## 2 Critical Biosynthetic Steps for Phytocannabinoid Formation
### The Formation of Olivetolic Acid
In this reaction, the tetraketide thioester intermediate must be hydrolyzed to the carboxylate prior to the aldol condensation while still being docked in the PKS active site [29; 30]. In fact, early release of hydrolyzed polyketide intermediate can also cause spontaneous lactonization via C1-O5 bond formation seen in the formation of many triacetic lactone (TAL) polyketides (Figure 2). While TAL products have not been reported in cannabis, they are found in other plant species such as _Gerbera hybrida_ (ornamental daisy) [27]. Without CsOAC, CsTKS proceeds with the regular aldol condensation mechanism to form olivetol (resorcinol skeleton) instead of OA (resorcinol skeleton). This carboxylate group is presented in the final cannabinoids such as A\({}^{9}\)-tetrahydrocannabinolic acid (THCA), which renders it non-psychoactive. When heated, THCA loses this carboxylate and transforms to the psychoactive THC. As such, it would appear that forming olivetol instead of OA is beneficial for producing the psychoactive THC instead to THCA. However, the presence of this carboxylate group is necessary for the enzyme activity of the terminal cyclases that forms various cannabinoids [31]. Therefore, OA formation via cyclization by CsOAC is required for cyclic cannabinoid biosynthesis. In rare cases, PKS can catalyze the C2-C7 aldol condensation prior to thiol esterification, which retains the terminal carbonyl without an additional cyclase. To date, there is only a handful such PKSs that are cloned and characterized, including the 2\({}^{\prime}\)-oxoxalkylresorylic acid synthase (ORAS) from the fugus _Neurospora crassa_[29], alkylresorylic acid synthase 1 and 2 (ARAS1, 2) from rice [30], and the stilbene carboxylic acid synthase (STCS) from _Hydrangea macrophulla_[32]. While these enzymes produce their respective resorylic acids as the major products, resorcinols were also produced, at least _in vitro_. It has also been reported that the PKS orcinol synthase from _Rhododendon dauricum_ produced small amounts of orsellinic acid (Figure 1). Orsellinic acid is an OA analog with a methyl side-chain instead of the pentyl group found in OA. It may be possible to engineer CsTKS to function directly as an OA synthase without the help of CsOAC. However, such modifications have yet to be reported, and CsOAC remains required for the biosynthesis of the final cyclized cannabinoids. A patent application describes the use of a type I fatty acid synthase/PKS from the amoeba _Dictyostelium discoideum_ for the biosynthesis of olivetol, 1-methylolivetol, and their geranylated products CBG and methyl-CBG (Figure 2) [33]. The DiPKS is a large multi-domain enzyme with \(\beta\)-ketoacyl-syntase, acyl transacetylase, dehydatase, \(C\)-methyl transferase, enoyl reductase, ketoreductase, acyl carrier protein, and polyketide synthase activities, which synthesized 1-methylolivetol directly from malonyl CoA, therefore sparing the need of additional engineering or exogenous supply of hexanoyl CoA. A G1516R mutation could effectively knock out the \(C\)-methyltransferase activity leaving olivetol as the only product, whereas G1516D_G1518A double mutation allows a mixture of methyl and non-methyl olivetol formation. When DiPKS was expressed in their engineered yeast _Saccharomyces cerevisiae_, 76 mg/L olivetol and 66 mg/L methyl-CBG de novo production was reported (Table 1). The prevalence of resorcinol structures in nature also suggests that other PKSs may be recruited for olivetol production. While CBG could not be cyclized to THC, CBD or other final cannabinoids by the characterized \(C\). _sativa_ cyclases [31], it is not known whether these enzymes can accept methyl-CBG as a substrate. Other than the implication of the DiPKS, all other publicly available peer-reviewed articles and patent applications for cannabinoid biosynthesis use CsTKS and CsOAC to our knowledge. The duo can accept a number of natural or modified aliphatic CoA starters to form the respective resorcylic acids of different side-chain lengths (e.g., C3-C6 etc.) [14; 22; 37].