## 2 Critical Biosynthetic Steps for Phytocannabinoid Formation
### Cannabigerolic Acid Formation by Aromatic Prenyltransferases  
Aromatic prenyltransferases are responsible for geranylating OA to form CBGA that is further cyclized to different cannabinoids. All known plant aromatic _C_-prenyltransferases are membrane-bound enzymes that are classified as _p_-hydroxybenzoic acid (PHB) and homogenistate (HG) PTs [57]. OA prenyltransferases for CBGA biosynthesis and chrysoeriol phenyltransferases for cannflavinA/B biosynthesis all belong to the HG family that are localized to plant plastids [38; 57; 58]. The plastid membrane localization strongly suggests the prenyl donors are supplied by the plastid 2-C-methyl-_D_-erythnitol 4-phosphate (MEP) pathway (Figure 2), and the hydrophobic interior of the phospholipid bilayers may also facilitate the hydrophobic product release after prenylation. OA prenyltransferase activity in _C. sativa_ was first described in the crude, soluble protein extract from hemp leaves [59]. Interestingly, the attempt of using membrane fractions from the crude protein was not successful. The crude, soluble proteins were able to prenylate OA at C3 with GPP or its _cis_-isomer aryl diphosphate (NPP), but could not prenylate Olivetol [59] (Figure 1). Since then, two membrane-bound OA prenyltransferases, namely CsPT1 and CsPT4 sharing 62% sequence identity at amino acid level, have been cloned from _C. sativa_ and characterized. While only accepting a GPP donor, CsPT1 could prenylate a broad array of aromatic acceptors including OA, Olivetol, phlorisovalerophenone (precursor to bitter acids in hops), naringenin (flavonoid), and resveratrol (stilbenoid) [60]. When OA was used, CBGA was reported as the major product with a minor product identified as 5-genanylolivetolic acid [60]. CsPT4 could geranylate OA and a number of its C6 alkyl substituted analogs to CBGA and analogs [37], suggesting this substitution does not significantly impact substrate binding or catalysis. However, its substrate specificity towards other aromatic compounds such as olivetol or prenyl donors such as NPP has not been reported. The remaining _C. sativa_ hypothetical aromatic prenyltransferases (CsPT2,3,5-10) were reported to be ineffective for the prenylation of OA [37; 38], while CsPT3 was identified as the cannflavin A/B synthase that prenylates chrysoeriol [58]. CsPT4 however showed markedly superior enzyme activity in vitro, with a Km for OA at 6.72 mM [37] comparing to 60 mM documented for CsPT1 [60], which raises the question as which PT is physiologically responsible for CBGA formation. There are enzymes with unusually high Km, such as the grape _N_-methylanthranilate synthase that has a Km of 15 mM for its co-substrate methanol. The plant makes up the deficiency by overproduction of the enzyme and through the high methanol content that occurs during fruit maturation [61]. The expression of CsPT4 in _C. sativa_ was enriched in glandular trichomes [38], whereas CsPT1 expression was also enriched in trichomes, flowers, and young leaves where cannabinoids were accumulated [38; 60]. While further evidence is required to determine which PT of these two is responsible for cannabinoid biosynthesis in planta, it is still puzzling considering the original report of this prenylation activity was discovered using soluble proteins from leaves. Nevertheless, both PTs were able to geranylate OA in vivo in _S. cerevisiae_ as demonstrated by OA feeding in several reports [34; 35; 36; 37; 51]. The activity of CsPT1 was also shown when expressed in _E. coli_[43; 46], with a CBGA yield of 1.2 mg/L in growth media by feeding OA and GPP (Table 1) [43]. However, the in vivo activity of CsPT1 was reported very poor or inactive in _S. cerevisiae_, the methylotrophic yeast _Komugataella phaffii_ (formerly known as _Pichia pastoris_) and _Nicotiana benthamiana_ (tobacco) [37; 38; 41]. Feeding OA to _S.