Abstract
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.
Highlights
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
The nafU overexpression did not affect the growth of nitrogenase-producing E. coli (Fig. S7). These results indicate that nafU is effective for maintaining nitrogenase activity under aerobic conditions in E. coli, suggesting that nafU is a useful gene for heterologous expression of nitrogenase under aerobic conditions in other microorganisms
We focused on the most upregulated gene, nafU with a previously unknown function[38], and revealed that this gene contributes to the maintenance of nitrogenase activity under aerobic conditions in A. vinelandii
Summary
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. 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. The protection of nitrogenase by the respiratory system is limited to low dissolved oxygen concentrations, and the consumption of oxygen at the cell surface is not very effective[30,31] These three mechanisms alone may not fully explain how A. vinelandii maintains high nitrogenase activity under aerobic conditions, suggesting the presence of additional important factors. The identification of such factors would advance the study of A. vinelandii, and lead to increased nitrogenase activity in heterologous expression under aerobic conditions since it is possible to introduce additional factors into the heterologous host of nitrogenase[32]
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