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Researcher

Regulating Secondary Metabolite Production in P.Cubensis

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Researcher

I noticed this at non-sponsor vendor page for Penis Envy, and took note as Workman is a well respected member of the community. I seriously doubt he would have made this up;

sporeworks2.jpeg

The 'therapy' described by Workman above, is surely from this 1973 paper;

HEAT THERAPY OF VIRUS-INFECTED CULTURES OF THE CULTIVATED MUSHROOM AGARICUS BISPORUS
https://www.researchgate.net/publication/248895443_Heat_therapy_of_virus-infected_cultures_of_the_cultivated_mushroom_Agaricus_Bisporus

After reading that the first thing that came to my mind was, if you did that to a Cubensis culture surely not every culture would even be able to survive and in those survivors it would definitely have triggered a heat shock response whereby they overexpress heat shock protein encoding genes that would in turn produce enough heat shock proteins and molecular chaperones to survive the oxidative attack brought on by the heat. Whether it was a random mutation induced by the heat or the literally thousands of different ways it could have actually happened,  the only one question of any real importance was;

Was PE a thermotolerant mutant?

I started looking into these genes on mycocosm (link to a general search for Cubensis' hsp's below), of particular note was(and you can check for yourself) is that
P.Cubensis has every gene that is mentioned in the paper below.

https://genome.jgi.doe.gov/pages/search-for-genes.jsf?organism=Psicub1_1

This paper came out a couple of months ago.....

Quote

Proteome and Transcriptome Reveal Involvement of Heat Shock Proteins and Indoleacetic Acid Metabolism Process in Lentinula Edodes Thermotolerance

The proteome results revealed that HSPs (heat shock proteins) such as Hsp40 (DnaJ), Hsp70,
Hsp90 and key enzymes involved in tryptophan and IAA metabolism process LeTrpE, LeTrpD,
LeTam-1, LeYUCCA were more highly expressed in S606 than in YS3357, demonstrating that
HSPs and tryptophan as well as IAA metabolism pathway should play an important role in
thermotolerance. Over-expression of LeDnaJ gene in S606 strains showed better tolerance to
heat stress.

Our KEGG analysis (Table S4) found that most of the DEGs were involved in metabolic pathways,
biosynthesis of secondary metabolites, biosynthesis of antibiotics, microbial metabolism in diverse
environments, MAPK(mitogen-activated protein kinase)signaling pathway - yeast, and oxidative
phosphorylation.

In our proteomic results, we found that the protein expression level of OABA synthase, one rate-
limiting enzyme in tryptophan biosynthesis, increased...

Nevertheless, the protein level of OABA synthase, an
important enzyme in tryptophan biosynthesis, in the thermotolerant L. edodes strain S606
after heat stress had a dramatical upregulation, while that of the heat-sensitive strain
YS3357 was not detected, indicating that LetrpE could play a pivotal role in L. edodes strain
thermotolerance.

Surprisingly, it was documented that
the anthranilate synthase (TrpE, LE01Gene02473) level in S606 had approximately 500-fold
increases after heat stress, respectively, but their levels in YS3357 were down-regulation (Fig.
5 and Table S7), demonstrating that they possess the potential in L. edodes thermotolerance.

Exogenous IAA maintained normal growth with the heat-sensitive strain YS3357 about
seven days after 24h of heat stress at 40°C (Fig. 9c), suggesting that a tryptophan downstream
event such as IAA biosynthesis may be another way by which L. edodes thermotolerance was
improved.

In fungi, IAA was detected
by chromatography and synthesized via IPA(indole-3-pyruvic acid), IAM(tryptamine),
and TAM(indole-3-acetonitrile) pathways, in Phanerochaete chrysosporium, Lentinus sajor-
caju, yeast, Pleurotus ostreatus, Tricholoma vaccinum and Fusarium graminearum [83-88].
Based on our proteomic results, those genes encoding IAA via tryptophan and IPA pathway
in S606 upregulated obviously after heat stress (Table S8), demonstrating that IAA in L.
edodes could be synthesized via tryptophan and IPA pathway and accumulated during heat
stress.[/b]
https://www.karger.com/Article/FullText/494784

 

suggesting that a tryptophan downstream event such as IAA biosynthesis may be another way by which L. edodes thermotolerance was improved

Now using that same nomenclature and reading Frickes work we know for sure that technically speaking psilocybin biosynthesis is also a tryptophan downstream event. I
am not proposing its sole purpose is this, but if it and it's biosynthesis does increase thermotolerance and also once completed has anti-oxidant properties(it is pretty easily oxidized to psilocin). And we know heat can unfold proteins but that most of the damage is done by oxidation which is possible because the heat provides the activation energy needed by the reactive oxygen species to push the reaction forward. We also know from Frickes recent work that cubensis posses a gene that encodes an enzyme which nearly instantly regenerates any psilocin present to psilocybin to a point that there is not really ever any quantifiable amounts of psilocin present in the cells of the fruiting body, restoring its anti-oxidant properties extremely quickly in a cycle that lasts as long  as that enzyme is present and there is something to oxidize psilocybin to psilocin.

Is PE a thermotolerant mutant?

If it is, it's within reason to be able to reproduce(or at least to create a thermotolerant clone as a proof of concept). No special methods or equipment are
needed. The paper below provides a great basis for any experiments, I also have some other very interesting papers I am gathering up the links to but I will let you
guys digest this first! One uses PABA instead of heat to induce this response. I have read a couple of papers so far that mention severly reduced pileus diameter as a
symptom of extreme shock.

The paper below notes increased secondary metabolism, Ganoderic Acid is not produced via the shikimic pathway. But that supports the evidence that enhanced
secondary metabolism is witnessed in these thermotolerant strains no matter what the pathway.

Quote

Heat Stress Modulates Mycelium Growth, Heat Shock Protein Expression, Ganoderic Acid Biosynthesis, and Hyphal Branching of
Ganoderma lucidum via Cytosolic Ca 2ⴙ
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4959220/

Quote

Ganoderma lucidum, a higher basidiomycete with bioactive secondary metabolites, has become a potential model system for


evaluating how environmental factors regulate the development and secondary metabolism of basidiomycetes. Heat stress (HS)
is an important environmental challenge. In this study, we found that HS inhibited mycelium growth, reduced hyphal branching, and
induced HSP expression and ganoderic acid biosynthesis in G. lucidum.

 

 

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Brad

Omg. I have some reading to do. I'm glad to see you here !)

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Researcher

You need any help getting your head around anything let me know.

P.S. Sorry with the micocosm link I gave I thought I had the form prefilled, you just have to enter 'heat shock' into the search engine to get an overview. You can also search for individual hsp's or chaperones there too.

Also a correction for above; psilocybin is not oxidized to psilocin its dephosphorylated to psilocin which is then oxidized hence its antioxidant properties (it was a lot to type above). So it is not cycled as quickly as I proposed above. But still does have antioxidant properties.

 

 

Edited by Researcher
added correction

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Researcher

Hopefully this will help;

On the forum Teladi said this;

Quote

It doesn't look like we know the full picture of the Psilocybin biosynthesis. As in whether or not or what
coenzymes/cofactors/inhibitors/activators if any are involved in the pathway. I know that the enzymes alone are fully sequenced, but I'm not sure we know their structures yet. Their structures can hint as whether or not other factors are involved. This is all a part of the question, "What affects the regulation of Psilocybin production?" It may be entirely genetically driven, with all the answers found in non codons, or driven only by environmental factors. It could have super stable enzymes that last the lifetime of the cell, so have really low expression. I need to dive back into my textbooks.

 

I tried to point out the extra potent penis envy came about via 'heat therapy' which rang a bell with me as I knew enough about fungal heat shock responses to wonder if there was something there? I had a quick look into it and was aware that it probably was a random mutation induced coincidentally by the heat. But I was getting some interesting results regarding heat shock responses.


Then this paper came out last month, I will put the quotes from the article and a quote of mine under it.

Quote

"Using heat-tolerant and heat-sensitive strains of L. edodes, we reported a combined proteome and transcriptome analysis of L. edodes response to 40°C heat stress for 24h."

L.edodes being Shiitake and it has a close enough match to cubensis in terms of the genes that control its heat shock responses to pay attention to.  In fact if you look in the second paper I cited later on you will see a close analogy between most basidiomycetes regarding heat shock responses.

So they grew a lot of strains and screened them for a strain that tolerated heat very well, and then screened for another that did very poorly in heat. Then they used these two differing strains to analyse the differences between them when they suffered heat shock.

Quote

The proteome results revealed that HSPs (heat shock proteins) such as Hsp40 (DnaJ), Hsp70, Hsp90 and key enzymes involved in tryptophan and IAA metabolism process LeTrpE, LeTrpD, LeTam-1, LeYUCCA were more highly expressed in S606 than in YS3357, demonstrating that HSPs and tryptophan as well as IAA metabolism pathway should play an important role in thermotolerance.

I can provide links to those genes on Cubensis' genome if anyone is curious, its Hsp genes are a close enough match to L.edodes that we can presume the pathways they use are similar if not the same except Shiitake obviously cannot go on to produce psilocybin.

And when they applied enough heat, it activated all these heat shock proteins (which is fair enough, it is it's defensive
mechanism to protect itself from the damage caused by heat). But the interesting part is that they not only noticed
these Hsp's were being more highly expressed in the strain that did well in the heat, after heat shock. They also noticed that it's tryptophan metabolic processes went into overdrive. And as we know psilocybin is a downstream product of tryptophan, and it's biosynthesis is a part of Cubensis' tryptophan metabolic process.

The tryptophan metabolic pathways they postulate(with some good science behind them) play an important role in thermotolerance.

So they apply heat and analyze the differences between the expression of genes in the strain that does well in heat
and the expression of genes in the strain that does not. And all the genes that are expressed about the same in both are forgotten, and the ones that are markedly different are labeled DEG's(differentially expressed genes). KEGG is a genome database.

Quote

Our KEGG analysis (Table S4) found that most of the DEGs were involved in metabolic pathways, biosynthesis of secondary metabolites, biosynthesis of antibiotics, microbial metabolism in diverse environments, MAPK (mitogen-activated protein kinase) signaling pathway - yeast, and oxidative phosphorylation.

Psilocybin is Cubensis' main secondary metabolite! So even if it played no role whatsoever in reducing heat stress, which I would find unusual as the ring hydroxylation alone removes a reactive oxygen from the system(pretty handy when under oxidative attack) the researchers in this paper (like the other paper cited and a lot more) noticed that secondary metabolism was increased significantly in thermotolerant strains(strains which express these hsp genes more than others) after heat shock. Also Psilocybin is notoriously thermally labile meaning it undergoes hydrolysis to psilocin pretty easily when in solution and heat is applied. It exists in the cell in solution so if much heat was applied it would break into a phosphate ion and psilocin which is then oxidized easily to the bluish/green compound we know (a heat activated
anti-oxidant? maybe not but an interesting thought?).

Quote

suggesting that a tryptophan downstream event such as IAA biosynthesis may be another way by which L. edodes thermotolerance was improved.

Quote

Nevertheless, most of genes related to tryptophan and IAA biosynthesis had an obvious upregulation in
term of protein content, yet their transcription levels had no change or downregulation (Table S9).

IAA being Indole-3-Acetic Acid another downstream tryptophan product.

Quote

In our proteomic results, we found that the protein expression level of OABA synthase, one rate-limiting enzyme in tryptophan biosynthesis, increased dramatically after 24h of heat(Table S9)

A rate-limiting enzyme is pretty much a bottleneck in the pathway, I just copied and pasted this as the official definition is better than mine;
"The slowest step in a metabolic pathway or series of chemical reactions, which determines the overall rate of the other reactions in the pathway."

So obviously a dramatic increase in the amount of this enzyme, equals a dramatic increase in the rate at which tryptophan the precursor of psilocybin can be produced inside the cell.

Quote

However, the thermotolerance of the L. edodes strain S606 was improved by the supplementation of exogenous tryptophan and OABA in the medium, indicating that the upstream and downstream events of tryptophan
biosynthesis played a positive role in improving L. edodes thermotolerance.

S606 being the thermotolerant strain.

So they are saying here that their experiments indicated that not only tryptophan downstream events but also the actual process of biosynthesis of tryptophan plays a positive role in improving thermotolerance.

The other quotes I put above are highlighting some of the notable cases of upregulation of genes involved in the shikimate pathway that ultimately produces psilocybin in cubensis.

Any thoughts?

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Researcher

I am putting up quotes from that paper like:

Quote

Surprisingly, it was documented that the anthranilate synthase (TrpE, LE01Gene02473) level in S606 had approximately 500-fold increases after heat stress, respectively, but their levels in YS3357 were down-regulation (Fig.5 and Table S7), demonstrating that they possess the potential in L. edodes thermotolerance.

That I am looking at saying wow look at this, and I think some are looking at and thinking so what?

For this quote for example;

Anthranilate synthase is an enzyme that produces Anthranilic acid, and is part of tryptophan biosynthesis (shikimate pathway).960px-Tryptophan_biosynthesis_(en)_svg.jpg.58929b35513bf13f5fdde4f9e1cdefb3.jpg

And this is the effect of enriching the substrate with either it or L-Serine (you see above serine is also significant because it is enzymatically condensed with indole to form tryptophan).

TLC–Densitometry analysis of indole compounds in mycelial culture of Imleria badia and Agaricus bisporus enriched with precursors — serine or anthranilic acid

Untitledttt1.thumb.jpg.39ab0b95989e11e1b86bf9a841f2c34f.jpg

https://akademiai.com/doi/full/10.1556/1326.2017.00325

As you can see above, the button mushroom usually produces 5-hydroxy-tryptophan as its main indole product. When they added either one of these precursors they started seeing pretty big increases in all the indole compounds the mushroom produced including tryptophan and tryptamine (both psilocybin precursors!).

 

Quote

This is the first report that shows that medium modification through serine or anthranilic acid supplementation affects the content of indole compounds in the resulting biomass. The concentration of the additive added to the medium is also important in the context of quantitative and qualitative content of these compounds in the resulting biomass. It indicates that modification of medium for mycelial cultures may be crucial for the potential production of lyophilized biomass containing specific indole compounds at suitable amounts that can be used in the supple-
mentation of indole derivatives.

So a 500 fold increase in the gene that produces anthranilic acid is noteworthy! And that 500 fold increase is inside the cell where it can be utilized instantly. And regulated by the fungi so to be at peak levels without having to add precise amounts of precursors for rate limiting steps.

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Doodlin

Hooray, I can finally chip in!  :D

Let me start right out by saying: this is a very cool idea. I have been musing for a while on how to create a cubensis growth system that places psilocin production under positive selective pressure, without having to actually assay every individual, and what you've come up with is by far the best route I've considered. I definitely think it's worth a try.

I'm not as sure as you that psilocin / psilocybin are actually involved in the HSR; I think it's just as likely that it's the increased trp flux that improves heat tolerance through downstream components, and the increased psiloc* levels are just a simple consequence of more trp.

Regardless... the question is how to actually translate this idea into a selective program. This is where I have to do more reading, as my experience is that this is quite organism-specific. I think the most ground would be covered by multiple cycles of mutagenesis-growth-selection, but the specifics of mutagen and selective pressure are very hand-wavy right now.

But in summary -- nice find!

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Researcher

Thanks for the feed back!

I am extremely glad to have you around, I find it important to have someone to bounce ideas off and get honest feedback.

OK, so back to business! If you are interested in collaborating on a program of sorts, I should let you know where we stand! I have a background in organic chemistry (most of my actual work has been in biology though). I have nearly finished putting together a TLC kit (still have not got the developing chamber yet), so I will have the capacity to actually assay every individual if needed. I have a pretty strict program to make sure the results can also be used to get rough quantitative estimates also, it is not elaborate but you do need good lab scales a decent pipettor that holds 1000μl and 2μl capillary tubes to make sure everything is exactly the same from test to test. Hence the results are not only comparative(which is good enough for our purposes), but also somewhat quantitative when compared against a known sample. For example make a 5% L-tryptophan solution and run that, you now have an accurate enough 5% reference spot to know when you are in the vicinity of 5%. It cost a couple of hundred in the end(bit more), if you want  my procedure (solvent systems ect) let me know and I will put it up. If somebody else stuck strictly to it we could get similar enough results for comparison. It is also great for getting an alkaloid profile of strains, which can be used to fingerprint strains and add to a database. I plan to share pics of the experiments,  so you will see all this anyway.....

 

Quote

I'm not as sure as you that psilocin / psilocybin are actually involved in the HSR; I think it's just as likely that it's the increased trp flux that improves heat tolerance through downstream components, and the increased psiloc* levels are just a simple consequence of more trp.

I am not 100% positive either, I was already looking into them for their possible role in increased potency. And then that paper came out, and I was taken back a bit. So I am also not sold yet, but think it is definitely one of the first ideas worth looking into. Also in a paper I was reading they heat shocked the young mycelium then let it recover and grew it out, and it produced a lot more secondary metabolites than the control. I was also thinking this was maybe a good application for some weaker potency varieties(anyway....).

Quote

Regardless... the question is how to actually translate this idea into a selective program.

This is really the million dollar question, I will also look into it. I am still a couple of weeks off actually getting spores to agar, so would be great to get a solid plan into action before then. Also if we created it intentionally as an open source program, so that anyone interested could participate I think we could advance it pretty quickly. For example screening for heat tolerance, if that route proved fruitful.

Or do we just start several large multispores from for example- PE6, KSSS, Puerto Rican(leaning towards PE6). Screen and select for potency, if they endup being heat tolerant (good for them...).

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