Study on biosynthesis of bufadienolide precursor through whole⁃cell catalysis

HU Yaoting; XIAO Chuanguang; CHEN Wenjie; WANG Rufeng; YANG Li; ZHAO Shujuan

doi:10.16306/j.1008-861x.2022.05.008

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Study on biosynthesis of bufadienolide precursor through whole⁃cell catalysis

Discipline Frontiers | 更新时间：2022-11-01

- Study on biosynthesis of bufadienolide precursor through whole⁃cell catalysis
- Academic Journal of Shanghai University of Traditional Chinese Medicine Vol. 36, Issue 5, Pages: 39-45(2022)
- 作者机构：
  
  1.上海中医药大学中药研究所，国家中医药管理局中药新资源与品质评价重点研究室，教育部中药标准化重点研究室，上海市中药复方重点实验室（上海　201203）
- 作者简介：
- 基金信息：
- DOI：10.16306/j.1008-861x.2022.05.008
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胡耀廷,肖传光,陈文杰等.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[J].上海中医药大学学报,2022,36(05):39-45.

HU Yaoting,XIAO Chuanguang,CHEN Wenjie,et al.Study on biosynthesis of bufadienolide precursor through whole⁃cell catalysis[J].Academic Journal of Shanghai University of Traditional Chinese Medicine,2022,36(05):39-45.
胡耀廷,肖传光,陈文杰等.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[J].上海中医药大学学报,2022,36(05):39-45. DOI： 10.16306/j.1008-861x.2022.05.008.

HU Yaoting,XIAO Chuanguang,CHEN Wenjie,et al.Study on biosynthesis of bufadienolide precursor through whole⁃cell catalysis[J].Academic Journal of Shanghai University of Traditional Chinese Medicine,2022,36(05):39-45. DOI： 10.16306/j.1008-861x.2022.05.008.

摘要

目的,2,将胆固醇生物转化成7α-羟基-4-胆甾烯-3-酮。,方法,2,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,2,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,2,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。

Abstract

Objective： To bioconvert cholesterol to 7α-hydroxy-4-cholesten-3-one.,Methods,2,Bufo bufo gargarizens, Cantor-derived 3β-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,2,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,2,Bg,-,HSD3B7, gene and ,Bg,-,CPR, gene were cloned from ,Bufo bufo gargarizans, Cantor and ,Rn,-,CYP7A1, 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.

关键词

中华大蟾蜍胆固醇工程菌3β羟基类固醇脱氢酶细胞色素P450还原酶

Keywords

Bufo bufo gargarizans Cantorcholesterolengineering bacteria3β-hydroxysteroid dehydrogenasecytochrome P450 reductase

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