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

Renewable Biomass MetabolicMetabolic to EngineeringEngineering Chemicals & Fuels

Dr. Lonnie O’Neal Ingram, Director

http://fcrc.ifas.ufl.edu polylactic acid solvents acids ethanol biodiesel power polylactic acid solvents acids ethanol biodiesel power in soil in soil Carbon • • • • • • • • • • • • Carbon Commodity chemicals Fuels Rural Employment Commodity chemicals Fuels Rural Employment – – – – – – Sequestration Sequestration Displacement of oil

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A e B 2 CO 2 RENEWABLE FUELS AND CHEMICALS CO 2 CO Newer carbon species Older carbon species PROPOSED BIOMASS-DERIVED COMPOUNDS

EthanolEthanol LacticLactic acidacid SuccinicSuccinic acidacid 1,21,2--PropandiolPropandiol 1,31,3--PropandiolPropandiol PolyhydroxybutyratePolyhydroxybutyrate

ReducedReducedReduced compoundscompoundscompounds producedproducedproduced underunderunder anaerobicanaerobicanaerobic conditionsconditionsconditions CONVERSION OF LIGNOCELLULOSICS TO ETHANOL

FEEDSTOCK PROCESS ETHANOL (CHEMICALS) 1. Choice 1. Recovery 2. Availability 2. Waste Disposal 3. Cost Solid 4. Quality Liquid

Depolymerization Biocatalyst 1. H+ 2. Cellulases 3. Hemicellulases 4. Inhibitors 1. Cellulose Cellulases Optimize with the Biocatalyst

2. Xylose Depolymerization Xylanases, Xylosidases

3. Glucuronoxylan α-Glucuronidase; Xylosidase

4. Acid Hydrolysis 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 BIOCATALYST 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 E.E. colicoli:: PotentialPotential IndustrialIndustrial PlatformPlatform forfor RenewableRenewable FuelsFuels andand ChemicalsChemicals

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

MicrobialMicrobial PlatformPlatform Embden-Meyerhof-Parnas Entner-Doudoroff Pentose Phosphate

Succinate X PEP PYRUVATEPYRUVATE (Zymomonas mobilis)

Lactate Dehydrogenase Pyruvate Pyruvate Decarboxylase 7.2 mM (ldhA) Formate-Lyase 0.4mM (pdc) 2 mM (pfl) Lactate Acetaldehyde + COCO2 Acetyl-CoA + Formate Alcohol Dehydrogenase (adhB)

Acetate Ethanol CO2 H2 EthanolEthanol >95%>95% ofof TheorTheor.. YieldYield E.E. colicoli BB (organic(organic acids)acids) andand KO11KO11 (ethanol)(ethanol)

10 10 100 100 Xylose (g/L) Xylose (g/L) 8 8 Cell Mass(g/liter) 80 Cell Mass(g/liter) 80

6 6 60 60 Ethanol (g/L)

40 4 40 4 Biomass (g/L)

20 2 20 Biomass (g/L) 2

Xylose and Ethanol (g/liter) Organic Acids Xylose and Ethanol (g/liter) 0 0 0 0 0 12 24 36 48 60 72 84 96 0 12 24 36 48 60 72 84 96 Time (h) Time (h)

Yield – 0.50 g ethanol and 0.49 g CO2 per g xylose

(10% Xylose, pH 6.5, 35C) PRODUCTIVITY IS RELATED TO CELL MASS

1.5

1 (g/liter.h)

0.5 Rate of Ethanol Production 0 0123456 Cell Mass (g) SUGAR UTILIZATION and SSCF

CELLULOSE: GLUCOSE

HEMICELLULOSE: XYLOSE

SEQUENTIAL – Catabolite Repression SIMULTANEOUS 13 13 Culture was grown with C1-glucose and C1- xylose at 37C in the NMR withour pH control.

TOLERANCE TO HIGHER LEVEL OF ETHANOL

Higher Product Yield

Lower Product Cost EthanolEthanol Tolerance:Tolerance: MutantsMutants reachreach overover 6.5%6.5% w/vw/v ethanolethanol (14% xylose, 35C, pH 6.5, 100 rpm, Luria Broth)

1500 > 65 g ethanol/liter

1250

1000 K011

750 Mut 1 Mut 2 500 50 g ethanol/liter Ethanol (mM)Ethanol

250

0 0 24 48 72 96 120 Time (h) 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 pta ackA Ethanol Acetaldehyde Acetyl-CoA Acetyl-P Acetate adhB

pdc fl p acs Pyruvate is g ATP lys ltA c AMP lyco itZ G + PPi Oxaloacetate Citrate

Malate Isocitrate

Fumarate 2-Ketoglutarate

Succinate Glutamate Genetic solution Glucose

pdc adhB Pyruvate Acetaldehyde adhE Ethanol

adhE Acetyl-CoA Acetyl~P X Acetate pta ackA CitZ Oxaloacetate (B. subtilis) Citrate

Malate Isocitrate

Fumarate 2-Ketoglutarate Glutamate (Osmoprotectant)

Succinate Fermentations with ∆ackA and ∆adhE

Acetate 2.0 • Deletion of ackA eliminates conversion ∆ ackA of acetyl-CoA to acetate. 1.5 • This resulted in a stimulation of growth 1.0 and ethanol production similar to acetate

Cell Mass (g/L) Mass Cell 0.5 KO11 supplementation. ∆ adhE 0.0 • Ethanol yield by ∆ackA, 0.47 g/g total 0 12 24 36 48 60 72 84 96 xylose (92%). Time (h) 50 • Average volumetric productivity for Acetate 40 ∆ ackA ∆ackA increased (0.57 g/L/h), compared to KO11 (0.33 g/L/h). 30

20 • Average specific productivity for ∆ackA

Ethanol (g/L) (0.38 g/g/h), similar to KO11 (0.36 g/g/h). 10 ∆ adhE KO11 0 • The combination (∆ackA ∆adhE) was no 0 12 24 36 48 60 72 84 96 better than the ∆ackA. Time (h) 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

2.5 50 2 g/L Acetate 2 g/L Acetate Bs citZ (pLOI2514) 2.0 40 Bs citZ (pLOI2514)

1.5 30

1.0 20 Ethanol (g/L)

Cell Mass (g/L) Cell 0.5 10 KO11 (TOPO) KO11 (TOPO) 0.0 0 0 12 24 36 48 60 72 84 96 0 12 24 36 48 60 72 84 96 Time (h) Time (h) Sugars, Oligosaccharides Microbial Zoo Ethanol & other (E. coli) 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 Who knows what ethyl acetate the future will bring? PRODUCTION OF OXIDIZED COMPOUNDS

Anaerobic: Redox Neutral or Reduced Compounds

C6H12O6 2 C3H6O3 or 2 C2H6O + 2 CO2 Glucose Lactic acid Ethanol

Aerobic: Oxidized Compounds

C6H12O6 2 C2H4O2 + 2 CO2 + 4H Glucose Acetic Acid OverviewOverviewOverview ofofof MetabolismMetabolismMetabolism ininin E.E.E. colicolicoli

AnaerobicAnaerobic AerobicAerobic

Glucose, C6H12O6 Glucose, C6H12O6

Cell Mass Cell Mass 5% of Carbon 50% of Carbon

¾ Up to 95 % of carbon converted to ¾ 50% of carbon converted to CO2 products (low CO2 production) ¾ 33 ATP (calc.) produced ¾ 2.5 ATP produced ¾ High growth rate ¾ Low growth rate ¾ External electron acceptor ¾ Internal electron acceptor Goal:Goal: CombineCombine thethe AttributesAttributes ofof AerobicAerobic && AnaerobicAnaerobic MetabolismMetabolism

Anaerobic High product yield SingleSingle Low cell yield Low cell yield BiocatalystBiocatalyst ++

High growth rate External e- acceptor Aerobic

NeutralNeutral oror OxidizedOxidized ProductsProducts Glucose ~P LactateLactate

~P NAD+ GlucoseGlucose MetabolismMetabolism Triose-P NADH ldhA NADH ~P CO2 + + NAD NADH NAD pykA PEP HCOOH pykF Pyruvate aceEF lpdA pflB + Cytb1(red) Cytb1(ox) 2 NADH 2 NAD poxB Yield: CO2 CO2 pta Acetyl-CoA ppc ackA >85% adhE EthanolEthanol AcetateAcetate ~P + + - Oxaloacetate H H O2 + e H2O NADH gltA

+ out NAD mdh Acetyl-CoA F F 0 Electron Transport Malate 0 System glcB aceB Citrate in fumABC atpIBEFHAGDC Glyoxylate acnB F Fumarate aceA F1 + UQH NAD NADH 2 Isocitrate FADH2 NADP+ sdhABCD frdABCD icdA + F UQ NADPH ADP H ATP 1 FAD+ CO + SuccinateSuccinate 2 22-Oxoglutarate-Oxoglutarate Pi sucDC sucAB NAD+ ADP ATP lpdA + ~P NADH Succinyl-CoA Pi ~P CO2 NEW RESEARCH AREAS ¾Limits for Glycolytic Flux? 5 Glucose ¾Control of Carbon 500 Ac etate Partitioning? TC36 ¾Limits for Growth Rate? Glucose Added 4 Cell Mass(g 400 ¾Maximum Cell Density?

3 300 Isogenic Strains:

. (Mixed acid, ethanol, lactate, 200 2 L

-1 acetate, pyruvate, glutamate, ) succinate, alanine, citrate) 100 1 Glucose & Acetate (mM) Acetate & Glucose ¾ATP/ADP? 0 0 ¾NADH/NAD? 0 6 12 18 24 30 36 ¾Metabolomics Time (h) ¾Proteomics ¾Transcriptome Analysis EngineeredEngineered E.E. colicoli TC44TC44 MetabolismMetabolism

- No ox-phos - e transport chain e + ½O2 H2O Glucose + UQH2 Acetate 2H NADH + H ~P ~P UQ poxBpoxB ~P - F1 CO2 Triose 3-P PEP Pyruvate

- CO2 HCO - 3 NADH + H ADP ATP +

Pi Oxaloacetate Acetyl-CoA NADH ¾¾ NADHNADH oxidizedoxidized byby ptapta electron transport Malate Citrate electron transport system.system. Acetyl-P Isocitrate ~P ¾¾ ~~ 22 ATPATP perper glucose.glucose. Fumarate NADPH2 ackAackA CO2 ¾ ~ 5-10% of glucose IncompleteIncomplete ¾¾ ~~ 55-10%-10% ofof glucoseglucose 2-Ketoglutarate carboncarbon isis convertedconverted toto TCATCA AcetateAcetate cellcell mass.mass. 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 BIOCATALYST 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 FutureFuture StudiesStudies

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)

Improvements for Ethanol and Other Chemicals: Ethanol tolerance, Process simplification BioRefinery 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