Biosynthesis of the nitrogenase active-site cofactor precursor NifB-co in <i>Saccharomyces cerevisiae</i>

Abstract

The radical <i>S</i>-adenosylmethionine (SAM) enzyme NifB occupies a central and essential position in nitrogenase biogenesis. NifB catalyzes the formation of an [8Fe-9S-C] cluster, called NifB-co, which constitutes the core of the active-site cofactors for all 3 nitrogenase types. Here, we produce functional NifB in aerobically cultured <i>Saccharomyces cerevisiae</i> Combinatorial pathway design was employed to construct 62 strains in which transcription units driving different expression levels of mitochondria-targeted <i>nif</i> genes (<i>nifUSXB</i> and <i>fdxN</i>) were integrated into the chromosome. Two combinatorial libraries totaling 0.7 Mb were constructed: An expression library of 6 partial clusters, including <i>nifUSX</i> and <i>fdxN</i>, and a library consisting of 28 different <i>nifB</i> genes mined from the Structure-Function Linkage Database and expressed at different levels according to a factorial design. We show that coexpression in yeast of the nitrogenase maturation proteins NifU, NifS, and FdxN from <i>Azotobacter vinelandii</i> with NifB from the archaea <i>Methanocaldococcus infernus</i> or <i>Methanothermobacter thermautotrophicus</i> yields NifB proteins equipped with [Fe-S] clusters that, as purified, support in vitro formation of NifB-co. Proof of in vivo NifB-co formation was additionally obtained. NifX as purified from aerobically cultured <i>S. cerevisiae</i> coexpressing <i>M. thermautotrophicus</i> NifB with <i>A. vinelandii</i> NifU, NifS, and FdxN, and engineered yeast SAM synthase supported FeMo-co synthesis, indicative of NifX carrying in vivo-formed NifB-co. This study defines the minimal genetic determinants for the formation of the key precursor in the nitrogenase cofactor biosynthetic pathway in a eukaryotic organism.