AUTHOR=Wang Minyang , Shang Yimin , Liu Xiaomeng , Chen Sanfeng 

TITLE=Assembly of nitrogenase biosynthetic pathway in Saccharomyces cerevisiae by using polyprotein strategy

JOURNAL=Frontiers in Microbiology

VOLUME=14

YEAR=2023

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2023.1137355

DOI=10.3389/fmicb.2023.1137355

ISSN=1664-302X

ABSTRACT=<p>Nitrogenase in some bacteria and archaea catalyzes conversion of N<sub>2</sub> to ammonia. To reconstitute a nitrogenase biosynthetic pathway in a eukaryotic host is still a challenge, since synthesis of nitrogenase requires a large number of <italic>nif</italic> (<italic>ni</italic>trogen <italic>f</italic>ixation) genes. Viral 2A peptide mediated “cleavage” of polyprotein is one of strategies for multigene co-expression. Here, we show that cleavage efficiency of NifB-2A-NifH polyprotein linked by four different 2A peptides (P2A, T2A, E2A, and F2A) in <italic>Saccharomyces cerevisiae</italic> ranges from ~50% to ~90%. The presence of a 2A tail in NifB, NifH, and NifD does not affect their activity. Western blotting shows that 9 Nif proteins (NifB, NifH, NifD, NifK, NifE, NifN, NifX, HesA, and NifV) from <italic>Paenibacillus polymyxa</italic> that are fused into two polyproteins <italic>via</italic> 2A peptides are co-expressed in <italic>S</italic>. <italic>cerevisiae</italic>. Expressed NifH from <italic>Klebsiella oxytoca</italic> NifU and NifS and <italic>P</italic>. <italic>polymyxa</italic> NifH fusion linked <italic>via</italic> 2A in <italic>S</italic>. <italic>cerevisiae</italic> exhibits Fe protein activity.</p>