title: Mutational analysis of the hyc-operon determining the relationship between hydrogenase-3 and NADH pathway in Enterobacter aerogenes.
abstract: In this study, the hydrogenase-3 gene cluster (hycDEFGH) was isolated and identified from Enterobacter aerogenes CCTCC AB91102. All gene products were highly homologous to the reported bacterial hydrogenase-3 (Hyd-3) proteins. The genes hycE, hycF, hycG encoding the subunits of hydrogenase-3 were targeted for genetic knockout to inhibit the FHL hydrogen production pathway via the Red recombination system, generating three mutant strains AB91102-E (ΔhycE), AB91102-F (ΔhycF) and AB91102-G (ΔhycG). Deletion of the three genes affected the integrity of hydrogenase-3. The hydrogen production experiments with the mutant strains showed that no hydrogen was detected compared with the wild type (0.886 mol/mol glucose), demonstrating that knocking out any of the three genes could inhibit NADH hydrogen production pathway. Meanwhile, the metabolites of the mutant strains were significantly changed in comparison with the wild type, indicating corresponding changes in metabolic flux by mutation. Additionally, the activity of NADH-mediated hydrogenase was found to be nil in the mutant strains. The chemostat experiments showed that the NADH/NAD(+) ratio of the mutant strains increased nearly 1.4-fold compared with the wild type. The NADH-mediated hydrogenase activity and NADH/NAD(+) ratio analysis both suggested that NADH pathway required the involvement of the electron transport chain of hydrogenase-3.
authors.affiliation: Key Laboratory of Molecular Biophysics, the Ministry of Education, Huazhong University of Science and Technology, Wuhan 430074, PR China; College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan 430074, PR China.
authors.forename: Jian
authors.initials: J
authors.lastname: Pi
authors.affiliation: Key Laboratory of Molecular Biophysics, the Ministry of Education, Huazhong University of Science and Technology, Wuhan 430074, PR China; College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan 430074, PR China.
authors.forename: Muhammad
authors.initials: M
authors.lastname: Jawed
authors.affiliation: Key Laboratory of Molecular Biophysics, the Ministry of Education, Huazhong University of Science and Technology, Wuhan 430074, PR China; College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan 430074, PR China.
authors.forename: Jun
authors.initials: J
authors.lastname: Wang
authors.affiliation: Key Laboratory of Molecular Biophysics, the Ministry of Education, Huazhong University of Science and Technology, Wuhan 430074, PR China; College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan 430074, PR China. Electronic address: xuli@hust.edu.cn.
authors.forename: Li
authors.initials: L
authors.lastname: Xu
authors.affiliation: Key Laboratory of Molecular Biophysics, the Ministry of Education, Huazhong University of Science and Technology, Wuhan 430074, PR China; College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan 430074, PR China. Electronic address: yanyunjun@hust.edu.cn.
authors.forename: Yunjun
authors.initials: Y
authors.lastname: Yan
mesh_terms: D000693:Anaerobiosis; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D056804:Biofuels; D002462:Cell Membrane / Q000201:enzymology; D004579:Electron Transport; D021902:Enterobacter aerogenes / Q000201:enzymology / Q000235:genetics*; D005285:Fermentation; D005560:Formate Dehydrogenases / Q000378:metabolism; D055786:Gene Knockout Techniques; D005798:Genes, Bacterial; D006859:Hydrogen / Q000378:metabolism*; D006864:Hydrogenase / Q000235:genetics* / Q000378:metabolism; D053858:Metabolic Networks and Pathways; D009097:Multienzyme Complexes / Q000378:metabolism; D009243:NAD / Q000378:metabolism*; D009876:Operon / Q000235:genetics*; D010084:Oxidation-Reduction; D021122:Protein Subunits / Q000172:deficiency / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011995:Recombination, Genetic
chemical_list: D001426:Bacterial Proteins; D056804:Biofuels; D009097:Multienzyme Complexes; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins; D009243:NAD; D006859:Hydrogen; D006864:Hydrogenase; D005560:Formate Dehydrogenases; C054017:formate hydrogenlyase
keywords: Enterobacter aerogenes; Formate hydrogen lyase (FHL); Knockout