Genetic Optimisation of C6 and C5 Sugar Fermentation with Saccharomyces cerevisiae

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1 Genetic Optimisation of C6 and C5 Sugar Fermentation with Saccharomyces cerevisiae Prof. Dr. Eckhard Boles Institute for Molecular Biosciences Goethe-University Frankfurt/Main

2 World Oil Production

3 Bio-refinery our contribution: yeast design fermentation CO 2

4 Biomass Lignocellulosic-ethanol production Pretreatment (Solubilisation of hemicellulose) Enzymatic hydrolysis (Conversion of cellulose to sugar) SSF Recirculation of process streams Energy and power production Lignin Fermentation (Conversion of sugars to ethanol) Distillation and evaporation Waste management Ethanol

5 Cellulose (33-51 %) LIGNOCELLULOSE (WOOD) Lignin (21-32 %) Extractives (1-5 %) Hemicellulose (19-34 %) HEXOSES HO HO CH 2 O CH 2 O CH 2 HO O HO CH 3 O β-d-glucopyranose β-d-mannopyranose α-d-galactopyranose α-l-rhamnopyranose FERMENTATION INHIBITORS PHENOLICS R R EXTRACTIVES Terpenoids etc. R FURALDEHYDES HO CH 2 5-hydroxymethyl- 2-furaldehyde (HMF) O O CHO 2-furaldehyde (furfural) CHO ETHANOL ALIPHATIC ACIDS CH 3 CO acetic acid HCO formic acid CH 3 COCH 2 CH 2 CO levulinic acid PENTOSES HO H 2 C URONIC ACIDS O CO O O H 3 CO CO HO O β-dxylopyranose α-l-arabinofuranose 4 O-methylα-D-glucuronopyranose α-d-galacturonopyranose

6 Mixed sugar utilisation in a very harsh environment

7 Bench-marking Ethanol production Water economy Inhibitor tolerance Product yield Productivity > 40 g ethanol / L > 0.4 g ethanol / g sugar > 0.5 g ethanol / g yeast x h Strategies for: 1. Improved C5 sugar fermentation 2. Enhancement of ethanol yields from total sugars 3. Inhibitor-tolerant yeasts

8 1. C5 sugar fermentation with Saccharomyces cerevisiae

9 Optimization of arabinose fermentation L-Arabinose Bacterial genes introduced into yeast Isomerase Kinase L-Ribulose L-Ribulose-5-P 4-Epimerase D-Xylulose-5-P Ethanol

10 Codon optimization improves arabinose fermentation Translation of genetic information into proteins Degenerated code Genetic code is degenerated: amino acids are encoded by more than one codon = synonymous codons Bacteria use different synonymous codons than yeast Yeast genes for sugar fermentation have a highly restricted codon usage Construction of synthetic arabinose fermentation genes with codon usage adapted to yeast sugar fermentation

11 Arabinose fermentation with codon-optimised yeast strain optimised strain old strain Complete consumption of arabinose Ethanol yield improved by 20%: 0.47 g / g arabinose Productivity improved but still very low (<0.01 g / g x h) Wiedemann and Boles, patent filed

12 Improving pentose uptake into S. cerevisiae Co-consumption Glucose Pentose High fermentation rate at low pentose concentrations

13 A pentose transporter from Pichia stipitis supports specific uptake of arabinose into S. cerevisiae cells Glucose Pentose Pentose transporter 2% arabinose empty vector Gal2 1 empty vector Gal2 2% glucose Keller and Boles, patent filed

14 2. Enhancement of ethanol yields from total sugars Nissen et al. (2000) Metab. Eng. 2, Bro et al. (2006) Metab. Eng. 8, NH4 + assimilation switched from NADPH consuming to NADH consuming 10% more ethanol 38% less glycerol Substitution of glycerol production with ethanol production by expression of an NADP + -GAPDH 3% more ethanol 40% less glycerol 25% more ethanol on glucose/xylose mixtures

15 Global transcription machinery engineering for increased ethanol tolerance and more efficient fermentation Alper et al. (2006) Science 314, Interconnected network of genes Increase in ethanol tolerance growth on 15% ethanol Increase in ethanol productivity: 41% (0.31 g / g dry mass x h)

16 3. Inhibitor-tolerant yeast strains Phenolic compounds Furaldehydes (furfural, HMF) Aliphatic acids (acetic acid) Directed adaptation method growth in the presence of inhibitors In-situ detoxification of the inhibitors Pentosephosphate pathway Glycolysis Liu (2006) Appl. Microbiol. Biotechnol. 73:27-36

17 Future perspectives: Stable integration of C5-converting pathways into industrial, inhibitor-tolerant yeast strains Integration of beneficial genes for enhanced ethanol yields Evolutionary engineering for improved co-consumption of C6 and C5 sugars in lignocellulosic, un-detoxified hydrolysates

18 Beate Wiedemann Eckhard Boles Marco Keller S. cerevisiae Christoph Schorsch

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