Biological nitrogen fixation by nitrogenase has attracted attention as an alternative method to chemical nitrogen fixation, which requires large amounts of fossil fuels. Azotobacter vinelandii, which produces an oxygen-sensitive nitrogenase, can fix nitrogen even under aerobic conditions; therefore, the heterologous expression of nif-related genes from A. vinelandii is a promising strategy for developing a biological nitrogen fixation method. We assembled 17 nif-related genes, which are scattered throughout the genome of A. vinelandii, into synthetic gene clusters by overlap-extension-PCR and seamless cloning and expressed them in Escherichia coli. The transcription and translation of the 17 nif-related genes were evaluated by RT-qPCR and LC-MS/MS, respectively. The constructed E. coli showed nitrogenase activity under anaerobic and microaerobic conditions. This strain would be a useful model for examining the effect of other genes from A. vinelandii on nitrogen fixation by expressing them in addition to the minimal set of nif-related genes.

中文翻译：

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从 Azotobacter vinelandii 构建生产固氮酶相关蛋白的重组大肠杆菌。

固氮酶的生物固氮作为需要大量化石燃料的化学固氮的替代方法而受到关注。Azotobacter vinelandii 产生一种对氧敏感的固氮酶，即使在有氧条件下也能固氮；因此，来自 A. vinelandii 的 nif 相关基因的异源表达是开发生物固氮方法的有前途的策略。我们通过重叠延伸 PCR 和无缝克隆将 17 个分散在 A. vinelandii 基因组中的 nif 相关基因组装成合成基因簇，并在大肠杆菌中表达。分别通过 RT-qPCR 和 LC-MS/MS 评估了 17 个 nif 相关基因的转录和翻译。构造的 E。大肠杆菌在厌氧和微需氧条件下均表现出固氮酶活性。除了最小的 nif 相关基因外，该菌株将成为检测来自 A. vinelandii 的其他基因对固氮作用的有用模型。