Production of biohydrogen by recombinant expression of [NiFe]- hydrogenase 1 in Escherichia coli

Background: Hydrogenases catalyze reversible reaction between hydrogen (H-2) and proton. Inactivation of hydrogenase by exposure to oxygen is a critical limitation in biohydrogen production since strict anaerobic conditions are required. While [FeFe]-hydrogenases are irreversibly inactivated by oxygen, it was known that [NiFe]-hydrogenases are generally more tolerant to oxygen. The physiological function of [NiFe]-hydrogenase 1 is still ambiguous. We herein investigated the H-2 production potential of [NiFe]-hydrogenase 1 of Escherichia coli in vivo and in vitro. The hyaA and hyaB genes corresponding to the small and large subunits of [NiFe]-hydrogenase 1 core enzyme, respectively, were expressed in BL21, an E. coli strain without H-2 producing ability. Results: Recombinant BL21 expressing [NiFe]-hydrogenase 1 actively produced H-2 (12.5 mL H-2/(h center dot L) in 400 mL glucose minimal medium under micro-aerobic condition, whereas the wild type BL21 did not produce H-2 even when formate was added as substrate for formate hydrogenlyase (FHL) pathway. The majority of recombinant protein was produced as an insoluble form, with translocation of a small fraction to the membrane. However, the membrane fraction displayed high activity (similar to 65% of total cell fraction), based on unit protein mass. Supplement of nickel and iron to media showed these metals contribute essentially to the function of [NiFe]-hydrogenase 1 as components of catalytic site. In addition, purified E. coli [NiFe]-hydrogenase 1 using his(6)-tag displayed oxygen-tolerant activity of similar to 12 nmol H-2/(min center dot mg protein) under a normal aeration environment, compared to [FeFe]-hydrogenase, which remains inactive under this condition. Conclusions: This is the first report on physiological function of E. coli [NiFe]-hydrogenase 1 for H-2 production. We found that [NiFe]-hydrogenase 1 has H-2 production ability even under the existence of oxygen. This oxygen-tolerant property is a significant advantage because it is not necessary to protect the H-2 production process from oxygen. Therefore, we propose that [NiFe]-hydrogenase can be successfully applied as an efficient biohydrogen production tool under micro-aerobic conditions.