From
Subject: MAPS: Molecular Entheogenetics: Biosynthesis of Mescaline and Tryptamines
From: dm_telvis@xxxxxxxxx
Date: Sun, 17 May 1998 19:27:43 -0400 (EDT)

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

Overview

I 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 or psilocin.

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 references).

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 time consuming, 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 hybrid strands

  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 products

  9. lysis of expanded pools

  10. extraction and restriction of plasmids and agarose gel separation of fragments

  11. ligation of characterized fragments to yeast plasmid

  12. sequential transformation of yeast cells (see references)

  13. selection of transformed yeast clones

Costs

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 the field.

None of these techniques are in the least speculative. They are the tried and proven workhorse tools of standard molecular biology. Only the application remains.

dm_telvis@yahoo.com

References

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 antitussives. 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 genetically engineered bacteria. BioTechnology 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, same authors and reference.

"Molecular Cloning : a Laboratory Manual" by Maniatis, Fritsch and Sambrooke, Academic Press, 1989




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