title: Large-Scale Transposition Mutagenesis of Streptomyces coelicolor Identifies Hundreds of Genes Influencing Antibiotic Biosynthesis.
abstract: Gram-positiveStreptomycesbacteria produce thousands of bioactive secondary metabolites, including antibiotics. To systematically investigate genes affecting secondary metabolism, we developed a hyperactive transposase-based Tn5transposition system and employed it to mutagenize the model speciesStreptomyces coelicolor, leading to the identification of 51,443 transposition insertions. These insertions were distributed randomly along the chromosome except for some preferred regions associated with relatively low GC content in the chromosomal core. The base composition of the insertion site and its flanking sequences compiled from the 51,443 insertions implied a 19-bp expanded target site surrounding the insertion site, with a slight nucleic acid base preference in some positions, suggesting a relative randomness of Tn5transposition targeting in the high-GCStreptomycesgenome. From the mutagenesis library, 724 mutants involving 365 genes had altered levels of production of the tripyrrole antibiotic undecylprodigiosin (RED), including 17 genes in the RED biosynthetic gene cluster. Genetic complementation revealed that most of the insertions (more than two-thirds) were responsible for the changed antibiotic production. Genes associated with branched-chain amino acid biosynthesis, DNA metabolism, and protein modification affected RED production, and genes involved in signaling, stress, and transcriptional regulation were overrepresented. Some insertions caused dramatic changes in RED production, identifying future targets for strain improvement.IMPORTANCEHigh-GC Gram-positive streptomycetes and related actinomycetes have provided more than 100 clinical drugs used as antibiotics, immunosuppressants, and antitumor drugs. Their genomes harbor biosynthetic genes for many more unknown compounds with potential as future drugs. Here we developed a useful genome-wide mutagenesis tool based on the transposon Tn5for the study of secondary metabolism and its regulation. UsingStreptomyces coelicoloras a model strain, we found that chromosomal insertion was relatively random, except at some hot spots, though there was evidence of a slightly preferred 19-bp target site. We then used prodiginine production as a model to systematically survey genes affecting antibiotic biosynthesis, providing a global view of antibiotic regulation. The analysis revealed 348 genes that modulate antibiotic production, among which more than half act to reduce production. These might be valuable targets in future investigations of regulatory mechanisms, for strain improvement, and for the activation of silent biosynthetic gene clusters.
authors.affiliation: State Key Laboratory of Microbial Metabolism, Joint International Research Laboratory of Metabolic & Developmental Sciences, and School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, China.
authors.forename: Zhong
authors.initials: Z
authors.lastname: Xu
authors.affiliation: State Key Laboratory of Microbial Metabolism, Joint International Research Laboratory of Metabolic & Developmental Sciences, and School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, China.
authors.forename: Yemin
authors.initials: Y
authors.lastname: Wang
authors.affiliation: Department of Molecular Microbiology, John Innes Centre, Norwich, United Kingdom.
authors.forename: Keith F
authors.initials: KF
authors.lastname: Chater
authors.affiliation: State Key Laboratory of Microbial Metabolism, Joint International Research Laboratory of Metabolic & Developmental Sciences, and School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, China.
authors.forename: Hong-Yu
authors.initials: HY
authors.lastname: Ou
authors.affiliation: Department of Biological Sciences, California State University, Los Angeles, California, USA.
authors.forename: H Howard
authors.initials: HH
authors.lastname: Xu
authors.affiliation: State Key Laboratory of Microbial Metabolism, Joint International Research Laboratory of Metabolic & Developmental Sciences, and School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, China.
authors.forename: Zixin
authors.initials: Z
authors.lastname: Deng
authors.affiliation: State Key Laboratory of Microbial Metabolism, Joint International Research Laboratory of Metabolic & Developmental Sciences, and School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, China tao_meifeng@sjtu.edu.cn.
authors.forename: Meifeng
authors.initials: M
authors.lastname: Tao
mesh_terms: D000900:Anti-Bacterial Agents / Q000096:biosynthesis*; D001482:Base Composition / Q000235:genetics; D001483:Base Sequence; D004251:DNA Transposable Elements / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial; D015723:Gene Library; D016254:Mutagenesis, Insertional; D011353:Prodigiosin / Q000031:analogs & derivatives* / Q000096:biosynthesis; D064210:Secondary Metabolism / Q000235:genetics*; D048372:Streptomyces coelicolor / Q000235:genetics*; D019895:Transposases / Q000235:genetics*
chemical_list: D000900:Anti-Bacterial Agents; D004251:DNA Transposable Elements; C473503:Tn5 transposase; C010307:prodiginine; C047347:undecylprodigiosin; D019895:Transposases; D011353:Prodigiosin
keywords: Streptomyces coelicolor; antibiotic biosynthesis; genome-wide; prodiginine; transposition mutagenesis