Methyl transfer in psilocybin biosynthesis.

Psilocybin, the natural hallucinogen produced by Psilocybe ("magic") mushrooms, holds great promise for the treatment of depression and several other mental health conditions. The final step in the psilocybin biosynthetic pathway, dimethylation of the tryptophan-derived intermediate norbaeocystin, is catalysed by PsiM. Here we present atomic resolution (0.9 Å) crystal structures of PsiM trapped at various stages of its reaction cycle, providing detailed insight into the SAM-dependent methylation mechanism. Structural and phylogenetic analyses suggest that PsiM derives from epitranscriptomic N6-methyladenosine writers of the METTL16 family, which is further supported by the observation that bound substrates physicochemically mimic RNA. Inherent limitations of the ancestral monomethyltransferase scaffold hamper the efficiency of psilocybin assembly and leave PsiM incapable of catalysing trimethylation to aeruginascin. The results of our study will support bioengineering efforts aiming to create novel variants of psilocybin with improved therapeutic properties.

Phylogenomics of the psychoactive mushroom genus Psilocybe and evolution of the psilocybin biosynthetic gene cluster.

Psychoactive mushrooms in the genus Psilocybe have immense cultural value and have been used for centuries in Mesoamerica. Despite the recent surge of interest in these mushrooms due to the psychotherapeutic potential of their natural alkaloid psilocybin, their phylogeny and taxonomy remain substantially incomplete. Moreover, the recent elucidation of the psilocybin biosynthetic gene cluster is known for only five of ~165 species of Psilocybe, four of which belong to only one of two major clades. We set out to improve the phylogeny of Psilocybe using shotgun sequencing of fungarium specimens, from which we obtained 71 metagenomes including from 23 types, and conducting phylogenomic analysis of 2,983 single-copy gene families to generate a fully supported phylogeny. Molecular clock analysis suggests the stem lineage of Psilocybe arose ~67 mya and diversified ~56 mya. We also show that psilocybin biosynthesis first arose in Psilocybe, with 4 to 5 possible horizontal transfers to other mushrooms between 40 and 9 mya. Moreover, predicted orthologs of the psilocybin biosynthetic genes revealed two distinct gene orders within the biosynthetic gene cluster that corresponds to a deep split within the genus, possibly a signature of two independent acquisitions of the cluster within Psilocybe.

Genome sequencing progenies of magic mushrooms (Psilocybe subaeruginosa) identifies tetrapolar mating and gene duplications in the psilocybin pathway.

Knowledge of breeding systems and genetic diversity is critical to select and combine desired traits that advance new cultivars in agriculture and horticulture. Mushrooms that produce psilocybin, magic mushrooms, may potentially be used in therapeutic and wellness industries, and stand to benefit from genetic improvement. We studied haploid siblings of Psilocybe subaeruginosa to resolve the genetics behind mating compatibility and advance knowledge of breeding. Our results show that mating in P. subaeruginosa is tetrapolar, with compatibility controlled at a homeodomain locus with one copy each of HD1 and HD2, and a pheromone/receptor locus with four homologs of the receptor gene STE3. An additional two pheromone/receptor loci homologous to STE3 do not appear to regulate mating compatibility. Alleles in the psilocybin gene cluster did not vary among the five siblings and were likely homozygous in the parent. Psilocybe subaeruginosa and its relatives have three copies of PsiH genes but their impact on production of psilocybin and its analogues is unknown. Genetic improvement in Psilocybe will require access to genetic diversity from the centre of origin of different species, identification of genes behind traits, and strategies to avoid inbreeding depression.

DNA Authentication and Chemical Analysis of Psilocybe Mushrooms Reveal Widespread Misdeterminations in Fungaria and Inconsistencies in Metabolites.

The mushroom genus Psilocybe is best known as the core group of psychoactive mushrooms, yet basic information on their diversity, taxonomy, chemistry, and general biology is still largely lacking. In this study, we reexamined 94 Psilocybe fungarium specimens, representing 18 species, by DNA barcoding, evaluated the stability of psilocybin, psilocin, and their related tryptamine alkaloids in 25 specimens across the most commonly vouchered species (Psilocybe cubensis, Psilocybe cyanescens, and Psilocybe semilanceata), and explored the metabolome of cultivated P. cubensis. Our data show that, apart from a few well-known species, the taxonomic accuracy of specimen determinations is largely unreliable, even at the genus level. A substantial quantity of poor-quality and mislabeled sequence data in public repositories, as well as a paucity of sequences derived from types, further exacerbates the problem. Our data also support taxon- and time-dependent decay of psilocybin and psilocin, with some specimens having no detectable quantities of them. We also show that the P. cubensis metabolome possibly contains thousands of uncharacterized compounds, at least some of which may be bioactive. Taken together, our study undermines commonly held assumptions about the accuracy of names and presence of controlled substances in fungarium specimens identified as Psilocybe spp. and reveals that our understanding of the chemical diversity of these mushrooms is largely incomplete. These results have broader implications for regulatory policies pertaining to the storage and sharing of fungarium specimens as well as the use of psychoactive mushrooms for recreation and therapy. IMPORTANCE The therapeutic use of psilocybin, the active ingredient in "magic mushrooms," is revolutionizing mental health care for a number of conditions, including depression, posttraumatic stress disorder (PTSD), and end-of-life care. This has spotlighted the current state of knowledge of psilocybin, including the organisms that endogenously produce it. However, because of international regulation of psilocybin as a controlled substance (often included on the same list as cocaine and heroin), basic research has lagged far behind. Our study highlights how the poor state of knowledge of even the most fundamental scientific information can impact the use of psilocybin-containing mushrooms for recreational or therapeutic applications and undermines critical assumptions that underpin their regulation by legal authorities. Our study shows that currently available chemical studies are mainly inaccurate, irreproducible, and inconsistent, that there exists a high rate of misidentification in museum collections and public databases rendering even names unreliable, and that the concentration of psilocybin and its tryptamine derivatives in three of the most commonly collected Psilocybe species (P. cubensis, P. cyanescens, and P. semilanceata) is highly variable and unstable in museum specimens spanning multiple decades, and our study generates the first-ever insight into the highly complex and largely uncharacterized metabolomic profile for the most commonly cultivated magic mushroom, P. cubensis.

Genetic Survey of Psilocybe Natural Products.

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.

Metabolic engineering of Saccharomyces cerevisiae for the de novo production of psilocybin and related tryptamine derivatives.

Psilocybin is a tryptamine-derived psychoactive alkaloid found mainly in the fungal genus Psilocybe, among others, and is the active ingredient in so-called "magic mushrooms". Although its notoriety originates from its psychotropic properties and popular use as a recreational drug, clinical trials have recently recognized psilocybin as a promising candidate for the treatment of various psychological and neurological afflictions. In this work, we demonstrate the de novo biosynthetic production of psilocybin and related tryptamine derivatives in Saccharomyces cerevisiae by expression of a heterologous biosynthesis pathway sourced from Psilocybe cubensis. Additionally, we achieve improved product titers by supplementing the pathway with a novel cytochrome P450 reductase from P. cubensis. Further rational engineering resulted in a final production strain producing 627 ± 140 mg/L of psilocybin and 580 ± 276 mg/L of the dephosphorylated degradation product psilocin in triplicate controlled fed-batch fermentations in minimal synthetic media. Pathway intermediates baeocystin, nor norbaeocystin as well the dephosphorylated baeocystin degradation product norpsilocin were also detected in strains engineered for psilocybin production. We also demonstrate the biosynthetic production of natural tryptamine derivative aeruginascin as well as the production of a new-to-nature tryptamine derivative N-acetyl-4-hydroxytryptamine. These results lay the foundation for the biotechnological production of psilocybin in a controlled environment for pharmaceutical applications, and provide a starting point for the biosynthetic production of other tryptamine derivatives of therapeutic relevance.

Taxonomic identification and bioactive compounds characterization of Psilocybe cubensis DPT1 to probe its antibacterial and mosquito larvicidal competency.

Mushrooms have an important role in sustainability since they have long been used as valuable food source and traditional medicine around the world. Regrettably, they are among the most rigorously affected populations, along with several plants and animals, due to the destructive activities of mankind. Thus the authentication and conservation of mushroom species are constantly needed to exploit the remarkable potential in them. In this perspective, an attempt has been made to identify and assess the biological attributes of psychedelic mushrooms collected from Kodaikanal, Tamil Nadu, India. The macromorphological features of the psychedelic mushroom DPT1 helped its presumptive identification and the molecular characters depicted by DNA marker revealed its close relationship with the genus Psilocybe. Accordingly, the psychedelic mushroom was identified as Psilocybe cubensis DPT1 and its crude ethyl acetate extract on analysis revealed the occurrence of phytoconstituents like alkaloids, flavonoids, tannins, saponins and carbohydrates. Moreover, it exhibited 80% larvicidal activity against Culex quinquefasciatus mosquito at 800 ppm concentration and an array of antibacterial effects with utmost susceptibility of Proteus vulgaris, and the identification of bioactive compounds by different analytical techniques substantiate that the bioactivities might be due to the presence of phytochemicals. The results of the study indicated that the extract of P. cubensis DPT1 having notable antibacterial and mosquito larvicidal efficacies which could be probed further for the isolation of medicinally important as well as bio-control compounds.

S-Adenosyl-l-Methionine Salvage Impacts Psilocybin Formation in "Magic" Mushrooms.

Psychotropic Psilocybe mushrooms biosynthesize their principal natural product psilocybin in five steps, among them a phosphotransfer and two methyltransfer reactions, which consume one equivalent of 5'-adenosine triphosphate (ATP) and two equivalents of S-adenosyl-l-methionine (SAM). This short but co-substrate-intensive pathway requires nucleoside cofactor salvage to maintain high psilocybin production rates. We characterized the adenosine kinase (AdoK) and S-adenosyl-l-homocysteine (SAH) hydrolase (SahH) of Psilocybe cubensis. Both enzymes are directly or indirectly involved in regenerating SAM. qRT-PCR expression analysis revealed an induced expression of the genes in the fungal primordia and carpophores. A one-pot in vitro reaction with the N-methyltransferase PsiM of the psilocybin pathway demonstrates a concerted action with SahH to facilitate biosynthesis by removal of accumulating SAH.

Monoamine Biosynthesis via a Noncanonical Calcium-Activatable Aromatic Amino Acid Decarboxylase in Psilocybin Mushroom.

Aromatic l-amino acid decarboxylases (AAADs) are a phylogenetically diverse group of enzymes responsible for the decarboxylation of aromatic amino acid substrates into their corresponding aromatic arylalkylamines. AAADs have been extensively studied in mammals and plants as they catalyze the first step in the production of neurotransmitters and bioactive phytochemicals, respectively. Unlike mammals and plants, the hallucinogenic psilocybin mushroom Psilocybe cubensis reportedly employs an unrelated phosphatidylserine-decarboxylase-like enzyme to catalyze l-tryptophan decarboxylation, the first step in psilocybin biosynthesis. To explore the origin of this chemistry in psilocybin mushroom, we generated the first de novo transcriptomes of P. cubensis and investigated several putative l-tryptophan-decarboxylase-like enzymes. We report the biochemical characterization of a noncanonical AAAD from P. cubensis ( PcncAAAD) that exhibits substrate permissiveness toward l-phenylalanine, l-tyrosine, and l-tryptophan, as well as chloro-tryptophan derivatives. The crystal structure of PcncAAAD revealed the presence of a unique C-terminal appendage domain featuring a novel double-ß-barrel fold. This domain is required for PcncAAAD activity and regulates catalytic rate and thermal stability through calcium binding. PcncAAAD likely plays a role in psilocybin production in P. cubensis and offers a new tool for metabolic engineering of aromatic-amino-acid-derived natural products.

Detection and Identification of Psilocybe cubensis DNA Using a Real-Time Polymerase Chain Reaction High Resolution Melt Assay.

Psilocybe cubensis, or "magic mushroom," is the most common species of fungus with psychedelic characteristics. Two primer sets were designed to target Psilocybe DNA using web-based software and NBCI gene sequences. DNA was extracted from eighteen samples, including twelve mushroom species, using the Qiagen DNeasy® Plant Mini Kit. The DNA was amplified by the polymerase chain reaction (PCR) using the primers and a master mix containing either a SYBR® Green I, Radiant™ Green, or LCGreen Plus® intercalating dye; amplicon size was determined using agarose gel electrophoresis. The PCR assays were tested for amplifiability, specificity, reproducibility, robustness, sensitivity, and multiplexing with primers that target marijuana. The observed high resolution melt (HRM) temperatures for primer sets 1 and 7 were 78.85 ± 0.31°C and 73.22 ± 0.61°C, respectively, using SYBR® Green I dye and 81.67 ± 0.06°C and 76.04 ± 0.11°C, respectively, using Radiant™ Green dye.

Enzymatic Synthesis of Psilocybin.

Psilocybin is the psychotropic tryptamine-derived natural product of Psilocybe carpophores, the so-called "magic mushrooms". Although its structure has been known for 60 years, the enzymatic basis of its biosynthesis has remained obscure. We characterized four psilocybin biosynthesis enzymes, namely i) PsiD, which represents a new class of fungal l-tryptophan decarboxylases, ii) PsiK, which catalyzes the phosphotransfer step, iii) the methyltransferase PsiM, catalyzing iterative N-methyl transfer as the terminal biosynthetic step, and iv) PsiH, a monooxygenase. In a combined PsiD/PsiK/PsiM reaction, psilocybin was synthesized enzymatically in a step-economic route from 4-hydroxy-l-tryptophan. Given the renewed pharmaceutical interest in psilocybin, our results may lay the foundation for its biotechnological production.

Fluorescent Random Amplified Microsatellites (F-RAMS) analysis of mushrooms as a forensic investigative tool.

The capability of Fluorescent Random Amplified Microsatellites (F-RAMS) to profile hallucinogenic mushrooms to species and sub-species level was assessed. Fifteen samples of Amanita rubescens and 22 samples of other hallucinogenic and non-hallucinogenic mushrooms of the genera Amanita and Psilocybe were profiled using two fluorescently-labeled, 5'degenerate primers, 5'-6FAM-SpC3-DD (CCA)5 and 5'-6FAM-SpC3-DHB (CGA)5, which target different microsatellite repeat regions. Among the two primers, 5'-6FAM-SpC3-DHB (CGA)5 provided more reliable data for identification purposes, by grouping samples of the same species and clustering closely related species together in a dendrogram based on amplicon similarities. A high degree of intra-specific variation between the 15 A. rubescens samples was shown with both primers and the amplicons generated for all A. rubescens samples were organized into three classes of amplicons (discriminant, private, and marker) based on their individualizing potential.