Pentose-Fermenting Zymomonas mobilis - EERE - U.S. Department ...

Zymomonas mobilis
Special Topics Session
Microbial Pentose Metabolism
25th Symposium on
Biotechnology for Fuels and Chemicals
May 5, 2003
Min Zhang
National Bioenergy Center
National Renewable Energy Laboratory

Glucose
Fructose
Sucrose
D-Xylose
L-Arabinose
Zymomonas mobilis
Ethanol

Metabolic Engineering for Pentose Fermentation
Pentose Metabolism Pathway
D-Xylose
Xylose Isomerase
L-arabinose isomerase
L-Ribulose
L-ribulokinase
D-Xylulose
Xylulokinase
ATP
ADP
L-Ribulose-5-P
ATP
ADP
L-ribulose-5-P 4-epimerase
D-Xylulose-5-P Ribulose-5-P Ribose-5-P
Sedoheptulose-7-P Glyceraldehyde-3-P
Erythrose-4-P
Transketolase
Transaldolase
Transketolase
L-Arabinose
Fructose-6-P
Fructose-6-P
Glyceraldehyde-3-P
Entner-Doudoroff Pathway
D-Glucose
Glucose-6-P
Gluconolactone-6-P
6-P-Gluconate
2-Keto-3-deoxy-6-P-Gluconate
Glyceraldehyde-3-P
1,3-P-Glycerate
ADP
ATP
3-P-Glycerate
ATP
ADP
2-P-Glycerate
ADP ATP
Phosphoenolpyruvate Pyruvate
Acetaldehyde + CO 2
Ethanol

Zymomonas Strains Developed
� Xylose-Fermenting Strains
– Host CP4: CP4(pZB4) and CP4(pZB5) etc.
– Host 39676: 39676(pZB4), 39676(pZB5) and 39676(pZB4L)
�Hydrolysate-adapted 39676(pZB4L)
�C25 (genomic integrated)
– Host 31821/ZM4: ZM4(pZB5)
�321(5), 2032 and 8b (genomic integrated)
� Xylose and Arabinose-Fermenting Strains
– Host 39676: 206C(pZB301) and 206C(pZB401)
� BC1(pZB301)
�AX strains (genomic integrated)
� Xylose, Arabinose and Mannose-Fermenting Strains

Highlights of Zymomonas mobilis
• Natural fermentative microorganism (GRAS)
• High ethanol yield from glucose (95-98% or 0.49-0.50 g/g)
• Low cell mass formation
• No oxygen requirement
• High ethanol tolerance (13% ethanol from 30% glucose)
• High specific productivity (2-6 g ethanol g dcw-1 . hr -1 )
• High sugar uptake rate (up to 10 g glucose g dcw-1 . hr –1 )

Cultivation Conditions
• Media:
− RM: 10 g/L YE, 2 g/L KH2PO4, 2% glucose (desired sugars)
or
Clarified CSL (cCSL) (1%) + sugars
− Fermentation Media using hydrolysate : cCSL (1%) + Hydrolysate,
overlimed or neutralized supplemented with extra glucose.
• Inoculum Size: 0.03-0.25 OD @600 nm (0.01 - 0.085 g cell/L)
• Fermentor: pH 5 or pH 6 controlled with KOH (2N)
• Temperature at 30°C or 37°C
• No aeration

Xylose, Ethanol (g/L)
120
100
80
60
40
20
0
Z. mobilis 8b on RM 10% xylose
Xylose 103 g/l
Ethanol 48 g/l
0 20 40 60 80 100 120
Time (hr)
1.2
1.0
0.8
0.6
0.4
0.2
0.0
DCM (g/L)
Y m: 0.47 g/g
Y p: 0.46 g/g
q E: 1.3 g/g/h
Vol. Prod.: 0.4 g/l/h

Concentration
(g/l)
Fermentation of Xylose-Fermenting Z. mobilis Strain
40
30
20
10
Z. mobilis 8b (genomically integrated)
0
0 10 20 30 40
Time (hr)
Glucose
Xylo s e
Ethanol
Cell Mass
RM media, pH=5.5, T=30 o C
5
4
3
2
1
0
Cell Mass (g/l)
Y m: 0.51 g/g
Y p: 0.51 g/g
q E: 1.13 g/g/h
Vol. Prod.: 1.5 g/l/h

Corn Stover Hydrolysates
Compound Concentration (g/L) Total Sugar Conc. (g/L)
Cellobiose 1.91 110
Glucose 15.65
Xylose 69.20
Arabinose 11.87
Galactose 6.63
Mannose 5.05
Acetic acid 10.98
Lactic acid 2.15
HMF 0.67
Furfural 1.14
Acid soluble lignin 10.77
Pretretment: using the flow-through mode of operation and at 25%
solids, 190 o C and 0.048 g acid/g dry biomass (Schell et al. 2002).

% Xylose Utilization
100
80
60
40
20
0
Increased Acetate Tolerance
Comparison of %Xylose Utilization by Strains C25 and 8b
at T 37°C, Grown on RMGX (2%:2%) in Baffled Shake Flask
0 4 6 8 10
Acetate (g/L)
C25
8b

Glucose, Xylose, Ethanol (g/l)
50
40
30
20
10
0
Fermentation of Strain Z. mobilis 8b
in cCSL+4% glu + 4 % xyl
in the presence of 8 g/l Acetate at 37 C at pH 6
0 10 20 30 40 50
Time (hr)
Glucose
Xylos e
Ethanol
DCM
Acetate
12
10
8
6
4
2
0
Acetate, DCM (g/l)
Y m: 0.52 g/g
Y p: 0.52 g/g
q E: 4.1 g/g/h
Vol. Prod.: 1.68 g/l/h

Glucose, Xylose,
Ethanol (g/l)
Fermentation profile of Z. mobilis 8b in 80% OL-
Corn Stover Hydrolysate spiked with glucose at
pH6 and 37C
80
70
60
50
40
30
20
10
0
0 20 40 60 80
Time (hr)
20
15
10
5
0
Acetate (g/l)
Glucose
Xylose
Ethanol
Acetate
Y m: 0.52 g/g
Y p: 0.42 g/g
Vol. Prod.: 2.1 g/l/h

Glucose, Xylose, Ethanol (g/l)
Fermentation of Z. mobilis 8b in 40% Neutralized CSH
spiked with glucose at 37 C pH6
45
40
35
30
25
20
15
10
5
0
0 20 40 60 80
Time (hr)
Glucose
Xylose
Ethanol
Acetate
5
4
3
2
1
0
Acetate (g/l)
Y m: 0.52 g/g
Y p: 0.51 g/g
Vol. Prod.: 1.45 g/l/h

Concentration (g/L)
Adaptation of Zymomonas 39676(pZB4L) to above 60% (v/v) OL Hydrolyzate
50
40
30
20
10
0
CSL=2% (v/v) D = 0.015 (1/hr)
Strain Improvement through Adaptation
Feed Mixture: Glucose (8 g/L) + Xylose (40 g/L)
T=33C, pH=5.8
glucose
xylose
ethanol
% hydrolyzate
0 10 20 30 40 50 60 70
Time (Days)
80
70
60
50
40
30
20
10
0
Hydrolysate (%, V/V)
Concentration (g/L)
50
40
30
20
10
0
Comparison of Adapted and Parent Strain 39676(pZB4L) Grown on cCSL (2%),
60% Sawdust Hydrolysate at pH 5.8, T 30°C, Initial Sugar Conc. (G:X 1%:5%)
Glucose
Xylose
Ethanol
Ethanol
Adapted Parent
Ethanol

Future Improvements
• Ferment all biomass sugars (including mannose and
galactose)
• Improve pentose fermentation yield and productivity
• Improve tolerance to hydrolysate toxicity

Metabolic Engineering Tools
• Transcriptomics
� Examine gene expression at global level
• Metabolomics
� Examine metabolites at global level
• High-throughput Enzyme Assays
� Compare strains
� Compare activities at different stages of growth
� Compare activities in different growth conditions
� Develop improved enzymes
• Mathematical approaches
− Metabolic flux analysis
− Kinetic modeling

Cytoplasmic membrane
Is Sugar Transport limiting?
Transporter protein
in
xylose
out
xylose
Facilitated Diffusion Transport
No energy required

Acknowledgements
The Office of the Biomass Program
of the US Department of Energy