[Development of a genetic modification system for caerulomycin producer Actinoalloteichus sp. WH1-2216-6].

In order to enable the caerulomyicn biosynthetic study by in vivo gene disruptions, it is crucial to develop a genetic modification system for the producer Actinoalloteichus sp. WH1-2216-6. The spore germination timing and the concentration of MgSO4 in the medium were investigated for the optimal conjugal transfer of exotic pSET152 DNA into Actinoalloteichus sp. WH1-2216-6. Using the PCR-targeting system, we disrupted a putative caerulomycin 2,3-dihydroxybenzoate-AMP ligase gene by "in-frame deletion" in E. coli, to afford the cosmid pCSG2104, which was then transferred into Actinoalloteichus sp. WH1-2216-6 by conjugation under optimized conditions. The putative caerulomycin 2,3-dihydroxybenzoate-AMP ligase in Actinoalloteichus sp. WH1-2216-6 was successfully disrupted by in-frame replacement with the aac3IV gene cassette. The resulting mutant strain was unable to produce caerulomycins. The presence of high concentration of MgSO4 in the medium can promote the conjugation efficiency between E. coli and Actinoalloteichus sp. WH1-2216-6 and lead to the successful development of a genetic modification system for Actinoalloteichus sp. WH1-2216-6, enabling the functional characterization of caerulomycin biosynthetic genes in vivo. A positive example was provided for other Actinobacteria recalcitrant to genetic modification.