Metabolic analysis of Escherichia coli in the presence and absence of the carboxylating enzymes phosphoenolpyruvate carboxylase and pyruvate carboxylase.

Fermentation patterns of Escherichia coli with and without the phosphoenolpyruvate carboxylase (PPC) and pyruvate carboxylase (PYC) enzymes were compared under anaerobic conditions with glucose as a carbon source. Time profiles of glucose and fermentation product concentrations were determined and used to calculate metabolic fluxes through central carbon pathways during exponential cell growth. The presence of the Rhizobium etli pyc gene in E. coli (JCL1242/pTrc99A-pyc) restored the succinate producing ability of E. coli ppc null mutants (JCL1242), with PYC competing favorably with both pyruvate formate lyase and lactate dehydrogenase. Succinate formation was slightly greater by JCL1242/pTrc99A-pyc than by cells which overproduced PPC (JCL1242/pPC201, ppc(+)), even though PPC activity in cell extracts of JCL1242/pPC201 (ppc(+)) was 40-fold greater than PYC activity in extracts of JCL1242/pTrc99a-pyc. Flux calculations indicate that during anaerobic metabolism the pyc(+) strain had a 34% greater specific glucose consumption rate, a 37% greater specific rate of ATP formation, and a 6% greater specific growth rate compared to the ppc(+) strain. In light of the important position of pyruvate at the juncture of NADH-generating pathways and NADH-dissimilating branches, the results show that when PPC or PYC is expressed, the metabolic network adapts by altering the flux to lactate and the molar ratio of ethanol to acetate formation.