Functional expression of an oxygen-labile nitrogenase in an oxygenic photosynthetic organism

Ryoma Tsujimoto,Konomi Yokomizo,Hiroya Kotani,Hisanori Yamakawa,Aoi Nonaka,Yuichi Fujita

doi:10.1038/s41598-018-25396-7

Abstract

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.

Highlights

Nitrogen fixation is the conversion of molecular nitrogen (N2) to ammonia, a more accessible nitrogen source for most organisms
We identified the cnfR gene encoding the transcriptional activator for nif genes in the 50-kb gene cluster of the L. boryana chromosome[29]
We examined whether nitrogen fixation can be conferred to Synechocystis 6803 by introduction of the 20-kb nif gene cluster together with the cnfR gene into its chromosome

Summary

Introduction

Nitrogen fixation is the conversion of molecular nitrogen (N2) to ammonia, a more accessible nitrogen source for most organisms. Chloroplast genomes of many algae and plants such as gymnosperms (e.g., Pinus thunbergii)[19] and mosses (e.g., Physcomitrella patens and Marchantia polymorpha)[20,21] encode three subunits of a nitrogenase-like enzyme, dark-operative protochlorophyllide oxidoreductase (DPOR), that shows similar oxygen sensitivity as nitrogenase[20]. This suggests that chloroplasts have the potential ability to express and accommodate active nitrogenase[14]

Methods

Results

Conclusion