The elucidation of forskolin biosynthesis

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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.