Maximizing Ethanol Production by Engineered Pentose-Fermenting Zymomonas mobilis

Maximizing Ethanol Production by Engineered Pentose-Fermenting Zymomonas mobilis Dhinakar S. Kompala Department of Chemical Engineering University of Colorado, Boulder James D. McMillan and Min Zhang Biotechnology Center for Fuels and Chemicals National Renewable Energy Laboratory Objective Develop robust microbial biocatalysts capable of effectively conversion of a variety of sugar feedstreams to fuels (and chemicals) Strategic Targets • Increase Ethanol Yield • Increase Ethanol Concentration • Reduce Fermentation Time Biomass Composition 10% Other 46% Cellulose 14% Other 36% Cellulose/ 25% 10% Lignin starch Lignin ARABINOSE XYLOSE XYLOSE 40% Hemicellulose 19% Hemicellulose l Hardwood Species l Agricultural Residues (i.e., Corn Fiber, Corn Stover) Fermentation Performance Criteria Fermentation Performance Criteria Desirable Traits Essential Microbial Traits High sugar consumption rate l High Conversion Yield High specific growth rate High volumetric productivity l High Ethanol Tolerance High specific productivity l Resistance to Inhibitors in C5/C6 cofermentation Hydrolysates Minimal nutrient requirements High salt tolerance (acetate) l No Oxygen Requirement Entner-Doudoroff pathway l Low Fermentation pH Facilitated sugar transport l Broad Substrate Utilization Range Non spore forming l GRAS Status Non-conjugative Amenable to scale-up Availability of "industrial"strains Compatibility with SSF Cellulase producer Thermotolerance High shear tolerance Available gene transfer system Promising Biocatalysts l Zymomonas l Recombinant Saccharomyces l Recombinant E. coli l Lactobacillus l Xylose-Assimilating Yeasts l Clostridium Highlights of Zymomonas mobilis • Advantages: – Natural fermentative microorganism (GRAS) – Near theoretical ethanol yield from glucose – Reduced yield loss to biomass formation – No oxygen requirement – Tolerant to inhibitors in hydrolysates – High ethanol tolerance – Fermentation at low pH – Grows at high sugar concentrations – High specific productivity • Limitations – Narrow substrate utilization range Pathways Required for Pentose Fermentation Pentose Metabolism Pathway Entner-Doudoroff Pathway D-Xylose L-Arabinose D- Glucose L -a r a b i n o s e i s o m e r a s e ATP Xylose Isomerase L-Ribulose ADP L -r i b u l o k i n a s e Glucose -6 - P D - Xylulose ATP ADP G l u c o n o l a c t o n e -6 - P Xylulokinase L - R i b u l o s e -5 - P ATP 6 -P- G l u c o n a t e L -ribulose-5 - P 4 -e p i m e r a s e ADP D - Xylulose-5 - P Ribose-5 - P 2 - Keto -3 - de o x y -6 -P - Gluconate R i b u l o s e -5 - P G l y c e r a l d e h y d e -3 - P Transketolase 1,3 -P- Glycerate S e d o h e p t u l o s e -7 - P G l y c e r a l d e h y d e -3 - P ADP ATP Transaldolase 3 -P- Glycerate 2 -P- Glycerate Erythrose -4 - P F r u c t o s e-6 - P ADP ATP F r u c t o s e-6 - P Phosphoenolpyruvate Pyruvate Transketolase G l y c e r a l d e h y d e -3 - P Acetaldehyde + CO2 Ethanol Cloning Strategy for Pentose Metabolism Pathway Xylose Assimilation Genes Arabinose Assimilation Genes Pentose Metabolism Genes Z. mobilis genome E. coli genome Z. mobilis genome E. coli genome Z. mobilis genome E. coli genome PCR Synthesis Subcloning PCR Synthesis PCR Synthesis X h o I/ NotI XhoI SalI NotI BglII XbaI XbaI BglII Pg a p Px y l Pg a p araB’ araB araA araD Pe n o tal tktA xylA xylB PCR-mediated overlap extension a n d Subcloning NotI BglII BglII NotI Pg a p Pg a p araB araA araD Pe n o tal tktA xylA xylB P g a p - xylA/ xylB Operon Pg a p - a r a B A D O p e r o n P e n o -tal/tktA O p e r o n Subcloning Plasmid Containing Xylose- and Arabinose-Fermenting Genes NotI Peno EcoRV AvaI Pgap EcoRI tal araB tkt araA araD AvaI pZB301 Pgap NotI 19.12 kb xylA Z. DNA xylB r AvaI Tc E.coli ori AvaI EcoRV EcoRV AvaI NotI Fermentation Profile by Z. mobilis 206C/pZB301 grown on Glu:Xyl:Ara (30:30:20 g/l) at pH = 5.5, T = 31.5°C 40 Concentration (g/l) glucose xylose arabinose 30 ethanol 20 10 0 0 20 40 60 80 100 120 Time (h) Specific Aims 1. Develop new genetic engineering tools for the control of gene dosage and expression, and construct improved strains of Z. mobilis for efficient ethanol fermentation. 2. Measure intracellular activity through enzymatic assays, HPLC and NMR spectroscopy and evaluate ethanol production by the new strains on mixed sugars. 3. Develop a structured kinetic model for the integrated pathway network and identify potential improvements through dynamic simulations. Strategy to Develop More Stable rZymomonas Strains ü Develop integration systems. ü Integrate xylose assimilation genes (xylA, xylB) pentose-phosphate pathway genes (tal, tkt) into the Zymomonas genome. ü Integrate xylose assimilation genes (xylA, xylB) arabinose assimilation genes (araA, araB, araD) pentose-phosphate pathway genes (tal,tkt) into the Zymomonas genome. Strategy for Gene Integration Gene Integration by Transposon Homologous Recombination Transposable Donor Tc r element Circular or linear Target Z.m. DNA Plasmid DNA Z.m. Chromosomal DNA Insertion Z.m. Chromosomal DNA Homologous recombination Tc r Repair synthesis loss of donor Site specific recombination Gene inactivation Random insertion Large DNA fragment Comparison of fermentation performance of plasmid- bearing and integrated Z. mobilis strains on mixed sugars (G:40, X:40, A:20 g/l) at pH=5.5, T=30 o C 50 50 206C(pZB301) AX101 40 40 Concentration (g/L) Concentration (g/L) Gluc. 30 Xyl. 30 Ara. 20 EtOH 20 10 10 0 0 0 20 40 60 80 100 120 0 20 40 60 80 100 120 Time (hrs) Time (hrs) Process yield on mixed sugars (G:40, X:40, A:20 g/l) at pH=5.5, T=30 o C 100 Process yield (%) 80 206C(pZB301) 60 AX101 AX1 40 G8 20 0 0 20 40 60 80 100 120 Time (hrs) Stability Test Ara+ Xyl+ strains Medium containing Inoculated glucose only Generation 0 40 80 120 160 Fermentation in flasks containing glucose + xylose + arabinose Xylose Utilization 100 Xylose Utilization (%) 90 80 70 60 Gen (0) 50 Gen (40) Gen (80) 40 Gen (120) Gen (160) 30 20 10 0 AX1 AX23 AX101 G8 Arabinose Utilization 100 Arabinose Utilization (%) 90 80 70 Gen (0) 60 Gen (40) Gen (80) Gen (120) 50 Gen (160) 40 30 20 10 0 AX1 AX23 AX101 G8 Progress along Specific Aim 1 • Integrated the seven genes necessary for xylose and arabinose fermentation into the Zymomonas mobilis genome and coordinately expressed the seven genes. • The chromosomal integrated strains demonstrated similar ethanol process yield (83%) to the plasmid- bearing strain from a mixture of glucose, xylose and arabinose. • The strains demonstrated stability for 160 generations on non-selective medium (in the absence of Tc).

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