# Biosynthesis of Nature-Inspired Unnatural Cannabinoidss
## 2 Olivetolic Acid Derivatives
### Protein Engineering  
Exploring the substrate selectivity profile of OLS allows us to understand the extent of \"how promiscuous\" the enzyme can be in accepting different substrates. However, there is a limit in an enzyme's substrate promiscuity profile that is governed by steric and polarity constraints. Protein engineering allows us to push the boundaries of an enzyme's innate substrate specificity by introducing favorable mutations that allows the enzyme in context to accept a different substrate that it was previously unable to accept, or even to alter its reaction mechanism. Traditional methods of protein engineering, such as directed evolution, require a selection method, such as a toxicity or biomarker screen, to facilitate the easy selection of clones with mutant proteins that carry the desired trait. However, it may not be easy to design a robust selection method for picking out mutants that produce novel products with unknown toxicity or pharmacological profiles. In such instances, a rational structure-based approach in protein engineering would be useful.  
As previously mentioned, close orthologues of OLS, chalcone synthase and stilbene synthase, catalyze similar reactions except for the difference in starter unit preference and cyclization mechanism. Structural alignment of these orthologues with OLS allows us to identify the non-conserved residues between the orthologues and may explain the differences in substrate specificity and cyclization patterns. Unfortunately, [PERSON] et al. was unable to identify the residues responsible for the differences in cyclization pattern among the orthologues when they performed targeted single mutational changes of the non-conserved residues in OLS to the corresponding residues in chalcone synthase [23]. Given the highly complex nature of the reaction catalyzed, it is likely that multiple residues were responsible for the catalytic differences in cyclization instead of single key residues. This is evident in the protein engineering project undertaken by [PERSON] et al. where 18 amino acid residues were mutated in order to convert an alfalfa CHS to a corresponding stilbene synthase catalyzing a C2-C7 decarboxylative aldol condensation [28]. The \"aldol switch\" identified, 18 amino acid residues along with a network of hydrogen-bonding configuration, was responsible for converting the chalcone-predominant product to a stilbene-predominant product. Future work investigating a more thorough set of the amino acid residue differences among these orthologues would be useful for future protein engineering work in creating novel OLA analogues.  
An interesting strategy for protein engineering of PKS enzymes governing substrate selectivity and catalytic activity is domain swapping. Domain swapping is the substitution of a domain in a monomeric protein with another domain of an identical or similar protein. Since the first instance of domain swapping was reported in diphthearia toxin dimeric form, 3D domain swapping as a protein engineering tool has been used to modify an enzyme's bioactivity function such as substrate specificity and catalytic activity [40]. [PERSON] and [PERSON] provided an excellent review on the current knowledge and possibilities of 3D domain swapping [41]. The term domain can be redefined as a functional unit of specific catalytic activity. In the context of PKS protein engineering, this is evidenced in many protein engineering studies of model PKSs such as the 6-deoxyerythronolide synthase (DEBS) where the swapping of domains within the enzyme produces varied products [42]. Future studies on OLS structure-function relationships will facilitate the engineering of catalytic domains into the enzyme such as introducing thioesterase activity in OLS to perform the C2-C7 aldol condensation reactions of the tetraketide product and factors governing substrate selectivity.