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

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Outline

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• 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

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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

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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

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Yeast is a processing aid

Similar to bioengineered enzymes; yeast is inactivated in the ethanol distillation process

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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)

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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.

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Regulatory Challenges

• Multiple agencies – Food, feed, environmental

• Other countries – Not all of the standards are harmonized

• Timeline – Can take ~2 yrs for product approval

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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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