Genetic Survey of Psilocybe Natural Products

Abstract

Psilocybe magic mushrooms are best known for their main natural product, psilocybin, and its dephosphorylated congener, the psychedelic metabolite psilocin. Beyond tryptamines, the secondary metabolome of these fungi is poorly understood. The genomes of five species (P. azurescens, P. cubensis, P. cyanescens, P. mexicana, and P. serbica) were browsed to understand more profoundly common and species-specific metabolic capacities. The genomic analyses revealed a much greater and yet unexplored metabolic diversity than evident from parallel chemical analyses. P. cyanescens and P. mexicana were identified as aeruginascin producers. Lumichrome and verpacamide A were also detected as Psilocybe metabolites. The observations concerning the potential secondary metabolome of this fungal genus support pharmacological and toxicological efforts to find a rational basis for yet elusive phenomena, such as paralytic effects, attributed to consumption of some magic mushrooms.

Keywords: aeruginascin; kinases; metabolome; psilocybin; secondary metabolism.

Figure 1

Chemical structures of Psilocybe natural products. A: tryptamines, B: β‐carbolines, C: N,N‐dimethyl‐l‐tryptophan, D: lumichrome and verpacamide A, i. e., the Psilocybe natural products identified by LC–MS in this study.

Figure 2

Gene arrangement in loci for psilocybin biosynthesis. The psiH gene encodes the cytochrome P450 monooxygenase for tryptamine 4‐hydroxylation, psiK encodes the 4‐hydroxytryptamine kinase, psiM the N‐methyltransferase, psiD the gateway l‐tryptophan decarboxylase, psiT denotes a hypothetical transporter gene.^[14a] Genes shown in light grey do not belong to the psilocybin biosynthesis.

Figure 3

LC–MS analysis of methanolic carpophore and mycelial extracts to detect tryptamines. A: carpophore extracts. Top trace: authentic standards of norbaeocystin (I), baeocystin (II), aeruginascin (III), psilocybin (IV), norpsilocin (V), and psilocin (VI) are shown as overlay of separate chromatograms. B: mass spectra of tryptamines, detected in carpophores. C: mycelial extracts. The inset above the chromatograms shows an extracted ion chromatogram for m/z 254 [M+H]⁺, i. e., the mass of cyclo(Arg‐Pro). Assignment of chromatograms to species: a: P. azurescens; b: P. cubensis FSU12407; c: P. cubensis FSU12410; d: P. cyanescens; e: P. mexicana; f: P. serbica.

Figure 4

LC–MS analysis of ethyl acetate extracts of Psilocybe mycelia. A: top trace: authentic standards of norharmane (I, t _R=2.2 min), racemate of enantiomeric cordysinins C and D (II, t _R=2.8 min), harmane (III, t _R=3.0 min), harmol (IV, t _R=3.4 min) and lumichrome (V, t _R=3.9 min) are shown as overlay of separate chromatograms. Below: chromatograms of fungal extracts, recorded at λ=340 nm. Trace a: P. azurescens; b: P. cubensis FSU12407; c: P. cubensis FSU12410; d: P. cyanescens; e: P. mexicana; f: P. serbica. B: Representative mass and UV/Vis spectra, extracted at the respective retention times of sample chromatograms.

Scheme 1

PsiK‐catalyzed ATP‐dependent phosphorylation of 4‐hydroxy‐N,N,N‐trimethyltryptamine to aeruginascin.

Figure 5

LC–MS analysis of PsiK in vitro assays. Chromatograms were recorded at λ=280 nm. Top trace: authentic standards of aeruginascin (I), psilocybin (II), psilocin (III), and 4‐OH‐TMT (IV) are shown as an overlay of separate chromatograms. Trace a: negative control with psilocin and heat‐inactivated enzyme; trace b: reaction with psilocin and native PsiK; trace c: negative control with 4‐OH‐TMT and heat‐inactivated enzyme; trace d: reaction with 4‐OH‐TMT and native PsiK. Mass spectra of product peaks show aeruginascin (m/z 299.1 [M]⁺) and psilocybin (m/z 285.1 [M+H]⁺) formation, respectively.