CoER : Biofuels
Department of Microbiology • Faculty of Natural Sciences
Construction of cellulolytic
Saccharomyces cerevisiae strains for
consolidated bioprocessing
WH van Zyl1, R Den Haan1, SH Rose1, DC la Grange1,
R van Rooyen1, JE McBride2 and LR Lynd2
1Department
of Microbiology, University of Stellenbosch,
Stellenbosch, South Africa
2Chemical and Biochemical Engineering Program, Thayer School of
Engineering, Dartmouth College, Hanover, NH 03755, USA
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Content
1. Next generation technologies for cellulose conversion
2. What is Consolidated Bioprocessing?
3. Conversion of amorphous cellulose to yeast biomass
4. Functional expression of cellobiohydrolases in yeast
5. Recent advances towards realizing CBP
6. Acknowledgments
7. SANERI Senior Chair of Energy Research : Biofuels
2
Next generation technologies
for cellulose conversion
3
Technologies for Cellulose Conversion
Lignocellulose composition
Non-fermentable
sugars
high energy
aromatics
Sugarcane bagasse
Lignin
28%
Arabinan
2%
Hexoses
Pentoses
(fermentable)
Cellulose
46%
(fermentable)
Xylan
25%
4
Technologies for Cellulose Conversion
Enzymatic hydrolysis of biomass
Ligninases
(laccases,
lignin peroxidases,
Mn-peroxidases)
Cellulases
(endoglucanases,
cellobiohydrolases,
-glucosidases)
Hemicellulases
(xylanases, -xylosidases
-arabinofuranosidases
-glucuronidases)
Esterases
(feruloyl esterases,
coumaroyl esterases)
5
Technologies for Cellulose Conversion
Biomass Processes for EtOH production
BiologicallyMediated
Event
Cellulase
Production
Enzyme Hydrolysis Processing Strategy1
(Each box represents a bioreactory - not to scale)
SHF
O2
SSF
O2
SSCF
CBP
O2
Lignocellulose
Hydrolysis
Hexose
Fermentation
Pentose
Fermentation
SHF: Separate Hydrolysis & Fermentation
SSF: Simultaneous Saccharification & Fermentation
SSCF: Simultaneous Saccharification & Co-Fermenation
6
CBP: Consolidated Bioprocessing
Consolidated BioProcessing (CBP)
7
Consolidated BioProcessing (CBP)
Fundamentals of
Microbial Cellulose Utilization
Microbiology and Molecular Biology Reviews 66: 506-577 (2002)
8
Consolidated BioProcessing (CBP)
Consolidated bioprocessing :
update
Current Opinion in Biotechnology 16:577–583 (2005)
9
Consolidated BioProcessing (CBP)
Consolidated bioprocessing :
update (2)
Advances in Biochemical Engineering / Biotechnology (2007)
10
Consolidated BioProcessing (CBP)
Consolidated bioprocessing :
update (3)
Applied Microbiology & Biotechnology (2010) [corrected proofs]
11
Consolidated BioProcessing (CBP)
Consolidated bioprocessing: update (3)
T. saccharolyticum
Lactic acid bacteria
C. acetobutylicum
Z. mobilis
E. coli
No activity
H. polymorpha
Very low activity
P. stipitis
S. cerevisiae
K. marxianus
Low activity
K. oxytoca
Good activity
WT GE WT WT GE WT WT GE WT GE WT GE WT GE WT GE WT GE WT GE
Breakdown crystalline cellulose
Breakdown amorphous cellulose
NA
Breakdown hemicellulose
Utilize cellobiose
NA NA
Utilize xylobiose
Grow on glucose
Grow on xylose
Ferment glucose to ethanol
Ferment xylose to ethanol
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
E
B
B
L
L
E
E
B
B
L
L
E
Resistant to hydrolysate inhibitors
NA NA NA NA NA NA
GRAS status
NA NA
Low pH
NA NA
NA NA
High temperature
Applied Microbiology & Biotechnology (2010) [corrected proofs]
12
Technologies for Ethanol Production
Ethanol production from sugar
Yeast
Sugarcane\
Sugarbeet
Sweet
sorghum
Fermentation
Alcohol
recovery
Distillation & dehydration
Storage
tank
Sugar
Storage
tank
Crashing
Sugar
extraction
Spent
yeast
Fuel
blending
13
Technologies for Ethanol Production
Ethanol production from cellulosics
Cellulases
Pre-treatment Saccharification
Agric Res
Woody
Material
Grasses
Water
Yeast
Fermentation
Alcohol
recovery
Distillation & dehydration
Steam
explosion
~200ºC
Storage
tank
mixing
tank
Chipping
Grinding
Cooling &
conditioning
Spent
material
Fuel
blending
14
Estimated Cellulase Cost
($/gal EtOH)
Largest Component of Recalcitrance Barrier:
Cost of Cellulase
10.00
d
2000
g
f
1.00
b
c
2007
2008
e
1997
0.10
2004
a
1991
0.01
1990
1995
2000
2005
2010
Year
a) Hinman et al. 1991. Appl. Biotechnol. Bioeng. 34/35:639-657.
b) Hettenhaus & Glassner, 1997 (http://www.ceassist.com/assessment.htm).
c) NREL, 1998. Bioethanol from the corn industry. DOE/GO-1009-577.
d) Schell, 2004. ASM Natl Meeting; McMillan, 2004. DOE/NASULGS Biomass & Solar Energy Workshops.
e) Genencor & Novozyme, 2004. Press releases (e.g. http://www.genencor.com/wt/groc/pr 109831360).
f) Petiot, Novozymes, Platts Cellulosic Ethanol & Second Generation Biofuels, 2007.
15
g) Sheridan (Novozymes) Nature Biotech, 2008.
Consolidated BioProcessing (CBP)
Glycosyl
Hydrolases
O
O
Xyl
Ara
O
O
O
Glu
Man
Gal
P
YFG
T
Ethanol + CO2
16
Technologies for Ethanol Production
Ethanol production from cellulosics
Cellulases
Pre-treatment Saccharification
Agric Res
Woody
Material
Grasses
Water
Yeast
Fermentation
Alcohol
recovery
Distillation & dehydration
Steam
explosion
~200ºC
Storage
tank
mixing
tank
Chipping
Grinding
Cooling &
conditioning
Spent
material
Fuel
blending
17
Technologies for Ethanol Production
Ethanol production from cellulosics
Cellulolytic Yeast
Pre-treatment
Agric Res
Woody
Material
Grasses
Water
Saccharification & Fermentation
Alcohol
recovery
Distillation & dehydration
Steam
explosion
~200ºC
Storage
tank
mixing
tank
Chipping
Grinding
Cooling &
conditioning
Spent
material
Fuel
blending
18
Conversion of amorphous cellulose
to yeast biomass
19
Cellobiose utilization in yeast
Cellobiose utilization by S. cerevisiae
2.0
max = 0.20
OD600
1.8
1.6
max = 0.21
1.4
1.2
max = 0.21
1.0
0.8
∑ host - glucose
∑ host - cellobiose
∑ySF1 - glucose
0.6
0.4
∑ySF1 - cellobiose
0.2
0
ΣySF1 = S. cerevisiae
expressing S.fibuligera
β-glucosidase (BGL1) gene
from 2μ plasmid.
SC media;
0
10
20
30
Time (hrs)
40
5g/L cellobiose; 30°C /
adjusted to 6.0
50 pH
(KOH)
Van Rooyen, R., B. Hahn-Hägerdal, D.C. La Grange, W.H. Van Zyl. 2005. Construction
and characterization of cellobiose-growing and fermenting Saccharomyces cerevisiae
strains. J. Biotechnol 20: 284 – 295.
20
Co-expression of endoglucanase
& β-glucosidase in S. cerevisiae
1:
2:
SC Glucose
SC cellobiose
3:
SC CMC
[REF] = Y294 [yEP352]::fur1;
[SFI] = Y294 [BGL1]::fur1;
4:
YPD PASC
[EG1] = Y294 [EG1]::fur1
[CEL5] = Y294 [EG1+BGL1]::fur1 21
Conversion of amorphous cellulose
Growth on amorphous cellulose (PASC)
[REF]
[SFI]
[EG1]
[CEL5]
Den Haan, R., S.H. Rose, L.R. Lynd, and W.H. Van Zyl. 2007. Hydrolysis and fermentation of amorphous
cellulose by recombinant Saccharomyces cerevisiae. Met. Eng. 9: 87–94.
22
Functional expression of
cellobiohydrolases in yeast
23
Expression of cellobiohydrolases in yeast
CBH expression in S. cerevisiae
Functional CBH expression: a long-standing but elusive goal!
pCBH1-4 =
pDLG100 =
ENO1p
ENO1
P
ENO1p
ENO1
P
P. chrysosporium CBH1-4
P. chrysosporium CBH1-4
pAZ21 =
ENO1P
ENO1
reesei CBH2
T.T.reesei
CBH2
pAZ22 =
ENO1p
ENO1
P
T.reesei CBH1
T.reesei
CBH1
pDLG82 =
ENO1p
ENO1
P
A.niger
niger CBHB
A.
CBHB
= XYNSEC secretion signal
ENO1tT
ENO1
ENO1tT
ENO1
ENO1tT
ENO1
ENO1tT
ENO1
ENO1tT
ENO1
= HisTag
24
Expression of cellobiohydrolases in yeast
CBH1 cellobiohydrolase production by yeast
MW (Da)
206,675
115,758
98,003
54,604
37,390
29,559
Y294[CBH1]
~150X conc.
20,366
7,036
Y294[CBH1] ~20X
conc.
Y294[CBH1]
~150X conc.
treated with
Pngase
+ control CBH1
~250 ng,
treated with
Pngase
Den Haan, R., J.E. Mcbride, D.C. La Grange, L.R. Lynd, and W.H. Van Zyl. 2007. Functional
expression of cellobiohydrolases in Saccharomyces cerevisiae towards one-step conversion of
25
cellulose to ethanol. Enzyme Microb. Technol. 40: 1291–1299.
Expression of cellobiohydrolases in yeast
Cellobiohydrolase production by yeast
mUnits/g dry cell weight
10000
2.6% of t.c.p.
1000
•CBH1 requirements
calculated based on
ratio of CBH1 to other
cellulase components in
T. reesei cellulase
mixtures to allow
growth rate of 0.02 hr-1
100
10
1
CBH1 CBH1-4 CBHB
CBH2
CBH1 CBH1
Aerobic Anaerobic
CBH expressed in yeast
Den Haan, R., J.E. Mcbride, D.C. La Grange, L.R. Lynd, and W.H. Van Zyl. 2007. Functional expression of
cellobiohydrolases in Saccharomyces cerevisiae towards one-step conversion of cellulose to ethanol. Enzyme
26
Microb. Technol. 40: 1291–1299.
Recent advances towards
realizing CBP
27
Mascoma Corporation
Technical facilities, Lebanon, NH, USA
(www.mascoma.com)
28
Leading Investment, Unprecedented Focus on CBP
Technical Focus: Overcoming the biomass recalcitrance barrier and enabling
the emergence of a cellulosic biofuels industry via pioneering CBP technology
integrated with advanced pretreatment
Partners in Mascoma’s CBP Organism Development Effort
• VTT
• BioEnergy Science Center
• Dartmouth College
• Department of Energy
• University of Stellenbosch
Three Platforms
1. T. saccharolyticum, thermophilic bacterium able to use non-glucose sugars
2. C. thermocellum, thermophilic cellulolytic bacterium
3. Yeast engineered to utilize cellulose and ferment glucose and xylose
Multiple chances to succeed near-term & long-term
29
Subsequent expression of cellobiohydrolases!
12% SDS-PAGE, silver staining
170
130
95
72
KDa
KDa
KDa
KDa
55 KDa
43 KDa
34 KDa
26 KDa
30
Cellulase (mg/g DCW)
Mascoma Cellulolytic Yeast
120
Cellulase expression in Mascoma Yeast
(robust C5/C6 fermenting) vs Time
Total cellulase (mg/g)
100
100
80
60
40
13
20
0.62
0
0.03
2.4
T. reesei cellulase T. reesei cellulase
Proprietary
Cellulase
Cellulase
in yeast
in Mascoma Yeast
cellulase in
expression
expression
(Reinikainen
(March '07)
Mascoma Yeast in Mascoma yeast in Mascoma yeast
et al., 1992)
(March '08)
(October '08)
(December '08)
Cellulase expression Time-line
Mar, 2007 to Dec, 2008: >3,000-fold improvement in expression levels
31
Enzyme Reduction on Hardwood
Mascoma CBP Strain (robust C5/C6 fermenting yeast) +
22% w/w unwashed Pretreated Hardwood + Commercial cellulase
45
Equivalent
performance
with 2.5-fold
less added
enzyme
Ethanol (g/L)
40
35
30
25
20
15
Mascoma non-cellulolytic yeast + baseline
commercial cellulase
Mascoma cellulolytic yeast + only 40% of
baseline commercial cellulase
10
5
Further
reduction
likely
0
0
20
40
60
80
100
120
Fermentation Time (hours)
140
160
32
Conversion of Paper Sludge to Ethanol:
Proof of CBP Concept
Mascoma CBP technology on 18% w/w paper sludge
Ethanol Yield (g/L)
(1 mg/g TS b-glucosidase and 1 mg/g TS xylanase added)
50 Mascoma non-CBP Yeast,
w/10 FPU loading
40 cellulases
Mascoma CBP Yeast,
no added cellulase
30
20
Non-CBP Yeast
10
0
0
20
40
60
80
100
Fermentation Time (hours)
120
140
85% cellulose conversion with production of recoverable
33
ethanol with no added cellulase!
Rome, NY Pilot & Demonstration Plant
January 2008
November 2008
34
Acknowledgements
Stellenbosch University, South Africa
Danie la Grange
Riaan den Haan
Shaunita Rose
Ronél van Rooyen
Dartmouth College, USA
John McBride
Lee Lynd
VTT, Finland
Marja Ilmen
Merja Penttilä
35
Chair of Energy Research : Biofuels (members)
Microbiology
Proc Eng
Chem Eng
36
Technologies for Cellulose Conversion
Cellulosics biofuels production value chain:
Primary
Biomass
Production
Biomass
Transport
Biomass
Biomass
Conversion Product
Primary
to Biofuels Distribution
Processing and By& Marketing
products
1. The CoER : Biofuels positions itself in the conversion
technologies, but acknowledges the importance of
establishing the whole value chain.
2. These includes both biochemical and thermochemical
processes and integration of the processes if
applicable
37
Technologies for Cellulose Conversion
Biomass Biorefinery Concept
Non-Biological
Processing
Biological
Processing
• Pretreatment
• Fermentation
• Separation
Cellulosic
Biomass
• Gasification
Residues • Fast pyrolysis
Steam
Exported Power
/Biofuels
• Power generation
• Synthesis & separation
Thermochemically
-derived
Chemicals
(potentially several)
Process Power
Ethanol
Biologically-derived
Chemicals
(potentially several)
Animal feed
Lynd et al. 2003. Plant Biomass Conversion to Fuels and Commodity Chemicals in South Africa: A Third
38
Chapter? South African Journal of Science 99: 499 – 507.
Biomass potential of Africa at large
Ratio of the energy content of the biomass on abandoned
agriculture lands relative to the current primary energy demand at
the country level. The energy content of biomass is assumed to be
20 kJ g−1. Source: Campbell et al. (2008)
39
South Africa’s potential:
Renewable biomass available
1. Residues
Agricultural
Maize stover
Sugar cane bagasse
Wheat straw
Sunflower stalks
Agricultural subtotal
Forest industry
Left in forest
Saw mill residue
Paper & board mill sludge
Forest industry subtotal
2. Energy crops
From 10% of available land
(Marrison and Larson, 1996)
3. Invasive plant species
Total, annual basis
6.7 Mt/a
(118 PJ/a)
3.3 Mt/a
(58 PJ/a)
1.6 Mt/a
(28 PJ/a)
0.6 Mt/a
(11 PJ/a)
12.3 Mt/a (214 PJ/a)
4.0 Mt/a
0.9 Mt/a
0.1 Mt/a
5.0 Mt/a
(69 PJ/a)
(16 PJ/a)
(2 PJ/a)
(87 PJ/a)
67 Mt/a (1 171 PJ/a)
8.7 Mt
(151 PJ)
93 Mt/a (1 622 PJ/a)
Lynd et al. 2003. Plant Biomass Conversion to Fuels and Commodity Chemicals in South Africa: A Third
40
Chapter? South African Journal of Science 99: 499 – 507.
South Africa’s potential:
Biofuels production
30000
20000
15000
10000
Maize to Ethanol = 430 L/ton
Biomass to upgraded bio-oils = 310 L/ton
Curr fuel
Strategy
target
Burned
grasses
Total
Energy
crops
Subtotal
Invasive
plants
Forest
biowaste
0
Agricult
biowaste
5000
Maize
Volumes in ML
25000
Petrol
Diesel
Ethanol (70% of residue)
Upgraded bio-oil (70% of residue)
BtL (50%)
Biomass to ethanol = 280 L/ton
Biomass to liquid (BtL) = 570 L/ton
41
Construction of cellulolytic S. cerevisiae
Thank you!
42