Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, 464-8601, Japan.
School of Agricultural Sciences, Nagoya University, Nagoya, 464-8601, Japan.
Sci Rep. 2018 May 9;8(1):7380. doi: 10.1038/s41598-018-25396-7.
Transfer of nitrogen fixation ability to plants, especially crops, is a promising approach to mitigate dependence on chemical nitrogen fertilizer and alleviate environmental pollution caused by nitrogen fertilizer run-off. However, the need to transfer a large number of nitrogen fixation (nif) genes and the extreme vulnerability of nitrogenase to oxygen constitute major obstacles for transfer of nitrogen-fixing ability to plants. Here we demonstrate functional expression of a cyanobacterial nitrogenase in the non-diazotrophic cyanobacterium Synechocystis sp. PCC 6803 (Synechocystis 6803). A 20.8-kb chromosomal fragment containing 25 nif and nif-related genes of the diazotrophic cyanobacterium Leptolyngbya boryana was integrated into a neutral genome site of Synechocystis 6803 by five-step homologous recombination together with the cnfR gene encoding the transcriptional activator of the nif genes to isolate CN1. In addition, two other transformants CN2 and CN3 carrying additional one and four genes, respectively, were isolated from CN1. Low but significant nitrogenase activity was detected in all transformants. This is the first example of nitrogenase activity detected in non-diazotrophic photosynthetic organisms. These strains provide valuable platforms to investigate unknown factors that enable nitrogen-fixing growth of non-diazotrophic photosynthetic organisms, including plants.
将固氮能力转移到植物，特别是作物中，是减少对化学氮肥依赖和缓解氮肥流失造成的环境污染的一种很有前途的方法。然而，将大量固氮（nif）基因转移和氮酶对氧气的极端脆弱性构成了将固氮能力转移到植物的主要障碍。在这里，我们展示了在非固氮蓝藻集胞藻 PCC 6803（集胞藻 6803）中功能性表达蓝藻氮酶。含有固氮蓝藻鱼腥藻 25 个 nif 和 nif 相关基因的 20.8kb 染色体片段与编码 nif 基因转录激活子的 cnfR 基因一起通过五步同源重组整合到集胞藻 6803 的中性基因组位点，以分离出 CN1。此外，还从 CN1 中分离出另外携带一个和四个基因的另外两个转化体 CN2 和 CN3。所有转化体中均检测到低但显著的氮酶活性。这是首次在非固氮光合生物中检测到氮酶活性。这些菌株为研究使非固氮光合生物（包括植物）能够进行固氮生长的未知因素提供了有价值的平台。
