Effect of inactivation of nuo and ackA-pta on redistribution of metabolic fluxes in Escherichia coli

The nuoA-N gene cluster encodes a transmembrane NADH:ubiquinone oxidoreduct ase (NDH-I) responsible for coupling redox chemistry to proton-motive force generation. Interactions between nuo and the acetate-producing pathway enc oded by ackA-pta were investigated by examining the metabolic patterns of s everal mutant strains under anaerobic growth conditions. In an ackA-pta str ain, the flux to acetate was decreased dramatically, whereas flux to lactat e was increased significantly when compared with its parent strain; the flu xes to pyruvate and ethanol also increased slightly. In addition, pyruvate was excreted. A strain carrying the nuo mutation showed metabolic flux dist ribution similar to the wild type. The ackA-pta-nuo strain showed a differe nt metabolic pattern. It not only exhibited reduced acetate accumulation bu t also significantly lower ethanol and formate synthesis. Metabolic flux di stribution analysis suggests that the excessive carbon flux was redirected at the pyruvate node through the lactate dehydrogenase pathway for lactate formation rather than the pyruvate formate-lyase (PFL) pathway for acetyl-C oA and formate production. The diminished capacity through the formate and ethanol (ADH) pathways was not the result of genetic disruption of function al PFL or ADH production. The introduction of a Bacillus subtilis acetolact ate synthase gene returned formate, ethanol, and lactate levels to those of the wild type (ackA(+)pta(+)nuo(+)) strain. Furthermore, transfer of a lac tate dehydrogenase mutation yielded a strain producing ethanol as the sole fermentation product. As confirmation of the nuo effect, cultures of the ac kA-pta strain, supplemented with an NDH-I inhibitor, produced intermediary levels of flux to ethanol and formate. Mutations in both ackA-pta and nuo a re required to significantly reduce the flux through the PFL pathway. (C) 1 999 John Wiley & Sons, Inc.