new yeasts for grain based ethanol · regulatory review requirements for bioengineered yeast •...
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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
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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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ConventionalDry
ConventionalSLY
GA-expressingyeast
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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+
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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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