Original article Effects of alkaloid precursor feeding on a Camptotheca acuminata cell line Andrea Silvestrini a , Gabriella Pasqua a, *, Bruno Botta b , Barbara Monacelli a , Robert van der Heijden c , Robert Verpoorte c a Department of Plant Biology, University “La Sapienza”, P.leAldo Moro 5, 00185 Rome, Italy b Department of Chemistry and Technology of Biological Active Molecules, University “La Sapienza”, P.le Aldo Moro 5, 00185 Rome, Italy c Division of Pharmacognosy, Leiden/Amsterdam Center for Drug Research, Leiden University, Gorlaeus Laboratories, P.O. Box 9502, 2300 RA Leiden, The Netherlands Received 28 November 2001; accepted 14 March 2002 Abstract To better understand the biosynthesis of Camptotheca acuminata alkaloids, the effect on camptothecin production of feeding with potential precursors of biosynthesis was studied (i.e., tryptamine and loganin combined, secologanin, and strictosidine). Two key enzymes in alkaloid biosynthesis [i.e., tryptophan decarboxylase (TDC; EC 4.1.1.28) and strictosidine synthase (STR; EC 4.3.3.2)] were also studied. The analyses were conducted using a C. acuminata CG1 cell line that does not produce alkaloids, which could be useful in better understanding the biosynthetic pathway and in identifying possible limiting factors. The activity of TDC was 5 pkat mg–1; the activity of STR was 1.1 pkat mg –1 . Feeding with strictosidine revealed that this precursor is easily biotransformed by two enzymes (i.e., a hydroxylase and a dehydrogenase) in hydroxystrictosidine and didehydrostrictosidine, but camptothecin was never detected. The indole pathway and the low level of STR activity could be limiting factors in the production of camptothecin in the cell line used. © 2002 Éditions scientifiques et médicales Elsevier SAS. All rights reserved. Keywords: Alkaloids; Camptotheca acuminata; Cell culture; Precursor feeding; Strictosidine synthase; Tryptophan decarboxylase 1. Introduction Camptotheca acuminata Decaisne (Nyssaceae) is a Chi- nese tree that produces camptothecin, a pentacyclic quino- line alkaloid, which was first isolated by Wall et al. [19]. Camptothecin is known for its remarkable inhibitory activ- ity against tumour cells (i.e., by blocking the eukaryotic topoisomerase I [20]) and for its activity against the human immunodeficiency virus (HIV) [13]. Although much is known about the pharmacological effects of camptothecin, little is known about its biosynthe- sis. In an attempt to produce camptothecin, plant-cell and tissue cultures have been used, yet the yield has been low [17]. The optimisation of culture medium, the supply of biosynthetic precursors and genetic engineering, are among the strategies adopted to increase alkaloid production in vitro [18]. Studies using plant-cell cultures are also of interest for the study of alkaloid biosynthesis. In Catharanthus roseus cell cultures, studies on the biosynthesis of terpenoid indole alkaloids have shown that tryptophan decarboxylase (TDC; EC 4.1.1.28) converts tryptophan into tryptamine by decarboxylation (Fig. 1) [10]. In C. roseus cell cultures treated with biotic and abiotic elicitors [9] or transferred to an alkaloid production medium [8], the activity of TDC has been shown to be correlated with the accumulation of monoterpenoid indole alkaloids. In transformed C. roseus roots cultured in vitro, TDC activity is correlated with vindoline accumulation [16]. TDC is also Abbreviations: CH 3 CN, acetonitrile; CPC, centrifugal partitioning chromatography; 2,4-D, 2,4-dichlorophenoxyacetic acid; DTT, 1,4- dithiothreitol; EDTA, ethylenediamino tetraacetic acid; FW, fresh weight; HPLC, high pressure liquid chromatography; kin, kinetin; NAA, naphtha- lene acetic acid; PVPP, polyvinylpolypyrrolidone; rpm, rotations per minute; STR, strictosidine synthase (EC 4.3.3.2); TDC, tryptophan decar- boxylase (EC 4.1.1.28); TFA, trifluoroacetic acid * Corresponding author. E-mail address: gabriella.pasqua@uniroma1.it (G. Pasqua). Plant Physiol. Biochem. 40 (2002) 749–753 www.elsevier.com/locate/plaphy © 2002 Éditions scientifiques et médicales Elsevier SAS. All rights reserved. PII: S 0 9 8 1 - 9 4 2 8 ( 0 2 ) 0 1 4 3 6 - 5