# Biosynthesis of Nature-Inspired Unnatural Cannabinoidss
## 4 Cannabinoid Synthases
### Orthologues of Interest  
Lastly, orthologues that evolved from different species can also be tested for bioactivity using CBGA and/or its analogues mentioned earlier as substrates. [PERSON] and [PERSON] provided an extensive review on phytocannabinoids found in different plant species [5]. However, we wanted to focus on the family of alkylrescornicnic acids produced by _Rloadedatron dauricum_ as previously mentioned. Like cannabinoid synthases, _R. dauricum_ also produces a flavoprotein oxidase that produces daurichromenic acid (DCA) from grifolic acid [81]. Daurichromenic acid chemical synthesis has been extensively studied in recent years [82; 83; 84] due to its value as a highly effective anti-HIV natural product [76]. In terms of substrate and products formed, DCA synthase is remarkably similar to the cannabinoid synthases. More specifically, the reaction catalyzed by DCA synthase is analogous to CBCA synthase in _C. sativa_ producing cannabichromenic acid (Figure 13A,B). DCA synthase, however, was unable to use CBGA as a substrate. This is possibly due to the pentyl chain on C6 of the resorcinolic ring, as it was able to catalyze cannabigerocinic acid, an analogue of CBGA with an acetyl group in place of the pentyl chain, to form the synthetic cannabinoid cannabinoidrepornicnic acid [85]. A further test on substrate promiscuity could be carried out on DCA synthase, using a library of different CBGA analogues with different chain lengths on C6 of the resorcinolic ring, to probe the limits of the enzyme's promiscuity profile. Interestingly, DCA synthase was observed to exhibit relaxed substrate specificity for substrate analogues with varied prenyl chain lengths, catalyzing the formation of cannabichromeocratic acid and diterpenodaurichromenic acid, from cannabigerocinic acid and 3-geranylgeranyl orsellinic acid as substrates, respectively (Figure 13C) [81]. This could represent a potential protein engineering opportunity for DCA synthase, analogous to the examples shown for the prenyltransferases, in engineering the enzyme to be able to catalyze the oxidative cyclization of CBGA analogues with different prenyl chain lengths. CBGA analogues with longer prenyl chain lengths, such as the C15 FPP and C20 GGPP, can be cyclized by orthologous sequences such as DCA synthase, that are able to act on the longer than native C10 geranyl prenyl chain. This potentially represents an interesting route of diversification in the phytocannabinoid chemical space,  
Figure 12: Structural alignment of THCA synthase crystal structure with homology models of CBDA and CBCA synthase showing amino acid differences in the active site. THCA synthase active site amino acid residues are shown in green, CBDA synthase residues shown in cyan and CBCA synthase residues shown in orange. CBGA ligand (shown in yellow) was docked into the THCA synthase active site while the FAD molecule is colored purple. based on the prenyl chain on C3 of the OLA resorcinolic acid ring, that has not been extensively explored. Cannabinoid synthases belong to a rapidly growing class of flavin oxidases named the vanillyl alcohol oxidase (VAO) protein family [86]. They share a highly conserved FAD-binding domain. Notable members of the group include the berberine bridge enzyme (BBE) from the psychoactive California poppy plant, _Eschscholzia californica_, which catalyzes the formation of (S)-scoulerine alkaloid from (S)-reticuline [87], and carbohydrate oxidases, _Ha_-CHOX and _Ls_-CHOX, from higher plants such as sunflower and lettuce, involved in antimicrobial activity against phytopathogens [88]. Currently identified THCA synthase, CBDA synthase, CBCA synthase and DCA synthase are oxidoreductase from two different species. An SSN generated by using THCA synthase as the query, with 40% sequence identity through the EFI-EST webtool [46], shown in Figure 14, depicts the vast number of other possible orthologues from various natural sources.