1. Construction of recombinant Escherichia coli producing nitrogenase-related proteins from Azotobacter vinelandii.
- Author
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Tatemichi, Yuki, Nakahara, Takeharu, Ueda, Mitsuyoshi, and Kuroda, Kouichi
- Subjects
NITROGEN fixation ,AZOTOBACTER ,ESCHERICHIA coli ,SYNTHETIC genes ,MOLECULAR cloning - Abstract
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. [ABSTRACT FROM AUTHOR]
- Published
- 2021
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