基于黄素的电子歧化在Clostridium autoethanogenum发酵合成气能量代谢中作用的研究

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.

"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。

C. autoethanogenum可发酵合成气生成乙酸、乙醇和2,3-丁二醇等化合物，是发酵合成气生产燃料和化学品的重要菌株，具有重要的工业应用价值。然而其经Wood-Ljungdahl途径发酵CO和固定CO2的生理代谢尤其是代谢中涉及的能量产生机理还不完全清楚。本项目首先对CO培养的C. autoethanogenum粗酶液中的氧化还原酶活力进行了详细调查，结合基因组分析，发现除了前期已经纯化鉴定的新型 “同时依赖NADP和Fd的氢化酶活力和甲酸脱氢酶复合物（HytA-E/Fdh）”外，还发现预期的电子歧化的NADH依赖的还原型铁氧还蛋白:NADP+氧化还原酶（Nfn）、 [FeFe]-氢化酶（[FeFe]-hyd-nuoF-nuoE）、亚甲基四氢叶酸还原酶（MetFV）、乳酸脱氢酶/EtfAB复合物（Ldh/EtfAB）、能量转换（engery-converting）的膜蛋白还原型铁氧还蛋白:NAD+氧化还原酶（Rnf复合物）和ATP合成酶（ATPase）等能量代谢关键酶。进一步经过异源表达纯化和生化测试，详尽研究了电子歧化的Nfn、MetFV和Ldh/EtfAB的生化性质，鉴定了它们催化的反应和生理功能；并对相关关键酶进行了转录水平分析。进一步综合分析 了C. autoethanogenum发酵CO过程中涉及的电子传递和能量产生问题，提出了新的观点：C. autoethanogenum能量代谢除底物水平磷酸化外，基于黄素的电子歧化酶和Rnf复合物等氧化还原酶组成的新型电子传递系统通过与ATPase的偶联来合成ATP是该菌能量产生的主要途径。在该过程中，CO氧化释放的电子经过CODH、Rnf复合物、电子歧化酶Nfn和HytA-E/Fdh以及Wood-Ljungdahl途径的相关酶的传递，用来固定CO2和合成醇化合物，同时推动Rnf向膜外侧泵出质子形成了跨膜电势差，经ATPase的偶联合成ATP。电子歧化的Ldh/EtfAB则形成了一个D-乳酸代谢循环用于解除高能荷时CO的还原压力和毒性。这不同于已有的微生物产能方式，因此电子歧化酶与Rnf等形成的电子传递与ATPase偶联的产能模式成为一种新的产能模式。本项目为理解自养产乙酸菌的基础代谢提供了新的认识，为菌株的进一步代谢工程改造提供了新的理论依据。