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 phosphoenolp yruvate carboxylase (PPC) and pyruvate carboxylase (PYC) enzymes were compa red under anaerobic conditions with glucose as a carbon source. Time profil es of glucose and fermentation product concentrations were determined and u sed to calculate metabolic fluxes through central carbon pathways during ex ponential cell growth. The presence of the Rhizobium etli pyc gene in E. co li (JCL1242/pTrc99A-pyc) restored the succinate producing ability of E. col i ppc null mutants (JCL1242), with PYC competing favorably with both pyruva te formate lyase and lactate dehydrogenase. Succinate formation was slightl y greater by JCL1242/pTrc99A-pyc than by cells which overproduced PPC (JCL1 242/pPC201,ppc(+)), even though PPC activity in cell extracts of JCL1242/pP C201 (ppc(+)) was 40-fold greater than PYC activity in extracts of JCL1242/ pTrc99a-pyc. Flux calculations indicate that during anaerobic metabolism th e pyc(+) strain had a 34% greater specific glucose consumption rate, a 37% greater specific rate of ATP formation, and a 6% greater specific growth ra te compared to the ppc(+) strain. In light of the important position of pyr uvate at the juncture of NADH-generating pathways and NADH-dissimilating br anches, the results show that when PPC or PYC is expressed, the metabolic n etwork adapts by altering the flux to lactate and the molar ratio of ethano l to acetate formation.