# Biosynthesis of Nature-Inspired Unnatural Cannabinoidss
## 3 Aromatic Prenyltransferases
### Alternative Soluble Prenyltransferases  
A popular solution for the membrane-associated nature of prenyltransferases from plants is to replace them with a soluble prenyltransferase from bacterial or fungal species. NphB is a well-characterized and soluble prenyltransferase from _Streptomyces_ sp. strain CL190 [58]. [PERSON] et al. characterized NphB to catalyze the transfer of the C10 prenyl group from GPP onto 1, 6-dihydroxyxnaphthalene (1, 6-DHN) via an Mg\({}^{2+}\)-dependent mechanism and solved the crystal structure of the _holo_ enzyme complexed with geranyl S-thiolodiphosphate (GGPP) and 1, 6-DHN. The presence of a large pocket at the aromatic substrate binding site may explain why NphB was substrate-promiscuous for its prenyl acceptors, as it was found to be able to catalyze similar prenyltransferase reactions with a number of different aromatic substrates, including OLA [58]. This gave rise to several reports of different research groups using NphB as a suitable replacement for the prenyltransferase step in the cannabinoid biosynthetic pathway [20; 52; 61]. However, when catalyzing the reaction between OLA and GPP, NphB was found to produce two products: CBGA, and a side product, 2-O-geranyl-olivetolic acid (2-O-GOA) [52]. This led to two separate accounts of NphB protein engineering in identifying mutants of the enzyme that has a higher selectivity in producing CBGA [61; 62]. Notably, [PERSON] et al. managed to obtain mutants of NphB (Y288A/G286S and Y288V/A232S) that only produces exclusively CBGA with a 1000-fold improvement in catalytic efficiency. They successfully went on to incorporate their improved prenyltransferase in a cell-free system that produced 1.25 g/L of CBGA from glucose [61]. Further in-depth structure-based studies into the catalytic mechanism of a promiscuous enzyme such as NphB will benefit future efforts in engineering new substrate combinations into the enzyme. The crystal structure of NphB showed that it exhibits a novel structure consisting of ten strands of anti-parallel \(\beta\)-sheets arranged in a cylindrical manner that was named a PT-barrel. This PT-barrel fold is formed by five repetitive \(\alpha\alpha\beta\beta\) structural elements and remained exclusive to the ABBA family [63] until the discovery of the dimethylallyl-tryptophan synthase (DMATS) family of indole prenyltransferases, involved in the biosynthesis of pharmaceutically important ergot alkaloids in several fungi belonging to the phylum Ascomycota [64; 65]. The ten \(\beta\)-strands arranging in an antiparallel manner forms a central \(\beta\)-barrel enclosing the active site in its spacious lumen, and the \(\alpha\)-helices form a solvent-exposed ring surrounding the barrel. Members of these two families exist only in bacteria and fungi, and all of them seem to catalyze the prenylation of aromatic substrates to generate secondary metabolites [66]. They do not contain (N/D)DxxD motifs, and some prenyltransferases such as CloQ are still active in the absence of Mg\({}^{2+}\) or other divalent cations [59]. These soluble aromatic prenyltransferases show remarkable promiscuity for their aromatic substrates and may be used to biosynthesize libraries of new prenylated aromatic analogues for use in drug development. Another interesting target of study is the aromatic prenyltransferase from _Aspergillus terreus_, named _At_aPT, as it was found to be highly promiscuous for both its prenyl donor (C5-C20) and aromatic acceptors. _At_aPT was reported to be able to catalyze the prenylations of at least 72 aromatic compounds with DMAPP, GPP and FFP [67]. Additionally, apart from the usual single prenylation event reported thus far, where one unit of aromatic substrate is prenylated with one unit of prenyl group, _At_aPT was found to be able to catalyze multiple prenylation events on a single aromatic substrate molecule (Figure 6) [68].