# Biosynthesis of Nature-Inspired Unnatural Cannabinoidss
## 2 Olivetolic Acid Derivatives
Several polyketide cyclases had previously been uncovered in bacteria, such as SnoaL from _Streptomyces no-galater_[31; 32], the tetracenomycin aromatase/cyclase (Tcm ARO/CYC) [33; 34] and the tetracenomycin F2 cyclase (TcmI) [35; 36] from _Streptomyces glaucscens_. These enzymes catalyze aldol condensation reactions, causing the aromatization of their substrates. A closer look at the structure of OAC reveals that it is structurally similar to bacterial polyketide cyclases from _Streptomyces_ sp., belonging to the dimeric \(\alpha+\beta\) barrel (DABB) protein  
Figure 3: Proposed OLS mechanism forming a tetraketide-CoA thioester product for OAC to cyclize into a resorcinolic ring such as OLA. family [37]. It was reported that OAC does not interact physically with OLS for its catalytic action, and it accepts the linear 3, 5, 7-trioxododecanoylI-CoA product from OLS as substrate [22]. By solving the X-ray crystal structure, and with insights obtained through a series of site-directed mutagenesis experiments, [PERSON] et al. proposed a reaction mechanism for OAC that uses an acid/base catalyst reaction to cyclize its substrate, as depicted in Figure 4[37]. Facilitated by Tyr72, His78 first conducts a nucleophilic attack on the C2 of the tetraketetide-CoA substrate to produce an enolate intermediate, as seen in Figure 4B. Next, the unstable enolate intermediate then attacks its C7 carbonyl carbon and a proton abstraction from His78 releasing water constitutes the aldol condensation reaction and cyclization. Subsequently, release of the CoA-linked intermediate from OAC followed by the spontaneous aromatization and cleavage of the CoA bond forms its product OLA. A few groups have attempted the heterologous biosynthesis of OLA and its analogues since the identification of OLS and OAC. In 2018, [PERSON] et al. published an article describing the synthetic production of OLA in _Escherichia coli_ from glycerol [18]. Using a \(\beta\)-oxidation reversal pathway and the acetyl-CoA carboxylase (ACC) for hexanoate and malonyl-CoA precursor supply, coupled with OLS and OAC enzymes, they were able to produce up to \(\sim\)80 mg/L of OLA in vivo using optimized fermentation conditions. [PERSON] et al. attempted OLA biosynthesis using the tobacco plant _Nicotiana benthamiana_ by heterologous expression of OLS and OAC and observed the production of OLA and a glucosyllated analogue, olivetolic acid glucoside. They postulated that this derivative occurs through the actions of uridine diphosphate glucosyltransferases (UDP-UGT), present in _N. benthamiana_, that are able to act on the OLA product as a substrate [38]. The glucosylation modification on OLA and its downstream cannabinoids may increase the aqueous solubility of these hydrophobic compounds and hence improve their bioavailability as medicines. With the biosynthetic pathway for OLA elucidated and well-characterized, efforts to produce novel cannabinoids downstream using novel cannabinoid precursors such as OLA analogues can be attempted. In order to serve as cannabinoid precursors, analogues of OLA will need to retain a few key features for the binding and activation of downstream CB receptors. The dihydroxystilbene acid feature with hydroxyl groups at C2 and C4, accorded by the use of malonyl-CoA as extenders, and a carboxylic acid attached to the resorcinolic  
Figure 4: Proposed OAC cyclization mechanism to form OLA. (**A**) Extraction of proton by His78 from C2 of substrate. (**B,C**) C2 to C7 aldol condensation facilitated by reabsorption of proton from His78 by carbonyl O on C7. (**D,E**) Aromatization, release of CoA thioester and subsequent release of OLA product. ring, is key for further downstream processing of prenylation and oxidative cyclization. The following sections discuss different strategies for producing OLA analogues with the intent of producing novel cannabinoids.