Optimized psilocybin production in tryptophan catabolism-repressed fungi

Abstract

The high therapeutic potential of psilocybin, a prodrug of the psychotropic psilocin, holds great promise for the treatment of mental disorders such as therapy-refractory depression, alcohol use disorder and anorexia nervosa. Psilocybin has been designated a 'Breakthrough Therapy' by the US Food and Drug Administration, and therefore a sustainable production process must be established to meet future market demands. Here, we present the development of an in vivo psilocybin production chassis based on repression of l-tryptophan catabolism. We demonstrate the proof of principle in Saccharomyces cerevisiae expressing the psilocybin biosynthetic genes. Deletion of the two aminotransferase genes ARO8/9 and the indoleamine 2,3-dioxygenase gene BNA2 yielded a fivefold increase of psilocybin titre. We transferred this knowledge to the filamentous fungus Aspergillus nidulans and identified functional ARO8/9 orthologs involved in fungal l-tryptophan catabolism by genome mining and cross-complementation. The double deletion mutant of A. nidulans resulted in a 10-fold increased psilocybin production. Process optimization based on respiratory activity measurements led to a final psilocybin titre of 267 mg/L in batch cultures with a space-time-yield of 3.7 mg/L/h. These results demonstrate the suitability of our engineered A. nidulans to serve as a production strain for psilocybin and other tryptamine-derived pharmaceuticals.

FIGURE 1

Schematic, simplified overview on l‐tryptophan metabolism. (A) Four‐enzyme psilocybin biosynthetic pathway, reconstituted in S. cerevisiae. (B) In S. cerevisiae, l‐tryptophan is deaminated to indole‐3‐pyruvic acid by the aminotransferases Aro8 and Aro9. A second catabolic route includes oxidative cleavage of the pyrrole ring of l‐tryptophan by the indoleamine‐2,3‐dioxygenase Bna2, to yield N‐formyl‐l‐kynurenine, that is a precursor for the biosynthesis of both kynurenic acid and quinolinic acid. The latter is required for nicotinamide adenine dinucleotide biosynthesis. (C) De novo biosynthesis of l‐tryptophan via the anthranilate phosphoribosyltransferase Trp4. l‐Ala, l‐alanine; l‐Glu, l‐glutamate; NAD⁺, nicotinamide adenine dinucleotide; PRPP, 5‐phospho‐α‐d‐ribose‐1‐diphosphate; SAH, S‐adenosyl‐l‐homocysteine; SAM, S‐adenosyl‐l‐methionine; α‐KG, α‐ketoglutaric acid.

FIGURE 2

Psilocybin production in S. cerevisiae mutant strains. (A) The phenotypic analysis of l‐tryptophan catabolism‐repressed S. cerevisiae confirmed their sensitivity to high l‐tryptophan (Trp) concentrations. (B) Genetic map of the expression plasmid pYes‐psi carrying the psi cluster expressed as a polycistron under the control of the constitutive TEF1 promoter. (C) Production of psilocybin, psilocin and baeocystin in S. cerevisiae wild type and tryptophan catabolism‐repressed mutants after 48 h of cultivation.

FIGURE 3

Identification and analysis of ARO‐like aminotransferases from A. nidulans. (A) Evolutionary distance among ARO‐like aminotransferases in Aspergilli. Clades are named based on the genes identified in A. fumigatus (AroH, AroI) and A. nidulans (TdiD). (B) qRT‐PCR analysis of the A. nidulans aro‐like genes in response to tryptophan (Trp). (C) Functional complementation analysis of the identified genes from A. nidulans in the S. cerevisiae Δaro8/Δaro9 mutant strain. BY4741 and Δaro8/Δaro9 contained the empty plasmid pYes2‐TEF1.

FIGURE 4

Identification and analysis of indoleamine 2,3‐dioxygenases from A. nidulans. (A) Phylogenetic tree of IDO‐like proteins identified in Aspergilli. The three main clades are named based on previous analyses made in A. fumigatus. Reported IdoA‐ and IdoB‐related proteins are shown in maroon and sand, respectively, which also show a conserved gene synteny (shown in panel B). IdoC‐like proteins are shown in violet, while proteins shown in light blue likely participate in secondary metabolism. The remaining IDO‐like proteins are depicted in grey. Scale bar indicates amino acid substitutions/site. (B) Genetic loci for the idoA and idoB genes in A. nidulans. (C) qRT‐PCR analysis of the A. nidulans idoA, idoB, kynU2 and idoC genes in response to tryptophan (Trp). (D) Functional complementation of S. cerevisiae Δbna2 and Δbna5 with idoA, idoB, idoC and kynU2, respectively. Corresponding masses are: 3‐HK [+m/z] 225.0870; KYN [+m/z] 209.0921; 3‐HA [+m/z] 138.0550. (E) Phenotypic analysis of A. nidulans ΔidoA on minimal media and in presence of nicotinamide (NAM) and tryptophan (Trp).

FIGURE 5

Psilocybin, psilocin and baeocystin production in A. nidulans mutant strains. Production of psilocybin, psilocin and baeocystin during shake flask cultivations of A. nidulans aroH single and double mutant strains expressing the psi cluster as a polycistron after 48 h (24 h post‐induction). Data are presented as averages and error bars indicate the standard error.

FIGURE 6

Process characterization and respiratory activity monitoring. (A) Online data of oxygen transfer rate (OTR) and carbon dioxide transfer rate (CTR) is shown for the A. nidulans double mutant ΔaroH1/ΔaroH2 and the control strain tJF03, both expressing the psi cluster as a polycistron. Induction was performed at 31 h upon decrease of metabolic activity observed by the decline in OTR signal. For improved enzyme production, the temperature was shifted to 30°C as indicated. (B) Offline data of glucose concentration, cell dry weight (CDW) and pH progression is shown over the cultivation time for both strains. (C) Production of psilocybin, psilocin and baeocystin over the time of cultivation for the wild type (tJF03) and double mutant backgrounds. Data are presented as averages from duplicate shake flask cultivations and triplicate product quantification with standard errors presented as error bars.