转化次丹参酮二烯酿酒酵母全细胞催化体系的构建  

Construction of Saccharomyces cerevisiae whole-cell biocatalyst system for conversion miltiradiene

作　　者：蔡媛[1,2] 郭娟 周雍进[2] 朱志伟[2] 吴文燕[1] 黄璐琦[1] 陈敏[1] 赵宗保[2] 

机构地区：[1]中国中医科学院中药资源中心,北京100700 [2]中国科学院大连化学物理研究所生物技术部,辽宁大连116023

出　　处：《药学学报》2013年第10期1618-1623,共6页Acta Pharmaceutica Sinica

基　　金：国家自然科学基金资助项目(81202871;81072990);北京市教育委员会科学研究与研究生培养共建项目(学科建设)

摘　　要：丹参酮是中药丹参的重要活性成分之一,其代谢途径尚未明确。快速筛选和鉴定丹参酮生物合成途径相关基因具有重要意义。铁锈醇作为丹参酮代谢途径中间产物,其产量相对较低,为得到大量铁锈醇用来作为下游P450功能鉴定的底物进行下游途径解析,迫切需要建立高效转化次丹参酮二烯至铁锈醇的体系。本文以酿酒酵母为宿主,构建了含有催化次丹参酮二烯生成铁锈醇的CYP76AH1及P450还原酶SmCPR1全细胞催化体系,并在DNA及mRNA水平对其进行了表征。通过透性剂处理,实现了一步生物转化合成铁锈醇,转化效率达到了69.9%。该研究证实含有CYP76AH1及来源于丹参的细胞色素还原酶SmCPR1的酿酒酵母能够将次丹参酮二烯转化为铁锈醇,研究结果不仅为生物转化生产铁锈醇提供了一个有效平台,还可以用于鉴定丹参酮下游合成的其他CYP450的功能。Tanshinones are the bioactive components of the Chinese medicinal herb Salvia miltiorrhiza, while its biosynthetic pathway remains to be characterized. Rapid identification and characterization of the genes correlated to tanshinones biosynthesis is very important. As one of the intermediates of tanshinones biosynthesis, the ferruginol content is relative low in both root and engineered bacteria. It is urgent to construct an efficient system for conversion of miltiradiene to ferruginol to obtain large amount of ferruginol as the substrates for further identifying other downstream genes involved in tanshinones biosynthesis. In this study, we constructed the whole-cell yeast biocatalysts co-expressing miltiradiene oxidase CYP76AH1 and cytochrome P450 reductases （SmCPR1） from Salvia miltiorrhiza, and then characterized it with RT-PCR. After permeabilization, the yeast whole-cell could catalyze turnover of miltiradiene to ferruginol efficiently through single-step biotransformation with a conversion efficiency up to 69.9%. The yeast whole-cell biocatalyst described here not only provide an efficient platform for producing ferruginol in recombinant yeast but also an alternative strategy for identifying other CYP genes involved in tanshinones biosynthesis.

关 键 词：丹参 CYP76AH1 酿酒酵母 全细胞催化 

分 类 号：R931[医药卫生—生药学]