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Re: Increasing alkaloid levels [Re: iudexk]
    #377763 -

i have found a nice way to increase the alkaliod levels. it is really a quite simple procedure and here it is. just eat more!!!! shrooms are pretty fucking abundant if you are growing them yourself which im guessing that you are so just eat more, if you want to trip harder. i dont see how soaking verm in dmt will increase the potency of youre shrooms, just waste dmt and time and money. and as im told time and money are two of the most important things to worry about these days, so you should be careful what you do with them. increasing the alkaliod level would be like telling you to grow grow a different hair color or something. genetics are already decided for the shroom and it would be hard, or even impossible to change it. i dont know much about the science of shrooms metabolism and the way they these fruitbodies just pop out of a "ball" of fungus either. it blows my mind.

one plus one plus one equals three


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the little kridders of nature; they dont know that thyre ugly!


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Re: Increasing alkaloid levels [Re: aluminum_can]
    #378456 -

increasing the alkaliod level would be like telling you to grow grow a different hair color or something. genetics are already decided for the shroom and it would be hard, or even impossible to change it.

It is well known that alkaloid levels in various plants can be manipulated. mescaline etc.

http://www.magickherbs.co.uk/


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http://www.magickherbs.co.uk/

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Re: Increasing alkaloid levels [Re: iudexk]
    #379772 -

true dat.


"i am a citizen of the world" diogenes

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Re: Increasing alkaloid levels [Re: iudexk] * 2
    #26312685 -

Adding oat bran as a supplement to the substrate, which is high in phosphorous, may serve to increase the psilocybin production (4-phosphoryloxy-dimethy,tryptamine) which is stable at room temperature and assures more lasting potency when the mushrooms are dried.  I know of no studies that confirm this hypothesis but anecdotally it seems to result in very potent Ps. mexicana mushrooms, which, per Stamets psilometric scale, is high in psilocybin and low in psilocin and has no baeocystin.  The Ps. mexicana also do not bruise blue, which is seen in mushrooms, such as Ps. cubensis, which has a higher psilocin content.


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γνῶθι σεαυτόν
gnōthi seauton
know thyself

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Re: Increasing alkaloid levels [Re: elfstone]
    #26342590 -

Ah, I love the bringing back of old posts. So, 18 years later has anything changed? Eflstone, you mentioned oat bran... I see you mentioned no studies but has this been tested by another grower?

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Re: Increasing alkaloid levels [Re: Schroomfairy] * 1
    #26345336 -

A lot has changed, and we now know that there’s nothing you can add to your substrate that will increase or decrease potency.

Cubes use ATP or Adenosine TriPhosphate as energy storage, while they may indeed like more phosphate, an increase in phosphate isn’t going to increase psilocybin production as phosphate isn’t the limiting factor.  Nor is tryptophan a limiting factor either.

We also know that Norbaeocystin and Baeocystin are intermediary steps in the organic synthesis of psilocybin and thus all species that use this pathway contain trace amounts.  Psilocin is a bit different as it it’s not in the synthesis but from exposure of psilocybin to PsiK a dephosphorelating enzyme.

Well so if none of that works, what does? 
Genetics.
The main limiting factor is the efficiency, speed and quantity of the enzymes that build psilocybin and its genetics that codes for the proteins that build the enzymes that builds the magic.  It’s that set of enzymes that takes common building blocks and puts them together in the right ways and those enzymes that E.coli formed when the psilocybin producing genetics was shot into it.

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Re: Increasing alkaloid levels [Re: StygianKnight]
    #26346885 -

StygianKnight said:
A lot has changed, and we now know that there’s nothing you can add to your substrate that will increase or decrease potency.

Cubes use ATP or Adenosine TriPhosphate as energy storage, while they may indeed like more phosphate, an increase in phosphate isn’t going to increase psilocybin production as phosphate isn’t the limiting factor.  Nor is tryptophan a limiting factor either.

We also know that Norbaeocystin and Baeocystin are intermediary steps in the organic synthesis of psilocybin and thus all species that use this pathway contain trace amounts.  Psilocin is a bit different as it it’s not in the synthesis but from exposure of psilocybin to PsiK a dephosphorelating enzyme.

Well so if none of that works, what does? 
Genetics.
The main limiting factor is the efficiency, speed and quantity of the enzymes that build psilocybin and its genetics that codes for the proteins that build the enzymes that builds the magic.  It’s that set of enzymes that takes common building blocks and puts them together in the right ways and those enzymes that E.coli formed when the psilocybin producing genetics was shot into it.



Thank you so much for your thoughtful reply. That clarified a lot of things for me. So the next question is your final statement in regards to the genetics. Even if we take a print and put it on agar with as many transfers as needed to obtain the perfect plate, is that the way to the perfect genetics?

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Re: Increasing alkaloid levels [Re: Schroomfairy]
    #26346913 -

There are a few things to consider when it comes to increasing alkaloid levels:

1. You can increase overall alkaloid levels by increasing yield. Yield is dependent on many factors (genetics, substrate composition, temperature, moisture levels, etc.).

2. You can increase alkaloid levels per unit of biomass by genetic selection. You can also grow species that reliably produce high alkaloid content.

3. As StygianKnight suggested, psilocybin-producing fungi are chemically complex. There are molecules other than psilocybin/psilocin that likely contribute to the physical and psychological effects that you experience when you consume mushrooms. Increasing psilocybin/psilocin content may come at the expense of other tryptamines that have a positive effect on the psychedelic experience.

My point is that there are many different ways to potentially increase alkaloid levels, but it's important to understand that psilocybin/psilocin are not the only alkaloids present in psilocybin-producing fungi.

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Re: Increasing alkaloid levels [Re: Mycoactive] * 1
    #26349473 -

As Mycoactive hit on, what are we talking about when we say increasing levels? Is that overall amount of alkaloids or ratio of alkaloids to fruit mass?

Getting a clean sample will indeed help to produce a larger amount of alkaloids than a dirty sample, but that’s not quite the same here since the solution to weak genetics, is just a larger dose.

Stronger genetics as in a higher amount of alkaloid to fruit mass comes about in a number of different ways, many of which we can’t measure easily at home.  So for at home hobbyist work the suggestion is to find the best growing fruits and clone them.  Take whatever is happiest in your environment and give it more room to grow.

When it comes to straight producing more enzymes it’s not known what the triggers are, or what causes production to slow down or stop. The issue is that enzymes are finicky and require specific local conditions to work their best.  Beyond just producing more enzymes it’s also about how much care is being taken by the mycelium to give them what they need to work efficiently. This is in part the reason for the suggestion above, it’s a presumption that fruits that are performing well in your environment are in their personal best environment to keep things stable inside themselves, and the enzymes working well.

Breeding genes that produce strong fruits with other genes that produce strong fruits can potentially help boost the production content, but it’s just dice rolls.  Just look at how famous Penis Envy and related strains are in part due to their potency increase.

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Re: Increasing alkaloid levels [Re: StygianKnight]
    #26360326 -

I’ve wanted to try to reproduce Gartz’s experiment for years. Shulgin certainly thought there was something to it. Sadly at this point it reads mostly as a “wive’s tail” in terms of implementation. Further study needed.


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‎שלום וְאור | PEACE & LIGHT

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Re: Increasing alkaloid levels [Re: Psilotyl]
    #26363611 -

What experiment is that?

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Re: Increasing alkaloid levels [Re: StygianKnight]
    #26363765 -

StygianKnight said:
Nor is tryptophan a limiting factor either.



Correct me, but if no presents of tryptophan(at all) nothing to convert to Psilocybin?

Learn more about Psilocybin



I believe this this is why no one could reproduce Gartz's study. I think his dung had super low to almost no tryptophan. Where when others tried almost all the spawn/subs have some amount of tryptophan.

Yes adding tryptophan will NOT increase potency. This debates pisses off a lot of old hands. The way I look at it is, you can't force a cube to bio tryptophan that's all on genetics, but I want to make sure some is there to work with.

Please jump in to correct me if needed.

Edited by murderlabz (12/05/19 07:39 PM)

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Re: Increasing alkaloid levels [Re: Schroomfairy] * 1
    #26363830 -

Schroomfairy said:
What experiment is that?


It’s already been posted earlier in this thread. Gartz supposedly added tryptamine HCL to his substrate, and the result was each flush containing progressively, obscenely higher levels of psilocin in the fruit bodies (up to 3.3% in the third flush) compared to the normally grown mushrooms used as the “control.” Psilocybin levels were lower than in the control batch. This was in the late 80’s. To my knowledge it has never been duplicated. Here’s the original study:

https://pdfs.semanticscholar.org/e648/5b3f89815404f8e0912507aa693a68f27f80.pdf

Also related: Alexander Shulgin wrote in TiKHAL (late ‘90’s) how if one added DMT to the substrate the mushroom would simply take it and convert it into 4-HO-DMT (psilocin), yielding higher potency levels of psilocin. He also spoke of “very interesting research done in Germany” showing that if specific tryptamine are added to substrate, such as DIPT, DET, MET, etc, the mushroom will add it’s four hydroxylated (“4-HO”) signature to it and create psychedelic substances unknown in nature such as 4-HO-DIPT, 4-HO-DET, 4-HO-MET, etc, in addition to or in lieu of psilocin/psilocybin. Hamilton Morris recently spoke of this, citing pretty much word for word from Shulgin, on the Joe Rogan Experience.

I’m the fence on this one. I’ve not seen any studies verifying what Shulgin wrote, but haven’t researched it much either. My understanding is that it is at least “in part” correct. As a chemist, I can assure you that weird shit can happen in a laboratory setting for all sorts of reasons. A one off study doesn’t always mean a lot. So which part or how much is true...who knows. It’s worth doing some deep internet digging about. It’s likely there are at least some legitimate studies that have at this point been conducted, but I couldn’t say for sure. If it’s true it says a lot about what mushrooms are capable of as an active “pharmacological vessel.”

As an experiment, I once tried adding a tryptamine (melatonin) to my grains before PCing. It had no effect whatsoever to the final potency. Possible reasons are that 1) the tryptamine was “killed” off and rendered inactive during the process of sterilization 2) melatonin is incompatible here due to the fact that it is a tryptamine already chemically structured as a five hydroxylated (“5-HO”) substance, whereas tryptamine like the ones listed above are not 3) the notion of tryptamine supplementation increasing alkaloid levels is all just hocus pocus bullshit. Couldn’t say which.

It’s an interesting, weird, and largely unexplored subject. The whole “DMT in the substrate to increase alkaloid levels” idea was almost certainly something Shulgin started by writing about it in TiKHAL. It’s important to remember he was a chemist and not a grower, and it might all be more theoretical than he lets on. If it’s true, a specific set of circumstances and processes as to how exactly to supplement with DMT are probably needed - as far as to when, how much, etc. Like much of PiKHAL and TIKHAL, it’s designed to encourage experimentation.

Unlike some on this forum, I am personally absolutely certain that there are things that can influence potency aside from genetics, namely relating to nitrogen and carbon presence in certain ratios. There is actually a study from way back in 1968 that demonstrably shows this, if it is to be believed.  Whether for positive or negative, alkaloid levels of psilocin, psilocybin, baecystin, and norbaecystin were all affected. I’ll find it and post it.


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‎שלום וְאור | PEACE & LIGHT

Edited by Psilotyl (12/05/19 09:33 PM)

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Re: Increasing alkaloid levels [Re: Psilotyl] * 1
    #26363851 -

Thank you for your excellent post. I will keep it for future reference.

When watching Hamilton Morris on the magic mushroom documentary, I found it interesting how the Mexican farmer talked about sugar cane and how it increased it's potency. Go to 14:48 [url=
Could be something to think about trying? He could be on to something? Or maybe even the possibility that adding to the substrate may actually cause something different. I am willing to complete some experiments for sure.

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Re: Increasing alkaloid levels [Re: Schroomfairy] * 1
    #26363901 -

Tryptophan
Yes, if there wasn’t any tryptophan then psilocybin couldn’t be produced, but it’s just animals that can’t produce our own tryptophan, plants and microbes can.  Grain contains tryptophan and many fungi can that build tryptophan from smaller blocks.

Gartz (1989)
Can be presumed to be incorrect based on modern knowledge,
“hydroxylated and methylated fed tryptamine to give psilocin in up to 3.3% dry mass of the obtained fruit bodies. By using HPLC and TLC, it was found that these mushrooms contain only a small amount of psilocybin (0.01-0.2% dry mass)” -Gartz

That the study got mostly psilocin and no psilocybin means something went wrong in their testing or prep.  The specific pathway has been found and it starts with phosphorylation, psilocin is only produced in mushrooms as an enzymatic breakdown of psilocybin into psilocin.  Besides it seems unwanted to get mushrooms with only the easily oxidized unstable compound.


DMT / DEPT / MET
Hypothetically yes adding DMT could reduce the amount of work the mycelium has to go through adding the two methyl groups, however it would need to be a sizable amount of DMT to end up in the right places without being oxidized by the oligermizing enzymes and you’re only skipping a single repeated step.

Similar with DEPT, etc. If you added enough that it found its way to the enzymes, they would probably do their work on them just fine, both the good ones and the bad ones.
This is something that we will probably see in the future but instead in psilocybin gene transferred e.coli bioreactors where the exact enzymes being produced can be controlled better.

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Re: Increasing alkaloid levels [Re: Schroomfairy]
    #26363926 -

Schroomfairy said:
Thank you for your excellent post. I will keep it for future reference.

When watching Hamilton Morris on the magic mushroom documentary, I found it interesting how the Mexican farmer talked about sugar cane and how it increased it's potency. Go to 14:48 [url=
Could be something to think about trying? He could be on to something? Or maybe even the possibility that adding to the substrate may actually cause something different. I am willing to complete some experiments for sure.


No problem. I think the sugarcane thing is entirely possible, “possible” being the key word there. Via YouTube, one can only guess what the nutritional contents of the soil actually are. If something is happening, the study I’m posting below would argue that it has to do with nitrogen and/or glucose levels. It’s the one I mentioned before, in my last post. Straight from MAPS:

https://bibliography.maps.org/resources/download/14256

I don’t want to discourage any experimentation, but I wouldn’t experiment with sugar alone in large amounts, if that’s what you’re saying. My current experiments are currently revolving around glycine/nitrogen supplementation. It’s too soon for me to say much but I have had one very encouraging result so far.


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‎שלום וְאור | PEACE & LIGHT

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Re: Increasing alkaloid levels [Re: StygianKnight] * 1
    #26363927 -

Some interesting information on the evolution of psilocin and it's role in nature. https://www.biorxiv.org/content/biorxiv/early/2018/07/27/374199.full.pdf


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Question everything, it's very responsive.


It's all downhill from here!
You ain't gonna learn what you don't want to know

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Re: Increasing alkaloid levels [Re: Nichrome] * 1
    #26364022 -

Damn this is a good thread. Sorry if already posted but good stuff.

Full Paper -> Enzymatic Synthesis of Psilocybin

Abstract:
Psilocybinis the psychotropictryptamine-derivednatural product of Psilocybe carpophores,the so-called“magicmushrooms”.Although its structure has been known for 60 years,the enzymatic basis of its biosynthesishas remain edobscure. We characterized four psilocybin biosynthesisenzymes,namelyi) PsiD,which represents  class of fungall-tryptophan decarboxylases,ii) PsiK,which catalyzes the phospho transfer step,iii)the methyltransferase PsiM, catalyzing it erativeN-methyltransferas the terminal biosynthetic step,and iv) PsiH, amonooxygenase. Inacombined PsiD/PsiK/PsiM reaction, psilocybin was synthesized enzy-matically in a stepeconomic route from 4-hydroxy-l-trypto-phan. Given the renewed pharmaceutical interest in psilocybin, our results may lay the foundation for its biotechnological production

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Full Paper -> Monoamine Biosynthesis via a Noncanonical Calcium-Activatable Aromatic Amino Acid Decarboxylase in Psilocybin Mushroom



Aromatic l-amino acid decarboxylases (AAADs) are a phylogenetically diverse group of enzymes responsible for the decarboxylation of aromatic amino acid substrates into their corresponding aromatic arylalkylamines. AAADs have been extensively studied in mammals and plants as they catalyze the first step in the production of neurotransmitters and bioactive phytochemicals, respectively. Unlike mammals and plants, the hallucinogenic psilocybin mushroom Psilocybe cubensis reportedly employs an unrelated phosphatidylserine-decarboxylase-like enzyme to catalyze l-tryptophan decarboxylation, the first step in psilocybin biosynthesis. To explore the origin of this chemistry in psilocybin mushroom, we generated the first de novo transcriptomes of P. cubensis and investigated several putative l-tryptophan-decarboxylase-like enzymes. We report the biochemical characterization of a noncanonical AAAD from P. cubensis (PcncAAAD) that exhibits substrate permissiveness toward l-phenylalanine, l-tyrosine, and l-tryptophan, as well as chloro-tryptophan derivatives. The crystal structure of PcncAAAD revealed the presence of a unique C-terminal appendage domain featuring a novel double-β-barrel fold. This domain is required for PcncAAAD activity and regulates catalytic rate and thermal stability through calcium binding. PcncAAAD likely plays a role in psilocybin production in P. cubensis and offers a new tool for metabolic engineering of aromatic-amino-acid-derived natural products.

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Full Paper -> Enzymatic route toward6-methylated baeocystin and psilocybin (2019)

Abstract
Methylated magic. The biocatalytic in vitro route to psilocybin was interrogated for substrate flexibility. 6‐Methylated substrates were accepted by the P. cubensis enzymes PsiD, PsiK, and PsiM and produced 6‐methylbaeocystin and 6‐methylpsilocybin. An in silico model of the methyltransferase PsiM identified nonconserved structural elements that likely govern the specificity of the methylation step.



------------------------------------------------------------------

Full Paper -> In vivo production of psilocybin in E. coli (2019)

Abstract
Psilocybin, the prodrug of the psychoactive molecule psilocin, has demonstrated promising results in clinical trials for the treatment of addiction, depression, and post-traumatic stress disorder. The development of a psilocybin production platform in a highly engineerable microbe could lead to rapid advances towards the bioproduction of psilocybin for use in ongoing clinical trials. Here, we present the development of a modular biosynthetic production platform in the model microbe, Escherichia coli. Efforts to optimize and improve pathway performance using multiple genetic optimization techniques were evaluated, resulting in a 32-fold improvement in psilocybin titer. Further enhancements to this genetically superior strain were achieved through fermentation optimization, ultimately resulting in a fed-batch fermentation study, with a production titer of 1.16 g/L of psilocybin. This is the highest psilocybin titer achieved to date from a recombinant organism and a significant step towards demonstrating the feasibility of industrial production of biologically-derived psilocybin.

---------------------------------------------------------------------

Full Paper -> Biosynthesis of bioactive natural products from Basidiomycota (2019)

Abstract
The Basidiomycota, also called club fungi, comprise a diverse group of fungi. Basidiomycota are strongly related to ecosystem functioning along with human life. These fungi display a wide range of bioactivities, and some are known to produce of deadly toxins or hallucinogens. Some Basidiomycota have be used as medicinal mushrooms for thousands of years. Recently, the biosyntheses of several classes of natural products from Basidiomycota have been reported. Here, we review recent studies on the biosynthetic pathways and enzymes of bioactive natural products from Basidiomycota fungi, with a focus on terpenoids, alkaloids, ribosomally synthesized and post-translationally modified peptides (RiPPs), and polyketides.



Edited by murderlabz (12/05/19 11:22 PM)

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Re: Increasing alkaloid levels [Re: murderlabz] * 1
    #26372448 -

I haven't fully read murderlabz' wonderful contribution but from what I know it seems that it can be relatively pointless to try and change a part of metabolism by upping one precursor or metabolite concentration.
Because metabolism consists of many equilibria of catalyzed reactions and feeding this system doesn't guarantee at all that it will end up in a useful way.

So yeah it makes sense to work on the enzymatic activity. If psilocybin is a form of storage for an oligomerization reaction and metabolically speaking psilocybin is kinda cornered by mainly the phosphorylation/dephosphorylation.. possibly it would help to modify the rate of phosphorylation as this may build up psilocybin concentration without it being broken down again through another path (hopefully there isn't one im too lazy to check right now).

If quantative analysis was much easier I guess we could just breed and select for it as we may pick up on mutants being generated and we could stabilize them without them just being noticed as a batch of above average potency. Or who knows, someone might crispr it.. >_>

And maybe there are agents which can induce PsiK and change the equilibrium of catalysis? Idk if that would be easier than increasing the expression. What regulates dephophorylation?


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My trade lists:
  • Fungi - Edible / Gourmet
  • Fungi - Medicinal/Insecticides/Bioluminescents
  • Fungi - Actives
  • Fungi - Old Exotic Spores (Actives)
  • Fungi - Misc Hunted Mushroom Spores
  • Plants
  • Other items - Supplies etc.


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    Re: Increasing alkaloid levels [Re: Solipsis] * 1
        #26372883 -

    Without sounding like a broken record, it’s important to remember that best way to achieve higher potency, or whatever you want with mushrooms, is indeed honing genetics. For instance, I have found the PES Hawaiian strain to be noticeably on the potent side, and worth further work. Cloning, agar isolation - these methods are tried and true. As RR said in Let’s Grow Mushrooms, agar work is great because it allows us to be “mad scientists.”

    With that said, I am a believer that grain, substrate, fruiting conditions, and harvesting protocol all affect end potency as well. Just because it’s not been empirically proven to be so doesn’t mean we shouldn’t experiment, anyway.

    Here is something interesting. Paul Staments recently found that the Polyphore mushroom extracts currently in development as a means of eradication of the “Varroa destructor” parasite and it’s RNA virus, the deformed wing virus (DWV) - which is the cause of widespread colony collapse among bees - have an efficacy of 10:1 when grown on corn, but 1000:1 ratio when grown on wood (I believe birch). The cause is of this is not known, nor has the active enzyme or whatever it is that is working so well to kill off these parasites and the RNA virus via the Polyphore mushroom even been identified...but it is known that SOMETHING in the substrate plays a part in greatly “up-regulating” it’s content. You can listen to him speak out this toward the end of his recent interview on the Joe Rogan Experience.

    Now admittedly we are dealing with very different mushrooms when we start talking about psilocybes. But it still begs the question: why should they, at least to an extent, be any different? Is it not possible to manipulate them similarly? This is the core of scientific inquiry: questioning, asking the proverbial “what if” even when we barely know where to even start. & I think the question is worth pursuing.

    I don’t argue with people who are adamant that substrate or supplementation techniques cannot effect potency. This has been their experience. And many like RR have damn good reasons as to why they hold to this. My differing opinion, for instance, that cubensis grown on manure are to some degree more potent than those grown on coir, is just that - an opinion. I have no empirical evidence. Which sucks, because locking in any empirical evidence here is damn near impossible.

    One mush remember, no matter what your stance, that we are dealing with an unfairly illegal substance, one that has been under tight prohibition by power hungry bastards  across the globe for half a century (with the exception of Brazil and some others). This directly effects the ability to carry out proper research in a serious way even for scientists, and especially for the average, inquiring Joe. To amass any real data scientifically here just has not really been very possible. Thankfully all this is changing, slowly but surely. I have hope there will eventually be some real answers.

    Murdelabz post gives us a lot to look at. I understand some of it but most of it, but most  is definitely over my head at least in terms of practical application. Great post though indeed :smile:


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    ‎שלום וְאור | PEACE & LIGHT

    Edited by Psilotyl (12/10/19 10:52 AM)

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    Mushrooms, Mycology and Psychedelics > Advanced Mycology
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