Effect of a pyruvate kinase (pykF-gene) knockout mutation on the control of gene expression and metabolic fluxes in Escherichia coli

Abstract

The metabolic regulation of Escherichia coli lacking a functional pykF gene was investigated based on gene expressions, enzyme activities, intracellular metabolite concentrations and the metabolic flux distribution obtained based on 13C-labeling experiments. RT-PCR revealed that the glycolytic genes such as glk, pgi, pfkA and tpiA were down regulated, that ppc, pckA, maeB and mdh genes were strongly up-regulated, and that the oxidative pentose phosphate pathway genes such as zwf and gnd were significantly up-regulated in the pykF mutant. The catabolite repressor/activator gene fruR was up-regulated in the pykF mutant, but the adenylate cyclase gene cyaA was down-regulated indicating a decreased rate of glucose uptake. This was also ascertained by the degradation of ptsG mRNA, the gene for which was down-regulated in the pykF mutant. In general, the changes in enzyme activities more or less correlated with ratios of gene expression, while the changes in metabolic fluxes did not correlate with enzyme activities. For example, high flux ratios were obtained through the oxidative pentose phosphate pathway due to an increased concentration of glucose-6-phosphate rather than to favorable enzyme activity ratios. In contrast, due to decreased availability of pyruvate (and acetyl coenzyme A) in the pykF mutant compared with the wild type, low flux ratios were found through lactate and acetate forming pathways.