Exploiting genetic diversity and gene synthesis to identify superior nitrogenase NifH protein variants to engineer N2-fixation in plants

Abstract Engineering nitrogen fixation in eukaryotes requires high expression of functional nitrogenase structural proteins, a goal that has not yet been achieved. Here we build knowledge-based library containing 32 nifH sequences from prokaryotes diverse ecological niches and metabolic features combine with rapid screening tobacco to identify superior NifH variants for plant mitochondria expression. Three outperform are further tested yeast. Hydrogenobacter thermophilus (Aquificae) is isolated large quantities yeast fulfills protein requirements efficient N 2 fixation, including electron transfer substrate reduction, P-cluster maturation, FeMo-co biosynthesis. H. expressed leaves shows lower activity than However, [Fe 4 S ] clusters NifU vitro increases 10-fold the tobacco-isolated NifH, revealing [Fe-S] cluster availability constitutes bottleneck engineer nitrogenases.