title: Biosynthesis of the pyrrolidine protein synthesis inhibitor anisomycin involves novel gene ensemble and cryptic biosynthetic steps.
abstract: The protein synthesis inhibitor anisomycin features a unique benzylpyrrolidine system and exhibits diverse biological and pharmacologic activities. Its biosynthetic origin has remained obscure for more than 60 y, however. Here we report the identification of the biosynthetic gene cluster (BGC) of anisomycin inStreptomyces hygrospinosusvar. beijingensis by a bioactivity-guided high-throughput screening method. Using a combination of bioinformatic analysis, reverse genetics, chemical analysis, and in vitro biochemical assays, we have identified a core four-gene ensemble responsible for the synthesis of the pyrrolidine system in anisomycin:aniQ, encoding a aminotransferase that catalyzes an initial deamination and a later reamination steps;aniP, encoding a transketolase implicated to bring together an glycolysis intermediate with 4-hydroxyphenylpyruvic acid to form the anisomycin molecular backbone;aniO, encoding a glycosyltransferase that catalyzes a cryptic glycosylation crucial for downstream enzyme processing; andaniN, encoding a bifunctional dehydrogenase that mediates multistep pyrrolidine formation. The results reveal a BGC for pyrrolidine alkaloid biosynthesis that is distinct from known bacterial alkaloid pathways, and provide the signature sequences that will facilitate the discovery of BGCs encoding novel pyrrolidine alkaloids in bacterial genomes. The biosynthetic insights from this study further set the foundation for biosynthetic engineering of pyrrolidine antibiotics.
authors.affiliation: State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200030, China.
authors.forename: Xiaoqing
authors.initials: X
authors.lastname: Zheng
authors.affiliation: State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200030, China.
authors.forename: Qiuxiang
authors.initials: Q
authors.lastname: Cheng
authors.affiliation: State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200030, China.
authors.forename: Fen
authors.initials: F
authors.lastname: Yao
authors.affiliation: State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200030, China.
authors.forename: Xiaozheng
authors.initials: X
authors.lastname: Wang
authors.affiliation: State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200030, China.
authors.forename: Lingxin
authors.initials: L
authors.lastname: Kong
authors.affiliation: State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200030, China.
authors.forename: Bo
authors.initials: B
authors.lastname: Cao
authors.affiliation: State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200030, China.
authors.forename: Min
authors.initials: M
authors.lastname: Xu
authors.affiliation: State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200030, China.
authors.forename: Shuangjun
authors.initials: S
authors.lastname: Lin
authors.affiliation: State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200030, China.
authors.forename: Zixin
authors.initials: Z
authors.lastname: Deng
authors.affiliation: School of Molecular Sciences, University of Western Australia, Perth, WA 6009, Australia; dlyou@sjtu.edu.cn yitheng.chooi@uwa.edu.au.
authors.forename: Yit-Heng
authors.identifier: 0000-0001-7719-7524
authors.initials: YH
authors.lastname: Chooi
authors.affiliation: State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200030, China; dlyou@sjtu.edu.cn yitheng.chooi@uwa.edu.au.|Joint International Research Laboratory of Metabolic and Developmental Sciences, Shanghai Jiao Tong University, Shanghai 200240, China.
authors.forename: Delin
authors.initials: D
authors.lastname: You
mesh_terms: D000841:Anisomycin / Q000096:biosynthesis* / Q000494:pharmacology; D000900:Anti-Bacterial Agents / Q000096:biosynthesis* / Q000494:pharmacology; D053898:Biosynthetic Pathways; D019295:Computational Biology; D016680:Genome, Bacterial; D057166:High-Throughput Screening Assays; D005810:Multigene Family; D013302:Streptomyces / Q000187:drug effects*
chemical_list: D000900:Anti-Bacterial Agents; D000841:Anisomycin
keywords: Streptomyces; anisomycin biosynthetic pathway; cryptic glycosylation; protein synthesis inhibitor; pyrrolidine antibiotics