## 2 Critical Biosynthetic Steps for Phytocannabinoid Formation
### FAD-Depended Cyclases Form Various Cannabinoids  
In _C. sativa_, CBGA is cyclized by several homologous flavin adenine dinucleotide (FAD)-dependent monoxygenases, namely THCA, CBDA, and CBCA synthase (THCAS, CBDAS, and CBCAS) which share ca. 80% amino acid sequence identities [50, 68, 69, 70, 71, 72]. By initiating a hydride shift to the oxidized FAD prosthetic group, these enzymes facilitate the oxidative cyclization of CBGA or its _cis_-isomer cannabinerolic acid (CBNRA) to the respective products. Molecular oxygen is reduced to hydrogen peroxide to regenerate the oxidized FAD in a reaction mechanism similar to that of the Berberine Bridge Enzyme (BBE) in the benzylisoquinoline alkaloid biosynthetic pathway [73]. These enzymes do not accept CBG as a substrate, since the interaction of carboxylate group with a histidine and an adjacent tyrosine residue is important for substrate binding as demonstrated by site directed mutagenesis and reported crystal structures [31]. Albeit forming the respective product as their name suggests, all three enzymes were shown to form small amounts of other cannabinoids when tested in vitro and in vivo, which was also influenced by pH [36, 72, 74]. The major by-product of THCAS is CBCA (ca. 10% at optimal pH), whereas the major by-products of CBDAS are THCA and CBCA (ca. 3-6% at optimal pH) [72]. The non-stringent product specificity is also likely an intrinsic feature of the native enzymes in _C. sativa_, because THC has always been detected in hemp varieties such as Finola that does not contain a THCAS in its genome [75]. In many cases, small amounts of enzyme by-products are not an issue. However, with THC being a controlled, psychoactive substance in most jurisdictions, the concomitant THCA production during CBDA production may be perceived as a problem for the cannabis industry who is looking for THC-void solutions. This intrinsic by-product production also suggests that creating a THC-void cannabis variety may not be fruitful without genetic modifications. Similar to other BBE-like enzymes, these cyclases are known for the requirement of protein _N_-glycosylation and intramolecular disulfide bridges, which are believed to be required for proper polypeptide folding and possibly the recruitment of the covalently bound FAD. This is supported by the fact that none of the BBE-like enzymes have been expressed as an active enzyme in _E. coli_, a prokaryote with an _N_-glycosylation process fundamentally different from that of eukaryotes. Expression of the recombinant proteins in tobacco was also only possible when they were tagged for secretion or endoplasmic reticulum (ER) localization, whereas the deletion of the signal peptides or replacement of a chloroplast targeting signal peptide did not yield detectable proteins [74]. However, the _N_-glycans are not needed for enzyme activity after protein maturation, since deglycosylated THCAS still showed comparable activity in vitro [39]. The known _C. sativa_ cyclases all contain a 28 amino acid, _N_-terminal signal peptide, and do not possess the classic C-terminal ER retention signal. The signal peptide directs the nascent polypeptides through the plant secretory pathway via ER translocation, _N_-glycosylation, disulfide bonds forming, FAD linking, and protein folding in ER lumen, _N_-glycan modifications in Golgi, and vesicle medicated secretion to the apoplastic region (i.e., outside of a cell) of the secretory cells of the glandular trichomes [76]. Such localization is perhaps evolutionarily preferential, as the hydrophobic environment in the glandular trichome storage cavity allows optimal enzymatic activity and accumulation of the hydrophobic products to concentrations that are cytotoxic to plant cells [76]. When expressed in tobacco leaves, the recombinant proteins were also detected in the chloroplast in addition to the apoplast [74]. Additionally, the signal peptide is cleaved in the mature enzyme.