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

• DOI: 10.3389/fmicb.2023.1137355
• Publication Date: 2023-03-02T04:32:27Z
• Authors: Minyang Wang; Yimin Shang; Xiaomeng Liu; Sanfeng Chen
• Keywords: polyprotein; 2A peptide; Saccharomyces cerevisiae; nitrogenase; Microbiology; QR1-502; co-expression

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