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
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