利用Tn5型转座突变系统筛选高产阿维菌素菌株  

Screening of High Avermectin-producing Strains via Tn5 Transposon Mediated Mutagenesis

作　　者：冯宝琪 冯娇 张苗 刘洋 曹睿 尹涵之 齐凤仙 李子龙[3] 尹守亮 FENG Bao-qi;FENG Jiao;ZHANG Miao;LIU Yang;CAO Rui;YIN Han-zhi;QI Feng-xian;LI Zi-long;YIN Shou-liang(School of Basic Medicine Sciences,North China University of Science and Technology,Tangshan 063210,China;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)

机构地区：[1]华北理工大学基础医学院,唐山063210 [2]华北理工大学生命科学学院,唐山063210 [3]中国科学院微生物研究所,微生物资源前期开发国家重点实验室,北京100101

出　　处：《中国生物工程杂志》2021年第7期32-41,共10页China Biotechnology

基　　金：河北省科技计划农业关键共性技术攻关专项(18222916);河北省自然科学基金(C2019209399)资助项目。

摘　　要：目的:转座突变技术是发现新功能基因和获得高产天然产物菌株的一种有效策略。通过理性设计和构建Tn5型转座突变系统,并将其应用于阿维链霉菌,筛选高产阿维菌素的工程菌株。方法:在转座突变载体pUCTN转座插入片段的上游和下游分别引入链霉菌常用的强启动子kasOp*和P21,强化插入位置上游和下游基因的转录表达;在插入片段两端分别添加双向转录终止子T1和T2,有效终止插入序列两端靶基因的转录,引入强启动子和终止子的目的在于增强对转座突变株生理代谢活动的扰动。结果:通过优化供体菌和受体菌的比例,转座效率显著提高。随机选择500株转座突变株进行发酵和阿维菌素产量测试,筛选到3株突变株的阿维菌素产量明显高于出发菌株产量的50%以上。结论:Tn5转座突变系统为研究阿维链霉菌的基因功能和生理代谢提供了有效的分子遗传工具。Objective:Transposition is an effective strategy for discovering new functional genes and developing high-titer production strains.The Tn5-based transposon mutagenesis system was rationally designed and constructed,and applied to Streptomyces avermitilis to screen the engineered strains with high avermectin production.Methods:In pUCTN,two strong promoters'sequence of kasOp*and P21,commonly used in Streptomyces,were introduced into the upstream and downstream of the transposable elements,respectively,which can enhance the transcription and expression level of the genes located at the insertion position.Two transcriptional terminators T1 and T2 were also added into the upstream and downstream of the transposable elements to terminate the transcription of genes adjacent to the insertion site.The purpose of introducing promoters and terminators is to enhance the disturbance to the physiological metabolism of the mutants.Results:To improve the transposition efficiency,the ratio of donor and recipient bacteria was optimized,and 500 transposon mutants were selected and tested for avermectin production,3 high avermectin-producing strains were screened,and the yield is significantly increased 50%than that of the parent strain.Conclusion:This Tn5 transposon mutation system established in this paper provides an effective molecular genetic tool for exploring the gene function and physiological metabolism of Streptomyces avermitilis.

关 键 词：阿维链霉菌 阿维菌素 Tn5转座突变 分子遗传工具 

分 类 号：Q819[生物学—生物工程]