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 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 offered a positive example for other Actinobacteria lacking an appropriate genetic manipulation system.