New Yeasts for Grain Based Ethanol

William R Kenealy, Ph.D. Principal Scientist Mascoma Corporation

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Pacific Rim Summit on Industrial Biotechnology and Bioenergy Stabilization in Fermentation Processes

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Facilities Research and Development Center in Lebanon

NH, proximate to Dartmouth College

History:

• Founded in 2006

• Roots at Dartmouth College L. Lynd, C. Wyman co-founders

• VC Funding: Khosla, Flagship, General Catalyst

• Strategic Investors: GM, Marathon, Valero

Mascoma: A State of the Art Biotechnology Company

Scale-up Facility in Rome, NY

Headquarters in Waltham, MA

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Mascoma’s Mission: Commercialize CBP

Polymeric

Sugars

Soluble Sugars

Yeast

Yeast-Secreted

Enzymes Added

Enzyme

Ethanol

Chemicals

Fuels

High yield and energy capture as product

Relatively low capital cost

Multiple product potential – fuels and chemicals

Consolidated Bioprocessing

(CBP)

Consolidated Bioprocessing improves the economics of bioconversion

Advantages of microbial conversion

New Yeast Reduced

Glucoamylase (GA) Enzymes

X

Fermentation FUEL ETHANOL

Example: Yeast Expressing Glucoamylase (GA)

GROUND CORN Jet cooker

Liquefaction Slurry

Alpha amylase enzymes

Distillation

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Application in Grain-Based Fuel Ethanol Production

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Insertion of a glucoamylase (GA) gene in yeast

Gene sequences identified from genomic databases

GA

Gene synthesis

Design of S. cerevisiae expression cassettes

GA pro ter

Transform DNA into yeast and select for recombination events

Integration on yeast chromosome Expression of GA

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Selection of Yeast Containing the GA gene

Host yeast TransFerm

Growth on starch

Screening of individual colonies for activity

Other selection criteria: - Growth rate - Thermo-robustness - Ethanol tolerance - Performance in application

Basic Fermentation Characteristics

Fermentation Results Host TransFerm

Ethanol yield (g/g) 0.40±0.01 0.41±0.03

Glycerol yield (g/g) 0.050±0.001 0.050±0.001

Acetic acid yield (g/g) 0.01±0.001 0.007±0.0008

Biomass (OD) 9.20±0.001 9.1±0.2

Fermentations were performed at 35C in 1 liter bioreactors. YPD100 (10 g/L Yeast Extract, 20 g/L Peptone and 100 g/L glucose). Initial pH = 6.0

Anaerobic fermentation of 100 g/L glucose

The GA-expressing yeast retains the basic fermentation characteristics of the unmodified host

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Enzyme Production During CBP Fermentation

Fermentation was performed at 35C in 1 liter bioreactor. YPD100 (10 g/L Yeast Extract, 20 g/L Peptone and 100 g/L glucose). Initial pH = 6.0

Anaerobic fermentation of 100 g/L glucose

The enzyme production is highly linked to cell biomass production

Fermentation of corn mash

Fermentation was performed at 4mL scale using 25% corn mash, 500 ppm urea and 35C

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CBP can help reduce costs and improve yield

Enzyme Reduction Yield Increase

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Corn mash fermentation results are improved with GA-expressing yeast

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From 4mL to 800,000 gal…

A key capability is to have a fermentation protocol which gradually scales up the volume and allows one to test a large pool of candidate strains.

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0

2

4

6

8

10

12

14

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ConventionalDry

ConventionalSLY

GA-expressingyeast

50

Hr

EtO

H (

% w

/v)

34.4 C

36.1 C

37.8 C

The modified strain behaves similarly to conventional yeast

Fermentations conducted on 34% initial TS industrial corn mash 1000ppm urea; 0.6 AGU/g GA for conventional; 0.3 AGU/g GA for TF Inoculum of 0.6 gDCW/l for dry; 0.3gDCW/l for liquid

Important parameters: - Propagation history - Nutrition - Timing of stress - Ethanol levels

Effect of High Temperature on Yeast

GA Producing Yeast has been a commercial success

• TransFerm launched industry-wide in 2012

• Currently in commercial use at >20 fuel ethanol facilities

• These facilities represent >1.5 Billion Gallons of annual production

• Over 500 Million Gallons of Ethanol produced to date using TransFerm

The first bioengineered yeast successfully introduced to the fuel ethanol industry

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GPD2

Metabolic Engineering Yeast for Reduced Glycerol

0.5 glucose

NADH

dihydroxyacetone

phosphate

ATP

glycerol

Glycerol-3-phosphate

Glycerol pathway

GPD1

Yeast make Glycerol 1) In response to stress 2) To re-oxidize NADH which is formed in

excess under anaerobic growth

Deleting GPD1/GPD2 results in: 1) No anaerobic growth 2) Poor robustness

Historically, attempts to reduce glycerol in industrial ethanol production strains have resulted in strains that lack the ability to grow under strict anaerobic conditions, and/or are unable to achieve high ethanol productivity

Metabolic Engineering Yeast for Reduced Glycerol

Bacterial ethanol pathway Glycerol pathway

Insertion of a bacterial ethanol pathway with an alternate electron acceptor, allows higher yield production of ethanol. Resulting strains grow under strict anaerobic conditions with GPD1/GPD2 knocked out.

Argyros, et al. WO2012138942

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• 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)

SUMMARY OF LARGE-SCALE TRIALS

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

Demonstrated 2.7% - 4.8% increase in ethanol yield compared to controls (over 100 fermentations)

Confidential 16

Summary of Large Scale trials with the Glycerol Reduction Yeast

How can Increased ethanol (7g/l) come from Decreased glycerol (3.3 g/l)? • Mascoma scientist Aaron Argyros observed:

– Glycerol production from glucose for anaerobic growth is redox balanced

– Glycerol production from glucose for stress response is not redox balanced

– Production of acetic acid, butanediol, acetoin, pyruvate, or acetaldehyde will be required to balance redox with stress response

• The conversion of glycerol to ethanol gives 0.5 g ethanol/gram glycerol

• The conversion of additional side products to ethanol (from stress response glycerol) would contribute additional ethanol

• Lower initial glucoamylase produces less osmotic stress-CBP effect

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Regulatory review requirements for bioengineered yeast

• U.S. Environmental Regulations

– Under the purview of the Environmental Protection Agency (EPA)

– Specific regulations regarding the large scale use of bioengineered microbes (MCAN)

• U.S. Food/Feed Regulations - In the U.S. is handled by Food and Drug Administration (FDA) in partnership with

Association of American Feed Control Officials (AAFCO)

- Yeast and enzymes are considered processing aids; not-labeled

- GRAS (Generally Recognized as Safe)

• Canadian Regulations New Substance Notification (NSN) encompasses both food/feed and environmental review

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Most other countries have a similar system

Advanced Yeasts can be the key to a more economic biofuels industry

• CBP Technology - Combines enzymatic hydrolysis and fermentation, reducing the need for expensive exogenous enzymes

• Metabolic Engineering - Enables higher yield conversion of the available sugars to ethanol or other products

Advanced Yeasts are coming now

• During only the last two years we have seen the first example of a bioengineered yeast successfully used in the fuel ethanol industry

• This paves the way for many future advances

Conclusions

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