# Biosynthesis of Nature-Inspired Unnatural Cannabinoidss
## 2 Olivetolic Acid Derivatives  
The amino acid sequence of the type III polyketide synthase, OLS, from _C. sativa_ was identified in 2009. It was noted then, that OLS does not produce OLA, and only forms three reaction products: a triktetide pyrone, a tetraketide pyrone and divetol, the decarboxylated alcohol form of OLA [22]. It was understood at that time that cannabinoid synthases producing THCA and CBDA produced downstream in the pathway from CBGA could not catalyze the reaction using the alcoholic form, CBG [26; 27]. Hence, this would mean that there is a gap in the pathway that produces the corresponding acidic polyketide upstream, OLA. The missing link in the upstream polyketide pathway was only uncovered with the identification of OAC in 2012 by [PERSON] et al. [24]. OAC was the first polyketide cyclase to be found in plants. It utilizes the putative tetraketide-CoA product from OLS as a substrate and cyclizes it to form OLA. This interplay between type III polyketide synthases and cyclase enzymes to produce structural diversity in natural products was an exciting development in the established field of polyketide synthases. The structure of OLS was finally resolved more than a decade after its initial characterization [23]. Type III PKSs have highly similar structural features, as observed in the well-characterized stilbene synthase [28] and chalcone synthase [29] (Figure 2). Both PKS mentioned along with OLS catalyze highly similar reactions and form a tetraketide-CoA thioester intermediate from three rounds of chain-elongating condensations. However, the reaction mechanism seems to diverge after the formation of the elongated tetraketide intermediate. Chalcone synthase (CHS) adopts a C6 to C1 Claisen condensation to form naringenin, a chalcone product, while OLS and stilbene synthase seems to adopt a spontaneous C2 to C7 decarboxylative aldol condensation to form the stilbene products, Olivetol and resveratrol, respectively. Figure 2: Superimposed structures of OLS (cyan, PDB 6 GW3 [23]), stilbene synthase (yellow, PDB 1U0U [28]), and chalcone synthase (purple, PDB 1 CGK [29]). Shown ligands CoA and naringenin are co-crystallized with OLS and chalcone synthase, respectively. The active site of OLS is bi-lobed, with each lobe accommodating the starter-molecule binding and the elongating polyketide chain. Using analogous information from previous type III PKSs [28; 29; 30], we proposed the following reaction mechanism for OLS involving the Cys157/His297/Asn330 catalytic triad (Figure 3). Upon binding of the starter-CoA unit at the active site, Cys157 activated by His297 attacks the thioester linkage between the starter unit and the CoA-moiety, forming a monoketide intermediate attached to Cys157. The first malonyl-CoA extender unit then diffuses into the active site and is decarboxylated to form an acetyl-CoA carbonion. The nucleophilic acetyl-CoA carbonion then attacks the thioester linkage between the starter unit and Cys157, thereby forming a diktetide-CoA intermediate. Subsequently, Cys157 recaptures the elongated diktetide unit and releases the CoA-moiety to reinitialize the process for two more rounds of elongation (Figure 3). However, unlike its orthologues, OLS lacks a specific cyclization mechanism for the elongated tetraketide-CoA product and releases it from its active site. The three reaction products found from OLS activity are postulated to be the result of a spontaneous C5-oxygen to C1 lactonization event that occurs in the triktetide-CoA intermediate from two rounds of elongation to form the triktetide pyrone, and differential spontaneous cyclization to form tetraketide pyrone and olivetol from a tetraketide-CoA product [22]. It was found later that OAC then utilizes this tetraketide-CoA product from OLS for a C2-C7 aldol cyclization without decarboxylation to produce OLA, as previously mentioned [24]. OAC was the first polyketide cyclase observed in plants.