Effect of ppc gene knockout on the metabolism of Escherichia coli in view of gene expressions, enzyme activities and intracellular metabolite concentrations.

Abstract

Physiological characteristics and regulatory mechanism were investigated based on comparative analyses of gene expressions, enzyme activities and intracellular metabolite concentrations between the ppc knockout Escherichia coli strain JWK3928 and its parent strain BW25113. RT-PCR was used to detect gene expressions in both strains. It was observed that the ppc knockout strain excreted little acetate and produced less carbon dioxide at the expense of a slower growth rate together with a lower glucose consumption rate, as compared with the parent strain. Accordingly, the biomass yield on glucose was improved in the ppc(-) mutant. It was found that genes such as gltA, icd, aceA and mdh involved in the glyoxylate shunt and part of the tricarboxylic acid cycle were significantly upregulated, whereas genes involved in glycolysis and the pentose phosphate pathway, such as pgi, fba, gapA, zwf and gnd, were significantly downregulated in response to ppc knockout. It was also found that the intracellular metabolites in glycolysis and the pentose phosphate pathway, such as phosphoenolpyruvate, glucose-6-phosphate, fructose-6-phosphate, fructose-1,6-phosphate and 6-phosphogluconate, increased, whereas oxaloacetate and acetyl-CoA concentrations decreased in ppc(-) mutant, as compared with those in the parent strain. Comparison between the gene expressions and enzyme activities implies that most of them were well correlated, except a few genes such as icd. It can be said from the present investigation that the combination of the information from gene expressions, enzyme activities and intracellular metabolite concentrations provides a comprehensive understanding of the physiological consequences and regulatory mechanisms for the specific gene-knockout E. coli.