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.

由rnfABCDGEH基因编码的Rnf（红细菌固氮）复合物是发挥固氮酶功能的关键酶。在这项工作中，我们通过用三个自发启动子表达rnf基因，研究了Rnf复合物表达水平对产氢细菌荚膜红细菌SB 1003中氢产生的影响。所述的取代RNF启动子与该的pucB导致一个1.59倍的增加RNF转录水平和在固氮酶活1.3倍的增加。在各种测试条件下，最大H ₂产量和生产率分别提高了12.4％–24.7％和6.5％–22.9％。当P _rnf :: P _pucB :: rnf融合构建体在背景菌株中表达时，Fe 2脱氮酶和Mo固氮酶同时表达，H ₂产量进一步增加。结果表明，Rnf络合物催化光发酵H ₂产生中的限速步骤。在弱光条件下或按比例放大的生物反应器中，Rnf复合物过表达的突变体比天然分离的菌株更具竞争力。