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

• DOI: 10.1007/s00253-014-6369-0
• Publication Date: 2015-01-20T06:27:27Z
• Authors: Pengwei Li; Jine Li; Zhengyan Guo; Wei Tang; Jianshan Han; Xiangxi Meng; Tingting Hao; Yaxin Zhu; Lixin Zhang; Yihua Chen
• Keywords: DNA, Bacterial; Genetics, Microbial; Recombination, Genetic; 0301 basic medicine; Genetic Vectors; Molecular Sequence Data; Sequence Analysis, DNA; Streptomyces; 03 medical and health sciences; Genes, Bacterial; Genes, Reporter; Gene Silencing; Genetic Testing; Molecular Biology; Crosses, Genetic; Piperidones; Plasmids

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.