产绿原酸内生菌细胞色素P450基因的克隆和功能研究  

Cloning and Function of Gene for Cytochrome P450 from a Chlorogenic Acid-producing Endophytic Bacterium

作　　者：王川[1] 李丽[1] 魏丕伟[1] 黄非[2] 

机构地区：[1]四川理工学院生物工程学院,自贡643000 [2]四川大学生命科学学院,成都610064

出　　处：《生物技术通报》2016年第6期181-186,共6页Biotechnology Bulletin

基　　金：四川省教育厅理工科重点项目(14ZA0211)

摘　　要：以一株产绿原酸的内生枯草芽孢杆菌为基础,对其绿原酸途径的关键酶基因进行克隆和功能研究。克隆该内生菌的细胞色素P450基因并进行原核表达,对重组蛋白进行肉桂酸羟基化酶(C4H)酶活测定。结果显示,从内生菌中克隆了4个细胞色素P450酶系基因并进行了原核表达,其中P-4重组蛋白具有C4H活性,产生的香豆酸与LC-MS检测的结果一致,该酶的最适温度范围较宽,产物香豆酸对该酶有抑制作用。该内生菌可能利用其细胞色素P450酶系作为C4H的同工酶从而将产物导入绿原酸合成途径。Based on a chlorogenic acid-producing endophytic Bacillus subtilis,cloning and function of a key gene in the synthetic pathway of chlorogenic acid was studied. The cytochrome P450 gene of the endophyte was cloned and expressed in prokaryotic vector,and the cinnamate 4-hydroxylase（C4H）activity of the recombinant proteins were determined. Four cytochrome P450 genes were cloned and expressed in prokaryotic vector,and the recombinant protein P-4 presented C4H activity. The coumarate produced by the C4H activity was the same as the result measured by LC-MS. The activity of P-4 existed in a broad optimal temperature and was inhibited by product coumarate. It is supposed that,taking cytochrome P450 as isozyme of C4H,the endophyte led the product coumarate to the synthetic pathway of chlorogenic acid.

关 键 词：绿原酸 细胞色素P450 C4H 表达 活性 

分 类 号：Q78[生物学—分子生物学] Q93