engineering saccharomyces cerevisiae for pentose fermentation · 2019-07-02 · engineering...
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Engineering Saccharomyces cerevisiaefor pentose fermentation
Ton van MarisDelft University of TechnologyDepartment of BiotechnologyDelft, the Netherlands
Detmold, April 25, 2007
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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
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sunlight
mining
burning
geologicalprocesses
biomass(plants)
CO2
photosynthesis
fuel(gasoline)
naturalreservoirs
oiloil refinery
(a few) years
(ethanol) €/$
naturalreservoirs
oil
mining
geologicalprocesses
oil refinery
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Ethanol Production: ~ 40 Mt.y-1 (2005)
starch, sucrose
glucose, fructose
hydrolysis
ethanol
S. cerevisiae(bakers’ yeast)
fermentation
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Agricultural residues and waste streams:Abundantly available
CheapNo competition with food production
Current Technology: No Utilization of Crop Residues
‘corn stover’cornstover bagasse wheatstraw
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Fuel Ethanol from Crop Residues: Strategy
Crop Residues
CelluloseHydrolysisPretreatment Fermentation
SugarMixture
Lignin
Ethanol
Combustion
Heat, electricity
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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
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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
?
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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)
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• 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
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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
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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
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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
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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
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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
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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
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< 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
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Further Improvements: ‘Evolution in the lab’
Generationtime~ 20 years
Generationtime~ 2 hour
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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
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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!
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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
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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
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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
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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
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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
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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
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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
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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
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A next challenge for metabolic engineering: pectine
+ Beetpulp
up to 30% pectine (galacturonic acid)
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Syntheticmedium
From the Lab… to the Real World
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cornstover bagasse wheatstraw
From the Lab to the Real World…
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Syntheticmedium
Planthydrolysate
From the Lab to the Real World and back again…
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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
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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….
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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
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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)
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• 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
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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
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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) €/$
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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
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• 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
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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
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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
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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
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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
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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)
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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
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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….
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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
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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
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maisloof tarwestro
Oil, Ethanol & Sugar
Ethanolproductie USA
18 billion litrein 2005!
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stop climate change
independence fromoil import
The petrochemical carbon cycle: Consequences
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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
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From the Real World back to the Lab:Effects of low pH and acetic acid
Syntheticmedium
Planthydrolysate
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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….
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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
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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