Engineering Nitrogen Fixation Activity in an Oxygenic Phototroph

Abstract

ABSTRACTBiological nitrogen fixation is catalyzed by nitrogenase, a complex metalloenzyme found only in prokaryotes. N2fixation is energetically highly expensive, and an energy generating process such as photosynthesis can meet the energy demand of N2fixation. However, synthesis and expression of nitrogenase is exquisitely sensitive to oxygen. Thus, engineering nitrogen fixation activity in photosynthetic organisms that produce oxygen is challenging. Cyanobacteria are oxygenic photosynthetic prokaryotes, and some of them also fix N2. Here, we demonstrate a feasible way to engineer nitrogenase activity in the non-diazotrophic cyanobacteriumSynechocystissp. PCC 6803 through the transfer of 35 nitrogen fixation (nif) genes from the diazotrophic cyanobacteriumCyanothecesp. ATCC 51142. In addition, we have identified the minimalnifcluster required for such activity inSynechocystis6803. Moreover, nitrogenase activity was significantly improved by increasing the expression levels ofnifgenes. Importantly, the O2tolerance of nitrogenase was enhanced by introduction of uptake hydrogenase genes, showing this to be a functional way to improve nitrogenase enzyme activity under micro-oxic conditions. To date, our efforts have resulted in engineeredSynechocystis6803 strains that remarkably have more than 30% N2-fixation activity compared to that inCyanothece51142, the highest such activity established in any non-diazotrophic oxygenic photosynthetic organism. This study establishes a baseline towards the ultimate goal of engineering nitrogen fixation ability in crop plants.IMPORTANCEApplication of chemically synthesized nitrogen fertilizers has revolutionized agriculture. However, the energetic costs of such production processes as well as the wide spread application of fertilizers have raised serious environmental issues. A sustainable alternative is to endow crop plants the ability to fix atmospheric N2in situ. One long-term approach is to transfer allnifgenes from a prokaryote to plant cells, and express nitrogenase in an energy-producing organelle, chloroplast or mitochondrion. In this context,Synechocystis6803, the non-diazotrophic cyanobacterium utilized in this study, provides a model chassis for rapid investigation of the necessary requirements to establish diazotrophy in an oxygenic phototroph.