浅蓝霉素产生菌海洋异壁放线菌WH1-2216-6 遗传操作体系的建立

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

摘要:

摘要: 【目的】建立二联吡啶类抗生素浅蓝霉素的产生菌海洋异壁放线菌Actinoalloteichus sp. WH1-2216-6的遗传操作体系，以便对浅蓝霉素的相关生物合成基因进行体内敲除和基因回补等遗传操作。【方法】以整合型质粒pSET152 为外源DNA，探索和优化了异壁放线菌WH1-2216-6 菌株与大肠杆菌进行接合转移实验的方法和条件，以此为基础，利用PCR-targeting 系统在体外构建了一个浅蓝霉素二羟基苯甲酸甲酯AMP 连接酶基因敲除的cosmid 质粒pCSG2104，并在优化后的

关键词:关键词: 异壁放线菌，遗传体系，浅蓝霉素，生物合成，二羟基苯甲酸甲酯AMP 连接酶

Abstract:

Abstract: ［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．

Key words:Keywords: actinoalloteichus，genetic manipulation system，caerulomycin，biosynthesis，2，3-dihydroxybenzoate-AMP ligase