Pentose-Fermenting Zymomonas mobilis - EERE - U.S. Department ...
Pentose-Fermenting Zymomonas mobilis - EERE - U.S. Department ...
Pentose-Fermenting Zymomonas mobilis - EERE - U.S. Department ...
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<strong>Zymomonas</strong> <strong>mobilis</strong><br />
Special Topics Session<br />
Microbial <strong>Pentose</strong> Metabolism<br />
25th Symposium on<br />
Biotechnology for Fuels and Chemicals<br />
May 5, 2003<br />
Min Zhang<br />
National Bioenergy Center<br />
National Renewable Energy Laboratory
Glucose<br />
Fructose<br />
Sucrose<br />
D-Xylose<br />
L-Arabinose<br />
<strong>Zymomonas</strong> <strong>mobilis</strong><br />
Ethanol
Metabolic Engineering for <strong>Pentose</strong> Fermentation<br />
<strong>Pentose</strong> Metabolism Pathway<br />
D-Xylose<br />
Xylose Isomerase<br />
L-arabinose isomerase<br />
L-Ribulose<br />
L-ribulokinase<br />
D-Xylulose<br />
Xylulokinase<br />
ATP<br />
ADP<br />
L-Ribulose-5-P<br />
ATP<br />
ADP<br />
L-ribulose-5-P 4-epimerase<br />
D-Xylulose-5-P Ribulose-5-P Ribose-5-P<br />
Sedoheptulose-7-P Glyceraldehyde-3-P<br />
Erythrose-4-P<br />
Transketolase<br />
Transaldolase<br />
Transketolase<br />
L-Arabinose<br />
Fructose-6-P<br />
Fructose-6-P<br />
Glyceraldehyde-3-P<br />
Entner-Doudoroff Pathway<br />
D-Glucose<br />
Glucose-6-P<br />
Gluconolactone-6-P<br />
6-P-Gluconate<br />
2-Keto-3-deoxy-6-P-Gluconate<br />
Glyceraldehyde-3-P<br />
1,3-P-Glycerate<br />
ADP<br />
ATP<br />
3-P-Glycerate<br />
ATP<br />
ADP<br />
2-P-Glycerate<br />
ADP ATP<br />
Phosphoenolpyruvate Pyruvate<br />
Acetaldehyde + CO 2<br />
Ethanol
<strong>Zymomonas</strong> Strains Developed<br />
� Xylose-<strong>Fermenting</strong> Strains<br />
– Host CP4: CP4(pZB4) and CP4(pZB5) etc.<br />
– Host 39676: 39676(pZB4), 39676(pZB5) and 39676(pZB4L)<br />
�Hydrolysate-adapted 39676(pZB4L)<br />
�C25 (genomic integrated)<br />
– Host 31821/ZM4: ZM4(pZB5)<br />
�321(5), 2032 and 8b (genomic integrated)<br />
� Xylose and Arabinose-<strong>Fermenting</strong> Strains<br />
– Host 39676: 206C(pZB301) and 206C(pZB401)<br />
� BC1(pZB301)<br />
�AX strains (genomic integrated)<br />
� Xylose, Arabinose and Mannose-<strong>Fermenting</strong> Strains
Highlights of <strong>Zymomonas</strong> <strong>mobilis</strong><br />
• Natural fermentative microorganism (GRAS)<br />
• High ethanol yield from glucose (95-98% or 0.49-0.50 g/g)<br />
• Low cell mass formation<br />
• No oxygen requirement<br />
• High ethanol tolerance (13% ethanol from 30% glucose)<br />
• High specific productivity (2-6 g ethanol g dcw-1 . hr -1 )<br />
• High sugar uptake rate (up to 10 g glucose g dcw-1 . hr –1 )
Cultivation Conditions<br />
• Media:<br />
− RM: 10 g/L YE, 2 g/L KH2PO4, 2% glucose (desired sugars)<br />
or<br />
Clarified CSL (cCSL) (1%) + sugars<br />
− Fermentation Media using hydrolysate : cCSL (1%) + Hydrolysate,<br />
overlimed or neutralized supplemented with extra glucose.<br />
• Inoculum Size: 0.03-0.25 OD @600 nm (0.01 - 0.085 g cell/L)<br />
• Fermentor: pH 5 or pH 6 controlled with KOH (2N)<br />
• Temperature at 30°C or 37°C<br />
• No aeration
Xylose, Ethanol (g/L)<br />
120<br />
100<br />
80<br />
60<br />
40<br />
20<br />
0<br />
Z. <strong>mobilis</strong> 8b on RM 10% xylose<br />
Xylose 103 g/l<br />
Ethanol 48 g/l<br />
0 20 40 60 80 100 120<br />
Time (hr)<br />
1.2<br />
1.0<br />
0.8<br />
0.6<br />
0.4<br />
0.2<br />
0.0<br />
DCM (g/L)<br />
Y m: 0.47 g/g<br />
Y p: 0.46 g/g<br />
q E: 1.3 g/g/h<br />
Vol. Prod.: 0.4 g/l/h
Concentration<br />
(g/l)<br />
Fermentation of Xylose-<strong>Fermenting</strong> Z. <strong>mobilis</strong> Strain<br />
40<br />
30<br />
20<br />
10<br />
Z. <strong>mobilis</strong> 8b (genomically integrated)<br />
0<br />
0 10 20 30 40<br />
Time (hr)<br />
Glucose<br />
Xylo s e<br />
Ethanol<br />
Cell Mass<br />
RM media, pH=5.5, T=30 o C<br />
5<br />
4<br />
3<br />
2<br />
1<br />
0<br />
Cell Mass (g/l)<br />
Y m: 0.51 g/g<br />
Y p: 0.51 g/g<br />
q E: 1.13 g/g/h<br />
Vol. Prod.: 1.5 g/l/h
Corn Stover Hydrolysates<br />
Compound Concentration (g/L) Total Sugar Conc. (g/L)<br />
Cellobiose 1.91 110<br />
Glucose 15.65<br />
Xylose 69.20<br />
Arabinose 11.87<br />
Galactose 6.63<br />
Mannose 5.05<br />
Acetic acid 10.98<br />
Lactic acid 2.15<br />
HMF 0.67<br />
Furfural 1.14<br />
Acid soluble lignin 10.77<br />
Pretretment: using the flow-through mode of operation and at 25%<br />
solids, 190 o C and 0.048 g acid/g dry biomass (Schell et al. 2002).
% Xylose Utilization<br />
100<br />
80<br />
60<br />
40<br />
20<br />
0<br />
Increased Acetate Tolerance<br />
Comparison of %Xylose Utilization by Strains C25 and 8b<br />
at T 37°C, Grown on RMGX (2%:2%) in Baffled Shake Flask<br />
0 4 6 8 10<br />
Acetate (g/L)<br />
C25<br />
8b
Glucose, Xylose, Ethanol (g/l)<br />
50<br />
40<br />
30<br />
20<br />
10<br />
0<br />
Fermentation of Strain Z. <strong>mobilis</strong> 8b<br />
in cCSL+4% glu + 4 % xyl<br />
in the presence of 8 g/l Acetate at 37 C at pH 6<br />
0 10 20 30 40 50<br />
Time (hr)<br />
Glucose<br />
Xylos e<br />
Ethanol<br />
DCM<br />
Acetate<br />
12<br />
10<br />
8<br />
6<br />
4<br />
2<br />
0<br />
Acetate, DCM (g/l)<br />
Y m: 0.52 g/g<br />
Y p: 0.52 g/g<br />
q E: 4.1 g/g/h<br />
Vol. Prod.: 1.68 g/l/h
Glucose, Xylose,<br />
Ethanol (g/l)<br />
Fermentation profile of Z. <strong>mobilis</strong> 8b in 80% OL-<br />
Corn Stover Hydrolysate spiked with glucose at<br />
pH6 and 37C<br />
80<br />
70<br />
60<br />
50<br />
40<br />
30<br />
20<br />
10<br />
0<br />
0 20 40 60 80<br />
Time (hr)<br />
20<br />
15<br />
10<br />
5<br />
0<br />
Acetate (g/l)<br />
Glucose<br />
Xylose<br />
Ethanol<br />
Acetate<br />
Y m: 0.52 g/g<br />
Y p: 0.42 g/g<br />
Vol. Prod.: 2.1 g/l/h
Glucose, Xylose, Ethanol (g/l)<br />
Fermentation of Z. <strong>mobilis</strong> 8b in 40% Neutralized CSH<br />
spiked with glucose at 37 C pH6<br />
45<br />
40<br />
35<br />
30<br />
25<br />
20<br />
15<br />
10<br />
5<br />
0<br />
0 20 40 60 80<br />
Time (hr)<br />
Glucose<br />
Xylose<br />
Ethanol<br />
Acetate<br />
5<br />
4<br />
3<br />
2<br />
1<br />
0<br />
Acetate (g/l)<br />
Y m: 0.52 g/g<br />
Y p: 0.51 g/g<br />
Vol. Prod.: 1.45 g/l/h
Concentration (g/L)<br />
Adaptation of <strong>Zymomonas</strong> 39676(pZB4L) to above 60% (v/v) OL Hydrolyzate<br />
50<br />
40<br />
30<br />
20<br />
10<br />
0<br />
CSL=2% (v/v) D = 0.015 (1/hr)<br />
Strain Improvement through Adaptation<br />
Feed Mixture: Glucose (8 g/L) + Xylose (40 g/L)<br />
T=33C, pH=5.8<br />
glucose<br />
xylose<br />
ethanol<br />
% hydrolyzate<br />
0 10 20 30 40 50 60 70<br />
Time (Days)<br />
80<br />
70<br />
60<br />
50<br />
40<br />
30<br />
20<br />
10<br />
0<br />
Hydrolysate (%, V/V)<br />
Concentration (g/L)<br />
50<br />
40<br />
30<br />
20<br />
10<br />
0<br />
Comparison of Adapted and Parent Strain 39676(pZB4L) Grown on cCSL (2%),<br />
60% Sawdust Hydrolysate at pH 5.8, T 30°C, Initial Sugar Conc. (G:X 1%:5%)<br />
Glucose<br />
Xylose<br />
Ethanol<br />
Ethanol<br />
Adapted Parent<br />
Ethanol
Future Improvements<br />
• Ferment all biomass sugars (including mannose and<br />
galactose)<br />
• Improve pentose fermentation yield and productivity<br />
• Improve tolerance to hydrolysate toxicity
Metabolic Engineering Tools<br />
• Transcriptomics<br />
� Examine gene expression at global level<br />
• Metabolomics<br />
� Examine metabolites at global level<br />
• High-throughput Enzyme Assays<br />
� Compare strains<br />
� Compare activities at different stages of growth<br />
� Compare activities in different growth conditions<br />
� Develop improved enzymes<br />
• Mathematical approaches<br />
− Metabolic flux analysis<br />
− Kinetic modeling
Cytoplasmic membrane<br />
Is Sugar Transport limiting?<br />
Transporter protein<br />
in<br />
xylose<br />
out<br />
xylose<br />
Facilitated Diffusion Transport<br />
No energy required
Acknowledgements<br />
The Office of the Biomass Program<br />
of the US <strong>Department</strong> of Energy