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.
概要
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。
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Zi-yue LI, Qing-ting BU, Jue WANG, and Yu LIU performed the experiments. Zi-yue LI and Qing-ting BU wrote the manuscript. Xin-ai CHEN, Xu-ming MAO, and Yong-Quan LI revised the manuscript. All authors read and approved the final manuscript. Therefore, all authors had full access to all the data in the study and take responsibility for the integrity and security of the data.
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Zi-yue LI, Qing-ting BU, Jue WANG, Yu LIU, Xin-ai CHEN, Xu-ming MAO, and Yong-Quan LI declare that they have no conflict of interest.
This article does not contain any studies with human or animal subjects performed by any of the authors.
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Project supported by the National Natural Science Foundation of China (Nos. 31520103901 and 3173002)
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Li, Zy., Bu, Qt., Wang, J. et al. Activation of anthrachamycin biosynthesis in Streptomyces chattanoogensis L10 by site-directed mutagenesis of rpoB. J. Zhejiang Univ. Sci. B 20, 983–994 (2019). https://doi.org/10.1631/jzus.B1900344
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DOI: https://doi.org/10.1631/jzus.B1900344