(194v) Characterization and Heterologous Expression of Iron Hydrogenase Ethha_0031 of Ethanoligenense harbinense in E. coli Blr(DE3)

Hydrogenase is a critical enzyme in hydrogen evolution. Finding hydrogenases with high ability to evolve hydrogen will make big significance for the improvement of fermentative hydrogen production. Ethanoligenens harbinense is a new hydrogen-producing bacteria isolated with high hydrogen production rate of 2.81 mole hydrogen per mole glucose. Therefore, the hydrogenases of Ethanoligenens harbinense has attracted wide attention.

In this study, iron hydrogenase Ethha_0031 (hyd1) was successfully cloned from the genome of Ethanoligenens harbinense Yuan-3^T. Hyd1 encodes 418 amino acids and is predicted to own a molecular weight of 45.91 kilodaltons. The amino acid sequence of hyd1 presents 63% identity and 58% identity with the iron hydrogenases of Clostridium cellulosi and Desulfosporosinus sp. OT, respectively. Hyd1 is a monomer consisting of four [4Fe-4S] clusters, one [2Fe-2S] cluster and one [2Fe-2S-5O-2H2O] cluster. It has a conserved domain named NuoI super family which is subunit of formate hydrogenlyase /NADH: ubiquinone oxidoreductase.

Hyd 1 gene fragment was inserted after T7 promoter to construct a recombinant plasmid pBENT-hyd1. The plasmid was then transferred into E. coli BLR(DE3), confirmed by resistance screening and PCR. SDS-PAGE confirmed the high-level expression of hyd 1. The mutants acquired the ability to evolve hydrogen as compared to the wild BLR(DE3). Furthermore, hydrogenase activity assay was tested and confirmed that hyd1 showed a high ability to evolve hydrogen in mutant. This research is a new addition to the existing knowledge for the construction of hydrogen-producing engineered strains.