Growth inhibition by ppc deletion is rescued by isocitrate dehydrogenase mutations in Escherichia coli.

Phosphoenolpyruvate carboxylase encoded by ppc catalyzes the anaplerotic reaction of oxaloacetate in the tricarboxylic acid (TCA) cycle in Escherichia coli. Deletion of ppc does not prevent the cells from replenishing oxaloacetate via the glyoxylate shunt, but the ppc-deletion strain almost did not grow on glucose. In the present study, we obtained evolved strains by deleting both ppc and mutS to increase the mutation rate and investigated the mechanisms for improving growth by analyzing the mutated genes. Genome resequencing revealed that the evolved strains have non-synonymous mutations in icd encoding isocitrate dehydrogenase (ICDH). The introduction of icd mutations rescued the growth defects caused by ppc deletion. ICDH activity was strongly reduced by the amino acid substitutions G205D or N232S. The evolved strains appeared to suppress the competitive pathway for increasing the glyoxylate shunt flux. In metabolic engineering, the deletion of iclR, which encodes a repressor of the aceBAK operon, has been used to activate the glyoxylate shunt. The growth rate of the ΔppcΔiclR strain slightly increased, but it was still much lower than that of the Δppc + icdG205D strains. This finding suggests that iclR deletion is not sufficient to enhance glyoxylate shunt flux and that inactivation of the competitive pathway by icd mutations is more effective.