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

Journal of Zhejiang University. Science. B 2021 May 15

filter terms:

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 (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 complemented pyrF gene coding for orotidine-5'-phosphate decarboxylase. The pyrF 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 pyrF along with pKC1132-pyrF into the genome of the mutant ΔpyrF at the targeted locus. Double-crossover recombinants were generated, from which the pyrF 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 pyrF-based screening system through deleting the otcR 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.

Citation

Yiying Yang, Qingqing Sun, Yang Liu, Hanzhi Yin, Wenping Yang, Yang Wang, Ying Liu, Yuxian Li, Shen Pang, Wenxi Liu, Qian Zhang, Fang Yuan, Shiwen Qiu, Jiong Li, Xuefeng Wang, Keqiang Fan, Weishan Wang, Zilong Li, Shouliang Yin. Development of a pyrF-based counterselectable system for targeted gene deletion in Streptomyces rimosus. Journal of Zhejiang University. Science. B. 2021 May 15;22(5):383-396