attB site disruption in marine Actinomyces sp M048 via DNA transformation of a site-specific integration vector

An efficient conjugation method has been developed for the marine Actinomyces sp. isolate M048 to facilitate the genetic manipulation of the chandrananimycin biosynthesis gene cluster. A phi C31-derived integration vector pIJ8600 containing oriT and attP fragments was introduced into strain M048 by bi-parental conjugation from Escherichia coli ET12567 to strain M048. Transformation efficiency was (6.38 +/- 0.41) x 10(-5) exconjugants per recipient spore. Analysis of eight exconjugants showed that the plasmid pIJ8600 was stably integrated at a single chromosomal site (attB) of the Actinomyces genome. The DNA sequence of the attB was cloned and shown to be conserved. The results of antimicrobial activity analysis indicated that the insertion of plasmid pIJ8600 seemed to affect the biosynthesis of antibiotics that could strongly inhibit the growth of E. coli and Mucor miehei (Tu284). HPLC-MS analysis of the extracts indicated that disruption of the attB site resulted in the complete abolition of chandrananimycin A-C production, proving the identity of the gene cluster. Instead of chandrananimycins, two bafilomycins were produced through disruption of the attB site from the chromosomal DNA of marine Actinomyces sp. M048.