Wang Di, Zhang Yaoping, Welch Emily, Li Jilun, Roberts Gary P
State Key Laboratory for Agrobiotechnology and Department of Microbiology and Immunology, College of Biological Sciences, China Agricultural University, Beijing 100193, P. R. China.
Int J Hydrogen Energy. 2010 Jul 1;35(14):7377-7385. doi: 10.1016/j.ijhydene.2010.04.183.
Nitrogenase not only reduces atmospheric nitrogen to ammonia, but also reduces protons to hydrogen (H(2)). The nitrogenase system is the primary means of H(2) production under photosynthetic and nitrogen-limiting conditions in many photosynthetic bacteria, including Rhodospirillum rubrum. The efficiency of this biological H(2) production largely depends on the nitrogenase enzyme and the availability of ATP and electrons in the cell. Previous studies showed that blockage of the CO(2) 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 H(2) 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 H(2) production of R. rubrum, especially in the presence of excess of ammonium. Furthermore, these mutants are able to produce H(2) over a much longer time frame than the wild type, increasing the potential of these recombinant strains for the biological production of H(2).
固氮酶不仅能将大气中的氮气还原为氨，还能将质子还原为氢气（H₂）。在包括深红红螺菌在内的许多光合细菌中，固氮酶系统是在光合和氮限制条件下产生H₂的主要方式。这种生物制氢的效率很大程度上取决于固氮酶以及细胞中ATP和电子的可用性。先前的研究表明，深红红螺菌中二氧化碳固定途径的阻断即使在有铵存在的情况下也能诱导固氮酶活性，推测这是为了去除细胞中多余的还原剂。我们在此报告对深红红螺菌中cbbM突变体的重新表征，以研究核酮糖-1,5-二磷酸羧化酶/加氧酶（Rubisco）对产氢的影响。我们新构建的cbbM突变体在厌氧条件下的苹果酸培养基中生长不佳。然而，引入组成型活性的固氮基因转录激活因子NifA（NifA*）可使cbbM突变体通过固氮酶系统消耗多余的还原剂并改善其生长。有趣的是，我们发现cbbM的缺失改变了固氮酶活性的翻译后调控，导致在有铵存在的情况下固氮酶部分活化。nifA、draT和cbbM中的突变组合极大地提高了深红红螺菌的产氢量，尤其是在铵过量的情况下。此外，这些突变体能够比野生型在更长的时间内产氢，增加了这些重组菌株生物制氢的潜力。
