Ammonia synthesis via an engineered nitrogenase assembly pathway in Escherichia coli

Heterologous expression of nitrogenase has been actively pursued because of the far-reaching impact of this enzyme on agriculture, energy and environment. Yet, isolation of an active two-component, metallocentre-containing nitrogenase from a non-diazotrophic host has yet to be accomplished. Here, we report the heterologous synthesis of an active Mo-nitrogenase by combining genes from Azotobacter vinelandii and Methanosarcina acetivorans in Escherichia coli. Metal, activity and EPR analyses demonstrate the integrity of the metallocentres in the purified nitrogenase enzyme; whereas growth, nanoSIMS and NMR experiments illustrate diazotrophic growth and ¹⁵N enrichment by the E. coli expression strain, as well as accumulation of extracellular ammonia upon deletion of the ammonia transporter that permits incorporation of thus-generated N into the cellular mass of a non-diazotrophic E. coli strain. As such, this study provides a crucial prototype system that could be optimized/modified to enable future transgenic expression and biotechnological adaptations of nitrogenase.