The pSG5-based thermosensitive vector family for genome editing and gene expression in actinomycetes.

Abstract

Actinomycetes are the most important producers of secondary metabolites for medical, agricultural and industrial applications. Efficient engineering of bacterial genomes to improve their biosynthetic capabilities largely depends on the available arsenal of tools and vectors. One of the most widely used vector systems for actinomycetes is derived from the Streptomyces ghanaensis DSM2932 plasmid pSG5. pSG5 is a broad host range multicopy plasmid replicating via a rolling circle mechanism. The unique feature of pSG5, which distinguishes it from other Streptomyces plasmids, is its naturally thermosensitive mode of replication. This allows the efficient elimination of the plasmid from its host by simply shifting the incubation temperature to non-permissive 37-39°C. This property makes pSG5 derivatives ideal facultative suicide vectors required for selection of gene disruption/gene replacement, transposon delivery or CRISPR/Cas9-mediated genome editing. Whereas these techniques depend on the fast elimination of the vector, stably replicating expression vectors for the production of recombinant proteins have been constructed more recently. This mini-review describes the generation and application of the pSG5 vector family, highlighting the specific features of the distinct vector plasmids.