# Biosynthesis of Nature-Inspired Unnatural Cannabinoidss
## 2 Olivetolic Acid Derivatives
### Orthologues of Interest  
Enzyme orthologues may behave differently when exposed to the same set of substrates. This was evidently observed in chalcone and stilbene synthases, which are both able to accept counaroyl-CoA and malonyl-CoA as the starter and extender unit, yet diverge to produce naringenin and resveratrol, respectively. This similar principle could be applied to screening efforts of a library of different OLS orthologues for the production of novel OLA analogues with a common resoncrily core that may serve as substrates for the following steps in the cannabinoid biosynthetic pathway. An example of such an orthologue is the type III PKS from _Rhododendon dauricum_, orcinol synthase (ORS), which catalyzes a similar reaction to OLS except that it uses acetyl-CoA instead of hexanoyl-CoA for its starter unit [43]. Interestingly, ORS alone also produces the decarboxylated rescorinol equivalent, orcinol as its major product, much like OLS producing olivetol as the major product. When OAC from _C. sativa_ was added in an in vitro reaction with ORS, the orsellinic acid (OSA) production increased significantly, which suggests that an unidentified polyketide cyclase much like OAC could be present in _R. dauricum_. It would be interesting to study amino acid differences between the cyclase from _R. dauricum_ and OAC from _C. sativa_ and elucidate how that contributes to substrate specificity. This phenomenon displays the close resemblance between the two enzymes separated by a speciation event that divides them into different species. Hydrangic acid and prelunularic acid are polyketides thought to be produced by putative stilbene synthases that utilizes counaroyl-CoA and dihydroxycoumaroyl-CoA, respectively, as the starter units, and three units of malonyl-CoA as the common extender unit. A unique property of these reactions is the ability of the putative type III PKS to catalyze C2-C7 aldol condensation with retention of the carboxylate functional group to form hydragic acid and prelunularic acid. This is presumed to be attributed to either the synergistic action of unidentified polyketide cyclases in _Hydrangea_ or to reduction via a ketoreductase (KR) domain in the enzyme due to the lack of the hydroxyl group at the C5 carbonyl [44; 5]. The ability of the PKS to perform a non-decarboxylase aldol condensation after chain elongation is important in cannabinoid biosynthesis, as the carboxylate functional group is vital in interacting with cannabinoid synthases later in the biosynthetic pathway [45]. Orthologues that may perform the rare non-decarboxylate aldol condensation step will circumvent the need for an additional cyclase step, improving yields in a heterologous metabolic pathway by reducing the extent of loss of intermediates. As mentioned previously, a replacement of OLS in the pathway producing a different product may require the synergistic action of a corresponding polyketide cyclase, such as OAC, to perform the C2-C7 non-decarboxylate aldol condensation to form the rescorcinolic acid scaffold for further downstream catalysis. The aforementioned biosynthesis of orsellinic acid by the action of ORS in \(R\). _dauricum_ still lacks clarity on an unidentified cyclase enzyme. Polyketide cyclases are relatively new to the world of polyketide biosynthesis and until more orthologous sequences are identified, this will remain a bottleneck in finding replacements for the PKS in the cannabinoid biosynthetic pathway. Webtools such as the Enzyme Similarity Tool from the Enzyme Function Initiative (EFI-EST) could aid in identifying orthologues of polyketide cyclases such as OAC that are not well-characterized or annotated. The web-tool uses databases such as UniProt to identify amino acid sequences related to the query sequence in terms of function and reaction catalysis. It generates a sequence similarity network (SSN) which allows for the easy visualization of the relationship between sequences across different species [46].