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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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