Applied Microbiology and Biotechnology

, Volume 63, Issue 4, pp 407–417 | Cite as

Metabolic flux analysis of pykF gene knockout Escherichia coli based on 13C-labeling experiments together with measurements of enzyme activities and intracellular metabolite concentrations

  • K. Al Zaid Siddiquee
  • M. J. Arauzo-Bravo
  • K. Shimizu
Original Paper

Abstract

Metabolic flux analysis based on 13C-labeling experiments followed by the measurement of intracellular isotope distribution using both 2D NMR and GC-MS was carried out to investigate the effect of pyruvate kinase (pyk) gene knockout on the metabolism of Escherichia coli in continuous culture. In addition, the activities of 16 enzymes, and the concentrations of 5 intracellular metabolites, were measured as a function of time in batch culture as well as continuous culture. It was found that flux through phosphoenol pyruvate carboxylase and malic enzyme were up-regulated in the pykF mutant as compared with the wild type, and acetate formation was significantly reduced in the mutant. In addition, flux through the phosphofructose kinase pathway was reduced and that through the oxidative pentose phosphate (PP) pathway increased in the mutant. This was evidenced by the corresponding enzyme activities, and the increase in the concentrations of phosphoenol pyruvate, glucose-6-phosphate and 6-phosphogluconate, etc. It was also found for continuous cultivation that the enzyme activities of the oxidative PP and Entner-Doudoroff pathways increased as the dilution rate increased for the pykF mutant. To clarify the metabolism quantitatively, it was found to be quite important to integrate the information on intracellular metabolic flux distribution, enzyme activities and intracellular metabolite concentrations.

Notes

Acknowledgement

It is acknowledged that this research was supported in part by a grant from New Energy and Industrial Technology Development Organization of Japan (NEDO) of the Ministry of Economy, Trade and Industry of Japan (Development of a Technological Infrastructure for Industrial Bioprocess Project).

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Copyright information

© Springer-Verlag 2004

Authors and Affiliations

  • K. Al Zaid Siddiquee
    • 1
  • M. J. Arauzo-Bravo
    • 1
  • K. Shimizu
    • 1
    • 2
  1. 1.Department of Biochemical Engineering and ScienceKyushu Institute of TechnologyFukuokaJapan
  2. 2.Institute for Advanced BiosciencesKeio UniversityYamagata Japan

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