Combined Assembly and Targeted Integration of Multigene for Nitrogenase Biosynthetic Pathway in Saccharomyces cerevisiae

Biological nitrogen fixation, a process unique to diazotrophic prokaryote, is catalyzed by the nitrogenase complex. There has been a long-standing interest in reconstituting a nitrogenase biosynthetic pathway in a eukaryotic host with the final aim of developing N₂-fixing cereal crops. In this study, we report that a nitrogenase biosynthetic pathway (∼38 kb containing 15 genes) was assembled in two individual one-step methods via in vivo assembly and integrated at δ and HO sites in Saccharomyces cerevisiae chromosome. Of the 15 genes, 11 genes (nifB, nifH, nifD, nifK, nifE, nifN, nifX, hesA, nifV, groES, groEL) were from Paenibacillus polymyxa WLY78 and 4 genes (nifS, nifU, nifF, nifJ) were from Klebsiella oxytoca. The 15-gene nitrogenase biosynthetic pathway was correctly assembled and transcribed in the recombinant S. cerevisiae. The NifDK tetramer with an identical molecular weight as that of P. polymyxa was formed in yeast and the expressed NifH exhibited the activity of Fe protein. This study demonstrates that it will be possible to produce active nitrogenase in eukaryotic hosts.