[Genetic manipulation system for tiacumicin producer Dactylosporangium aurantiacum NRRL 18085].

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.