羊踯躅中木藜芦烷型微量镇痛活性成分生物合成基因挖掘与代谢途径在酿酒酵母中的重构

Rhododendron molle belongs to Ericaceae family and is a traditional toxic Chinese medicine with the function of relieving pain, etc. Modern studies showed that the grayanane diterpenoids are the main active components of the herb, which have strong analgesic effect but are non-opioid receptor agonists. Therefore these compounds are potent candidates for developing a new generation of analgesic drug. However, the natural content of these compounds in the plant is very low and the traditional isolation method is far from meeting the demand of the pharmacological and clinical studies. Moreover, the biosynthetic pathway, together with the catalytic enzymes, is unclear. In this project, we will mine the unknown and the key biosynthetic genes, such as the diterpene synthases (diTPSs) and CYP450 genes, through transcriptome sequencing technique and bioinformatics analysis. Heterologous expression of the genes will be used to obtain the intermediate products, and the structures of the products will be determined by spectroscopy analysis. Thus, the enzyme functions can be elucidated and the biosynthetic pathway can be proposed. Meanwhile, we will construct the chassis cells of Saccharomyces cerevisiae by the metabolic engineering approach, i.e., to increase the innate GGPP concentration, a common precursor of the diterpenoids, via the strategy of “broadening source and reducing expenditure”, and to increase the innate heme concentration via introducing the heme3 gene expression cassette into the host. Then, we will reconstruct the biosynthetic pathway of the grayanane diterpenoids in the yeast chassis cells, resulting in the episomal engineered strain followed by the integrative engineered strain. Finally, we will optimize the fermentation conditions to prepare the grayanane products or their intermediates for further pharmacological research and new drug development.

羊踯躅是杜鹃花科具缓解疼痛等功能的传统有毒中药，现代研究表明其镇痛活性成分主要为木藜芦烷型二萜化合物且为非阿片受体激动剂，有望被开发成为新一代镇痛药物。但其天然含量低微，传统的提取方式远远不能满足药理学和临床研究需要，且其生物合成途径未知、相关酶系信息缺乏。本项目利用转录组测序技术和生物信息学分析方法挖掘该类化合物未知的二萜合酶、P450氧化还原酶关键基因，通过重组表达获取中间产物，对中间产物进行波谱解析、酶功能验证和解析其生物合成途径。构建酿酒酵母底盘细胞，通过“开源节流”提高二萜生物合成通用前体GGPP胞内浓度；通过导入内源性血红素生物合成heme3基因表达盒提高P450氧化还原酶辅基血红素的生成量。在酵母底盘细胞中重构木藜芦烷型二萜化合物生物合成途径，依次获得附加体型和整合型工程菌，通过发酵条件优化制备木藜芦烷型二萜化合物或其中间体，为深入开展后续的药理学研究及新药开发奠定基础。

现代研究表明传统有毒中药羊踯躅的镇痛活性成分主要为木藜芦烷型二萜且为非阿片受体激动剂，有望被开发成为新一代镇痛药物。但其天然含量低微，无法满足药理学和临床研究需要，且其生物合成途径未知、相关酶系信息缺乏。本项目开展羊踯躅基因组测序及组装、转录组测序和生物信息学分析，开展关键酶萜类合酶和P450酶的筛选，期望解析木藜芦烷生物合成途径；开展酿酒酵母底盘细胞构建工作，期望在酵母中重构木藜芦烷生物合成途径生产木藜芦烷二萜或其中间体，为深入开展其新药研发奠定基础。.已完成羊踯躅各器官转录组测序、基因组测序与精细组装和生物信息学分析；分别挖掘和功能鉴定了能够催化GGPP环化生成对映-柯巴基焦磷酸（ent-CPP）的关键II型二萜合酶RmTPS1和RmTPS4；挖掘和功能鉴定了能够催化ent-CPP进一步环化为重要中间产物16α-hydroxy-ent-kaurane的关键I型二萜合酶RmTPS3，据此提出了木藜芦烷型二萜生物合成假设即：通用前体GGPP在II型萜二萜合酶化下产生ent-CPP进而在I型二萜合酶催化下产生16α-hydroxy-ent-kaurane，后者在一种P450酶催化下结构重排为木藜芦烷型骨架并经其他P450酶等修饰生成结构复杂的木藜芦烷型产物。.采用基于序列同源性筛选、羊踯躅植物组织培养和P450候选基因在烟草系统中瞬时表达等策略筛选关键P450酶。锁定属于CYP71 和CYP76 亚家族的CYP450候选基因35个；分别用高效合成GGPP的酵母底盘细胞和烟草瞬时表达系统筛选P450候选基因，已在烟草表达系统的代谢产物中发现分子量为630和452的LC/MS特征峰，有可能为期待的被糖基化的骨架氧化产物，后续工作将逐步缩小候选基因范围直至获得目的基因；首次实现用羊踯躅叶片快速形成愈伤组织(R培养基+α-萘乙酸NAA 1mg/L+玉米素ZT 1.5mg/L+维生素C 100mg/L，培养温度25C，无光照培养，20d)和初步完成液体悬浮细胞培养，为筛选P450酶等关键基因提供了另一种技术手段。此外我们还鉴定1个负责倍半萜selina-6-en-4-ol合成的基因簇。.一旦挖掘到关键P450酶，即可在已建的酵母底盘细胞中重构木藜芦烷骨架生物合成途径，这对于应用合成生物学技术生产微量活性的木藜芦烷具有重要理论与实际意义。