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

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.

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。

丹参酮是丹参中二萜类化合物，具有治疗心脑血管疾病的显著疗效.但丹参酮生物合成途径尚未被解析，尤其是多个参与丹参酮下游合成途径的细胞色素P450（P450）仍未被鉴定。本项目主要研究目标即鉴定到参与丹参酮生物合成的新的P450。本项目的主要研究内容包括候选P450的基因克隆及序列分析、基因表达谱分析、基因过表达和RNAi载体构建及丹参毛状根遗传转化、筛选毛状根阳性株系、并利用UPLC等方法去检测毛状根阳性株系中的丹参酮类化合物含量变化情况、检测丹参酮类化合物生物合成途径关键酶基因表达变化、以及候选基因的酵母表达等。本项目已完成8个可能参与丹参酮生物合成的P450的功能鉴定。我们首次明确了这些P450s具有促进或抑制丹参酮生物合成的作用。我们共鉴定到3个促进丹参酮合成的新P450（SmCYP81C16、CYP71BE37、SmCYP76AK3.1）、4个抑制丹参酮合成的新P450（CYP72A395、CYP76AK5v2、CYP72A394v2、CYP71AU52）和一个不参与丹参酮生物合成过程的P450（CYP71BE51）。其中，SmCYP81C16促进丹参酮生物合成。CYP71BE37促进丹参酮类化合物生物合成且更可能直接参与催化隐丹参酮和丹参酮IIA的合成过程。SmCYP76AK3.1促进丹参酮IIA生物合成。CYP72A395抑制丹参酮生物合成，尤其更显著地影响二氢丹参酮I和隐丹参酮的合成；CYP76AK5v2、CYP72A394v2和CYP71AU52均抑制丹参酮类化合物的积累。这4个P450可能参与丹参酮合成途径的竞争途径，或是该类化合物的降解途径。CYP71BE51不影响丹参酮类化合物的生物合成，可能参与丹参中其他类化合物的合成或代谢途径中。本项目研究结果为进一步解析丹参酮生物合成途径分子机制提供了崭新的视角和扎实的数据，具有重要科学意义；同时也为丹参优良种质选育提供候选标记基因，具有良好应用前景。