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psilocybin/psilocin biosynthesis
    #323203 -

this post is intended for those who are interested in the biosynthesis of psilocybin and psilocin, and related factors, such as sbustrate supplementation, enzymes involved, genetic differences between species, etc..., and to explore these issues by exchanging ideas and knowledge.
it is my intent to share relevant scientific information that i find and hope that others will do so as well.

please do not turn this into a useless flamed thread as related posts such as superpotent shrooms are possible, doubters of the dmt substrate... have become

no posts such as "dude, if i add phalaris grass to my casing will i have superpotent mushrooms?" and/or anything resembling a dirtmaster post.

hopefully that will be enough administrative bullshit, let the science commence


c12h16n24ohdmt

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Re: psilocybin/psilocin biosynthesis [Re: gray1]
    #323209 -

does anyone have access to the "journal of basic microbiology"?
if you're at a university, check the science library or easier yet, check the list of journals your library subscribes to online at the lib. webpage.
i think this article would be interesting to read, all i have is the abstract from pubmed:

J Basic Microbiol 1989;29(6):347-52 Related Articles, Books

Biotransformation of tryptamine derivatives in mycelial cultures of Psilocybe.
Gartz J.
Institut fur Biotechnologie der AdW, Leipzig.
Mycelial cultures of Psilocybe cubensis capable of forming psilocybin and psilocin de novo display a high capacity for hydroxylation of tryptamine derivatives at the 4-position. A specific biotransformation of added synthetic N,N-diethyl-tryptamine was found. Thus high amounts of 4-hydroxy-N,N-diethyltryptamine (up to 3.3%) and a minor quantity of 4-phosphoryloxy-N,N-diethyltryptamine (0.01-0.8%) were isolated from fruiting bodies of Psilocybe cubensis in corresponding experiments. This is the first example of a directed biosynthesis of tryptamine substances by fungi. An effective biotransformation of N-methyltryptamine was also demonstrated with surface cultures of Psilocybe semilanceata. Baeocystin, a possible natural precursor of psilocybin, was detected and quantified in the biomasses. No alkaloids could be found in the culture medium.

also, see my last post in the DMT doubters thread for an interesting article reference

gray1

c12h16n24ohdmt

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Re: psilocybin/psilocin biosynthesis [Re: gray1]
    #323211 -

is anyone fluent in german? it would be interesting to navigate through the Leipzig University site in search of the institute of biotechnology homepage, where it appears that j. gartz is doing his research, and find out some information about his lab, what he is currently working on, a list of publications, etc...
http://www.uni-leipzig.de/

c12h16n24ohdmt

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Re: psilocybin/psilocin biosynthesis [Re: gray1]
    #323813 -

gray1 please e-mail me.i am also very interested in getting hold of papers such as these as i woul like to find out exactly what enzymes catalyse DMT and NN DMT to psilocin and then to psilocybin.Im at uni and may have access to some of the papers you require.

Want to trade? edibles and psychoactives, spores and slants.


--------------------
visit the Australasian free seed ring
www.shaman-australis.com.au/seedring

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Re: psilocybin/psilocin biosynthesis [Re: aNaPhylaktik]
    #324651 -

i can get hold of the journal of micro from 1973 onward i believe via an inter university reciprocal borrowing doo da thingy. so yeah i will go and do some photocopying soon

Want to trade? edibles and psychoactives, spores and slants.


--------------------
visit the Australasian free seed ring
www.shaman-australis.com.au/seedring

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Re: psilocybin/psilocin biosynthesis [Re: gray1] * 1
    #324660 -

gray1.. have you ever worked w/ any bioinformatic systems? Do they contain DNA models for mushrooms?

Sorry if this is one of those off-the-wall posts you said not to make. I couldn't tell for sure.

* Contributor to the Free Spore Ring


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Where there's skill, there's a better way..

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Re: psilocybin/psilocin biosynthesis [Re: egghead]
    #324864 -

not at all, very good post.
i've used several programs that allow for manipulation / analysis of genetic material, however, i have yet to discover (or look for that matter) any mushrooms that have been sequenced.
before any bioinformatic research can be done, there must be a genome sequence. there may verywell be sequenced mushroom genomes, but i'm sure that p. cubensis is not one of them

once you had a sequence, you could then look for possible proteins by comparing sequences to other known proteins, human or otherwise. at least the active domains should bear some resemblence. so, it is also useful to determine what enzymes are used in the conversions that make psilocybin from tryptophan, things like hydroxylases, aminotransferases..., then get their sequences, hopefully from several different organisms, and run a search of the genome in question.
would be interesting to look. i'll give it a try later


c12h16n24ohdmt

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Re: psilocybin/psilocin biosynthesis [Re: gray1]
    #325007 -

Cool.. As I've told you before, I'm no chemist/biologist. I'm reading a book on bioinformatics to get a look into the data/research publishing/sharing technology and how it's used. My hope is to apply the best-practices of bioinformatics to other fields of endevor.

Are you using GenBank or PDB? Do you have access to private databases? From what I've seen so far, the industry is just now beginning to crawl. I mean they're still coming to terms w/ predicting/modeling a protein structure from it's sequence. Would you say bioinformatics will evolve w/ the genetic research industry or is there already another more unified research/data organization methodology that folks are standardizing on or around?

* Contributor to the Free Spore Ring


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Where there's skill, there's a better way..

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Re: psilocybin/psilocin biosynthesis [Re: egghead]
    #325078 -

yes, bioinformatics is increasingly useful for the analysis of genomes, attempting to predict the proteins that they will produce, and the structures of those proteins. however, it is merely a guide, and will never be able to replace tangible research and visualization techniques such as NMR and protein crystallography.
one important thing to remember when using bioinformatics to predict gene and protein function / and or compare sequences between different organisms based on genetic or protein sequence is that there may be inherrent differences in the actual final product or structure due to endogenous processing differences
ie: the same exact sequence of nucleotides/dna could produce a completely different protein in two different organisms. a protein has structural characteristics that result in its ability to bind substrates and have a certain cellular activity, and completely different structures can be derived from the same dna sequence:
there are three levels of protein structure: primaary level is the sequence of amino acids itself. the secondary level is the way in which the sequence is arranged in two dimensional space (is it linear, does it loop back and make a u shape, etc...) and the third level is it's actuall shape in three dimensions, all of these structural arrangements play a role in the characteristics of a protein, as do cellular environmental conditions, binding of metal ions, pH, etc...
so, in summary, while bioinformatics can be a good predictor, and is an excellent place to start theoretical research, it is not tangible.
if you're interested in this you may want to do some research on biotech companies and the work that they do, specifically there are numerous companies that specialize in bioinformatics.
a new direction that is considered to be under the umbrella of bioinformatics is called proteomics, and is involved in characterising the functions of proteins. this is increasingly important as more and more genomes are being sequenced (importantly the human genome) because function needs to be assigned to thesequences of dna/protein that are discovered. afterall, function is what is interesting, therapeutically and for the general desire to better understand living organisms.

i intend to use both genbank and pdb through the ncbi homepage
i have no access to private databases, but doubt that there are many that have pertinent information about mushrooms. most private databases focus on human info and model organisms that are routinely used in science (fruit flies, worms, yeast, bacteria, some plants...)
i have no idea yet whether there will be mushroom sequence information. i'm not too hopeful.

anyone want to sequence the p. cubensis genome?
that would be huge.

c12h16n24ohdmt

Edited by gray1 on 05/24/01 03:00 PM.


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Re: psilocybin/psilocin biosynthesis [Re: gray1]
    #325814 -

best thing is it probably wouldnt be ;)
it should have a fairly small genome compared to more complex organisms
im in .will have to find out whats involved...

Want to trade? edibles and psychoactives, spores and slants.


--------------------
visit the Australasian free seed ring
www.shaman-australis.com.au/seedring

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Re: psilocybin/psilocin biosynthesis [Re: aNaPhylaktik]
    #326017 -

I think the only way to get the genome of a P.Cub would be to either pay alot of money, or work for Genentech or some other biotechnology company. once you have the code clipped from the mushroom DNA it shouldnt be hard to duplicate and insert it. I just wanna know where your getting shroom DNA code from?



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_______________________________________
CSC

Life's a garden, Dig it!
~Joe Dirt

Off Topic Website

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Re: psilocybin/psilocin biosynthesis [Re: Cow Shit Collector]
    #326170 -

Hi there,

I cant find any P cube or other psilocybian shroom sequences in the public domain. There are some BAC sequences for Amanita but this is just raw sequence. I dont think that a psilocybin synthase has been identified yet.

What we need folks is a simple plate assay for psilocybin.
Thus we construct a genomic library of P cube and zap this into yeast and plate on our 'assay plates' for our gene. Any ideas?

Sequencing the p cube genome is completely unrealistic, It would take years and $100,000s or $1,000,000s. Then the relevant gene would still have to be identified.

By the way has anyone thought of zapping in a fungicide resistance marker into thier P cube strains? should make it easy to grow without contams

If anyone out there has some good pics of P cube mycelium growing on agar, please let me know as Im struggling to isolate a strain and dont really know what to look for.

Thanks a lot.
Mad.

Edited by madscientist on 05/27/01 09:32 AM.



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Instead of the dove as the symbol of peace we should have a pillow. Its got more feathers but doesnt have that nasty sharp beak......

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Re: psilocybin/psilocin biosynthesis [Re: gray1]
    #327410 -

I checked out the Leipzig University site and did a search of whos who in biotechnology and turned up nothing on Gartz. The seach does list researchers as well as faculty, but I found nothing... Sorry.

I am interested in that article about causing alkaloid production in mycellium...

Some parts of Gartz's formula would have to be mearly for nutrition, and the rest caused early production of psilocybin. A combination of good thinking and "trial and error" could easily determine which components are responsible for this effect, and those ingredients could simply be added to whatever substrate you want.

OPI


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Re: psilocybin/psilocin biosynthesis [Re: madscientist]
    #327486 -

look on the fungi perfecti site the button/pic with the mycelium - thats p cubensis.it morphs with age but in its prime its white rhizomorphic and just luvely :)

i agree on a lot of points made here. We definitely dont need to sequence the cubes entire genome
if all were interested in is the tryptamine production.for instance we dont need to know all the genes (the majority ) that simply deal with intracellular goings on- probably identical to every other fungus and likely us too
my raw idea is that if you could induce a state where no psilocin/psilocybin is produced then you could then create the cDNA from the RNA and look for the missing elements in normal mycelium cDNA. after all were not even really that interestedf in the enzymes you need to get to DMT or NN DMT just the handful responsible from there on...
if you werent sure you could dideoxysequence it and analyse the sequence to see waht its likely to be
best of all with the mRNA approach you bypass all the shit between the genes.
then.. clone it and try to get a product and so on..

fungicide resistance marker.. good idea but not sure if id want to be eating the shroom unless i had that same resistance!?
however if we id'd the DMT hydroxylase or wahterver itll be we could clone it into a non ps producing fungus
like say pan campanulatus or psilocybe coprophila and see if it make it active and we could dtermine transf9ormation efficiecy with this marker

Want to trade? edibles and psychoactives, spores and slants.


--------------------
visit the Australasian free seed ring
www.shaman-australis.com.au/seedring

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Re: psilocybin/psilocin biosynthesis [Re: aNaPhylaktik]
    #327833 -

I have an even more intriguing question for you all. Are there any other species (ie non psilo species) that will 4-hydroxylate something? For example, lets say some agaricus species would do this, but lacked the ability to break tryptophan into tryptamine, or some other step on the biosynthetic route to our desired tryptamines. It would never produce psilocybin in nature, but it might still turn out psilo cin/cybin if you fed it a DMT containing substrate.

OPI


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Re: psilocybin/psilocin biosynthesis [Re: Opi]
    #328599 -

very interesting idea. my preliminary feelings are that the later, highly specific conversion in the metabolic pathway wouldn't exist in absence of a prior, more basic one, but in the grand scheme of things, it can't be known without trying it out.

i also like the idea of a simple gene introduction into a mushroom that has "incomplete" (with respect to our interest) metabolic capabilities. what kind of vector would deliver the genes in a non-transient transfection? viral?

however, primarily, theoretically, i'm most interested in research about inducing other organisms to produce psilocybin/psilocin.

here's an outline of the most basic steps that need to be taken:
identify the types of enzymes that are involved in the process
target organism: search for what enzymes it has that are related, what enzymes it needs...
isolate and introduce the enzymes
grow and analyze for content

could enzymes with the same function, say hydroxylation, be used in place of the actual enzymes from the fungal genome? that would be interesting to see, or is it something specific about the fungal enzymes that end up in the production of indole alkaloids.

without sequence, i guess mRNA analysis is the only way to go, but developing, not to mention working with, a cDNA library is costly, time consuming and hard.

describe the plate assay in more detail...

gray1

c12h16n24ohdmt

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Re: psilocybin/psilocin biosynthesis [Re: gray1]
    #328608 -

The plate assay was actually hypothetical. I dont know how those other guys assayed for psilostuff but I would guess they did GC/HPLC/Mass Spec or something else involving horrendously expensive and difficult to use pieces of equipment. My idea is just to whack the psilosyhnethic gene into yeast and use this instead of going to all the hassle of growing shrooms which is a pain the arse and difficult to keep secret:) Yeast on the other hand can be grown up within 24-48 hrs ("hey, wanna trip on saturday? yeh, Ill just inoculate a flask now and it should be ready...). So, what is needed is a simple plate assay that would give for example, a colour change around colonies producing psilostuff. A psilocybe genomic library could easily be created and screened using this assay. No cDNAs. No sequencing. Producing such yeast would then be within the realms of possibility.....



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Instead of the dove as the symbol of peace we should have a pillow. Its got more feathers but doesnt have that nasty sharp beak......

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Re: psilocybin/psilocin biosynthesis [Re: madscientist]
    #330232 -

i like that idea.
my idea was eventually to have psilocybin /psilocin (pref psilocybun - it keeps better) expressed in the leaf of a plant
any plant that make decent amounts of DMT or NN DMT - for example acacias or other legumes
something nice and commonplace and easy to grow - it would make it impossible to police.Im thinking about
what we are going to do when the war on drugs beast takes a taste for us shroomers

off topic - doesnt Mucuna pruriens make l dopa in its seeds? then wouldnt it be possible to
manipulate it to make mescaline instead- in the seeds - a new meaning for mescal bean :)

Want to trade? edibles and psychoactives, spores and slants.


--------------------
visit the Australasian free seed ring
www.shaman-australis.com.au/seedring

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Re: psilocybin/psilocin biosynthesis [Re: aNaPhylaktik]
    #330465 -

ponder this philosophical quesdtion:
for whatreason and purpose have fungi evolved to produce psilocin/psilocybin?
it could be useful in two ways, endogenously or exogenously.
from what we know, it isn't used endogenously, fungi doen't have a eukaryotic nervous transmission system. so it must be produced in order to support it's life/perpetuation of genes and species, like a tree produces tasty fruit that humans and other animals eat and thereby spread the seeds contained within. so in a sense, fungi produce psilocybin so that we will want to cultivate and perpetuate the species. but is there a more basic level to this, i would assume of course that these mushrooms evolved before humans. do other animals enjoy hallucenogenic mushrooms? does psilocybin actually play an internal physiological role for the mushroom? (a simple genetic deletion study could determine this if we had the genome in an artificial vector construct like a bac)
please bear with me while i pose such questions, i think that by really evaluating the existence of psilocybin and the mushrooms abiity to produce it, we might have new insight as to where our theoretical experiments should be heading, open us up to new ideas.


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Re: psilocybin/psilocin biosynthesis [Re: gray1]
    #332200 -

I think it highly unlikely that the shroom produces these compounds just for our enjoyment. Bear in mind that fruit trees and crops etc have been selectivly bred for millenia to provide the best fruit and grain. While it may be true that some plants evolved such nice fruit to entice animals to eat and spread the seed, mushrooms deposit spores locally and thus have nothing to gain, everything to lose by being eaten. Thus any species that expends energy to no purpose would be at a selective disadvantage to ones that dont and on to thier eventual extinction! No, these psychoactives must have some role to play. Bear in mind that although shrooms have no nervous system they do have complex signalling pathways (like plants). I would venture a guess that Psilostuff is some sort of fungal hormone or perhaps a precursor for something else.

About creating workable transgenics: Acaia is not an option. Too slow growing and needs too much space, difficult to transform 30 odd years seed to seed?. The best option would be to go for eukaryotics that are fast and easy to grow. Arabidopsis is the best option for this I think. Small, takes only 6 weeks seed to seed and easy to transform.

But i think that yeast is the best option.

Edited by madscientist on 06/03/01 08:49 AM.



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Instead of the dove as the symbol of peace we should have a pillow. Its got more feathers but doesnt have that nasty sharp beak......

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Re: psilocybin/psilocin biosynthesis [Re: madscientist]
    #333882 -

good post.
so if psilocybin and psilocin are chemical mediators for cellular mechanisms, acting like signalling molecules or hormones, what could their function be? there must be receptors to psilocybin and psilocin somewhere in the fungi, and presumably these receptors would be similar in structure to the receptors in our brains that bind psilocybin and psilocin.
so, it would be interesting, if in fact psilocin and psilocybin are indeed important mlecules for the physiology of the fungi, to do knockout studies which would render the mushrooms unable to produce psilocybin and psilocin and see what effect this has on growth. also, what about mushrooms that don't produce psilocybin and psilocin naturally?
so many questions, so little actual research that has ever been done. lets hope that the MAPS study that is getting under way will break new ground and develop more interest in the musshrooms. then maybe more scientific studies about the biology of the mushrooms, not just the psychological impact on the human consciousness, will be done.
gray1


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Re: psilocybin/psilocin biosynthesis [Re: gray1]
    #334329 -

Alexander Shulgin, if you haven't heard of him, is one of the most important chemists of our century. Here is something that he has to say about mushrooms under the 4-HO-DET entry.

Some fascinating studies have been done in Germany where the metabolically active mycelium of some Psilocybe species have been administered diethyltryptamine as a potential diet component. Normally, this mushroom species dutifully converts N,N-dimethyltryptamine (DMT) to psilocin, by introducing a 4-hydroxyl group into the molecule by something that is probably called an indole 4-hydroxylase by the biochemists. You put DMT in, and you get 4-hydroxy-DMT out, and this is psilocin. Maybe if you put Mickey Mouse in, you would get 4-hydroxy-Mickey Mouse out. It is as if the mushroom psyche didn't really care what it was working with, it was simply compelled to do its sacred duty to 4-hydroxylate any tryptamine it came across. It was observed that if you put N,N-diethyltryptamine (DET, not a material found in nature) into the growing process, the dutiful and ignorant enzymes would hydroxylate it to 4-hydroxy-N,N-diethyltryptamine (4-HO-DET) a potent drug also not known in nature. This is the title drug of this commentary. What a beautiful burr to thrust into the natural versus synthetic controversy. If a plant (a mushroom mycelium in this case) is given a man-made chemical, and this plant converts it, using its natural capabilities, into a product that had never before been known in nature, is that product natural? What is natural? This is the stuff of many long and pointless essays.

Check out http://www.erowid.org/library/books_online/tihkal/ for tryptamines and http://www.erowid.org/library/books_online/pihkal/ for all of the phenylethylamines.

I come from brilliance and I return to brilliance. What is this?


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I come from brilliance and I return to brilliance. What is this?

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Re: psilocybin/psilocin biosynthesis [Re: ionic]
    #334988 -

i've just finished reading the first sections in pihkal (personal memoirs...) and now from time to time i pick it up and read an entry or two in the back section.
has anyone read thikal? please comment on its virtue

i remember reading that about 4-hydroxylation. would certainly be interesting to try and reproduce the results by adding det to a mycelium culture. however, what is the availability of DET? i'm assuming, without looking at the pihkal entry or the structure, that it would have to be synthesized, and that the materials needed to do this are scarce. additionally, if you had the materials necessary, why not just complete the process chemically...

for the philosophical question: in my opinion, the product is one of a natural process, regardless that the det is not natrually occuring and introduced to the organism, and therefore is a natural product. could be debated in several ways though.

gray1


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Re: psilocybin/psilocin biosynthesis [Re: gray1]
    #335261 -

This might be pushing it a bit for the cultivation section, but... I don't know how many people here have heard of quantum coherence/superconductivity in human brain cells but here is a very simple run down. Microtubules were once believed only to be used for structural integrity in the cells but research found that these microtubules exhibit some fascinating properties. These tubules vibrate at between 100-650 Hz, they shimmer when metabolically active, and seem to exhibit a fascinating quantum computing like property. The tubules are made up of a certain protein that has three distinctive electrical states. These states are like a quantum computer almost 10^7 tubulins per neuron. This would possibly explain some of our difficulties in locating consciousness, and the interesting part to mushrooms is in explaining the relatively intelligent activity of eukaryotic cells. These cells, with no real nervous center, have excellent survival tactics, feeding procedures etc. I don't know much of mycology and cell differentiation, but a bioligist here would be quite helpful. I think that neuropeptide receptors on certain mushrooms cells would bind the psilo's and possibly activate some sort of new quantum state for the tubulin. The consciousness of mushrooms eh? Go to http://www.consciousness.arizona.edu for more and better info. If there is anyone here who knows about cell differentiation and the peptides involved in this process in mushrooms, we may be on to something quite interesting.

I come from brilliance and I return to brilliance. What is this?


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_______________________________________________
I come from brilliance and I return to brilliance. What is this?

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Re: psilocybin/psilocin biosynthesis [Re: ionic]
    #335278 -

i'm a biochemist and this is beyond my scope of knowledge, but i do find it interesting.
my first thoughy in response to your post is that fungi don't have a nervous system, therefore, there aren't any neurotransmitters, no action potential, no neuropeptide receptors.
however, there are definately, and must be, chemical mediators that signal all kinds of things.

what are you getting at? what makes up, and for that matter, if there is, some sort of connective conscious in organisms like plants and fungi?

elaborate please


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Re: psilocybin/psilocin biosynthesis [Re: gray1]
    #335538 -

I may be mistaken on this account, but I believe that each of our cells, or at least most, have receptors, some of which are for serotonin in the heart etc. I think that the cells don't really depend upon the CNS to communicate, but can communicate amongst themselves somewhat. What I am proposing is basically, that something way beyond our current ideas of chem and or life is going on. All od the ancient myths about Teonacactl, the mushroom god, may be more than just human conjecture. There were experiments done, I'll get the name of the scientist who led them, about conscious plants. Some plant was placed in an empty room and one person came in, ripped off a leaf, and left. A second came in, stroked the plant and talked nicely. Now this whole time an EEG or something is hooked up to it, and the eletrical signals are being recroded. The people, came into the room, and the plants electrical signals changed. It knew the difference between the two people. So basically I am saying, there is a whole lot going on behind what we see. Like, we have some idea of what certain neurotransmitters do, but we really have know idea if our emotions change them, or if we change the chemicals. Its a question of causality, and so to say what these psilo's are really there for is tough. So, in conclusion, something much beyond what we currently understand is most likely going on. The physiological necessity of these psilo's may never be understood, because possibly their purpose can not be determined pathologically, but only by experiencing them directly. I see them as the plant world, in total awareness, trying to reconnect itself with other species. Mushrooms help to show us a proper way to live, to further our existence, to love them, and to enjoy the mystery of existence. This post should be in the philosophy section, but anyway.

I come from brilliance and I return to brilliance. What is this?


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_______________________________________________
I come from brilliance and I return to brilliance. What is this?

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Re: psilocybin/psilocin biosynthesis [Re: ionic]
    #336136 -

This is all explained in the book "The Secret Life of Plants" One of the best books I have ever read. It really made me think about vegetarians who want to save the pretty animals so they only eat plants. Plants are responsible for everything we have.. Most are only producers. They provide food, air, water, shelter, etc. But they don't "think" the way we do, so we kill them much more easily than a cow... Amazing.

OPI


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Re: psilocybin/psilocin biosynthesis [Re: Opi]
    #336360 -

i'm not sure that i totally buy into what you're saying yet but i'm absolutely fascinated and intrigued. i'd very much like to read more, please post any references that you reccomend.
opi: private lives of plants is a great book indeed, my father is a botanist and several years ago we both unknowingly gave it to each other as a christmas gift. haven't read it in a while though, could you remind me where it talks about this subject? thanks.
i drove with some colleagues from ny to philadelphia for a work related suymposium today and this evening on the return trip through the industrial wasteland that is new jersey these words came into my mind:
what is this beast called man? so intelligent that it is constantly striving to advance it's technology in an effort to produce a higher quality of life, and yet so stupid that in the process of this strife it is constantly counteracting its efforts by destroying the natural ecosystems which gave rise to man, continue to support all of life, and in reality, it is getting back in touch with nature that could really imporve our quality of life, both physically(physiologically), mentally, and most of all, spiritually.

gray1


hopefully, this thread won't be moved to the philosophy section because it starts off on the scientific/experimental side and have no doubt that it will get back there at some point.


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Re: psilocybin/psilocin biosynthesis [Re: gray1]
    #336434 -

I thnk a major cause for our self-destruction has been a terrible abstraction of the nature of time. As we moved out of caves and to more stable agrarian societies, time began to lose its cyclical nature. We forgot that all is now, because there is an upcoming harvest we must prepare for. The psychological effects of being in a purely agrarian society are very, very much less than that of our industrial society. People live their entire lives tomorrow, two weeks from now, or three years ago. We feel extremely pressed to do something, leave a lasting effect, because there is only so much time left. We want to give something to generations to come. It is funny how our mind has lead us so far off track. We are constantly receiving a flood of information, but we never question it. One of the most profound effects of hallucinogens for me is the destruction of all notions of time. There is only now, and there are constantly myriad things to experience. I really, truly feel that these fungi/plants are producers in so many aspects. I wonder, sometimes, why the kingdom animalia evolved whatsoever. It seems so senseless, the plants do not need us, although they have adapted to us. In order to attempt to save themselves, and the existence of life on this planet they have devised various way of grabbing our consciousness. We can marvel at their beauty, we can use them to feed our children, and we can become one with them during the hallucinogenic experience. Plants are jumping out at me, on every front, saying, "Look, we are here! No reason to fret, be content to exist, be now, grow and flourish." By involving myself in their lifecycles through this cultivation process, and examining biosynthesis of their alkaloids I can gain a very one sided knowledge into their nature. When I am reading or writing about the enzymes and various reactions I always feel something is missing. A glaring, WHY? is looking me in the face. Why do I even have this thrist for knowledge? It's senseless, and is really only lessennig my experience of existence. It helps in many ways, but as Mark Twain said: Now that we understand a rainbow it has really lost its magic. This doesn't mean I'm going to quit yet though. I constantly find amazing things in the realm of science, and by sitting on the edge between science and the mystery of existence, I hope to one day fall over. This quote from deoxy.org seems to fit perfectly:

People have been standing for centuries before a worm-eaten door, making pinholes in it with increasing ease. The time has come to kick it down, for it is only on the other side that everything begins. Raoul Vaneigem

I come from brilliance and I return to brilliance. What is this?


--------------------
_______________________________________________
I come from brilliance and I return to brilliance. What is this?

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Re: psilocybin/psilocin biosynthesis [Re: ionic]
    #336700 -

Ionic.. My god!!! Thank you for that site on conciousness. I have been working on a immense project dealing with Quantum mechanics and conciousness, and that site fills in a lot of missing theory for me.. Basically I have developed a model in which QM explains nearly every type of "paranormal" activity. My missing information is basically explaining a feasible physiological basis for the ability of conciousness to select one eigenstate over another. There is a lot to wade through, but I have a feeling I found what I've been looking for. Thanks a lot!

Opi


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Re: psilocybin/psilocin biosynthesis [Re: Opi]
    #6264080 -

I'm fairly confident the reason psilocybes evolved psilocin/psilocybin was the not for animals enjoyment but rather to deter animals from eating it. example if a rat was walking along and came across the mushroom it may eat it then totally trip out and be too scared to go near it again. fruit benefit from being eaten as it fertilizes their seeds but mushroom spore do not need this and in fact would be killed from being eaten

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Re: psilocybin/psilocin biosynthesis [Re: leftover_crack]
    #6264223 -

That's ridiculous. Psilocybin is not an effective deterrent in the least. It doesn't kick in for 30 min or so and it's not lethal or even unpleasant. An animal would have no idea what caused the trip.

Effective deterrents are either extremely distasteful or poisonous. Also mushrooms want to be eaten. It's advantageous to be eaten by animals as it helps spread and fertilize the spores.

The deterrent theory has no reasonable basis whatsoever.


-FF

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Re: psilocybin/psilocin biosynthesis [Re: fastfred]
    #6267406 -

destroying spores by digestion to me seems like a odd way to spread. no matter how far they are spread if they are destroyed hey won't grow. also animal do understand the relation ship between food they have eaten and side effects (whether its sickness or tripping). rats for example are quite good at making those connections which is why rat poision is often not effective when trying to get rid of rats because with new food they eat a bit to try it out and if there are bad side effects they won't eat it again. this includes other rats around not just the one that tried it. animals are not stupid. if they eat a foos type they haven't had before and then 0.5 an hour to an hour later they begin to trip out they will make the connection. this is what i learnt when i was studying natural plant defenses at uni. and i know mushrooms aren't plants but the same evolutionary forces are applied to both knigdoms.

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Re: psilocybin/psilocin biosynthesis [Re: leftover_crack]
    #6276484 -

I believe the spores are able to survive the digestive tract.

Animals aren't going to be able to put together what they ate a half hour ago and any strange effects they are having. If they ate a bunch they might taste and smell the mushrooms in their puke and avoid them in the future, but they're unlikely to eat enough to ever have any highly unpleasant effects.

It is to the mushrooms advantage to be eaten, so the deterrent theory just doesn't make any sense. The effects are also non-lethal, not unpleasant, and slow acting, so it wouldn't act as an effective deterrent.


-FF

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Re: psilocybin/psilocin biosynthesis [Re: fastfred]
    #6283921 -

So Fred, you wake up...go get a drink of orange juice(not knowing i dosed it the night before :smile:)....15 mins later(common for onset effects) you start to trip balls. would you make the connection? Id hope you would. Animals are no different...you are an animal!!! Most humans that had not encountered the sensations before would think that they were poisoned. Humans eat mushrooms with anticipation of the effects. If you were a feral child wondering around foraging for food, ate a grip of mushrooms and tripped....you wouldn't likely eat them again. Oh and my cat bit into a glow stick the other week...he freaked out...now if he is bad all I have to do is show him a dead glow stick and he runs and hides. He remembers the glow stick, he isn't likely to play with one again, it wasn't poisonous, didn't make him sick, no adverse effects except it tasted bad. Deterrents can be non lethal and considered pleasant by humans.
Woodchuck Solution
The safe repellent critters find so offensive. Woodchucks, chipmunks, gophers, rabbits, raccoons, moles, voles, squirrels, field mice, armadillos and skunks are all discouraged from feeding and burrowing by this unique formula, which is so objectionable to all rodents but smells wonderful to humans.
Works by smell, affects sinuses and upsets digestive tracks. Won't harm animals but makes the local environment mighty uncomfortable. Primary "AI" is Rosemary, Thyme and Caster Oil. Re-apply every 100 days.


--------------------
Save Marc

www.emeryseeds.com
Holy dog shit! Texas? Only steers and queers come from Texas, Private Cowboy. And you don't look much like a steer to me so that kinda narrows it down. Do you suck dicks?
-Gunnery Sergeant Hartman(Full-Metal Jacket)

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Re: psilocybin/psilocin biosynthesis [Re: ESanceOfCyan]
    #6284009 -

<<If you were a feral child wondering around foraging for food, ate a grip of mushrooms and tripped....you wouldn't likely eat them again>>

What? :smile: How do you think it happened in the first place? :smile:

But what I have observed is that even higher animals (chickens, turtles, deer..) will take various amounts of different mushrooms. I have seen chickens take a single peck or two from jack o lanterns - is this how they benefit annually by worming themselves?

<<Animals aren't going to be able to put together what they ate a half hour ago and any strange effects they are having.>>

That seems to be true in real life here.

<<If they ate a bunch they might taste and smell the mushrooms in their puke and avoid them in the future, but they're unlikely to eat enough to ever have any highly unpleasant effects.>>

Not altogether true. Poor animals sometimes die out here from plant poisoning.

Sorry - off topic but towards ending "animal conjecture" at least! :smile:


--------------------
Yours in the Natural State Land of Enchantment!

"Sometimes the only way for a man to find true happiness is to take drugs in a group" - Cochise

No makin funna my pomes!

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Re: psilocybin/psilocin biosynthesis [Re: curenado]
    #6284835 -

I think you're giving grazing animals way too much credit in the cogniative department. I say that the cow probably wouldn't ever eat more than a stray bite if it accidentially grazed a little too close to it's own pile of shit. That one tiny bite would then go into it's massive stomach along with perhaps 10 pounds of digesting grass. How much effect do you think it's going to have on a 1000 pound animal?

None. That means no deterrent effect. It also doesn't deter slugs and insects, so I can't see where you see any deterrent effect. The effects aren't unpleasant, so if there are any animals that are deterred then there are probably just as many that are encouraged. It's current evolutionary advantage is it's attractive properties to man, and I don't see any reason to think that that hasn't always been the case.

As for your cat example... If you always beat your cat 15 minutes after he did something would he ever learn not to do it? No.


-FF

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Re: psilocybin/psilocin biosynthesis [Re: fastfred]
    #6306825 -

in my experience magic mushrooms are eaten considerably less by slugs and shit the non magic ones. i rarely ever see partially eaten magics but quite often see half eaten normal mushrooms. also humans have not been around long enough to have had anything to do with the evolution of magic mushrooms. it is highly likely that all magic mushrooms evolved from the same ancestor. the odds of each species evolving separately are so amazingly unlikely. magic mushrooms are present on many continents which proves that the first magic species originated before the continents divided perhaps in gondwana land. that was hundreds of millions of years ago. the first even remotely human like species arose no where near that long ago. hence humans liking mushrooms can not possibly have anything at all to do with their evolution. its just not possible.

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Re: psilocybin/psilocin biosynthesis [Re: leftover_crack]
    #6324053 -

Lets get a project together.

1) Sequence the genome of P. Cubensis.
2) Determine the genes that code for all enzymes responsible for the biosynthesis of psilocin from tryptophan.
3) Splice these genes into a vector.
4) Transoform a bacteria with the vector and an antibiotic selection marker.
5) Culture the bacteria on a tryptophan medium!

Science fiction, or in the near future?

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Re: psilocybin/psilocin biosynthesis [Re: kmIL]
    #6324090 -

The biosynthesis of tryptophan to psilocin by P. Cubensis very likely is the result of evolution. However, the idea that P. Cubensis evolved this trait because humans enjoy to eat them to achieve inebriation is counterintuitive to the theory of evolution.

While one may argue that the mushrooms want to be consumed by ambulatory mammals to spread their spores, there is a more logical explanation.

I believe that psilocin is a toxin that is supposed to discourage animals from eating P. Cubesnsis. I'm 99% confident that any mammal other than humans would not enjoy the inebriation caused by Psilocin. When an animal eats something like P. Cubsensis and starts to trip out 20 minutes later, they are able to associate the poisoning with the food they have eaten, causing them to remember not to eat that particular item. This is the most likely evolutionary factor that caused these mushrooms to produce this chemical.

Using psilocin to encourage animals to consume the fruits to spread their spores isn't the most effective way by which psilocin increases a fungus' "fitness". "Fitness" is an ecological term that refers to an organisms ability to reproduce. Evolution revolves around random mutations that result in traits that increase an organisms fitness; traits which are then seen in higher frequency due to the increased reproduction of this particular organism.

Psilocin, then, is likely a defense mechanism to deter animals from eating the fruits so that they can mature and produce spores.

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Re: psilocybin/psilocin biosynthesis [Re: kmIL]
    #6325452 -

I'd go with yeast. easy to grow in large amounts. think about home brew kits. thats what they grow. also it is a eukaryote which means it will perform post-translation modifications such as proper protein folding and phosphorylation. in theory it should work fine. i don't even think you need to sequence the genome. knowing the sequence doesn't get you the coding sequence in DNA form. isolating the cDNA would be the best (or at least a good) way. you would need to use a lot of anti-biotic markers because the enzyme genes would be to big to put all in one vector hence multiple transformations would need to be done.

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Re: psilocybin/psilocin biosynthesis [Re: gray1]
    #6364768 -

I have searched for information on the biosynthetic pathway involved in psilc(yb)in production but have never been able to find any. I'm a little surprised, because the biosynthetic pathway for a load of ergot alkaloids is known in great detail.

The best I can find are partial sequences. I got the Genbank numbers from:

Nugent, K G. and Saville, B J. Forensic analysis of hallucinogenic fungi: a DNA-based approach. Forensic Science International. 140, pp. 147 - 157. (2004)

The relevant GenBank accession numbers for P. Cubensis are:

AY129351, AY129376, AY129342, AY129377.

You can access GenBank through PubMed.

These are relatively small sequences, for example, AY129351 is 274 bp of a ribosomal RNA sequence. GenBank seems to have 7 sequences for P. Cubensis available. Related databases do not seem to have any protein sequences or structures available.

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Re: psilocybin/psilocin biosynthesis [Re: Chemiker]
    #6364786 -

These may be articles of interest:

Aqurell, S. et al. Biosynthesis of psilocybin in submerged culture of Psilocybe cubensis. 1. Incorporation of labelled tryptophan and tryptamine. Acta Chemica Scandinavica. 3, pp. 37 - 44. (1966)

Aqurell, S. and Nilsson, J L. Biosynthesis of psilocybin. II. Incorporation of labelled tryptamine derivatives. Acta Chemica Scandinavica. 22, pp. 1210 - 1218. (1968)

Aqurell, S. and Nilsson, J L. A biosynthetic sequence from tryptophan to psilocybin. Tetrahedron Letters. 9, pp. 1063 - 1064. (1968)

There is no abstract for the last one available on Pubmed, but I wouldn't get too excited about it. It's very short, so it's probably a proposed mechanism based on the labelling studies and not a fully mapped out pathway.

I'll dig them up at the library the next time I get a chance. Some of the articles have been cited by other articles, so with a little more digging, I'm sure I could find something interesting. Give me about a week and I'll see what I can find.

Let me know if anyone finds either a peptide sequence or (even better) crystal structure of any of the enzymes involved in psiloc(yb)in biosynthesis.

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Re: psilocybin/psilocin biosynthesis [Re: Chemiker]
    #6364992 -

I've read the above papers. They all propose a tryptophan -> tryptamine -> N-methyltryptamine -> N,N-dimethyltryptamine -> psilocin -> psilocybin mechanism.

An interesting thing I would mention is that 2 of the papers reference another study by the same authors that was waiting to be published. As far as I can tell it never was. I dug for quite some time and couldn't find any sign of it. I assume that it was just too repetitive of their earlier work to get published.


I've discussed the published sequences and the phylogeny constructed from them in past posts, you might be interested to search those out.

The only published sequences are ITS1, ITS2, and LSU regions. It will be a long time before anyone sequences anything else. No sequences = no protein sequences. Sequencing isn't really a useful tool for doing much anyway.

You don't find (specific) genes by sequencing genomes. It would be nice to have a psychedelic yeast that made a nice psychedelic brew though. It wouldn't be all that hard to do. Extract and chop up the PC DNA, slap it into YACs (yeast artificial chromosomes), transform some yeast, then screen perhaps 10,000 yeast colonies.

If you could figure out a nifty trick or two it wouldn't be all that tough.


-FF

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Re: psilocybin/psilocin biosynthesis [Re: fastfred]
    #6364995 -

Here are a couple posts on the published sequences and the phylogeny derived from them. I think I've posted a few other times on the topic, but you'd have to do a little searching to find them.

http://www.shroomery.org/forums/showflat.php/Cat/0/Number/5748260

http://www.shroomery.org/forums/showflat.php?Cat=0&Board=Forum4&Number=5707531


-FF

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Re: psilocybin/psilocin biosynthesis [Re: fastfred]
    #6365699 -

Yeah, I knew that, what I mean is that I'd be interested in the amino acid sequence of a protein involved in psiloc(yb)in biosynthesis, not the nucleotide sequence. The amino acid sequence can be derived from the nucleotide sequence, provided we know where the gene lies, but what I'm particularly interested in are the structures of any enzymes involved. At least with an amino acid sequence, I could search for overlapping sequences and we could learn something about the active site, possibly.

I suppose the genetic stuff would be interested. I'm sure that all the mycologists out there would love to know how psiloc(yb)in production was regulated; feedback mechanisms; etc.


fastfred said:
You don't find (specific) genes by sequencing genomes.


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Re: psilocybin/psilocin biosynthesis [Re: Chemiker]
    #6621648 -

knowing the nucleotide sequence would indeed be helpful because then primers could be designed to amplify the specific genes by PCR which could then be put into an appropriate vector and then into yeast.

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Re: psilocybin/psilocin biosynthesis [Re: leftover_crack]
    #6622989 -

You're still putting the cart before the horse there.

If you sequence the entire genome all you have is a bunch of characters stored in a database. It will give you no clue as to what genes are important in the biosynthesis.

You need to clone the gene first, then you sequence it. There are also ways to manipulate genes that don't involve sequencing them.

If you are doing random inserts and then screening for the gene product or some sort of activity then you don't really need to sequence the gene once you find it because you'll already know what primers to use based on what sort of vector you used to clone it in the first place.


-FF

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Re: psilocybin/psilocin biosynthesis [Re: fastfred]
    #6690544 -

yeah i agree. sequencing the genome would be a waste of time. but knowing the sequence of the enzymes involved would indeed be useful. the main issue is isolating the correct genes. i have a few ideas about that but can't be bothered going into that at the moment

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Re: psilocybin/psilocin biosynthesis [Re: gray1]
    #7017917 -

I'm new to the shroomery. I hope that it's not bad etiquette to revive a dead thread. I reasoned that it would be better to add to the existing thread on this topic than to create a redundant new thread.

I've read through the discussion, and I agree that yeast is the preferred system for transgenic expression. I imagine that one could generate transgenic yeast to carry out only the last few steps of the biosynthetic pathway in tryptophan-supplemented medium.

This would require cloning the genes for an unknown number of enzymes in Psilocybe spp. Can anyone propose a feasible cloning strategy? A heroic (read: practically impossible) strategy would be a standard forward genetic screen:

(1) mutagenesis by random insertion of tagged DNA
(2) selection for mutants that fail to produce psilocybin
(3) probe for the inserted tag and sequence the locus of mutation

Difficulties:
(1) Is it possible to get Psilocybe spp. to produce psilocybin in test-tube culture?
(2) Is it possible to make the cells competent for insertion of exogenous DNA?
(3) Is there a fast assay for the absence of psilocybin?

Another possibility would be to chop up the Psilocybe genome into a cDNA library, express them in bacteria, and hope that one of the lines produces psilocybin. But this is unlikely to work, because there are probably multiple enzymes required for successful biosynthesis of psilocybin.

If anyone has expertise, I would love to hear about it.

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Re: psilocybin/psilocin biosynthesis [Re: chairmanK]
    #7018111 -

> Can anyone propose a feasible cloning strategy?

The screen you proposed should work fine. You could also make and screen a cDNA library.

> Is it possible to get Psilocybe spp. to produce psilocybin in test-tube culture?

Not really. Psilocybin isn't really produced until the hyphal knot stage. That takes a lot longer in a test tube than on any sort of solid media. You would be better off using plates. You'd want to plate it out frequently anyway.

> Is it possible to make the cells competent for insertion of exogenous DNA?

Yes. You first need to prepare protoplasts, then probably electroporate them. You take an osmoticaly balanced solution, add chitinase, add whatever DNA you want to transform them with, electroporate the solution, then regenerate the protoplasts.

An alternative would be to use agrobacterium to transfect the cells. A lot of people don't know that, but you can use agrobacterium on fungi.

> Is there a fast assay for the absence of psilocybin?

You could check for a lack of bluing, but that might lead you astray. Most reagents like Ehrlich's will react with most indoles, so that probably wouldn't help too much as it would probably react with tryptamine on up.

You'd probably have to use TLC. That would probably be the cheapest and easiest method.


-FF

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Re: psilocybin/psilocin biosynthesis [Re: fastfred]
    #7019363 -

Has anyone published a successful protocol for transforming Psilocybe and obtaining viable colonies? I doubt that the same voodoo tricks that are used to transform Saccharomyces will be effective on Psilocybe. (Nature is a cruel mistress; She rarely cooperates with our investigations.) Tweaking the process to achieve high efficiency could take a year of work.

TLC is cheap and effective, and nowadays one can automate it for high-throughput screening; however, preparing the samples is annoying when your organism grows on plates. This is why I asked whether Psilocybe fungi can be coaxed to produce psilocybin in liquid suspension culture; it's much less tedious to pipette conditioned liquid media from a rack of test tubes (hurrah for fluid-handling robotics!) than to scrape spots from plates.

As an alternative approach, I wonder whether an immunoblot assay would work. There are good monoclonal antibodies against serotonin and LSD, and I found a paper (in a mediocre Japanese journal) in which the authors claim to have raised an antibody against psilocin. Assume that it is possible to construct a selective monoclonal antibody that is fused to a reporter enzyme. Then one could blot the whole plate on a membrane, incubate with antibody, workup with reporter substrate, and look for spots which lack signal.

Over how much space does a hyphae colony need to grow out in order to start producing psilocybin? This constrains the minimum grid spacing between spots on the plate, which sets the per-plate throughput of the immunoblot assay.

Edited by chairmanK (06/07/07 01:40 PM)

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Re: psilocybin/psilocin biosynthesis [Re: chairmanK]
    #7019488 -

> Has anyone published a successful protocol for transforming Psilocybe and obtaining viable colonies?

Not that I'm aware of. It's been awhile since I searched the literature.

> I doubt that the same voodoo tricks that are used to transform Saccharomyces will be effective on Psilocybe.

Probably not. Competent yeast is usually used. For multicellular fungi you probably need to prepare protoplasts or use agrobacterium to get your DNA in.

There is a decent amount of literature on transforming fungi though. You should be able to get mediocre efficiency right off the bat. A little tweaking and you should be good to go.

> preparing the samples is annoying when your organism grows on plates.

No doubt. I would try to find a way to assay right on the plate. One method might be to rupture some cells on the plate and look for an absence of bluing. If you have a colony picker you could probably automate the process and have your picker picking white spots. I mention that because you just might have one if you've got a liquid handling robot.

> As an alternative approach, I wonder whether an immunoblot assay would work.

Sounds like a good idea, but probably a lot of work. One thing I do remember is a post here where somebody created a spray that would cause active species to fluoresce. You might try looking that up.

EDIT: Ah! Ha! Found it.
http://www.shroomery.org/forums/showflat.php/Number/6607853

Looks like that might be the ticket for your assay. Be sure to post if you dig into it.

> Over how much space does a hyphae colony need to grow out in order to start producing psilocybin?

That's not easy to say. It depends on a number of factors. RR could probably give you a good idea. It depends on the myc reaching the hyphal knot stage. Reaching that stage is accelerated by nutrient constraints. Once the space runs out and/or the nutrients are depleted the myc begins to enter it's fruiting stage.

If you just allowed it to run it would take quite awhile to start producing actives.

I would look through the literature on transforming and screening fungi. I bet there are some innovative methods out there. Good luck and be sure to let us know if you find anything interesting.


-FF

Edited by fastfred (06/07/07 02:15 PM)

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Re: psilocybin/psilocin biosynthesis [Re: fastfred]
    #7019704 -

Very nice. Thanks for the tip.

Click here for the abstract. Unfortunately, this isn't an open-access journal, so I will not post the full PDF. (I have little regard for Blackwell-Synergy's copyright, but I do respect the rules of the Shroomery, which prohibit me from posting copyrighted materials. Message me if you want details from the paper.)

EDIT: This method is not promising. It's useful for detecting low concentrations of psilocin and psilocybin once these compounds are already resolved by chromatography. But starting with an unresolved mix of indoleamines, this assay will not reveal whether psilocyin is specifically missing or present in this mix.

Edited by chairmanK (06/07/07 03:36 PM)

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Re: psilocybin/psilocin biosynthesis [Re: chairmanK]
    #7025269 -

> ...once these compounds are already resolved by chromatography.

Bummer. The article made it sound like you could spray down mushroom patches and ID the glowing ones.

I came up with a list of possible deleloping reagents awhile back...

Metol (photographic chemical, Kodak's Elon)
Keller's reagent (glacial acetic acid, ferrous chloride, and concentrated sulfuric acid)
Ehrlich's reagent (4-dimethyl-aminobenzaldehyde in 50 ml of 90% ethanol and 50 ml of 38% hydrochloric acid)
Marquiss Test [1 volume formaldehyde solution (formalin) with 9 volumes of sulphuric acid]
pDMAB (p-dimethylaminobenzaldehyde)
Van Urk’s reagent (10% p-dimethylaminobenzaldehyde in conc. HCl)
para-dimethylaminocinnamaldehyde
potassium permanganate
pine tree shavings in HCl
One drop of 37% formaldehyde to 15 drops of concentrated sulfuric acid.
p-toluenesulfonic acid in methanol


If you figure anything out be sure to post.


-FF

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Re: psilocybin/psilocin biosynthesis [Re: fastfred]
    #7071181 -

check this paper from the American Society for Microbiology journal (2000)

"A Fruiting Body Tissue Method for Efficient Agrobacterium-Mediated Transformation of Agaricus bisporus"

it should work on psilocybe's

its free on pubmed i think

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Re: psilocybin/psilocin biosynthesis [Re: leftover_crack]
    #7075802 -

http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=92332
A Fruiting Body Tissue Method for Efficient Agrobacterium-Mediated Transformation of Agaricus bisporus
Xi Chen, Michelle Stone, Carl Schlagnhaufer, and C. Peter Romaine
Appl Environ Microbiol. 2000 October; 66(10): 4510–4513.

We describe a modified Agrobacterium-mediated method for the efficient transformation of Agaricus bisporus. Salient features of this procedure include cocultivation of Agrobacterium and fruiting body gill tissue and use of a vector with a homologous promoter. This method offers new prospects for the genetic manipulation of this commercially important mushroom species.

We have devised a highly efficient, convenient, and expeditious genetic transformation system for the button mushroom Agaricus bisporus.
[...]
The unavailability of a practical gene transfer system is the single largest obstacle precluding the use of molecular approaches for the genetic improvement of mushrooms. Despite considerable interest in the development of a transformation scheme (3, 12, 14, 17, 18), no method is in general use today, owing to low efficiency or lack of utility and convenience.
[...]
The agro-transformation method described in this paper offers a practical means for exploiting transgenic approaches for the genetic manipulation and improvement of mushrooms.

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Re: psilocybin/psilocin biosynthesis [Re: kmIL]
    #7305903 -

It sounds like everyone has a good idea whats necessary, but very few people have any idea how to do that.
Lets look at the facts: you want to put the genes from an entire biosynthetic pathway into another organism, and expecting that it will produce abundant amounts what you want. Yeast isn't even in the same kingdom!
we don't have an assay for psilocin (I have no idea whats involved, I'm not a biochemist)
We don't have purified psilocin, so even if we had an assay, we wouldn't know if it worked.
We don't have the sequence (it would take years)
We don't know the genes involved (it would take years, maybe decades after that)
If you tried to skip ahead, and just shotgun chunks of DNA into yeast, you have no idea whats going to happen. The chances of getting something are laughable. Molecular genetics is not a random art. It works when you know whats going on, you know the sequence and the changes you are making and you constantly constantly constantly verify it.

The only thing that even has a chance is if someone came up with an excellent assay for psilocin, an enormous feat in itself, something that could be done on a plate, in high throughput to thousands of mutants and we tried to grow up yeast with fragments of the genome from a cDNA on different media's containing the intermediate tryp iso's. If you find one enzyme that makes the one final change which allows you to go from N,N-whatever in the media to psilocin, then you will have just assured yourself fame for life (at least you'll be MY hero!). But like I said: no idea where the genes are, no idea how many are involved... could be unique post translational modification, could be hundreds of multifunction enzymes working in tandum, for all I know it could be the will of God. And believe me, I play God on a day to day basis, so an admission like this from a molecular geneticist who works in the field should be evidence that you are going about this the wrong way.

Try something easier, like growing bigger mushrooms faster. Thats your best shot at getting more bang for your buck.

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Re: psilocybin/psilocin biosynthesis [Re: gray1]
    #7327450 -

> Yeast isn't even in the same kingdom!

Ummm... You might want to go back to Micro 101 and bone up on your phylogeny. Yeast are unicellular fungi in the kingdom fungi, the same kingdom as all mushrooms.

> we don't have an assay for psilocin

You can run a HPLC for it, you could observe the natural bluing reaction, use TLC, or use a colorimetric reagent such as pDMAB.

> We don't have purified psilocin, so even if we had an assay, we wouldn't know if it worked.

It would be easy enough to produce some purified psilocin using an AB extraction. You could further purify it on a column if desired. You really don't need purified psilocin anyways. If you really want a positive control then just use an active mushroom.

> We don't have the sequence (it would take years)

Not needed. As I pointed out earlier, it really wouldn't be much help anyway. Using forward genetics and other techniques you only have to sequence small amounts that are well within the range and budget of even small labs.

Even if you wanted the sequence it wouldn't take "years" just a good chunk of money. Genomes of this size are sequenced in weeks or months, not years, nowdays.

> We don't know the genes involved (it would take years, maybe decades after that)

It wouldn't be much of a task if everything was already known. You seem to overlook the fact that this sort of stuff is done all the time and it's often the case that little is known at the start.

There is only 4-5 steps involved and the first is already well known. In three unknown steps you're at psilocin. Finding three genes and cloning them really isn't that hard. It's not some complex mechanism, you're only locating and cloning three genes that are involved in producing a single product. It's not like you're trying to cure a complicated disease or anything.

> If you tried to skip ahead, and just shotgun chunks of DNA into yeast, you have no idea whats going to happen.

Sure you do. You're either going to start producing psilocin or you're not. One method, one screen. It seems like a good way to do an easy brute force attack at the problem.

> The chances of getting something are laughable.

That's why you spend a little time getting your transformation efficiency as good as you can, then you start transforming large amounts of yeast. Being unicellular you would obviously be transforming millions or billions of yeast per attempt. Do it right and your chances are far from laughable.

> Molecular genetics is not a random art.

I would not agree with you there. There is plenty of chance involved and you have little direct control. In fact almost everything about molecular biology is pretty random. The basis of almost every technique is transforming large numbers and then screening them. There isn't really any "art" to transforming organisms. You take a culture tube, insert DNA, and you get your low transformation efficiency. 1E-8 is not uncommon with fungi.

As far as using cDNA, I say Bahhhh. People are too into the whole cDNA thing nowdays. You need larger chunks. Otherwise you'd need to build a cDNA library, and that's pretty expensive.

I think bigger chunks with YAC's would be a good way to go. Otherwise I don't see why it would be all that difficult using forward genetics.

I don't really see it as being that difficult of a task, just one requiring some money.

Also, considering the fact that both PCR and the structure of DNA were discovered as a result of psychedelic usage, it's quite reasonable to hope for some divine intervention from the mushroom gods.


-FF

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Re: psilocybin/psilocin biosynthesis [Re: fastfred]
    #20899509 -

So yeast would be preferred to bacteria?

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