Investigation of the Cyanothece nitrogenase cluster in Synechocystis : a blueprint for engineering nitrogen fixing photoautotrophs

The nitrogenase gene cluster of unicellular diazotrophic cyanobacteria such as Cyanothece is frequently selected by nature for nitrogen fixing partnerships with eukaryotic phototrophs. The essential cluster components that confer an advantage in such partnerships remain under explored. To use this cluster for the development of synthetic, phototrophic nitrogen-fixing systems, a thorough and systematic analysis of its constituent genes is necessary. An initial effort to assess the possibility of engineering this cluster into non-diazotrophic phototrophs led to the generation of a Synechocystis 6803 strain with significant nitrogenase activity. In the current study, a refactoring approach was taken to determine the dispensability of the non-structural genes in the cluster and to define a minimal gene set for constructing a functional nitrogenase for phototrophs. Using a bottom-up strategy, the nif genes from Cyanothece 51142 were re-organized to form new operons. The genes were then seamlessly removed to determine their essentiality in the nitrogen fixation process. We demonstrate that besides the structural genes nifHDK , nifBSUENPVZTXW as well as hesAB are important for optimal nitrogenase function in a phototroph. We also show that optimal expression of these genes is crucial for efficient nitrogenase activity. Our findings provide a solid foundation for generating synthetic systems that will facilitate solar powered conversion of atmospheric nitrogen into nitrogen rich compounds, a stride towards a greener world.