## 2 Critical Biosynthetic Steps for Phytocannabinoid Formation
### FAD-Depended Cyclases Form Various Cannabinoids
Although the recruitment process for FAD is not clear, considerable knowledge has been made with regard to the _N_-glycosylation and disulfide formation that involve numerous chaperones and enzymes, which are nicely summarized in several reviews [77, 78, 79, 80]. While mechanistically the _N_-glycosylation process in ER lumen is highly conserved among plants, animals, and fungi, the recognition and glycan attachments may differ significantly in different systems, which impacts the expression of _C. sativa_ cyclases in fungi such as yeast. Incorrect or insufficient _N_-glycosylation can result in protein sorting into ER-associated degradation (ERAD) [78], and overexpression of secretory proteins may overload the _N_-glycosylation system which results in ER stress and unfolded protein response (UPR) that limits the recombinant protein expression [81]. Expression of these cyclases has been demonstrated using insect _Spodoptera frugiperda_ (Sf9) cell culture [50], yeast (_K. phaffii_[71], _S. cerevisiae_[37; 40]), plant (_N. benthamiana_[38; 50; 74], and also claimed in a number of patents and patent applications. In all above systems, active enzymes can be produced without complications. However, the in vivo activity could be significantly improved by a number of bioengineering approaches as seen in two recent reports. [PERSON] et al. [72] systematically examined the cyclase activity in yeast _K. phaffii_ crude lysate by site-directed mutagenesis. Replacement of all seven Asn residues deemed necessary for _N_-glycosylation to Gln resulted in undetectable THCAS activity in cell lysate, whereas N89Q_N499Q double substitutions instead resulted in 2-fold increase of THCAS activity. At the enzyme active site, the triple substitution A414V_A46V_T47A was able to increase CBDAS activity 4.1 fold. The same group also looked into improving cyclase activity by enhancing protein folding in the ER [42]. ER chaperones (KAR2 and CNE), FAD synthetases (FAD1), foldases, protein disulfide isomerases (PDII), and transcription factors (HAC1) were overexpressed in _K. phaffii_, which led to an impressive 20-fold in vivo THCAS activity increase. The result joins other reports in suggesting that improving ER protein folding is a viable solution to higher protein titer. It should be noted that the THCAS signal peptide was replaced with a vacuole protease A signal peptide in the above work to direct THCAS inside of vacuole instead of being secreted. A systematic method for cannabinoid production engineered in yeast _S. cerevisiae_ was also demonstrated in a patent application with similar approaches [36]. CBDA biosynthesis with full-length CBDAS harboring its native signal peptide was enabled by feeding OA to the yeast strain which was engineered with GPP production and other necessary cannabinoid biosynthetic genes (Table 1). Overexpression of proteins enhancing ER protein folding dramatically increased the basal CBDA titer of 5 mg/L (3-day feeding) to 179 mg/L (7-day feeding) with overexpression of KAR2, IRE1 (HAC1 activation), PDI1, ERO1 (PDI1 activation) and downregulation of ROT2 (ER glucosidase), PEP4 (protease). Site-directed mutagenesis in CBDAS also positively influenced enzyme activities shown by the improvement from the basal 162 mg/L to 234 mg/L (N196Q), 229 mg/L (L132M), or 224 mg/L (N57D) collected from testing 68 variants. Approximately 6-10% of the CBDAS product is THCA in these experiments. Similarly, THCA titer from OA feeding was improved from 131 mg/L with wild-type THCAS to 320 mg/L with N196Q substitution consistent with previous finding (Table 1) [72]. It is worth noting that wild type and most tested THCAS variants expressed in _S. cerevisiae_ did not produce detectable amounts of CBCA, which was quite different from the THCAS produced from _K. phaffii_ (10% CBCA) [72].