Large-Scale Transposition Mutagenesis of Streptomyces coelicolor Identifies Hundreds of Genes Influencing Antibiotic Biosynthesis

Gram-positive Streptomyces bacteria produce thousands of bioactive secondary metabolites including antibiotics. To systematically investigate genes affecting secondary metabolism, we developed a hyperactive transposase-based Tn 5 transposition system and employed it to mutagenize the model species Streptomyces 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. Base composition of the insertion site and its flanking sequences compiled from the 51,443 insertions inferred 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 Tn 5 transposition targeting in the high GC Streptomyces genome. 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. Importance High-GC gram positive streptomycetes and related actinomycetes have provided more than 100 clinical drugs used as antibiotics, immunosuppressants, and antitumors. Their genomes harbour biosynthetic genes for many more unknown compounds with potential as future drugs. Here we developed a useful genome-wide mutagenesis tool for the study of secondary metabolism and its regulation, based on transposon Tn 5 . Using Streptomyces coelicolor as a model strain, chromosomal insertion was relatively random, except for some hotspots, 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, in strain improvement, and in the activation of silent biosynthetic gene clusters.