Check this out....it is.....astounding...
Preliminary Feasibility Study for
The Biological Production of L-Dopa, Mescaline
and Tryptamines by Intact Recombinant Yeast Cells
Using Only Common Amino Acids as Precursors to Bioenzymatic Synthesis
OverviewI intend to present here a culling of existing and proven laboratory
techniques for the recombinant transformation of microorganisms to the
purpose of their production of pharmaceutical compounds which at the
present time are only being made via organic syntheses using legally
restricted and costly chemicals, and chemical procedures both difficult
and dangerous to utilize for the layman not extensively trained in
traditional organic synthetic methods.
Genetically transformed yeasts and e. coli cultures are currently
harnessed on an industrial level to make such varied compounds as human
insulin, opiates (see references) and cytokines and other immunological
artifacts. There is every reason to believe that provided with a
moderately equipped recombinant genetics lab and the use of these proven
techniques, that yeast cells could be made to produce in high yield
tryptamines such as DMT and psilocybin, mescaline and other drugs. I chose
to start with tryptamines and phenethylamines as they are simple
molecules, which are almost identical to their biosynthetic precursors:
the ubiquitous amino acids tryptophan and tyrosine. THC, LSA, and other
restricted compounds could also be made in this fashion, but with much,
much greater difficulty as they are very much more complex molecules and
their biosynthesis is not explored in great detail at this time. With
such a recombinant yeast (and I chose yeast over e. coli due the
simplicity of yeast culture, i.e., bread and brewing techniques
considered-- and also due to the total lack of pathogenicity of cervesia
yeasts in general and the fact that yeasts are eukaryotic) all an
untrained layman would need to produce these
compounds in a pure yield would be: one transformed yeast cell, a bucket,
warm water, sugar, amino acids and 12 hours-- and simple acid base
extraction techniques to separate the pure psychedelic compounds from the
waste materials. Even given theoretical restrictions on amino acids such
as those currently imposed on tryptophan, these amino acids are present in
almost all living tissue and could be obtained from such. The yeast would
utilize its foreign dna inserts to code enzymes that would biochemically
substitute the molecular groups that create mescaline or DMT from their
amino acid precursors as part of the transformed yeast's metabolic
routine. It is not unrealistic to expect a gram or two of pure material
from such a 'brewing' effort, in theory--and perhaps more with the use of
a pH balanced, aerated fermentation chamber.
In addition, several useful pharmaceuticals could potentially be
made by precursor strains of such a yeast, including L-DOPA, a valuable
medicinal compound. L-DOPA is one hydroxyl group (one cactus enzyme) away
from tyrosine; it is one further aromatic hydroxyl group and three methyl
groups and a decarboxylation away from mescaline. A yeast transformed with
only one of the cactus enzymes would produce L-DOPA. I hold out the
potential to royalties from such an organism to a prospective funding
agency as another reason to support this project: biologically
manufactured L-DOPA would be hundreds of times less costly than that which
is currently made by pharmaceutical companies using expensive traditional
methods which, unlike enzymatic synthesis, create a toxic waste stream.
Bioenzymatic synthesis is totally clean and extremely efficient. Also
enzymes almost always produce a single enantiomeric species, though that
is not a consideration here. Considerations
The following techniques would most easily produce tryptamines, as DMT
is created from tryptophan by the work of only two enzymes: one enzyme
decarboxylates the L-alkylaminocarboxy chain to an ethylamino chain which
is then N-methylated by the second enzyme. The decarboxylation gene is
already known and commercially available, and would also work with the
alkylamine chains of tyrosine and phenylalanine. The second enzyme would
have to be isolated by reverse cloning of fungal mrna into cdna, which
would be tagged with synthetic linkers, methylated and inserted into a
restricted plasmid appropriate for a yeast strain. A third enzyme would be
required for DMT to psilocin, a 4-hydroxyl transferase.
Transforming the yeast cell with the ligated plasmid dna and foreign
vector dna is actually the easiest and simplest step of the entire
process: it can be accomplished with electroporation or cheap and simple
chemical methods employing buffering agents and lithium acetate (see
references). The bulk of the work is involved in identifying the appropriate
reading frame for the cloned mrna to cdna fragment which codes a single
enzyme for a specific chemical transformation, again two such genes in the
case of tryptophan to N-methyltryptamine --and in choosing appropriate
marker genes to identify the transformed vs. wild type cells. Somewhere
between three and possibly five unkown genes will have to be characterized
and inserted for a yeast to produce mescaline from tyrosine ( not
including the known decarboxylation enzyme gene) so it is a more difficult
transformation to attempt by far. But I have other reasons to consider as
an initial project in this area the choosing of mescaline over DMT orpsilocin.
First, the mrna to get the reverse cloned cdna is easily obtainable
from the semi-legal and widely available "San Pedro" cactus, T. pachanoi,
which typically produces . 12% mescaline from its dry weight. It is not
feasible to use restricted genomic dna, as it is almost impossible to know
where to begin a reading frame, or if a restriction site lies in the
desired frame. Mrna clones only for the active enzymes involved in the
current metabolism of the plant or other organism, and it can be
relatively easily cloned back into double stranded cdna which is easily
inserted in a plasmid with the appropriate promoters and markers (see
My main reason for not choosing tryptamines to work with initially is
not a technical one, as I have shown the tryptamines to be more
technically simple. It is the legal issue and unwillingness to deal with
Schedule I fungal materials that I am concerned with. And while it would
also be possible to use semi-legal DMT plant sources for such a
transformation, I have had great difficulty obtaining such materials in a
fresh condition at any affordable cost. And there is the L-DOPA side
reaction to consider, which will only come from working on the mescaline
synthetic route. The L-DOPA synthesis could also be used as a factor to
legitimize the research, on the road to perhaps an understated goal of
mescaline synthesis. Technique and Protocol
Several of the obstacles I have faced in planning this project have
revolved around ways to avoid extra expenses while not contaminating the
integrity of the process. Traditional molecular biology laboratories
utilize freely much expensive equipment such as computerized DNA
synthesizers and analytical equipment.
Barring that perhaps some of this could be borrowed, I propose that
possibly much of the project could be done with more tedious and timeconsuming,
but far less expensive methods. For example, rather than using
radiolabeled synthetic oligionucleotide probes based on purified enzyme
analysis to identify the correct cdna fragments that clone a specific
enzyme gene, perhaps cdna fragments could simply be inserted at random (after
adding synthetic linkers and methylation to avoid a restriction site
within the reading frame, and also making it easy to identify and recover
the vector dna from the e. coli for insertion into yeast ) and the
resulting pools of bacteria subdivided and tested until a strain is
identified that performs a single enzymatic task. Growth and lysis of
these strains after large scale culture would be used in place of
polymerase chain reaction artificial amplification of the cdna. It may be
unavoidable to utilize some pcr amplification of the extracted mrna, as it
is typically isolated as a very small sample.
Some unusual options may be open in the case of mescaline: the
aromatic hydroxylase activities of many bacteria are under intense
scrutiny right now in general for the search for bacteria capable of
degrading toxic waste. I have found one such gene described (see
references) but it specifically will not work here as it does not
recognize phenylalanine as a substrate and has very low activity with
tyrosine. There are probably other bacterial genes already recognized
that could 3,5 hydroxylate the aromatic ring of tyrosine. As with standard
chemical procedures, once a ring has one hydroxyl present this will
catalyze the formation of further hydroxylations. The human gene which
converts the hydroxy free phenylalanine into 4-OH phenylalanine (aka
tyrosine) requires a cofactor, tetrahydrobiopterin, a relative of folic
acid. In general, the cofactors required for bioenzymatic synthesis (as
opposed to cell free enzymatic synthesis) are often present (NADP, NADH
and various metal ions are common examples) in the cell. But it is
distantly possible that one or more of these enzyme cofactors will be
required to be cloned also.
Two hydroxylase genes will have to be isolated from the cactus to
complement the 4-hydroxyl group present in tyrosine. The already known
decarboxylase gene will be easy to transform, as it is identified and
characterized. This leaves the three methyl groups to transfer to the
3,4,5 hydroxyl intermediate. It is possible that either one enzyme will
transfer a methyl to each of these groups to make 3,4,5
trimethoxyphenethylamine (mescaline) --but it also possible that three
different enzymes methylate these aromatic hydroxyls. This brings the
total of unknown genes to be cloned from cactus mrna to a possible high of
five, and a possible low of three. The above is a slightly simplified
overview of the process, leaving out routine details of constant analyisis
of the progress of transformation through agarose gel electrophoresis, and
nmr confirmation of target structure at every point. The references I have
included will fill in those gaps. This is a preliminary report, and I was
rushed to get it together by the 1st, May when the board of the Heffter
Organization meets. More detail can and will be provided, if necessary.
Rough Summary of Technique (see references attached for details)
1. extraction of active mrna from fungus or cactus
2. cloning of mrna into cdna clones or possible use of mrna/cdna hybridstrands
3. attachment of synthetic linkers to cdna fragments
4. methylation ofcdna fragments
5. restriction of plasmid using sites corresponding to synthetic linkers
6. ligation of plasmid and vector dna using T4 ligase
7. transformation of e. coli pools with ligation plasmid and
identification of marker genes on plasmid (typically antibiotic resistance)
8. subdividision of e. coli pools until single unknown genes are
characterized by expression products9. lysis of expanded pools
10. extraction and restriction of plasmids and agarose gel separation of
fragments11. ligation of characterized fragments to yeast plasmid
12. sequential transformation of yeast cells (see references)
13. selection of transformed yeast clonesCosts
I have not had time to run every detail of the cost of such a project, and
much depends on the availability of borrowed equipment and technical
expertise, as I have no current lab access beyond some organic equipment.
Also much could be purchased used, saving considerably. I have previously
mentioned some methods which could save money at the expense of time. The
bulk of the costs are tied up in the high end microfuge and centrifuge
required, Sorval type rotors capable of 12,000 g. It may be possible to
substitute a slower centrifuge used for longer spin periods. Also, other
minor items will be needed such as a shaker and gel electrophoresis kit. I
have also not looked into pre-prepared kits specifically designed for
recombinant engineering. The inorganic chemicals and antibiotics and
restriction enzymes will also be costly. I have been given an estimate
of from $10,000 to $15,000 for the whole project by an expert in thefield.
None of these techniques are in the least speculative. They are the tried
and proven workhorse tools of standard molecular biology. Only the
Nucleotide sequence and over-expression of morphine dehydrogenase, a
plasmid encoded gene from Pseudomonas putida M10. J Biochem, 290, 539-544
A M Hailes and N C Bruce (1993)
The biological synthesis of the analgesic, hydromorphone :
an intermediate in the metabolism of morphine by Pseudomonas putida M10.
Appl Environ Microbiology, 59, 2166-2170 G W W Cameron and N C Bruce (1993)
Towards enginering pathways for the synthesis of analgesics antitussives.
Ann N Y Acad Sci, Vol 721, 85-89
G W W Cameron, K N Jordan, P J Holt, P B Baker, C R Lowe and N C Bruce(1994)
Pathway Engineering for the biological synthesis of analgesics and
Conference Proceedings on Applied Catalysis, Biotechnology '94, UK
Institute of Chemical Engineers, pp 50-52 N C Bruce and M T Long, (1994)
Biological production of semisynthetic opiates using geneticallyengineered
bacteria. Bio/Technology vol 13, 674-676 N C Bruce and M T Long (1995)
Engineering microbial transformation pathways for the synthesis of
morphine alkaloids. Trends in Biotechnology, 13, 200-205
M T Long, A M Hailes, G W Kirby and N C Bruce (1995)
Morphinone reductase : characterization, cloning and application to
biocatalytic hydromorphone production. Ann N Y Acad Sci, In Press
A M Hailes, C E French, D A Rathbone and N C Bruce (1996)
Engineering pathways in E. Coli for the synthesis of morphine alkaloid
analgesics and antitussives Ann N Y Acad Sci, In Press
D A Rathbone, P-J Holt, C R Lowe and N C Bruce (1996) also Towards
the redesign of morphine dehydrogenase with improved properties, sameauthors
and reference. "Molecular Cloning : a Laboratory Manual" by
Maniatis, Fritsch and Sambrooke, Academic Press, 1989
This would not be as cheap or as simple as Most Hated thinks it would be. I wouldn't use his transformation methods either, I doubt they would be effective considering the amount of DNA to be added. Yeast artifical chromosomes could be a viable option, but I envision a 100-300k outlay and at least 4 years of full-time research.
I would definately agree with KRZ
Research on that scale is much more expensive than the lay person would assume.
Just the equipment that you listed would run you way over your $15,000 estimate.
Now a fully equiped molecular biologist could possibly pull this off in his spare time.
Hey bees why don't one of you do this and start selling the yeasties
The fermentative conditions required for ideal performance might sill require a bioreactor, and once it was produced there would ensue a pretty involved extraction process to obtain the pure compound. Certainly an advantageous project for someone with the desire to produce multi-tonne batches of mescaline on an industrial scale. I'll see what I can't do about it though ;-)
heh, I m just throwing it out there... I emailed the guy about it.. he said that he hasn't been able to do any reserach because of the money factor.. Once the yeast was altered,,.. you would be able to clone it.. so as soon as its done it would be very easy to reproduce...and it would be very advantagous for someone to do this.. not only for illegal purposes but legal ones too...
l-dopa can be made enzymatically from tyrosine quite easily. The enzyme used in this process is tyrozine hydroxylase or polyphenol hydroxylase and can be procurred from potatoes, apples and bananas. This is the same copper dependent enzyme responsible for the production of dopamine in the hippocampus and I believe that when the Cu stores are depleted in that region dopamine production is halted. There have been studies in fish that have shown that Cu depletions in the hippocampus have caused a decrease in the amount of l-dopa and ultimatly dopamine produced in the fishes brain. And since essentially many believe that we are a bipedal cod fish I don't see why things would be different for us. They have shown in humans that depletion of tyrosine hydroxylase is caused by injestion and metabolism of amphetamines. This causing chemically induced parkinsonism. Not to turd on your subject though it is only a fact and should be considered when using stims. This enzyme is only available in a uncooked fruit or vegtable. I doubt though that it will make it past the hydrolysing HCl in the stomach though making thus hindering its way to the CNS neurons.
nice to see ya KrZ...the amount of DNA is rarely a problem now though, once you know what sequence(s) you need and how to get them, simply amplify the product. In the case of yeast, there are numerous papers which use E. coli as their amplification species...PCR if you have the $
-Indoletheylamine N-methyltransferase (the enzyme you described in tryptamine N-methylation) was studied almost 20 years ago and was recently purified to a great enought degree to produce cystals, which were subsequently described via X-ray crystallography from humans (yes we've got the enzyme ourselves...)
-The enzyme for 4-hydroxylation was also recently extracted and it's corresponding mRNA isolated in a thesis which Lilienthal can probably give you the reference for (thanks again Lili )
-A number of hydroxylation-type enzymes as used in mesc. production have been described, although at the moment I can't recall if any specific to hydroxylations of phenethylamines (phenylalanine hydroxylase has) are included in this list.
-There are numerous other ways to identify mRNA fragments, why bother randomly selecting fragments? PCR probably would never have to be used with the amplification I mentioned to KrZ. Just isolate the mRNA of interest from a sample by a chemical test on a library...voila
The information is coming in now, in bits and pieces
Alright, molecular biology on the hive. When someone gets some constructs for mescaline production, immediatly send me a few microliters of it. What was wrong with those transformations, electroporation is as simple as it gets. But for those that cannot afford an electroporader heat shock is the way to go. From ice your bacteria with the DNA goes at 42C for 45-55s, then back one ice for two minutes, LB is added and the bacteria is incubated for an hour at 37C. It will take longer if you do not buy those kits, but will be a hell of lot cheaper. Some are necessary though, like your bacculavirus kit(or whatever means you use). You ever check the price on most of those.
There are quite a few creative people out there on the hive. Maybe some tweeker can rig up an efficient thermal cycler so that you do not have to buy a super overpriced PCR machine. Maybe you could pay a tweeker to spin tubes over their head at 13 G's so that you do not have to buy a centrifuge.
We must stop making drugs and enslave some pretty bacteria or yeast to a life of clandestine biochemistry.
bee all that you can bee
That would be BEAUTIFUL, since it would make that drug as easy to come by as alcohol.
Why not one up this? Make a virus that'll permeate the blood-brain barrier and manufacture dmt/mesc/whatever? One dose'd do you . . . any volunteers?
If you desire, do what thou wilt.
This isn't really my specialty, but I recall some studies in neuropsychology which looked at the possibility of endogenous DMT-related compounds being responsible for some mental illnesses. I guess this would be the next logical step; skip em all and jump to this one
Begin with the dissolution of superfluous matters
So that desire and consciousness are free
What kind of endogenous tryptamines are we talking of, and what kind of mammal has these? Extraction from meat is fun, nothing like blending a hunk of meat in a blender with solvent, mmm mmm good. A guess it could just maybeebee possible, just look at that compared to serotonin, but they never taught us about that in that metabolism class.
Shouldn't you bee asleep by now?
What type of organism?...your type..
Ayahoasca: an experimental psychosis that mirrors the transmethylation hypothesis of schizophrenia
Pomilio AB, Vitale AA, Ciprian-Ollivier J, Cetkovich-Bakmas M, Gomez R, Vazquez G
JOURNAL OF ETHNOPHARMACOLOGY
65: (1) 29-51 APR 1999
Document type: Review Language: English Cited References: 125 Times Cited: 0
The experimental psychosis observed after drinking Ayahoasca, a South American hallucinogenic beverage from the Amazon Indians, reproduces the pathologic transmethylation theory of schizophrenia. This theory postulates a decrease in the monoamine oxidase (MAO) activity, which results in the accumulation of methylated indolealkylamines, such as bufotenin (5-hydroxy-N,N-dimethyltryptamine), N,N-dimethyltryptamine (DMT) and 5-methoxy-N,N-dimethyltryptamine. These substances are strong hallucinogens as has been previously confirmed experimentally. On the other hand, it is known that Ayahoasca is a beverage usually prepared by boiling two plants, one of them rich in beta-carbolines, which are naturally occurring strong inhibitors of MAO, and the other with high quantities of DMT. This particular combination reproduces what is supposed to occur under pathologic conditions of different psychoses. The effects of Ayahoasca were studied in subjects, assessing urine levels of DMT by gas chromatography-mass spectrometry (GC-MS) before and after the intake of the beverage. The results of this study confirm that the hallucinogenic compounds detected in the healthy subjects' (post-Hoasca, but not before) urine samples are the same as those found in samples from acute psychotic unmedicated patients. The chemical composition of the Ayahoasca beverage, and of the plant material used for its preparation are also reported as well as psychometric and neuroendocrine subject parameters. (C) 1999 Elsevier Science Ireland Ltd. All rights reserved.
There's something in there producing this for you, or at least there is the possibility in some genotypes:
Transmethylation hypothesis of schizophrenia(Stam et al., 1969; Smythies, 1983) proposes that,due to enzymatic disturbances (Buscaý´no et al.,1966, 1969), schizophrenic patients produce high amounts of methylated indolealkylamines, such as bufotenin (5-hydroxy-N,N-dimethyltryptamine)(Fulle
McLeod, 1990). In spite of this high turn-over,methylated indolealkylamines have been reported in urine samples from psychiatric patients, not only schizophrenics (Tanimukai et al., 1970; Saavedra and Axelrod, 1972; Strahilevitz et al.,
1975). In our previous work (Ciprian-Ollivier et al., 1986, 1988; Ciprian-Ollivier, 1991), in agree-ment with other authors (Rodnight et al., 1978; Murray et al., 1979; Checkley et al., 1980), it has been proposed that these compounds are related to perceptual disturbances, remarking that not only true hallucinations but more subtle percep-tual disturbances are present in several entities.
Therefore, methylated indolealkylamines may play the role of ‘state markers’ for clinical or subclinical psychoses rather than being a trait of any diagnostic category. Their accumulation in patients could be caused either by an acceleration in the kinetics of their production or, and most probably, by a decrease in the kinetics of the
enzyme (MAO) responsible for the breakdown of the methylated indolealkylamines (Mc Geer et al., 1978; Ra¨isa ¨nen and Ka¨rkka¨inen, 1978, 1979). Many reports are known of decreased MAO ac-tivity in schizophrenia, which are thus in agree-ment with this theory (Davis et al., 1982).
Decreased MAO activity allows the accumulation of indolealkylamines, crossing the blood brain barrier (BBB) and acting on the central nervous system (CNS), due to the fact that these com-pounds are not necessarily produced within CNS. mystic states that clearly mirrors this situation. Ayahoasca or Hoasca tea (the Brazilian name for
Ayahuasca; see Section 1.1) is essentially made by boiling two plants, Banisteriopsis caapi and Psy-chotria 6iridis. The first is rich in b-carbolines derivatives, which are strong natural MAO in-hibitors, and the second contains high amounts of DMT, being an important natural source of this compound (Rivier and Lindgren, 1972; McKenna
et al., 1984; McKenna and Towers, 1985; McKenna et al., 1986). In an empirical way, Amazon shamans discovered, many years ago, that in order to have the hallucinogenic effect of one of the plants, Psychotria sp., the presence of
the other, B. caapi, was needed. Therefore, pe-ripheral MAO inhibition by b-carbolines allows the concentration of DMT and further BBB crossing, thus exerting their hallucinogenic effects in the CNS.
btw-I have no clue why those random dashes copied in when I pasted this...??
Begin with the dissolution of superfluous matters
So that desire and consciousness are free
Biological production of pharmaceuticals is on the horizon. At least two obstacles remain:
1)discovery of the appropriate enzymes to do the chemistry
2)purification of the product from the biological cocktail
The first obstacle is nearly complete.
We've discussed this in the novel forum for phenethylamines regarding
Phenylalanine or Tyrosine->MDMA or MDA
Post 38437 (MrGreen: "Re: Not insects! Trees!", Novel Discourse)
This really interests the closet molecular biologist in me.
Once the bacteria is created by just one person, it could be distributed to all other bees who have the purification set-up, etc.
I have to say that I've been thinking about this a lot lately, esp. in regards to psilocybin (because of the haploid nature of the fungus).
Even if the enzymes could be isolated, it would be amazing. P. Cub. has an interesting feature, in that it puts a 4 HO on any tryptamine fed to it . . . this must be the result of a single enzyme.
Tryptophan --+ Tryptamine
Tryptamine --+ n methyl tryptamine
n methyl trypt --+ dmt
dmt --+ psilocin ( 4 ho dmt)
Wouldn't it be wonderful to find a chem that would n-methylate tryptamines? Or add an HO to the 4? A little tinkering, and it could be altered to say, n-methylate phenethylamines or amphetamine . . . maybe add an Ho to the say, 5 pos. Who knows?
I would be willin' to wager that the enzymes that are responsible for this are the same or similar in the many dmt plants, so isolation could be a bit easier, considering that there would repition in the genomic libraries.
The fungus could be subjected to mutagenic agents, and analyzed in different media. If the fungus grew, one could then extract to see if alkaloids were being produced. if they were, nothing special. If not, then the strain could be transferred to three differing media. The first would be a tryptophan media, the sec. tryptamine, the third n methyl tryptamine, the third dimethyltryptamine. If any of these media produced alkaloids, then the genes triggering the various enzymes could be isolated, cut, and inserted into a yeast cell, or e coli, or whatever.
Meme is insane, imaginary, and lying.
Pysilocybe cubensis is a haploid?
Could mutagenisis by simple treatment (of vegetative cells in culture) with colchicine produce a heritable polyploid line? Fatter finer shrooms?
turning science fact into <<science fiction>>
It's haploid, but dikaryotic. The first phase of the fungus is haploid monokaryotic, but it must achieve somotogamy with a similar organism, wheras the two (both haploid) dual-control the fungus (thus dikaryotic).
I don't know if you could produce a polyploid line quite so easily. The haploid/dikayotic evolutionay plan is responsible for the fungus being sexual, and yet moneocious (sp?), having only one sex.
The spores each contain a single bundle of dna, which makes things easier. I am unaware of any fungus that are polyploid, though.
I'm going to start a new thread, I feel of topic.
Today is opposites day. Everything I say, I mean the opposite.