Heterologous or homologous expression of an entire tautomycetin biosynthetic gene cluster in Streptomyces hosts using E. coli-Streptomyces shuttle bacterial artificial chromosome system

Developments of next-generation sequencing technologies have brought recognition of microbial genomes as a rich source of diverse natural products. Functional expression of an entire secondary metabolite biosynthetic pathway gene cluster is an attractive alternative to facilitate production improvement and biosynthetic modification of a potentially-valuable natural product derived from various genetically-recalcitrant Streptomyces species. Using a versatile Escherichia coli-Streptomyces shuttle Bacterial Artificial Chromosomal (BAC) for genome engineering techniques as well as PCR-targeted gene manipulation, here we show the both homologous and heterologous expressions of an entire biosynthetic gene cluster of tautomycetin (TMC), a protein phosphatase PP1/PP2A inhibitor and T-cell-specific immunosuppressant. Unique XbaI restriction sites were first inserted at the both border regions of the TMC biosynthetic gene cluster (approximately 100 kb) in the chromosome of Streptomyces sp. CK4412, followed by site-specific recombination of the modified pSBAC into the flanking region of the TMC gene cluster. The entire TMC gene cluster was then recovered as a giant single recombinant pSBAC by the XbaI digestion of the chromosomal DNA and the subsequent self-ligation. The recombinant pSBAC construct containing the entire TMC cluster in E. coli was conjugated into the model Streptomyces strains such as S. lividans or S. coelicolor, resulting in the fast and enhanced TMC production. Moreover, re-introduction of the TMC cluster-containing pSBAC into the wild-type Streptomyces sp. CK4412 as well as the TMC cluster-containing S. coelicolor resulted in a tandem repeat of an entire TMC cluster in the chromosome with increased TMC productivities.