## 2 Critical Biosynthetic Steps for Phytocannabinoid Formation
### Cannabigerolic Acid Formation by Aromatic Prenyltransferases
cerevisiae_ expressing CsPT4 has led to 96 mg/L and 216 mg/L CBGA production in the growth media from two patent applications (Table 1) [34; 35], whereas in vivo CBGA formation by CsPT1 was either reported much lower or not described with titers. A green fluorescent protein (GFP)-dPT4(1-246 nt deletion) fusion protein or full-length PT4 were used in each case. The yield differences may have resulted from the de novo GPP supply in each background, although other factors might be involved too. The first strain included a mutant ScERG20 (F96W, N127W) as GPP synthase (GPPS), an _N_-terminal-truncated 3-hydroxy-3-methyl-glutaryl-coenzyme A synthase (tScHMG1), and the deletion of yeast native ERG20 promoter [34]. The second strain had extensive engineering in the mevalonate pathway, which included overexpression of seven _S. cerevisiae_ genes (tHMG1, ERG8, 10, 12, 13, 19, isopentenyl diphosphate isomerase) and the pyruvate decarboxylase (PDC) from _Zymomonas mobilis_, in addition to ERG20(F96W, N127W) [35]. It was evident that ERG20(F96W, N127W) was optimal in yeast compared to other tested plant GPPS such as those from _Abies grandis_ (grand fir) and _Phalanenopsis bellina_ (orchid), which afforded as high as 15 mg/L CBGA from OA feeding [35]. Site-directed mutagenesis has not been reported for either preryltransferases, yet it was mentioned that an upcoming patent application describes the CsPT4 _N_-terminal engineering and domain swapping with aromatic preryltransferase from hops to improve its activity [35]. In addition to the two _C. sativa_ membrane-bound aromatic preryltransferases, a soluble aromatic preryltransferase NphB from the bacteria _Streptomyces_ strain CL109 was shown to geranylate OA to CBGA [41]. StNphB was discovered and characterized from a gene cluster related with the biosynthesis of naphterpin, a polyketide with cyclized geranyl chain similar to that of THC [62]. Although the native substrate of NphB has not been determined [63], it exhibited high degree of substrate promiscuity, catalyzing both _C_- and _O_-geranylation of a number of aromatic compounds including hydroxynaphthalenes, flavonoids, stilbenoids, and olivetol (K\({}_{\text{m}}\) 0.52 mM, _kcat_ 0.027\(\times\)10\({}^{3}\)/s) [64; 62]. When expressed in the yeasts _S. cerevisiae_ and _K. phaffii_, StNphB could catalyze the OA geranylation at both C3 forming CBGA (minor) and O2 forming 2-O-geranylolivetolic acid (2 OG, major) (Figure 2) [41]. Prerylation on other positions of the aromatic ring was also reported [65]. Co-expression of vacuole-targeted THCA synthase (THCAS) and StNphB of multiple genomic copies in _K. phaffii_ enabled the in vivo biotransformation of OA and GPP to CBGA and THCA [41]. The wild-type (WT) StNphB was reported to have much lower in vivo and in vitro activity towards OA than CsPT4 when expressed in yeast [33; 37; 41; 52] and _E. coli_ (K\({}_{\text{m}}\) 0.64 mM, _kcat_ 0.0021/min) [54; 55]. Several groups have worked on enhancing its product specificity and kinetics. Mutants of Y288A_G286S or Y288V_A232S dramatically improved StNphB kinetics resulting K\({}_{\text{m}}\) 0.45 mM, _kcat_ 1.58/min, and K\({}_{\text{m}}\) 0.12 mM, _kcat_ 1.30/min for OA [54; 55]. Such point mutations also rendered almost exclusive production of CBGA, which together led to 21-fold increase in CBGA titer (744 mg/L) in a cell free system with OA feedstock and de novo produced GPP (Table 1). By continuously removing CBGA from the cell free reaction with nonane, the group was able to push the CBGA titer to 1.25g/L. Further mutations were performed to improve the mutant StNphB thermal stability [56]. Improvement of CBGA product specificity was also achieved by Q295F mutation resulting in 20-fold increase of in vivo activity in _E. coli_ and concomitant CBGA/2 OG ratio shift from 1:5 to 20:1 with GPP/OA feedstock [66]. A large-scale mutagenesis of StNphB also revealed altered activity and regiospecificity of the aromatic ring prerylation [65].