 Ady2-Rv ATTACCCTTTCAGTAGATGG vectors MK- 343 PanRasA ATGTCTAACTTGTTCATCACTAACG Random mutagenese of rspA _Fw MK- 344 PanRasA TCATGGTCTCCAAACAGTACC Random mutagenese of rspA _Rv MK-HR- AAAAACAAAAAGTTTTTTTAATTTTAATCAAAAAGTTAA 345 PanRasA Random mutagenese of rspA CATGTCTAACTTGTTCATCACTAACG _Fw MK-HR- TGTAAGCGTGACATAACTAATTACATGACTCGAGGTC 346 PanRasA Random mutagenese of rspA GACTCATGGTCTCCAAACAGTACC _Rv ATGTACCCATACGACGTTCCAGACTACGCTGCTGGT Ilv3-Ha- Cloning of ILV3 with HA-tag 351 TCTCCAGGTTCTGGTGCTAAGAAGTTGAACAAGTACT into pRS62 vectors N_Fw C ACCAGAACCTGGAGAACCAGCAGCGTAGTCTGGAAC p76N-Ha- Cloning of ILV3 or ILV5 with 352 GTCGTATGGGTACATGTTAACTTTTTGATTAAAATTAA HA-tag into pRS62 vectors N_Rv AAAAACTTTTTGTTTTTG GCTGGTTCTCCAGGTTCTGGTTACCCATACGACGTT p62N-Ha- Cloning of ILV3 or ILV5 with 353 CCAGACTACGCTTAATAAGTCGACCTCGAGTCATGTA HA-tag into pRS62 vectors C_Fw ATTAG Ilv3-Ha- TTATTAAGCGTAGTCTGGAACGTCGTATGGGTAACCA Cloning of ILV3 with HA-tag 354 C_Rv GAACCTGGAGAACCAGCAGCGTCCAAAACACAACC into pRS62 vectors Ilv5-Ha- TTATTAAGCGTAGTCTGGAACGTCGTATGGGTAACCA Cloning of ILV5 with HA-tag 355 C_Rv GAACCTGGAGAACCAGCACCAGCAACAGCAATTCTC into pRS62 vectors Ani_DgdA AGTTTTTTTAATTTTAATCAAAAAGTTAACATGGTTAA Cloning of A2RAU0 (Enolase) 359 _Fw GATCAAGTCTATCGAATAC into pRS62 vectors Ani_DgdA ACATAACTAATTACATGACTCGAGGTCGACTCACCAT Cloning of A2RAU0 (Enolase) 360 _Rv TCTCTGATACCACCGTC into pRS62 vectors CC_PDR CCCTTGCAGCGGCACATTCTGTTTTAGAGCTAGAAAT PDR12 targeting seq for 367 12-Fw AGCAAGTTAAAATAAGG CrisprCas CC_PDR AGAATGTGCCGCTGCAAGGGGATCATTTATCTTTCAC PDR12 targeting seq for 368 12-Rv TGCGGAG CrisprCas DR_PDR GGTTTACAGATTTATTGTTATTGTTCTTATTAATAAAA 369 AAACAAGCAAGCAAATATATTTGTATTTCGTGGTTTAA PDR12 Donor (CrisprCas) 12-Fw CACA DR_PDR TGTGTTAAACCACGAAATACAAATATATTTGCTTGCTT 370 GTTTTTTTATTAATAAGAACAATAACAATAAATCTGTA PDR12 Donor (CrisprCas) 12-Rv AACC A1- 371 TCGATCTTTCGGCTATGG PDR12 deletion control PCR PDR12 A4- 372 GCGACAGACATTTGTTGG PDR12 deletion control PCR PDR12 STIp- CAACCTATACGCAAGAAAGGGATCCATGTACACTGTT 373 Build of pWG112 KivD_Fw GGTGACTACTTG KivD- TACATTACTTATGTGATGCGGATCCTTAAGACTTGTTT 374 Build of pWG113 GUSt_Rv TGTTCAGCGAAC TGAGCTAAGAGGAGATAAATACAACAGAATCAATTTT DR_Ilv2- ILV2 Donor (CrisprCas) - 375 CAAAGACATGTCATTGTTACAACGGGTGTGGGGCAA CenPK Fw_Neu CATCAAA TTTGATGTTGCCCCACACCCGTTGTAACAATGACATG DR_Ilv2- ILV2 Donor (CrisprCas) - 376 TCTTTGAAAATTGATTCTGTTGTATTTATCTCCTCTTA CenPK Rv_Neu GCTCA 120
PRIMER LIST
Ilv6- GACATAACTAATTACATGACTCGAGTTACAAAACAGC Cloning of ILV6D into pRS62 388 D149_Rv GTAAACTGGAAC vectors Ilv3- CAGAGCCTGAGCCTGCTGGACTAGTAGCGTCCAAAA Cloning of ILV3 (synZip1 C-t) 389 L3_Rv CACAACC into pRS62 vectors TCCAGCAGGCTCAGGCTCTGCTAGCAATCTAGTCGC Cloning of ILV3 (synZip1 C-t) 390 L3-Z1_Fw TCAGCTAGAG into pRS62 vectors Z1- AACTAATTACATGACTCGAGTCATTCCTCAATCTTTTT Cloning of ILV3 (synZip1 C-t) 391 pRS62K_ CCTTAAG into pRS62 vectors Rv pRS62K- TTTTTAATTTTAATCAAAAAATGAATCTAGTCGCTCAG Cloning of ILV3 (synZip1 N-t) 392 Z1_Fw CTAG into pRS62 vectors CAGAGCCTGAGCCTGCTGGACTAGTTTCCTCAATCTT Cloning of ILV3 (synZip1 N-t) 393 Z1-L3_Rv TTTCCTTAAGTTAGC into pRS62 vectors L3- TCCAGCAGGCTCAGGCTCTGCTAGCATGGCTAAGAA Cloning of ILV3 (synZip1 N-t) 394 Ilv3_Fw GTTGAACAAGTAC into pRS62 vectors TCCAGCAGGCTCAGGCTCTGCTAGCGCCAGAAATGC Cloning of ILV3 (synZip2 C-t) 395 L3-Z2_Fw ATACTTAAGG into pRS62 vectors Z2- AACTAATTACATGACTCGAGTTATTGTTCATGAGAGG Cloning of ILV3 (synZip2 C-t) 396 pRS62K_ CCACC into pRS62 vectors Rv pRS62K- TTTTTAATTTTAATCAAAAAATGGCCAGAAATGCATAC Cloning of ILV3 (synZip2 N-t) 397 Z2_Fw TTAAGG into pRS62 vectors CAGAGCCTGAGCCTGCTGGACTAGTTTGTTCATGAG Cloning of ILV3 (synZip2 N-t) 398 Z2-L3_Rv AGGCCACC into pRS62 vectors Ilv5- CAGAGCCTGAGCCTGCTGGACTAGTACCAGCAACAG Cloning of ILV5 (synZip1 C-t) 399 L3_Rv CAATTCTC into pRS62 vectors L3- TCCAGCAGGCTCAGGCTCTGCTAGCATGGCTAACTA Cloning of ILV5 (synZip1 N-t) 400 Ilv5_Fw CTTCAACACC into pRS62 vectors TTTTTAATTTTAATCAAAAAATGGCTTTGTCTAGAGTT Cloning of ilvHm into pRS62 408 IlvHm_Fw ATCG vectors AACTAATTACATGACTCGAGTCAAGAAACTCTCAAAA Cloning of ilvHm into pRS62 409 IlvHm_Rv CGTC vectors CC-Yro2- TTCACCATGGCTTCTAACTTGTTTTAGAGCTAGAAAT YRO2 and MRH1 targeting 410 Mrh1_Fw AGCAAGTTAAAATAAGG seq for CrisprCas CC-Yro2- AAGTTAGAAGCCATGGTGAAGATCATTTATCTTTCAC YRO2 and MRH1 targeting 411 Mrh1_Rv TGCGGAG seq for CrisprCas ATAACTTAAAGTGACACTATTTTTTAAAAAAAGCATCA 412 DR-Yro2 AAATGCTTTGATCTGCTGATGTAGCAATGTTTTTTTCC YRO2 Donor (CrisprCas) TTCT CATCTTCCTTTAACCCACAGAACAAAGAAGAAAAATA 413 DR-Mrh1 ACAGCCAATCGTCATGTCAATTATGTGCTACTTTCTT MRH1 Donor (CrisprCas) CTTTCT CC- TCTCCTGTTAGTGATCTTTAGTTTTAGAGCTAGAAATA TPO2 and TPO3 targeting seq 414 Tpo2n3_F GCAAGTTAAAATAAGG for CrisprCas w CC- TAAAGATCACTAACAGGAGAGATCATTTATCTTTCAC TPO2 and TPO3 targeting seq 415 Tpo2n3_ TGCGGAG for CrisprCas Rv CATCTAATAACTAATCACAAAAATAATACACAAAACAA 416 DR-Tpo2 ATGCTGGTGCAAGTTTCCGGTAAAAATAATGATGTTC TPO2 Donor (CrisprCas) TAGTC AATTTTGCATTAGTACTCCTCTAGCCAAAGATAAACA 417 DR-Tpo3 GAAGAGTAACGATTTTACGAATCGAGCATAATAATGA TPO3 Donor (CrisprCas) AAAATT 421 A1-Yro2 GATTCCTCATCGGGTTGTTG YRO2 deletion control PCR 422 A4-Yro2 TTACGGACCCGAATGGATAG YRO2 deletion control PCR 423 A1-Mrh1 GGATCTCTTCACCTGGTTTC MRH1 deletion control PCR 424 A4-Mrh1 TCCTCCAGCACTAAACCTTC MRH1 deletion control PCR 425 A1-Tpo2 GAACAATATACAACTTCCTTCTCC TPO2 deletion control PCR 426 A4-Tpo2 TGTGTCGTCTTCGAATAAGC TPO2 deletion control PCR 427 A1-Tpo3 CGCCTAAGAACATTTGTCTTTG TPO3 deletion control PCR
121
PRIMER LIST
428 A4-Tpo3 AATTCCGACCCTTTGTTTAGTG TPO3 deletion control PCR CC-Hxt9- GGGGCAATCAACTTTTACTAGTTTTAGAGCTAGAAAT HXT9, HXT11 and HXT12 431 11-12_Fw AGCAAGTTAAAATAAGG targeting seq for CrisprCas CC-Hxt9- TAGTAAAAGTTGATTGCCCCGATCATTTATCTTTCACT HXT9, HXT11 and HXT12 432 11-12_Rv GCGGAG targeting seq for CrisprCas ACAATTGAGTACTAAAAGCTTTCGTATCTTACCCAATA 433 DR-Hxt9 TCTCTTCCGGTTTTTAGTAACTGGAAAAAAATACATG HXT9 Donor (CrisprCas) AACTT CACAATTTAGTTCTAAACGCTTTTGTTATTACTCAATA 434 DR-Hxt11 TCTCTTCTAGTTTTCGGTAAATTGGTAAAAAAGCAAAA HXT11 Donor (CrisprCas) AAAA ACAATTGAGTACTAAAAGCTTTCGTATCTTACCCAATA 435 DR-Hxt12 TCTCTTCTAGTTTTCGGTAAATTGGTAAAAAAGCAAAA HXT12 Donor (CrisprCas) AAAA HXT9 deletion control PCR 436 A4-Hxt9 CCACCCTTCGGAACGTTAC (A1: HDP439) HXT11 deletion control PCR 437 A1-Hxt11 GGTCTAGGCTCATCTTTCTCAG (A4: PJP06) co- TTTTTAATTTTAATCAAAAAATGGCTCCAGTTACTATC 438 Build of pWG136 Aro10_Fw G co- AACTAATTACATGACTCGAGTTACTTCTTGTTTCTCTT 439 Build of pWG136 Aro10_Rv CAAAGC CC- TGCACATGCCAGTCCGATGCGTTTTAGAGCTAGAAAT QDR1 targeting seq for 444 Qdr1_Fw AGCAAGTTAAAATAAGG CrisprCas CC- GCATCGGACTGGCATGTGCAGATCATTTATCTTTCAC QDR1 targeting seq for 445 Qdr1_Rv TGCGGAG CrisprCas TTTAGTAGAAACTCTGCTCTCAAACTTGAGTACTGCA DR-Qdr1- Donor for QDR1 and QDR2 446 ACGGTAAAAATTCAGAAAAAGTTTCGACGTGAAGAAA simultaneous deletion 2 AAAAGT A1-Qdr1- PCR control of QDR1 and 447 TGATGTCGTATGAGCTACTG 2 QDR2 deletion A4-Qdr1- PCR control of QDR1 and 448 TTATGCCCATTCAACATCCG 2 QDR2 deletion CC-Pdr5- GATGCTGTTGTTGGTGTTGCGTTTTAGAGCTAGAAAT PDR5 and PDR15 targeting 449 15_Fw AGCAAGTTAAAATAAGG seq for CrisprCas CC-Pdr5- GCAACACCAACAACAGCATCGATCATTTATCTTTCAC PDR5 and PDR15 targeting 450 15_Rv TGCGGAG seq for CrisprCas AAGTTTTCGTATCCGCTCGTTCGAAAGACTTTAGACA 451 DR-Pdr5 AAATAGAATTTTGAATTTGGTTAAGAAAAGAAACTTAC PDR5 Donor (CrisprCas) CAAGA CACACACACAAGCAAACACACTTATAATTATCAAAAA 452 DR-Pdr15 CCTTGACGTTATTTTCCTTTTTTTTAGTTATATTATCTT PDR15 Donor (CrisprCas) TTTA 453 A1-Pdr5 AATACAAACAAGGCCTCTCC PDR5 deletion control PCR 454 A4-Pdr5 AACCGTAAGGCACAGTTAAG PDR5 deletion control PCR 455 A1-Pdr15 AGGTATGGCACGATGGTAAG PDR15 deletion control PCR 456 A4-Pdr15 AAAGGCCGGTACGAGTTTAG PDR15 deletion control PCR CC- CTGCGGCATACCCGGGAGATGTTTTAGAGCTAGAAA MCH5 targeting seq for 457 Mch5_Fw TAGCAAGTTAAAATAAGG CrisprCas CC- ATCTCCCGGGTATGCCGCAGGATCATTTATCTTTCAC MCH5 targeting seq for 458 Mch5_Rv TGCGGAG CrisprCas TAAAAAGCTCTGATTTTACTATAGATTAAAAAGAAAAA 459 DR-Mch5 TAAAAGTAAGACAGTGCCCTTTATAGATCTTCAAAAA MCH5 Donor (CrisprCas) AGTAA 460 A1-Mch5 CCTAGGCGGTATTGTATGAG MCH5 deletion control PCR 461 A4-Mch5 CTGAATGGCTGAAGTTGAAG MCH5 deletion control PCR CC- GAAAATCTATGTACCCACGCGTTTTAGAGCTAGAAAT BDH1 targeting seq for 462 Bdh1_Fw AGCAAGTTAAAATAAGG CrisprCas CC- GCGTGGGTACATAGATTTTCGATCATTTATCTTTCAC BDH1 targeting seq for 463 Bdh1_Rv TGCGGAG CrisprCas
122
PRIMER LIST
DR- Donor for BDH2 and BDH1 GCAATAAGAATAACAATAAATTCATTGAACATATTTCA 464 Bdh2pro_ deletion (exchanged by IlvHm GAGTACCGGCCGCAAATTAAAG Fw cassette) DR- Donor for BDH2 and BDH1 TACAAATGAGCCGCGAGGGGCCCCAAATATTATTTT 465 Bdh1ter_ deletion (exchanged by IlvHm GTCAAATACCGCATGAGCTCGTAG Rv cassette) PCR control of BDH2 (+BDH1) 466 A1-Bdh2 TGACTGTGTTTGTGGTTCTC deletion PCR control of BDH1 (+BDH2) 467 A4-Bdh1 TCGTCTTTGTTCCCACATTC deletion Glk1p-Z2- ACAACTCTATCATACACAAGGGCGCGCCATGGCCAG Cloning of zipped ilvC6E6 into 468 IlvC_Fw AAATGCATACTTAAG pWG135 CC- TATATGGGGTTTTGCGTGTTTTAGAGCTAGAAATAGC FLR1 targeting seq for 483 FLR1_Fw AAGTTAAAATAAGG CrisprCas CC- GCAAAACCCCATATAGGCGATCATTTATCTTTCACTG FLR1 targeting seq for 484 FLR1_Rv CGGAG CrisprCas GCTATTATAAAAGAGTATCCGTTGAACGACAATCTCC 485 DR-FLR1 ACTAAAGCCGATGTTCTTACCATGAAAATTTGAATTAT FLR1 Donor (CrisprCas) AATAA 486 A1-FLR1 GGTAAGGAGCGATAACAGTG PCR control of FLR1 deletion 487 A4-FLR1 CATCCGTATGGTAAGCAAAG PCR control of FLR1 deletion CC- AGTTAGCGGTGGTCTGGGTTTTAGAGCTAGAAATAG AQR1 targeting seq for 488 AQR1_F CAAGTTAAAATAAGG CrisprCas w CC- AGACCACCGCTAACTGTGGATCATTTATCTTTCACTG AQR1 targeting seq for 489 AQR1_Rv CGGAG CrisprCas TTTTGAGAATCCAAGCTAGATTCAGAAAGTCGAATCA 490 DR-AQR1 GCATTGGCATTCTTCAATTTGATAGACACTTATCCCT AQR1 Donor (CrisprCas) GCATAT 491 A1-AQR1 CCGTAATGACTCGGAAACTC AQR1 deletion control PCR 492 A4-AQR1 GACCCAGCCAAATACAACAG AQR1 deletion control PCR CC-Leu4- ACAATGACCGTGGTTGGTTTTAGAGCTAGAAATAGCA LEU4 and LEU9 targeting seq 494 9_Fw AGTTAAAATAAGG for CrisprCas CC-Leu4- CAACCACGGTCATTGTGACAGATCATTTATCTTTCAC LEU4 and LEU9 targeting seq 495 9_Rv TGCGGAG for CrisprCas TACTGTAGACTTTTTCCTTACAAAAAGACAAGGAACA 496 DR-Leu4 ATCGAACTTTTCTGTATTTCAGGACTTATTCGCTTCTA LEU4 Donor (CrisprCas) TTTAT GGATAATACTATCGGCACATTATCATTTAGCCGCGTA 497 DR-Leu9 GCCTAGAAAGGAGTAGCTTATGATTACTCATGTTATA LEU9 Donor (CrisprCas) TATATA 498 A1-Leu4 TTGTACAGTAACGGCCAGTC LEU4 deletion control PCR 499 A4-Leu4 TTCGTCACTAACCGCCAAAC LEU4 deletion control PCR 500 A1-Leu9 GGTAACGGTCGTAGTGAATG LEU9 deletion control PCR 501 A4-Leu9 TGTTCTCCCTTCACAAAGTC LEU9 deletion control PCR tPGK- TCAATTCAATTCAATCCCGGGTTAAACCTTGGGATTG 507 Build of pWG139 Leu3_Fw AACGC Leu3D60 Build of pWG139 (Leu3 508 TCTTAAGCTCGTCGAGGGAACAACTGAATCATGC 1_Fw truncation 601) Leu3D60 Build of pWG139 (Leu3 509 GATTCAGTTGTTCCCTCGACGAGCTTAAGAGCG 1_Rv truncation 601) Leu3- TAATACATATTCAAACCCGGGATGGAAGGAAGATCA 510 Build of pWG139 pFBA_Rv GATTTTG TTAATCAAAAAGCGGCCGCATGCTTAAGGACCCTTC 511 Leu4_Fw Build of pWG139 CTC AAGATGGAATGGACACATCACCAGAATGGATAATATT Leu4mut- Build of pWG139 (Leu4 mut 512 GTTAATGGTGGCAAAGATGGCTCTCACTGAAGAATC D578Y) tCYC_Rv ACCGACATATTCGGAGACACCTACACC TGTCCATTCCATCTTTGGCCGAGGTCGAAGGTAAGA Leu4mut- Build of pWG139 (Leu4 mut 513 ATGCTGCGGCATCTGGCTCTGCATAACTCGAGTCAT D578Y) tCYC_Fw GTAATTAGTTATGTC
123
PRIMER LIST
CC- GAAGAACTGTGCTCCCGGTTTTAGAGCTAGAAATAG ECM31 targeting seq for 516 Ecm31_F CAAGTTAAAATAAGG CrisprCas w CC- GGAGCACAGTTCTTCAATGATCATTTATCTTTCACTG ECM31 targeting seq for 517 Ecm31_R CGGAG CrisprCas v DR- ATTAGCTTGCCATAAAATTAGGGAAATTTTTACTCACA 518 ATAATATATAGATAAAAATCACTGCATAGGGAAAAAA ECM31 Donor (CrisprCas) Ecm31 ACTTT A1- PCR control of ECM31 519 ATGTACACGACAGACATTCC Ecm31 deletion A4- PCR control of ECM31 520 TATTATAAAGCGGCCAGCTC Ecm31 deletion CC- TACTTTACCCGACGTCCCGTTTTAGAGCTAGAAATAG ILV1 targeting seq for 521 Ilv1_Fw CAAGTTAAAATAAGG CrisprCas CC- GACGTCGGGTAAAGTAACGATCATTTATCTTTCACTG ILV1 targeting seq for 522 Ilv1_Rv CGGAG CrisprCas CAAGCCACATTTAAACTAAGTCAATTACACAAAGTTA 523 DR-Ilv1 GTGAACCGACAATTTACTTTATAAATTTACGCAACAA ILV1 Donor (CrisprCas) CTTGTT 524 A1-Ilv1 AATTCACTAGCGGCTCCTTG ILV1 deletion control PCR 525 A4-Ilv1 ATGGCTATGTGGAAGAAGTC ILV1 deletion control PCR STIp- TATACGCAAGAAAGGGATCCATGTACACTGTTGGTG 526 Build of pWG134 KdcA_Fw ACTACTTG KdcA- TACTTATGTGATGCGGATCCCTACTTGTTTTGTTCAG 527 Build of pWG135 GUSt_Rv CGAACAAC AACATCTTTAACATACACAAACACATACTATCAGAATA ALD6 Donor (CrisprCas from 535 DR-Ald6 CATGTACCAACCTGCATTTCTTTCCGTCATATACACA Schadeweg) AAATA CC- ATTGCTGGAGTCGGCGGTTGGTTTTAGAGCTAGAAA AZR1 targeting seq for 536 Azr1_Fw TAGCAAGTTAAAATAAGG CrisprCas CC- CAACCGCCGACTCCAGCAATGATCATTTATCTTTCAC AZR1 targeting seq for 537 Azr1_Rv TGCGGAG CrisprCas AGTAACACCACGTCTACATTAGTATCTAAATTGCAGC 538 DR-Azr1 CCAGTTAAGCACGATTTAGAAGGTGGCGAGCCTCAA AZR1 Donor (CrisprCas) AGTGTAA 539 A1-Azr1 TCTTGCACTGTTACCATAGG PCR control of AZR1 deletion 540 A4-Azr1 AAGGTGACATAGAGGCATTG PCR control of AZR1 deletion CC- CCAGGAAACTTTGCAGATCGGTTTTAGAGCTAGAAAT ARR3 targeting seq for 541 Arr3_Fw AGCAAGTTAAAATAAGG CrisprCas CC- CGATCTGCAAAGTTTCCTGGGATCATTTATCTTTCAC ARR3 targeting seq for 542 Arr3_Rv TGCGGAG CrisprCas CAAGAGAACCCAACCAACAAATCATCAGGTTAGTAGA 543 DR-Arr3 ATATTGTTGACTCACCAAAAAATTACTTGGGCACCAA ARR3 Donor (CrisprCas) TGAATA 544 A1-Arr3 ATTACGCTTGCTGGATTGTC ARR3 deletion control PCR 545 A4-Arr3 ACAGTTTGCCACTTGAGTTC ARR3 deletion control PCR CC- ACAAGAGCTTGGCCAGGGTTTTAGAGCTAGAAATAG BAT2 targeting seq for 557 Bat2_Fw CAAGTTAAAATAAGG CrisprCas CC- CCTGGCCAAGCTCTTGTGGCGATCATTTATCTTTCAC BAT2 targeting seq for 560 Bat2_Rv TGCGGAG CrisprCas AAATTTAAGGGAAAGCATCTCCACGAGTTTTAAGAAC 562 DR-Bat2 GATAGTATCGCTATTGCTACGTAAAGTAATTAAAAGT BAT2 Donor (CrisprCas) TAAAAA 565 A1-Bat2 GTGAGAGGAGATCCGAAATGAG PCR control of BAT2 deletion 566 A4-Bat2 TCCACCGACATTACGGAAAC PCR control of BAT2 deletion REHp- TTTACATTTTGAAAGCCGCGGATGAAGGCTGCTGTTG 567 AdhARE1 Build of pWG140 TTAGAC _Fw AdhARE1 GGGGCGAGAGTGAACCCCGCGGTTACTTAGTGAAGT 568 Build of pWG140 -TUBt_Rv CGATAACCATTCTAC 124
PRIMER LIST
CC- ATAGGCAACGGGGGCCCAAGGTTTTAGAGCTAGAAA HXT5 targeting seq for 574 Hxt5_Fw TAGCAAGTTAAAATAAGG CrisprCas CC- CTTGGGCCCCCGTTGCCTATGATCATTTATCTTTCAC HXT5 targeting seq for 575 Hxt5_Rv TGCGGAG CrisprCas ATTTTTCTAGAAAAAAGAATATATTAGAGGTAAAGAAA 576 DR-Hxt5 GATCTCTTGTAGTGTAGGATCAACTATTTTCGCATAC HXT5 Donor (CrisprCas) TTGCA 577 A1-Hxt5 AGAAAGGAGGGCCAGAATC HXT5 deletion control PCR 578 A4-Hxt5 GTTGTTAGGCGTAGCAACC HXT5 deletion control PCR
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ZUSAMMENFASSUNG (KURZ)
APPENDIX C: ZUSAMMENFASSUNG (KURZ)
Saccharomyces cerevisiae ist ein natürlicher Produzent von Isobutanol, das als Biokraftstoff zahlreiche Vorteile gegenüber Ethanol besitzt. Isobutanol besitzt eine höhere Energiedichte, ist weniger korrosiv und ist weniger hygroskopisch als Ethanol. Isobutanol wird durch eine Kombination der Valinbiosynthese und des Ehrlich Stoffwechselweges produziert. In dieser Arbeit wurde ein Industriestamm von S. cerevisiae für die Isobutanolproduktion verwendet, bei dem die Valinbiosynthese in das Cytosol relokalisiert wurde. Die Valinbiosynthese aus Zuckern in Hefe besitzt ein Ungleichgewicht an Cofaktoren, da in der Glykolyse NADH produziert, in der Valin- Biosynthese (Ilv5) jedoch NADPH verbraucht wird. Um die Cofaktorspezifität auszugleichen, wurde Ilv5 durch die NADH-verbrauchende Mutante IlvC6E6 ausgetauscht. Zusätzlich wurden Ilv6-Varianten zur Steigerung der Isobutanolproduktion getestet, da dieses Protein die Rückkoppelungsinhibition der Valinbiosynthese kontrolliert. Keine der getesteten Ilv6-Varianten konnte die Isobutanolproduktion steigern. Da die Isobutanolausbeute nicht erhöht werden konnte, wurden die Limitierungen dieses Stoffwechselwegs eingehender untersucht. Die Umwandlung von 2,3-Dihydroxyisovalerat (DIV) zu 2-Ketoisovalerat (KIV) wurde als Hauptlimitierung des Isobutanolstoffwechselweges identifiziert, da hohe Konzentrationen von Acetoin, 2,3-Butandiol und insbesondere DIV im Überstand des Mediums gemessen wurden, jedoch kein KIV oder Isobutyraldehyd. Diese Reaktion wird durch Ilv3 katalysiert, die für ihre Aktivität Eisen-Schwefel-Cluster (FeS) benötigt. Die Assemblierung und Bereitstellung von FeS könnte unzureichend sein und daher sollte die Assemblierung und Bereitstellung von FeS im Cytosol gesteigert werden. Die Aktivität von Ilv3Δ19 konnte hierdurch nicht gesteigert werden. Hiernach wurden Alternativen für die Substitution von Ilv3Δ19 getestet. Heterologe Ilv3- Orthologe mit möglichen Vorteilen wurden untersucht, aber Ilv3Δ19 war von allen untersuchten Kandidaten die vielversprechendste Auswahl. Zusätzlich wurden auch Zuckersäure-Enolasen als Alternativen für Ilv3Δ19 untersucht. Diese Enolasen katalysieren die Dehydration ihres Substrats in der gleichen Art und Weise wie Ilv3, verwenden jedoch Mg2+ als Cofaktor. Eine der untersuchten Enolasen konnte die Valinauxotrophie einer iv3Δ-Mutante komplementieren, sie vermittelte jedoch nur sehr langsames Wachstum und die Aktivität konnte nicht durch Mutagenesemethoden gesteigert werden. Interessanterweise wurde festgestellt, dass DIV aus der Zelle 126
ZUSAMMENFASSUNG (KURZ) sekretiert wird und nicht wieder aufgenommen werden kann, was die Isobutanolproduktion zusätzlich erniedrigt. Um den Verlust von DIV zu vermindern, sollten mögliche DIV-Exporter deletiert und Substrate Channeling von DIV zwischen IlvC6E6 und IlvΔ19 etabliert werden. Es wurde in einer Transkriptomanalyse ein Stamm, der hohe Mengen an DIV sekretiert, mit einem Stamm, der kein DIV produziert, verglichen, um mögliche DIV-Exportproteine zu finden. In dem DIV-sekretierenden Stamm wurden zahlreiche hochregulierte Transporter gefunden, aber Deletionsanalysen ergaben keine eindeutigen Hinweise auf einen DIV-Exporter. Für die Substrate Channeling Strategie wurden künstliche Enzymnetze durch Fusion von IlvC6E6 und Ilv3Δ19 mit synthetischen Zippern konstruiert. Die Zipper binden mit hoher Affinität aneinander und die Enzyme bilden Oligomere. Die Verwendung dieses Enzymnetzes steigerte die Isobutanolproduktion um 17% und zusätzlich die Produktausbeute von 3-Methyl-butanol um 25%. Neben der Limitierung durch die Ilv3-Aktivität wird die Isobutanolproduktion durch die hohe Produktion von Ethanol als Nebenprodukt beeinflusst. Die Pyruvatdecarboxylase-Gene PDC1 und PDC5 wurden deletiert, um die Ethanolproduktion zu unterbinden. Zusätzlich wurden auch die Butandioldehydrogenase-Gene BDH1 und BDH2 deletiert, um eine NADH-treibende Kraft in Richtung Isobutanolproduktion zu erzeugen. Die Isobutanolausbeute von diesem Stamm war jedoch geringer als im Stamm ohne diese Deletionen. In dem Deletionsstamm wurde eine hohe Produktion von Isobuttersäure beobachtet. Das Einbringen unterschiedlicher KIV-Decarboxylasen und Isobutanoldehydrogenasen konnte die Isobutanolproduktion nicht steigern. Anschließend wurden andere Stoffwechselwege (Valin-, Leucin-, Isoleucin-, und Panthetonatbiosynthese) unterbunden, um den Fluss von Intermediaten in Richtung Isobutanolproduktion zu verstärken. Dennoch waren die Isobutanolausbeuten gering und die Hauptnebenprodukte waren Glycerin, Acetoin, DIV und Isobuttersäure. Ohne eine Steigerung der Dihydroxysäure-Dehydratase Aktivität scheinen die weiteren Optimierungen der Isobutanolproduktion keinen entscheidenden Effekt zu zeigen.
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ACKNOLEDGEMENTS
APPENDIX D: ACKNOWLEDGEMENTS
I would like to thank Prof. Dr. Eckhard Boles for the support and positivism with the project and my ideas. I really appreciated the moments of discussions, sometimes more than isobutanol, but very important for my future life and career.
I would like to thank Prof. Dr. Jörg Soppa by friendly accept to be my second reviewer.
I am thankful for the support of the people that helped me in this work, namely, Prof. Dr. Eugen Proschak, Dr. Mislav Oreb, Lisa Eberle, Sabrina Zöller, Elizabeth Gehr, Alexander Bissel, Laura Pöschel, Navadurka Navabalasingam and Andrea Schudok.
I would like to express how fortunate I was to be in the AK Boles. I am really glad for all the support and fellowship of the secretary, postdocs, technicians and students, which became friends that I will bring for my whole life. Individually, I would like to thank Feline, Manuela, Stefan and Thomas for the support and the friendship.
I also would like to thank the other friends outside the working group that were very important in these four years. I do not want to mention names, in order to not forget any one, but just know that if one day we shared a beer and laughs together, you were important to make Frankfurt my home.
I would like to thank the Brazilian Counsel of Technological and Scientific Development (CNPq) by the scholarship and the support in these years.
And my special thanks goes to some people from the other side of the ocean: Neide and Antônio Carlos (my parents), Wellington (my brother), Maria and Adelina (my grandmothers). I am thankful for the strength and comprehension, always lifting and supporting me during these four years of adventure. [ E meus especiais agradecimentos vão às pessoas que estavam do outro lado do oceano: Neide e Antônio Carlos (meus pais), Wellington (meu irmão), Maria e Adelina (minhas avós). Eu os agradeço por terem segurado a saudade, serem fortes e compreensivos, sempre me dando força e suporte durante estes quatro anos de aventura. ] 128
CURRICULUM VITAE
APPENDIX E: CURRICULUM VITAE
Wesley Cardoso Generoso
Birth: 19.05.1987 – Birigui/São Paulo – Brazil
Address: Rua Basílio Troncoso, 653. Jardim Marister. Birigui/SP 16204-255 Brazil
Education / Titration
Ph.D. in Natural Science. 2013 - current Title: Exploring the limitations of isobutanol production by engineered industrial Saccharomyces cerevisiae strains. Supervisor: Eckhard Boles Johann Wolfgang Goethe Universität Frankfurt, Frankfurt am Main, Germany
2010 - 2012 Master in Evolutionary Genetics and Molecular Biology. Title: Recombinant expression and characterization of an undescribed endoxylanase from Trichoderma harzianum, Supervisor: Flávio Henrique da Silva Federal University of São Carlos, São Carlos/SP, Brazil
2006 - 2010 Undergraduate in Biotechnology (Emphasis in Agriculture). Federal University of São Carlos, São Carlos/SP, Brazil
1998 - 2009 Elementary and high school. Ricardo Peruzzo state school, Birigui/SP, Brazil
Articles published in journals
Generoso WC, Gottardi M, Oreb M, Boles E. 2016. Simplified CRISPR-Cas genome editing for Saccharomyces cerevisiae. Journal of Microbiological Methods, v. 127, p. 203-205.
Generoso WC, Malagó-jr W, Pereira-jr N, Henrique-Silva F. 2016. Triggering the expression of cellulolytic genes using a recombinant endoxylanase from Trichoderma harzianum IOC-3844. BioEnergy Research, v. 9 (3), p. 931–941.
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CURRICULUM VITAE
Generoso WC, Schadeweg V, Oreb M, Boles E. 2015. Metabolic engineering of Saccharomyces cerevisiae for production of butanol isomers. Current Opinion in Biotechnology, v. 33, p. 1-7.
Urbaczek AC, Ximenes VF, Afonso A, Generoso WC, Nogueira CT, Tansini A, Cappelini LT, Malagó-jr W, Henrique-Silva F, Fonseca LM, Costa PI. 2015. Recombinant hepatitis C virus- envelope protein 2 interactions with low-density lipoprotein/CD81 receptors. Memórias do Instituto Oswaldo Cruz (Online).
Urbaczek AC, Ribeiro LCA, Ximenes VF, Afonso A, Nogueira CT, Generoso WC, Alberice JV, Rudnicki M, Ferrer R, Fonseca LM, Costa PI. 2014. Inflammatory response of endothelial cells to hepatitis C virus recombinant envelope glycoprotein 2 protein exposure. Memórias do Instituto Oswaldo Cruz (Online).
Vizoná-Liberato M, Generoso WC, Malagó-Jr W, Henrique-Silva F, Polikarpov I. 2012. Crystallization and preliminary X-ray diffraction analysis of endoglucanase III from Trichoderma harzianum. Acta Crystallographica section F, v. 68, p. 306-309.
Generoso WC, Malagó-jr W, Pereira-jr N, Henrique-Silva F. 2012. Recombinant expression and characterization of an endoglucanase III (cel12a) from Trichoderma harzianum (Hypocreaceae) in the yeast Pichia pastoris. Genetics and Molecular Research, v. 11, p. 1544-1557.
Generoso WC, Oliveira VH, Kano AM, Santos BK, Eto DK, Verruma-Bernardi MR. 2012. Avaliação sensorial de iogurtes probióticos comerciais. Nutrição Brasil, v. 11, p. 161-168-168.
Generoso WC, Borges MTMR, Ceccato-Antonini SR, Marino ALF, Silva MVM, Nassu RT, Verruma-Bernardi MR. 2009. Avaliação microbiológica e físico-química de açúcares mascavo comerciais. Revista do Instituto Adolfo Lutz, v. 68, p. 259-268.
Recent event participations
2016. 14th International Congress on Yeast. Oral and poster presentations: Isobutanol production with Saccharomyces cerevisiae is limited by secretion of a pathway intermediate. Awaji Yumebutai, Japan.
2014. Summer School Biotransformations 2014. Poster presentation: Isobutanol production by an industrial Saccharomyces cerevisiae strain. Bad Herrenalb, Germany.
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2013. PhD course "Industrial Biotechnology for Lignocellulose-Based Processes". Gothenburg, Sweden.
2013. 10th Metabolic Engineering. Poster presentation: Isobutanol production by an industrial Saccharomyces cerevisiae strain. Vancouver, Canada.
2013. 26th International Conference on Yeast Genetics and Molecular Biology. Frankfurt am Main, Germany.
2012. 41th Annual Meeting of the Brazilian Society for Biochemistry and Molecular Biology. Poster presentation: Glycosylation influences the enzymatic activity of the recombinant Xyn3 from Trichoderma harzianum expressed in Pichia pastoris. Foz do iguaçu/PR, Brazil.
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