School of Life Sciences, North China University of Science and Technology, Tangshan 063210, China.
State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China.
J Zhejiang Univ Sci B. 2021 May 15;22(5):383-396. doi: 10.1631/jzus.B2000606.
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 is highly desirable. In this study, we developed a screening system for targeted gene knockout using a uracil auxotrophic host (Δ) 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 gene coding for orotidine-5'-phosphate decarboxylase. The 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 along with pKC1132-pyrF into the genome of the mutant Δ at the targeted locus. Double-crossover recombinants were generated, from which the 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 based screening system through deleting the gene, which encodes the cluster-situated regulator that directly activates oxytetracycline biosynthesis in M4018. This system provides a new genetic tool for investigating the genetic characteristics of species.
产生了许多具有临床和药物应用价值的重要生物分子。因此，开发简单高效的基因编辑工具来遗传修饰是非常需要的。在这项研究中，我们开发了一种使用尿嘧啶营养缺陷型宿主（Δ）的靶向基因敲除筛选系统，该宿主对由 PyrF 转化的 5-氟乳清酸（5-FOA）的高度毒性尿嘧啶类似物具有抗性，并且携带非复制载体 pKC1132-pyrF 携带编码乳清酸-5'-磷酸脱羧酶的互补 基因。 基因在遗传修饰过程中作为重组体的正选择和负选择标记。通过将 连同 pKC1132-pyrF 一起重新引入到突变体 Δ的靶向基因座的基因组中，在不含尿嘧啶的最低培养基上选择单交换同源整合突变体。通过同源重组交换从这些突变体中产生双交换重组体，其中切除了 基因、质粒骨架和靶向基因。这些重组体通过筛选剂 5-FOA 进行快速筛选。我们通过删除 基因证明了该基于筛选系统的可行性和优势，该基因编码簇定位的调节剂，该调节剂直接激活 M4018 中的土霉素生物合成。该系统为研究 种的遗传特征提供了一种新的遗传工具。
