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

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.

FIG. 1

Biochemical master network with reactions for identified genes or enzyme activities in E. coli. The information was compiled from the EcoCyc database (17) and other sources (11, 20). The reaction sets of glycolysis, the PP pathway, the TCA cycle (including the glyoxylate shunt), and C₁ metabolism are shaded, and enzymes catalyzing key reactions are indicated in italics. The hatched arrow highlights the TCA cycle-replenishing anaplerotic reaction, and the broken arrows indicate the anaerobic pyruvate formate-lyase, which is interrupted in strain KO20. The grey arrows indicate the reactions catalyzed by the enzymes encoded on the pet operon of Z. mobilis. The intact carbon fragment patterns of boxed metabolites were directly determined by ¹³C-¹H COSY of proteinogenic amino acids. Abbreviations: F6P, fructose-6-phosphate; Ru5P, ribulose-5-phosphate; X5P, xylulose-5-phosphate; E4P, erythrose-4-phosphate; S7P, seduheptulose-7-phosphate; PGA, 3-phosphoglycerate; SER, serine; GLY, glycine; AAD, acetaldehyde; CIT, citrate; ICT, isocitrate; OGA, oxoglutarate; SUC, succinate; GOX, glyoxylate; and DH, dehydrogenase.

FIG. 2

Origins of metabolic intermediates (A to P) during aerobic growth of E. coli MG1655 in glucose-limited or ammonia-limited chemostats. In certain cases, the NMR data permit the determination only of upper bounds (ub) or lower bounds (lb) on the origin of intermediates. The experimental error (error bars) was estimated from the analysis of redundant ¹³C scalar coupling fine structures and the signal-to-noise ratio of the ¹³C-¹H COSY spectra by use of the Gaussian law of error propagation. The fraction of the total pool for a particular metabolite quantifies the ratio of this metabolite derived from a specified substrate to the sum of all other substrates that contribute to the pool of this metabolite. In cases where only two reactions contribute to one metabolite, e.g., OAA from PEP and PEP from OAA, the remaining fraction of the total pool can be attributed to the competing reaction. Abbreviations are explained in the text and in the legend to Fig. 1; rev., reversibly.

FIG. 3

Growth of E. coli ATCC 11303 in aerobic batch cultures. The line represents the best fit to the exponential growth phase data, and the arrows indicate the times of biomass sampling for the METAFoR analysis.

FIG. 4

Origin of metabolic intermediates (A to P) during aerobic exponential growth of various E. coli strains. For more details, see the legend to Fig. 2.

FIG. 5

Origin of metabolic intermediates (A to N) during anaerobic exponential growth of various E. coli strains. For more details, see the legend to Fig. 2.