"基于黄素的电子歧化"是近年在厌氧微生物中发现的一种全新的节能方式。该类电子歧化酶结合黄素，催化一个放能反应和一个吸能反应的偶联反应。Clostridium autoethanogenum已被用来发酵合成气（主成分为CO）产燃料乙醇，但其能量代谢关键机理尚不明晰，亟待回答。申请人前期对其基因组和粗酶液调查、两个电子歧化酶"依赖铁氧还蛋白的转氢酶（Nfn）"和"同时依赖NADP和铁氧还蛋白的甲酸脱氢酶-氢化酶复合物（Hyt/Fdh）"研究显示，其能量代谢不同于产乙酸模式菌，可能经Nfn和Hyt/Fdh等的电子歧化与能量转换膜蛋白Rnf联合作用来产能。本项目据此假设拟在前期工作基础上深入研究挖掘相关电子歧化酶，尤其是预期为新的电子歧化酶亚甲基四氢叶酸还原酶，敲除关键电子歧化酶基因，从生化和转录水平揭示它们在CO发酵中的角色，阐明电子歧化在该菌能量代谢中的作用，为合成气发酵技术提供坚实依据。

Flavin-based electron bifurcation is a novel mode of energy conservation discovered recently in anaerobic bacteria and archaea. This type of electron bifurcating enzymes contain flavin and catalyze an exergonic reaction coupling with an endergonic reaction. Acetogen Clostridium autoethanogenum has been used for fermenting syngas (CO as main component) to produce fuel ethanol. However, the key mechanism of energy metabolism involved in the CO fermentation in the bacterium is still not well understood. We have done the camparative analysis of several acetogens' genomes, investigated the oxidoreductases in the crude extracts of CO-growing Clostridium autoethanogenum, and characterized two electron-bifurcating enzyems: ferredoxin-dependent transhydrogenase (NfnAB) and NADP- and ferredoxin- dependent formate dehydrogenase-[FeFe]-hydrogenase complex (HytA-E/Fdh). The results indicate that Clostridium autoethanogenum represents a new type of energy metabolism in acetogens, which is different from the model acetogens. We predicts that the energy in Clostridium autoethanogenum is mainly produced by coupling of electron bifurcation catalyzed by enzymes such as Nfn and HytA-E/Fdh to the membrane-bound energy-converting enzyme Rnf. In this proposal, we make a plan based on the hypothesis to continue to characterize the biochemical properties of the related electron-bifurcating oxidoreductase, especially methylene-tetrahydrofolate reductase, which is expected to be another electron-bifurcating enzyme for energy conservation in the bacterium. In addition, we would disrupt the genes of key electron-bifurcating enzymes and investigate the effects of the disruption at both transcriptional and biochemical levels to explore their roles in the energy metabolism. By this way, we will elucidate the function of the electron bifurcation in the syngas fermentation by Clostridium autoethanogenum, and provide new insights and biological basis for the syngas fermation technology.