Abstract
The effect of poxB gene knockout on metabolism in Escherichia coli was investigated in the present paper based on the growth characteristics and the activities of the enzymes involved in the central metabolic pathways. The absence of pyruvate oxidase reduced the glucose uptake rate and cell growth rate, and increased O2 consumption and CO2 evolution. The enzyme assay results showed that although glucokinase activity increased, the flux through glycolysis was reduced due to the down-regulation of the other glycolytic enzymes such as 6-phosphofructosekinase and fructose bisphosphate aldolase in the poxB mutant. TCA cycle enzymes such as citrate synthase and malate dehydrogenase were repressed in the poxB mutant when the cells were cultivated in LB medium. The pyruvate oxidase mutation also resulted in the activation of glucose-6-phosphate dehydrogenase and acetyl-CoA synthetase. All these results suggest that pyruvate oxidase is not only a stationary-phase enzyme as previously known, and that the removal of the poxB gene affects the central metabolism at the enzyme level in E. coli.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Abdel-Hamid AM, Attwood MM, Guest JR (2001) Pyruvate oxidase contributes to the aerobic growth efficiency of Escherichia coli. Microbiology 147:1483–1498
Brown TDK, Jones-Mortimer MC, Kornberg HL (1977) The enzymic interconversion of acetate and acetyl-coenzyme A in Escherichia coli. J Gen Microbiol 102:327–336
Baba T, Ara T, Hasegawa M, Takai Y, Okumura Y, Baba M, Datsenko KA, Tomita M, Wanner BL, Mori H (2006) Construction of Escherichia coli K-12 in frame, single-gene knockout mutants: the Keio collection. Mol Syst Biol msb4100050-E1
Chang YY, Cronan JE Jr (1983) Genetic and biochemical analyses of Escherichia coli strains having a mutation in thestructural gene (poxB) for pyruvate oxidase. J Bacteriol 154:757–762
Chang YY, Cronan JE Jr (1984) An Escherichia coli mutant deficient in pyruvate oxidase activity due to altered phospholipid activation of the enzyme. Proc Natl Acad Sci USA 81:4348–4352
Carter K, Gennis RB (1985) Reconstitution of the ubiquinone dependent pyruvate oxidase system of Escherichia coli with the cytochrome o terminal oxidase complex. J Biol Chem 260:10986–10990
Chang YY, Wang AY, Cronan JE Jr (1994) Expression of Escherichia coli pyruvate oxidase (PoxB) depends on the sigmafactor encoded by the rpoS (katF) gene. Mol Microbiol 11:1019–1028
Causey TB, Shanmugam KT, Yomano LP, Ingram LO (2004) Engineering Escherichia coli for efficient conversion of glucose to pyruvate. Proc Natl Acad Sci USA 101:2235–2240
Datsenko KA, Wanner BL (2000) One-step inactivation of chromosomal genes in Escherichia coli K12 using PCR products. Proc Natl Acad Sci USA 97:6640–6645
El-Mansi EM, Holms WH (1989) Control of carbon flux to acetate excretion during overflow in Escherichia coli. Biotechnol Bioeng 35:732–738
Grabau C, Cronan JE Jr (1984) Molecular cloning of the gene (poxB) encoding the pyruvate oxidase of Escherichia coli, a lipid-activated enzyme. J Bacteriol 160:1088–1092
Han K, Lim HC, Hong J (1992) Acetic acid formation in Escherichia coli fermentation. Biotech Bioeng 39:663–671
Jiang GR, Nikolova S, Clark DP (2001) Regulation of ldhA gene, encoding the fermentative lactate dehydrogenase of Escherichia coli. Microbiology 147:2437–2446
Kitagawa M, Ara T, Arifuzzaman M, Ioka-Nakamichi T, Inamoto E., Toyonaga H, Mori H (2005) Complete set of ORF clones of Escherichia coli ASKA library (A complete set of E. coli K-12 ORF Archive): unique resources for biological research. DNA Res 12:291–299
Li M, Ho PY, Yao SJ, Shimizu K (2006) Effect of lpdA gene knockout on the metabolism in Escherichia coli based on enzyme activities, intracellular metabolite concentrations and metabolic flux analysis by 13C-labeling experiments. J Biotechnol 122:254–266
Peng LF, Shimizu K (2003) Global metabolic regulation analysis for Escherichia coli K12 based on protein expression by 2-dimensional electrophoresis and enzyme activity measurement. Appl Microbiol Biotechnol 61:163–178
Peng LF, Arauzo-Bravo MJ, Shimizu K (2004) Metabolic flux analysis for a ppc mutant Escherichia coli based on 13C-labelling experiments together with enzyme activity assays and intracellular metabolite measurements. FEMS Microbiol Lett 235:17–23
Siddiquee LAZ, Arauzo-Bravo MJ, Shimizu K (2004) Metabolic flux analysis of pykF gene knockout Escherichia coli based on 13C-labeling experiments together with measurements of enzyme activities and intracellular metabolite concentrations. Appl Microbiol Biotechnol 63:407–417
Tao H, Gonzalez R, Martinez A, Rodriguez M, Ingram LO, Preston JF, Shanmugam KT (2001) Engineering a homo-ethanol pathway in Escherichia coli: Increased glycolytic flux and levels of expression of glycolytic genes during xylose fermentation. J Bacteriol 183:2979–2988
Tarmy EM, Kaplan NO (1968) Kinetics of Escherichia coli B D-Lactate dehydrogenase and evidence for pyruvate-controlled change in conformation. J Biol Chem 243:2587–2596
Vemuri GN, Minning TA, Altman E, Eiteman MA (2005) Physiological response of central metabolism in Escherichia coli to deletion of pyruvate oxidase and introduction of heterologous pyruvate carboxylase. Biotechnol Bioeng 90:64–76
Acknowledgements
It is acknowledged that the research was supported in part by a grant from New Energy and Industrial Technology Development Organization (NEDO) of the Ministry of Economy, Trade and Industry of Japan (Development of a Technological Infrastructure for Industrial Bioprocess Project).
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Li, M., Yao, S. & Shimizu, K. Effect of poxB gene knockout on metabolism in Escherichia coli based on growth characteristics and enzyme activities. World J Microbiol Biotechnol 23, 573–580 (2007). https://doi.org/10.1007/s11274-006-9267-5
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11274-006-9267-5