# 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  
Cannabinoid biosynthesis by means of biotechnological approaches and independently of the plant has been boosted by: (i) its interest as pharmaceutical and bioactive compounds; (ii) the limiting production of synthetic THC and; (iii) the legal regulatory issues for the cultivation of _C. sativa_ in most countries. Thus, numerous papers and patents describing biotechnological procedures for the microbial production of cannabinoids (mainly in yeasts) have been reported in the recent years, as it is summarized below. The identification of the cannabinoids biosynthetic genes and enzymes [68; 69; 70; 24; 71; 72] paved the way to reconstruct the biosynthetic pathway and engineer the metabolism in suitable heterologous systems [39], mainly yeasts (Figure 2). Early reports focused on the recombinant expression of THCAS in different hosts, such as tobacco hairy roots, insect cell cultures, and _K. phaffii_ cultures [73; 71]. The expression  
Figure 2: Main pathways engineered in yeasts in order to produce cannabinoids from different precursors. Abbreviations: AAE1, acyl activating enzyme; CBDAS, cannabidiolic acid synthase; CBGA, cannabigerolic acid; CBGAS, CBGA synthase; CsPT4, _C. sativa_ prenyltransferase 4; Erg20, GPP synthase; NphB, aromatic prenyltransferase from _Streptomyces_ sp. CL190 strain; OA, Olivetolic acid; OAC, OA cyclase; OLS, Olivetol synthase; THCAS, \(\Delta^{9}\)-tetrahydrocanabinolic acid synthase. of THCAS in these hosts was very low, although it opened the door to the possibility of biotechnological \(\Delta^{9}\)-THCA production by means of biotechnological platforms. _E. coli_ was initially used as a potential host microorganism, but it was quickly discarded, since no functional expression of THCAS was achieved in this microorganism [74; 75], thereby suggesting that a eukaryotic expression system was necessary for appropriate enzyme folding and maturation. Initial drawbacks of the THCAS scale-up processes regarding the water solubility of CBGA and the membrane localization of CBGAS led scientists to also focus on the intracellular expression of THCAS in _S. cerevisiae_ and _K. phaffii_ cells and the possible application of a whole-cell production system for \(\Delta^{9}\)-THCA [75]. These authors used the signal peptide of the vacuolar proteinase A to target THCAS in the cell vacuole and found that the highest enzyme activity was obtained in cultures from proteinase A knockout strains of _K. phaffii_. The further optimization of THCAS expression in _K. phaffii_ dramatically increased enzyme activity in relation to previous studies, and the bioconversion of CBGA in whole yeast cells led to the production of 1 mM \(\Delta^{9}\)-THCA [75]. The CBGAS expression problems related to its membrane localization led scientists to use the soluble aromatic prepryltransferase from _Streptomyces_ sp. CL190 strain (StNphB) instead of plant preyltransferase. The partial reconstruction of the \(\Delta^{9}\)-THCA biosynthetic pathway in _K. phaffii_ gave rise to the successful synthesis of this compound from OA and GPP. Although the side-product 2-O-geranyl OA was also produced because of StNPhB activity, these results suggested that NphB was a promising candidate for the substitution of plant-derived membrane-bound preprtransferases in the heterologous system [76]. Although _K. phaffii_ was found to be a good host for the heterologous expression of cannabinoid biosynthetic enzymes, several bottlenecks attributed to THCAS protein folding were also observed. They were counteracted by the co-production of different proteins, including foldases and chaperones.