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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。
Tanshinone is one of the important active ingredients of traditional Chinese medicine Salvia miltiorrhiza, its metabolic pathway is not yet clear. Rapid screening and identification of tanshinone biosynthetic pathway related genes have important significance. As a middle product of tanshinone metabolic pathway, rust alcohol has a relatively low yield. In order to obtain a large amount of rust alcohol for downstream function analysis of downstream P450 function identification, it is urgent to establish a system to efficiently convert tanshinone to rust rust alcohol. In this paper, we constructed the whole-cell catalytic system of CYP76AH1 and P450 reductase SmCPR1, which catalyses the production of rust alcohol by dichlorethane-diolefin, using Saccharomyces cerevisiae as host, and characterized by DNA and mRNA levels. Through the treatment of the permeable agent, one-step biotransformation into the rust alcohol was achieved, and the conversion efficiency reached 69.9%. This study demonstrated that Saccharomyces cerevisiae containing CYP76AH1 and SmCPR1 derived from Salvia miltiorrhiza can convert cis-danshensu-dienol into rustal alcohol. The results not only provide an effective platform for biotransformation to produce rust alcohol, but also can be used to identify tanshinone Downstream synthesis of other CYP450 functions.