Improvement of hydrogen yield at hydrogen fermentation is expected for hydrogen society. Enterobacter aerogenes, a facultative anaerobic bacterium, performs hydrogen-fermentation under anaerobic condition and produces 1 mol-H₂ from 1 mol-glucose. E.aerogenes evolves hydrogen from NADH through membrane-bound hydrogenase. The reaction is like follows; NADH+H⁺→NAD⁺+H₂. Therefore the more NADH remains unused for the metabolite production, the more hydrogen is evolved. Maximum NADH production is only 4 moles under anaerobic condition. But under aerobic condition 10 mol-NADH is produced. These NADH are re-oxidized by O₂ and H₂O is produced at electron transport chain under aerobic condition. This reaction is catalyzed by ComplexI. Inhibition of ComplexI is expected to increase unused NADH, and hydrogen yield will be able to increase to 10 mol-H₂/mol-glucose. In study of Escherichia coli, ComplexI is encoded by 14 structural genes from nuoA to nuoN. In particular, nuoG is essential for the function of NADH re-oxidization in ComplexI. In this study, we decided nuoG as the target gene and planned to break down the gene by homologous DNA recombination. At this moment, gene disruption cassette which contains homologous region of the target gene was constructed by means of the fusion PCR method and gene disruption vector was constructed by TAcloning method. Heat-shock method was carried out to shot gene disruption vector into E.aerogenes. Some colonies were obtained but unfortunately any mutant which is improved in hydrogen yield has not been obtained yet.