Blog
About

14
views
0
recommends
+1 Recommend
1 collections
    0
    shares
      • Record: found
      • Abstract: found
      • Article: found

      Construction of a mutant of Actinoplanes sp. N902-109 that produces a new rapamycin analog

      Read this article at

      ScienceOpenPublisherPubMed
      Bookmark
          There is no author summary for this article yet. Authors can add summaries to their articles on ScienceOpen to make them more accessible to a non-specialist audience.

          Abstract

          In the present study, we introduced point mutations into Ac_rapA which encodes a polyketide synthase responsible for rapamycin biosynthesis in Actinoplanes sp. N902-109, in order to construct a mutant with an inactivated enoylreductase (ER) domain, which was able to synthesize a new rapamycin analog. Based on the homologous recombination induced by double-strand breaks in chromosome mediated by endonuclease I-SceI, the site-directed mutation in the first ER domain of Ac_rapA was introduced using non-replicating plasmid pLYERIA combined with an I-SceI expression plasmid. Three amino acid residues of the active center, Ala–Gly–Gly, were converted to Ala–Ser–Pro. The broth of the mutant strain SIPI-027 was analyzed by HPLC and a new peak with the similar UV spectrum to that of rapamycin was found. The sample of the new peak was prepared by solvent extraction, column chromatography, and crystallization methods. The structure of new compound, named as SIPI-rapxin, was elucidated by determining and analyzing its MS and NMR spectra and its biological activity was assessed using mixed lymphocyte reaction (MLR). An ER domain–deficient mutant of Actinoplanes sp. N902-109, named as SIPI-027, was constructed, which produced a novel rapamycin analog SIPI-rapxin and its structure was elucidated to be 35, 36-didehydro-27- O-demethylrapamycin. The biological activity of SIPI-rapxin was better than that of rapamycin. In conclusion, inactivation of the first ER domain of rapA, one of the modular polyketide synthase responsible for macro-lactone synthesis of rapamycin, gave rise to a mutant capable of producing a novel rapamycin analog, 35, 36-didehydro-27- O-demethylrapamycin, demonstrating that the enoylreductase domain was responsible for the reduction of the double bond between C-35 and C-36 during rapamycin synthesis.

          Related collections

          Author and article information

          Journal
          CJNM
          Chinese Journal of Natural Medicines
          Elsevier
          1875-5364
          20 March 2018
          : 16
          : 3
          : 210-218
          Affiliations
          1Shanghai Institute of Pharmaceutical Industry, Shanghai 201203, China
          2Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China
          Author notes
          *Corresponding author: HU Hai-Feng, Tel/Fax: 86-21-55880207, E-mails: haifenghu88@ 123456163.com ; HUANG He, huanghe@ 123456sibs.ac.cn

          These authors have no conflict of interest to declare.

          Article
          S1875-5364(18)30049-9
          10.1016/S1875-5364(18)30049-9
          29576057
          Copyright © 2018 China Pharmaceutical University. Published by Elsevier B.V. All rights reserved.
          Funding
          Funded by: National Major Scientific Instruments and Equipment Development Projects
          Award ID: 2011YQ15007208
          Funded by: Shanghai Science and Technology Support Project
          Award ID: 12431901102
          This work was supported by the National Major Scientific Instruments and Equipment Development Projects (No. 2011YQ15007208) and Shanghai Science and Technology Support Project (No. 12431901102).

          Comments

          Comment on this article

          Similar content 101