Promoting photo-fermentative hydrogen production performance by substituting the rnf promoter in Rhodobacter capsulatus

The Rnf (Rhodobacter nitrogen fixation) complex encoded by rnfABCDGEH genes is a crucial enzyme for the functioning of nitrogenase. In this work, we studied the effect of Rnf complex expression level on hydrogen production in the hydrogen-producing bacterium Rhodobacter capsulatus SB 1003 by expressing rnf genes with three autologous promoters. Substitution of the rnf promoter with that of pucB resulted in a 1.59-fold increase in rnf transcription level and 1.3-fold increase in nitrogenase activity. Under various test conditions, maximum H₂ yields and production rates were increased by 12.4%–24.7% and 6.5%–22.9%, respectively. There was a further increase in H₂ production when the P_rnf::P_pucB::rnf fusion construct was expressed in a background strain with both the alternative Fe nitrogenase and the Mo nitrogenase co-expressed. The results demonstrate that the Rnf complex catalyzes a rate-limiting step in photo-fermentative H₂ production. The mutants with overexpressed Rnf complex are more competitive than naturally isolated strains under low light condition or in scale-up bioreactors.