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

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

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.

摘要

目的

以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。

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Acknowledgments

This work is supported by the Natural Science Foundation of Hebei Province (No. C2019209399), Tangshan Science and Technology Project (No. 20130208b), the Science and Technology Program of Hebei (No. 18222916), and the Research Fund for Top Discipline Construction of North China University of Science and Technology (No. 18060720), China.

We wish to thank Professor Meijin GUO (East China University of Science and Technology, Shanghai, China) for providing Streptomyces rimosus M4018. We dedicate this article to the memory of our friend and mentor Keqian YANG (Institute of Microbiology, Chinese Academy of Sciences, Beijing, China), who made important contributions to understanding the biosynthesis and function of secondary metabolites in Streptomyces.

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Correspondence to Zilong Li or Shouliang Yin.

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Author contributions

Yiying YANG, Qingqing SUN, and Yang LIU were responsible for the methodology, data curation, and formal analysis. Hanzhi YIN, Wenping YANG, and Yang WANG contributed to strain resources and supervision. Ying LIU and Yuxian LI contributed to the software and sequence alignment. Shen PANG, Wenxi LIU, Qian ZHANG, Fang YUAN, and Shiwen QIU contributed to the strain fermentation and composition analysis by HPLC. Jiong LI and Xuefeng WANG performed the fermentation experiments. Keqiang FAN, Weishan WANG, Zilong LI, and Shouliang YIN contributed to the investigation, project administration, supervision, writing, review, and editing. All authors have read and approved the final manuscript and, therefore, have full access to all the data in the study and take responsibility for the integrity and security of the data.

Compliance with ethics guidelines

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, and Shouliang YIN declare that they have no conflict of interest.

This article does not contain any studies with human or animal subjects performed by any of the authors.

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Table S1; Figs. S1 and S2

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Yang, Y., Sun, Q., Liu, Y. et al. Development of a pyrF-based counterselectable system for targeted gene deletion in Streptomyces rimosus. J. Zhejiang Univ. Sci. B 22, 383–396 (2021). https://doi.org/10.1631/jzus.B2000606

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Key words

  • Counterselectable system
  • pyrF
  • 5-Fluoroorotic acid (5-FOA)
  • Gene deletion
  • Streptomyces rimosus

关键词

  • 反选敲除系统
  • pyrF
  • 5-氟乳清酸
  • 基因敲除
  • 龟裂链霉菌