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  • Ian Bollinger

Elucidating psilocybin biosynthesis: Known and Novel pathways

Updated: Dec 19, 2020


Scientists (Ali R. Awan et al) generated and compiled DNA and RNA data that investigated the proposed chromosomal structures of psilocybin production in mushrooms. They showed that psilocybin production is NOT unique to Psilocybe species and that the genes associated with psilocybin production were observed to have at least four (4) different cluster arrangements that likely came about independently through convergent evolution or possible horizontal gene transfer.

#genetics #psilocybin #psilocybe #research #psilocybinresearch #biosynthesis

Hypotheses and Takeaways

The scientists started with one main question that was investigated: “The evolutionary advantages conferred to mushrooms by psilocybin remain uncharacterised.” It can be inferred from this statement, as well as the conclusions of the paper, that the two main hypotheses were 1) to determine if psilocybin production in mushrooms arose independently, in different branches of family tree and 2) producing psilocybin provides an evolutionary advantage to the mushroom in the form of reduced grazing from insects.

Based on the compiled data presented the scientists propose that the genes associated with psilocybin production are likely subtelomeric, meaning that they are pseudogenes (transcribed genes producing RNA sequences not translated into protein) and gene families. Subtelomeric genes in humans typically code for olfactory receptors, immunoglobulin heavy chains, and zinc-finger proteins. They propose that “[t]he lack of…transposable elements in the assembly … is readily explained by the lack of long sequencing reads…able to resolve repetitive regions during assembly. This subtelomeric placement would be consistent with increased chromosomal rearrangement and expansion of the psilocybin cluster in Psiocybe [sic] cyanescens relative to the other psilocybin-producing species shown (Fig 1)[5,6]”

Insight into Compound Production

Psilocybin, a tryptamine alkaloid that acts on serotonin 5-HT2A/C receptor sites

· Derived from the amino acid tryptophan

· Many organisms (including humans) enzymatically metabolize tryptophan into tryptamine, the precursor to the neurotransmitters serotonin and melatonin

· Addition of the Hydroxy group to the 4-position is a defining characteristic of Psilocybin-like compounds (baeocystin, aeruginascin, etc); unlike the addition of a Hydroxy group to the 5-position, which leads to serotonin (5-Hydroxytryptamine)

· The enzyme PsiM can add a Methyl group to the amine ‘arm’ multiple times, creating mono-methyl (baeocystin), di-methyl (psilocybin), and tri-methyl (aeruginascin) analogs.

Conclusion and Caveats

Psilocybin producing clusters observed in P. cyanescens and P. cubensis contain an additional kinase and major facilitator superfamily-type (MFS-type) trans-membrane small solute transporter compared to the cluster in Gymnopilus dilepis and Pluteus salicinus (Fig 1a). While in P. cyanescens there is observed a second monooxygenase (labeled PsiH2) which is different from the one observed in all five species; more uniquely, a third MFS-type transporter is present. All of these P. cyanescens specific genes were observed being expressed at the RNA level (Fig 1a). As the scientists put “These findings hint at the exciting possibility that mushrooms from the Psilocybe genus, and particularly P. cyanescens, could be producing novel psilocybin-like molecules.”

The scientists do note that the theory of psilocybin production presenting as an evolutionary advantage to prevent grazing is based in empirical data, but fundamentally denounce it. There exists a “dearth of easily observed features of psilocybin-containing mushrooms that distinguish them from non-psilocybin-containing mushrooms and allow for rapid recognition and learned avoidance or attraction by mycophagous animals is paradoxical.” This is strongly based on their rearing experiments which demonstrated that psilocybin does not provide complete protection from flies that utilize mushrooms as brood sites. Although they do give the caveat that whether “mycophagous animals exhibit innate or learned avoidance of or attraction to psilocybin containing substrates, or experience decreased or increased fitness from using them, remains to be tested.”

Accept or Reject Proposed Hypotheses?

1) Psilocybin production in mushrooms arose independently, in different branches of family tree (Sup Fig 4) and were chemically observed in unrelated species (Fig 2a & 2d)

2) Producing psilocybin provides an evolutionary advantage to the mushroom in the form of reduced grazing insects (Fig 3b).

Reviewing the data and the conclusions put forth it can be stated that the first hypotheses (convergent evolution of psilocybin production) proposed could be accepted; while the second (psilocybin production deters insect grazing) could not. The reason for acceptance and rejection are quoted below (emphasis and parentheticals mine):

Evidence For Hypothesis 1)

“To explore this seeming discrepancy, we confirmed psilocybin production in a single specimen of I. corydalina (Fig 2a), and then used that same specimen to resequence the genome at greater depth.”

“A search of the I. corydaline genome for alternative psilocybin biosynthetic clusters revealed a single candidate, containing all four types of biosynthetic enzyme necessary for the conversion of tryptophan to psilocybin, plus an MFS-transporter (Fig 2d).”

Evidence Against Hypothesis 2)

“However, analyses of our genome sequencing and RNA-seq reads for nonfungal sources of genetic material revealed hundreds of predicted insect contigs and proteins (Fig 3a, Tables S12-13), many of which belonged to the Order Diptera (true flies). This result suggested that insects of the order Diptera were present in the Psilocybe cyanescens fruiting bodies used for genomic and RNA-seq library construction. Indeed, genomic sequences were found that unequivocally belonged to the species Exechia fusca, a species of 'fungus gnat' in the family Mycetophilidae (Table S1)

“Several fruiting bodies of Psilocybe cyanescens and of the co-occurring psilocybin nonproducer Stropharia aeruginosa were collected from the same small patch of wood chips. The individual mushrooms were separately washed thoroughly to remove any surface insects, and these mushrooms were placed into two separate glass jars, separated by species. After several days, 4-5 larvae emerged in each jar, followed by pupation and the emergence of a single fly in each jar by two weeks. The flies were isolated separately and identified as both belonging to the family Sciaridae, commonly known as dark-winged fungus gnats, which are common pests of the commercial mushroom industry”.

“[T]he fact that orthologues of the psilocybin cluster genes are present in the termite [mutualist] fungus Fibularhizoctonia sp.[2], we suggest the alternative hypothesis that psilocybin’s evolutionary benefit may lie in facilitating mutualism between fungi and insects.”


1) Lin HC, Hewage RT, Lu YC, Chooi YH. Biosynthesis of bioactive natural products from Basidiomycota. Org Biomol Chem. 2019 Jan 31;17(5):1027-1036. doi: 10.1039/c8ob02774a. PMID: 30608100.

2) Reynolds, H. T. et al. Horizontal gene cluster transfer increased hallucinogenic mushroom diversity. Evolution Letters 2, 88–101 (2018).

3) Wellinger, R. J. & Sen, D. The DNA structures at the ends of eukaryotic chromosomes. Eur. J. Cancer 33, 735–749 (1997).

4) Biscotti, M. A., Olmo, E. & Heslop-Harrison, J. S. Repetitive DNA in eukaryotic genomes. Chromosome Res. 23, 415–420 (2015).

5) Brown, C. A., Murray, A. W. & Verstrepen, K. J. Rapid expansion and functional divergence of subtelomeric gene families in yeasts. Curr. Biol. 20, 895–903 (2010).

6) Anderson, M. Z., Wigen, L. J., Burrack, L. S. & Berman, J. Real-Time Evolution of a Subtelomeric Gene Family in Candida albicans. Genetics 200, 907–919 (2015).

7) Fricke, J., Blei, F. & Hoffmeister, D. Enzymatic Synthesis of Psilocybin. Angew. Chem. Int. Ed. 56, 12352–12355 (2017).

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