Plant Science 166 (2004) 381–385 Olivetol as product of a polyketide synthase in Cannabis sativa L Tri J. Raharjo a,b , Wen-Te Chang b , Young Hae Choi b , Anja M.G. Peltenburg-Looman b , Robert Verpoorte b,∗ a Department of Chemistry, Gadjah Mada University, Yogyakarta, Indonesia b Division of Pharmacognosy, Section of Metabolomics, Institute of Biology, Leiden Amsterdam Center for Drug Research, Leiden University, Gorlaeus Laboratories, P.O Box 9502, 2300 RA Leiden, The Netherlands Received 30 May 2003; received in revised form 26 September 2003; accepted 30 September 2003 Abstract A polyketide synthase (PKS) was suggested to catalyze the first step of cannabinoid biosynthesis, leading to olivetolic acid. An activity of a PKS was detected in the protein extract of Cannabis sativa flowering top. The enzyme converts one molecule of n-hexanoyl-CoA and three molecules of malonyl-CoA to olivetol. The product was identified by its UV-spectrum, mass spectrometry analysis and comparison with reference compound. The activity of the enzyme was also found in the upper leaves, but the activity occurring there is lesser than in the one occurring in the flowers. The activity of chalcone synthase (CHS), another PKS enzyme, was also found in the protein extract. © 2003 Elsevier Ireland Ltd. All rights reserved. Keywords: Cannabis sativa L; Olivetol; Olivetolic acid; Polyketide synthase; Stilbene synthase; Stilbene carboxylate synthase 1. Introduction Olivetolic acid is the first intermediate involved in the cannabinoid biosynthesis in Cannabis sativa. Prenylation of olivetolic acid by geranyl diphosphate or neryl diphosphate results in cannabigerolic acid or cannabinerolic acid respec- tively [1]. These compounds lead to the acidic forms of the major cannabinoids such as  9 -tetrahydrocannabinolic acid and cannabinolic acid [2]. It makes olivetolic acid an impor- tant metabolite in cannabinoid biosynthesis. Biosynthesis of olivetolic acid is not well known. Based on the chemical structure this compound might be syn- thesized by cyclization of a polyketide compound. This polyketide is formed by condensation of one molecule of n-hexanoyl-CoA with three molecules of malonyl-CoA as shown in Fig. 1. Condensation and cyclization reactions are catalyzed by a polyketide synthase (PKS). This hypothesis is among others based on the knowledge on the biosynthesis of hop bitter acid. In hops (Humulus lupulus), a plant of the same family as C. sativa, the first step is the condensation of one molecule isovaleryl-CoA or isobutyryl-CoA with three molecules of malonyl-CoA, resulting in phloroisovalerophe- ∗ Corresponding author. Tel.: +31-071-5274528; fax: +31-71-5274511. E-mail address: verpoort@lacdr.leidenuniv.nl (R. Verpoorte). none or phloroisobutyrophenone [3]. These products similar to olivetolic acid in cannabis [1], are then prenylated to give bitter acids. A way to prove a biosynthetic pathway is to detect the en- zyme activities going on in the plant. Based on the structure of olivetolic acid, the enzyme that is involved in this path- way should be a stilbene synthase (STS). Assays for other types of PKS enzymes, such as chalcone synthase (CHS) and chalcone synthase like (CHSL) have been developed [4,5]. The mechanisms of CHS and STS are slightly different. Both enzymes catalyze polyketide formation in a similar way but differently in the cyclization step. The CHS cycliza- tion is a Claisen condensation while the STS cyclization is an Aldol condensation. For STS, with the cyclization, the tetraketide is also decarboxylated [6,7]. However, a gene encoding stilbenecarboxylate synthase (STCS) has been cloned from Hydrangea macrophyla. This enzyme cat- alyzes the formation of lunularic acid by the condensation of dihydro-p-coumaryl-CoA and malonyl-CoA [8]. This enzyme is an STS enzyme without decarboxylation activ- ity. Such a reaction raises the interesting and challenging question of the occurrence of a similar kind of enzyme in other plants also. In case of C. sativa a STCS-like enzyme is thought to be responsible for the formation of olivetolic acid. In this 0168-9452/$ – see front matter © 2003 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.plantsci.2003.09.027