{"title":"Characterization of the Azotobacter vinelandii nitrogenase complex expressed in Escherichia coli toward further activity improvement.","authors":"Yusuke Ito, Daisuke Yoshidome, Makoto Hidaka, Yasuko Araki, Kotaro Ito, Saori Kosono, Makoto Nishiyama","doi":"10.2323/jgam.2024.12.001","DOIUrl":null,"url":null,"abstract":"<p><p>We previously constructed an Escherichia coli strain expressing 16 nitrogen fixation (nif) and 2 nif-related genes from Azotobacter vinelandii and improved nitrogenase activity to some extent by enhancing NifH-related functions. In the present study, we analyzed the formation of dinitrogenase, a heterotetrameric NifD<sub>2</sub>K<sub>2</sub>, produced in E. coli, using gel-filtration chromatography and blue native PAGE to gain insight into further increases in nitrogenase activity. A certain proportion of NifD and NifK proteins produced in E. coli were present as the complete NifD<sub>2</sub>K<sub>2</sub> component, but some remained in the intermediate stages of maturation. Overexpression of nafY, which is involved in holo-NifD<sub>2</sub>K<sub>2</sub> formation, effectively increased nitrogenase activity.</p>","PeriodicalId":15842,"journal":{"name":"Journal of General and Applied Microbiology","volume":" ","pages":""},"PeriodicalIF":0.8000,"publicationDate":"2025-01-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Journal of General and Applied Microbiology","FirstCategoryId":"99","ListUrlMain":"https://doi.org/10.2323/jgam.2024.12.001","RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q4","JCRName":"BIOTECHNOLOGY & APPLIED MICROBIOLOGY","Score":null,"Total":0}
引用次数: 0
Abstract
We previously constructed an Escherichia coli strain expressing 16 nitrogen fixation (nif) and 2 nif-related genes from Azotobacter vinelandii and improved nitrogenase activity to some extent by enhancing NifH-related functions. In the present study, we analyzed the formation of dinitrogenase, a heterotetrameric NifD2K2, produced in E. coli, using gel-filtration chromatography and blue native PAGE to gain insight into further increases in nitrogenase activity. A certain proportion of NifD and NifK proteins produced in E. coli were present as the complete NifD2K2 component, but some remained in the intermediate stages of maturation. Overexpression of nafY, which is involved in holo-NifD2K2 formation, effectively increased nitrogenase activity.
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JGAM is going to publish scientific reports containing novel and significant microbiological findings, which are mainly devoted to the following categories: Antibiotics and Secondary Metabolites; Biotechnology and Metabolic Engineering; Developmental Microbiology; Environmental Microbiology and Bioremediation; Enzymology; Eukaryotic Microbiology; Evolution and Phylogenetics; Genome Integrity and Plasticity; Microalgae and Photosynthesis; Microbiology for Food; Molecular Genetics; Physiology and Cell Surface; Synthetic and Systems Microbiology.
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