# Biosynthesis of Nature-Inspired Unnatural Cannabinoidss
## 3 Aromatic Prenyltransferases
### Alternative Soluble Prenyltransferases
Such an unprecedented promiscuity profile is fascinating as it represents exciting opportunities as a replacement in many biosynthetic pathways that requires a prenyltransferase step, allowing users to produce a vast array of different chemical structures. A comparison of the aromatic acceptor-binding pocket of _At_aPT with those of other DMATS-type prenyltransferases, such as AnaPT from _Neosartorya fischeri_[69] and FgaPT2 from _Aspergillus fumigatus_[60], showed that _At_aPT has an exceptionally spacious hydrophobic substrate-binding pocket, formed by multiple hydrophobic residues with relatively shorter side chains (Gly106, Cys175, Ser192, Gly251, and Gly326) compared to the other DMATS-type prenyltransferases [67]. This provides more flexibility to accommodate various prenyl donors and acceptors, and the hydrophobic nature of the pocket may aid to enhance the stability of prenyl carbocation intermediates, thus facilitating prenylation at multiple sites. Mutagenesis studies of Gly326 in _At_aPT to methionine, to mimic the corresponding Met364 residue in FgaPT2, resulted in a mutant that could no longer accept GPP or FPP as prenyl donors when (+)-butyrolactone II was used as the aromatic acceptor [67]. This demonstrates the possibility of engineering its substrate specificity via the manipulation of active site residues by site-directed mutagenesis. Figure 6: _At_aPT catalyzes predominantly C-monoprenylation of acylphoroglucnols but can also form _gem_-diprenylated products with DMAPP as the prenyl donor, as well as C- and O-diprenylated derivatives. Soluble and promiscuous aromatic prenyltransferases, such as NphB and _At_aPT, represent only a few of the many sequences found in nature that may catalyze the prenyltransferase step in the cannabinoid biosynthetic pathway. Other uncharacterized prenyltransferase sequences from various sources can be screened for novel functions in relation to cannabinoid biosynthesis. Novel cannabinoid derivatives with different structures can be produced enzymatically; these derivativizations may alter or complement the bioactivity of existing cannabinoid therapeutics. The EFI-EST webtool provides users with an easy way of identifying these related sequences and their relation to each other through the creation of an SSN. Using NphB as a reference sequence, we generated an SSN of related sequences with at least 40% sequence identity to each other in the UniProt database. This allowed us to identify potential orthologous sequences in the database that may perform similar functions, and hence serve as enzymatic replacements in the pathway, or produce novel compounds either from the same substrates or a different set of substrates. Figure 7 shows an SSN generated using the NphB query sequence, identifying 176 sequences, most of which are from bacterial and fungal species. Ongoing studies in our lab have identified several orthologues that are able to catalyze similar reactions using OLA and GPP as substrates to produce CBGA. Additionally, some of these orthologues were found to produce novel products using the same set of substrates, suggesting the ability to prenylate OLA at different positions to give novel CBGA analogues such as 4-\(O\)-geranyl olivetolic acid and 5-geranyl olivetolic acid, as depicted in Figure 8 (unpublished data). Further studies are underway to uncover prenyltransferase orthologues that are able to catalyze the prenylation using various isoprenoid groups such as DMAPP, FPP and GGPP to an OLA rescorinolic acid scaffold at the C3 position, as depicted in Figure 9. Novel CBGA analogues with either a different side chain on C6 of the OLA resorcinolic acid scaffold, or longer prenyl groups on the C3 position, provide unique cannabinoid scaffolds for testing with downstream cannabinoid synthases in the biosynthetic route. Figure 7: SSN showing putative NphB orthologs belonging to IPR036239 with at least 40% sequence identity to each other.