摘要
【目的】建立新型大环内酯类抗生素台勾霉素的生产菌指孢囊菌Dactylosporangium aurantiacum NRRL18085的遗传操作体系,实现台勾霉素相关生物合成基因的敲除突变。【方法】以整合型质粒pSET152为载体,建立了外源DNA通过接合转移进入指孢囊菌NRRL18085的操作方法和培养条件,利用PCR-targeting系统在体外构建了一个台勾霉素卤化酶基因敲除的cosmid质粒,通过接合转移转入到指孢囊菌NRRL18085野生菌中。【结果】获得了台勾霉素卤化酶基因敲除的指孢囊菌NRRL18085的双交换突变株,该突变株失去了产生台勾霉素的能力。【结论】成功建立和优化了指孢囊菌NRRL18085菌株的遗传操作体系,使得在体内分析和鉴定台勾霉素生物合成基因的功能成为可能,同时也为建立其他类似放线菌的遗传操作体系提供了参考。
[Objective] To optimize the production of tiacumicin B in Dactylosporangium aurantiacum NRRL 18085,we developed a genetic manipulation system for disrupting genes involved in tiacumicin biosynthesis.[Methods ] We developed a method of conjugation to transfer exotic DNA pSET152 into D.aurantiacum NRRL 18085.Using the PCRtargeting system,we disrupted a putative tiacumicin halogenase gene in vitro by＂in-frame deletion＂in E.coli,and then the resulting cosmid was transferred into D.aurantiacum NRRL 18085 by conjugation.[Results]The putative tiacumicin halogenase gene in D.aurantiacum NRRL 18085 was disrupted by in-frame deletion from a double-crossover recombination event.The resulting mutant strain lost the ability to produce tiacumicin B.[Conclusion]We developed a genetic manipulation system for D.aurantiacum NRRL 18085,enabling the functional characterization of tiacumicin biosynthetic genes in vivo,and we offered a positive example for other Actinobacteria lacking an appropriate genetic manipulation system.
出处
《微生物学报》
CAS
CSCD
北大核心
2010年第8期1014-1022,共9页
Acta Microbiologica Sinica
基金
中国科学院知识创新工程重要方向项目(KZCX2-YW-216)
国家自然科学基金青年科学基金(30900035)
中国博士后科学基金(20090460836)
中国科学院百人计划项目(08SL111002)~~
关键词
指孢囊菌
遗传体系
台勾霉素
生物合成
卤化酶
Dactylosporangium
genetic manipulation system
tiacumicin
biosynthesis
halogenase
