Molecular characterization of the pentacyclic triterpenoid biosynthetic pathway in Catharanthus roseus

Catharanthus roseus is an important medicinal plant and the sole commercial source of monoterpenoid indole alkaloids (MIA), anticancer compounds. Recently, triterpenoids like ursolic acid and oleanolic acid have also been found in considerable amounts in C. roseus leaf cuticular wax layer. These simple pentacyclic triterpenoids exhibit various pharmacological activities such as anti-inflammatory, anti-tumor and anti-microbial properties. Using the EST collection from C. roseus leaf epidermome ( http://www.ncbi.nlm.nih.gov/dbEST ), we have successfully isolated a cDNA (CrAS) encoding 2,3-oxidosqualene cyclase (OSC) and a cDNA (CrAO) encoding amyrin C-28 oxidase from the leaves of C. roseus. The functions of CrAS and CrAO were analyzed in yeast (Saccharomyces cerevisiae) systems. CrAS was characterized as a novel multifunctional OSC producing α- and β-amyrin in a ratio of 2.5:1, whereas CrAO was a multifunctional C-28 oxidase converting α-amyrin, β-amyrin and lupeol to ursolic-, oleanolic- and betulinic acids, respectively, via a successive oxidation at the C-28 position of the substrates. In yeast co-expressing CrAO and CrAS, ursolic- and oleanolic acids were detected in the yeast cell extracts, while the yeast cells co-expressing CrAO and AtLUP1 from Arabidopsis thaliana produced betulinic acid. Both CrAS and CrAO genes show a high expression level in the leaf, which was consistent with the accumulation patterns of ursolic- and oleanolic acids in C. roseus. These results suggest that CrAS and CrAO are involved in the pentacyclic triterpene biosynthesis in C. roseus.