the fact that the pure drug-type plants, such as the Mexican strain, do not contain
CBDA (Shoyama et al. 1975 ). Wefirst attempted to detect the enzyme that cat-
alyzes this reaction using the crude enzyme extract prepared from a drug-type plant
(Mexican strain). However, we could not identify the isomerase, despite testing
various extraction and assay conditions. In contrast, THCA producing activity was
confirmed in the soluble fraction from leaf bud tissues when CBGA was incubated
as the substrate. Therefore, THCA appears to actually be biosynthesized from
CBGA via the stereoselective oxidative cyclization of a geranyl group by the action
of a novel enzyme, THCA synthase (Fig.8.3) (Taura et al. 1995 ).
THCA synthase was purified using column chromatography to a homogeneous
protein of which the partial amino acid sequences were determined by protein
sequencing. The gene encoding THCA synthase was cloned by degenerate PCR
and the rapid amplification of cDNA ends. The gene consists of a 1635-nucleotide
open reading frame, encoding a 545-amino acid polypeptide of which thefirst 28
amino acids constitute the signal peptide. This was thefirst gene involved in
cannabinoid biosynthesis to be cloned (Sirikantaramas et al. 2004 ).
Fig. 8.3 Biosynthesis of tetrahydrocannabinolic acid (THCA) by THCA synthase. Cannabidiolic
acid (CBDA) is not a precursor of THCA. Tetrahydrocannabinol (THC) is formed by the
non-enzymatic decarboxylation of THCA.CBGAcannabigerolic acid
8 Cannabinoids: Biosynthesis and Biotechnological Applications 187