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Probing and Engineering the Fatty Acyl Substrate Selectivity of Starter Condensation Domains of Nonribosomal Peptide Synthetases in Lipopeptide Biosynthesis
Author(s) -
Liu Qian,
Fan Wenjie,
Zhao Yajin,
Deng Zixin,
Feng Yan
Publication year - 2020
Publication title -
biotechnology journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.144
H-Index - 84
eISSN - 1860-7314
pISSN - 1860-6768
DOI - 10.1002/biot.201900175
Subject(s) - nonribosomal peptide , lipopeptide , biosynthesis , starter , substrate (aquarium) , chemistry , metabolic engineering , peptide , biochemistry , selectivity , substrate specificity , combinatorial chemistry , biology , bacteria , enzyme , catalysis , food science , ecology , genetics
Lipopeptides are produced by nonribosomal peptide synthetases (NRPSs) and contain diverse fatty acyl moieties that are major determinants of antibiotic potency. The lipid chains are incorporated into peptidyl backbones via lipoinitiation, a process comprising free fatty acid activation and the subsequent starter condensation domain (C1)‐catalyzed conjugation of fatty acyl moieties onto the aminoacyl substrates. Thus, a thorough understanding of lipoinitiation biocatalysts would significantly expand their potential to produce novel antibiotics. Here, biochemical assays, in silico analysis, and mutagenesis studies are used to ultimately identify the specific amino acid residues that control the fatty acyl substrate selectivity of C1 in lipopeptide A54145. In silico docking study has identified four candidate amino acids, and subsequent in vitro assays confirmed their functional contribution to the channel that controls substrate selectivity. Two engineered variants with single point mutations in C1 are found to alter the substrate selectivity toward nonnatural fatty acyl substrates. The detailed mechanistic insights into the catalytic contribution of C1 obtained from the present study will facilitate future NPRS biocatalyst efforts