Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kitashirakawa Oiwake-cho, Sakyo-ku, Kyoto, 606-8502, Japan.
Research and Development Division, Kikkoman Corporation, 338 Noda, Noda, Chiba, 278-0037, Japan.
Sci Rep. 2022 Mar 9;12(1):4182. doi: 10.1038/s41598-022-08007-4.
Since nitrogenase is irreversibly inactivated within a few minutes after exposure to oxygen, current studies on the heterologous expression of nitrogenase are limited to anaerobic conditions. This study comprehensively identified genes showing oxygen-concentration-dependent expression only under nitrogen-fixing conditions in Azotobacter vinelandii, an aerobic diazotroph. Among the identified genes, nafU, with an unknown function, was greatly upregulated under aerobic nitrogen-fixing conditions. Through replacement and overexpressing experiments, we suggested that nafU is involved in the maintenance of nitrogenase activity under aerobic nitrogenase activity. Furthermore, heterologous expression of nafU in nitrogenase-producing Escherichia coli increased nitrogenase activity under aerobic conditions by 9.7 times. Further analysis of NafU protein strongly suggested its localization in the inner membrane and raised the possibility that this protein may lower the oxygen concentration inside the cells. These findings provide new insights into the mechanisms for maintaining stable nitrogenase activity under aerobic conditions in A. vinelandii and provide a platform to advance the use of nitrogenase under aerobic conditions.
由于固氮酶在暴露于氧气后几分钟内会不可逆失活，因此目前关于固氮酶的异源表达研究仅限于厌氧条件下进行。本研究全面鉴定了在好氧固氮菌根瘤菌中仅在固氮条件下表现出氧浓度依赖性表达的基因。在所鉴定的基因中，具有未知功能的 nafU 在好氧固氮条件下被强烈上调。通过替换和过表达实验，我们表明 nafU 参与了好氧固氮条件下维持固氮酶活性。此外，在产固氮酶的大肠杆菌中异源表达 nafU 可使固氮酶活性在好氧条件下提高 9.7 倍。对 NafU 蛋白的进一步分析强烈表明其定位于内膜上，并提出了该蛋白可能降低细胞内氧气浓度的可能性。这些发现为根瘤菌在好氧条件下维持稳定固氮酶活性的机制提供了新的见解，并为在好氧条件下使用固氮酶提供了一个平台。
