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

Hsien Tai Chiu, Chien Pao Weng, Yu Chin Lin, Kuan Hung Chen

研究成果: Article同行評審

6 引文 斯高帕斯(Scopus)

摘要

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.

原文English
頁(從 - 到)1988-2006
頁數19
期刊Organic and Biomolecular Chemistry
14
發行號6
DOIs
出版狀態Published - 2016 一月 1

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Physical and Theoretical Chemistry
  • Organic Chemistry

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