Global metabolic response of Escherichia coli to gnd or zwf gene-knockout, based on 13C-labeling experiments and the measurement of enzyme activities
- 982 Downloads
An integrated study on cell growth, enzyme activities and carbon flux redistribution was made to investigate how the central metabolism of Escherichia coli changes with the knockout of genes in the oxidative pentose phosphate pathway (PPP). Mutants deficient in glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase were constructed by disrupting the zwf and gnd genes and were grown in minimal media with two different carbon sources, such as glucose or pyruvate. It was shown that the knockout of either gnd or zwf gene did not affect the cell growth rate significantly, but the cellular metabolism was changed. While the specific substrate uptake rate and the specific carbon dioxide evolution rate for either mutant grown on glucose were higher than those obtained for the parent strain, these two rates were markedly decreased in mutants grown on pyruvate. The measurement of enzyme activities implied a significant change in metabolism, when alternative pathways such as the Entner–Doudoroff pathway (EDP) and the malic enzyme pathway were activated in the gnd mutant grown on glucose. As compared with the parent strain, the activities of phosphoglucose isomerase were increased in mutants grown on glucose but decreased in mutants grown on pyruvate. The metabolic flux redistribution obtained based on 13C-labeling experiments further indicated that the direction of the flux through the non-oxidative PPP was reversed in response to the gene knockout. Moreover, the knockout of genes caused an increased flux through the tricarboxlic acid cycle in mutants grown on glucose but caused a decrease in the case of using pyruvate. There was also a negative correlation between the fluxes through malic enzyme and isocitrate dehydrogenase in the mutants; and a positive correlation was found between the fluxes through malic enzyme and phosphoenolpyruvate carboxylase.
KeywordsParent Strain Pentose Phosphate Pathway Flux Distribution Metabolic Flux Analysis Oxidative Pentose Phosphate Pathway
This research was supported in part by a grant from New Energy and Industrial Technology Development Organization of the Ministry of Economy, Trade and Industry of Japan (Development of a Technological Infrastructure for Industrial Bioprocess Project).
- Colowick SP (1963) Preparation and assay of enzymes. Methods Enzymol 6:1–640Google Scholar
- Lim SJ, Jung YM, Shin HD, Lee YH (2002) Application of the NADPH-related genes zwf and gnd for the Oddball biosynthesis of PHB in an E. coli transformant harbouring a cloned phbCAB operon. J Biosci Bioeng 93:543–549Google Scholar
- Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275Google Scholar
- Marx A, Graaf AA, Wiechert W, Eggeling L, Sahm H (1996) Determination of the fluxes in the central metabolism of Corynebacterium glutamicum by nuclear magnetic resonance spectroscopy combined with metabolite balancing. Biotechnol Bioeng 49:111–129Google Scholar
- Neidhardt FC, Ingraham JL, Schaechter M (1990) Physiology of the bacterial cell: a molecular approach. Sinauer Associates, Sunderland, Mass.Google Scholar
- Park SM, Sinskey AJ, Stephanopoulos G (1997) Metabolic and physiological studies of Corynebacterium glutamicum mutants. Biotechnol Bioeng 55:864–878Google Scholar
- Schmidt K, Nielsen J, Villadsen J (1997) Modeling isotopomer distributions in metabolic networks using isotopomer mapping matrices. Biotechnol Bioeng 55:831–840Google Scholar