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Applied Microbiology and Biotechnology

, Volume 64, Issue 1, pp 91–98 | Cite as

Global metabolic response of Escherichia coli to gnd or zwf gene-knockout, based on 13C-labeling experiments and the measurement of enzyme activities

  • J. Zhao
  • T. Baba
  • H. Mori
  • K. Shimizu
Original Paper

Abstract

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.

Keywords

Parent Strain Pentose Phosphate Pathway Flux Distribution Metabolic Flux Analysis Oxidative Pentose Phosphate Pathway 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Notes

Acknowledgement

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).

Supplementary material

Appendix 1

appendix1.pdf (104 kb)
(PDF 106 KB)

Appendix 2

appendix2.pdf (68 kb)
(PDF 70 KB)

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Copyright information

© Springer-Verlag 2004

Authors and Affiliations

  1. 1.Department of Biochemical Engineering & ScienceKyushu Institute of TechnologyIizukaJapan
  2. 2.Metabolome Unit, Institute for Advanced BiosciencesKeio UniversityTsuruokaJapan
  3. 3.Genome Engineering Unit, Institute for Advanced BiosciencesKeio UniversityTsuruokaJapan

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