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An efficient blue-white screening based gene inactivation system for Streptomyces

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Abstract

Streptomyces is studied intensively for its outstanding ability to produce bioactive secondary metabolites and for its complicated morphological differentiation process. A classical genetic manipulation system for Streptomyces has been developed and widely used in the community for a long time, using antibiotic resistance markers to select for double-crossover mutants. The screening process is always laborious and time-consuming. However, the lack of a suitable chromogenic reporter for Streptomyces has limited the use of color-based screening system to simplify the selection process for double-crossover mutants. In this study, a blue reporter system for Streptomyces has been established by mining an indigoidine synthetase gene (idgS) from Streptomyces lavendulae CGMCC 4.1386, leading to the development of a time-saving gene inactivation system for Streptomyces by simple blue-white screening. A series of Streptomyces suicide and temperature-sensitive plasmids containing the idgS reporter cassette were constructed and used successfully to inactivate genes in Streptomyces, allowing a simple and efficient screening method to differentiate the colonies for double-crossover (white) and single-crossover (blue) mutants. Inactivation of the putative γ-butyrolactone synthase gene afsA-y via the idgS-based blue-white screening method revealed that the paulomycin production is negatively controlled by afsA-y in Streptomyces sp. YN86.

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Acknowledgments

We thank Dr. Mervyn J. Bibb, the John Innes Center, Norwich, UK, for providing the S. coelicolor M1146 strain; Dr. Zhaoxin Lu, Nanjing Agriculture University, Nanjing, China, for providing the Streptomyces sp. GB-2 strain; and Dr. Gongli Tang, Shanghai Institute of Organic Chemistry, CAS, Shanghai, China, for providing the plasmid pTGE33. This work was supported in part by the MOST of China (2013CB734003 and 2015CB150602). L.Z. is an awardee for the National Distinguished Young Scholar Program in China. Y.C. is an awardee for the “Hundred Talents Program” of CAS.

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Authors and Affiliations

  1. State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, NO.1 Beichen West Road, Chaoyang District, Beijing, 100101, China

    Pengwei Li, Jine Li, Zhengyan Guo, Wei Tang, Jianshan Han, Xiangxi Meng, Tingting Hao, Yaxin Zhu & Yihua Chen

  2. CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100101, China

    Lixin Zhang

  3. University of Chinese Academy of Sciences, Beijing, 100049, China

    Pengwei Li, Wei Tang, Jianshan Han, Xiangxi Meng & Tingting Hao

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  1. Pengwei Li
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Correspondence to Yihua Chen.

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Li, P., Li, J., Guo, Z. et al. An efficient blue-white screening based gene inactivation system for Streptomyces . Appl Microbiol Biotechnol 99, 1923–1933 (2015). https://doi.org/10.1007/s00253-014-6369-0

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