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Background  Direct cloning combined with heterologous expression of a secondary metabolite biosynthetic gene cluster has become a useful strategy for production improvement and pathway modification of potentially valuable natural products present at minute quantities in original isolates of actinomycetes. However, precise cloning and efficient overexpression of an entire biosynthetic gene cluster remains challenging due to the ineffectiveness of current genetic systems in manipulating large-sized gene clusters for heterologous as well as homologous expression.    Results  A versatile  Escherichia coli - Streptomyces  shuttle bacterial artificial chromosomal (BAC) conjugation vector, pSBAC, was used along with a cluster tandem integration approach to carry out homologous and heterologous overexpression of a large 80-kb polyketide biosynthetic pathway gene cluster of tautomycetin (TMC), which is a protein phosphatase PP1/PP2A inhibitor and T cell-specific immunosuppressant. Unique  Xba I restriction sites were precisely inserted at both border regions of the TMC biosynthetic gene cluster within the chromosome of TMC-producing  Streptomyces  sp. CK4412, followed by site-specific recombination of pSBAC into the flanking region of the TMC gene cluster. The entire TMC gene cluster was then rescued as a single giant recombinant pSBAC by  Xba I digestion of the chromosomal DNA as well as subsequent self-ligation. Next, the recombinant pSBAC construct containing the entire TMC cluster in  E. coli  was directly conjugated into model  Streptomyces  strains, resulting in rapid and enhanced TMC production. Moreover, introduction of the TMC cluster-containing pSBAC into wild-type  Streptomyces  sp. CK4412 as well as a recombinant  S. coelicolor  strain resulted in a chromosomal tandem repeat of the entire TMC cluster with 14-fold and 5.4-fold enhanced TMC productivities, respectively.    Conclusions  The 80-kb TMC biosynthetic gene cluster was isolated in a single integration vector, pSBAC. Introduction of TMC biosynthetic gene cluster in TMC non-producing strains has resulted in similar amount of TMC production yield. Moreover, over-expression of TMC biosynthetic gene cluster in original producing strain and recombinant  S. coelicolor  dramatically increased TMC production. Thus, this strategy can be employed to develop a custom overexpression scheme of entire metabolite pathway clusters present in actinomycetes.    Electronic supplementary material  The online version of this article (doi:10.1186/s12934-015-0325-2) contains supplementary material, which is available to authorized users.

Precise cloning and tandem integration of large polyketide biosynthetic gene cluster using Streptomyces artificial chromosome system