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.

The evolution and ecology of psilocybin in nature.

Fungi produce diverse metabolites that can have antimicrobial, antifungal, antifeedant, or psychoactive properties. Among these metabolites are the tryptamine-derived compounds psilocybin, its precursors, and natural derivatives (collectively referred to as psiloids), which have played significant roles in human society and culture. The high allocation of nitrogen to psiloids in mushrooms, along with evidence of convergent evolution and horizontal transfer of psilocybin genes, suggest they provide a selective benefit to some fungi. However, no precise ecological roles of psilocybin have been experimentally determined. The structural and functional similarities of psiloids to serotonin, an essential neurotransmitter in animals, suggest that they may enhance the fitness of fungi through interference with serotonergic processes. However, other ecological mechanisms of psiloids have been proposed. Here, we review the literature pertinent to psilocybin ecology and propose potential adaptive advantages psiloids may confer to fungi.

In vivo production of psilocybin in E. coli.

Psilocybin, the prodrug of the psychoactive molecule psilocin, has demonstrated promising results in clinical trials for the treatment of addiction, depression, and post-traumatic stress disorder. The development of a psilocybin production platform in a highly engineerable microbe could lead to rapid advances towards the bioproduction of psilocybin for use in ongoing clinical trials. Here, we present the development of a modular biosynthetic production platform in the model microbe, Escherichia coli. Efforts to optimize and improve pathway performance using multiple genetic optimization techniques were evaluated, resulting in a 32-fold improvement in psilocybin titer. Further enhancements to this genetically superior strain were achieved through fermentation optimization, ultimately resulting in a fed-batch fermentation study, with a production titer of 1.16 g/L of psilocybin. This is the highest psilocybin titer achieved to date from a recombinant organism and a significant step towards demonstrating the feasibility of industrial production of biologically-derived psilocybin.

Facile assembly and fluorescence-based screening method for heterologous expression of biosynthetic pathways in fungi.

Heterologous expression of multi-gene biosynthetic pathways in eukaryotic hosts is limited by highly regulated individual monocistrons. Dissimilar to prokaryotes, each eukaryotic gene is strictly controlled by its own regulatory elements, such as promoter and terminator. Consequently, parallel transcription can occur only when a group of genes is synchronously activated. A strategy to circumvent this limitation is the concerted expression of multiple genes as a polycistron. By exploiting the "stop-carry on" mechanism of picornaviruses, we have designed a sophisticated, yet easy-to-assemble vector system to heterologously express multiple genes under the control of a single promoter. For facile selection of correctly transformed colonies by basic fluorescence microscopy, our vector includes a split gene for a fluorescent reporter protein. This method was successfully applied to produce the psychotropic mushroom alkaloid psilocybin in high yields by heterologous expression of the entire biosynthetic gene cluster in the mould Aspergillus nidulans.

Identification of members of the genera Panaeolus and Psilocybe by a DNA test. A preliminary test for hallucinogenic fungi.

Abuse of hallucinogens produced by the fungal genera Psilocybe and Panaeolus are a growing problem. Five species from each of the two genera were examined in this preliminary research and a method that will unambiguously identify fungal samples as being of one of these two genera has been developed. The method uses genus specific DNA sequences within the Internal Transcribed Spacer of the ribosomal gene complex. Amplification of a common DNA product and a genus specific product results in two identifiable products, which facilitates the unambiguous identification of material from these two fungi to generic level.

Evolution of the toxins muscarine and psilocybin in a family of mushroom-forming fungi.

Mushroom-forming fungi produce a wide array of toxic alkaloids. However, evolutionary analyses aimed at exploring the evolution of muscarine, a toxin that stimulates the parasympathetic nervous system, and psilocybin, a hallucinogen, have never been performed. The known taxonomic distribution of muscarine within the Inocybaceae is limited, based only on assays of species from temperate regions of the northern hemisphere. Here, we present a review of muscarine and psilocybin assays performed on species of Inocybaceae during the last fifty years. To supplement these results, we used liquid chromatography-tandem mass spectrometry (LC-MS/MS) to determine whether muscarine was present in 30 new samples of Inocybaceae, the majority of which have not been previously assayed or that originated from either the tropics or temperate regions of the southern hemisphere. Our main objective is to test the hypothesis that the presence of muscarine is a shared ancestral feature of the Inocybaceae. In addition, we also test whether species of Inocyabceae that produce psilocybin are monophyletic. Our findings suggest otherwise. Muscarine has evolved independently on several occasions, together with several losses. We also detect at least two independent transitions of muscarine-free lineages to psilocybin-producing states. Although not ancestral for the family as a whole, muscarine is a shared derived trait for an inclusive clade containing three of the seven major lineages of Inocybaceae (the Inocybe, Nothocybe, and Pseudosperma clades), the common ancestor of which may have evolved ca. 60 million years ago. Thus, muscarine represents a conserved trait followed by several recent losses. Transitions to psilocybin from muscarine-producing ancestors occurred more recently between 10-20 million years ago after muscarine loss in two separate lineages. Statistical analyses firmly reject a single origin of muscarine-producing taxa.