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

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

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

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

Introduction: starch CBP -amylase CH OH 2 O OH -amylase glucoamylase Amylose pullulanase isoamylase CH OH 2 O OH Amylopectin CH -amylase glucoamylase -amylase

Results: Screening amylolytic genes Glucoamylases Aspergillus awamori (glaA) Rhizopus oryzae (glaR) Humicola grisea thermoidea (gla1) Saccharomycopsis fibuligera (gluI) Thermomyces lanuginosis (TLG) α-Amylases Aspergillus oryzae (AMYLIII) Lipomyces kononenkoae (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

Results: Screening amylolytic genes Soluble Starch Raw Starch Glucoamylase (Soluble Starch) (Raw Starch) Alpha-amylase AGA/AOA 0.52 U/ml 0.047 U/ml 0.15 U/ml TLG/SFA 1.187 U/ml 0.576 U/ml 0.812 U/ml

Results: Raw starch batch fermentations 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

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

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

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

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

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

Results: CBH expression screening CBH1 (modified) CBH2 H. grisea cbh1 T. aurantiacus cbh1 T. emersonii cbh1 N. fischerii cbh1 P. janthinellum cbh1 G. zeae cbh1 N. haematococca cbh1 F. poae cbh1 As. terreus cbh1 P. chrysogenum cbh1 N. crassa cbh1 C. thermophilum cbh1 Ac. thermophilun cbh1 T. reesei cbh1 Tecbh1-TrCBM-C Tecbh1-HgCBM-C Tecbh1-CtCBM-C Tecbh1-TrCBM-N Tecbh1-TrCBM-N2 Tecbh1-TrCBM-C2 C. heterostrophus cbh2 G. zeae cbh2 I. lacteus cbh2 V. volvacea cbh2 Piromyces sp. cbh2 T. emersonii cbh2 T. reesei cbh2 C. lucknowense cbh2 A. cellulolyticus cbh2 C. thermophilum cbh2

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

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

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

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

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

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 β-Glucosidase activity U/mg DCW 10 20 30 40 50 60 70 80 90 100 Ref Cel3A Cel3A-SOD1 Cel3A-PSE1 Cel3A-PSE1/SOD1 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

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 ΔMNN11 had 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

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

Thank you! Acknowledgments: Lee Lynd John McBride Elena Brevnova Allan Froehlich Alan Gilbert Heidi Hau Erin Wiswall Hoowen Xu Merja Penttilä Marja Ilmen Anu Koivula Sanni Voutilainen Emile van Zyl Riaan den Haan Marlin Mert Danie La Grange Maryna Saayman Marko Viktor Heinrich Kroukamp

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

Introduction: xylan CBP Colins et al, 2005

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

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