Development of a pyrF-based counterselectable system for targeted gene deletion in Streptomyces rimosus

Streptomyces produces many valuable and important biomolecules with clinical and pharmaceutical applications. The development of simple and highly efficient gene editing tools for genetic modification of Streptomyces is highly desirable. In this study, we developed a screening system for targeted gene knockout using a uracil auxotrophic host(Δpyr F) resistant to the highly toxic uracil analog of 5-fluoroorotic acid(5-FOA) converted by Pyr F, and a non-replicative vector p KC1132-pyr F carrying the complemented pyr F gene coding for orotidine-5'-phosphate decarboxylase. The pyr F gene acts as a positive selection and counterselection marker for recombinants during genetic modifications. Single-crossover homologous integration mutants were selected on minimal medium without uracil by reintroducing pyr F along with p KC1132-pyr F into the genome of the mutant Δpyr F at the targeted locus. Double-crossover recombinants were generated, from which the pyr F gene, plasmid backbone, and targeted gene were excised through homologous recombination exchange. These recombinants were rapidly screened by the counterselection agent, 5-FOA. We demonstrated the feasibility and advantage of using this pyr F-based screening system through deleting the otc R gene, which encodes the cluster-situated regulator that directly activates oxytetracycline biosynthesis in Streptomyces rimosus M4018. This system provides a new genetic tool for investigating the genetic characteristics of Streptomyces species.