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

【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,并在优化后的条件下通过接合转移将其转入异壁放线菌WH1-2216-6野生型菌株。【结果】获得了异壁放线菌WH1-2216-6菌株的浅蓝霉素二羟基苯甲酸甲酯AMP连接酶基因缺失的双交换突变株,发酵结果显示该突变株失去了产生浅蓝霉素A和浅蓝霉素D的能力。【结论】高浓度的MgSO4对大肠杆菌与异壁放线菌的接合转移有促进作用,介导了异壁放线菌WH1-2216-6菌株的遗传操作体系的成功建立,为进一步研究浅蓝霉素的生物合成基因和对浅蓝霉素结构进行组合生物合成改造奠定了基础,同时也为其他类似放线菌的遗传操作体系的建立提供了借鉴和参考。

[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.