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 the environment. However, 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 molybdenum-nitrogenase by combining genes from Azotobacter vinelandii and Methanosarcina acetivorans in Escherichia coli. Metal, activity and electron paramagnetic resonance analyses demonstrate the integrity of the metallocentres in the purified nitrogenase enzyme; whereas growth, nanoscale secondary ion mass spectrometry and nuclear magnetic resonance experiments illustrate diazotrophic growth and 15N enrichment by the E. coli expression strain, and accumulation of extracellular ammonia upon deletion of the ammonia transporter that permits incorporation of thus-generated nitrogen 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. Heterologous expression of an active, metallocentre-containing nitrogenase in a non-diazotrophic host is challenging. Now, the heterologous biosynthetic pathway of Mo-nitrogenase is pieced together in Escherichia coli using genes from Azotobacter vinelandii and Methanosarcina acetivorans.