The elucidation of forskolin biosynthesis
毛喉素生物合成的阐明
基本信息
- 批准号:12672060
- 负责人:
- 金额:$ 2.24万
- 依托单位:
- 依托单位国家:日本
- 项目类别:Grant-in-Aid for Scientific Research (C)
- 财政年份:2000
- 资助国家:日本
- 起止时间:2000 至 2002
- 项目状态:已结题
- 来源:
- 关键词:
项目摘要
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.
1. [1-对<13>毛喉鞘蕊花毛状根在B5液体培养基中培养2周后给予[2C]葡萄糖。然后,将毛状根培养2周并收获。根据常规方法用EtOAc萃取MeOH萃取物。将EtOAc提取物进行硅胶色谱,然后通过重复的反相HPLC纯化,得到13 <13>C-标记的毛喉素及其类似物。[1-^<13>C]葡萄糖掺入这些化合物的模式表明,在C。毛喉菌毛状根通过非甲羟戊酸途径生物合成。此外,桦木酸来源于[1-^<13>C]葡萄糖的标记模式与通过甲羟戊酸途径生物合成的标记模式一致。很明显,甲羟戊酸和非甲羟戊酸途径在C。毛喉菌毛状根2.为了分离毛喉素生物合成的中间体,我们研究了C.从毛状根中分离得到17个二萜类化合物,其中4个为新化合物。根据分离得到的化合物的结构,提出了forskolin的生物合成途径.根据已克隆的柯巴基二磷酸合成酶(CPS)cDNA序列,通过RT-PCR方法克隆了二萜环化酶基因。因此,没有获得与CPS具有相似性的克隆。
项目成果
期刊论文数量(0)
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科研奖励数量(0)
会议论文数量(0)
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ASADA Yoshihisa其他文献
ASADA Yoshihisa的其他文献
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