Development of Bioengineered Yeast for the Grain Ethanol Industry

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1 Development of Bioengineered Yeast for the Grain Ethanol Industry Overcoming Challenges in Regulation and Intellectual Property Track 3: Advanced Biofuels and Biorefinery Platforms Session 5: Tuesday, December 10-4:00 PM - 5:30 PM Kevin S. Wenger, Ph.D. William Kenealy, Ph.D. Mascoma Corporation 1

2 Outline Mascoma Background TransFerm and TransFerm Yield+ Regulatory Approaches 2

3 Mascoma Overview Facilities Corporate Headquarters in Waltham, MA Research and Development Center in Lebanon, NH Scale-up Facility in Rome, NY (Pretreatment up to 20 TPD, and fermentation up to 19,000L) Engineering in Toronto, Ontario

4 Business Strategy Focused on Three Main Segments Mascoma Grain Technology Transform existing assets TransFerm TransFerm Yield+ Future Products Partnership with Lallemand Americas Region Cellulosic Enable 2 nd Gen Ethanol CBP5x CBP56x Hardwood to ethanol projects in MI & Alberta Mascoma Chemicals Address long-term demand picture Developing partnerships Our Business Strategy, and Commercialization plan are Focused on Establishing our Leading CBP Bioconversion Platform as the BioConversion Technology of Choice across all Biomass to Petroleum Replacement Applications 4

5 Mascoma s CBP Technology Platform Polymeric Sugars from Starch, Cellulose, Hemicellulose Metabolic Engineering to Convert Soluble Sugars To End Products at High Yield Mascoma Yeast Fermentation Products Secretion of Enzymes To Break Down Polymeric Sugars Into Soluble Sugars High yield and energy capture as product, Additional enzyme production at zero cost, Multiple product potential fuels and chemicals 5

6 TransFerm, A New Yeast Expressing Glucoamylase The first bioengineered yeast successfully launched in commercial ethanol production Alpha amylase enzymes Reduced Glucoamylase (GA) Enzymes X TransFerm GROUND CORN Jet cooker Fermentation FUEL ETHANOL Slurry Liquefaction Distillation

7 Our MGT Product Line Creates Significant Potential Value for Customers TransFerm TransFerm Yield + MGT 1.2 and Future Generations GA Enzyme Displacement Cost Savings TransFerm + 2%-4% Additional Yield Improved Conversion Of Sugar TransFerm Yield + plus Additional Yield Further Advancement In Enzymes And Metabolism Gross Incremental Value ( /gallon) Key Assumption: Ethanol Price of $2.50/gal $ TransFerm $ Transferm Yield + $ Under Development Future Gen 7

8 TransFerm has been a commercial success Over 1 Billion Gallons Cumulatively Produced 8

9 TransFerm Yield+ Provides Increased Ethanol Yield Wild Type Cells: Produce glycerol for osmotic stress and redox balance Ethanol yield is reduced due to glycerol production Sugars Salts Organic acids NAD+ NADH Glucose NAD+ NADH Glycerol Ethanol CO 2 Cells TransFerm Yield+ Balances the redox during anaerobic growth by using an alternative electron acceptor Makes sufficient glycerol to balance osmotic stress NAD+ NAD+ NADH Ethanol NADH Glucose Alternative e - acceptor NAD+ NADH Glycerol Ethanol CO 2 Cells

10 Ethanol (w/v %) Glycerol(w/v %) Pilot scale testing to demonstrate TransFerm Yield Ethanol profile Conventional yeast (100% GA) TransFerm Yield+ (70% GA) Final Glycerol (60 hours) Time (hours) Conventional yeast (100% GA) TransFerm Yield+ (70% GA) Note: pilot data based on average of n=3 for conventional yeast; n=5 for TransFerm Yield+ Fast kinetics Higher ethanol/solids Reduced glycerol 10

11 TransFerm Yield+ Commercial Scale Trials Show 2-4% Yield Increase PLANT GA REDUCTION GLYCEROL REDUCTION YIELD BOOST NUMBER OF FERMENTATIONS 1) PILOT -30% -30% +4.1% 5 2) PLANT 1-40% -23% +2.9% 67 3) PLANT 2-35% -30% +4.8% 15 4) PLANT 3-50% -43% +3.1% 14 5) PLANT 4-30% -28% +2.7% 3 SUMMARY -30% to -50% -23% to -43% +2.7 to +4.8% 104 TransFerm Yield+ demonstrated 2.7% - 4.8% increase in ethanol yield compared to controls (over 100 fermentations) 11

12 Regulatory Framework FDA Regulation of Feed ingredients Generally Recognized as Safe (GRAS) Feed Additive Petition (FAP) AAFCO Ingredient Definition EPA Biotechnology Manufacturing Microbial Commercial Activity Notice (MCAN) 12

13 Regulatory review status Product Status Generally Recognized as Safe (GRAS) Ingredient Definition for Distillers CoProduct Microbial Commercial Activity Notice (MCAN)

14 AAFCO Ingredient Definition Process Association of American Feed Control Officials AAFCO maintains definitions of feed ingredients in an Official Publication (OP) AAFCO works with FDA (Division of Animal Feeds) under a Memorandum of Understanding Petitions for new feed ingredient definitions are reviewed by AAFCO officials in collaboration with FDA scientists. After review FDA will provide a letter of concurrence regarding the suitability of the feed ingredient. 14

15 AAFCO Process Overview Good case scenario: 36 months from submission to publication mos 3-6 mos 6 mos 12 mos minimum CVM Review Biannual meeting cycle Annual publication cycle Submission CVM Letter AAFCO Ingredients Definition Cmte Vote AAFCO General Membership Vote Publication in the next Official Publication CVM & AAFCO MOU: The 2 organizations work together under a Memorandum of Understanding that currently expires Sept

16 Cell count, million CFU per ml Yeast is a processing aid Similar to bioengineered enzymes; yeast is inactivated in the ethanol distillation process Thermal inactivation of yeast strains at 140F from fermentation beer, 24 and 48 hrs into fermentation (60C) Beer EtOH MGT - 24h MGT - 48h Time, min Beer Stripper 80-90C 2-5 mins Stillage Results were confirmed at commercial facilities 16

17 Environmental persistence The bioengineered yeast persists similarly to host strain in simulated water and soil environments Sterilized Soil Water Dotted = modified strain Solid = Host Non- Sterilized Soil 17

18 Both host and donors have safe use history Saccharomyces cerevisiae Saccharomyces cerevisiae is a common human food and animal feed ingredient with a long history of safe use. Saccharomyces cerevisiae is used in a number of applications in animal feed, as a direct fed microbial, as a source of single cell protein, and as a processing aid in the production of distillers co-products. Saccharomycopsis fibuligera Has been used to produce single-cell protein. Originally isolated from bread. Naturally found on many food sources. Bifidobacterium adolescentis Commensal microorganism. Approved direct-fed microbial in the AAFCO OP. 18

19 Regulatory Challenges Multiple agencies Food, feed, environmental Other countries Not all of the standards are harmonized Timeline Can take ~2 yrs for product approval 19

20 Summary TransFerm and TransFerm Yield+ are the first bioengineered yeasts for ethanol production that have gone through formal FDA and EPA review. This application of biotechnology will have a significant impact on the production cost of biofuels in the future. The regulatory paths in the US are relatively clear, and one can expect that we will see more microorganisms coming through regulatory approval processes in the future. 20

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