# 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
Several critical aspects that require systematic optimization were detected after the analysis of metabolic bottlenecks, such as: (i) insufficient hexanoic acid formation in the fatty acid biosynthesis; (ii) low acetyl-CoA precursor delivery to the hexanoic acid biosynthesis and the mevalonate pathway; (iii) the limited catalytic activity of NphB or CsPT and THCAS; (iv) insufficient ATP and NADPH regeneration and; (v) ethanol production by Crabtree effect [79]. In another study, _S. cerevisiae_ was engineered by introducing plant uridine diphosphate glycosyltransferases into its genome in order to produce OA glucoside and cannabinoid acid glucoside. This could be of interest due to the high hydrophobicity of naturally occurring cannabinoids, which affects their use as pharmaceuticals [80]. Due to the importance of yeast peroxis in different metabolic pathways, such as the \(\beta\)-oxidation of fatty acids leading to the creation of a pool of acetyl-CoA that can be used in the formation of derivatives [81] (e.g., isoprenoid via the mevalonate pathway), these organelles were engineered for the more efficient production of CBGA. Thus, GPP synthase Erg20p (N127W) and geranyl diphosphate:olivetolate geranyltransferase CsPT4 were targeted to the yeast peroxisome together with the mevalonate pathway proteins. Under galactose-induced conditions and after supplementation with OA, the production of CBGA reached 0.82 mg/L, thereby confirming that yeast organelles can act as synthetic biology devices [82]. In addition to the scientific papers indicated above, which represent some of the research carried out in this field, several patents have been published dealing with the biosynthesis of cannabinoids in yeast. Some of these patents are described below. In a patent application filed in 2016 [83], transgenic yeasts (_S. cerevisiae_ and _Kluyveromyces marxianus_) capable of reconstituting the CBDA metabolic pathway of _C. sativa_ were generated. This biosynthetic process was claimed to be sustainable and more environmentally friendly than synthetic production. To circumvent the low production of the precursor GPP in the yeasts, an improved mutant prenyltransferase Erg20 (K179E) that shifted the GPP:FPP ratio towards the first compound (70:30) was used in both yeasts. In _S. cerevisiae_, CBDA was produced upon the addition of hexanoic acid. In contrast, _K. marxianus_ was engineered to produce hexanoyl-CoA from glucose, since the genes encoding the enzymes required for this route were introduced in this microorganism in addition to those involved in CBDA biosynthesis. In another patent, _S. cerevisiae_ was engineered to incorporate the cannabinoid biosynthetic pathway, including mutant variants of Erg20. Depending on the feedstock used (OA or hexanoic acid), the \(\Delta^{9}\)-THCA titers were 84 mg/L and 23 mg/L, respectively. An improvement in CBGA production was achieved due to the greater availability of hexanoic acid when the endogenous FOX1 gene, which controls the \(\beta\)-oxidation of long-chain fatty acids, was disrupted in this yeast strain [84]. In order to produce water-soluble cannabinoids, thereby improving traditional cannabinoid production, other inventors generated a yeast expression system based on the methylotrophic yeast _K. phaffii_, which was engineered to express glycosyltransferases from different organisms. The intracellular expression of NtGT4 (UGT 73-like glycosyltransferase from _Nicotiana tabacum_) led to the highest level of glycosylation of CBDA after the addition of 27 \(\mu\)M CBDA [85]. In another patent application, _S. cerevisiae_ was genetically modifiedfor the production of phytocannabinoids and phytocannabinoid analogs using a type I fatty acid synthase/PKS from the amoeba _Dictyostelium discoideum_ (DiPKS).