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[Development of a genetic modification system for caerulomycin producer Actinoalloteichus sp. WH1-2216-6]
Qinheng Lin1, Guangtao Zhang, Sumei Li
1Key Laboratory of Marine Bio-resources Sustainable Utilization, RNAM Center for Marine Microbiology, Guangdong Key Laboratory of Marine Materia Medica, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou 510301, China. linqinheng@163.com
Objective:
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.
Methods:
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.
Results:
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.
Conclusion:
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.
