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

Nitrogenase in some bacteria and archaea catalyzes conversion of N 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 ( trogen ixation) genes. Viral 2A peptide mediated "cleavage&quo...

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Published inFrontiers in microbiology Vol. 14; p. 1137355
Main Authors Wang, Minyang, Shang, Yimin, Liu, Xiaomeng, Chen, Sanfeng
Format Journal Article
LanguageEnglish
Published Switzerland Frontiers Media S.A 02.03.2023
Subjects
2A peptide
co-expression
Microbiology
nitrogenase
polyprotein
Saccharomyces cerevisiae
co-expression
2A peptide
Saccharomyces cerevisiae
nitrogenase
polyprotein
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Summary:Nitrogenase in some bacteria and archaea catalyzes conversion of N 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 ( trogen 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 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 that are fused into two polyproteins 2A peptides are co-expressed in . . Expressed NifH from NifU and NifS and . NifH fusion linked 2A in . exhibits Fe protein activity.
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Reviewed by: Shekhar Mishra, University of Illinois at Urbana-Champaign, United States; David Mulder, National Renewable Energy Laboratory (DOE), United States; Qiang Yan, DSM, United States
This article was submitted to Microbiotechnology, a section of the journal Frontiers in Microbiology
Edited by: Mingfeng Cao, Xiamen University, China
ISSN:1664-302X
1664-302X
DOI:10.3389/fmicb.2023.1137355