The biosynthesis of the cannabinoids

Abstract

Cannabis has been integral to Eurasian civilization for millennia, but a century of prohibition has limited investigation. With spreading legalization, science is pivoting to study the pharmacopeia of the cannabinoids, and a thorough understanding of their biosynthesis is required to engineer strains with specific cannabinoid profiles. This review surveys the biosynthesis and biochemistry of cannabinoids. The pathways and the enzymes' mechanisms of action are discussed as is the non-enzymatic decarboxylation of the cannabinoic acids. There are still many gaps in our knowledge about the biosynthesis of the cannabinoids, especially for the minor components, and this review highlights the tools and approaches that will be applied to generate an improved understanding and consequent access to these potentially biomedically-relevant materials.

Keywords: C. sativa; Cannabinoid biosynthesis; Decarboxylation; Enzymatic mechanism; Enzymatic transformation.

Fig. 1

Structures of important Cannabis sativa cannabinoid classes: Δ⁹-tetrahydrocannabinol (Δ⁹-THC or THC) and its acidic counterparts (THCA-A/THCA-B), tetrahydrocannabivarin (THCV), cannabidiol (CBD) and its acidic counterpart (CBDA), cannabigerol (CBG), cannabichromene (CBC), and cannabinol (CBN)

Fig. 2

Biosynthesis of cannabinoids. a Proposed cannabinoid biosynthetic pathway for Δ⁹-tetrahydrocannabinol (Δ⁹-THC), cannabidiol (CBD), and cannabichromene (CBC) including by-product formation (pentyl diacetic lactone (PDAL), hexanoyl triacetic acid lactone (HTAL), and olivetol shown in the dotted box) and highlighting the chemical conversion of CBD into THC, long thought to be the source of THC, but this conversion does not occur in vivo. b Synthesis of geranyl pyrophosphate (GPP) from dimethylallyl pyrophosphate (DMAPP) and isopentenyl pyrophosphate (IPP) catalyzed by geranyl pyrophosphate synthase

Fig. 3

X-ray diffraction structures of three key enzymes implicated in cannabinoid biosynthesis. Key active site residues are highlighted in green, and interaction diagrams, generated by the authors using the Schrödinger computational software suite (Maestro 2020), of a) tetrameric tetraketide synthase (TKS) from C. sativa in complex with CoenzymeA (CoA, 6GW3), CoA is orange, with the four tetramers in red, orange, light green and cyan respectively (Kearsey et al. 2020); b) expansion of the active site; c) Olivetolic acid cyclase (OAC) from C. sativa (5BO9) (Yang et al. 2016), the pentyl-binding pocket and its key residues are gray, olivetolic acid (OLA) is orange, chain A is gray and chain B is in light orange; d) expansion of the active site, inverted; e) tetrahydrocannabinolic acid synthase (THCAS) from C. sativa (3VTE) bound to flavin adenine dinucleotide (FAD) and without ligand. FAD is orange and the protein navy; and f) expansion of the active site (Shoyama et al. 2012)

Fig. 4

Proposed mechanism for the formation of olivetolic acid (OLA) by olivetolic acid cyclase (OAC)

Fig. 5

Reaction mechanism for the conversion of cannabigerolic acid (CBGA) into tetrahydrocannabinolic acid (THCA) proposed by Taura and cowerkers (2019). a Covalently incorporated flavin adenine dinucleotide (FAD) in black, b THCA synthase pathway is shown in green, and c cannabidiolic acid (CBDA) synthase pathway is shown in purple; CBDA and THCA in red. The red box represents the active site of the enzyme

Fig. 6

Comparison of cannabidiolic acid synthase (CBDAS) and tetrahydrocannabinolic acid synthase (THCAS) and the metabolism of cannabinoids. a Homology model of CBDAS developed from THCAS (3VTE); residues conserved from THCAS are purple while variant residues are cyan, sequence insertions are red, and FAD is green; b active site of these enzymes highlighted with a cartoon showing conversion to tetrahydrocannabinolic acid (THCA), cannabidiolic acid (CBDA) and cannabichromenic acid (CBCA) from cannabigerolic acid (CBGA); c experimentally demonstrated oxidation (ox) and isomerization (is) reactions and metabolic fates (encircled) for Δ⁹-THCA and Δ⁹-Tetrahydrocannabinol (Δ⁹-THC)