9,10-环甲基十七烷酸对灵芝三萜合成的诱导机制

Triterpenoids is one of the key medicinal compostions of Ganodema lingzhi (syn. G. lucidum), which has important pharmacological activities. However, the biosynthesis pathway and synthesis regulation mechanism of G. lingzhi triterpenoids are not clear. In our previous studies, we found that the triterpenoids biosynthesis amount was remarkably promoted by 9,10-methylenehexadecanoic acid (9,10-CMA) from the insect, Catharsius molossus, however, the induction mechanisms is not known. Therefore, the aim of the object is to elucidate the induction mechanisms of 9,10-CMA on triterpenoids biosynthesis of Ganoderma lingzhi, and research the relationship between 9,10-CMA and metabolisms of G. lingzhi fungus..Major works and related methods: The differences of gene transcription and related enzymes and functional proteins of triterpenoids biosynthesis with and without adding 9,10-CMA would be studied by using differential proteomics techniques and transcriptome technology. The key enzymes and action sites in process of triterpenoids biosynthesis would be studied by using monitoring technology of enzyme activity kinetics and metabolic flux technology. The signal molecules involved in the induction effect and related intracellular signal transduction mechanisms would be analyzed and elucidated. .Research significance: The results will reveal the induced molecular mechanism of exogenous on triterpenoids biosynthesis in G. lingzhi cells, elucidate the key enzymes, metabolism nodes and signaling molecules related to triterpenoids biosynthesis, and provide scientific basis for the regulation of the triterpenoids biosynthesis of G. lucidum, and also lay a theoretical foundation for the efficiently oriented production of G. lingzhi triterpenoids.

三萜是灵芝的关键活性成分，具有重要的药理作用，但其生物合成途径和合成调控机制尚不明了。前期研究发现昆虫蜣螂中的9,10-环甲基十七烷酸（9,10-CMA）可极显著诱导灵芝三萜的生物合成，但其诱导机制尚不清楚。为此，本项目拟通过灵芝三萜关键合成酶动力学监测、代谢通量分析和转录组、蛋白组分析等技术，分析诱导条件下灵芝三萜合成相关基因转录的差异，明确诱导三萜合成相关的合成酶和功能蛋白，解析诱导灵芝三萜合成代谢的作用位点，阐明参与诱导效应的胞内信号分子（途径）和转导机制。研究结果将揭示外源9,10-CMA诱导灵芝细胞内三萜生物合成的分子机制，解析诱导灵芝三萜合成的代谢节点、相关关键酶及胞内信号应答分子，为灵芝三萜的合成调控网络提供科学依据，并为灵芝三萜的高效定向生产奠定理论基础。

灵芝三萜的生物合成途径和合成调控机制尚不明了，是其规模化合成和生产应用的源头瓶颈。9,10-环甲基十七烷酸（9,10-CMA）可显著诱导灵芝三萜的生物合成，但其诱导机制尚不清楚。为此，本项目从发酵动力学的角度，考察9,10-CMA诱导下灵芝三萜在发酵过程中合成的动态差异，并采用全基因组测序及差异转录组学、代谢组学技术分析诱导灵芝三萜合成的关键合成酶基因、关键调控转录因子，并从信号转导的角度解析介导9,10-CMA诱导三萜合成的胞内信号分子（途径）。.本项目研究证明，添加9,10-CMA后，灵芝菌丝体细胞对底物葡萄糖的利用速度加快，细胞内三萜酸的比合成速率显著提高。.项目完成了灵芝全基因组测序，对灵芝三萜生物合成相关的基因进行了注释，发现灵芝中存在13个基因编码的11种灵芝三萜合成酶，构建了新的灵芝三萜合成途径。添加9,10-CMA后，灵芝三萜合成相关的3‐羟基‐3‐甲基戊二酰辅酶A还原酶基因hmgr、细胞色素P450单加氧酶CYP5150L8基因、鲨烯合酶基因sqs的上调幅度最大，表明添加9,10-CMA后，灵芝三萜合成的甲羟戊酸途径得到加强，同时，更多的中间产物法呢酰二磷酸酯向三萜重要前体鲨烯方向合成。进一步鉴定了灵芝中bHLH zip转录因子固醇调节元件结合蛋白（SREBP），然后通过SREBP过表达菌株构建及差异转录组学、代谢组学分析，证明SREBP是直接调控灵芝三萜合成基因表达的转录因子。用9,10-CMA诱导后，灵芝菌株中SREBP基因转录水平显著上调，且菌株中的三萜合成前体甲羟戊酸、重要骨架中间产物羊毛甾醇，以及7种不同三萜酸的含量均显著升高，证明转录因子SREBP是9,10-CMA诱导灵芝三萜合成的关键调控因子。此外，在9,10-CMA诱导下，NO可作为灵芝菌丝体中PAL活化和CYP450产生和三萜合成的上游信号分子起作用。.本项目研究结果对于分析灵芝三萜合成代谢途径，以及解析诱导灵芝三萜合成的相关关键酶、转录调控机制具有重要科学意义，同时也为灵芝三萜的高效定向生产奠定理论基础。