# Biotechnological Fungal Platforms for the Production of Biosynthetic Cannabinoids
## 4 Development of Biotechnological Fungal Platforms for the Biosynthesis of Cannabinoids
### Biosynthesis of Cannabinoids in Yeasts
Thus, the highest THCAS activity was obtained with the co-production of the spliced gene version of: (i) basic leucine zipper transcription factor Hac1s (involved in the regulation of several genes involved in protein folding, secretion, ER quality control, glycosylation, and ER-associated degradation); (ii) CNE1 (an ER membrane protein that functions as a constituent of the ER quality control apparatus); and (iii) FAD1 (FAD synthetase, involved in the production of FAD from FMN). This strain showed improved productivity in comparison to previous strains and reached 3.05 g/L THCA from the bioconversion of CBGA within 8 h of incubation [77]. The same authors also carried out site-directed mutagenesis in THCAS and CBDAS in order to elucidate the structure-function relationship and improve the enzyme properties for biotechnological cannabinoid production. After the recombinant expression of different mutant enzymes in _K. phaffi_, the enzyme bearing the double substitution N89Q + N499Q (lacking two glycosylation sites) gave rise to a 2.0-fold increase in THCAS activity, whereas the variant A414V (mutant in the active site) was able to increase the catalytic activity for the production of CBDA by 3.3 times [78]. In parallel, _S. cerevisiae_ was engineered to generate strains capable of performing the complete biosynthesis of CBGA, \(\Delta^{9}\)-THCA, CBDA, \(\Delta^{9}\)-tetrahydrodcannabivarinic acid, and cannabidivarinic acid from the simple sugar galactose [23]. In this biosynthetic platform, hexanoyl-CoA was produced either by feeding hexanoic acid as a substrate for the acyl-activating enzyme (encoded by CsAAE1 from _C. sativa_) or from acetyl-CoA (derived from sugars) due to the introduction of a multiorganism heterologous biosynthetic pathway comprising RebktB from _Ralstonia eutropha_, CrpaaH1 from _Cupriavidus necator_, Caxtt from _Clostridium acetobutylicum_, and Tdter from _Treponema danticicola_. In addition to this, the native mevalonate pathway was modified, including a mutant version of Erg20 (F69W/N127W) to overproduce GPP. The introduction of the OA biosynthetic genes CsTKS and CsOAC, a gene encoding a previously undiscovered enzyme with geranylpyrophosphate:olivetolate geranyltransferase activity (CsPT4) involved in CBGA biosynthesis, together with THCAS and CBDAS (with a vacuolar localization tag), allowed _S. cerevisiae_ to produce 1.1 mg/L \(\Delta^{9}\)-THCA or 4.3 ug/L CBDA from hexanoic acid, or 2.3 mg/L \(\Delta^{9}\)-THCA or 4.2 ug/L CBDA from galactose. These strains also produced \(\Delta^{9}\)-tetrahydrocannabivarinic acid and cannabidivarinic acid. The titers of \(\Delta^{9}\)-THCA and \(\Delta^{9}\)-tetrahydrocannabivarinic acid were further increased up to 8.0 mg/L (\(\Delta^{9}\)-THCA) and 4.8 mg/L (\(\Delta^{9}\)-tetrahydrocannabivarinic acid) after the overexpression of additional single copies of the biosynthetic genes CsTKS, CsOAC, and THCAS. Moreover, unnatural cannabinoid analogs with modifications in the part of the molecule that is known to alter receptor binding affinity and potency were obtained by feeding different fatty acids (e.g., pentanoic acid, heptanoic acid, and 5-hexenoic acid) to the _S. cerevisiae_ engineered strains [23]. The reconstruction of the complete \(\Delta^{9}\)-THCA and CBDA biosynthetic pathway in baker's yeast represented a milestone in the field of the heterologous biosynthesis of cannabinoids, but the titers were considered low from an industrial point of view. Therefore, bioengineering studies and the modeling of the heterologous biosynthesis of \(\Delta^{9}\)-THCA were carried out in this yeast chassis.