# 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  
To date, there have been no reports on the biosynthetic production of cannabinoids in filamentous fungi. To the best of our knowledge, there is only one report describing the biosynthesis of OA in _Aspergillus nidulans_ using fungal tandem polykteide synthases without the requirement of CsOLS and CsOAC [93], which suggested that new synthetic biology strategies can be implemented for the biosynthesis of microbial cannabinoids in filamentous fungi. The successful trajectory of _P. chrysogenum_ as industrial producer of penicillin, and the large titers achieved by this microorganism under industrial conditions [94], together with the availability of different molecular and omics tools for the genetic engineering of this filamentous fungus [54; 55; 57], make it very attractive as cell factory for secondary metabolite production. Based on this, we decided to use _P. chrysogenum_ as a platform for cannabinoid biosynthesis and production. Therefore, we aimed to test the feasibility of filamentous fungi as cannabinoid producers by developing _A. chrysogenum_-based biotechnological platform that allows the production of cannabinoids in submerged cultures. In order to divert the high metabolic flux resulting from penicillin biosynthesis through the heterologous cannabinoid biosynthetic pathway, a strain lacking a region that includes the penicillin biosynthetic gene cluster (_pcbAB_, _pcbC_, and _penDE_ genes) and several ORFs flanking this cluster, was chosen as the parental strain. In addition, to facilitate selection upon transformation, a uridine auxotroph of this strain was used (_P. chrysogenum_ Wis54-1255 npe10 pyrG\(-\)(Apen)) [95; 96]. Due to the availability of commercial CBGA and OA, we proposed a synthetic biology strategy consisting of the gradual introduction of heterologous genes required for \(\Lambda^{9}\)-THCA biosynthesis, starting with the late biosynthetic gene THCA (Figure 3a). The sequence of the gene encoding the THCAS of _C. sativa_ was optimized from the point of view of fungal codon usage. Previous studies executed _in silico_ with the SignalP-5.0 program [97] indicated that the first 28 amino acids of this enzyme constitute a signal peptide. This signal peptide was replaced with the 19 amino acid signal peptide of the _P. chrysogenum_ Pc21g02370 (with a strong similarity to aspergillus apnS from _Aspergillus phoenicis_) [98]. Therefore, the cDNA of THCAS was designed to include 57 nucleotides encoding the above-mentioned signal peptide and was subcloned into the pBluescript II  
Figure 3: Production of \(\Delta^{9}\)-THCA in _P. chrysogenum_ after feeding CBGA to the culture medium. (**a**) Schematic representation of the strategy followed to produce \(\Delta^{9}\)-THCA from CBGA in _P. chrysogenum_. Plasmid pSK-F-THCAS included: _PgdhA_ (_A. aavnori_ glutamate dehydrogenase gene promoter), _Tcyc1_ (transcriptional terminator of the _S. cerevisiae cyc1_ gene), LP (signal peptide), _thas_ (\(\Delta^{9}\)-tetrahydrocannabinolic acid synthase gene), and PyrG expression cassette. (**b**) Representative chromatogram at 270 nm of one of the transformants of the F-THCAS strain showing the formation of \(\Delta^{9}\)-THCA (blue line). Cannabinoid standards are included (red line). SK plasmid under the control of the _A. awamori gdhA_ (glutamate dehydrogenase) gene promoter and the transcriptional terminator of the _S. cerevisiae cyc1_ gene. This construct also included the PyrG expression cassette to allow selection upon transformation and was named pSK-F-THCAS (Figure 3a). The transformations were tested by PCR to confirm the integration of the THCAS expression cassette (not shown), and genetically modified _P. chrysogenum_ transformants were named F-THCAS. These transformants were grown in PMMY medium [99] for 48 h at 26 \({}^{\circ}\)C and 250 rpm and supplemented with 0.1 mM CBGA. After 48 h of additional growth, mycelia were collected, broken down in liquid nitrogen to a fine powder, and resuspended in methanol.