Polyprotein strategy for stoichiometric assembly of nitrogen fixation components for synthetic biology

Significance The 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.