Yang Y T, Bennett G N, San K Y
Departments of Bioengineering and Chemical Engineering, Rice University, PO Box 1892, MS 142, Houston, Texas 77251-1892, USA.
Biotechnol Bioeng. 1999 Nov 5;65(3):291-7.
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.
nuoA - N基因簇编码一种跨膜NADH：泛醌氧化还原酶（NDH - I），负责将氧化还原化学与质子动力产生相偶联。通过检测几种突变菌株在厌氧生长条件下的代谢模式，研究了nuo与由ackA - pta编码的乙酸生成途径之间的相互作用。在ackA - pta菌株中，与亲本菌株相比，乙酸通量显著降低，而乳酸通量显著增加；丙酮酸和乙醇通量也略有增加。此外，丙酮酸被分泌。携带nuo突变的菌株显示出与野生型相似的代谢通量分布。ackA - pta - nuo菌株表现出不同的代谢模式。它不仅乙酸积累减少，而且乙醇和甲酸合成也显著降低。代谢通量分布分析表明，过量的碳通量在丙酮酸节点处通过乳酸脱氢酶途径重新定向用于乳酸形成，而不是通过丙酮酸甲酸裂解酶（PFL）途径用于乙酰辅酶A和甲酸生成。通过甲酸和乙醇（ADH）途径能力的降低不是功能性PFL或ADH产生的基因破坏的结果。引入枯草芽孢杆菌乙酰乳酸合酶基因使甲酸、乙醇和乳酸水平恢复到野生型（ackA(+)pta(+)nuo(+)）菌株的水平。此外，转移乳酸脱氢酶突变产生了一种以乙醇作为唯一发酵产物的菌株。作为对nuo效应的证实，用NDH - I抑制剂补充的ackA - pta菌株培养物产生了中等水平的乙醇和甲酸通量。ackA - pta和nuo两者的突变都需要才能显著降低通过PFL途径的通量。
