## 2 Critical Biosynthetic Steps for Phytocannabinoid Formation
### The Formation of Olivetolic Acid  
The first committed step in cannabinoid biosynthesis in _C. sativa_ is formation of a polyketide intermediate and its cyclization to form olivetolic acid (OA) (Figure 2). In _C. sativa_, this reaction is catalyzed by a type III polyketide synthase named tetraketide synthase (CsTKS) or olivetol synthase (CsOLS), and a unique cyclase named olivetolic acid cyclase (CsOAC), which are both enriched in the glandular trichomes [22; 23; 24; 25]. In the reaction of CsTKS, malonyl coenzyme A (CoA) is first decarboxylated and then the resulting acetyl forms a C-C bond with the carbonyl of the enzyme-bound hexanoyl CoA. After three rounds of condensation, the labile tetraketide intermediate is released, which is cyclized by OAC via C2-C7 aldol condensation (-H\({}_{2}\)O) followed by spontaneous aromatization to form the resorcylic acid skeleton (Figure 1) while still retaining the CoA thioester. Spontaneous ester hydrolysis occurs to give the final product OA (Figure 2). Plant polyketide biosynthesis is largely catalyzed by type III PKSs that condense several malonyl CoA with various starting CoA esters followed by a cyclization. Type III PKSs are considered the minimal PKS as they are simple homodimers with only polyketide synthase and cyclase activities. In contrast, type I PKSs are large polypeptides with several domains such as acyl carrier proteins, acyltransferases, PKSs, dehydrogenase, and keto-reductases that catalyze a series of reactions. Type II PKSs are instead individual enzymes that forms a complex functionally similar to Type I PKSs. More detailed biochemistry of PKS can be found in these excellent reviews [26; 27; 28]. Figure 2: The native and engineered biosynthetic pathway for phytocannabinoids in _C. sativa_. Enzymes in italics are mutant enzymes. The abbreviations are listed as follows. The cannabinoid pathway: TKS, tetraketide synthase; OAC, olivetolic acid synthase; PKS, polyktetide synthase; NphB, haplterpin biosynthetic cluster gene B; PT, prenyltransferase; THCAS, tetrahydrodecannabinolic acid synthase; CBDAS, cannabinoidic acid synthase; CBCAS, cannabinoidic acid synthase. MVA pathway: mvaE, acetoacetyl-CoA thiolase/HMG-CoA reductase; mvaS, HMG-CoA synthase; ERG12, mevalonate kinase; ERG8, phosphomevalonate kinase; ERG19, diphosphomevalonate decarboxylase; IDI1 isopentenyl diphosphate: dimethylallyl diphosphate isomerase; ERG20, farnesyl pyrophosphate synthetase. MEP pathway: DXS, deoxyxyululose 5-phosphate synthase; DXR, deoxyxyululose 5-phosphate reductio isomerase; IspD, 4-diphosphocytidyl-2-C-methyleryththird (CDP-ME) synthase; IspE, CDP-ME kinase; IspF, 2-C-methyl-D-eryththird 2,4-cyclodiphosphate synthase; IspC, (E)-4-Hydroxy-3-methyl-but-2-enyl pyrophosphate (HMB-PP) synthase; IspH, HMB-PP reductase. Fatty acid biosynthesis: BktB, \(\beta\)-keto thiolase; ACC, acetyl CoA carboxylase; MCT1: malonyl CoA-acyl carrier protein transacylase; HBD/PaaH1, \(\beta\)-hydroxybutyryl-CoA dehydrogenase; CRT: 3-hydroxybutyryl-CoA dehydatase; Ter, trans-enoyl-CoA reductase; FAA2, Long-chain-fatty-acid-CoA ligase 2; KmTES1, _Kluyveromyces_ _maxxinus_ acyl-CoA thioesterase; AAE: acyl activating enzyme. Species abbreviations: Cs, _Cannabis sativa_; St, _Streptomyces_ strain CL109; Di, _Dictyostelium discoideum_; Ef, _Enterococcus faecalis_; Re, _Ralstoni eutropha_; Ca, _Clostridium acetobutylcium_; Td, _Tropenema denticola_; Sc, _Saccharomyces cerevisiae_; Km, _Kluyveromyces marxinus_. The most ubiquitous type III PKS in plants is perhaps chalcone synthase (CHS), the first committed enzyme in the biosynthesis of the flavonoid metabolite class, which is found in all plants. After forming the labile polyketide, CHS cyclizes it to form the chalcone skeleton via C1-C6 Claisen condensation (Figure 2), which is one of the two most common cyclization mechanisms among type III PKSs. In contrast, stilbene synthase cyclizes the polyketide chain via the C2-C7 aldol condensation with concomitant elimination of the terminal carbonyl as CO2.