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Metabolic Engineering for Fuels and Chemicals K.T. Shanmugam and Lonnie O. Ingram Dept. of Microbiology and Cell Science University of Florida Gainesville, Florida Florida Center for Renewable Chemicals and Fuels Metabolic Engineering Renewable Biomass to Chemicals & Fuels Dr. Lonnie O’Neal Ingram, Director http://fcrc.ifas.ufl.edu RENEWABLE FUELS AND CHEMICALS CO2 CO2 CO2 Displacement of oil ov b A n u o r eg d – Commodity chemicals • polylactic acid • solvents • acids – Fuels • • • ethanol biodiesel power – Rural Employment Newer carbon species Older carbon species Below ground Carbon Sequestration in soil PROPOSED BIOMASS-DERIVED COMPOUNDS Ethanol Lactic acid Succinic acid 1,2-Propandiol 1,3-Propandiol Polyhydroxybutyrate Reduced Reduced compounds compounds produced produced under under anaerobic anaerobic conditions conditions CONVERSION OF LIGNOCELLULOSICS TO ETHANOL FEEDSTOCK 1. 2. 3. 4. PROCESS Choice Availability Cost Quality Depolymerization 1. 2. 3. 4. H+ Cellulases Hemicellulases Inhibitors ETHANOL (CHEMICALS) 1. Recovery 2. Waste Disposal Solid Liquid Biocatalyst 1. Cellulose Cellulases Optimize with the Biocatalyst Depolymerization 2. Xylose Xylanases, Xylosidases 3. Glucuronoxylan α-Glucuronidase; Xylosidase 4. Acid Hydrolysis 1. 2. 3. 4. BIOCATALYST 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. High Growth Rate High Cell Yield High Product Yield Volumetric Productivity Specific Productivity Purity of the Product Optical Chemical Minimal Growth Requirements Metabolic Versatility Co-utilization of Various Sugars Tolerate High Sugar Concentration Resistance to Inhibitors Insensitive to Product Inhibition High-value Co-products Amenable to Genetic Engineering Robust Cellulases Xylan degradation E. coli: Potential Industrial Platform for Renewable Fuels and Chemicals 1. Safety, reliability, and industrial experience. 2. Uses broad range of sugars derived from biomass (hexose, pentose, sugar alcohol and sugar acid; expanded to – cellobiose and xylobiose). 3. Simple nutrient requirements. 4. Well understood physiology and established tools for genetic manipulation. + HEXOSES PENTOSES Microbial Platform Embden-Meyerhof-Parnas Entner-Doudoroff Succinate X PYRUVATE PEP Lactate Dehydrogenase 7.2 mM (ldhA) Pyruvate Formate-Lyase 2 mM (pfl) Lactate Acetyl-CoA Acetate Ethanol + Formate Pentose Phosphate (Zymomonas mobilis) Pyruvate Decarboxylase 0.4mM (pdc) Acetaldehyde + CO2 Alcohol Dehydrogenase (adhB) CO2 H2 Ethanol >95% of Theor. Yield Xylose (g/L) 8 6 60 4 40 Biomass (g/L) 2 20 Organic Acids 0 0 0 12 24 36 48 60 Time (h) 72 84 96 10 100 Xylose (g/L) 8 80 60 6 Ethanol (g/L) 4 40 20 Biomass (g/L) 2 0 0 0 12 24 36 48 60 72 84 96 Time (h) Yield – 0.50 g ethanol and 0.49 g CO2 per g xylose (10% Xylose, pH 6.5, 35C) Cell Mass(g/liter) 80 Xylose and Ethanol (g/liter) 10 100 Cell Mass(g/liter) Xylose and Ethanol (g/liter) E. coli B (organic acids) and KO11 (ethanol) PRODUCTIVITY IS RELATED TO CELL MASS 1.5 (g/liter.h) Rate of Ethanol Production 2 1 0.5 0 0 1 2 3 4 Cell Mass (g) 5 6 SUGAR UTILIZATION and SSCF CELLULOSE: GLUCOSE HEMICELLULOSE: XYLOSE SEQUENTIAL – Catabolite Repression SIMULTANEOUS Culture was grown with 13C1- glucose and 13C1- xylose at 37C in the NMR withour pH control. TOLERANCE TO HIGHER LEVEL OF ETHANOL Higher Product Yield Lower Product Cost Ethanol Tolerance: Mutants reach over 6.5% w/v ethanol (14% xylose, 35C, pH 6.5, 100 rpm, Luria Broth) Ethanol (mM) 1500 > 65 g ethanol/liter 1250 1000 K011 750 Mut 1 50 g ethanol/liter 500 Mut 2 250 0 0 24 48 72 Time (h) 96 120 FERMENTATIONS AT HIGH SUGAR CONCENTRATIONS Expect: Higher Product Yield Observed: Lower Growth Rate and Cell Yield of KO11 Cause: Osmotic Effect Limiting Acetyl-CoA Pool NAD+ NADH adhE Acetaldehyde Ethanol adhB is s y l o c Gly Acetyl-CoA Acetyl-P Acetate pfl pdc ackA pta acs Pyruvate glt A citZ Oxaloacetate Citrate ATP AMP + PPi Isocitrate Malate Fumarate 2-Ketoglutarate X Succinate Glutamate Glucose Genetic solution Pyruvate pdc Acetaldehyde adhB adhE Ethanol adhE Acetyl-CoA pta Acetyl~P X ackA Acetate CitZ Oxaloacetate (B. subtilis) Citrate Isocitrate Malate Fumarate 2-Ketoglutarate Glutamate (Osmoprotectant) Succinate Fermentations with ∆ackA and ∆adhE Cell Mass (g/L) • Deletion of ackA eliminates conversion of acetyl-CoA to acetate. Acetate ∆ ackA 2.0 1.5 • This resulted in a stimulation of growth and ethanol production similar to acetate supplementation. 1.0 KO11 0.5 ∆ adhE 0.0 0 12 24 36 48 60 72 84 96 Time (h) • Average volumetric productivity for ∆ackA increased (0.57 g/L/h), compared to KO11 (0.33 g/L/h). Ethanol (g/L) 50 Acetate ∆ ackA 40 30 • Average specific productivity for ∆ackA (0.38 g/g/h), similar to KO11 (0.36 g/g/h). 20 10 ∆ adhE KO11 0 0 12 24 • Ethanol yield by ∆ackA, 0.47 g/g total xylose (92%). 36 48 60 Time (h) 72 84 96 • The combination (∆ackA ∆adhE) was no better than the ∆ackA. E. coli Citrate Synthase Inhibited by NADH & 2-ketoglutarate 70% inhibition at 50µM NADH and 0.16mM Acetyl-CoA (Weitzman, PDJ. 1966. Biochim. Biophys. Acta 128:213-215) B. subtilis Citrate Synthase Inhibited by ATP 2 mM NADH – No effect Expression of B. subtilis citZ in KO11 50 2 g/L Acetate Bs citZ (pLOI2514) 2.0 2 g/L Acetate Ethanol (g/L) Cell Mass (g/L) 2.5 1.5 1.0 0.5 40 Bs citZ (pLOI2514) 30 20 10 KO11 (TOPO) KO11 (TOPO) 0.0 0 12 24 36 48 60 Time (h) 72 84 0 96 0 12 24 36 48 60 Time (h) 72 84 96 Sugars, Oligosaccharides Microbial Zoo (E. coli) Erwinia Klebsiella ~33kb secretion genes 2 PTS cellobiose genes 2 cellulases 2 xylobiose genes pectate lyase Pseudomonas esterase for ethyl acetate Ethanol & other products Bacillus Zymomonas citrate synthase PDC+ADH Who knows what the future will bring? PRODUCTION OF OXIDIZED COMPOUNDS Anaerobic: Redox Neutral or Reduced Compounds C6H12O6 2 C3H6O3 Glucose Lactic acid Aerobic: or 2 C2H6O + 2 CO2 Ethanol Oxidized Compounds C6H12O6 Glucose 2 C2H4O2 + 2 CO2 + 4H Acetic Acid Overview of Metabolism in E. coli Anaerobic Glucose, C6H12O6 Aerobic Glucose, C6H12O6 Cell Mass Cell Mass 5% of Carbon 50% of Carbon ¾ Up to 95 % of carbon converted to products (low CO2 production) ¾ 2.5 ATP produced ¾ Low growth rate ¾ Internal electron acceptor ¾ 50% of carbon converted to CO2 ¾ 33 ATP (calc.) produced ¾ High growth rate ¾ External electron acceptor Goal: Combine the Attributes of Aerobic & Anaerobic Metabolism Anaerobic High product yield Low cell yield Single Biocatalyst + High growth rate External e- acceptor Aerobic Neutral or Oxidized Products Glucose Lactate ~P ~P Triose-P NAD+ NADH ~P + NAD PEP Yield: >85% CO2 ldhA NADH CO2 NADH NAD+ pykA pykF Pyruvate Cytb1(red) Cytb1(ox) CO2 Glucose Metabolism poxB ppc aceEF lpdA pflB 2 NADH 2 NAD+ Acetyl-CoA pta ackA HCOOH Ethanol adhE Acetate ~P NADH NAD+ Oxaloacetate H+ gltA Malate glcB aceB F0 Citrate Glyoxylate Fumarate aceA UQH2 Electron Transport System atpIBEFHAGDC F1 acnB NAD+ NADH Isocitrate NADP+ sdhABCD icdA Succinate CO2 22-Oxoglutarate -Oxoglutarate sucAB lpdA sucDC NAD+ ~P ~P NADH Succinyl-CoA CO2 F1 + NADPH ADP H ATP UQ FAD+ F0 in fumABC frdABCD H2O out mdh Acetyl-CoA FADH2 H+ O2 + e- + Pi ADP + Pi ATP NEW RESEARCH AREAS 500 4 Glucose Added 400 3 300 200 2 100 1 Cell Mass (g . L-1) Glucose & Acetate (mM) 5 Glucose Acetate TC36 ¾Limits for Glycolytic Flux? ¾Control of Carbon Partitioning? ¾Limits for Growth Rate? ¾Maximum Cell Density? Isogenic Strains: (Mixed acid, ethanol, lactate, acetate, pyruvate, glutamate, succinate, alanine, citrate) ¾ATP/ADP? 0 0 0 6 12 18 Time (h) 24 30 36 ¾NADH/NAD? ¾Metabolomics ¾Proteomics ¾Transcriptome Analysis Engineered E. coli TC44 Metabolism e- transport chain No ox-phos e- + ½O2 Glucose UQH2 NADH + H ~P ~P Triose 3-P ~P PEP poxB CO2NADH + H Acetyl-CoA NADH Malate Fumarate Incomplete TCA Citrate Isocitrate NADPH2 CO2 2-Ketoglutarate F1 CO2- Pyruvate HCO3Oxaloacetate 2H+ Acetate UQ H2O pta Acetyl-P ackA ~P Acetate ADP + Pi ATP ¾ NADH NADH oxidized oxidized by by electron transport system. ¾ ~ 2 ATP per glucose. ¾ ~ 55-10% -10% of glucose carbon is converted to cell mass. 1. 2. 3. 4. BIOCATALYST 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. High Growth Rate High Cell Yield High Product Yield Volumetric Productivity Specific Productivity Purity of the Product Optical Chemical Minimal Growth Requirements Metabolic Versatility Co-utilization of Various Sugars Tolerate High Sugar Concentration Resistance to Inhibitors Insensitive to Product Inhibition High-value Co-products Amenable to Genetic Engineering Robust Cellulases Xylan degradation Future Studies Gene Array Investigations: Global regulators for carbon metabolism (mutations in mlc, crp, csrA) Global regulators for redox control (mutations in fnr, arcA) Prolonging the growth phase and metabolism (comparing ethanol/lactic acid) BioRefinery Improvements for Ethanol and Other Chemicals: Ethanol tolerance, Process simplification Carbon partitioning/production costs Rates and yields Cellulases, cellobiose/triose; Xylanases, xylobiose/triose Metabolic Engineering for Higher Value Products: L(+)-lactic acid and D(-)-lactic acid Acetic acid, pyruvic acid, succinate, glutamate, citrate Dependence on petroleum remains as the single most important factor affecting the world distribution of wealth, global conflict, human health, and environmental quality. Reversing this dependence would increase employment, preserve our environment, and facilitate investments that improve the health and living conditions for all. Professor Ohta conducting fermentation studies at the University of Florida