A new-engineered integrative tool to target the terminal compartment of the Streptomyces chromosome

Phages are a valuable resource for the genetic engineering of Streptomyces antibiotic-producing bacteria. Indeed, a few integrative vectors based on phage integrase are available to insert transgene at specific genomic loci. Chromosome conformation captures previously demonstrated that the Streptomyces linear chromosome is organized in two spatial compartments: The central compartment encompassing most conserved and highly expressed genes in exponential phase, and the terminal compartments enriched in poorly conserved sequences including specialized metabolite biosynthetic gene clusters. This study introduces a new integrative tool based a recently described phage, Samy, which specifically targets the terminal compartment of its native host chromosome. Samy is related to PhiC31 phage and, like this latter, encodes a serine integrase. Whereas PhiC31 targets a site generally located near the origin of replication, the Samy integration site is one of the farthest known attB sites from it. We demonstrated that the Samy integrase efficiently mediates the specific integration of a non-replicating plasmid in six Streptomyces strains from distinct clades. Bioinformatic analyses revealed that the Samy att B site is rather conserved, and located in the terminal compartment of most Streptomyces chromosomes. Unusually for a serine integrase, Samy-based integration system exhibits slight excision. The Samy orthogonal site-specific recombination system extends the range of genetic tools available for engineering the Streptomyces chromosome by broadening the range of targeted locations, especially within the terminal spatial compartment.