Reconstruction and minimal gene requirements for the alternative iron-only nitrogenase in Escherichia coli

Significance To date, three different nitrogenase systems [molybdenum (MoFe), vanadium (VFe), and iron-only (FeFe)] have been found in nature. The MoFe nitrogenase has been studied extensively, but the alternative vanadium-dependent (Vnf) and iron-only (Anf) systems are less well characterized, particularly with respect to components required for their biosynthesis and activity. We have engineered an artificial FeFe nitrogenase system in Escherichia coli that combines anf structural genes with accessory nitrogen fixation genes ( nif ) to provide a minimal 10-gene cluster that supports the biosynthesis and activity of the FeFe nitrogenase. Our findings underscore the potential for the future engineering of nitrogen fixation in eukaryotes because the Anf system can bypass limitations in molybdenum availability in plant organelles.