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Development of Bioengineered Yeast for the Grain Ethanol Industry
Kevin S. Wenger, Ph.D. William Kenealy, Ph.D. Mascoma Corporation
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
1
Outline
2
• Mascoma Background
• TransFerm and TransFerm Yield+
• Regulatory Approaches
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
Mascoma Overview
Americas Region Cellulosic
Mascoma Grain Technology
Mascoma Chemicals
Transform existing assets TransFerm TransFerm Yield+ Future Products Partnership with Lallemand
Enable 2nd Gen Ethanol CBP5x CBP56x Hardwood to ethanol projects in MI & Alberta
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
Business Strategy Focused on Three Main Segments
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Polymeric Sugars from Starch, Cellulose, Hemicellulose
Fermentation Products
Mascoma’s CBP Technology Platform
High yield and energy capture as product, Additional enzyme production at zero cost, Multiple product potential – fuels and chemicals
Metabolic Engineering to Convert Soluble Sugars To End
Products at High Yield
Secretion of Enzymes To Break Down Polymeric Sugars Into
Soluble Sugars
Mascoma Yeast
5
TransFerm Reduced
Glucoamylase (GA) Enzymes
X
Fermentation FUEL ETHANOL
TransFerm®, A New Yeast Expressing Glucoamylase
GROUND CORN Jet cooker
Liquefaction Slurry
Alpha amylase enzymes
Distillation
The first bioengineered yeast successfully launched in commercial ethanol production
TransFerm 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
“MGT 1.2” and Future
Generations
TransFerm Yield +
Gross Incremental Value (¢/gallon) Key Assumption: Ethanol Price of $2.50/gal
Our MGT Product Line Creates Significant Potential Value for Customers
0
2
4
6
8
10
12
14
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TransFerm TransfermYield +
Future Gen
$0.04-0.06
$0.01-0.02
$0.08-0.10
Under Development
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TransFerm has been a commercial success
Over 1 Billion Gallons Cumulatively Produced
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TransFerm® Yield+ Provides Increased Ethanol Yield
• Produce glycerol for osmotic stress and redox balance
• Ethanol yield is reduced due to glycerol production
Wild Type Cells:
TransFerm Yield+
• Balances the redox during anaerobic growth by using an alternative electron acceptor
• Makes sufficient glycerol to balance osmotic stress
• Sugars
• Salts
• Organic acids Glucose
Glycerol Ethanol Cells CO2
NAD+
NADH NAD+
NADH
Glycerol Ethanol Cells CO2
NAD+
NADH NAD+
NADH
Ethanol Alternative e-
acceptor
Glucose
NAD+
NADH
• Fast kinetics
• Higher ethanol/solids
• Reduced glycerol
Note: pilot data based on average of n=3 for conventional yeast; n=5 for TransFerm Yield+
11.0
11.5
12.0
12.5
13.0
13.5
0 6 12 18 24 30 36 42 48 54 60
Eth
ano
l (w
/v %
)
Time (hours)
Conventional yeast (100% GA)
TransFerm Yield+ (70% GA)
Ethanol profile
Pilot scale testing to demonstrate TransFerm Yield+
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1.200
1.300
1.400
1.500
1.600
1.700
Conventional yeast(100% GA)
TransFerm Yield+ (70%GA)
Gly
cero
l(w
/v %
)
Final Glycerol (60 hours)
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)
TransFerm® Yield+ Commercial Scale Trials Show 2-4% Yield Increase
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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)
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Product Status
Generally Recognized as Safe (GRAS)
Ingredient Definition for Distillers CoProduct
Microbial Commercial Activity Notice (MCAN)
Regulatory review status
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.
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AAFCO Process Overview
12-18 mos 3-6 mos 6 mos 12 mos minimum
Submission
CVM Review
CVM Letter
AAFCO Ingredients Definition Cmte Vote
Good case scenario: 36 months from submission to publication
Biannual meeting cycle Annual publication cycle
CVM & AAFCO MOU: The 2 organizations work together under a Memorandum of Understanding that currently expires Sept 2015
Publication in the “next” Official
Publication
AAFCO General Membership
Vote
15
Yeast is a processing aid
Similar to bioengineered enzymes; yeast is inactivated in the ethanol distillation process
0
20
40
60
80
100
120
140
160
180
0 0.5 1 1.5 2 2.5
Ce
ll co
un
t, m
illio
n C
FU p
er
ml
Time, min
Thermal inactivation of yeast strains at 140F from fermentation beer, 24 and 48 hrs into fermentation
MGT - 24h
MGT - 48h
Results were confirmed at commercial facilities
Beer
Stripper
80-90C
2-5 mins
Beer
Stillage
EtOH
(60C)
16
Environmental persistence
The bioengineered yeast persists similarly to host strain in simulated water and soil environments
Water
Sterilized
Soil
Non-
Sterilized
Soil
Dotted = modified strain
Solid = Host
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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
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
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.
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