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

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.

Membrane-Located Expression of Thioesterase From Acinetobacter baylyi Enhances Free Fatty Acid Production With Decreased Toxicity in Synechocystis sp. PCC6803

“…We constructed two Synechocystis strains named AcT and mAcT. For the AcT strain, we inserted the 546-bp ‘AcTesA with the N-terminus 6XHis tag along with the kanamycin resistance cassette (KanR) into the neutral site ( slr1311 ) ( Lagarde et al, 2000 ; Kuchmina et al, 2012 ; Bentley et al, 2014 ; Tsujimoto et al, 2018 ) of the Synechocystis genome under IPTG inducible promoter, Ptrc (Figure 1A ). This strain was developed to express ‘AcTesA in the cytosol to facilitate random hydrolysis of intracellular acyl-ACP.…”

Unicellular Cyanobacteria Exhibit Light-Driven, Oxygen-Tolerant, Constitutive Nitrogenase Activity Under Continuous Illumination

“…Recent attempts to transfer a functional complement of nitrogenase genes to Synechocystis sp. 6803 have demonstrated the complexities engineering BNF into a non-diazotrophic phototroph 28,29 . The engineered cells exhibited nitrogenase activity at anaerobic levels, but cells exposed to 1% oxygen quickly lost ~90% of activity.…”

Transfer of Nitrogen Fixation (nif) Genes to Non‐diazotrophic Hosts

“…PCC 6803. [51] Under anaerobic conditions, the nitrogenase activity was detected in transformants, but activity was only about 0.26 %o ft hose of L. boryana. Furthermore, 35 nif and nif-related genes from the diazotrophic cyanobacterium Cyanothece sp.…”