Entheome Foundation release Psilocybe genomes to the community

The current public fascination with psychedelic-assisted psychotherapy has meant that species of mushrooms from the genus Psilocybe have gained a lot of attention. This new attention is helping fill the gaps of a very patchy story resulting from decades of this genus being neglected by academics due to prohibition. 

With every new fungi season, interesting and novel fungi are being found, and thanks to citizen science platforms such as iNaturalist.org, many of these finds are being documented for science. In addition to tracking location and dates, iNaturalist also allows other data to be collected, such as habitat data, and barcoding genes.

While barcoding can help determine the relationships between genera and, in some cases, species, there is also a push within citizen science communities for full genome data to investigate these relationships at a deeper level. Genome sequencing also allows us to look at genes responsible for secondary alkaloid production. As sequencing becomes cheaper and more readily available, genetic data is being generated at increasingly better quality, and importantly, often publicly available. 

Founded in 2021, The Entheome Foundation (Entheome) was created with the explicit aim of sequencing the genomes of all entheogenic plants, fungi, and animals in order to create open and publicly accessible data for the benefit of everyone. Entheome announced their first major publication in late December 2024, published in Microbiology Resource Announcements Volume 14, Issue 2. 

The purpose of the publication was to notify the mycological community about the release of high-quality draft genomes for five ecologically and geographically diverse Psilocybe species: Psilocybe semilanceata, the holotype of the genus, Psilocybe gandalfiana nom. ​prov., and Psilocybe caeruleorhiza, newly published in 2024. Also included in the announcement were genomes of Psilocybe azurescens and Psilocybe allenii, species known to be conspecific with Psilocybe subaeruginosa.

The year 2024 proved to be a significant year for papers describing the genus Psilocybe, including “Phylogenomics of the psychoactive mushroom genus Psilocybe and evolution of the psilocybin biosynthetic gene cluster” by Alexander Bradshaw et al. from the Dentinger lab published in Proceedings of the National Academy of Sciences (PNAS), and Australian researcher Alistair McTaggart and coauthors’ long awaited paper “Wood-loving magic mushrooms from Australia are saprotrophic invaders in the Northern Hemisphere” being published in Fungal Systematics and Evolution. These papers contributed significantly to our understanding of the evolution of Psilocybe, taxonomy, and a deeper understanding of the Psilocybin Biosynthesis Gene Cluster (BGC). [The preprint of the McTaggart et al. paper, which includes the results of the analysis of the Psilocybin BGC in Psilocybe subaeruginosa,  can be found here.]

The biosynthesis of psilocybin is the result of a series of enzyme-driven chemical reactions. The enzymes that catalyse the reactions are encoded in the highly conserved gene region, referred to as the Psilocybin BGC, occurring primarily within species of Psilocybe but also known to occur within other genera from the same ecological niche via Horizontal Gene Transfer as described in Reynolds and colleagues’ 2018 paper “Horizontal gene cluster transfer increased hallucinogenic mushroom diversity”. Fricke and colleagues in their 2017 paper, “Enzymatic Synthesis of Psilocybin”, describe how the biosynthetic pathway (Figure 1) synthesizes psilocybin using biogenetic enzymes (PsiD, PsiK, PsiM, PsiH) encoded by the psilocybin gene cluster. The biosynthesis begins with tryptophan, which PsiD decarboxylates to transform it into tryptamine. The cytochrome P-450 monooxygenase PsiH hydroxylates tryptamine at the 4-position of the indole backbone, resulting in the formation of 4-hydroxytryptamine.

PsiK phosphorylates 4-hydroxytryptamine to yield norbaeocystin. PsiM methylates the amine to produce baeocystin, which is then converted to psilocybin through additional methylation of the amine. Psilocybin is dephosphorylated to psilocin, which can revert to psilocybin through the action of PsiK (Figure 2).

Psilocybe is thought to have evolved approximately 65 Million Years Ago, after the KPg extinction event that wiped out the dinosaurs and many other forms of life. Approximately 55 Mya, the genus diverged into two distinct Clades - I & II.  Clade I contains Section Semilanceateae, Section Zapotecorum, Section Mexicanae, and others. Clade II includes Section Subaeruginosae, Section Cubensae, and Section Aztecorum. Each clade, and then some sections within the two clades, have rearrangements of the Psilocybin BGC, and in some cases duplications of specific genes. Given how conserved these genes are, it does raise questions about additional changes.

The Entheome Foundation has been playing an exciting role in this growing narrative by publishing high-quality genomes with impressive BUSCO and N50 values. Genomes are assembled from tiny fragments of overlapping genetic data collected during the sequencing process, so BUSCO (Benchmarking Universal Single-Copy Orthologs) serves as a helpful tool for assessing the overall completeness of the genomic data. BUSCO scores provide a valuable measurement that is important for evaluating the quality of genome assemblies. With these genomes, we're looking forward to a deeper exploration of the psilocybin gene cluster and enhanced taxonomic resolution within the Psilocybe genus. The genomes were sequenced using both Illumina and Oxford Nanopore Technologies, and they range from about 39 to 80 Mbp (for context, a human genome is roughly 3.2 gigabases).

Psilocybe subaeruginosa, Image by Jonathan Carmichael

The psilocybin gene clusters largely matched the gene clusters published by Bradshaw et al. 2024 having mapped the psilocybin gene clusters from a number of species. The new data from the announcement further highlights the difference between Clade I and Clade II of the Psilocybe phylogeny as first described by Ramirez Cruz et al. in the 2013 paper “Phylogenetic inference and trait evolution of the psychedelic mushroom genus Psilocybe sensu lato (Agaricales)”, and confirmed in the Bradshaw et al. 2024 paper. The Psilocybin Biosynthesis Gene Clusters (BGC) from P. allenii and P. azurescens - two species recently shown to be conspecific with P. subaeruginosa - match the Psilocybin Biosynthesis Gene Clusters from P. subaeruginosa, further highlighting their shared taxonomy.

As we gather more genomes of Psilocybe, so too will we be in a better position to understand the reasons behind variations in the psilocybin gene clusters, but also better understand a variety of ecological adaptations, and potentially unusual effects such as wood lover paralysis.

In line with Entheome’s core aim, the data is publicly available on GenBank BioProject PRJNA1013220.

References:

Bollinger IM, Singer H, Jacobs J, et al. (2024) High-quality draft genomes of ecologically and geographically diverse Psilocybe species. Microbiology Resource Announcements 14(2): e00250-00224.

Bradshaw AJ, Ramírez-Cruz V, Awan AR, et al. (2024) Phylogenomics of the psychoactive mushroom genus Psilocybe and evolution of the psilocybin biosynthetic gene cluster. Proceedings of the National Academy of Sciences 121(3): e2311245121.

Fricke J, Blei F, Hoffmeister D (2017) Enzymatic Synthesis of Psilocybin. Angew. Chem. Int. Ed. 56(40): 12352-12355.Ghosal S, Dutta SK, Sanyal AK, et al. (1969) Arundo donax. Phytochemical and pharmacological evaluation. Journal of Medicinal Chemistry 12(3): 480-483. 

McTaggart AR, Scarlett K, Slot JC, et al. (2024) Wood-loving magic mushrooms from Australia are saprotrophic invaders in the Northern Hemisphere. Fungal Systematics and Evolution 14: 209-217. 

Reynolds HT, Vijayakumar V, Gluck-Thaler E, et al. (2018) Horizontal gene cluster transfer increased hallucinogenic mushroom diversity. Evolution Letters 2(2): 88-101. 

Ramírez-Cruz V, Guzmán G, Villalobos-Arámbula AR, et al. (2013) Phylogenetic inference and trait evolution of the psychedelic mushroom genus Psilocybe sensu lato (Agaricales). Botany. 91(9): 573-591. https://doi.org/10.1139/cjb-2013-0070