Evaluation of vector system in Saccharopolyspora erythraea and construction of new replicative vector

Saccharopolyspora erythraea is an industrially important actinobacteria and a model in polyketide biosynthesis studies. However, its genetic toolbox remains limited. Here, we evaluated the properties of integrative and replicative vectors in Sacch. erythraea DSM40517. Three actinophage-based integrative vectors (φC31, φBT1, VWB) were efficiently transferred into Sacch. erythraea. The attB sites for these vector systems were identified. In case of φBT1- and VWB-based vectors, unique integration sites exists within the chromosome of the strain. In case of pSET152 (φC31), at least four different loci were identified with different efficiency of recombination. Our data revealed that all three integrative vectors are excised from the chromosome of the strain with varying frequencies, highlighting the need for selective pressures for their stable maintenance. Commonly used replicative vectors with Streptomyces replicons (pIJ101 and pSG5) have low transfer efficiency in Sacch. erythraea. To solve this problem, we constructed a shuttle vector pYS191 based on the replicon of plasmid pJV1. This vector was efficiently transferred into Sacch. erythraea and other actinobacteria including Streptomyces, Saccharothrix and Couchioplanes species. pYS191 is maintained at a low copy number under selective conditions, and is rapidly lost without antibiotic pressure. Neither integrative nor replicative vectors significantly affected erythromycin production. This study provides a comprehensive assessment of vector system in Sacch. erythraea and reports a new replicative vector based on rarely exploited pJV1 replicon for transient gene expression and genome engineering. These results expand the genetic toolbox for Saccharopolyspora and facilitate its use in natural products research and industrial biotechnology.  KEY POINTS: • Actinobacterial integrative vectors are suited for gene cloning in Sacch. erythraea. • attB sites for three integrative vectors identified within Sacch. erythraea genome. • A new replicative vector pYS191 for use in Sacch. erythraea was constructed.