催化丹参酮合成的细胞色素P450新基因的系统筛选与功能鉴定

丹参酮是丹参中二萜类天然产物，具有治疗人类心脑血管疾病的显著疗效，有多个细胞色素P450（P450）参与其生物合成途径。目前，除CYP76AH1外，尚未鉴定到催化丹参酮合成的其他P450新基因，是解析该途径分子机制的主要障碍。本项目申请人基于课题组前期完成的丹参全基因组图谱及19个RNA-seq转录组数据：①已发现15个与丹参酮合成关键酶基因显著共表达的P450s，它们是最可能催化丹参酮合成的候选基因。②将利用GC-MS检测候选P450过表达和RNAi转基因株系中萜类代谢物指纹图谱变化，鉴定催化丹参酮合成P450新基因的产物及底物；结合核磁共振谱分析其催化位点。③构建P450体外酶促反应体系并添加特异底物，GC-MS检测P450酶活性及动力学特征。④在生产铁锈醇的菌株中转入新鉴定的P450，构建生产目标产物的工程菌株。本项目为阐明丹参酮合成途径分子机制及开展丹参酮合成生物学研究奠定基础。

Tanshinones, belonging to diterpenoids of Salvia miltiorrhiza, possess significant efficacy in the treatment of human cardiovascular disease. Several cytochrome P450s (P450s) are involved in tanshinone biosynthesis. Except of CYP76AH1, the other new P450s have not been identified, leading to impede the illustration of tanshinone biosynthetic mechanisms. In this project, according to the genes involved in terpenoid biosynthetic pathways with co-expression patterns, the applicant used the S.miltiorrhiza genome, which was completed by our group firstly and RNA-seq transcriptome data generated from 19 different organs/tissues/MeJA treatment cDNA libraries to analyze the differential expressed genes and discovered 15 P450s co-expressed with SmCPS, SmKSL and CYP76AH1, the key enzyme-coding genes involved in tanshinone biosynthesis. Based on the analysis of different diterpene metabolite fingerprints between the over-expression and RNAi transgenic lines for these candidate P450s using GC-MS method, we can identify the specific P450 involved in tanshinone biosynthesis and the catalytic function of the P450. The activity and kinetic characteristics of the P450 enzymes will be detected by adding specific substrate into the P450 vitro reaction system. Importantly, the specific catalytic sites and products will also be detected by NMR in detail. The engineering yeast strain will be constructed by transforming these new identified P450 in this project into the ferruginol-product yeast strain. This study will lay the foundation for the illustration of the molecular mechanism of tanshinone biosynthesis and the research of synthetic biology of tanshinones in S.miltiorrhiza.