Engineering Nitrogen Fixation Activity in an Oxygenic Phototroph

Biological nitrogen fixation is catalyzed by nitrogenase, a complex metalloenzyme found only in prokaryotes. N(2) fixation is energetically highly expensive, and an energy-generating process such as photosynthesis can meet the energy demand of N(2) fixation. However, synthesis and expression of nitrogenase are exquisitely sensitive to the presence of 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 N(2). Here, we demonstrate a feasible way to engineer nitrogenase activity in the nondiazotrophic cyanobacterium Synechocystis sp. PCC 6803 through the transfer of 35 nitrogen fixation (nif) genes from the diazotrophic cyanobacterium Cyanothece sp. ATCC 51142. In addition, we have identified the minimal nif cluster required for such activity in Synechocystis 6803. Moreover, nitrogenase activity was significantly improved by increasing the expression levels of nif genes. Importantly, the O(2) tolerance 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 engineered Synechocystis 6803 strains that, remarkably, have more than 30% of the N(2) fixation activity of Cyanothece 51142, the highest such activity established in any nondiazotrophic oxygenic photosynthetic organism. This report establishes a baseline for the ultimate goal of engineering nitrogen fixation ability in crop plants.