Metabolic flux ratio analysis of genetic and environmental modulations of Escherichia coli central carbon metabolism

The response of Escherichia coli central carbon metabolism to genetic and e nvironmental manipulation has been studied by use of a recently developed m ethodology for metabolic flux ratio (METAFoR) analysis; this methodology ca n also directly reveal active metabolic pathways. Generation of fluxome dat a arrays by use of the METAFoR approach is based on two-dimensional C-13-H- 1 correlation nuclear magnetic resonance spectroscopy with fractionally lab eled biomass and, in contrast to metabolic flux analysis, does not require measurements of extracellular substrate and metabolite concentrations. META FoR analyses of E. coli strains that moderately overexpress phosphofructoki nase, pyruvate kinase, pyruvate decarboxylase, or alcohol dehydrogenase rev ealed that only a few flux ratios change in concert with the overexpression of these enzymes. Disruption of both pyruvate kinase isoenzymes resulted i n altered flux ratios for reactions connecting the phosphoenolpyruvate (PEP ) and pyruvate pools but did not significantly alter central metabolism. Th ese data indicate remarkable robustness and rigidity in central carbon meta bolism in the presence of genetic variation. More significant physiological changes and flux ratio differences were seen in response to altered enviro nmental conditions. For example, in ammonia-limited chemostat cultures, com pared to glucose-limited chemostat cultures, a reduced fraction of PEP mole cules was derived through at least one transketolase reaction, and there wa s a higher relative contribution of anaplerotic PEP carboxylation than of t he tricarboxylic acid (TCA) cycle for oxaloacetate synthesis. These two par ameters also showed significant variation between aerobic and anaerobic bat ch cultures. Finally, two reactions catalyzed by PEP carboxykinase and mali c enzyme were identified by METAFoR analysis; these had previously been con sidered absent in E. coli cells grown in glucose-containing media. Backward flux from the TCA cycle to glycolysis, as indicated by significant activit y 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.