Abstract
In order to rationally manipulate the cellular metabolism of Escherichia coli for d-lactate production, single-gene and multiple-gene deletions with mutations in acetate kinase (ackA), phosphotransacetylase (pta), phosphoenolpyruvate synthase (pps), pyruvate formate lyase (pflB), FAD-binding d-lactate dehydrogenase (dld), pyruvate oxidase (poxB), alcohol dehydrogenase (adhE), and fumarate reductase (frdA) were tested for their effects in two-phase fermentations (aerobic growth and oxygen-limited production). Lactate yield and productivity could be improved by single-gene deletions of ackA, pta, pflB, dld, poxB, and frdA in the wild type E. coli strain but were unfavorably affected by deletions of pps and adhE. However, fermentation experiments with multiple-gene mutant strains showed that deletion of pps in addition to ackA-pta deletions had no effect on lactate production, whereas the additional deletion of adhE in E. coli B0013-050 (ackA-pta pps pflB dld poxB) increased lactate yield. Deletion of all eight genes in E. coli B0013 to produce B0013-070 (ackA-pta pps pflB dld poxB adhE frdA) increased lactate yield and productivity by twofold and reduced yields of acetate, succinate, formate, and ethanol by 95, 89, 100, and 93%, respectively. When tested in a bioreactor, E. coli B0013-070 produced 125 g/l d-lactate with an increased oxygen-limited lactate productivity of 0.61 g/g h (2.1-fold greater than E. coli B0013). These kinetic properties of d-lactate production are among the highest reported and the results have revealed which genetic manipulations improved d-lactate production by E. coli.
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References
Baba T, Ara T, Hasegawa M et al (2006) Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Mol Syst Biol 2:8–18
Bloor AE, Cranenburgh RM (2006) An efficient method of selectable marker gene excision by Xer recombination for gene replacement in bacterial chromosomes. Appl Environ Microbiol 72:2520–2525
Bunch PK, Mat-Jan F, Lee N et al (1997) The ldhA gene encoding the fermentative lactate dehydrogenase of Escherichia coli. Microbiology 143:187–195
Chang YY, Wang AY, Cronan JE (1994) Expression of Escherichia coli pyruvate oxidase (PoxB) depends on the sigma factor encoded by the rpoS (katF) gene. Mol Microbiol 11:1019–1028
Datsenko KA, Wanner BL (2000) One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci USA 97:6640–6645
Grabar TB, Zhou S, Shanmugam KT et al (2006) Methlglyoxal bypass identified as source of chiral contamination in L(+) and D(–)-lactate fermentations by recombinant Escherichia coli. Biotechnol Lett 28:1527–1535
Ma XJ, Shu YL (2005) Short protocols in molecular biology, 4th edn. Science Preess, Bei**g, p 2
Martinez A, Grabar TB, Shanmugam KT et al (2007) Low salt medium for lactate and ethanol production by recombinant Escherichia coli B. Biotechnol Lett 29:397–404
Wee YJ, Kim JN, Ryu HW (2006) Biotechnological production of lactic acid and its recent applications. Food Technol Biotechnol 44:163–172
Zhou S, Causey TB, Hasona A et al (2003) Production of optically pure d-lactic acid in mineral salts medium by metabolically engineered Escherichia coli W3110. Appl Environ Microbiol 69:399–407
Zhou S, Shanmugam KT, Yomano LP et al (2006) Fermentation of 12% (w/v) glucose to 1.2 M lactate by Escherichia coli strain SZ194 using mineral salts medium. Biotechnol Lett 28:663–670
Zhu J, Shimizu K (2004) The effect of pfl gene knockout on the metabolism for optically pure d-lactate production by Escherichia coli. Appl Microbiol Biotechnol 64:367–375
Zhu J, Shimizu K (2005) Effect of a single-gene knockout on the metabolic regulation in Escherichia coli for d-lactate production under anaerobic condition. Metab Eng 7:104–115
Zhu Y, Eiteman MA, De Witt K et al (2007) Homolactate fermentation by metabolically engineered Escherichia coli strains. Appl Environ Microbiol 73:456–464
Zhu Y, Eiteman MA, Altman E (2008) Indirect monitoring of acetate exhaustion and cell recycle improve lactate production by non-growing Escherichia coli. Biotechnol Lett 30:1943–1946
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This work was partly funded by the Sino-South Africa Cooperation Program 2009DFA31300 and the National Natural Science Foundation of China No. 21006039.
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Zhou, L., Zuo, ZR., Chen, XZ. et al. Evaluation of Genetic Manipulation Strategies on d-Lactate Production by Escherichia coli . Curr Microbiol 62, 981–989 (2011). https://doi.org/10.1007/s00284-010-9817-9
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DOI: https://doi.org/10.1007/s00284-010-9817-9