Functional Nitrogenase Cofactor Maturase NifB in Mitochondria and Chloroplasts of Nicotiana benthamiana

Engineering plants to synthesize nitrogenase and assimilate atmospheric N will reduce crop dependency on industrial N fertilizers. This technology can be achieved by expressing prokaryotic nitrogen fixation gene products for the assembly of a functional nitrogenase in plants. NifB is a critical nitrogenase component since it catalyzes the first committed step in the biosynthesis of all types of nitrogenase active-site cofactors. Here, we used a library of 30 distinct sequences originating from different phyla and ecological niches to restore diazotrophic growth of an Azotobacter vinelandii mutant. Twenty of these variants rescued the mutant phenotype despite their phylogenetic distance to A. vinelandii. Because multiple protein interactions are required in the iron-molybdenum cofactor (FeMo-co) biosynthetic pathway, the maturation of nitrogenase in a heterologous host can be divided in independent modules containing interacting proteins that function together to produce a specific intermediate. Therefore, functional modules composed of a variant, together with the A. vinelandii NifS and NifU proteins (for biosynthesis of NifB [Fe S ] clusters) and the FdxN ferredoxin (for NifB function), were expressed in Nicotiana benthamiana chloroplasts and mitochondria. Three archaeal NifB proteins accumulated at high levels in soluble fractions of chloroplasts (Methanosarcina acetivorans and Methanocaldococcus infernus) or mitochondria ( and Methanothermobacter thermautotrophicus). These NifB proteins were shown to accept [Fe S ] clusters from NifU and were functional in FeMo-co synthesis . The accumulation of significant levels of soluble and functional NifB proteins in chloroplasts and mitochondria is critical to engineering biological nitrogen fixation in plants. Biological nitrogen fixation is the conversion of inert atmospheric dinitrogen gas into nitrogen-reactive ammonia, a reaction catalyzed by the nitrogenase enzyme of diazotrophic bacteria and archaea. Because plants cannot fix their own nitrogen, introducing functional nitrogenase in cereals and other crop plants would reduce our strong dependency on N fertilizers. NifB is required for the biosynthesis of the active site cofactors of all nitrogenases, which arguably makes it the most important protein in global nitrogen fixation. NifB functionality is therefore a requisite to engineer a plant nitrogenase. The expression of genes from a wide range of prokaryotes into the model diazotroph Azotobacter vineland