Genetic modification system for tiacumicin producer Dactylosporangium aurantiacum NRRL 18085

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.