Progress and perspectives in nitrogenase engineering in plants and yeast

Crop productivity relies on synthetic nitrogen fertilizers, which entail significant economic and environmental costs. Engineering crop plants to produce nitrogenase allowing them to acquire nitrogen from the atmosphere offers a transformative approach to sustainable agriculture. However, engineering the nitrogenase pathway into eukaryotic cells poses significant challenges due to the enzyme metal clusters, high energy demands, and extreme sensitivity to oxygen. This review summarizes recent advances in nitrogenase engineering in yeast and plants. Progress has been made using a stepwise modular approach and a functional validation pipeline for core and ancillary nitrogenase proteins. Key milestones include recovery of NifH with in vitro activity from mitochondria and chloroplasts, production of NifB that is active upon isolation or after in vitro cluster reconstitution, production of NifB-co in yeast mitochondria, in vitro FeMo-co biosynthesis using proteins purified from yeast mitochondria, and recovery of nitrogenase components from transgenic rice with activity demonstrated in vitro. Complete nitrogenase activity in engineered yeast or plants has not yet been demonstrated. We discuss the importance of organelle targeting, the exploitation of natural diversity and engineered proteins, and synthetic biology tools for the assembly of multigene pathways. Finally, we present the main remaining obstacles, such as the assembly of functional NifDK in plants, integration with host metabolism and coordinated regulation, and we outline the prospects for the development of nitrogen-fixing crops.