On the distribution of recombinant organisms; I had a discussion about psilocin biosynthesis in the tryptamine forum some time back. E. Coli. has been discussed as a potential expression system, which is all well and good, but why try switching from eukaryote to prokaryote and risk any possible disulfide bridges and glycosylations? An edible plant expression system might work well, but is more complicated than yeast expression... But how to get the genes out of P. Cubensis? (which appears to be so good at the pathway, since it can produce 3% by dry weight of these products) Well. A quick GenBank search for tryptophan decarboxylase turned up some hits. 6 to be exact. While this is pretty minimal, a BLAST search turned up some interesting stuff. It seems that among the tryp./DOPA/tyrosine decarboxylases, the large large majority (A dozen or so across numerous species) show this highly conserved sequence; gacgcagcttacgc. Generally we would prefer an 18 base probe, but 14 will still work pretty well, with an average occurence of 4^14 it should be relatively specific. Some additional BLAST work on INMTs showed a conserved sequence among several divergent species. This one is even longer, located near the middle of most of these cDNAs; tcctgcgtggccctggagaa. The genome of Aspergeillus Nidulans is around 28 mb, I have no earthly idea how big P. Cubensis' is, but lets assume it's 150 mb just to be safe. From N=1-(1-f)^P, we would need 15000 cosmids (45kb) to cover at 0.99 P. Then probe, isolate those transformants with our gene of interest, and check their cDNA products. Anyway, the DMT producing enzymes in the pathway should be relatively easy to isolate. Affinity binding, protein sequencing, and tRNA preference/low redundancy (or 0) regions could further elucidate the pathway enzymes. I have a friend in mycology who does nothing but make DNA libraries all day, and she seemed to find the idea sound, although quite time consuming for a renegade anarchist Humans deserve the right to ingest and experiment with whatever compounds they like, and this really takes it to the non-scientists. This general schema could be applied to numerous other natural psychadelic products, tyrosine to mescaline, DMT, psilocin, psilocybin, amphetamine/methylamphetamine (bleah, not worth the trouble), etc.. What others can you think of? I know that these products can be cultured already, from their natural hosts, and subsequent extraction, but this opens up alot of possibilities, not all of which are expressed in sufficient quantities to be practical/easily grown. I pulled out some of the best parts for you quicksilver, neat stuff; Psilocybin biosynthesis 4-Hydroxylated or 4-methoxylated indoles are very rare in nature. The only known examples beside the psilocybin-type alkaloids are the 4-hydroxylation of indole acetic acid by Aspergillus niger strains (54), methoxylated b-carbolines from Banisteriopsis argentea and Picrasma javanica, the reserpine analog venenatine from Alstonia venenata, the yohimbine analog mitragynine from Mitragyna speciosa (79, 82 p. 703), and the aminopyrimidyl-indolic meridianines from the tunicate Aplidium meridianum (25). Psilocybin has a simple structure in contrast to the former alkaloids. It is formally derived from tryptophan in five distinct biosynthetic reactions, i.e. decarboxylation, indole-hydroxylation, two N-methylations, and O-phosphorylation (Table 1). Feeding experiments with putative intermediates, analogs of them, or radioactive precursors supported the view that tryptophan decarboxylation is the first biosynthetic step and that O-phosphorylation is the final step. The sequence of the remaining intermediate reaction steps is still unclear. Some authors even suppose a biosynthetic grid with multiple routes to psilocybin (Figure 1) (13, 5, 78). Abbreviation Full name Decarboxylase Tryptophan Decarboxylase Hydroxylase Tryptamine 4-Monooxygenase, Tryptamine : Oxygen Oxidoreductase, 4-hydroxylating Methylase Tryptamine-w-amino-methyltransferase Phosphorylase Psilocin-O-phosphotransferase Table 1. The psilocybin synthesizing enzymes. The full name follows standard enzyme nomenclature while the abbreviated name is used throughout this text. The enzymes may actually consist of several related enzyme with slightly different substrate specifities. Expression cloning procedure For a definitive evaluation of psilocybin biosynthesis, the participating enzymes need to be isolated and tested for their substrate specifities and activities. The feasible way to do this was by genetically cloning and heterologous expression. The cloning methodology chosen is an alternative to PCR based procedures and has been devoloped and used succesfully to clone a broad range of fungal enzymes (21, 20, 15, 14, 53). It consists of ligating a cDNA library into an expression vector (pYes2), transforming a host organism (S. cerevisiae), and a screening the resulting colonies for enzyme activity (Figure 2). The success of such expression cloning procedures depends on reliable and sensitive enzyme assays for the colony screening step. Indeed it was possible to find such assays for all enzymes of the psilocybin biosynthesis pathway (Table 2). A second important requirement is the use of biomaterial containing high amounts of the respective enzymes mRNA. Usually this is a growing tissue containing the metabolites of interest. In this study still developing Psilocybe tampanensis sclerotia were used for mRNA extraction. They grew rapidly and reliably on a special medium and contained high amounts of psilocybin and psilocin. Total RNA isolation from enzyme producing biomaterial \/ mRNA preparation \/ cDNA synthesis \/ Ligation into E. coli / S. cerevisiae shuttle expression vector (pYes2) \/ Amplification in E. coli \/ Colony pooling and plasmid preparation (20 pools of 5,000 colonies each) \/ Yeast transformations \/\/\/\/ Enzyme activity screenings of colonies by color reactions after inducing expression (galactose) \/ Rescreening positive clones \/ Cross-transformation of E. coli, insert sequencing and analysis Figure 2. Expression cloning flow scheme. As used by Dalbøge et al. to clone a broad range of fungal enzymes. Enzyme Screening procedure Decarboxylase 5-fluoro-tryptophan resistance Hydroxylase feeding tryptamines, Keller's reaction Methylase feeding tryptamines, derivatization and removal of substrate, radioactive detection Phosphorylase feeding psilocin, derivatization of substrate, Keller's reaction after phosphatase treatment Table 2. Enzyme activity screening procedures. All tests can be performed on colonies to allow a parallel screening procedures. The Keller's reaction is a very sensitive and specific color reaction for hydroxylated indoles. Mushroom media and culturing Media ingredients and sources: dried unrefined sugar-cane extract (Rapadura, organic food or third world stores), sugar-beet syrup (75% dry matter, food stores), mixed pollen (organic or health food stores), dry yeast extract (Oxoid, England), peptone (Difco Laboratories), agar (Merck, Germany), commercial malt extract / yeast solutions "Salvator" (containing 18.3% stammwuerze) and "Hefe Weissbier dunkel" (dark unfiltered wheat beer, containing 12.4% stammwuerze, both from Paulaner, Munich, Germany). The mycelia were cultured on Parafilm sealed MEY plates (6% malt extract syrup, 0.6% yeast extract, 1.5% agar) at 28°C ± 2°C in an incubator (6). For propagation 1 cm x 1 cm blocks were cut out and transferred to the middle of a new agar plate under sterile conditions. Plant hormones were added as ethanolic solutions. Media containing KH2PO4, pollen, acetic acid, citric acid, and ascorbic acid medium were autoclaved after adjusting the pH. Media from beer (pH around 5.5) were autoclaved for 40 min. Mushroom extraction For a simple but efficient extraction of psilocin as well as psilocybin the following method was applied (98, 58, 45). 7.5 mg lyophilized mushroom material was homogenized in a 1 ml glass mortar with 250 µl methanolic extraction solvent (75% MeOH, 0.1% ascorbic acid). This suspension and 2 x 125 µl methanolic rinsings were pooled in a microcentrifuge tube. After agitation for 10 min at RT the tube was centrifuged for 2 min at 14,000 rpm at RT (5415C centrifuge), the supernatant was transferred to a fresh tube, and the pellet was resuspended in 250 µl ethanolic extraction solvent (75% EtOH, 0.1% ascorbic acid). After agitation for 10 min at RT and centrifugation as above the supernatants were pooled and stored at -20°C in an airtight microcentrifuge tube. Reagents and procedures from the cDNA Synthesis System Kit (Gibco BRL) were used in combination with a vector primed cDNA synthesis literature method (85). This approach was not succesful. Here's a cool transformant screening procedure; 5-Fluoro-tryptophan decarboxylase screening For tryptophan decarboxylase screening transformed FY 73 cells were plated directly onto SC-gal containing 0.5 - 1.5 mM 5-fluoro-tryptophan and were incubated up to 6 days at 30°C. No-Go for submerged culture; The tested Psilocybe strains showed variable preferences for the tested media. In general, the defined Leatham’s media, sugar-cane medium, and beer-based media were poor substrates compared to malt extract based media. The submerged culture produced the highest amounts of biomass, followed by the surface cultures on soft agar (0.2%). But under both conditions the mycelia did not produce measurable amounts of alkaloids. The same was true for Ps. tampanensis submerged cultures in 6% malt extract with no supplement, 0.3% yeast extract, 2% pollen, or both added (data not shown). Ps. cubensis, Ps. tampanensis, Ps. azurescens, and Ps. cyanescens were grown in different media. After 14 days the mycelium was harvested, weighed, extracted, standardized, and analyzed for psilocin and psilocybin content. Ps. cubensis produced psilocin, but no psilocybin under the conditions analyzed. In contrast Ps. tampanensis and Ps. azurescens produced both alkaloids. Ps. azurescens and Ps. cyanescens were growing very slowly on all media tested. Psilocin and psilocybin Rf values Psilocin and psilocybin references (in slightly acidic solution, isolated from Ps. cyanescens fruiting bodies) were TL-chromatographed using various solvent systems. The plates were stained with Van Urk’s reagent (DMCA modification) and Rf values were observed (Table
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Chrom. 593: 201 (1992) Tryptophan->Tryptamine DEFINITION C.roseus tdc gene for tryptophan decarboxylase. ACCESSION X67662 /translation="MGSIDSTNVAMSNSPVGEFKPLEAEEFRKQAHRMVDFIADYYKN VETYPVLSEVEPGYLRKRIPETAPYLPEPLDDIMKDIQKDIIPGMTNWMSPNFYAFFP ATVSSAAFLGEMLSTALNSVGFTWVSSPAATELEMIVMDWLAQILKLPKSFMFSGTGG GVIQNTTSESILCTIIAARERALEKLGPDSIGKLVCYGSDQTHTMFPKTCKLAGIYPN NIRLIPTTVETDFGISPQVLRKMVEDDVAAGYVPLFLCATLGTTSTTATDPVDSLSEI ANEFGIWIHVDAAYAGSACICPEFRHYLDGIERVDSLSLSPHKWLLAYLDCTCLWVKQ PHLLLRALTTNPEYLKNKQSDLDKVVDFKNWQIATGRKFRSLKLWLILRSYGVVNLQS HIRSDVAMGKMFEEWVRSDSRFEIVVPRNFSLVCFRLKPDVSSLHVEEVNKKLLDMLN STGRVYMTHTIVGGIYMLRLAVGSSLTEEHHVRRVWDLIQKLTDDLLKEA" DEFINITION Oryctolagus cuniculus indolethylamine N-methyltransferase (INMT) mRNA, complete cds. ACCESSION AF077826 /translation="MEGGFTGGDEYQKHFLPRDYLNTYYSFQSGPSPEAEMLKFNLEC LHKTFGPGGLQGDTLIDIGSGPTIYQVLAACESFKDITLSDFTDRNREELAKWLKKEP GAYDWTPALKFACELEGNSGRWQEKAEKLRATVKRVLKCDANLSNPLTPVVLPPADCV LTLLAMECACCSLDAYRAALRNLASLLKPGGHLVTTVTLQLSSYMVGEREFSCVALEK EEVEQAVLDAGFDIEQLLYSPQSYSASTAPNRGVCFLVARKKPGS" This can also catalyze the second methylation according to quicksilver (who has been alot of help, and I am very appreciative of). Complete mRNAs for every step, nothing too gigantic either...