# Phytocannabinoids Biosynthesis in Angiosperms, Fungi, and Liverworts and Their Versatile Role
## 3 Biosynthesis of Phytocannabinoids
### Cannabis sativa  
Phytocannabinoids are prenylated polyketides, i.e., terpenophenolic compounds, which are derived from isoprenoid and fatty acid precursors. Phytocannabinoid biosynthesis occurs in different cellular compartments: gland cells cytosol, the plastids, and the extracellular storage cavity. In the cytosol, oxidative cleavage of fatty acid such as palmitic acid yields hexanoic acid; it further synthesizes olivetolic acid (OA). The next step is prenylation of phenolic moiety (the polyketide derivatives, 5-pentenyl resorcinolic acid, and OA) with the terpenoid geranyl pyrophosphate (GPP). This step originates from the methylerythritol-4-phosphate (MEP) pathway in plastids. Cyclization (oxidative) and storage of the final products take place outside the gland cells. Transport proteins and vesicle trafficking participate in mobilizing intermediates across the morphologically highly specialized interface between the gland cells and storage cavity [30; 31; 32]. In _C. sativa_, phytocannabinoids biosynthesis is divided into three important places: cytosol (for polyketide pathway), plastids (MEP pathway for prenylation), and apoplastic spaces (oxidocyclization and storage) (Figure 5). Inside cytosol, biosynthesis of phytocannabinoids participates in the integration of major steps in polyketide and isoprenoid metabolism. Fatty acids (C18) are sequentially desaturated, peroxygenated, and cleaved into the hexanoic acid (C6) and C12 product via enzyme desaturase, lipoxygenase (LOX), and hydroperoxide lyases, respectively. Hexanoic acid is converted into thioester hexanoylCoA; this reaction is catalyzed by acyl-activated enzyme 1 (AAE1). Later, hexanoyl-CoA and malonyl-CoA (C2 donor) together via the action of Olivetol synthase (OLS) and olive-tolic acid cyclase (OAC) synthesizes OA. Moreover, it was reported that OAC is the dimeric (\(\alpha\)+\(\beta\)) barrel protein, it is the first plant enzyme that catalyzes (C2-C7) intramolecular aldol condensation along with carboxylate retention; OAC contains distinctive active-site bearing the pentyl-binding hydrophobic pocket and polyketide binding site, whereas it is devoid of aromatase and thioesterase activities [31; 33; 34]. Figure 5: Phytocannabinoids biosynthesis in _Cannabis sativa_. Abbreviations: CBCA, cannabinoidacid; CBCAS, cannabinoidic acid synthase; CBDA, cannabinoidic acid; CBDAS, cannabinoidic acid synthase; \(\Delta^{9}\)-THCA, \(\Delta^{9}\)-tetrahydrocannabinolic acid; \(\Delta^{9}\)-THCAS, \(\Delta^{9}\)-tetrahydrocannabinolic acid synthase. Inside plastids, the MEP pathway synthesizes GPP. It prenylates OA, which forms the intermediate branch-point and first cannabinoid which is cannabinoid acid (CBGA), and this reaction is catalyzed by cannabigerolic acid synthase (CBGAS). CBGA is an essential cannabinoid because it acts as the precursor of several cannabinoids with an alkylic pentyl side chain. In contrast, CBGAS is a transmembrane aromatic premyltransferase (PT) that transfers plastid signals. Then CBGA is converted into \(\Delta^{9}\)-THCA and cannabidiotic acid (CBDA) with the help of two enzymes which are CBDAS and \(\Delta^{9}\)-tetrahydrocannabinolic acid synthase (\(\Delta^{9}\)-THCAS). This conversion continues by reducing oxygen (O\({}_{2}\)) into hydrogen peroxide (H\({}_{2}\)O\({}_{2}\)) via oxidative cyclization reactions. Additionally, CBDAS and \(\Delta^{9}\)-THCAS are necessary flavoprotein enzymes that are dependent on O\({}_{2}\) (electron acceptor) [1; 30; 31]. Another important enzyme, cannabichromenic acid synthase (CBCAS), dependent from FAD and O\({}_{2}\), takes part in the synthesis of cannabichromenic (CBCA). Additionally, enzymes \(\Delta^{9}\)-THCAS and CBCAS have high sequence similarity at about a 96% nucleotide level. Both remain active inside resin space, which shows that CBCAS participates as an O\({}_{2}\) dependent flavoprotein that converts CBGA to cannabichromenic acid (CBCA) with H\({}_{2}\)O\({}_{2}\) as the side product via an oxidocyclization reaction.