Development of efficient Escherichia coli succinate production strains

Development of efficient Escherichia coli succinate production strains Ka-Yiu San Department of Bioengineering Department of Chemical and Biomolecular Engineering Rice University, Houston, Texas International Conference Renewable Resources and Biorefineries Ghent Belgium September, 20, 2005

Top Value Added Chemicals from Biomass (2004 DOE report )

Top Value Added Chemicals from Biomass (2004 DOE report )

Top Value Added Chemicals from Biomass (2004 DOE report )

Production of succinic acid Non-recombinant microorganisms : A. succiniproducens A. succinogenes E. coli succinic acid minor fermentation product (~7.8% of total) Metabolically Engineered E. coli: Deletion of ldh overexpression of C/ malic enzyme or PYC Deletion of ldh and pfl NZN111 Deletion of ldh/pfl/ptsg APF111

Current challenges Existing strains have low productivity Low yield Low production rate (weak strain) Existing strains produce succinate anaerobically need two stage fermentation Product purity mixed acids fermentation

High yield anaerobic succinic acid production system Design rationale

High yield anaerobic succinic acid production system Limitation

Central anaerobic pathway in E. coli 2NAD + 2 Biomass Malate OAA Formate Glucose Glucose-6-P Fructose 1,6-diP P i + NAD + Glycerate-1,3-diP C CoA LDH NAD + limitation Max theoretical Yield 1 mole/mole Lactate H 2 Acetyl- CoA Ethanol 2 2NAD + PTA Acetyl-P ACK Acetate

1. Deletion of competing pathways to conserve and carbon atoms Glucose Glucose-6P - Fructose 1,6-diP Glyceraldehyde-3 P P i + Glycerate 1,3-diP Dihydroxyacetone -P Fumarate Malate OAA PYK PFL Acetyl- CoA LDH Formate PTA ADH 2 2 Acetyl-P Ethanol Lactate H 2 CO2 ACK Acetate

iclr acebak 2. Deletion of iclr to relieve the repression of the expression of the enzymes involved in the glyoxylate pathway Glucose Glucose-6P - Fructose 1,6-diP OAA Malate Fumarate Glyceraldehyde-3 P P i + Glycerate 1,3-diP PYK PFL Acetyl- CoA LDH Formate PTA ADH 2 2 Dihydroxyacetone -P Acetyl-P Ethanol Lactate H 2 CO2 ACK Acetate

OAA Malate aceb Acetyl -CoA Fumarate iclr acebak 2. `Deletion of iclr to relieve the repression of the expression of the enzymes involved in the glyoxylate pathway hence opening the acetyl-coa flow though this pathway glyoxylate acea Citrate Isocitrate acea Glucose Glucose-6P - Fructose 1,6-diP Glyceraldehyde-3 P P i + Glycerate 1,3-diP PYK PFL Acetyl- CoA LDH Formate PTA ADH 2 2 Dihydroxyacetone -P Acetyl-P Ethanol Lactate H 2 CO2 ACK Acetate

3. `Deletion of ack-pta to conserve acetyl- CoA and help channel it through the glyoxylate pathway 4. Heterologous overexpression of PYC to increase OAA pool OAA Malate aceb Acetyl -CoA Fumarate iclr acebak glyoxylate acea Citrate acea PYC Isocitrate Glucose Glucose-6P - Fructose 1,6-diP Glyceraldehyde-3 P P i + Glycerate 1,3-diP PYK PFL Acetyl- CoA LDH Formate PTA ADH 2 2 Dihydroxyacetone -P Acetyl-P Ethanol Lactate H 2 CO2 ACK Acetate

iclr acebak Glucose Glucose-6P - 5. Heterologous overexpression of B. subtilis citrate synthase (citz gene) assures no reaction limiting effects in the synthesis of citrate OAA Malate aceb Acetyl -CoA Fumarate glyoxylate acea Citrate Isocitrate acea PYC Fructose 1,6-diP Glyceraldehyde-3 P P i + Glycerate 1,3-diP PYK PFL Acetyl- CoA LDH Formate PTA ADH 2 2 Dihydroxyacetone -P Acetyl-P Ethanol Lactate H 2 CO2 ACK Acetate

Resulting pathway design Glucose Glucose-6P - Fructose 1,6-diP Glyceraldehyde-3 P P i + Glycerate 1,3-diP Dihydroxyacetone -P OAA Malate aceb Acetyl -CoA Fumarate glyoxylate acea Citrate PYC Isocitrate PYK PFL Acetyl- CoA Formate acea Fermentative pathway Glyoxylate pathway

Experimental results Strain SBS550MG ( adhe ldha iclr ackpta::cm R ) phl413 - PYC, Ap R phl531 - Citrate Synthase, Km R Fermentation Time: 24 hours

Conc (mm) 180 160 140 120 100 80 60 40 20 0 Strain SBS550MG ( adhe ldha iclr ackpta::cm R ) Glucose Used Acetate Formate Metabolites phl413+pdhk29 (pyc, control) phl413+phl531 (pyc, glt)

Typical HPLC metabolite output succinate

(Sanchez et al., Metabolic Engineering,, 2005, 7(3):229-39) 39) Max usage of biocatalyst - Repeated feeding experiments Conc. (mm) 400 350 300 250 200 150 100 50 0 succinate 2.0 1.8 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 Cumulative Yield (mol/mol) 20 18 16 14 12 10 8 6 4 2 0 + Optical density, (OD 600) 0 20 40 60 80 100 Time (hrs) Glucose Formate Acetate O Cumulative succinate yield + Optical density

WILD TYPE E. coli MG1655 with PYC vs SBS550MG with PYC STRAIN MG 550MG υ 1 100 100 Glucose υ 1 υ2 Biomass Glucose-6-P STRAIN υ 3 υ 3 MG 98 550MG 101 Glyceraldehyde-3-P STRAIN υ 4 MG 550MG STRAIN MG 550MG υ 4 196 203 υ 2 2 0 STRAIN MG 550MG υ 6 0 0 89% vs 69% MG 550MG υ 13 84 121 STRAIN MG 550MG υ ES 93 160 Lactate STRAIN υ 5 LDH MG 84 υ C 6 550MG 121 υ 5 = υ C +υ PYC υ 8 CO PFL υ 7 H 2 Formate 2 PYC υ RF STRAIN υ 12 Acetyl-CoA PTA MG 75 υ 9 ACK 550MG 83 υ OAA υ 11 12 υ 11 Acetyl-P CS ACDH Citrate Malate 2 ADH AceB Acetyl-CoA 2 υ 10 υ 11 Isocitrate Fumarate glyoxylate AceA Ethanol STRAIN υ υ 11 STRAIN υ 11 13 MG 9 MG AceA 550MG 38 550MG STRAIN υ 11 υ ES iclr υ 10 19 0 STRAIN MG 550MG Formate Acetate υ 8 12 36 STRAIN MG 550MG acebak STRAIN MG 550MG υ 9 74 7 11% vs 31% υ 7 112 83 υ RF 100 46

120 120 0 Lactate 80 0 20 Formate 0 Acetyl- CoA Ethanol 100 PATHWAY DESIGN THEORETICAL OPTIMIZATION 40 0 Acetate Oxaloacetate Citrate 40 80 Acetyl- CoA Isocitrate Malate Glyoxylate 40 Fumarate Glucose 100 G6P 100 G3P 200 0 100 40 OAA split: 33% flux to Glyoxylate New theoretical yield= 1.6 mol/mol PATHWAY IMPLEMENTATION CALCULATED FLUXES FROM EXPERIMENTAL DATA 121 121 121 0 83 Formate 0 Acetyl- CoA 38 Lactate 38 7 Acetate Oxaloacetate Citrate 38 83 Acetyl- CoA Isocitrate Malate Glyoxylate 38 Fumarate Glucose 100 G6P 101 G3P 203 36 +H 2 46 Ethanol OAA split: 31% flux to Glyoxylate Experimental yield= 1.6 mol/mol (Cox et al., Metabolic Engineering,, Published online (2005)

Summary (anaerobic) We have successfully designed and implemented a very robust dual-route succinate production system This system converts glucose to succinate at fast rates and high yields with minimal by-products The yield* is very close to the maximum theoretical value The requirement is reduced relative to the sole fermentative pathway, from two moles of per mole of succinate to ~ 1.25 therefore maximizing succinate production * during production phase

Aerobic succinate production system

Reconstructing the TCA cycle and glyoxylate bypass DNA Techniques: λ Red recombinase P1 phage transduction Verified by genomic PCR Max. theoretical yield: 1.0 (mol succinate/ mol glucose) Malate Fumarate OAA sdhab Glucose ptsg G6P G3P Acetyl-CoA Acetyl-CoA aceb glyoxylate acea poxb Citrate acea acka-pta Acetate Isocitrate icd Acetate 2-ketoglutarate iclr acebak sdhab - 2 iclr - 5 poxb - 6 acka-pta - 7 Succinyl-CoA

Two routes engineered for succinate production Glucose Strain HL2765k ptsg G6P Strain HL27659k Genes inactivated: sdhab - 2 acka-pta - 7 poxb - 6 G3P Genes inactivated: sdhab - 2 acka-pta - 7 poxb - 6 iclr - 5 Acetyl-CoA iclr - 5 ptsg - 9 OAA Malate aceb Acetyl-CoA glyoxylate Citrate Isocitrate acea icd 2-ketoglutarate Succinyl-CoA

HPLC metabolite output for strain HL27659k(pKK313) Strain HL27659k(pKK313) is the aerobic succinate production system

Fed batch Fed batch culture culture of strain HL27659k(pKK313) 600 1.10 mm 550 500 450 400 350 300 250 200 150 100 50 succinate 1.00 0.90 0.80 0.70 0.60 0.50 0.40 0.30 0.20 0.10 yield yield (mol/mol glucose) ( )( (mm( mm) ) ( )( Glucose (mm( mm) ) ( )( (mm( mm) ) ( )( Acetate (mm( mm) ) ( ) 0 0 10 20 30 40 50 60 0.00 hr 58.25 g/l (493.63 mm) ) succinate produced in 59 hrs Average succinate yield was 0.94 ± 0.07 mol/mol glucose Average productivity was 1.08 ± 0.06 g/l-hr Average specific productivity was 89.77 ± 3.40 mg/g-hr. (Lin et al., Biotechnol. Bioeng., 2005, 20;90(6):775-9 9 )

Summary (aerobic) We have successfully designed and implemented a very robust dual-route aerobic succinate production system This system converts glucose to succinate at fast rates and high yields with minimal by-products The synthesis of succinate through these two pathways does not require any

Hybrid succinate production systems

Construction of a hybrid succinate production system DNA Techniques: λ Red recombinase P1 phage transduction Verified by genomic PCR Malate OAA Glucose ptsg G6P G3P Acetyl-CoA Acetyl-CoA aceb ldha Glyoxylate poxb acka-pta Citrate acea Lactate Acetate adhe Isocitrate Acetate Ethanol iclr acebak Fumarate sdhab acea icd 2-ketoglutarate Succinyl-CoA

Three routes engineered for succinate production Glucose ptsg G6P G3P Acetyl-CoA Strain SBS552 Genes inactivated: sdhab acka-pta poxb iclr ptsg ldha adhe Fumarate OAA Malate aceb Acetyl-CoA Glyoxylate Citrate Isocitrate acea icd 2-ketoglutarate Succinyl-CoA (anaerobic) (anaerobic & aerobic) (aerobic)

Overall Summary We have developed: an efficient aerobic succinate production system with a molar yield of close to 1 a high yield anaerobic succinate production system with a molar yield of close to 1.6 A hybrid system that is capable of producing succinate under aerobic and anaerobic conditions Same technology can be used to produce other C-4 compounds, such as fumarate and malate

Acknowledgments Collaborator: Graduate Students: George N. Bennett Ailen M. Sanchez Henry Lin Irene Martínez Susana Berrios-Rivera This work was supported by grants from the National Science Foundation (BES-0222691, BES-0000303, and BES-0420840)