# Biosynthesis of Nature-Inspired Unnatural Cannabinoidss
## 3 Aromatic Prenyltransferases  
\"Prenylation\" is the enzymatic transfer of the hydrophobic side chain from a terpene diphosphate to another biomolecule [49]. Prenyl groups can be small, five-carbon units (C5) such as the dimethylallyl or isopentenyl moieties, or larger components with multiple five-carbon units such as the geranyl (C10) or farnesyl (C15) groups. Although a lot of research has been carried out on protein prenyltransferases and the significance of this posttranslational modification step in cellular signaling activities [50], not much is known about small aromatic molecule prenyltransferases. Aromatic prenylation plays a critical role in the biosynthesis of diverse bioactive secondary metabolites in plants, fungi and bacteria, with the prenyl group enhancing the uptake and biological activities of the aromatic core residue [51]. CBGA is the first cannabinoid produced in the cannabinoid biosynthetic pathway in _C. sativa_, and is formed by a C-C Friedel-Craft alkylation of OLA at the C3 position [52].  
CBGA, the first branch-point in the cannabinoid biosynthetic pathway, was found to be produced from OLA and GPP by GOT in the _Cannabis_ plant more than two decades ago [25], and a _Cannabis_ GOT (_Cs_PT1) was patented 16 years later [53]. In the patent, _Cs_PT1 was cloned and expressed in _Spodoptera frugiperda_ 9 insect cells, and was able to produce CBGA and the side product 5-geranyl divetolate when insect cell microsomes containing _Cs_PT1 were incubated with GPP, OLA and MgCl\({}_{2}\)[53]. Nevertheless, several groups tried to express and characterize a functional _Cs_PT1 in other hosts but were unsuccessful [20; 38]. By analyzing published _Cannabis_ transcriptomes, [PERSON] et al. found a functionally active prenyltransferase, named CsPT4, from _C. sativa_, that biosynthesizes CBGA from GPP and OLA. Interestingly, _Cs_PT4 only shared 62% homology to _Cs_PT1, and is predicted to have eight transmembrane helices [20]. _Cs_PT4 was also found to be substrate-promiscuous for its aromatic prenyl-acceptor as it could accept OLA analogues derived from butanoic acid, pentanoic acid, 5-hexenoic acid, heptanoic acid, 4-methylhexanoic acid, and 6-heptynoic acid, while using GPP as the prenyl-donor producing CBGA analogues with varying side chains at the C6 position. Although the heterologously expressed _Cs_PT4 can localize to the purified microsomal fraction from yeast and facilitate the determination of enzyme kinetics when the microsomal preparation was incubated with OLA and GPP, the lack of a crystal structure hinders efforts to gain mechanistic insights into its substrate selectivity. Concurrently, research by another group showed that _Agrobacterium tamefaciens_ mediated the transient expression of _Cs_PT4 in _N. benthamiana_, which resulted in CBGA production when GPP and OLA were delivered as substrates by leaf infiltration [38]. The gene loci of _Cs_PT1 and _Cs_PT4 are in close proximity, and both are highly and selectively expressed in _Cannabis_ trichomes where cannabinoids accumulate, suggesting that _Cs_PT1 and _Cs_PT4 might be functionally redundant in _C. sativa_.