Construction of over-expression shuttle vectors in Streptomyces

Two high-copy number vectors, pL97 and pL98, that contain the strong constitutive ermEp* from Saccharopolyspora erythraea and the ssrA promoter (ssrAp) from Streptomyces coelicolor, respectively, and an inducible high-copy vector, pL99, with the PnitA-NitR system from Rhodococcus rhodochrous, were constructed based on the pIJ101 replicon. These vectors have pUC18 and pIJ101 replication origins for high-copy plasmid number in Escherichia coli and Streptomyces, respectively, and the oriT (RK2) allows the efficient and convenient plasmid transfer from E. coli to Streptomyces. The transformants can be easily selected with apramycin. There is also a multiple cloning site (MCS) between promoter and terminator convenient for heterologous gene cloning. The enhanced green fluorescent protein (EGFP) gene and redD, a pathway-specific regulatory gene for the production of undecylprodigiosin in S. coelicolor, were inserted into these plasmids and could be over-expressed in S. coelicolor. Western blotting revealed that the expression of EGFP was dramatically increased compared to the cell with a single copy of EGFP. Furthermore, the production of undecylprodigiosin was also greatly enhanced in the cells constitutively over-expressing redD. Hence, the high-copy plasmids could be readily used to express foreign proteins and for production of secondary metabolites, especially the antibiotics, potentially for industrial applications.