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 ExplosiveMango
HallucinogenusDigitallus
 
Registered: 07/12/05
Posts: 3,222
Last seen: 2 years, 6 months
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 Norbaeocystin -> Baeocystin -> Psilocybin? 
#6175335 - 10/16/06 01:30 PM (5 years, 7 months ago) |
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I have never heard that this process occurs in a mushroom.
I have never heard that this process does not occur in a mushroom.
What prevents the addition of the methyl groups to the molecule after the 4 position has been phosphorylated?
Maybe this could be why we see such tiny levels of DMT in a mushroom?
(It would not rule out the DMT->Psilocin->Psilocybin either)
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Know your self.
Know your substance.
Know your source.
The most distorted perspective possible is the perspective that yours is not distorted.
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Viper
half man, half amazing
Registered: 10/09/06
Posts: 36
Loc: Serbia
Last seen: 9 months, 3 days
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Re: Norbaeocystin -> Baeocystin -> Psilocybin? [Re: ExplosiveMango]
#6175377 - 10/16/06 01:42 PM (5 years, 7 months ago) |
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When the indole ring is phosphorylated, the nitrogen atom is positively charged. This is because one of the acid protons attacks the free electron couple on nitrogen, and thus the positive methyl radical can not react with the nitrogen.
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I've come to restore what they stole from you, cure the cold in you, reverse what was told to you!
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 fastfred
Old Hand
Registered: 05/17/04
Posts: 6,242
Loc: Dark side of the moon
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Re: Norbaeocystin -> Baeocystin -> Psilocybin? [Re: ExplosiveMango]
#6175435 - 10/16/06 02:02 PM (5 years, 7 months ago) |
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> What prevents the addition of the methyl groups to the molecule after the 4 position has been phosphorylated?
Phosphorylation changes the charge and pH of the molecule. In my pic above I have baeocystin included, but it probably doesn't work that way. Also, if it did, it would go straight to psilocybin rather than through psilocin.
IMHO norbaeocystin is probably the dead end product that happens when tryptamine gets 4-hydroxylated. Because then it gets phosphorylated it probably can't be n-methylated. I think baeocystin happens when N-methyltryptamine is 4-hydroxylated, rather than norbaeocysitn --> baeocystin via n-methylation. So I think they are both just small amounts of dead end products that happen when 4-hydroxylase reacts with the wrong molecule.
> It would not rule out the DMT->Psilocin->Psilocybin either
That is exactly what is believed to happen. My diagram is pretty much correct with the exception of baeocystin being shown to be phosphorylated to psilocin, and of course the phosphorylase arrow to baeocystin. It would have to be hydroxylated first.
-FF
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ExplosiveMango
HallucinogenusDigitallus
Registered: 07/12/05
Posts: 3,222
Last seen: 2 years, 6 months
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Re: Norbaeocystin -> Baeocystin -> Psilocybin? [Re: fastfred]
#6175467 - 10/16/06 02:17 PM (5 years, 7 months ago) |
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Quote:
That is exactly what is believed to happen. My diagram is pretty much correct with the exception of baeocystin being shown to be phosphorylated to psilocin, and of course the phosphorylase arrow to baeocystin. It would have to be hydroxylated first.
Yeah, this is the process I am familiar with, with the baeocystin's being the guys who 'got lost along the way'. (phosphorylated early)
I just find it interesting that there is such a small amount of DMT in mushrooms. The phosphorylation must simply be a much more aggressive process than the methylation (is methylation a correct term?).
Upon realizing the molecular charge effect of the phosphorylation, it is quite understandable that the methyl group will not bond.
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Know your self.
Know your substance.
Know your source.
The most distorted perspective possible is the perspective that yours is not distorted.
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fastfred
Old Hand
Registered: 05/17/04
Posts: 6,242
Loc: Dark side of the moon
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Re: Norbaeocystin -> Baeocystin -> Psilocybin? [Re: ExplosiveMango]
#6177367 - 10/17/06 01:41 AM (5 years, 7 months ago) |
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Evidence indicates that it is the tryptophan decarboxylation is the limiting step. There is a decent and variable psilocybin/psilocin ratio, so it would seem that the phosphorylation step isn't all that fast. It's the 4-hydroxylation speed that would lead to small amounts of DMT. The 4-hydroxylation step is fairly fast and somewhat non-specific. That's why you get (nor)baeocystin.
Alexander Shulgin believed that many different indoles would be 4-hydroxylated if present in the mycellium. I'm not sure if that's been proven, but it's been talked about a lot.
The precursor to the limiting step in the pathway is going to be the one present in decent quantities. The rest will be in rather small quantities. In this case it's tryptamine that is the limiting factor and it's precursor is tryptophan, which is highly regulated in all biological systems. That keeps everything in check.
If you could find a way to increase the tryptophan decarboxylase it would probably lead to some amazing results.
-FF
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Feelers
Anti-Myth-Rhythm-Rock-Shocker
Registered: 06/18/02
Posts: 1,691
Loc: Land of Oz
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Re: Norbaeocystin -> Baeocystin -> Psilocybin? [Re: fastfred]
#6177383 - 10/17/06 02:07 AM (5 years, 7 months ago) |
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What promoters do they use for GE'ing fungi? Can you use the Cauliflower mosaic virus or is that only a plant thing?
How hard would it be to work out the sequence for the enzymes involved in these processes? What is involved in this ?
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fastfred
Old Hand
Registered: 05/17/04
Posts: 6,242
Loc: Dark side of the moon
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Re: Norbaeocystin -> Baeocystin -> Psilocybin? [Re: Feelers]
#6178782 - 10/17/06 12:56 PM (5 years, 7 months ago) |
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> What promoters do they use for GE'ing fungi?
In the experiments I've read they haven't gotten that advanced. Well, actually I remember an experiment where they created a button mushroom that was resistant to some fungicide. They used a standard plasmid for that I believe. So they may have chosen the promoter.
I've see more instances where they use other techniques. I was interested to learn that they use agrobacterium tumifaciens (sp?) to insert foreign DNA into fungi. In that process they cut up some DNA and make a clone library which they screen for the gene of interest. Then they insert that into the agrobacterium and use it to insert the DNA into fungi.
So they don't usually choose the promoter unless they are using a standard, well known gene. For most instances they are just using whatever promoter is upstream of the gene in the source organism's DNA.
That usually works alright since you already know it works and sometimes it will even provide a useful regulatory mechanism for the gene. If you just throw a hot promoter in front of the gene it often fails because the gene will be overexpressed and just kill the organism or make it waste too much time making the gene product. OTOH it's very useful to use an inducible promoter, that way you can grow up the organism and then induce gene expression at the levels you want.
> Can you use the Cauliflower mosaic virus or is that only a plant thing?
Viri are usually pretty specific to their hosts. They are much harder to work with and they can only carry a very small amount of DNA.
> How hard would it be to work out the sequence for the enzymes involved in these processes?
Not really all that hard, but it would take a good chunk of cash and a decent lab.
> What is involved in this?
First you would cut up the organism's DNA and make a clone library from it. You could also create a cDNA library and possibly save some time and effort, but it's more complicated.
Then you would either screen the clone library directly or insert it into a host and screen that. When you've found the genes required you would have to insert them into the host organism. There are several methods to transform fungi. You can use protoplasts transformation, electroporation, agrobacterium, etc. Or you could sequence the inserts of the clones of interest. But once you have the clones you already have the tools to do your GEing, so I don't see why you'd be overly concerned with getting the sequence.
So for PC actives you'd want tryptophan decarboxylase, 4-hydroxylase, N-methylase, and phosphorylase.
Tryptophan decarboxzylase has already been engineered into plants using agrobacterium, so that would be easy. And both TDC and phosphorylases are present in quite a few organisms, so you might not need to even worry about them. 4-hydroxylase and N-methylase would be the harder ones. I would guess 4-hydroxylase is pretty specific to active species.
The problem with N-methylase is that it's not been conclusively proven that this is the biosynthetic route, even though everything points to it and it is a highly likely route. It's hard to say much about it since not much is known. It's not known if there are one or two different N-methylases (for the first and second N-methylations).
So that's the story in my opinion. The first and last steps would be pretty easy, but the two middle ones would require developing a screen for and finding them in the clone library that you would have to make to do so. Genetics doesn't work like chemistry. You have to deal with low transformation efficiency, low rates of expression, and a huge volume of DNA to screen. Combine that with the fact that bacteria can only hold a small amount of DNA and our eukaryotic fungi have a comparatively huge amount of DNA and you see that it is a good sized task.
Of course you can break it down into smaller bits, and if you're smart about it you could profit humanity at each step.
How you ask? Well, TDC is decently studied and it luckily seems to be the limiting step in production. If you're lucky you might be able to get some of the agrobacterium from one of those researchers and all you'd have to do is use it to infect some myc and then somehow screen the myc for tryptamine levels (or actives). At that point you would have produced a more potent mushie.
So then another step would become the limiting factor and you could find that gene and insert a few extra copies and then you would again increase potency. Or you could insert some phosphorylase genes to alter the ratio in favor of psilocybin vs. psilocin.
The TDC thing would be so simple if you could get that already transformed agrobacterium. Perhaps they filed a voucher sample, or would be willing to supply some to an interested researcher. If I had the time and still worked with actives I would be so on top of that!
-FF
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anonjon
Partially Right
Registered: 11/03/08
Posts: 6,322
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Re: Norbaeocystin -> Baeocystin -> Psilocybin? [Re: fastfred]
#10744065 - 07/26/09 03:26 PM (2 years, 9 months ago) |
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I know this is an old thread, but it's still a fascinating conversation.
If one were able to acquire the bacteria that produce TDC, one wouldn't need to get those genes into the fungus. (a task for real genetic engineers)
One could simply breed a strain of the bacteria that survives pasteurization temps and colonize bulk sub with it. Yes?
One could even stick the tdc expressing bacteria in a blender with thermophilic yeast. I understand that bacteria frequently exchange genes spontaneously.
I know RR hates this topic, but TDC has yet to be ruled out as a potency enhancing substrate additive, unlike tryptophan/tryptamine. Fast Fred's post's on the subject deserve reading.
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