Site - Specific Recombination On The Genetic Modification Of Streptomyces And The Cloning Of Antibiotic Gene Clusters

Streptomyces are known for producing a number of important natural compounds by secondary metabolism, especially the antibiotics with biological activities. Currently, gene function studies involved with antibiotic-biosynthesizing are generally through homologous genomic recombination by gene-targeting vectors. Here, we present a rapid and efficient method for construction of gene-targeting vectors. This approach is based on Streptomyces phage φBT1integrase-mediated multisite in vitro site-specific recombination. The destination gene-targeting vector could be generated by a one-step reaction. This method is time-effective to perform the replacement of the target gene. After targeted modification of the genome, the selectable markers could be precisely excised from the chromosome by φC31integrase-mediated in vivo site-specific recombination between pre-placed attB and attP sites. Thus, we are able to implement genetic modification of the genome repeatedly. Using this method, we deleted part of the calcium-dependent antibiotic (CDA) and actinorhodin (Act) biosynthetic gene clusters in Streptomyces coelicolor M145, and the synthesis of CDA and Act was disrupted. On this basis, the rrdA encoding RrdA, a negative regulator of Red production, was further deleted to generate the mutant strain ZB8. The final undecylprodigiosin production of the engineered strain was over five times that of the wild-type strain.The technology of site-specific recombination presents huge potential for rapid gene-targeting vector construction and marker removal in streptomycetes. Additionally, in our work, we also present a different site-specific recombination strategy for the cloning of antibiotic biosynthetic gene cluster based on φBT1integrase. The whole process could be completed by two steps. First, the recognition sites attB and attP were inserted into both sides of the target gene cluster by homologous recombination; then the gene cluster was cyclized by φBT1integrase catalyzing site-specific recombination reaction in vitro between attB and attP. The approach has been validated by successfully cloning55kb-size erythromycin biosynthetic gene cluster. The gene cluster was further integrated into the chromosome by attP site of φC31integrase and expressed in engineering Streptomyces coelicolor and other heterologous hosts. The new compounds were discovered by mass spectrometric detection.In summary, our methods provide new tools for the genetic modification of the genome and new opportunities for functional gene characterization in Streptomyces.