Improvement of hydrogen production by over-expression of a hydrogen-promoting protein gene in Enterobacter cloacae

The gene of a hydrogen-promoting protein (which we term HPP) from Enterobacter cloacae IIT-BT 08 was cloned and over-expressed in E. cloacae CICC10017 for the first time in this study, and the overall hydrogen yield was greatly improved using the recombinant strain. A recombinant plasmid containing the gene in-frame with Glutathione-S-Transferase (GST) gene was transformed into a hydrogen producing strain of E. cloacae CICC10017 to produce a GST-fusion protein. SDS-PAGE and western blot analysis confirm the successful expression of the GST-tagged protein. An in vitro assay of cell lysates indicates hydrogenase activity of the recombinant strain is 534.78 ± 18.51 ml/(g-DW·h), nearly 2-fold higher than the wild strain. The hydrogen yield of the recombinant strain is 2.55 ± 0.1 mol/mol-glucose, also 2-fold higher than the wild strain. The recombinant strain produces more acetate and butyrate during hydrogen fermentation, but less ethanol, due to the higher hydrogenase activity with the over-expression of the hydrogen-promoting protein. Together, the results demonstrate that successful expression of a single structural gene improves the overall yield of hydrogen by directing metabolic fluxes away from formation of products that compete for NADH.