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Metabolic flux analysis of pykF gene knockout Escherichia coli based on 13C-labeling experiments together with measurements of enzyme activities and intracellular metabolite concentrations.
- Source :
-
Applied microbiology and biotechnology [Appl Microbiol Biotechnol] 2004 Jan; Vol. 63 (4), pp. 407-17. Date of Electronic Publication: 2003 Jun 12. - Publication Year :
- 2004
-
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.
- Subjects :
- Acetic Acid metabolism
Adenosine Diphosphate metabolism
Adenosine Triphosphate biosynthesis
Carbohydrates analysis
Carbon Dioxide metabolism
Cytoplasm chemistry
Escherichia coli genetics
Escherichia coli metabolism
Gas Chromatography-Mass Spectrometry
Glucose-6-Phosphate analysis
Glycolysis
Magnetic Resonance Spectroscopy
Malate Dehydrogenase physiology
Pentose Phosphate Pathway physiology
Phosphoenolpyruvate metabolism
Phosphoenolpyruvate Carboxylase physiology
Pyruvic Acid metabolism
Escherichia coli enzymology
Gene Deletion
Genes, Bacterial
Pyruvate Kinase genetics
Pyruvate Kinase metabolism
Subjects
Details
- Language :
- English
- ISSN :
- 0175-7598
- Volume :
- 63
- Issue :
- 4
- Database :
- MEDLINE
- Journal :
- Applied microbiology and biotechnology
- Publication Type :
- Academic Journal
- Accession number :
- 12802531
- Full Text :
- https://doi.org/10.1007/s00253-003-1357-9