Integration of an [FeFe]-hydrogenase into the anaerobic metabolism of Escherichia coli.
Integration of an [FeFe]-hydrogenase into the anaerobic metabolism of Escherichia coli.
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DOI：
10.1016/j.btre.2015.10.002
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发表时间：
2015-12
期刊：
影响因子：
--
通讯作者：
Sargent F
中科院分区：
文献类型：
--
作者：
Kelly CL;Pinske C;Murphy BJ;Parkin A;Armstrong F;Palmer T;Sargent F
工程微生物生物制氢可能具有生物技术应用。设计了一种编码NADH连接的[FeFe]-氢化酶的合成操纵子。该酶是异源产生、激活和表征的。铁氧还蛋白和丙酮酸氧化还原酶的加入是必要的体内活性。生物制氢是微生物能量代谢的一种潜在有用产物。在细菌中工程化生物氢生产的一种方法是在宿主细胞例如大肠杆菌中产生非天然氢化酶活性。在一些微生物中，氢化酶通过利用NAD+/NADH辅因子的心肌黄酶直接与中心代谢相关联。在这项工作中，假设异源产生的NAD+/NADH连接的氢化酶可以连接E.大肠杆菌宿主直接参与其中枢代谢。为了测试这一点，设计了一种合成操纵子，其特征在于编码一种来自Caldanaerodynus subarcus的明显依赖于NADH的氢释放[FeFe]-氢化酶。合成的操纵子稳定整合到大肠杆菌中。大肠杆菌染色体，并显示产生活性氢化酶，但没有观察到H2的生产。随后，发现来自海栖热袍菌的丙酮酸盐：：铁氧还蛋白氧化还原酶和铁氧还蛋白的异源共生产对于通过该系统驱动H2生产是必需的。这项工作提供了遗传学证据，证明Ca. subarcus [FeFe]-氢化酶可能在体内作为电子聚集酶起作用。
Engineering microbial biohydrogen production may have biotechnological applications. A synthetic operon encoding an NADH-linked [FeFe]-hydrogenase was designed. The enzyme was heterologously produced, activated and characterised. The addition of ferredoxin and pyruvate oxidoreductase was necessary for in vivo activity. Biohydrogen is a potentially useful product of microbial energy metabolism. One approach to engineering biohydrogen production in bacteria is the production of non-native hydrogenase activity in a host cell, for example Escherichia coli. In some microbes, hydrogenase enzymes are linked directly to central metabolism via diaphorase enzymes that utilise NAD+/NADH cofactors. In this work, it was hypothesised that heterologous production of an NAD+/NADH-linked hydrogenase could connect hydrogen production in an E. coli host directly to its central metabolism. To test this, a synthetic operon was designed and characterised encoding an apparently NADH-dependent, hydrogen-evolving [FeFe]-hydrogenase from Caldanaerobacter subterranus. The synthetic operon was stably integrated into the E. coli chromosome and shown to produce an active hydrogenase, however no H2 production was observed. Subsequently, it was found that heterologous co-production of a pyruvate::ferredoxin oxidoreductase and ferredoxin from Thermotoga maritima was found to be essential to drive H2 production by this system. This work provides genetic evidence that the Ca.subterranus [FeFe]-hydrogenase could be operating in vivo as an electron-confurcating enzyme.