Using synthetic biology to express nitrogenase biosynthesis pathway in rice and to overcome barriers of nitrogenase instability in plant cytosol.

Engineering nitrogen fixation in cereals could reduce usage of chemical nitrogen fertilizers. Here, a nitrogenase biosynthesis pathway comprising 13 genes (nifB nifH nifD nifK nifE nifN nifX hesA nifV nifS nifU groES groEL) was introduced into rice by transforming multigene vectors and subsequently by sexual crossing between transgenic rice plants. Genome sequencing analysis revealed that 13 nif genes in F hybrid rice lines L12-13 and L8-17 were inserted at two loci on rice chromosome 1. Eleven nitrogen fixation (Nif) proteins were produced and stable NifDK tetramer was formed in rice cytosol. NifH in rice cytosol was unstable and NifH-S18 was found to be a key residue that conferred susceptibility to proteinase degradation. NifH variants with Fe protein activity and resistance to plant endoproteinase cleavage were obtained. This study provides an efficient approach for introducing multiple nif genes into plants and also helps to pre-evaluate the stability of prokaryotic proteins in plant cytosol.