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

In this study, we developed a screening system for targeted gene knockout using a uracil auxotrophic host ( ΔpyrF) 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 complementedpyrF gene coding for orotidine-5 ′-phosphate decarboxylase. ThepyrF 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 reintroducingpyrF along with pKC1132-pyrF into the genome of the mutant ΔpyrF at the targeted locus. Double-crossover recombinants were generated, from which thepyrF 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 thispyrF-based screening system through deleting theotcR gene, which encodes the cluster-situated regulator that directly activates oxytetracycline biosynthesis inStreptomyces rimosus M4018. This system provides a new genetic tool for investigating the genetic characteristics ofStreptomyces species.
Source: Journal of Zhejiang University. Science. B. - Category: Universities & Medical Training Source Type: research