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Engineering yeasts for next generation ethanol production

Engineering yeasts for next generation ethanol production. Riaan den Haan 1 , D . C. la Grange 1 , M. Mert 1 , H . Kroukamp 1 , M. Saayman 1 , M. Viktor 1 , J.E. McBride 3 , L.R. Lynd 3 , M. Ilmen 4 , M. Penttilä 4 , J.F. Görgens 2 , M. Bloom 1 , W.H. van Zyl 1

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Engineering yeasts for next generation ethanol production

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  1. Engineering yeasts for next generation ethanol production Riaan den Haan1, D.C. la Grange1, M. Mert1, H. Kroukamp1, M. Saayman1, M. Viktor1, J.E. McBride3, L.R. Lynd3,M. Ilmen4, M. Penttilä4, J.F. Görgens2, M. Bloom1, W.H. van Zyl1 (1) Depts. of Microbiology, and (2) Process Engineering Stellenbosch University, South Africa (3) Mascoma Corporation, Lebanon, NH (4) VTT Technical Research Centre of Finland

  2. Introduction • Biofuels such as ethanol have gained significant interest due to environmental concerns and issues such as energy security - resulting in the current first generation ethanol market • Most of the ethanol produced worldwide is produced from starch • The development of a yeast that converts raw starch to ethanol in one step (CBP) could yield significant cost reductions in 1st generation bioethanol production from corn starch • 2nd Generation bioethanol produced from lignocellulosic biomass has great benefits in terms of energy balance, food security, etc. • Organisms that hydrolyse the cellulose and hemicelluloses in biomass and produce a valuable product such as ethanol at a high rate and titre would significantly reduce the costs of current biomass conversion technologies

  3. Thermostable α-amylase Alcohol recovery Glucoamylase Yeast Saccharification Fermentation Distillation & dehydration Corn Wheat Triticale Rye Jet Cooker >100ºC >5 - 8 min Water Storage tank Slurry tank Grinding Fuel blending Secondary Liquefaction 95ºC, ~90 min DDGS Ethanol production from starch Liquefaction

  4. Alcohol recovery Maize Wheat Triticale Rye Water Water Slurry tank Slurry tank Grinding Amylolytic Yeast! Ethanol production from starch Saccharification & Fermentation Distillation & dehydration Storage tank Fuel blending DDGS

  5. Introduction: starch CBP Amylose -amylase CH OH CH OH CH OH CH OH CH OH 2 2 2 2 2 O O O O O OH OH OH OH OH O O O O O OH OH OH OH OH OH glucoamylase -amylase Amylopectin -amylase glucoamylase CH OH CH OH CH OH CH OH 2 2 2 2 O O O O OH OH OH OH O O O OH O OH OH OH OH pullulanaseisoamylase CH OH CH OH CH OH CH OH CH 2 2 2 2 2 O O O O O OH OH OH OH OH O O O O O OH OH OH OH OH OH -amylase

  6. Results: Screening amylolytic genes • Glucoamylases • Aspergillus awamori (glaA) • Rhizopusoryzae (glaR) • Humicolagriseathermoidea (gla1) • Saccharomycopsis fibuligera (gluI) • Thermomyceslanuginosis (TLG) • α-Amylases • Aspergillusoryzae (AMYLIII) • Lipomyceskononenkoae (LKA) • Saccharomycopsis fibuligera (SFA) • Genes were cloned into episomal plasmids and activity assayed in lab strains • Best candidates were cloned into vectors to allow multicopy chromosomal integration in industrial yeast strains • Patent nr. WO 2011/128712 A1

  7. Results: Screening amylolytic genes Raw Starch Soluble Starch

  8. Results: Raw starch batch fermentations • 2% Raw Starch • 0.05% Glucose • Inoculate with 0.3 g/L Dry Weight Cells • Weight loss – 0.94 g • EtOH production – 8.08 g/L • 87.85% conversion

  9. Results: Raw starch batch fermentations • 10% Raw Starch • 0.05% Glucose • Inoculate with 20 g/L Wet Weight Cells • Max EtOH produced – 56.596 g/L , thus ~95% conversion

  10. Discussion: Starch CBP • Raw starch conversion was possible with no added enzymes or with reduced enzyme loadings; fermentation times must be improved • Current and future prospects: • Screen yeast strains with superior fermentation capacities • Screen a wider array of α-amylase encoding genes • Create strain with higher copy numbers of genes

  11. Introduction: Lignocellulose CBP • Lignocellulosic biomass consisting of mainly lignin, cellulose and hemicellulose, is an abundant, renewable & sustainable source of fuels etc. • The main barrier that prevents widespread utilization of this resource for production of commodity products is the lack of low-cost technologies to overcome the recalcitrance of lignocellulose

  12. Introduction: Lignocellulose CBP • Conversion of biomass to ethanol is a complex process and advances are required at several stages for efficiency and cost effectiveness • The CBP microbe thus converts pretreated biomass directly to ethanol • “Widely considered to be the ultimate low-cost configuration of cellulose hydrolysis and fermentation” – DOE/USDA Joint research Agenda • No ideal CBP organisms exists Biomass pretreatment Enzyme production Feedstock hydrolysis CBP Hexose fermentation(mainly glucose) Pentose fermentation(mainly xylose) ETHANOL

  13. Elements required for CBP with S. cerevisiae • EG and BGL expression successful • CBH expression problematic • This study: screen several CBH candidates for expressibility in S. cerevisiae • Genes were cloned into episomal plasmids and activity assayed in lab strains

  14. Results: CBH expression screening

  15. Results: CBH expression • Growth of strains in minimal media to examine secreted proteins: • N-glycosylation observed • Large variation in protein levels produced • Protein levels not necessarily reflecting activity levels – not all produced protein active • Candidate producing superior levels identified

  16. Results: CBH1 & CBH2 co-expression • Several well expressed CBH1s and CBH2s combined in the same strain • Though lower levels of either CBHs were observed in co-expression, higher levels of crystalline cellulose hydrolysis resulted – likely due to synergy % Avicel degradation μM MU released per minute

  17. Results: Avicel conversion • To test conversion of avicel to ethanol by CBH producing yeasts: • Strains cultured in YPD • 2% Avicel added • Novozyme 188 (BGL) added • Cultures producing CBHs converted Avicel to cellobiose in the absence of BGL • Cultures producing CBHs converted Avicel to ethanol in the presence of BGL • ~30% of theoretical maximum

  18. Discussion: cellulose CBP • High level secretion of exoglucanases is required for crystalline cellulose utilization - major hurdle in CBP yeast development • Indentified gene candidates compatible with expression in yeast • T.e.CBH1 and its T.r.CBM attached derivative yielded 100-200 mg/liter in shake flasks and ~300 mg/liter in HCD conditions • The highest CBH level secreted, ~1 g/liter C.l.CBH2b (~4% tcp) exceeded any previous reports on CBH production in S. cerevisiae • Thus S. cerevisiae is capable of secreting CBHs at high levels that compare well with the highest heterologous protein production levels described for S. cerevisiae

  19. Introduction: strain engineering • The innate low secretion capacity of S. cerevisiae, even when compared to other yeast species represents a drawback in its development as a CBP organism • Over-expression of genes encoding foldases, chaperones or other parts of the secretion pathways or knockouts of genes encoding negative regulators have been shown to increase secretion capacity in fungi • We aimed to improve the secretion of hydrolases by S. cerevisiae through strain engineering

  20. Results: strain engineering • Enhanced secretion of native proteins was reported when the protein secretion enhancer 1 protein (PSE1) of S. cerevisiae was overexpressed • Pse1 was overproduced in a strain expressing S.f.bgl1 • Pse1 overproduction yielded an almost 4-fold improvement of BGL activity • Sod1 co-overproduction yielded a further ~20% increase • The effect of these genes were reporter protein specific as less effect was seen on T.r.Cel7B and N.p.Cel6A 100 90 80 70 60 β-Glucosidase activity U/mg DCW 50 40 30 20 10 0 Cel3A Ref Cel3A-PSE1 Cel3A-SOD1 Cel3A-PSE1/SOD1

  21. Results: strain engineering • Knock-out of MNN-genes in S. cerevisiae have been shown to have a general effect on secretion enhancement • Two N-glycosylation mutants, ΔMNN10 and ΔMNN11had significantly higher extracellular enzyme activity for both Cel7A and invertase • Changes in cell wall structure or the degree of enzyme glycosylation may have contributed to this enhanced secretion phenotype

  22. Conclusion • Fermentation of raw starch by recombinant S. cerevisiae strains was demonstrated without the addition of commercial enzymes • S. cerevisiae was shown to be capable of expression of levels of CBHs that would overcome the barrier of sufficiency for conversion of cellulosic biomass to ethanol • Simultaneous expression of CBHs with EG and β-glucosidase enabled S. cerevisiae to directly convert cellulosic substrates to ethanol and to grow on cellulose under CBP conditions • S. cerevisiae strains could be manipulated to allow improved secretion of hydrolase enzymes • Combining optimal gene candidates in enhanced host strains will lead to improved strains for CBP applications

  23. Thank you! Acknowledgments: Lee Lynd John McBride Elena Brevnova Allan Froehlich Alan Gilbert Heidi Hau Erin Wiswall HoowenXu MerjaPenttilä Marja Ilmen AnuKoivula SanniVoutilainen Emile van Zyl Riaan den Haan Marlin Mert Danie La Grange MarynaSaayman Marko Viktor Heinrich Kroukamp

  24. Barriers to lignocellulose CBP with S. cerevisiae • Consumption of all major sugar constituents of biomass • High level expression of cellulases, especially cellobiohydrolases • Expression of the diverse enzymes required to hydrolyze biomass • Production of enzymes and consumption of sugars in toxic process conditions

  25. Introduction: xylan CBP Colinset al, 2005

  26. Introduction: xylan CBP 48 h 72 h 136 h • Xylanase & xylosidase • T. reesei xyn2 and A. nigerxlnD • Demonstrated degradation of birchwoodxylan to D-xylose • Xyloseisomerase • Synthetic codon optimisedB. thetaiotaomicronxylA • Xylose used as sole carbon source • Construct strain YMX1 • xylA integrated • xyn2 & xlnD episomal Xylose Xylobiose Xylotrose Xylanase Xylanase Xylanase Marker Marker Marker Control Control Control Xylanase/Xylo Xylanase/Xylo Xylanase/Xylo Biomass (OD600) 0 1 2 3 4 5 6 7 8 9 10 24 48 72 96 120 144 168 192 216 Time (hours)

  27. Results: xylan CBP • YP-Xylan (50 g/L beechwood) • YMX1 strain pre-culture grown on xylose • 10% innoculum • Growth of S. cerevisiae on xylan as sole carbohydrate was achieved but growth rate has to be improved

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