Development of a -based counterselectable system for targeted gene deletion in .

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 is highly desirable. In this study, we developed a screening system for targeted gene knockout using a uracil auxotrophic host (Δ) resistant to the highly toxic uracil analog of 5-fluoroorotic acid (5-FOA) converted by PyrF, and a non-replicative vector pKC1132-pyrF carrying the complemented gene coding for orotidine-5'-phosphate decarboxylase. The 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 along with pKC1132-pyrF into the genome of the mutant Δ at the targeted locus. Double-crossover recombinants were generated, from which the 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 based screening system through deleting the gene, which encodes the cluster-situated regulator that directly activates oxytetracycline biosynthesis in M4018. This system provides a new genetic tool for investigating the genetic characteristics of species.