Olivetol as product of a polyketide synthase in Cannabis sativa L

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.

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 respectively [1]. These compounds lead to the acidic forms of the major cannabinoids such as Δ⁹-tetrahydrocannabinolic acid and cannabinolic acid [2]. It makes olivetolic acid an important metabolite in cannabinoid biosynthesis.

Biosynthesis of olivetolic acid is not well known. Based on the chemical structure this compound might be synthesized 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 phloroisovalerophenone 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 enzyme activities going on in the plant. Based on the structure of olivetolic acid, the enzyme that is involved in this pathway 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 cyclization 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 catalyzes 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 activity. 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 paper, the identification of this enzyme in the C. sativa, was done using an HPLC in vitro assay.