灵芝三萜的生物合成途径解析及其异源生物合成研究

Ganoderma lucidum is the most famous medicinal fungus with the most economic value in China. Triterpenoids are one of the major group of bioactive components found in G. lucidum. Modern pharmacological research has demonstrated that G. lucidum triterpenoids have multiple therapeutic actions, including antitumor and immunomodulatory activities, suggesting they are important source for R&D of novel drugs. However, the difficulties in separation and chemical synthesis in large scale have greatly limited R&D of G. lucidum triterpenoid-derived drugs. We have constructed the fine draft of G. lucidum genome and found and curated all candidate genes encoding the key enzymes involved in the biosynthesis of G. lucidum triterpenoids, such as cytochrome P450 and so on. To narrow down the candidate genes, we detect the variation of their expression levels among the different samples using DNA microarray technology. The genes coexpressed with the lanosterol synthase gene are selected for further study. The selected genes are cloned and expressed in Saccharomyces cerevisiae, and then the enzymes are characterized through detecting the enzymatic products with LC-MS. The elucidation of the G. lucidum triterpenoid biosynthetic pathway lays the foundation for the heterologous biosynthesis of the triterpenoids. Guided with the principle of synthetic biology, we modify the host strain to enhance the metabolic flux towards lanosterol biosynthesis, and then reconstruct the downstream triterpenoid biosynthetic module in S. cerevisiae, using internal lanosterol as intermediate to produce G. lucidum triterpenoids. This study will provide abundant resources for R&D of G. lucidum triterpenoid-derived drugs and valuable clues for the heterologous biosynthesis of other natural medicines.

灵芝(Ganoderma lucidum)是我国最著名和最具经济价值的药用真菌，灵芝三萜是灵芝的主要有效成分之一，具有抗肿瘤和免疫调节等多种功效，是新药研发的重要源泉。但是其单体难以通过化学分离或合成方法大量获得已成为灵芝三萜类药物研发的主要瓶颈。本课题组已完成灵芝基因组精细图，发现并校正了细胞色素P450等所有可能参与灵芝三萜合成的候选基因。本研究将利用DNA芯片等技术检测不同灵芝样本中上述候选基因的表达变化,筛选出与羊毛甾醇合成酶基因共表达的基因，并在酿酒酵母中表达，通过LC-MS检测酶促反应产物来完成酶的鉴定。在充分解析灵芝三萜合成途径的基础上，以合成生物学理念为指导，优化酿酒酵母自身的羊毛甾醇合成模块以增加其产量，并导入灵芝三萜下游合成模块，实现以羊毛甾醇为中间体底物的灵芝三萜异源生物合成，为灵芝三萜类药物的研发提供原料和物质基础，并为其他天然药物的异源生物合成研究提供有益的借鉴。

灵芝(Ganoderma lucidum)是我国最著名和最具经济价值的药用真菌，灵芝三萜是灵芝的主要有效成分之一，具有抗肿瘤和免疫调节等多种功效，是新药研发的重要源泉。但是其单体难以通过化学分离或合成方法大量获得已成为灵芝三萜类药物研发的主要瓶颈。本项目对测序完成的灵芝基因组序列进行挖掘，获得197个细胞色素P450基因，通过与已知的灵芝三萜合成关键酶羊毛甾醇合酶（LAS）基因的共表达分析，选出相关性最高的10个基因进行进一步研究。在酿酒酵母中表达这些候选基因，并通过LC-MS或GC-MS检测酶促反应产物来鉴定酶的催化活性。以合成生物学理念为指导，优化酿酒酵母自身的羊毛甾醇合成模块以增加其产量，并导入灵芝三萜下游合成模块，实现以羊毛甾醇为中间体底物的灵芝三萜异源生物合成，为灵芝三萜类药物的研发提供原料和物质基础，并为其他天然药物的异源生物合成研究提供有益的借鉴。本项目共发表学术论文10篇，出版专著一部，申请发明专利4项。