杀粉蝶菌素A1生物合成基因簇中甲基转移酶PieB2的功能  

Function of methyltransferase gene pieB2 in the biosynthetic cluster of piericidin A1

作　　者：赵震宇[1] 刘倩[1] 由德林[1] 

机构地区：[1]上海交通大学生命科学技术学院,微生物代谢国家重点实验室,上海200240

出　　处：《微生物学报》2016年第7期1186-1193,共8页Acta Microbiologica Sinica

基　　金：国家自然科学基金(31170085);国家“973计划”(2012CB721004)~~

摘　　要：【目的】研究杀粉蝶菌素A1产生菌中甲基转移酶基因pieB2的功能。【方法】利用接合转移和同源重组双交换的方法,构建pieB2基因缺失突变株,以及利用接合转移的方法,构建回补菌株。通过高保真PCR克隆pieB2基因到表达载体pET28a上,构建质粒pJTU5997,转化入大肠杆菌E.coliBL21(DE3)/pLysE中诱导表达。利用高效液相色谱检测PieB2的体外酶活。【结果】获得了pieB2基因缺失的双交换突变株。发酵结果显示,该突变株不再产生杀粉蝶菌素A1,而是积累了一种脱甲基产物。N-末端融合组氨酸标签的PieB2在大肠杆菌中获得可溶性表达,通过体外催化证明了PieB2甲基转移酶的功能。【结论】体内遗传实验和体外生化实验证明了PieB2作为甲基转移酶在杀粉蝶菌素A1合成中的作用。[Objective] In order to obtain piericidin intermediates of low toxicity by metabolic engineering,we studied the function of methyltransferase gene pieB2 in the biosynthetic cluster of piericidin A1.[Methods] The methyltransferase pieB2 gene disrupted Streptomyces piomogeues var.Hangzhouwanensis was constructedby double crossover recombination.The methyltransferase gene pieB2 was also PCR amplified and cloned into the plasmid p ET28 a for overexpressing N-（His）6-tag pieB2 in E.coli BL21（DE3）/p Lys E.The recombinant PieB2 was purified by affinity chromatography via AKTA FPLC system.The pieB2 catalyzed reactions were performed using SAM and demethyl-piericidinas substrates.[Results] The disruption mutant LQ-9 produced demethyl-piericidininstead of piereicidin A1,which was restored by in trans complementation of the pieB2 gene.The N-（His）6-tag pieB2 was expressed in E.coli in soluble form and was successfully purified via Ni-（2＋） mediated affinity chromatography.In vitro biochemical experiments showed that PieB2 could convert demethyl-piericidininto piereicidin A1 in the presence of SAM.The demethyl-piericidin intermediat showed an attractive biological activities as well as piericidin A1.[Conclusion] We confirmed that pieB2 is function as a SAM dependent methyltransferase in the biosynthetic gene cluster of piericidin A1.

关 键 词：杀粉蝶菌素A1 甲基转移酶 基因敲除 生物合成 

分 类 号：TQ453[化学工程—农药化工] Q78[生物学—分子生物学]