类芽孢杆菌中固氮酶电子传递途径的鉴定和功能分析

电子传递在生物固氮过程中起着非常重要作用。但类芽孢杆菌中固氮酶的电子传递途径尚不清楚。我们通过转录组学发现一些电子传递基因在固氮条件下表达显著上调，其中fer和fldA（编码黄素氧还蛋白）及pfoAB（编码丙酮酸:铁氧化还原酶）能提高重组大肠杆菌78-7（携带类芽孢杆菌固氮基因簇nifBHDKENXhesAnifV）的固氮酶活性。但fldA和fer编码的黄素氧还蛋白是固氮酶的直接电子供体吗？pfoAB编码的丙酮酸:铁氧化还原酶直接为固氮酶产生电子吗？在固氮条件下表达显著上调的flr（黄素氧蛋白）及yqjM3、yqjM5、yqiG（NADH:黄素氧化还原酶）在固氮酶的电子传递中有什么功能呢？是否还有更能提高固氮效率的电子传递基因呢？有几个电子传递途径呢？本项目拟通过基因突变、电子传递功能鉴定等，阐明类芽孢杆菌中固氮酶的电子传递途径，研究结果对提高固氮效率、扩大宿主范围等都具有重要意义。

The reduction of N2 to two ammonium molecules by nitrogenase requires eight electrons and eight protons. Thus, the electron transport pathway to nitrogenase plays a very important role in nitrogen fixation. Generally, oxidoreductase generates electrons by degrading carbohydrate and then flavodoxin or ferredoxin mediates electron to nitrogenase. However, the electron transport pathway in N2-fixing Paenibacillus is unknown. Our previous studies on a global transcriptional profiling analysis of Paenibacillus polymyxa WLY78 demonstrated that some genes encoding electron transporters were highly up-regulated in N2-fixing condition compared to non-N2-fixing condition. Furthermore, we demonstrated that fer and fldA encoding flavodoxin, and pfoAB encoding pyruvate-ferredoxin oxidoreductase could enhance the nitrogenase activity of the recombinant Escherichia coli 78-7 which carries a nif gene cluster (nifBHDKENXhesAnifV) of P. polymyxa WLY78. However, we do not know whether the flavodoxin encoded by fer or fldA is the direct electron donor to nitrogenase and whether the pyruvate-ferredoxin oxidoreductase encoded by pfoAB generates specifically electron for nitrogenase. Also, the genes yqiG,yqjM3,yqjM5 and ywcH3（encoding NADH:flavin oxidoreductases）and flr (encoding flavodoxin) were highly expressed in N2-fixing condition compared to non-N2-fixing condition, but their roles in the electron transports to nitrogenase need to be determined. In this study, the functions of the above-described genes in the electron transports to nitrogenase will be determined. This study will help us to find which gene(s) encoding flavodoxin or ferredoxin is (are) electron donor for nitrogenase and which gene(s) encoding pyruvate-ferredoxin oxidoreductase or NADH:flavin oxidoreductases generates electron for nitrogenase. The results will be of great use in enhancing nitrogen efficiency and transferring non-N2-fixing organisms into N2-fixing organisms.