CoER : Biofuels Department of Microbiology • Faculty of Natural Sciences
UNIVERSITEIT • STELLENBOSCH • UNIVERSITY
jou kennisvennoot • your knowledge partner
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 Africa2Chemical and Biochemical Engineering Program, Thayer School of
Engineering, Dartmouth College, Hanover, NH 03755, USA
2
Content
4. Functional expression of cellobiohydrolases in yeast
3. Conversion of amorphous cellulose to yeast biomass
6. Acknowledgments
1. Next generation technologies for cellulose conversion
2. What is Consolidated Bioprocessing?
5. Recent advances towards realizing CBP
7. SANERI Senior Chair of Energy Research : Biofuels
3
Next generation technologies for cellulose conversion
4
Lignocellulose composition
Sugarcane bagasseLignin28%
Arabinan2%
Xylan25%
Cellulose46%
Hexoses(fermentable) Pentoses
(fermentable)
Non-fermentablesugars
high energy aromatics
Technologies for Cellulose Conversion
5
Enzymatic hydrolysis of biomass
Ligninases(laccases, lignin peroxidases,Mn-peroxidases)
Cellulases(endoglucanases, cellobiohydrolases,-glucosidases)
Hemicellulases(xylanases, -xylosidases
-arabinofuranosidases-glucuronidases)
Esterases(feruloyl esterases,coumaroyl esterases)
Technologies for Cellulose Conversion
6
Biomass Processes for EtOH productionBiologically-Mediated
Event
Enzyme Hydrolysis Processing Strategy1
(Each box represents a bioreactory - not to scale)
CellulaseProduction
LignocelluloseHydrolysis
HexoseFermentation
PentoseFermentation
SHF: Separate Hydrolysis & Fermentation
SHF
O2
SSF: Simultaneous Saccharification & Fermentation
SSF
O2
SSCF: Simultaneous Saccharification & Co-Fermenation
SSCF
O2
CBP: Consolidated Bioprocessing
CBP
Technologies for Cellulose Conversion
7
Consolidated BioProcessing (CBP)
8
Microbiology and Molecular Biology Reviews 66: 506-577 (2002)
Fundamentals of Microbial Cellulose Utilization
Consolidated BioProcessing (CBP)
9
Current Opinion in Biotechnology 16:577–583 (2005)
Consolidated BioProcessing (CBP)Consolidated bioprocessing :
update
10
Advances in Biochemical Engineering / Biotechnology (2007)
Consolidated bioprocessing : update (2)
Consolidated BioProcessing (CBP)
11
Applied Microbiology & Biotechnology (2010) [corrected proofs]
Consolidated BioProcessing (CBP)Consolidated bioprocessing :
update (3)
12Applied Microbiology & Biotechnology (2010) [corrected proofs]
Consolidated BioProcessing (CBP)Consolidated bioprocessing: update (3)
Good activity
Low activity
Very low activity
No activity
WT GE WT WT GE WT WT GE WT GE WT GE WT GE WT GE WT GE WT GE
NA
Utilize cellobiose
Utilize xylobiose NA NA
E E E E E E E E E E E E B B L L E
E E E E E E E E E E B B L L ENA NA NA NA NA NA
NA NA NA NA
NA NA
High temperature
K.m
arx
ian
us
T. sa
ccha
roly
ticu
m
Grow on xylose
Ferment glucose to ethanol
Ferment xylose to ethanol
Resistant to hydrolysate inhibitors
GRAS status
Low pH
Lac
tic
acid
bac
teri
a
Breakdown crystalline cellulose
Breakdown amorphous cellulose
Breakdown hemicellulose
C. a
ce
tob
uty
lic
um
Grow on glucose
H. p
oly
mo
rph
a
S.c
ere
vis
iae
E.c
oli
Z.m
ob
ilis
K.o
xy
toc
a
P.s
tip
itis
13
Ethanol production from sugar
Spent
yeast
Crashing
Sugar
extraction
Sugarcane\
Sugarbeet
Sweet
sorghum
Fuel
blending
Alcoholrecovery
Distillation & dehydration
Storage
tank
Yeast
Fermentation
Technologies for Ethanol Production
Sugar
Storage
tank
14
Ethanol production from cellulosics
Spent
material
Pre-treatment
Cooling &
conditioning
Chipping
Grinding
Agric Res
Woody
Material
Grasses
Water
mixing
tank
Steam explosion
~200ºC
Cellulases YeastAlcoholrecovery
Fuel
blending
Saccharification Fermentation Distillation & dehydration
Storage
tank
Technologies for Ethanol Production
Largest Component of Recalcitrance Barrier:Cost of Cellulase
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.
g) Sheridan (Novozymes) Nature Biotech, 2008.
0.01
0.10
1.00
10.00
1990 1995 2000 2005 2010
Es
tim
ate
d C
ellu
las
e C
os
t
($/g
al E
tOH
)
Year
2008
g
2007
f
e
2004
d
2000
c
1997
b
a
1991
15
16
Consolidated BioProcessing (CBP)
OO
O
O
O
Glu Man Gal
Xyl Ara
Ethanol + CO2
P TYFG
Glycosyl
Hydrolases
17
Ethanol production from cellulosics
Spent
material
Pre-treatment
Cooling &
conditioning
Chipping
Grinding
Agric Res
Woody
Material
Grasses
Water
mixing
tank
Steam explosion
~200ºC
Cellulases
Fuel
blending
Saccharification
Alcoholrecovery
Distillation & dehydration
Storage
tank
Yeast
Fermentation
Technologies for Ethanol Production
18Spent
material
Pre-treatment
Cooling &
conditioning
Chipping
Grinding
Agric Res
Woody
Material
Grasses
Water
mixing
tank
Steam explosion
~200ºC
Fuel
blending
Alcoholrecovery
Distillation & dehydration
Storage
tank
Cellulolytic Yeast
Saccharification & Fermentation
Technologies for Ethanol Production
Ethanol production from cellulosics
19
Conversion of amorphous celluloseto yeast biomass
20
∑ host - glucose
Cellobiose utilization by S. cerevisiae
SC media;5g/L cellobiose; 30°C / pH adjusted to 6.0 (KOH)
∑ySF1 - cellobiose
∑ host - cellobiose
∑ySF1 - glucose
0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
2.0
0 10 20 30 40 50
OD
60
0
Time (hrs)
max = 0.20
max = 0.21
max = 0.21
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.
Cellobiose utilization in yeast
ΣySF1 = S. cerevisiae expressing S.fibuligeraβ-glucosidase (BGL1) gene from 2μ plasmid.
21
Co-expression of endoglucanase & β-glucosidase in S. cerevisiae
[REF] = Y294 [yEP352]::fur1; [EG1] = Y294 [EG1]::fur1[SFI] = Y294 [BGL1]::fur1; [CEL5] = Y294 [EG1+BGL1]::fur1
1: 2:
3: 4:
SC Glucose SC cellobiose
SC CMC YPD PASC
22
[REF] [EG1]
[SFI] [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.
Growth on amorphous cellulose (PASC)
Conversion of amorphous cellulose
23
Functional expression of cellobiohydrolases in yeast
24
CBH expression in S. cerevisiae
ENO1p ENO1tA. niger CBHBENO1P ENO1TA. niger CBHBpDLG82 =
ENO1 ENO1tT. reesei CBH2ENO1P ENO1TT. reesei CBH2pAZ21 =
ENO1p ENO1tT.reesei CBH1 ENO1P ENO1TT.reesei CBH1 pAZ22 =
= XYNSEC secretion signal
P. chrysosporium CBH1-4ENO1p ENO1tENO1P ENO1TpDLG100 =
P. chrysosporium CBH1-4ENO1p ENO1tENO1P ENO1TpCBH1-4 =
= HisTag
Functional CBH expression: a long-standing but elusive goal!
Expression of cellobiohydrolases in yeast
25
Y294[CBH1] ~20X conc.
Y294[CBH1] ~150X conc.
Y294[CBH1] ~150X conc. treated with
Pngase
+ control CBH1 ~250 ng,
treated with Pngase
206,675115,75898,003
54,604
37,390
29,559
20,366
7,036
MW (Da)
CBH1 cellobiohydrolase production by 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 Microb. Technol. 40: 1291–1299.
Expression of cellobiohydrolases in yeast
26
CBH expressed in yeast
mU
nits
/g d
ry c
ell w
eigh
t
1
10
100
1000
10000
CBH1 CBH1-4 CBHB CBH2 CBH1Aerobic
CBH1Anaerobic
•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
2.6% of t.c.p.
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
Microb. Technol. 40: 1291–1299.
Cellobiohydrolase production by yeast
Expression of cellobiohydrolases in yeast
27
Recent advances towards realizing CBP
28
Mascoma CorporationTechnical facilities, Lebanon, NH, USA
(www.mascoma.com)
Leading Investment, Unprecedented Focus on CBP
Technical Focus: Overcoming the biomass recalcitrance barrier and enablingthe emergence of a cellulosic biofuels industry via pioneering CBP technology integrated with advanced pretreatment
Partners in Mascoma’s CBP Organism Development Effort
• Dartmouth College
• University of Stellenbosch
• VTT • BioEnergy Science Center
Three Platforms
1. T. saccharolyticum, thermophilic bacterium able to use non-glucose sugars2. C. thermocellum, thermophilic cellulolytic bacterium3. Yeast engineered to utilize cellulose and ferment glucose and xylose
• Department of Energy
Multiple chances to succeed near-term & long-term29
Subsequent expression of cellobiohydrolases!
12% SDS-PAGE, silver staining
170 KDa130 KDa
95 KDa
72 KDa
55 KDa
43 KDa
34 KDa
26 KDa
30
Mascoma Cellulolytic Yeast
Mar, 2007 to Dec, 2008: >3,000-fold improvement in expression levels
Cellulase expression in Mascoma Yeast(robust C5/C6 fermenting) vs Time
T. reesei cellulase
in yeast
(Reinikainen
et al., 1992)
T. reesei cellulase
in Mascoma Yeast
(March '07)
Proprietary
cellulase in
Mascoma Yeast
(March '08)
Cellulase
expression
in Mascoma yeast
(October '08)
Cellulase expression Time-line
Cell
ula
se (
mg
/g D
CW
)
0.62 0.03 2.413
100
20
40
60
80
100
120Total cellulase (mg/g)
0Cellulase
expression
in Mascoma yeast
(December '08)
31
Enzyme Reduction on Hardwood
Equivalent performance with 2.5-fold less added enzyme
Further reduction likely
Mascoma CBP Strain (robust C5/C6 fermenting yeast) + 22% w/w unwashed Pretreated Hardwood + Commercial cellulase
0
5
10
15
20
25
30
35
40
45
0 20 40 60 80 100 120 140 160
Fermentation Time (hours)
Eth
an
ol (g
/L)
Mascoma non-cellulolytic yeast + baseline
commercial cellulase
Mascoma cellulolytic yeast + only 40% of
baseline commercial cellulase
32
Conversion of Paper Sludge to Ethanol: Proof of CBP Concept
85% cellulose conversion with production of recoverable ethanol with no added cellulase!
Mascoma CBP Yeast, no added cellulase
Mascoma non-CBP Yeast, w/10 FPU loadingcellulases
Non-CBP Yeast
Mascoma CBP technology on 18% w/w paper sludge (1 mg/g TS b-glucosidase and 1 mg/g TS xylanase added)
0
10
20
30
40
50
0 20 40 60 80 100 120 140
Fermentation Time (hours)
Eth
an
ol Y
ield
(g
/L)
33
Rome, NY Pilot & Demonstration Plant
January 2008
November 200834
35
Shaunita Rose
Riaan den Haan
Acknowledgements
Danie la Grange
Stellenbosch University, South Africa
Dartmouth College, USA
Ronél van Rooyen
John McBride Lee Lynd
VTT, Finland
Marja Ilmen Merja Penttilä
36
Microbiology
Chem EngProc Eng
Chair of Energy Research : Biofuels (members)
37
Cellulosics biofuels production value chain:
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
Technologies for Cellulose Conversion
Primary
Biomass
Production
Biomass
Transport
Biomass
Primary
Processing
Biomass
Conversion
to Biofuels
and By-
products
Product
Distribution
& Marketing
38
BiologicalProcessing• Pretreatment• Fermentation• Separation
Non-BiologicalProcessing
• Gasification• Fast pyrolysis• Power generation• Synthesis & separation
Biomass Biorefinery Concept
Steam
Process Power
Ethanol
CellulosicBiomass
Biologically-derivedChemicals (potentially several)
Thermochemically-derivedChemicals(potentially several)
Animal feed
Exported Power/BiofuelsResidues
Technologies for Cellulose Conversion
Lynd et al. 2003. Plant Biomass Conversion to Fuels and Commodity Chemicals in South Africa: A Third
Chapter? South African Journal of Science 99: 499 – 507.
39
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)
40
South Africa’s potential:Renewable biomass available
1. ResiduesAgriculturalMaize stover 6.7 Mt/a (118 PJ/a)Sugar cane bagasse 3.3 Mt/a (58 PJ/a)Wheat straw 1.6 Mt/a (28 PJ/a)Sunflower stalks 0.6 Mt/a (11 PJ/a)Agricultural subtotal 12.3 Mt/a (214 PJ/a)Forest industryLeft in forest 4.0 Mt/a (69 PJ/a)Saw mill residue 0.9 Mt/a (16 PJ/a)Paper & board mill sludge 0.1 Mt/a (2 PJ/a)Forest industry subtotal 5.0 Mt/a (87 PJ/a)
2. Energy crops From 10% of available land 67 Mt/a (1 171 PJ/a)
(Marrison and Larson, 1996)3. Invasive plant species 8.7 Mt (151 PJ)
Total, annual basis 93 Mt/a (1 622 PJ/a)Lynd et al. 2003. Plant Biomass Conversion to Fuels and Commodity Chemicals in South Africa: A Third
Chapter? South African Journal of Science 99: 499 – 507.
Tot
al
Ene
rgy
crop
s
Bur
ned
gras
ses
41
South Africa’s potential:Biofuels production
Maize to Ethanol = 430 L/ton Biomass to ethanol = 280 L/ton
Biomass to liquid (BtL) = 570 L/tonBiomass to upgraded bio-oils = 310 L/ton
BtL (50%)
0
5000
10000
15000
20000
25000
30000
Diesel
Ethanol
Petrol
Volum
es
in M
L
Mai
ze
For
est
bio
was
te
Inva
sive
plan
ts
Sub
tota
l
Cur
r fu
el
Str
ategy
targ
et
Agr
icul
tbio
was
te
Upgraded bio-oil (70% of residue)
(70% of residue)
42
Construction of cellulolytic S. cerevisiae
Thank you!