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‘Magic mushroom’ enzyme mystery solved
Researchers unravel the biosynthesis of the psychoactive drug psilocybin, making large-scale production a possibility
By Stephen K. Ritter

The euphoria and hallucinations induced from eating Psilocybe “magic mushrooms” have earned the fungi a cult following. Sandoz chemist Albert Hofmann isolated and determined the structure of psilocybin, the main ingredient in mushrooms that leads to the psychedelic effects, nearly 60 years ago. That discovery and subsequent mind-altering experiments by Harvard University psychologist Timothy F. Leary have left scientists longing to develop a large-scale synthesis of the compound for medical uses, which include treating anxiety and depression in terminal cancer patients and treating nicotine addiction. Yet no one has been able to unravel the enzymatic pathway the mushrooms use to make psilocybin, until now.
Janis Fricke, Felix Blei, and Dirk Hoffmeister of Friedrich Schiller University Jena have identified and characterized to the greatest extent so far the four enzymes that the mushrooms use to make psilocybin. The team then developed the first enzymatic synthesis of the compound, setting the stage for its possible commercial production (Angew. Chem. Int. Ed. 2017, DOI: 10.1002/anie.201705489).
During their study, Hoffmeister and coworkers sequenced the genomes of two mushroom species to identify the genes that govern fungal enzymatic production of psilocybin. They further used engineered bacteria and fungi to confirm the gene activity and exact order of synthetic steps. This process includes a newly discovered enzyme that decarboxylates tryptophan, an enzyme that adds a hydroxyl group, an enzyme that catalyzes phosphorylation, and an enzyme that mediates two sequential amine methylation steps. With that knowledge in hand, the team designed a one-pot reaction using three of the enzymes to prepare psilocybin from 4-hydroxy-L-tryptophan.
Medicinal chemist Courtney Aldrich of the University of Minnesota, Twin Cities, praises Hoffmeister and his coworkers for their painstaking efforts to elucidate the biosynthesis of psilocybin. “Our knowledge of the biosynthesis of fungal natural products has lagged behind our understanding of the corresponding bacterial biosynthetic pathways owing to a number of unique challenges,” Aldrich says. These include greater genome complexity in fungi compared with bacteria, many fungi are still not amenable to genetic manipulation, and cultivating fungi to produce sufficient amounts of desired metabolites is not always straightforward. “The new work lays the foundation for developing a fermentation process for production of this powerful psychedelic fungal drug, which has a fascinating history and pharmacology,” Aldrich adds.
“The publication by Hoffmeister and colleagues highlights a terrific example of genomics-based biocatalyst-pathway discovery,” adds natural products researcher Jon S. Thorson of the University of Kentucky. “While psilocybin biosynthesis derives from a series of fairly simple chemical transformations, this new study identifies the contributing genes and biocatalysts for the first time and, importantly, provides strong evidence to support a revision of the order of the key steps proposed more than five decades ago. This work clearly sets the stage for bioengineered psilocybin production and/or for analogs that may serve as compelling alternatives to existing synthetic strategies.”

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Nature Boy said:
The line that caught my attention was the one that said "an enzyme that mediates two sequential amine methylation steps."  This infers that there is the potential for a wholly enzymatic synthesis of dimethyltryptamine (DMT).  Would love to read the paper that sets THAT process out in detail. 

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24sevenZed said:
What I find more interesting about this is that it could allow one to develop knock out methods for these enzymes in, e.g. Psilocybe Cubensis, and to finally trade Cubensis (and other psilocybe) cultures legally.

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36fuckin5 said:
Why is that interesting? The only cool things about Psilocybes are that they're active. Take that away and they're not worth eating.

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24sevenZed said:
What I find more interesting about this is that it could allow one to develop knock out methods for these enzymes in, e.g. Psilocybe Cubensis, and to finally trade Cubensis (and other psilocybe) cultures legally.

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Nature Boy said:
The line that caught my attention was the one that said "an enzyme that mediates two sequential amine methylation steps."  This infers that there is the potential for a wholly enzymatic synthesis of dimethyltryptamine (DMT).  Would love to read the paper that sets THAT process out in detail. 

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Inspir3 said:

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Nature Boy said:
The line that caught my attention was the one that said "an enzyme that mediates two sequential amine methylation steps."  This infers that there is the potential for a wholly enzymatic synthesis of dimethyltryptamine (DMT).  Would love to read the paper that sets THAT process out in detail. 

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meaning it allows step 2 to go to step 4, skipping 3?

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Our results therefore imply that 2 (Psilocin) may not be a true substrate en route to 1 (Psilocybin). Given that PsiK readily turns over 2 (Psilocin) to 1 (Psilocybin), we propose that this activity represents a protective mechanism to rephosphorylate the instable 2 to the stable 1 in case of intracellular ester cleavage

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Wiiiiilson said:
An exciting prospect from this research is a PCR to detect these enzymes and give instant confirmation of active species vs non-actives. I've seen papers that use a PCR for a non coding satellite repeat region from psilocybe species to detect 'relatedness' - which correlated well with psychoactivity, but not a direct PCR for the enzymes responsible for psychoactive metabolite production.

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what a fabulous black box that would make. Get two enzyme preperations, one that can methylation s-adenosyl-homocysteine to S-adenosyl-methionine and another that can regenerate s-adenosyl-homocysteine by transferring a methyl group to the available amine. Two catalysts in a chamber heated to 37°c, with a spigot adding tryptamine at the top and another releasing DMT out the bottom. That's the science, I'll leave the details to the engineers.

-shulgin; TIHKAL; DMT is everywhere chapter

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Wiiiiilson said:
Alan, I tried looking for the actual sequence but there is no accession listed in the paper and there are no full genomes for P. cubensis on Genbank yet, which is quite suspicious for a published paper. They dont even list any of the primers they used in the paper to make their clones. It makes me wonder how it passed review really - all we have is their word.

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I was curious to know if this operon was on a mobile element - which is why I was searching for the sequence. Do you know if it actually is passed by HGT?

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Wiiiiilson said:
Well as their names suggest, L-tryptophan decarboxylase is specialized to the role of decarboxylating L-tryptophan. A homolog of this may be a decarboxylase of a different, similar substrate - probably a different amino acid like tyrosine, for use in a different pathway.

Kinases are common - they add phosphates to other molecules. Phosphorylation is important in all sorts of signalling pathways (various environmental sensors and stress responses).

S-adenosyl-L-methionine (SAM)-dependent N-methyltransferases are involved in methylation of other molecules. Methylation is important for regulation of gene expression. In humans, methylation is typically associated with gene silencing. Too much or unregulated demethylation can cause cancer, so they're pretty important and pretty common.

Monooxygenases I don't know about really, but wiki says that they add -OH groups to other molecules and are involved in a wide variety of metabolic pathways. The addition of an -OH to a signalling molecule can deactivate it.

It makes you wonder then, if psilocybin isn't some sort of signalling molecule? Perhaps it helps in detecting physical damage to the fruit body (the bluing when you bruise the mushroom), as it is caused by rapid oxidation - it would probably be a decent indicator of cellular damage? Who knows.

Life is very complex!

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Warrk said:
Is it known how psilocybin and its related analogues function in magic mushrooms? What purpose do they serve?

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Wiiiiilson said:
Interesting stuff - but again, no solid molecular biology to look at there (the link in the year old post just says 'permission denied').

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It makes you wonder then, if psilocybin isn't some sort of signalling molecule? Perhaps it helps in detecting physical damage to the fruit body (the bluing when you bruise the mushroom), as it is caused by rapid oxidation - it would probably be a decent indicator of cellular damage? Who knows.

Life is very complex!

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In any case, it makes me wonder how hard it is to cross a non-active with a compatible active and get a new active species?
They are genetically quite distinct, so it probably wouldn't work, but imagine crossing cubes with oysters and getting a super fast growing active oyster!

If it could be demonstrated with one active non-active pair, you could use a phylogenetic tree to choose a series of species to breed and gain the psilocybin genes until you have it in a faster growing species like Oyster.
Or king oysters! Big fat active kings!

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Alan Rockefeller said:

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Warrk said:
Is it known how psilocybin and its related analogues function in magic mushrooms? What purpose do they serve?

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No.  Does not appear to serve any function - however not everything in nature serves a function, useless traits can stick around if they don't cause the organism to expend too much work.

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Wiiiiilson said:
The paper isnt bad, I just find a suspicious lack of detail. It says it's a communication though, not a full article - perhaps there is more to come soon.

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Alan - As for psilocybin as a potential (completely hypothetical) signalling molecule, how can you say that they don't seem to do anything with the information?

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Here's some more - potential antioxidant, precursor to another compound, metabolite of some other process, foraging attractant? It has to offer some benefit otherwise the operon would not survive.

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Crossing species - I'm not saying you could go straight from cubes to oysters, I'm saying you could (albeit very slowly and with a lot of luck) cross closely related species in series until you eventually get it into something more vigorous. Have a look at the phylo tree in the article you linked from your colleagues PhD thesis:
http://www.nrcresearchpress.com/na101/home/literatum/publisher/nrc/journals/content/cjb/2013/cjb.2013.9109/cjb-2013-0070/20130903/images/large/cjb-2013-0070f4.jpeg

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There could be enough genetic similarity between psilocybes and say...Hebeloma.. for a cross.

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Look how closely gyms and galerina are - There's probably already some active galerina, but it wouldnt be useful.

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There's very likely more genera that aren't included in the table which would bridge the gap between the actives in the top hlaf of the tree and the Strophariaceae in the lower half.

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These analyses suggest a psilocybin
cluster origin in Agaricales by horizontal transfer of
multiple genes from Atheliales, and a horizontal
cluster transfer from dung-inhabiting Psilocybe to
Panaeolus. The patterns of dispersal of this cluster
may reflect at least one role for psilocybin as a defense
against mycophagous invertebrates.

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