Confidential 1

Advanced Yeast Biocatalysts for 2nd Generation Ethanol ProductionBIO Pacific Rim; December 8, 2014

John McBride, Ph.D.

Complex Sugars Simple SugarsEnzymes

Value of Advanced Yeasts

glucose

glycerol

ethanol

New pathways

Native pathways

xylose

acetate

arabinose

glucose

glycerol

ethanol

Complex Sugars Simple SugarsEnzymes

Native pathways

Potential value created by bioengineered Yeast:

Assumptions:• $1.75/gal

EtOH selling price

• $12/Kg enzyme selling price

Mill Size (MGY): 40 80 20

• 2 products being used at commercial scale• >3 Billion gallons of EtOH made with

Mascoma strains to date

0

5

10

15

20

25

Cane Sugar Corn Cellulosic

$M

M/y

r

Enzymes

Yield Increase

0

5

10

15

20

25

Mar

ket

Pe

ne

trat

ion

(%

)Months In Market Place

3

Application in Grain-Based Fuel Ethanol Production: Transferm

Concept Lab Scale Demonstration

Scale-Up Rapid Commercial Adoption

Liquid Yeast dosing & Storage

TransFerm® Yield+, Increased Yields and GA for Corn Ethanol

TransFerm Yield+

• Balances the redox during anaerobic growth with a new metabolic pathway

• Makes sufficient glycerol to balance osmotic stress

Note: pilot data based on n=3 for conventional yeast; n=5 for TransFermYield+

0.0

2.0

4.0

6.0

8.0

10.0

12.0

14.0

16.0

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)

11.0

11.5

12.0

12.5

13.0

13.5

24 36 48 60Time (hours)

• 4.5% Yield increase

• Same Kinetics

Pilot Scale Data:

TransFerm® Yield+ Commercial Scale Results

CustomerPlant Scale

(MGPY)

Design Type

N=Ethanol Yield

Increase Achieved

Glycerol Reduction

Enzyme Reduction

Estimated Total Value

per Year (MPY)

1 110 ICM 13 2.9% 21% 45% $6.3

2 125 ICM 10 1.6% 40% 30% $3.9

3 115 ICM 29 2.0% 37% 25% $3.6

4 60 ICM 53 2.0% 30% 40% $2.1

5 35 Delta T 88 1.7% 26% 28% $0.9

• Robust commercial scale results, kinetics

• Average value of $0.04/gal delivered

• >$500M value creation for the U.S. ethanol industry from this product10.0

10.5

11.0

11.5

12.0

12.5

13.0

13.5

14.0

30 hrs 36 hrs 42 Hrs 48 Hrs Drop

Eth

ano

l (%

w/v

)

Transferm Yield+, 55% GA, n=13

Baseline Yeast, 100% GA, n=22

Distillation and Storage

Pretreatment

Proprietary Enzyme Cocktail

Conditioning

Fermentation

Hydrolysis

Feedstock Supply

Acid

Base

Gen2 Yeast: Drop-in Value

Mascoma Gen2Y

Xylose, Galactose, Mannose, Arabinose, Acetic Ferm.

CBP Cellulases & Hemicellulases

CelluloseHemicellulose Enzyme from hydrolysis

Glucose Fermentation

XX

X

XAA

G

G

G

G

GG

M

Gal

X

X

G

G

G

OH

O

OH

OOH

O

OH

O

Technology for Bagasse Sugar Fermentation Yield +

Xylose Fermentation 33%

Glycerol Reduction 5%

Acetate Conversion to Ethanol 5%

Arabinose Utilization 5%

Cellulase and Hemicellulase Secretion >10%

Xylose Fermentation

A. Isolation of unique xylose isomerasesB. Optimization of core genesC. Identification of new mutations

• Finishes fermentation in ~30 hours from small inoculum

• 0.45 g/g ethanol yield• Directly Engineered, no adaptation

Commercial corn stover hydrolyzate fermentation

0

2

4

6

8

10

12

14

16

0

10

20

30

40

50

60

0 12 24 36 48

CO

2 P

rod

uce

d (

L)

Co

nce

ntr

atio

n (

g/L)

Fermentation Time (hours)

Glucose Xylose Ethanol CO2 (L)

Arabinose Fermentation

8

• 93% arabinose utilized• 0.45 g/g yield on added arabinose • Directly engineered, no adaptation

AraT

AraA

AraB

AraD

L-arabinose

L-ribulose

L-ribulose-5-P

D-xylulose-5-P

L-arabinose

TKL1TAL1RKI1RPE1

Pentose phosphate pathway

ethanolglycolysis

L-arabinitolGRE3

0

5

10

15

20

0

5

10

15

20

25

30

35

wild type round 3 eng. round 4 eng.

eth

ano

l g/L

arab

ino

se g

/Lstrain

5 g/L glucose + 30 g/L arabinose

9

Acetate Ethanol

NAD+NADH

Biomass Glucose

adhE

Acetate Ethanol

NAD+NADH

CO2 Glucose

adhE

~10% via Glycerol reduction

~90% via new pathway

0

10

20

30

40

50

60

70

Wild Type Engineered

g/L

glycerol acetate ethanol

• 9 g/L acetic acid consumed• 9.3% yield increase• Yield on glucose is 0.513 g/g

Novel Pathway for Acetic Acid Fermentation120 g/L glucose + 10 g/L acetate

Acetate Fermentation

10

0%

20%

40%

60%

80%

100%

N=0 N=1 N=3 N=6 N=7 N=8 N=10 N=11

% H

ydro

lysi

s

0%

20%

40%

60%

80%

100%

% O

f En

zym

e S

yste

m B

y En

zym

e

Typ

e

% Glucan Related % Mannan Related % Xylan Related

Screened activities

xylanase

xylosidase

acetyl xylan esterase

glucuronyl esterase

acetyl esterase

endoglucanases

α-galactosidase

β-glucosidase

α-glucuronidase

β-mannanase

β-mannosidase

others

Unique Enzyme Combinations:

Yeast Secreted Hemicellulase Enzymes

0

5

10

15

20

25

30

0 50 100

EtO

HC

on

cen

trat

ion

(g

/L)

Fermentation Time (hours)

CBP Yeast

Conventional Xylose Yeast

Applied in Pilot Scale Fermentations:

Need for Enzyme Eliminated

Conventional Enzymes

Direct Fermentation of Hardwood Xylo-oligomers from 2-stage Pretreatment with no Enzyme:

Cellulase Secretion

non-CBP CBP

Impact of secreting cellulases:

Enzymatic Activities Expressed in Yeast

cellobiohydrolase I CBH I chimeras* cellobiohydrolase II

endoglucanases arabinofuranosidase feruloyl esterase

swollenin α-expansin β-expansin

* See Ilmen et al. “High Level Secretion of Cellobiohydrolases by Saccharomyces cerevisiae.”

Biotechnol. Biofuels 4, no. 1: 30.

Source: NREL.

Highest Reported CBH1

and CBH2 Expression by

10X

30

35

40

45

50

55

60

2 4 6 8 10

Co

nce

ntr

atio

n a

t 9

6 H

ou

rs (

g/L)

Enzyme Loading (mg/g TS)

M3799 Ethanol M5477 (CBP) EthanolXylose Utilizing Background Strain

+CBH1, 2, and 6 accessory enzymes

31% Enzyme savings

Hydrolysis of cellulose

~$0.16/gal savings @ $0.50/gal enzyme cost

Hemicellulase Secretion in Corn Stover HHF Process

12

10% Yield improvement by hemicellulase enzyme expression

0

10

20

30

40

50

60

0 20 40 60 80 100 120

Eth

ano

l/Su

gars

in S

up

ern

atan

t, g

/L

Process Time, hr

Baseline Yeast

Engineered Yeastglucose

xylose

EtOH

@20MM gal scale, Value = +2 MM gal x $3/gal = $6M/yr = $0.30/gal

Hydrolysis Fermentation

Co-Fermentation to Extend 1G Capital

13

0

2

4

6

8

10

12

14

50

60

70

80

90

100

110

120

130

0 24 48 72

Xyl

ose

Co

nce

ntr

atio

n (

g/L)

Eth

ano

l Tit

er

(g/L

)

Fermentation Time (hrs)

Corn Mash and Xylose: Molasses and Xylose:

0

10

20

30

40

50

60

70

80

0 2 4 6 8 10

Co

nce

ntr

atio

n (

g/L)

Fermentation Time (hrs)

ethanol

glucose

fructose

xylose

Dash = wild type Solid = C5 strain

xylose

ethanol

Summary

Proven ability to deploy GM Yeasts for commercial fuel production

Suite of technologies developed for 2G ethanol production (xylose, glycerol, acetic acid, enzyme secretion)

Yield increases of 25% and cost savings of ≥$0.30/gal above xylose utilizing strains

Novel process configurations to extend 1G fermentation capital are now possible

14