USOO7927859B2 (12) United States Patent (10) Patent No.: US 7927,859 B2 San et al. (45) Date of Patent: Apr. 19, 2011 (54) HIGH MOLARSUCCINATEYIELD FOREIGN PATENT DOCUMENTS BACTERIA BY INCREASING THE WO WO99.06532 * 2/1999 INTRACELLULAR NADHAVAILABILITY WO WO 2007 OO1982 1, 2007 OTHER PUBLICATIONS (75) Inventors: Ka-Yiu San, Houston, TX (US); George N. Bennett, Houston, TX (US); Ailen Branden et al. Introduction to Protein Structure, Garland Publishing Inc., New York, p. 247, 1991.* Sánchez, Houston, TX (US) ExPASy. Formate Dehydrogenase.* Vemuri et al. Effects of growth mode and pyruvate carboxylase on (73) Assignee: Rice University, Houston, TX (US) Succinic acid production by metabolically engineered strains of Escherichia coli. Appl Environ Microbiol. Apr. 2002:68(4): 1715 (*) Notice: Subject to any disclaimer, the term of this 27.3 patent is extended or adjusted under 35 Goodbye et al. Cloning and sequence analysis of the fermentative alcohol-dehydrogenase-encoding gene of Escherichia coli. Gene. U.S.C. 154(b) by 791 days. Dec. 21, 1989;85(1):209-14.* Datsenko et al. One-step inactivation of chromosomal genes in (21) Appl. No.: 10/923,635 Escherichia coli K-12 using PCR products. Proc Natl AcadSci USA. Jun. 6, 2000:97(12):6640-5.* (22) Filed: Aug. 20, 2004 Berrios-Rivera et al. Metabolic engineering of Escherichia coli: increase of NADHavailability by overexpressing an NAD(+)-depen (65) Prior Publication Data dent formate dehydrogenase. Metab Eng. Jul. 2002;4(3):217-29.* Gupta et al. Escherichia coli derivatives lacking both alcohol US 2005/OO42736A1 Feb. 24, 2005 dehydrogenase and phosphotransacetylase grow anaerobically by lactate fermentation. J Bacteriol. Jul. 1989; 171(7):3650-5.* Related U.S. Application Data Alam, Ketal. Anaerobic fermentation balance of E. coli as observed by in vivo nuclear magnetic resonance spectroscopy; J. of Bacteriol (60) Provisional application No. 60/497, 195, filed on Aug. ogy, vol. 171 (11), pp. 6213-7, Nov. 1989. 22, 2003. Aristidou AA, San Ky, Bennett GN. Metabolic engineering of Escherichia coli to enhance recombinant protein production through (51) Int. Cl. acetate reduction. Biotechnol Prog. Jul.-Aug. 1995; 11(4):475-8. CI2N I/20 (2006.01) Aristidou AA, San Ky, Bennett GN. Metabolic flux analysis of CI2N 9/02 (2006.01) Escherichia coli expressing the Bacillus subtilis acetolactate CI2N 9/04 (2006.01) Synthase in batch and continuous cultures. Biotechnol Bioeng. Jun. 20, 1999:63(6): 737-49. CI2N 9/10 (2006.01) Gokarn, R. R.; Eiteman, M. A.; Altman, E. Expression of pyruvate CI2N IS/00 (2006.01) carboxylase enhances Succinate production in Escherichia coli with CI2P 7/64 (2006.01) out affecting glucose uptake rate. Biotech. Let. 1998, 20, 795-798. CI2P 2L/04 (2006.01) U.S. Appl. No. 10/923,635, filed Aug. 20, 2004, San et al. CI2O I/68 (2006.01) U.S. Appl. No. 10/987,511, filed Nov. 12, 2004, San et al. CI2O I/00 (2006.01) U.S. Appl. No. 10/286,326, filed Nov. 2, 2002, San et al. C7H 2L/04 (2006.01) U.S. Appl. No. 60/604,922, filed Aug. 27, 2004, San et al. C7H 2L/02 (2006.01) U.S. Appl. No. 60/599.956, filed Aug. 9, 2004, San et al. U.S. Appl. No. 60/610,750, filed Sep. 17, 2004, San et al. (52) U.S. Cl. ................... 435/252.33; 435/189: 435/190; U.S. Appl. No. 60/638,765, filed Dec. 22, 2004, San et al. 435/.440; 435/252.3:435/320.1; 435/4; 435/6; Gokarn, R. R.; Eiteman, M. A.; Altman, E. Metabolic analysis of 435/193:435/135; 435/69.1:536/23.2:536/23.1 Escherichia coli in the presence and absense of the carboxylating (58) Field of Classification Search ........................ None enzymes phosphoenolpyruvate carboxylase and pyruvate See application file for complete search history. carboxylase. Appl Environ Microbiol. 2000, 666, 1844-1850. (56) References Cited (Continued) U.S. PATENT DOCUMENTS Primary Examiner —Yong D Pak 6,159,738 A 12/2000 Donnelly et al. (74) Attorney, Agent, or Firm — Boulware & Valoir 6,448,061 Bl 9, 2002 Panet al. 6,455.284 B1 9, 2002 Gokarn et al. (57) ABSTRACT 2003, OO87381 A1 5, 2003 Gokarn et al. 2006,0040368 A1 2/2006 San et al. The invention relates to increasing the yield of Succinate in 2006,004.6288 A1 3, 2006 Ka-Yiu et al. bacteria by increasing the intracellular availability of cofac 2006, OO73577 A1 4/2006 Ka-Yiu et al. tors such as NADH. 2006.0128001 A1 6, 2006 Yukawa et al. 2006, O141594 A1 6, 2006 San et al. 18 Claims, 4 Drawing Sheets US 7,927,859 B2 Page 2 OTHER PUBLICATIONS membrane-driven fumarate reduction and energy conservation; J. of Bacteriology, vol. 181 (8), pp. 2403-2410, Apr. 1999. Hahm, D. H.; Pan, J. G.; Rhee, J. S. Characterization and evaluation Phillips, G. J.; Park, S. K.; Huber, D. High copy number plasmids of a pta (phosphotransacetylase) negative mutant of Escherichia coli compatible with commonly used cloning vectors. Biotechniques. HZB101 as a production host of foreign lipase. Appl Microbiol 2000, 28, 400-408. San KY, Bennett GN. Berrios-Rivera SJ, Vadali RV.YangYT, Horton Biotechnol. 1994, 42, 100-107. E. Rudolph FB, Sariyar B, Blackwood K. Metabolic engineering Holms, W. H. The central metabolic pathways in Escherichia coli: through cofactor manipulation and its effects on metabolic flux redis relationship between flux and control at a branchpoint, efficiency of tribution in Escherichia coli. Metab Eng. Apr. 2002:4(2): 182-92. conversion to biomass, and excretion of acetate. Curr Top Cell Regul. Sanchez, A. M.; Bennett, G. N.; San, K.-Y. Efficient Succinate pro 1986, 28, 69-105. duction from glucose through overexpression of pyruvate Hong, S. H.; Lee, S.-Y. Importance of redox balance on the produc carboxylase in an Escherichia coli alcohol dehydrogense and lactate tion of Succinic acid by metabolically engineered Escherichia coli. dehydrogenase mutant. Submitted. 2004a. Appl Microbiol Biotechnol. 2002, 58,286-290. Vemuri, G.N.: Eiteman, M. A.; Altman, E. Effect of growth mode and Leonardo, M. et al., Anaerobic regulation of the adhE gene, encoding pyruvate carboxylase on Succinic acid production by metabolically the fermentative alcoholdehydrogenase of E. coli: J. of Bacteriology, engineered strains of Escherichia coli. Appl Environ Microbiol. vol. 175(3), pp. 870-8, Feb. 1993. 2002, 68, 1715-1727. Levanon SS, San KY, Bennett GN. Effect of oxygen on the Yang YT, Aristidou AA, San KY, Bennett GN. Metabolic flux analy Escherichia coli ArcA and FNR regulation systems and metabolic sis of Escherichia coli deficient in the acetate production pathway responses. Biotechnol Bioeng. Mar. 5, 2005;89(5):556-64. and expressing the Bacillus subtilis acetolactate synthase. Metab Lin H. San KY, Bennett GN. Effect of Sorghum vulgare Eng. Jan. 1999; 1(1):26-34. phosphoenolpyruvate carboxylase and Lactococcus lactis pyruvate Yang YT, Bennett GN. San KY. Effect of inactivation of nuo and carboxylase coexpression on Succinate production in mutant strains ackA-pta on redistribution of metabolic fluxes in Escherichia coli. of Escherichia coli. Appl Microbiol Biotechnol. Nov. 24, 2004; Biotechnol Bioeng. Nov. 5, 1999:65(3):291-7. Epub ahead of print. Yang YT. Bennett GN. San KY. The effects offeed and intracellular Lin H. Vadali RV. Bennett GN. San KY. Increasing the acetyl-CoA pyruvate levels on the redistribution of metabolic fluxes in pool in the presence of overexpressed phosphoenolpyruvate Escherichia coli. Metab Eng. Apr. 2001:3(2): 115-23. carboxylase or pyruvate carboxylase enhances succinate production Yang YT, Peredelchuk M. Bennett GN, San KY. Effect of variation of in Escherichia coli. Biotechnol Prog. Sep.-Oct. 2004:20(5): 1599 Klebsiella pneumoniae acetolactate synthase expression on meta 604. bolic flux redistribution in Escherichia coli. Biotechnol Bioeng. Jul. Luli. G. W.; Strohl, W. R. Comparison of growth, acetate production, 20, 2000:69(2): 150-9. and acetate inhibition of Escherichia coli strains in batch and fed San, Ka-Yiu et al. Metabolic Engineering through Cofactor Manipu batch fermentations. Applied and Environmental Microbiology. lation and its Effects on Metabolic Flux Redistribution in Escherichia 1990, 56, 1004-1011. coli, Metabolic Engineering 4, pp. 182-192,(2002). Park. D. et al., Utilization of electrically reduced neutral red by Actinobacillus succinogenes: physiological functio of neutral red in * cited by examiner U.S. Patent Apr. 19, 2011 Sheet 1 of 4 US 7927,859 B2 Glucose PEP PTSG Pyruvate Glucose-6-P - ATP ADP Fructose 1,6-diF w Glyceraldehyde-3-P --- Dihydroxyacetone-P Succinate P+NAD+ NADH Glycerate-1,3-diP 2NAD- v ADP NADH Co, - ATP ls1. y:-ADP OAA NN -r LDH Lactate PYC Pyruvate NADH NAD PFL Formate Acetyl-CoA - -- Acetyl-P PTA CO 2 --" ALDH ACK ADP ADH 2NADH ATP H2 2NAD Acetate Ethanol FIG. I. U.S. Patent Apr. 19, 2011 Sheet 2 of 4 US 7927,859 B2 Succinate Yield O 100 Fomate Conc. (mM) SBS110 (pDHC30+pHL413) is SBS110 (pASF2+pHL413) B C D Lactate Yield Acetate Yield as2 1.0 m -os----------------...s.n.m.- : 10 a.m...a...a.................... a2 0.25 0.8 is 0.20 -- S 0.6 a 0.15 3 3 g 0.4 3 0.10 02 0.05 g D 0.0 000 O 10 Fomate Conc. (mM) Formate Conc (mM) Formate Conc. (mM) SBS11 O(pDHC30-pHL413) SBS11 OcpASF2-phLA13) FIG 2 U.S. Patent Apr. 19, 2011 Sheet 3 of 4 US 7927,859 B2 Ghucose Consumed 40 120 100 Formate Acetate 11 ESBS110MG(pTRC99A) SBS110MG(pHLA 13) OSBS220MG(pHLA 13) Succinate Formate Acetate FIG. 3 U.S. Patent Apr. 19, 2011 Sheet 4 of 4 US 7927,859 B2 A B Ghucose Consumed 140 140 a loo120 S.gol 120 E 80 2 80 - - 60 9 60 2 40 5 40 Od 20 O 20 0 O 48 96 168 48 96 168 C 100 to 80 e a 60 5 40 2 3 20 O O 48 96 168 Time (hrs) Time (hrs) E OSBS110MG(pHLA 13) an SBS880MGK(pHLA 13) s .32 > Time (hrs) FIG.