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【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】 The purpose of this study was to investigate the function of the methyltransferase gene pieB2 in the production of gadobiserin A1. 【Method】 PieB2 gene deletion mutant was constructed by conjugation transfer and homologous recombination double exchange, and the complementary strain was constructed by conjugation and transfer. The pieB2 gene was cloned into the expression vector pET28a by high-fidelity PCR to construct the plasmid pJTU5997, which was transformed into E.coli BL21 (DE3) / pLysE to induce expression. Detection of PieB2 in Vitro Enzyme Activity by High Performance Liquid Chromatography. 【Result】 The double-crossover mutant with deletion of the pieB2 gene was obtained. Fermentation results showed that the mutant no longer produce to kill siphonin A1, but the accumulation of a demethylation product. PieB2 with histidine tag fused to the N-terminus obtained soluble expression in E. coli, demonstrating the function of PieB2 methyltransferase by in vitro catalysis. 【Conclusion】 In vivo genetic experiments and in vitro biochemical experiments demonstrated the role of PieB2 as a methyltransferase in the synthesis of siphonin A1.