title: Target-specific identification and characterization of the putative gene cluster for brasilinolide biosynthesis revealing the mechanistic insights and combinatorial synthetic utility of 2-deoxy-l-fucose biosynthetic enzymes.
abstract: Brasilinolides exhibiting potent immunosuppressive and antifungal activities with remarkably low toxicity are structurally characterized by an unusual modified 2-deoxy-l-fucose (2dF) attached to a type I polyketide (PK-I) macrolactone. From the pathogenic producer Nocardia terpenica (Nocardia brasiliensis IFM-0406), a 210 kb genomic fragment was identified by target-specific degenerate primers and subsequently sequenced, revealing a giant nbr gene cluster harboring genes (nbrCDEF) required for TDP-2dF biosynthesis and those for PK-I biosynthesis, modification and regulation. The results showed that the genetic and domain arrangements of nbr PK-I synthases agreed colinearly with the PK-I structures of brasilinolides. Subsequent heterologous expression of nbrCDEF in Escherichia coli accomplished in vitro reconstitution of TDP-2dF biosynthesis. The catalytic functions and mechanisms of NbrCDEF enzymes were further characterized by systematic mix-and-match experiments. The enzymes were revealed to display remarkable substrate and partner promiscuity, leading to the establishment of in vitro hybrid deoxysugar biosynthetic pathways throughout an in situ one-pot (iSOP) method. This study represents the first demonstration of TDP-2dF biosynthesis at the enzyme and molecular levels, and provides new hope for expanding the structural diversity of brasilinolides by combinatorial biosynthesis.
authors.affiliation: Department of Chemistry, National Cheng Kung University, No. 1, University Road, Tainan 701, Taiwan. hchiu@mail.ncku.edu.tw.
authors.forename: Hsien-Tai
authors.initials: HT
authors.lastname: Chiu
authors.affiliation: Department of Chemistry, National Cheng Kung University, No. 1, University Road, Tainan 701, Taiwan. hchiu@mail.ncku.edu.tw.
authors.forename: Chien-Pao
authors.initials: CP
authors.lastname: Weng
authors.affiliation: Department of Chemistry, National Cheng Kung University, No. 1, University Road, Tainan 701, Taiwan. hchiu@mail.ncku.edu.tw and Department of Biological Science and Technology, National Chiao Tung University, Hsinchu 300, Taiwan.
authors.forename: Yu-Chin
authors.initials: YC
authors.lastname: Lin
authors.affiliation: Department of Biological Science and Technology, National Chiao Tung University, Hsinchu 300, Taiwan.
authors.forename: Kuan-Hung
authors.initials: KH
authors.lastname: Chen
mesh_terms: D000595:Amino Acid Sequence; D055162:Biocatalysis; D018942:Macrolides / Q000737:chemistry / Q000378:metabolism*; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D005810:Multigene Family / Q000235:genetics*; D048630:Polyketide Synthases / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment
chemical_list: D018942:Macrolides; C111141:brasilinolide A; C406050:brasilinolide B; D048630:Polyketide Synthases