title: Effect of inactivation of nuo and ackA-pta on redistribution of metabolic fluxes in Escherichia coli.
abstract: The nuoA-N gene cluster encodes a transmembrane NADH:ubiquinone oxidoreductase (NDH-I) responsible for coupling redox chemistry to proton-motive force generation. Interactions between nuo and the acetate-producing pathway encoded by ackA-pta were investigated by examining the metabolic patterns of several mutant strains under anaerobic growth conditions. In an ackA-pta strain, the flux to acetate was decreased dramatically, whereas flux to lactate was increased significantly when compared with its parent strain; the fluxes to pyruvate and ethanol also increased slightly. In addition, pyruvate was excreted. A strain carrying the nuo mutation showed metabolic flux distribution similar to the wild type. The ackA-pta-nuo strain showed a different metabolic pattern. It not only exhibited reduced acetate accumulation but also significantly lower ethanol and formate synthesis. Metabolic flux distribution 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-CoA and formate production. The diminished capacity through the formate and ethanol (ADH) pathways was not the result of genetic disruption of functional PFL or ADH production. The introduction of a Bacillus subtilis acetolactate synthase gene returned formate, ethanol, and lactate levels to those of the wild type (ackA(+)pta(+)nuo(+)) strain. Furthermore, transfer of a lactate dehydrogenase mutation yielded a strain producing ethanol as the sole fermentation product. As confirmation of the nuo effect, cultures of the ackA-pta strain, supplemented with an NDH-I inhibitor, produced intermediary levels of flux to ethanol and formate. Mutations in both ackA-pta and nuo are required to significantly reduce the flux through the PFL pathway.
authors.affiliation: Departments of Bioengineering and Chemical Engineering, Rice University, PO Box 1892, MS 142, Houston, Texas 77251-1892, USA.
authors.forename: Y T
authors.initials: YT
authors.lastname: Yang
authors.forename: G N
authors.initials: GN
authors.lastname: Bennett
authors.forename: K Y
authors.initials: KY
authors.lastname: San
mesh_terms: D042967:Electron Transport Complex I; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000378:metabolism*; D005285:Fermentation; D005810:Multigene Family; D009154:Mutation; D009247:NADH, NADPH Oxidoreductases / Q000037:antagonists & inhibitors / Q000235:genetics*
chemical_list: D004791:Enzyme Inhibitors; D009247:NADH, NADPH Oxidoreductases; D042967:Electron Transport Complex I