標題: | Target-specific identification and characterization of the putative gene cluster for brasilinolide biosynthesis revealing the mechanistic insights and connic enzymes |
作者: | Chiu, Hsien-Tai Weng, Chien-Pao Lin, Yu-Chin Chen, Kuan-Hung 生物科技學系 Department of Biological Science and Technology |
公開日期: | 2016 |
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. |
URI: | http://dx.doi.org/10.1039/c5ob02292d http://hdl.handle.net/11536/133735 |
ISSN: | 1477-0520 |
DOI: | 10.1039/c5ob02292d |
期刊: | ORGANIC & BIOMOLECULAR CHEMISTRY |
Volume: | 14 |
Issue: | 6 |
起始頁: | 1988 |
結束頁: | 2006 |
顯示於類別: | 期刊論文 |