Metabolic Engineering for Fuels and Chemicals

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1 Metabolic Engineering for Fuels and Chemicals K.T. Shanmugam and Lonnie O. Ingram Dept. of Microbiology and Cell Science University of Florida Gainesville, Florida

2 Florida Center for Renewable Chemicals and Fuels Metabolic Engineering Renewable Biomass to Chemicals & Fuels Dr. Lonnie O Neal Ingram, Director

3 RENEWABLE FUELS AND CHEMICALS CO 2 CO 2 CO 2 Above ground Displacement of oil Commodity chemicals polylactic acid solvents acids Fuels ethanol biodiesel power Rural Employment Newer carbon species Older carbon species Below ground Carbon Sequestration in soil

4 PROPOSED BIOMASS-DERIVED COMPOUNDS Ethanol Lactic acid Succinic acid 1,2-Propandiol 1,3-Propandiol Polyhydroxybutyrate Reduced compounds produced under anaerobic conditions

5 CONVERSION OF LIGNOCELLULOSICS TO ETHANOL FEEDSTOCK PROCESS ETHANOL (CHEMICALS) 1. Choice 2. Availability 3. Cost 4. Quality 1. Recovery 2. Waste Disposal Solid Liquid Depolymerization Biocatalyst 1. H + 2. Cellulases 3. Hemicellulases 4. Inhibitors

6 1. Cellulose Cellulases Optimize with the Biocatalyst Depolymerization 2. Xylose Xylanases, Xylosidases 3. Glucuronoxylan α-glucuronidase; Xylosidase 4. Acid Hydrolysis

7 BIOCATALYST 1. High Growth Rate 2. High Cell Yield 3. High Product Yield Volumetric Productivity Specific Productivity 4. Purity of the Product Optical Chemical 5. Minimal Growth Requirements 6. Metabolic Versatility 7. Co-utilization of Various Sugars 8. Tolerate High Sugar Concentration 9. Resistance to Inhibitors 10. Insensitive to Product Inhibition 11. High-value Co-products 12. Amenable to Genetic Engineering 13. Robust 14. Cellulases 15. Xylan degradation

8 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.

9 HEXOSES + PENTOSES Microbial Platform Embden-Meyerhof-Parnas Entner-Doudoroff Pentose Phosphate Succinate X PEP PYRUVATE (Zymomonas mobilis) Lactate Dehydrogenase 7.2 mm (ldha) Lactate Acetate Acetyl-CoA + Ethanol Pyruvate Formate-Lyase 2 mm (pfl) Formate CO 2 H 2 Pyruvate Decarboxylase 0.4mM (pdc) Acetaldehyde + CO 2 Alcohol Dehydrogenase (adhb) Ethanol >95% of Theor. Yield

10 E. coli B (organic acids) and KO11 (ethanol) Xylose and Ethanol (g/liter) Xylose (g/l) Biomass (g/l) Organic Acids Cell Mass(g/liter) Xylose and Ethanol (g/liter) Xylose (g/l) Ethanol (g/l) Biomass (g/l) Cell Mass(g/liter) Time (h) Time (h) Yield 0.50 g ethanol and 0.49 g CO 2 per g xylose (10% Xylose, ph 6.5, 35C)

11 PRODUCTIVITY IS RELATED TO CELL MASS Rate of Ethanol Production (g/liter.h) Cell Mass (g)

12 SUGAR UTILIZATION and SSCF CELLULOSE: GLUCOSE HEMICELLULOSE: XYLOSE SEQUENTIAL Catabolite Repression SIMULTANEOUS

13 Culture was grown with 13 C 1 -glucose and 13 C 1 - xylose at 37C in the NMR withour ph control.

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15 TOLERANCE TO HIGHER LEVEL OF ETHANOL Higher Product Yield Lower Product Cost

16 Ethanol Tolerance: Mutants reach over 6.5% w/v ethanol (14% xylose,, 35C, ph 6.5, 100 rpm, Luria Broth) Ethanol (mm) > 65 g ethanol/liter 50 g ethanol/liter K011 Mut 1 Mut Time (h)

17 FERMENTATIONS AT HIGH SUGAR CONCENTRATIONS Expect: Higher Product Yield Observed: Lower Growth Rate and Cell Yield of KO11 Cause: Osmotic Effect

18 Ethanol NAD + NADH adhb Limiting Acetyl-CoA Pool Acetaldehyde adhe Acetyl-CoA pta Acetyl-P acka Acetate Glycolysis pdc pfl Pyruvate Oxaloacetate glta citz acs Citrate AMP + PP i ATP Malate Isocitrate Fumarate 2-Ketoglutarate X Succinate Glutamate

19 Glucose Genetic solution Pyruvate pdc Acetaldehyde adhb adhe Ethanol adhe Acetyl-CoA pta Acetyl~P X acka Acetate Oxaloacetate CitZ (B. subtilis) Citrate Malate Isocitrate Fumarate Succinate 2-Ketoglutarate Glutamate (Osmoprotectant)

20 Fermentations with acka and adhe Cell Mass (g/l) Ethanol (g/l) Acetate acka KO11 adhe Acetate acka Time (h) adhe KO Time (h) Deletion of acka eliminates conversion of acetyl-coa to acetate. This resulted in a stimulation of growth and ethanol production similar to acetate supplementation. Ethanol yield by acka, 0.47 g/g total xylose (92%). Average volumetric productivity for acka increased (0.57 g/l/h), compared to KO11 (0.33 g/l/h). Average specific productivity for acka (0.38 g/g/h), similar to KO11 (0.36 g/g/h). The combination ( acka adhe) was no better than the acka.

21 E. coli Citrate Synthase Inhibited by NADH & 2-ketoglutarate 70% inhibition at 50µM NADH and 0.16mM Acetyl-CoA (Weitzman, PDJ Biochim. Biophys. Acta 128: ) B. subtilis Citrate Synthase Inhibited by ATP 2 mm NADH No effect

22 Expression of B. subtilis citz in KO11 Cell Mass (g/l) g/l Acetate Bs citz (ploi2514) KO11 (TOPO) Ethanol (g/l) g/l Acetate Bs citz (ploi2514) KO11 (TOPO) Time (h) Time (h)

23 Sugars, Oligosaccharides Microbial Zoo (E. coli) Ethanol & other products Erwinia Klebsiella Bacillus Zymomonas ~33kb secretion genes 2 PTS cellobiose genes citrate synthase PDC+ADH 2 cellulases 2 xylobiose genes pectate lyase Pseudomonas esterase for ethyl acetate Who knows what the future will bring?

24 PRODUCTION OF OXIDIZED COMPOUNDS Anaerobic: Redox Neutral or Reduced Compounds C 6 H 12 O 6 2 C 3 H 6 O 3 or 2 C 2 H 6 O + 2 CO 2 Glucose Lactic acid Ethanol Aerobic: Oxidized Compounds C 6 H 12 O 6 Glucose 2 C 2 H 4 O CO 2 + 4H Acetic Acid

25 Overview of Metabolism in E. coli Anaerobic Glucose, C 6 H 12 O 6 Cell Mass 5% of Carbon Aerobic Glucose, C 6 H 12 O 6 Cell Mass 50% of Carbon Up to 95 % of carbon converted to products (low CO 2 production) 2.5 ATP produced Low growth rate Internal electron acceptor 50% of carbon converted to CO 2 33 ATP (calc.) produced High growth rate External electron acceptor

26 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

27 Glucose Yield: >85% ~P Triose-P NAD + NADH ~P NADH CO 2 ppc PEP pyka pykf Cyt b1(red) CO 2 Oxaloacetate ~P poxb Acetate NAD + NADH Lactate Pyruvate Cyt b1(ox) ~P glta ldha acka CO 2 NAD + NADH HCOOH aceef lpda pta Glucose Metabolism pflb Acetyl-CoA H + 2 NADH adhe 2 NAD + H + O 2 + e - Ethanol H 2 O FADH 2 frdabcd FAD + NAD + Malate fumabc Fumarate ~P mdh sdhabcd UQH 2 UQ glcb aceb Succinate sucdc ~P Acetyl-CoA Glyoxylate Succinyl-CoA acea sucab lpda Citrate acnb Isocitrate NADP + icda NAD + NADH CO 2 2-Oxoglutarate CO 2 NADPH out in ADP + P i F F 0 0 F 1 H + atpibefhagdc ATP Electron Transport System ADP + P i NAD + F 1 ATP NADH

28 NEW RESEARCH AREAS Glucose & Acetate (mm) Glucose Acetate TC36 Glucose Added Cell Mass (g. L -1 ) Limits for Glycolytic Flux? Control of Carbon Partitioning? Limits for Growth Rate? Maximum Cell Density? Isogenic Strains: (Mixed acid, ethanol, lactate, acetate, pyruvate, glutamate, succinate, alanine, citrate) ATP/ADP? Time (h) 0 NADH/NAD? Metabolomics Proteomics Transcriptome Analysis

29 ~P Glucose Engineered E. coli TC44 Metabolism NADH + H ~P e - transport chain Triose 3-P PEP Pyruvate ~P UQH 2 UQ No ox-phos Acetate CO 2 - poxb e - + ½O 2 F 1 2H + H 2 O HCO - 3 Oxaloacetate NADH Malate Fumarate Incomplete TCA Citrate Isocitrate NADPH 2 CO 2 2-Ketoglutarate CO 2 - Acetyl-CoA pta acka Acetyl-P ~P Acetate NADH + H ADP + P i ATP NADH oxidized by electron transport system. ~ 2 ATP per glucose. ~ 5-10% 5 of glucose carbon is converted to cell mass.

30 BIOCATALYST 1. High Growth Rate 2. High Cell Yield 3. High Product Yield Volumetric Productivity Specific Productivity 4. Purity of the Product Optical Chemical 5. Minimal Growth Requirements 6. Metabolic Versatility 7. Co-utilization of Various Sugars 8. Tolerate High Sugar Concentration 9. Resistance to Inhibitors 10. Insensitive to Product Inhibition 11. High-value Co-products 12. Amenable to Genetic Engineering 13. Robust 14. Cellulases 15. Xylan degradation

31 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

32 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