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On-line Access: 2021-05-07

Received: 2022-04-22

Revision Accepted: 2020-12-23

Crosschecked: 2022-04-22

Cited: 0

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Citations:  Bibtex RefMan EndNote GB/T7714

 ORCID:

Zilong LI

https://orcid.org/0000-0002-4568-6902

Shouliang YIN

https://orcid.org/0000-0001-5087-3289

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

http://doi.org/10.1631/jzus.B2000606


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",
volume="22",
number="5",
pages="383-396",
year="2021",
publisher="Zhejiang University Press & Springer",
doi="10.1631/jzus.B2000606"
}

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%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 Qian ZHANG
%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

TY - JOUR
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


Abstract: 
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.

基于pyrF作为反选标记构建适用于龟裂链霉菌的基因敲除系统

目的:pyrF作为反选标记基因构建基因敲除系统,快速、高效敲除龟裂链霉菌的靶基因。
创新点:在链霉菌种属内首次采用pyrF作为反选标记基因,以5-氟乳清酸作为筛选因子,实现靶基因的敲除。与传统的敲除方法相比,该敲除系统获得同源双交换突变株的时间大大缩短。
方法:基于pyrF反选标记的基因敲除系统包括一个尿嘧啶营养缺陷型宿主(ΔpyrF)和一个携带互补基因pyrF的非自主复制型的敲除载体(pKC1132-pyrF)。第一步,将携带有互补基因pyrF的敲除质粒导入到尿嘧啶营养缺陷型宿主(ΔpyrF)中,只有发生单交换重组的突变株,才能在缺少尿嘧啶的基本培养基中生长;第二步,将发生单交换的突变株接种到含有5-氟乳清酸的基本培养基中,只有发生第二次同源重组,且被敲除的靶基因、pyrF基因及质粒骨架发生重组丢失的突变株才能在含有5-氟乳清酸的培养基中生长。经过两步筛选,快速、高效实现靶基因的敲除。
结论:本文通过生物信息学比对、基因敲除和回补实验验证pyrF基因为乳清酸核苷-5’-磷酸脱羧酶的编码基因。利用pyrF基因作为反选标记,以5-氟乳清酸作为筛选因子,快速、高效的实现了龟裂霉菌的靶基因otcR的敲除。本文构建的基因敲除系统为链霉菌的分子遗传操作提供了一种新的遗传工具。

关键词:反选敲除系统;pyrF;5-氟乳清酸;基因敲除;龟裂链霉菌

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

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