# Biotechnological Fungal Platforms for the Production of Biosynthetic Cannabinoids
## 4 Development of Biotechnological Fungal Platforms for the Biosynthesis of Cannabinoids
### Biosynthesis of Cannabinoids in Filamentous Fungi: The Paradigm of P. chrysogenum
HPLC analysis was carried out following the protocol previously described in [100] with some modifications. Chromatographic separations were achieved using a Core-Shell Kinetex(r) EVO C18 (Phenomenex, Torrance, CA, USA) analytical column (3 \(\upmu\)m, 150 mm \(\times\) 4.6 mm i.d.). The mobile phase consisted of a gradient of (methanol: acetonitrile 75:25)/water, containing 0.1% formic acid. The flow rate was set to 0.5 mL/min, and the injection volume was 20 \(\upmu\)L. All experiments were carried out at 50 \({}^{\circ}\)C. Full spectra were recorded in the range of 200-400 nm. As a result of the bioconversion of CBGA, \(\Delta^{9}\)-THCA was detected intracellular (Figure 3b). The authenticity of \(\Delta^{9}\)-THCA was confirmed by mass spectrometry using a Triple Quad 6500 System (AB Sciex LLC, Framingham, MA, USA). In a second step, we evaluated the ability of the F-THCAS strain to carry out the prenylation of OA and biosynthesis \(\Delta^{9}\)-THCA (Figure 4a). To achieve this goal, we codon-optimized and overexpressed in these strains a mutant variant (Y288A/G2865) of the soluble aromatic prenyltransferase from _Streptomyces_ sp. CL19 strain (StNphB), which has been reported to show improved enzyme kinetics [101], together with a codon-optimized mutant version of Erg20 (F69W/N127W), the latter leading to GPP overproduction [23]. The cDNAs encoding these two proteins were included in the pBluescript II SK plasmid under the control of the bidirectional _pcbAB-pcbC_ gene promoter (controlling the expression of the _pcbAB_ and _pcbC_ genes of the penicillin biosynthetic gene cluster and absent in the _P. chrysogenum_ Wis54-1255 npe10 PyrG\(-\)strain) and the transcriptional terminator of the _pyrG_ gene. For the selection of transformants, this construct also included the phleomycin resistance cassette, thus giving rise to pSK-F-erg20-nphB (Figure 4a). The transformants were tested by PCR to confirm the integration of the DNA fragment including the _StNphB\({}^{Y288A/G2865}\)_ and _Erg20F\({}^{69W/N127W}\)_ genes and the bidirectional _pcbAB-pcbC_ promoter (not shown). The genetically modified _P. chrysogenum_ transformants obtained in this step were named F-THCAS-erg20-nphB and were grown in PMMY medium. After 80 h of growth at 26 \({}^{\circ}\)C and 250 rpm, the mycelia were collected and broken down in liquid nitrogen to a fine powder to assess the formation of \(\Delta^{9}\)-THCA in the lysates in the presence of 1 mM OA and 0.1 mM GPP in a reaction carried out as previously described [76]. As a result of the bioconversion of OA into CBGA and the latter into \(\Delta^{9}\)-THCA, this cannabinoid was detected by HPLC analysis (Figure 4b) following a protocol similar to that indicated above with a different gradient. The authenticity of \(\Delta^{9}\)-THCA was confirmed by mass spectrometry using a Triple Quad 6500 System (SCIEX). These preliminary results confirm that _P. chrysogenum_ can biosynthesize cannabinoids using the codon-optimized plant biosynthetic genes, thus demonstrating the feasibility of filamentous fungi as cannabinoid producers. Due to the strong background of _P. chrysogenum_ in the pharmaceutical industry as penicillin producer, the genetic engineering of this Ascomycete with the rest of the cannabinoid biosynthetic genes could give rise to an interesting platform for the industrial production of these compounds, although several optimization steps are still required to make it attractive for this purpose.