Biotechnology Letters 26: 793–798, 2004. © 2004 Kluwer Academic Publishers. Printed in the Netherlands. 793 Alkaloid accumulation in Catharanthus roseus cell suspension cultures fed with stemmadenine Magdi El-Sayed 1,2 , Young H. Choi 1 , M. Fr´ ed´ erich 1 , Sittiruk Roytrakul 1 & Rob Verpoorte 1,∗ 1 Department of Pharmacognosy, Section of Metabolomics, Institute of Biology Leiden, Leiden University, Leiden, The Netherlands 2 Department of Botany, Faculty of Science, South Valley University, Aswan, Egypt ∗ Author for correspondence (Fax: +31 71 5274511; E-mail: verpoort@chem.leidenuniv.nl) Received 3 February 2004; Revisions requested 9 February 2004; Revisions received 8 March 2004; Accepted 8 March 2004 Key words: alkaloid accumulation Catharanthus roseus, cell culture condylocarpine, stemmadenine, tabersonine- catharanthine pathway Abstract Feeding stemmadenine to Catharanthus roseus cell suspension culture resulted in the accumulation of catharanth- ine, tabersonine and condylocarpine. Condylocarpine is not an intermediate in the pathway to catharanthine or tabersonine when it is fed to the cultures. The results support the hypothesis that stemmadenine is an intermediate in the pathway to catharanthine and tabersonine. Introduction The tropical plant Madagaskar Periwinkle [Cathar- anthus roseus (L.) G.Don] is a rich source of indole alkaloids which have a high medicinal and economic value, especially the anti-tumor alkaloids, vinblastine and vincristine, and the anti-hypertensive compound, ajmalicine (Creasey 1994, Verpoorte et al. 1997). Terpenoid indole alkaloid biosynthesis starts with the condensation of tryptamine and secologanin to form the key intermediate strictosidine. Strictosidine is then hydrolyzed by strictosidine β -glucosidase producing cathenamine as the main product. Besides cathenam- ine, the related carbinolamine and epi-cathenamine have been detected by NMR in the reaction mix- ture (Stevens 1994). Other studies proved indirectly that an equilibrium exists between cathenamine and 4,21-dehydrogeissoschizine (Heinstein et al. 1979). As cathenamine is the main product of strictosidine conversion by the enzyme strictosidine β -glucosidase, Stevens (1994) concluded that this equilibrium was more favored towards cathenamine rather than 4,21- dehydrogeissoschizine. At some point in this reac- tion equilibrium, routes diverge into different indole alkaloid pathways. Lounasmaa & Hanhinen (1998) presented the ‘La Ronde’ scheme for the intercon- version of cathenamines in three steps: cyclization, epimerization and isomerization. The intermediate branch-points leading to the different classes of al- kaloids, though, were not as well-defined. Brown et al. (1971), suggested that geissoschizine could be converted to stemmadenine or akuammicine through some intermediates such as formylstrictamine and in- dolenine. Qureshi & Scott (1968a) reported the first evidence for formation of secodine as an interme- diate when stemmadenine was refluxed in glacial acetic acid for 34 h yielding a mixture of tabersonine (12%), (±)catharanthine (9%) and pseudocatharanth- ine (16%). The same authors (Qureshi & Scott 1968b) claimed that tabersonine, after refluxing in glacial acetic acid for 16 h was converted into catharanth- ine and pseudocatharanthine. However, this work was not reproducible and contradictory results have been reported by Brown et al. (1969): when tabersonine was treated under the same conditions it failed to pro- duce catharanthine or pseudocatharanthine. Battersby & Hall (1969) reported that, corynanthine aldehyde and geissoschizine fed to Catharanthus roseus plants were incorporated into catharanthine. From these ex- periments, the pathway was suggested as going from