Elimination of Rubisco alters the regulation of nitrogenase activity and increases hydrogen production in Rhodospirillum rubrum

Nitrogenase not only reduces atmospheric nitrogen to ammonia, but also reduces protons to hydrogen (H2). The nitrogenase system is the primary means of H2 production under photosynthetic and nitrogen-limiting conditions in many photosynthetic bacteria, including Rhodospirillum rubrum. The efficiency of this biological H2 production largely depends on the nitrogenase enzyme and the availability of ATP and electrons in the cell. Previous studies showed that blockage of the CO2 fixation pathway in R. rubrum induced nitrogenase activity even in the presence of ammonium, presumably to remove excess reductant in the cell. We report here the re-characterization of cbbM mutants in R. rubrum to study the effect of Rubisco on H2 production. Our newly constructed cbbM mutants grew poorly in malate medium under anaerobic conditions. However, the introduction of constitutively active NifA (NifA*), the transcriptional activator of the nitrogen fixation (nif) genes, allows cbbM mutants to dissipate the excess reductant through the nitrogenase system and improves their growth. Interestingly, we found that the deletion of cbbM alters the posttranslational regulation of nitrogenase activity, resulting in partially active nitrogenase in the presence of ammonium. The combination of mutations in nifA, draT and cbbM greatly increased H2 production of R. rubrum, especially in the presence of excess of ammonium. Furthermore, these mutants are able to produce H2 over a much longer time frame than the wild type, increasing the potential of these recombinant strains for the biological production of H2.