A Perspective for Assembling SALP(Streptomyces Artificial Linear Plasmid): A Potential New Genetic Tool of Gene Manipulation System for Producing Bioactive Secondary Metabolites in Streptomyces

Abstract

Genetic systems for combinatorial biosynthesis of bioactive products including antibiotic have been developed in Streptomyces genus. Several shuttle vectors, including circular plasmid, cosmid, BAC (Bacterial Artificial Chromosome)/PAC (P1-derived Artificial Chromosome) between E. coli-Streptomyces have been used for manipulation of Streptomyces gene cluster for producing bioactive chemicals. Usually normal circular plasmids can carry only 1–20 kb DNA size. Cosmids are able to carry 37 to 52 kb of DNA, it was reported that cosmids have been successfully used for construction of Streptomyces genome libraries to clone biosynthetic gene clusters [2-7]. Nonetheless, most biosynthetic gene clusters for natural products are larger than the average capacity of common cosmid vectors, thus, vectors with large capacity such as BAC or PAC have been used to construct E. coli-Streptomyces artificial chromosomes for heterologous production of secondary metabolites [8-14]. The DNA fragments insertion loading capacity for BAC/PAC is from 100 kb up to 100-300 kb in E. coli cell, the large capacity of BAC/ PAC allows the gene manipulation of complete biosynthesis gene cluster of secondary metabolites in bacteria E. coli cell. However, E coli is not an ideal host for heterologous gene overexpression and production of Streptomyces biosynthesis gene cluster due to its lower genomic GC content compare to Streptomyces’s. In addition, BAC capacity can be up to 100 kb and more, but the copy number usually is low. Therefore, a shutter capability for the vector to introduce BAC back to Streptomyces host is required for efficient production. Intergeneric conjugation is a major approach for manipulating DNA shuttered between Streptomyces and E. coli [11].