基于全细胞催化的蟾蜍二烯内酯前体化合物生物合成研究

Study on biosynthesis of bufadienolide precursor through whole-cell catalysis

目的:将胆固醇生物转化成7α-羟基-4-胆甾烯-3-酮。方法:PCR扩增中华大蟾蜍来源的3β羟基类固醇脱氢酶基因(Bg-HSD3B7)、细胞色素P450还原酶基因(Bg-CPR)、大鼠来源的细胞色素P450酶基因(Rn-CYP7A1),生物信息学网站ExPASy、TMHMM预测理化性质,采用不同的构建组合方式构建工程菌;以胆固醇为底物进行全细胞催化,高效液相色谱检测是否产生7-α羟基胆固醇中间产物和7α-羟基-4-胆甾烯-3-酮终产物。结果:克隆并以单顺反子方式构建到pETM6载体,获得带有3个基因Rn-CYP7A1、Bg-CPR、Bg-HSD3B7的大肠杆菌工程菌BL21(DE3),该工程菌可以全细胞催化方式将胆固醇转化生成7α-羟基-4-胆甾烯-3-酮,且没有中间产物。结论:克隆得到中华大蟾蜍来源的Bg-HSD3B7基因、Bg-CPR基因、大鼠来源的Rn-CYP7A1基因,构建了具有全细胞催化功能的工程大肠杆菌,可以催化胆固醇转化生成7α-羟基-4-胆甾烯-3-酮。

Objective:To bioconvert cholesterol to 7α-hydroxy-4-cholesten-3-one.Methods:Bufo bufo gargarizensCantor-derived3β-hydroxysteroid dehydrogenase gene(Bg-HSD3B7),cytochrome P450 reductase gene(Bg-CPR) and Rattus norvegicus-derived cytochrome P450 enzyme gene(Rn-CYP7A1)were amplified by PCR. Physicochemical properties were predicted by bioinformatics website ExPASy,TMHMM. The engineered bacterias were obtained by using different construction and combination methods. Whole-cell catalysis was performed using cholesterol as a substrate,and high-performance liquid chromatography was used to detect whether the intermediate product of 7α-hydroxycholesterol and the final product of 7α-hydroxy-4-cholesten-3-one were produced.Results:Three genes were cloned and constructed into pETM6 vector in a monocistronic manner and E. coli engineered bacteria BL21(DE3) with Rn-CYP7A1, Bg-CPR and Bg-HSD3B7 genes was obtained. This engineered bacteria could convert cholesterol to 7α-hydroxy-4-cholesten-3-one by whole-cell catalytic method without any intermediate.Conclusion:Bg-HSD3B7gene and Bg-CPR gene were cloned from Bufo bufo gargarizans Cantor and RnCYP7A1 gene was cloned from Rattus norvegicus. And the engineered E. coli with whole-cell catalytic function was constructed,which could catalyze cholesterol to 7-hydroxy-4-cholesten-3-one.