All diazotrophic organisms sequenced to date encode a molybdenum-dependent nitrogenase, but some also have alternative nitrogenases that are dependent on either vanadium (VFe) or iron only (FeFe) for activity. In Azotobacter vinelandii, expression of the three different types of nitrogenase is regulated in response to metal availability. The majority of genes required for nitrogen fixation in this organism are encoded in the nitrogen fixation (nif) gene clusters, whereas genes specific for vanadium-or irondependent diazotophy are encoded by the vanadium nitrogen fixation (vnf) and alternative nitrogen fixation (anf) genes, respectively. Due to the complexities of metal-dependent regulation and gene redundancy in A. vinelandii, it has been difficult to determine the precise genetic requirements for alternative nitrogen fixation. In this study, we have used Escherichia coli as a chassis to build an artificial iron-only (Anf) nitrogenase system composed of defined anf and nif genes. Using this system, we demonstrate that the pathway for biosynthesis of the iron-only cofactor (FeFe-co) is likely to be simpler than the pathway for biosynthesis of the molybdenum-dependent cofactor (FeMo-co) equivalent. A number of genes considered to be essential for nitrogen fixation by FeFe nitrogenase, including nifM, vnfEN, and anfOR, are not required for the artificial Anf system in E. coli. This finding has enabled us to engineer a minimal FeFe nitrogenase system comprising the structural anfHDGK genes and the nifBUSV genes required for metallocluster biosynthesis, with nifF and nifJ providing electron transport to the alternative nitrogenase. This minimal Anf system has potential implications for engineering diazotrophy in eukaryotes, particularly in compartments (e.g., organelles) where molybdenum may be limiting.

SignificanceThe requirement of maintaining balanced expression of a large number of gene products represents a major challenge to the engineering of nitrogen fixation in cereal crops, necessitating reiterative combinatorial assembly cycles to optimize monocistronic gene expression. In this study, we have explored a “fuse-and-cleave” virus-derived polyprotein strategy to reduce gene numbers and achieve balanced expression of protein components required for nitrogenase biosynthesis and activity. After testing and regrouping assemblies on the basis of expression profiles, cleavage patterns, and activity, 14 essential genes were selectively assembled into 5 giant genes that enable growth on dinitrogen. This strategy has potential advantages, not only for transferring nitrogen fixation to plants, but also for the engineering of other complex systems of profound agronomic and ecological importance.

A large number of genes are necessary for the biosynthesis and activity of the enzyme nitrogenase to carry out the process of biological nitrogen fixation (BNF), which requires large amounts of ATP and reducing power. The multiplicity of the genes involved, the oxygen sensitivity of nitrogenase, plus the demand for energy and reducing power, are thought to be major obstacles to engineering BNF into cereal crops. Genes required for nitrogen fixation can be considered as three functional modules encoding electron-transport components (ETCs), proteins required for metal cluster biosynthesis, and the "core" nitrogenase apoenzyme, respectively. Among these modules, the ETC is important for the supply of reducing power. In this work, we have used Escherichia coli as a chassis to study the compatibility between molybdenum and the iron-only nitrogenases with ETC modules from target plant organelles, including chloroplasts, root plastids, and mitochondria. We have replaced an ETC module present in diazotrophic bacteria with genes encoding ferredoxin-NADPH oxidoreductases (FNRs) and their cognate ferredoxin counterparts from plant organelles. We observe that the FNR-ferredoxin module from chloroplasts and root plastids can support the activities of both types of nitrogenase. In contrast, an analogous ETC module from mitochondria could not function in electron transfer to nitrogenase. However, this incompatibility could be overcome with hybrid modules comprising mitochondrial NADPH-dependent adrenodoxin oxidoreductase and the Anabaena ferredoxins FdxH or FdxB. We pinpoint endogenous ETCs from plant organelles as power supplies to support nitrogenase for future engineering of diazotrophy in cereal crops.nitrogen fixation | electron transport | plant organelles | nitrogenase engineering