台勾霉素生产菌指孢囊菌NRRL 18085遗传操作体系的建立

Genetic modification system for tiacumicin producer Dactylosporangium aurantiacum NRRL 18085

摘要:

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 NRRL 18085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NRRL 18085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NRRL 18085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NRRL 18085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NRRL 18085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。

Abstract:

Abstract: [Objective] To optimize the production of tiacumicin B in Dactylosporangium aurantiacum NRRL 18085, we developed a genetic modification system by 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 PCR-targeting 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 offer a positive example for other Actinobacteria lacking an appropriate genetic manipulation system.