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Journal of Bacteriology, November 1999, p. 6679-6688, Vol. 181, No. 21
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Metabolic Flux Ratio Analysis of Genetic and Environmental Modulations of Escherichia coli Central Carbon Metabolism

Uwe Sauer,1,* Daniel R. Lasko,1,dagger Jocelyne Fiaux,2 Michel Hochuli,2 Ralf Glaser,2 Thomas Szyperski,2,Dagger Kurt Wüthrich,2 and James E. Bailey1

Institut für Biotechnologie1 and Institut für Molekularbiologie und Biophysik,2 ETH Zürich, CH-8093 Zürich, Switzerland

Received 30 April 1999/Accepted 23 August 1999

The response of Escherichia coli central carbon metabolism to genetic and environmental manipulation has been studied by use of a recently developed methodology for metabolic flux ratio (METAFoR) analysis; this methodology can also directly reveal active metabolic pathways. Generation of fluxome data arrays by use of the METAFoR approach is based on two-dimensional 13C-1H correlation nuclear magnetic resonance spectroscopy with fractionally labeled biomass and, in contrast to metabolic flux analysis, does not require measurements of extracellular substrate and metabolite concentrations. METAFoR analyses of E. coli strains that moderately overexpress phosphofructokinase, pyruvate kinase, pyruvate decarboxylase, or alcohol dehydrogenase revealed that only a few flux ratios change in concert with the overexpression of these enzymes. Disruption of both pyruvate kinase isoenzymes resulted in altered flux ratios for reactions connecting the phosphoenolpyruvate (PEP) and pyruvate pools but did not significantly alter central metabolism. These data indicate remarkable robustness and rigidity in central carbon metabolism in the presence of genetic variation. More significant physiological changes and flux ratio differences were seen in response to altered environmental conditions. For example, in ammonia-limited chemostat cultures, compared to glucose-limited chemostat cultures, a reduced fraction of PEP molecules was derived through at least one transketolase reaction, and there was a higher relative contribution of anaplerotic PEP carboxylation than of the tricarboxylic acid (TCA) cycle for oxaloacetate synthesis. These two parameters also showed significant variation between aerobic and anaerobic batch cultures. Finally, two reactions catalyzed by PEP carboxykinase and malic enzyme were identified by METAFoR analysis; these had previously been considered absent in E. coli cells grown in glucose-containing media. Backward flux from the TCA cycle to glycolysis, as indicated by significant activity of PEP carboxykinase, was found only in glucose-limited chemostat culture, demonstrating that control of this futile cycle activity is relaxed under severe glucose limitation.

* Corresponding author. Mailing address: Institut für Biotechnologie, ETH Zürich, CH-8093 Zürich, Switzerland. Phone: 41-1-633 3672. Fax: 41-1-633 1051. E-mail: sauer{at}biotech.biol.ethz.ch.

dagger Present address: Genetics Institute, Andover, MA 01810.

Dagger Present address: Department of Chemistry, State University of New York, Buffalo, NY 14260.

Journal of Bacteriology, November 1999, p. 6679-6688, Vol. 181, No. 21
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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