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Engineering Saccharomyces cerevisiaefor pentose fermentation
Ton van MarisDelft University of TechnologyDepartment of BiotechnologyDelft, the Netherlands
Detmold, April 25, 2007
sunlight
mining
burning
geologicalprocesses
biomass(plants)
CO2
photosynthesis
fuel(gasoline)
naturalreservoirs
oiloil refinery
Mill
ion
s of
yea
rs
250
270
290
310
330
350
370
390
1850
atm
osph
eric
CO
2 (p
pm)
1900 1950 2000 2050
Year
The petrochemical carbon cycle
sunlight
mining
burning
geologicalprocesses
biomass(plants)
CO2
photosynthesis
fuel(gasoline)
naturalreservoirs
oiloil refinery
(a few) years
(ethanol) €/$
naturalreservoirs
oil
mining
geologicalprocesses
oil refinery
Ethanol Production: ~ 40 Mt.y-1 (2005)
starch, sucrose
glucose, fructose
hydrolysis
ethanol
S. cerevisiae(bakers’ yeast)
fermentation
Agricultural residues and waste streams:Abundantly available
CheapNo competition with food production
Current Technology: No Utilization of Crop Residues
‘corn stover’cornstover bagasse wheatstraw
Fuel Ethanol from Crop Residues: Strategy
Crop Residues
CelluloseHydrolysisPretreatment Fermentation
SugarMixture
Lignin
Ethanol
Combustion
Heat, electricity
Non-fermentable by S. cerevisiae
Sugars in Crop Residues: the Pentose Challenge
Corn stover Wheat straw Bagasse
Sugars (%)glucose 34.6 32.6 39.0
mannose 0.4 0.3 0.4
galactose 1.0 0.8 0.5
xylose 19.3 19.2 22.1
arabinose 2.5 2.4 2.1
uronic acids 3.2 2.2 2.2
Other (%)lignin 17.7 16.9 23.1
Grohmann & Bothast 1994Lee et al. 1997www.ecn.nl
D-xylose
Biochemistry of xylose metabolism
ethanolD-xylulose
PPP
pyruvate
D-xylulose-5-P
glucose
ATP
NADH
CO2
ATP
glucose-6-P
fructose-6-P
fructose-1,6-biP
DHAPG-3-P
PEP
NADH
ATP
ATP
ATP
glycerol
NADH
CO2
2 NADPH
xylulokinase
?
PPP
pyruvate
D-xylulose-5-P
D-xylose
xylitol
D-xylulose ethanol
glucose
NADPH
NADH
ATP
NADH
CO2
ATP
glucose-6-P
fructose-6-P
DHAP
fructose-1,6-biP
G-3-P
PEP
NADH
ATP
ATP
ATP
glycerol
NADH
CO2
2 NADPH
I
II
Xylose metabolism in non-Saccharomyces yeasts
• xylose-fermenting yeasts(e.g. Pichia stipitis)
• two-step conversion of xyloseinto xylulose:
I Xylose Reductase (XR)II Xylitol Dehydrogenase (XDH)
• Cloning of Pichia stipitisstructural genes for XRand XDH in S. cerevisiae
• Slow (aerobic) growth on xylose
• Ethanol production accompaniedby extensive production ofxylitol
Metabolic Engineering of S. cerevisiae for xylose fermentation: the beginning
6 xylose 10 ethanol + 10 CO2 + 10 ATP12 xylose 9 ethanol + 12 CO2 + 9 ATP + 6 xylitol6 xylose + 3 O2 9 ethanol + 12 CO2 + 9 ATP + 6 H2O
6 xylose6 xylitol
The XR-XDH Approach: Redox Constraints
3 O26 H2O
XR
6 ATP
6 xylose 6 xylitol 6 xylulose
6 C5P
6 NADH6 NAD
6 NADP6 NADPH
1 C6P
3 C6P
3 CO2
3 C5P
2 C6P
1 C3P 2 C3P4 CO2
8 ATP
1 ATP2 ATP
1 ethanol
4 C3P
4 ethanol
2 C3P
2 ethanol 2 ethanol
2 CO2
4 ATP1 CO2
2 ATP2 CO2
4 ATP
XDH
Research on XR-XDH strategy(1990 – present):
• Important insights in kinetics of xylose metabolism
- role of xylulokinase- xylose transport- role of pentose-phosphate-pathway enzymes- role of aldose reductase (Gre3p)- inhibitor sensitivity- aeration studies
Bärbel Hahn-Hägerdal Nancy HoThomas Jeffries
et al., and coworkers
• Redox constraints remain a challenge
PPP
pyruvate
D-xylulose-5-P
D-xylose
D-xylulose ethanol
glucose
ATP
NADH
CO2
xylitolNADPH
NADH
ATP
glucose-6-P
fructose-6-P
DHAP
fructose-1,6-biP
G-3-P
PEP
NADH
ATP
ATP
ATP
glycerol
NADH
CO2
2 NADPH
The ‘bacterial’ pathway for xylose fermentation
XI
• xylose isomerase (XI) catalysesxylose/xylulose isomerisation
• common in Bacteria, Archaea
6 xylosebacterial & ArchaealXI genes known,but no efficientexpression in yeast
6 xylose 10 ethanol + 10 CO2 + 10 ATP
xylose isomerase
6 xylulose
6 C5P
4 C6P
8 C3P
8 ethanol
2 C3P
2 ethanol
6 ATP
4 ATP
8 CO2
16 ATP2 CO2
4 ATP
pentose phosphate pathway
glycolysis
Xylose Isomerase: no redox constraints
A Novel, Fungal Xylose Isomerase Gene
Research at Nijmegen University, The Netherlands:
• Anaerobic Piromyces fungus from elephant dung• Isolation of first fungal xylose isomerase gene (XylA)
Harhangi et al. 2003Arch. Microbiol. 180:134
Kuyper et al. 2003FEMS Yeast Research 4:69
Follow-up in Delft:
• High expression levels in S. cerevisiae• XI expression not sufficient for fast xylose fermentation
+ Xyl A
wt
Metabolic Engineering of Xylose Conversion Overexpression of 5 S. cerevisiae genes (pentose-phosphate pathway)
Deletion of GRE3 (aspecific aldose reductase)
6 xylulose
6 xylulose-5P
2 C3P + 4 C6P
6 xylose
glycolysis
xylitol
ethanol
Xylulokinase (XKS1)
Transketolase (TKL1)
Ribose-5P-isomerase (RKI1)
Transaldolase (TAL1)
Ribulose-5P-epimerase (RPE1)
Aldose reductase (GRE3)
XylA
Kuyper et al. 2005FEMS Yeast Research 5:399-409
< 0.01Xylitol yield (g.g-1)
0.42Ethanol yield (g.g-1)
0.10μ xylose anaerobic (h-1)
Xylose Fermentation by Engineered S. cerevisiae
Kuyper et al. 2005FEMS Yeast Research 5:399-409
Anaerobic batch cultivation of the XI-expressing, metabolicallyengineered S. cerevisiae strain RWB 217 on xylose
xylose
glycerol
(xylitol)
CO2ethanol
XylA + XKS1↑ TAL1↑ TKL1↑ RPE1↑ RKI1↑ gre3Δ
Further Improvements: ‘Evolution in the lab’
Generationtime~ 20 years
Generationtime~ 2 hour
Selection for Improved Xylose Affinity
Kuyper et al. 2005FEMS Yeast Research 5: 925-934
Long-term cultivation of RWB217 in anaerobic, xylose-limited chemostat(D = 0.06 h-1): decrease of residual xylose concentration
CO2
ethanol
xylose
glucose
Anaerobic Fermentation of a glucose-xylose Mixture
Kuyper et al. 2005FEMS Yeast Research 5: 925-934
glucose
At industrially relevant rates and yields!
Non-fermentable by S. cerevisiae
The pentose challenge continued: L-arabinose
Corn stover Wheat straw Bagasse
Sugars (%)glucose 34.6 32.6 39.0
mannose 0.4 0.3 0.4
galactose 1.0 0.8 0.5
xylose 19.3 19.2 22.1
arabinose 2.5 2.4 2.1
uronic acids 3.2 2.2 2.2
Other (%)lignin 17.7 16.9 23.1
Grohmann & Bothast 1994Lee et al. 1997www.ecn.nl
PPP
pyruvate
(XKS1 / XYL3)
D-xylulose-5-P
D-xylose
D-xylulose
L-arabinose
(xylA)ethanol
glucose
ATP
NADH
CO2
L-ribulose L-ribulose-5-PATP
(araB)
(araD)
(araA)
ATP
glucose-6-P
fructose-6-P
fructose-1,6-biP
DHAPG-3-P
PEP
NADH
ATP
ATP
ATP
glycerol
NADH
CO2
2 NADPH
(GRE3 /XYL1)L-arabinitol
L-xylulose
xylitol
(XYL2)
NADH
NADPH
NADH
(lxr1)
(lad1)
NADPH
Pathways for L-arabinose fermentation
Arabinose fermentation by engineered S. cerevisiae
M.E. strategy Ethanol productivity
Fungal pathway
P. stipidis XYL1 + XYL2, T. reesei lad1 + lxr1, S. cerevisiae XKS1
0.35 mg g-1h-1 Richard et al. (2003)
Bacterial pathway
E. coli AraABD - Sedlak & Ho (2001)
B. subtilis AraA, E. coli AraBD+ evolution
60-80 mg g-1h-1 Becker & Boles (2003)
• Bacterial pathway most promising (Becker & Boles 2003)• Challenges: rate, anaerobicity, arabinitol formation
Strain construction
pAKXAD11440 bps
T_cyc1pMB1 ori
AmpR
URA3
P_TDH3AraA
T_ADH1P_HXT7
AraD
T_PGI1
2mu
P_TPI XylA
pRS305XKS1LpAraB11286 bps
T_ADH1
AraBP_PGI1
P_ADH1
XKS1
T_CYC
LEU2
Amp
• Host: XylA-expressing S. cerevisiae strain optimized for xylose fermentation
• Expression of AraA, AraB and AraD from Lactobacillus plantarum
Wouter Wisselink et al.unpublished
0 20 40 60 80 100 120 140 160 180 2000.00
0.05
0.10
0.15
time (days)
μ ( h
−1 )
Serial transfer in shake-flask cultures
serial transfers in synthetic medium with 2% (w/v) arabinose
Wouter Wisselink et al.unpublished
Evolutionary engineering for growth on L-arabinose
0 20 40 60 80 100 120 140 160 180 2000.00
0.05
0.10
0.15
time (days)
μ ( h
−1 )
Serial transfer in shake-flask cultures
Anaerobic SBR
Strain IMS0001
Single cell isolateIMS0002
Evolutionary engineering for growth on L-arabinose
serial transfers in synthetic medium with 2% (w/v) arabinose
Wouter Wisselink et al.unpublished
Characterisation of single-cell line:anaerobic batch cultures on L-arabinose and
glucose/L-arabinose mixture
glucose arabinose ethanol CO2 glycerol
0 10 20 30 40 50 60 70 800
20
40
60
80
100
120
140
160
0
100
200
300
400
500
time (h)
arab
inos
e, g
lyce
rol (
mM
)
etha
nol,
CO
2 (m
M)
0 10 20 30 40 50 60 70 800
20
40
60
80
100
120
140
160
0
100
200
300
400
500
time (h)
gluc
ose,
ara
bino
se, g
lyce
rol (
mM
)
etha
nol,
CO
2 (m
M)
qara= 0.66 ± 0.01 g g-1 h-1
qeth= 0.27 ± 0.01 g g-1 h-1qara= 0.46 ± 0.03 g g-1 h-1
qeth= 0.21 ± 0.02 g g-1 h-1
Wouter Wisselink et al.unpublished
Non-fermentable by S. cerevisiae
The challenge continued: uronic acids?
Corn stover Wheat straw Bagasse
Sugars (%)glucose 34.6 32.6 39.0
mannose 0.4 0.3 0.4
galactose 1.0 0.8 0.5
xylose 19.3 19.2 22.1
arabinose 2.5 2.4 2.1
uronic acids 3.2 2.2 2.2
Other (%)lignin 17.7 16.9 23.1
Grohmann & Bothast 1994Lee et al. 1997www.ecn.nl
A next challenge for metabolic engineering: pectine
+ Beetpulp
up to 30% pectine (galacturonic acid)
Syntheticmedium
From the Lab… to the Real World
cornstover bagasse wheatstraw
From the Lab to the Real World…
Syntheticmedium
Planthydrolysate
From the Lab to the Real World and back again…
Fed-batch Batch
0
1
2
3
4
5 20
10
10
ethanol
glucose
xylose
20 30 40 50
Suga
r (g
/L)
Etha
nol (
g/L)
Ethanol yield:0.47 g ethanol/g sugar238 L ethanol/ton dry
biomass
Ethanol Production from Wheat-Straw Hydrolysate(‘academic’ xylose-fermenting strain)
De Laat et al. 2006unpublished
0 6 12 18 24 30 360
5
10
15
20
25
0
2
4
6
8
10
Glucose Xylose Glycerol Acetic AcidEthanol BIomass
Time (hr)
Met
abol
ites
(g/L
)B
iomass (O
D 660 nm
)
Fermentation of glucose-xylose mixture by S. cerevisiae RWB218pH 3.5
Anaerobic batch culture, synthetic medium, 20 g.l-1 glucose and 20 g.l-1 xylose
Bellissimi et al. 2006unpublished
Low pH as such is not a problem forxylose-fermenting S. cerevisiae….
0 6 12 18 24 30 36 42 480
5
10
15
20
25
0
2
4
6
8
10
Glucose Xylose Glycerol Acetic AcidEthanol Biomass
Time (hr)
Met
abol
ites
(g/L
) Biom
ass (660 nm)
Fermentation of glucose-xylose mixture by S. cerevisiae RWB218pH 3.5, 3 g.l-1 acetic acid
Anaerobic batch culture, synthetic medium, 20 g.l-1 glucose and 20 g.l-1 xylose
Bellissimi et al. 2006unpublished
….but acetic acid specifically inhibitsxylose fermentation
• ethanol yieldon xylosenot affected
0 6 12 18 24 30 36 42 480
4
8
12
16
20
24
0
10
20
30
40
50
60
Glucose Xylose Acetic AcidEthanol BiomassGlycerol
Time (hr)
Met
abol
ites
(g/L
) Ethanol (g/L)
Continuous glucosefeed (2.4 g.l-1.h-1)
Bellissimi et al. 2006unpublished
Glucose co-feeding alleviates acetic acid inhibition ofxylose fermentation (pH 3.5 + 3 g.l-1 acetic acid)
Similar to simultaneous saccharification and fermentation (SSF)
• metabolic engineering and evolutionary engineering are powerful tools in industrial microbiology
• Isomerase-based pathways are the key for robust pentose fermentation by S. cerevisiae
• Inhibitor tolerance: experiments with real-life plant biomass hydrolysates (wheat straw, corn stover) are promising
• Future: Towards industrial scale fermentations?
Conclusions
AcknowledgementsDelft Industrial Microbiology
Derek AbbottMarinka AlmeringEleonora BellissimiJoost van den Brink Jean-Marc DaranPascale Daran-LapujadeLeonie van DijkHans van DijkenAndreas GombertDiana HarrisLucie HazelwoodEline HuisjesErik de HulsterZita van der KrogtMarko KuyperMarijke LuttikTon van Maris Jack PronkIshtar SnoekMaurice ToirkensHan de WindeWouter WisselinkRintze Zelle
sunlight
mining
burning
geologicalprocesses
biomass(plants)
CO2
photosynthesis
fuel(gasoline)
naturalreservoirs
oiloil refinery
Mill
ion
s of
yea
rs
250
270
290
310
330
350
370
390
1850 1900 1950 2000 2050
Year
atm
osph
eric
CO
2 (p
pm)
(a few) years
(ethanol) €/$
Research on XR-XDH strategy(1990 – present):
• Important insights in kinetics of xylose metabolism
- role of xylulokinase- xylose transport- role of pentose-phosphate-pathway enzymes- role of aldose reductase (Gre3p)- inhibitor sensitivity- aeration studies
Bärbel Hahn-Hägerdal Nancy HoThomas Jeffries
et al., and coworkers
• Redox constraints remain a challenge
• Cloning of Pichia stipitisstructural genes for XRand XDH in S. cerevisiae
• Slow (aerobic) growth on xylose
• Ethanol production accompaniedby extensive production ofxylitol
Metabolic Engineering of S. cerevisiae for xylose fermentation: the beginning
Anaerobic Mixed-Substrate Utilisation
xylose
CO2
glucose
ethanol
Suboptimal kinetics of xyloseutilisation: an affinity problem?
Kuyper et al. 2005FEMS Yeast Research 5:399-409
Anaerobic growth of S. cerevisiae RWB217 on 20 g/L glucose and 20 g/L xylose
CEN.PK113-7D RWB217 RWB218Reference Genetically Evolved
engineered
Glucose Glucose Glucose
Xylose Xylose
Transcriptome analysis
• Affymetrix GeneChips • triplicate chemostat cultures(anaerobic, C-limited, D = 0.05 h-1)for each strain/condition
Transcriptome Analysis (1)Genes involved in hexose transport
Bellissimi et al. unpublished
Transcript LevelsGenes
Parental strainRWB 217
Evolved strainRWB 218
HXT 1 14.2 ± 3.87 91.7 ± 26.3 6.44 0.034
HXT 2 394.9 ± 131 2645.4 ± 756
HXT 3 2194 ± 816 39.3 ± 12.8
9.5 0.025
-39 0.18
HXT 4 325 ± 258 1872 ± 430
HXT 5 394.9 ± 46.9 109.9 ± 32.3
8.4 0.008
-3.59 0.002
HXT 6 4144.9 ± 359 3696 ± 459 -1.12 0.26
HXT 7 3573 ± 201 3824.8 ± 441 1.07 0.44
HXT 8 12.03 ± 0.058 13.6 ± 2.14 1.13 0.34
HXT 9 12 12 1.0 -
HXT 10 12.23 ± 0.4 14.13 ± 3.7 1.16 0.47
HXT 12 56.4 ± 6.5 43.2 ± 11.1 -1.3 0.17
HXT 14 12.97 ± 1.67 12.83 ± 1.27 -1.01 0.92
HXT 16 834.4 ± 231.5 26.6 ± 2.51 -31 0.026
GAL 2 12.03 ± 0.058 12.1 ± 0.17 1.01 0.58
RGT 2 36.6 ± 7.80 55.1 ± 8.51 1.39 0.08
SNF 3 29.6 ± 3.66 25.2 ± 3.46 -1.17 0.21
Fold Change
p-value
Altered kinetics of U-14C xylose transport
CEN.PK113-7D RWB217 RWB218Reference Genetically Evolved
engineered
Glucose Glucose Glucose
Xylose Xylose
Transcriptome analysis
• Affymetrix GeneChips • triplicate chemostat cultures(anaerobic, C-limited, D = 0.05 h-1)for each strain/condition
RWB 217(genetic engineering only)
70 transcripts higher on xylose70 transcripts higher on xylose25 transcripts higher on glucose25 transcripts higher on glucose
100/6000 = 1.7 % of transcriptome100/6000 = 1.7 % of transcriptome
Transcriptome analysis (2)Transcriptional responses to carbon source (glucose, xylose)
2-fold threshold, statistical analysis with SAM algorithm
Bellissimi, Kuyper et al. 2006unpublished
0 transcripts higher on xylose0 transcripts higher on xylose6 transcripts higher on glucose6 transcripts higher on glucose
6/6000 = 0.1 % of transcriptome6/6000 = 0.1 % of transcriptome
RWB 218(genetic engineering &evolutionary engineering)
Strain construction
pAKXAD11440 bps
T_cyc1pMB1 ori
AmpR
URA3
P_TDH3AraA
T_ADH1P_HXT7
AraD
T_PGI1
2mu
P_TPI XylA
pRS305XKS1LpAraB11286 bps
T_ADH1
AraBP_PGI1
P_ADH1
XKS1
T_CYC
LEU2
Amp
• Host: XylA-expressing S. cerevisiae strain optimized for xylose fermentation
• Expression of AraA, AraB and AraD from Lactobacillus plantarum
Wouter Wisselink et al.unpublished
0 6 12 18 24 30 360
5
10
15
20
25
0
2
4
6
8
10
Glucose Xylose Glycerol Acetic AcidEthanol BIomass
Time (hr)
Met
abol
ites
(g/L
)B
iomass (O
D 660 nm
)
Fermentation of glucose-xylose mixture by S. cerevisiae RWB218pH 3.5
Anaerobic batch culture, synthetic medium, 20 g.l-1 glucose and 20 g.l-1 xylose
Bellissimi et al. 2006unpublished
Low pH as such is not a problem forxylose-fermenting S. cerevisiae….
0 6 12 18 24 30 36 42 480
5
10
15
20
25
0
2
4
6
8
10
Glucose Xylose Glycerol Acetic AcidEthanol Biomass
Time (hr)
Met
abol
ites
(g/L
) Biom
ass (660 nm)
Fermentation of glucose-xylose mixture by S. cerevisiae RWB218pH 3.5, 3 g.l-1 acetic acid
Anaerobic batch culture, synthetic medium, 20 g.l-1 glucose and 20 g.l-1 xylose
Bellissimi et al. 2006unpublished
….but acetic acid specifically inhibitsxylose fermentation
• ethanol yieldon xylosenot affected
Why is xylose fermentation more sensitive to acetic acidthan glucose fermentation ?
CH3COO- + H+ ↔ CH3COOH
CH3COO- + H+ ↔ CH3COOH pH 3.5pH 7
H+
ATP
ADPADP
ATP
glucose/xylose
ethanol + CO2
Bellissimi et al. 2006unpublished
maisloof tarwestro
Oil, Ethanol & Sugar
Ethanolproductie USA
18 billion litrein 2005!
stop climate change
independence fromoil import
The petrochemical carbon cycle: Consequences
stop climate change
independence fromoil import
The Bio-ethanol Boom: a Weird & Wonderful Coalition
new marketsfor farmers
This corresponds to 30 million tonnesof fuel ethanol per year
Target: 5,75 % replacement of all petrol and diesel for transport purposes by December 31, 2010
From the Real World back to the Lab:Effects of low pH and acetic acid
Syntheticmedium
Planthydrolysate
0 6 12 18 24 30 360
5
10
15
20
25
0
2
4
6
8
10
Glucose Xylose Glycerol Acetic AcidEthanol BIomass
Time (hr)
Met
abol
ites
(g/L
)B
iomass (O
D 660 nm
)
Fermentation of glucose-xylose mixture by S. cerevisiae RWB218pH 3.5
Anaerobic batch culture, synthetic medium, 20 g.l-1 glucose and 20 g.l-1 xylose
Bellissimi et al. 2006unpublished
Low pH as such is not a problem forxylose-fermenting S. cerevisiae….
0 6 12 18 24 30 36 42 480
5
10
15
20
25
0
2
4
6
8
10
Glucose Xylose Glycerol Acetic AcidEthanol Biomass
Time (hr)
Met
abol
ites
(g/L
) Biom
ass (660 nm)
Fermentation of glucose-xylose mixture by S. cerevisiae RWB218pH 3.5, 3 g.l-1 acetic acid
Anaerobic batch culture, synthetic medium, 20 g.l-1 glucose and 20 g.l-1 xylose
Bellissimi et al. 2006unpublished
….but acetic acid specifically inhibitsxylose fermentation
• ethanol yieldon xylosenot affected
Why is xylose fermentation more sensitive to acetic acidthan glucose fermentation ?
CH3COO- + H+ ↔ CH3COOH
CH3COO- + H+ ↔ CH3COOH pH 3.5pH 7
H+
ATP
ADPADP
ATP
glucose/xylose
ethanol + CO2
Bellissimi et al. 2006unpublished