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

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. coli BL21(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。

关键词:杀粉蝶菌素A1;甲基转移酶;基因敲除;生物合成

Abstract:

[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 pET28a for overexpressing N-(His)₆-tag PieB2 in E. coli BL21(DE3)/pLysE. 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 Ni²⁺ 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.

Key words:piericidin A1;methyltransferase;gene disruption;biosynthesis