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Suppl. Mater.: 

CLC number: Q756

On-line Access: 2019-11-21

Received: 2019-06-26

Revision Accepted: 2019-09-18

Crosschecked: 2019-11-01

Cited: 0

Clicked: 2308

Citations:  Bibtex RefMan EndNote GB/T7714

 ORCID:

Yong-Quan Li

https://orcid.org/0000-0001-6013-4068

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Journal of Zhejiang University SCIENCE B 2019 Vol.20 No.12 P.983-994

http://doi.org/10.1631/jzus.B1900344


Activation of anthrachamycin biosynthesis in Streptomyces chattanoogensis L10 by site-directed mutagenesis of rpoB


Author(s):  Zi-Yue Li, Qing-Ting Bu, Jue Wang, Yu Liu, Xin-Ai Chen, Xu-Ming Mao, Yong-Quan Li

Affiliation(s):  Institute of Pharmaceutical Biotechnology & First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou 310058, China; more

Corresponding email(s):   lyq@zju.edu.cn

Key Words:  Streptomyces, Cryptic gene cluster, Site-directed mutagenesis, Secondary metabolism


Zi-Yue Li, Qing-Ting Bu, Jue Wang, Yu Liu, Xin-Ai Chen, Xu-Ming Mao, Yong-Quan Li. Activation of anthrachamycin biosynthesis in Streptomyces chattanoogensis L10 by site-directed mutagenesis of rpoB[J]. Journal of Zhejiang University Science B, 2019, 20(12): 983-994.

@article{title="Activation of anthrachamycin biosynthesis in Streptomyces chattanoogensis L10 by site-directed mutagenesis of rpoB",
author="Zi-Yue Li, Qing-Ting Bu, Jue Wang, Yu Liu, Xin-Ai Chen, Xu-Ming Mao, Yong-Quan Li",
journal="Journal of Zhejiang University Science B",
volume="20",
number="12",
pages="983-994",
year="2019",
publisher="Zhejiang University Press & Springer",
doi="10.1631/jzus.B1900344"
}

%0 Journal Article
%T Activation of anthrachamycin biosynthesis in Streptomyces chattanoogensis L10 by site-directed mutagenesis of rpoB
%A Zi-Yue Li
%A Qing-Ting Bu
%A Jue Wang
%A Yu Liu
%A Xin-Ai Chen
%A Xu-Ming Mao
%A Yong-Quan Li
%J Journal of Zhejiang University SCIENCE B
%V 20
%N 12
%P 983-994
%@ 1673-1581
%D 2019
%I Zhejiang University Press & Springer
%DOI 10.1631/jzus.B1900344

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T1 - Activation of anthrachamycin biosynthesis in Streptomyces chattanoogensis L10 by site-directed mutagenesis of rpoB
A1 - Zi-Yue Li
A1 - Qing-Ting Bu
A1 - Jue Wang
A1 - Yu Liu
A1 - Xin-Ai Chen
A1 - Xu-Ming Mao
A1 - Yong-Quan Li
J0 - Journal of Zhejiang University Science B
VL - 20
IS - 12
SP - 983
EP - 994
%@ 1673-1581
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PB - Zhejiang University Press & Springer
ER -
DOI - 10.1631/jzus.B1900344


Abstract: 
Genome sequencing projects revealed massive cryptic gene clusters encoding the undiscovered secondary metabolites in Streptomyces. To investigate the metabolic products of silent gene clusters in Streptomyces chattanoogensis L10 (CGMCC 2644), we used site-directed mutagenesis to generate ten mutants with point mutations in the highly conserved region of rpsL (encoding the ribosomal protein S12) or rpoB (encoding the RNA polymerase β-subunit). Among them, L10/RpoB (H437Y) accumulated a dark pigment on a yeast extract-malt extract-glucose (YMG) plate. This was absent in the wild type. After further investigation, a novel angucycline antibiotic named anthrachamycin was isolated and determined using nuclear magnetic resonance (NMR) spectroscopic techniques. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis and electrophoretic mobility shift assay (EMSA) were performed to investigate the mechanism underlying the activation effect on the anthrachamycin biosynthetic gene cluster. This work indicated that the rpoB-specific missense H437Y mutation had activated anthrachamycin biosynthesis in S. chattanoogensis L10. This may be helpful in the investigation of the pleiotropic regulation system in Streptomyces.

恰塔努加链霉菌L10中rpoB基因突变激活蒽塔恰霉素生物合成基因簇的研究

目的:运用核糖体技术激活恰塔努加链霉菌Streptomyces chattanoogensis L10中的隐性基因簇,进一步研究突变菌株的次级代谢产物并初步探索其对应的生物合成基因簇激活机制.
创新点:首次分离得到了蒽塔恰霉素,并初步探索了RpoB突变株中蒽塔恰霉素生物合成基因簇的激活机制.
方法:采用核糖体工程技术,对S.chattanoogensis L10的RpsL和RpoB的高度保守区域定点突变,使用高效液相色谱法(HPLC)检测突变株的代谢产物.运用一维和二维核磁共振(1D NMR、 2D NMR)解析L10/RpoB (H437Y)的次级代谢产物蒽塔恰霉素的化学结构,通过铁离子还原法(FRAP)和ABTS自由基清除等实验研究其抗氧化活性.采用实时荧光定量聚合酶链式反应(qRT-PCR)和凝胶电泳迁移率分析(EMSA)探索蒽塔恰霉素生物合成基因簇的激活机制.
结论:L10/RpoB (H437Y)中蒽塔恰霉素的生物合成基因簇被激活.qRT-PCR和EMSA结果表明:RNA聚合酶β亚基结构改变,可能影响全局性调控基因的转录水平,并激活蒽塔恰霉素生物合成基因簇.

关键词:链霉菌;隐性基因簇;定点突变;次级代谢产物

Darkslateblue:Affiliate; Royal Blue:Author; Turquoise:Article

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