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Zilong LI


Shouliang YIN


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Journal of Zhejiang University SCIENCE B 2021 Vol.22 No.5 P.383-396


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

Author(s):  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

Affiliation(s):  School of Life Sciences, North China University of Science and Technology, Tangshan 063210, China; more

Corresponding email(s):   lizl@im.ac.cn, yinsl@ncst.edu.cn

Key Words:  Counterselectable system, pyrF, 5-Fluoroorotic acid (5-FOA), Gene deletion, Streptomyces rimosus

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[J]. Journal of Zhejiang University Science B, 2021, 22(5): 383-396.

@article{title="Development of a pyrF-based counterselectable system for targeted gene deletion in Streptomyces rimosus",
author="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",
journal="Journal of Zhejiang University Science B",
publisher="Zhejiang University Press & Springer",

%0 Journal Article
%T Development of a pyrF-based counterselectable system for targeted gene deletion in Streptomyces rimosus
%A Yiying YANG
%A Qingqing SUN
%A Yang LIU
%A Hanzhi YIN
%A Wenping YANG
%A Yang WANG
%A Ying LIU
%A Yuxian LI
%A Shen PANG
%A Wenxi LIU
%A Fang YUAN
%A Shiwen QIU
%A Jiong LI
%A Xuefeng WANG
%A Keqiang FAN
%A Weishan WANG
%A Zilong LI
%A Shouliang YIN
%J Journal of Zhejiang University SCIENCE B
%V 22
%N 5
%P 383-396
%@ 1673-1581
%D 2021
%I Zhejiang University Press & Springer
%DOI 10.1631/jzus.B2000606

T1 - Development of a pyrF-based counterselectable system for targeted gene deletion in Streptomyces rimosus
A1 - Yiying YANG
A1 - Qingqing SUN
A1 - Yang LIU
A1 - Hanzhi YIN
A1 - Wenping YANG
A1 - Yang WANG
A1 - Ying LIU
A1 - Yuxian LI
A1 - Shen PANG
A1 - Wenxi LIU
A1 - Qian ZHANG
A1 - Fang YUAN
A1 - Shiwen QIU
A1 - Jiong LI
A1 - Xuefeng WANG
A1 - Keqiang FAN
A1 - Weishan WANG
A1 - Zilong LI
A1 - Shouliang YIN
J0 - Journal of Zhejiang University Science B
VL - 22
IS - 5
SP - 383
EP - 396
%@ 1673-1581
Y1 - 2021
PB - Zhejiang University Press & Springer
ER -
DOI - 10.1631/jzus.B2000606

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.




Darkslateblue:Affiliate; Royal Blue:Author; Turquoise:Article


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