The elucidation of forskolin biosynthesis

1. [1-^<13>C]葡萄糖在B5液体培养基中培养2周。毛状根培养2周后收获。用乙酸乙酯按常规方法提取甲醇提取物。乙酸乙酯提取物经硅胶层析，再经反复反相高效液相色谱纯化得到^<13> c标记的福斯克林及其类似物。[1-^<13>C]葡萄糖在这些化合物中的掺入模式表明，毛状根中的二萜是通过非甲羟戊酸途径合成的。此外，由[1-^<13>C]葡萄糖衍生的白桦酸的标记模式与甲羟戊酸途径的生物合成一致。很明显，甲羟戊酸和非甲羟戊酸途径在毛状根中起作用。为了分离出福斯柯林生物合成的中间体，我们对福斯柯林毛状根的成分进行了研究，得到了17个含4个新化合物的二萜。根据分离化合物的结构，提出了福斯克林的生物合成途径。采用RT-PCR技术，根据已克隆cDNA的共酚二磷酸合成酶（copalyl diphosphate synthase， CPS）的氨基酸比对，克隆了二萜环化酶。因此，没有获得与CPS相似的克隆。

1. [1-^<13>C]glucose was administered to the Coleus forskohlii hairy roots cultured for 2 weeks in B5 liquid medium. Then, the hairy roots were cultured for 2 weeks and harvested. The MeOH extract was extracted with EtOAc according to a conventional method. The EtOAc extract was subjected to silica gel chromatography and then purified by repeated reversed-phase HPLC to give ^<13>C-labeled forskolin and its analogs. The incorporation patterns of [1-^<13>C] glucose into these compounds demonstrated that diterpenes in the C. forskohlii hairy root were biosynthesized via non-mevalonate pathway. Furthermore, labeling pattern of betulinic acid derived from [1-^<13>C] glucose was consistent with that biosynthesizing via mevalonate pathway. It became clear that mevalonate and non-mevalonate pathways are working in C. forskohlii hairy roots.2. For the isolation of intermediates in forskolin biosynthesis, we investigated the constituents of C. forskohlii hairy roots to give 17 diterpenes containing 4 new compounds. On the basis of the structures of isolated compounds, the biosynthetic pathway of forskolin was proposed.3. The cloning of diterpene cyclase was performed by RT-PCR on the basis of the amino acid alignment of copalyl diphosphate synthase (CPS) which cDNA has already been cloned. Consequently, the clone which has similarity with CPS was not obtained.