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

Abstract

Nitrogenase in some bacteria and archaea catalyzes conversion of N2 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 nif (nitrogen fixation) 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 Saccharomyces cerevisiae 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 Paenibacillus polymyxa that are fused into two polyproteins via 2A peptides are co-expressed in S. cerevisiae. Expressed NifH from Klebsiella oxytoca NifU and NifS and P. polymyxa NifH fusion linked via 2A in S. cerevisiae exhibits Fe protein activity.

Nitrogen Fixation by Rhizobacterial Nif Mechanism: An Advanced Genetic Perspective

“…In Klebsiella pneumoniae, these twenty (20) genes are enclosed within seven nif operons [11]. They play a crucial role in nitrogen fixation and are primarily present in nitrogen-fixing bacteria [12]. Klebsiella pneumoniae exists as a free-living bacterium, within its chromosome, there is a specific region spanning 24 kilobases (24-Kb) that contains a total of 20 nif genes [13].…”

“…The nitrogenase (nif) genes encompass a range of components, including the nitrogenase structural genes responsible for forming the nitrogenase enzyme [17]. Additionally, there are genes involved in activating the Fe protein, molybdenum nitrogenase biosynthesis, iron-molybdenum cofactor biosynthesis, electron donation to nitrogenase, and regulatory genes essential for the expression of nif genes [12]. The nif regulon consists of factors that can activate or inhibit the production of proteins required for nitrogen fixation based on the prevailing environmental conditions [3].…”

“…nifK, nifE, nifN, nifX, hesA, and nifV) from Paenibacillus polymyxa (WLY78) is effectively achieved in yeast. Moreover, yeast nifH, co-expressed with nifH from P. polymyxa, and nifS and nifU from Klebsiella oxytoca demonstrate the activity of the Fe protein[12].…”