丹参酮类化合物结构修饰关键CYP450酶Miltiradiene-12-Hydroxylase的功能研究

丹参酮的生物合成主要包括前体形成、骨架构建和后修饰三个阶段，目前其生物合成途径已经推进到了碳骨架结构次丹参酮二烯，在此基础上通过一系列后修饰酶的催化形成结构各异的丹参酮类化合物。细胞色素P450酶介导的次丹参酮二烯羟基化反应作为结构修饰的第一步起关键作用。本项目在转录组文库的基础上克隆得到14个诱导表达显著上调的CYP450基因，通过实时定量PCR分析筛选到了7个在丹参根中表达较高的基因，利用RNAi、酵母表达和酶促反应以次丹参酮二烯作为底物对其催化功能进行研究，最后将功能基因转入内源产生次丹参酮二烯的酵母工程菌进行体内功能验证。通过筛选和功能分析得到催化次丹参酮二烯C12位羟基化的CYP450酶（次丹参酮二烯-12-羟化酶），进一步解析丹参酮生物合成途径，为定向遗传育种以及活性成分的批量生产奠定基础。

The biosynthesis of tanshinone includes three consecutive processes: the formation of precursor, the construction of skeleton and post-modification of the skeleton. Cyclization of GGPP to miltiradiene was followed by a series of post-modification enzymes to produce various tanshinones. The first oxygenation step is the CYP450-mediated hydroxylation of the diterpene olefin precursor miltiradiene. In this study, fourteen up-regulated CYP450s were cloned for further analysis based on the transcriptome. Real-time PCR indicated that seven of them was highly expressed in the rhizome of S.miltiorrhiza. The seven candidates were functional expressed in yeast, and miltiradiene was used as substrate for catalyze analysis. RNAi was used to silence gene expression. The catalyzed characterization was further confirmed by transforming into the engineered yeast strain which produce miltiradiene endogenously. Cloning and functional characterization of CYP450 (Miltiradiene-12-Hydroxylase) catalyzing hydroxylation of miltiradiene is useful for directional genetic breeding and heterologous production of tanshinones.