Metabolic Flux Ratio Analysis of Genetic and Environmental Modulations of <i>Escherichia coli</i> Central Carbon Metabolism

TLDR

Escherichia coli central carbon metabolism has been examined under genetic and environmental perturbations using a novel metabolic flux ratio (METAFoR) method that directly identifies active pathways. METAFoR generates fluxome data from two‑dimensional 13C‑1H NMR of fractionally labeled biomass, eliminating the need for extracellular metabolite measurements. Flux ratio analysis showed that moderate overexpression of key glycolytic enzymes changes only a few ratios, while disrupting pyruvate kinase isoenzymes alters PEP‑pyruvate fluxes without major metabolic disruption, and environmental shifts such as ammonia limitation or glucose scarcity produce more pronounced changes, including increased anaplerotic PEP carboxylation, altered transketolase contribution, and activation of previously undetected PEP carboxykinase and malic enzyme reactions, demonstrating robust central metabolism with context‑dependent flexibility.

Abstract

ABSTRACT 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 13 C- 1 H 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.