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Offlinegray1
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psilocybin/psilocin biosynthesis
    #323203 - 05/22/01 12:04 PM (15 years, 6 months ago)

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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Offlinegray1
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Re: psilocybin/psilocin biosynthesis [Re: gray1]
    #323209 - 05/22/01 12:07 PM (15 years, 6 months ago)

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

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Offlinegray1
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Re: psilocybin/psilocin biosynthesis [Re: gray1]
    #323211 - 05/22/01 12:13 PM (15 years, 6 months ago)

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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OfflineaNaPhylaktik
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Re: psilocybin/psilocin biosynthesis [Re: gray1]
    #323813 - 05/23/01 12:41 AM (15 years, 6 months ago)

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.

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OfflineaNaPhylaktik
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Re: psilocybin/psilocin biosynthesis [Re: aNaPhylaktik]
    #324651 - 05/23/01 11:33 PM (15 years, 6 months ago)

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

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Offlineegghead
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Re: psilocybin/psilocin biosynthesis [Re: gray1]
    #324660 - 05/23/01 11:45 PM (15 years, 6 months ago)

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.

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Offlinegray1
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Re: psilocybin/psilocin biosynthesis [Re: egghead]
    #324864 - 05/24/01 08:38 AM (15 years, 6 months ago)

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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Offlineegghead
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Re: psilocybin/psilocin biosynthesis [Re: gray1]
    #325007 - 05/24/01 01:10 PM (15 years, 6 months ago)

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?

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Offlinegray1
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Re: psilocybin/psilocin biosynthesis [Re: egghead]
    #325078 - 05/24/01 02:55 PM (15 years, 6 months ago)

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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OfflineaNaPhylaktik
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Re: psilocybin/psilocin biosynthesis [Re: gray1]
    #325814 - 05/25/01 04:29 AM (15 years, 6 months ago)

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...

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InvisibleCow Shit Collector
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Re: psilocybin/psilocin biosynthesis [Re: aNaPhylaktik]
    #326017 - 05/26/01 04:15 AM (15 years, 6 months ago)

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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Offlinemadscientist
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Re: psilocybin/psilocin biosynthesis [Re: Cow Shit Collector]
    #326170 - 05/26/01 11:34 AM (15 years, 6 months ago)

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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OfflineOpi
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Re: psilocybin/psilocin biosynthesis [Re: gray1]
    #327410 - 05/27/01 11:09 PM (15 years, 6 months ago)

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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OfflineaNaPhylaktik
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Re: psilocybin/psilocin biosynthesis [Re: madscientist]
    #327486 - 05/28/01 01:19 AM (15 years, 6 months ago)

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.


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OfflineOpi
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Re: psilocybin/psilocin biosynthesis [Re: aNaPhylaktik]
    #327833 - 05/28/01 02:42 PM (15 years, 6 months ago)

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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Offlinegray1
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Re: psilocybin/psilocin biosynthesis [Re: Opi]
    #328599 - 05/29/01 09:43 AM (15 years, 6 months ago)

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

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Offlinemadscientist
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Re: psilocybin/psilocin biosynthesis [Re: gray1]
    #328608 - 05/29/01 10:00 AM (15 years, 6 months ago)

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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OfflineaNaPhylaktik
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Re: psilocybin/psilocin biosynthesis [Re: madscientist]
    #330232 - 05/31/01 01:11 AM (15 years, 6 months ago)

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.


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www.shaman-australis.com.au/seedring


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Offlinegray1
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Re: psilocybin/psilocin biosynthesis [Re: aNaPhylaktik]
    #330465 - 05/31/01 09:03 AM (15 years, 6 months ago)

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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Offlinemadscientist
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Re: psilocybin/psilocin biosynthesis [Re: gray1]
    #332200 - 06/02/01 07:52 AM (15 years, 6 months ago)

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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