[Development of a genetic modification system for caerulomycin producer Actinoalloteichus sp. WH1-2216-6].
作者信息
Lin Qinheng, Zhang Guangtao, Li Sumei, Zhang Haibo, Ju Jianhua, Zhu Weiming, Zhang Changsheng
机构信息
Key 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.
出版信息
Wei Sheng Wu Xue Bao. 2011 Aug;51(8):1032-41.
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.
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