title: Characterization of tiacumicin B biosynthetic gene cluster affording diversified tiacumicin analogues and revealing a tailoring dihalogenase.
abstract: The RNA polymerase inhibitor tiacumicin B is currently undergoing phase III clinical trial for treatment of Clostridium difficile associated diarrhea with great promise. To understand the biosynthetic logic and to lay a foundation for generating structural analogues via pathway engineering, the tiacumicin B biosynthetic gene cluster was identified and characterized from the producer Dactylosporangium aurantiacum subsp. hamdenensis NRRL 18085. Sequence analysis of a 110,633 bp DNA region revealed the presence of 50 open reading frames (orfs). Functional investigations of 11 orfs by in vivo inactivation experiments, preliminarily outlined the boundaries of the tia-gene cluster and suggested that 31 orfs were putatively involved in tiacumicin B biosynthesis. Functions of a halogenase (TiaM), two glycosyltransferases (TiaG1 and TiaG2), a sugar C-methyltransferase (TiaS2), an acyltransferase (TiaS6), and two cytochrome P450s (TiaP1 and TiaP2) were elucidated by isolation and structural characterization of the metabolites from the corresponding gene-inactivation mutants. Accumulation of 18 tiacumicin B analogues from 7 mutants not only provided experimental evidence to confirm the proposed functions of individual biosynthetic enzymes, but also set an example of accessing microbial natural product diversity via genetic approach. More importantly, biochemical characterization of the FAD-dependent halogenase TiaM reveals a sequentially acting dihalogenation step tailoring tiacumicin B biosynthesis.
authors.affiliation: CAS Key Laboratory of Marine Bio-resources Sustainable Utilization, RNAM Center for Marine Microbiology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, 164 West Xingang Road, Guangzhou 510301, PR China.
authors.forename: Yi
authors.initials: Y
authors.lastname: Xiao
authors.forename: Sumei
authors.initials: S
authors.lastname: Li
authors.forename: Siwen
authors.initials: S
authors.lastname: Niu
authors.forename: Liang
authors.initials: L
authors.lastname: Ma
authors.forename: Guangtao
authors.initials: G
authors.lastname: Zhang
authors.forename: Haibo
authors.initials: H
authors.lastname: Zhang
authors.forename: Gaiyun
authors.initials: G
authors.lastname: Zhang
authors.forename: Jianhua
authors.initials: J
authors.lastname: Ju
authors.forename: Changsheng
authors.initials: C
authors.lastname: Zhang
mesh_terms: D000617:Aminoglycosides / Q000096:biosynthesis* / Q000737:chemistry* / Q000494:pharmacology; D000900:Anti-Bacterial Agents / Q000096:biosynthesis* / Q000737:chemistry* / Q000494:pharmacology; D001419:Bacteria / Q000187:drug effects; D003001:Cloning, Molecular; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism; D000077732:Fidaxomicin; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D054879:Halogenation; D008826:Microbial Sensitivity Tests; D008848:Micromonosporaceae / Q000201:enzymology / Q000235:genetics / Q000378:metabolism; D005810:Multigene Family / Q000235:genetics*; D009154:Mutation; D010088:Oxidoreductases / Q000235:genetics / Q000378:metabolism*; D012118:Resorcinols / Q000737:chemistry; D017422:Sequence Analysis, DNA
chemical_list: D000617:Aminoglycosides; D000900:Anti-Bacterial Agents; D012118:Resorcinols; C501612:orsellinic acid; D005182:Flavin-Adenine Dinucleotide; D003577:Cytochrome P-450 Enzyme System; D010088:Oxidoreductases; D000077732:Fidaxomicin