The Hive > Tryptamine Chemistry
Enzyme Sequences
bioboy:
1: Plant Mol Biol 1997 Aug;34(6):935-48
Molecular cloning and characterization of desacetoxyvindoline-4-hydroxylase, a
2-oxoglutarate dependent-dioxygenase involved in the biosynthesis of vindoline in
Catharanthus roseus (L.) G. Don.
A 2-oxoglutarate-dependent dioxygenase (EC 1.14.11.11) which catalyzes the 4-hydroxylation of
desacetoxyvindoline was purified to homogeneity. Three oligopeptides isolated from a tryptic digest of the
purified protein were microsequenced and one oligopeptide showed significant homology to hyoscyamine 6
beta-hydroxylase from Hyoscyamus niger. A 36-mer degenerate oligonucleotide based on this peptide sequence was used to screen a Catharanthus roseus cDNA library and three clones, cD4H-1 to -3, were isolated. Although none of the three clones were full-length, the open reading frame on each clone encoded a putative protein containing the sequence of all three peptides. Primer extension analysis suggested that cD4H-3, the longest cDNA clone, was missing 156 bp at the 5' end of the clone and sequencing of the genomic clone,
gD4H-8, confirmed these results. Southern blot analysis suggested that d4h is present as a single-copy gene in C. roseus which is a diploid plant, and the significant differences in the sequence of the 3'-UTR between
cD4H-1 and -3 suggest that they represent dimorphic alleles of the same hydroxylase. The identity of the clone
was further confirmed when extracts of transformed Escherichia coli expressed D4H enzyme activity. The D4H
clone encoded a putative protein of 401 amino acids with a calculated molecular mass of 45.5 kDa and the
amino acid sequence showed a high degree of similarity with those of a growing family of
2-oxoglutarate-dependent dioxygenases of plant and fungal origin.
KrZ:
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 8).
Solvent system Psilocin Rf Psilocybin Rf
H2O: MeOH: AcOH (50 + 50 + 1) 0.92 0.88
MeOH 0.54 0.11
Acetone 0.30 0.06
MeOH: AcOH (1 + 1) 0.33 0.13
H2O 0.88 0.47
H2O: AcOH (1 + 1) 0.88 0.76
nPrOH: H2O: AcOH (10 + 3 + 3) 0.71 0.39
CH2Cl2 0.00 0.00
Best Host;
Yeast as host organism
An important factor for a succesful expression cloning procedure is the choice of an adequate host organism. Yeasts, especially S. cerevisiae, are easy to culture and grow rapidly. As well efficient transformation techniques in common with bacteria have been developed. Otherwise they have many compartments and posttranslational processing and transport systems in common with complex multicellular eukaryotes, especially with other fungi like mushrooms. For the crosstransformation from positive clones into E. coli simple methods exist (90, 84).
REFERENCES
1. Brassinosteroids. G. Adam, V. Marquardt. Phytochem. 25: 1787 (1986)
2. Biosynthesis of psilocybin in submerged culture of Psilocybe cubensis. S. Agurell, S. Blomkvist, P. Catalfomo. Acta Pharm. Suecica 3: 37 (1966)
3. Biosynthetic studies on ergot alkaloids and related indoles. S. Agurell. Acta Pharm. Suecica 3: 71 (1966)
4. A biosynthetic sequence from tryptophan to psilocybin. S. Agurell, J. G. L. Nilsson. Tet. Lett. 9: 1063 (1968)
5. Biosynthesis of psilocybin. S. Agurell, J. G. L. Nilsson. Acta Chem. Scand. 22: 1210 (1968)
6. The influence of temperature of mycelial growth of Psilocybe, Paneolus, and Copelandia. R. W. Ames. Mycopath. et Mycol. Appl. 9: 268 (1958)
7. Occurrence of psilocybin and psilocin in certain Conocybe and Psilocybe species. R. G. Benedict, L. R. Brady, A. H. Smith, V. E. Tyler. Lloydia. 25: 156 (1972)
8. Galerina steglichii spec. nov., ein halluzinogener Häubling. H. Besl. Z. Mycol. 59: 215 (1993)
9. Quantitative analysis of psilocybin and psilocin in Psilocybe baeocystis (Singer and Smith) by high-performance liquid chromatography and by thin layer chromatography. M. W. Beug, J. Bigwood. J. Chrom. 207: 379 (1981)
10. Psilocybin and psilocin levels in twenty species from seven genera of wild mushrooms in the pacific northwest, USA. M. W. Beug, J. Bigwood. J. Ethnopharmacol. 5: 271 (1982)
11. Tryptophan als biogenetische Vorstufe des Psilocybins. A. Brack, A. Hofmann, F. Kalberer, H. Kobel, J. Rutschmann. Arch. Pharm. 294: 230 (1961)
12. The production of psilocybin in submerged culture by Psilocybe cubensis. P. Catalfomo, V. E. Tyler. Lloydia 27: 53 (1964)
13. Psilocin, bufotenine and serotonin: historical and biosynthetic observations. W. C. Chilton, J. Bigwood, R. E. Jensen. J. Psyched. Drugs 11: 61 (1979)
14. Expression cloning, purification and characterization of a b-1,4-galactanase from Aspergillus aculeatus. S. Christgau, S. Sandal, L. V. Kofod, H. Dalbøge. Curr. Genet. 27: 135 (1995)
15. Pectin methyl esterase from Aspergillus aculeatus: expression cloning in yeast and characterization of the recombinant enzyme. S. Christgau, L. V. Kofod, T. Halkier, L. N. Andersen, M. Hockauf, K. Dörreich, H. Dalbøge, S. Kauppinen. Biochem. J. 319: 705 (1996)
16. Determination of psilocybin in Psilocybe semilanceata using high-performance liquid chromatography on a silica column. A. L. Christiansen, K. E. Rasmussen, F. Tønnesen. J. Chrom. 210: 163 (1981)
17. Screening of hallucinogenic mushrooms with high-performance liquid chromatography and multiple detection. A. L. Christiansen, K. E. Rasmussen. J. Chrom. 270: 293 (1983)
18. Ultra-fast alkaline lysis plasmid extraction (UFX). R. S. Cormack, I. E. Somssich. Elsevier Techn. Tips Online (1997)
19. Carbon source dependent differences in the composition of the cell walls of the basidiomycete Picnoporus cinnabarinus. J. A. Cury, D. Amaral. Can. J. Microbiol. 23: 1313 (1977)
20. Using molecular techniques to identify new microbial biocatalysts. H. Dalbøge, L. Lange. Trends Biotech. 16: 265 (1998)
21. A novel method for efficient expression cloning of fungal enzyme genes. H. Dalbøge, H. P. Heldt-Hansen. Mol. Gen. Genetics 243: 253 (1994)
22. Second-strand cDNA synthesis with E. coli DNA polymerase I and RNase H: the fate of information at the mRNA 5’ terminus and the effect of E. coli DNA ligase. J. M. D’Alessio, G. F. Gerard. Nucl. Acid Res. 16: 1999 (1988)
23. Ligation of EcoRI endonuclease generated DNA fragments into linear and circular structures. A. Dugaiczyk, H. W. Boyer, H. M. Goodman. J. Mol. Biol. 96: 171 (1975)
24. A highly sensitive and non-lethal beta-galactosidase plate assay for yeast. H. M. Duttweiler. Trends Gen. 12: 340 (1996)
25. Indole alkaloids from the tunicate Aplidium meridianum. L. H. Franco, E. B. Joffe, L. Puricelli, M. Tatian, A. M. Seldes, J. A. Palerma. J. Nat. Prod. 61: 1130 (1998)
26. Der erste Nachweis des Vorkommens von Psilocybin in Rißpilzen. J. Gartz, G. Drewitz. Z. Mycol. 51: 199 (1985)
27. Zur Untersuchung von Psilocybe semilanceata (Fr.) Kumm. J. Gartz. Pharmazie 40: 506 (1985)
28. Vergleichende dünnschichtchromatographische Untersuchung zweier Psilocybe- und einer halluzinogenen Inocybeart. J. Gartz. Pharmazie. 40: 134 (1985)
29. Dünnschichtchromatographische Analyse der Inhaltsstoffe von Pilzen der Gattung Stropharia. J. Gartz. Pharmazie. 40: 134 (1985)
30. Quantitative Bestimmung der Indolderivate von Psilocybe semilanceata (Fr.) Kumm. J. Gartz. Biochem. Physiol. Pflanzen 181: 117 (1986)
31. Vorkommen von Psilocybin und Baeocystin in Fruchtkörpern von Pluteus salicinus. J. Gartz. Planta Med. 290 (1987)
32. Variation der Indolalkaloide von Psilocybe cubensis durch unterschiedliche Kulturbedingungen. J. Gartz. Beiträge zur Kenntnis der Pilze Mitteleuropas 3: 257 (1987)
33. Biotransformation of tryptamine in fruiting body of Psilocybe cubensis. J. Gartz. Planta Medica 55: 249 (1988)
34. Biotransformation of tryptamine derivatives in mycelial cultures of Psilocybe. J. Gartz. J. Basic Microbiol. 29: 347 (1989)
35. Analysis and cultivation of fruit bodies and mycelia of Psilocybe bohemica. J. Gartz, G. K. Müller. Biochem. Physiol. Pflanzen. 184: 337 (1989)
36. Analyse der Indolderivate in Fruchtkörpern und Mycelien von Paneolus subalteatus (Berk. & Br) Sacc. J. Gartz. Biochem. Physiol. Pflanzen 184: 171 (1989)
37. Bildung und Verteilung der Indolalkaloide in Fruchtkörpern, Mycelien und Sklerotien von Psilocybe cubensis. J. Gartz. Beiträge zur Kenntnis der Pilze Mitteleuropas 5: 167 (1989)
38. Growth-promoting effect of a brassinosteroid in mycelial cultures of the fungus Psilocybe cubensis. J. Gartz, G. Adam, H.-M. Vorbrodt. Naturw. 77: 388 (1990)
39. Einfluß von Phosphat auf Fruktifikation und Sekundärmetabolismen der Myzelien von Psilocybe cubensis, Psilocybe semilanceata und Gymnopilus purpuratus. J. Gartz. Z. Mycol. 57: 149 (1991)
40. Ethnomycology, biochemistry, and cultivation of Psilocybe samuiensis Guzmán, Bandala and Allen, a new psychoactive fungus from Koh Samui, Thailand. J. Gartz, J. W. Allen, M. D. Merlin. J. Ethnopharmacol. 43: 73 (1994)
41. Occurence of psilocybin and psilocin in Psilocybe pseudobullacea (Petch) Pegler from the Venezuelan Andes. V. Marcano, A. Morales Méndez, F. Castellano, F. J. Salazar, L. Martinez. J. Ethnopharmacol. 43: 157 (1994)
42. Transforming yeast with DNA. R. D. Gietz, R. H. Schiestl. Meth. Mol. Cell. Biol. 5: 255 (1995)
43. A chimaeric tryptophan decarboxylase gene as a novel selectable marker in plant cells. O. J. Goddijn, P. M. van der Duyn-Schouten, R. A. Schilperoort, J. H. Hoge. Plant Mol. Biol. 22: 907 (1993)
44. The plant growth regulator methyl jasmonate inhibits aflatoxin production by Aspergillus flavus. M. Goodrich-Tanrikulu, N. E. Mahoney, S. B. Rodriguez. Microbiol. (reading) 141: 2831 (1995)
45. Determination of psilocin and 4-hydroxyindole-3-acetic acid in plasma by HPLC-ECD and pharmacokinetic profiles of oral and intravenous psilocybin in man. F. Hasler, D. Bourquin, R. Brenneisen, T. Bär, F. X. Vollenweider. Pharm. Acta. Helv. 72: 175 (1997)
46. The occurence of psilocybin in Gymnopilus species. G. M. Hatfield, L. J. Valdes, A. H. Smith. Lloydia 41: 140 (1978)
47. Verfahren zur Herstellung und Gewinnung von Psilocybin und Psilocin. R. Heim, A. Hofmann, A. Brack, H. Kobel, R. Cailleux. DBP patent 1087321 (1959)
48. LSD, mein Sorgenkind. A. Hofmann. Klett-Cotta, Stuttgart, Germany (1979),
engl. transl.: LSD, my problem child. McGraw-Hill, New York (1980)
49. Psilocybin and Psilocin, zwei psychotrope Wirkstoffe aus mexikanischen Rauschpilzen. A. Hofmann, R. Heim, A. Brack, H. Kobel, A. Frey, H. Ott, Th. Petrzilka, F. Troxler. Helv. Chim. Acta 52: 1557 (1959)
50. Dephosphorylation psilocybin to psilocin by alkaline phosphatase. A. Horita, L. J. Weber. Proc. Soc. Exp. Biol. Med. 106: 32 (1961)
51. High efficiency transformation of Escherichia coli with plasmids. H. Inoue, H. Nojima, H. Okayama. Gene 96: 23 (1990)
52. Isolation of psilocybin from Psilocybe argentipes and ist determination in specimens of some mushrooms. Y. Koike, K. Wada, G. Kusano, S. Nozoe. J. Nat. Prod. 44: 362 (1981)
53. Cloning and characterization of two structurally and functionally divergent rhamnogalacturonases from Aspergillus aculeatus. L. V. Kofod, S. Kauppinen, S. Christgau, L. N. Andersen, H. P. Heldt-Hansen, K. Dörreich, H. Dalbøge. J. Biol. Chem. 269: 29182 (1994)
54. Hydroxylation of indolyl-3-acetic acid by the fungus Aspergillus niger IBFM-F-12. K. A. Koshcheenko, T. G. Baklashova, A. G. Kozlavskii, M. U. Arinbasarov, G. K. Skriabin. Prikl. Biokhim. Mikrobiol. 13: 248 (1977)
55. Sprühreagentien. K. G. Krebs, D. Heusser, H. Wimmer. in: Dünnschichtchromatographie, Ed. E. Stahl, Springer Verlag Berlin, Heidelberg, New York, 2. ed., p. 813 (1967)
56. Gymnopilus purpuratus, ein psilocybinhaltiger Pilz adventiv im Bezirk Rostock. H. Kreisel, U. Lindequist. Z. Mycol. 54: 73 (1988)
57. High-performance liquid chromatographic determination of some psychotropic indole derivatives. R. Kysilka, M. Wurst. J. Chrom. 21: 435 (1989)
58. A novel extraction procedure for psilocybin and psilocin determination in mushroom samples. R. Kysilka, M. Wurst. Planta Med. 56: 327 (1990)
59. H. Laatsch, Department of Organic Chemistry, University of Göttingen, Germany. preliminary communication
60. A chemically defined medium for the fruiting of Lentinus edodes. G. F. Leatham. Mycologia 75: 905 (1983)
61. Effects of growth regulating substances on fungi. K. M. Leelavathy. Can. J. Microbiol. 15: 713 (1968)
62. Production of psilocybin in Psilocybe baeocystis saprophytic culture. A. Y. Leung, A. H. Smith, A. G. Paul. J. Pharm. Sci. 54: 1576 (1965)
63. Baeocystin, a mono-methyl analog of psilocybin from Psilocybe baeocystis saprophytic culture. A. Y. Leung, A. G. Paul. J. Pharm. Sci. 56: 146 (1967)
64. Baeocystin and norbaeocystin: new analogs of psilocybin from Psilocybe baeocystis. A. L. Leung, A. G. Paul. J. Pharm. Sci. 57: 1667 (1968)
65. The relationship of carbon and nitrogen nutrition of Psilocybe baeocystis to the production of psilocybin and ist analogs. A. Y. Leung, A. G. Paul. Lloydia 32: 66 (1969)
66. Molecular cloning. J. Sambrook, E. F. Fritsch, T. Maniatis. Cold Spring Harbour Laboratory Press (1989)
67. Dextran blue as an aid for DNA precipitation and gel loading. U. Matysiak-Scholze, S. Dimmeler, M. Nehls. Elsevier Techn. Tips Online (1996)
68. Psilocybe semilanceata (Fr.) Quel. (Spitzkegeliger Kahlkopf). H. Michaelis. Z. Pilzkunde 43: 305 (1977)
69. Multiple Molecular forms of diarylpropane oxygenase, an H2O2 requiring, lignin degrading enzyme from Phanerochaete chrysosporium. V. Renganathan, K. Miki, M. H. Gold. Arch. Biochem. Biophys. 241: 304 (1985)
70. Permeabiliation of microorganisms by Triton X-100. G. F. Miozzari, P. Niederberger, R. Hütter. Anal. Biochem. 90: 220 (1978)
71. Interrelationship of phosphate nutrition, nitrogen metabolism, and accumulation of key secondary metabolites in saprophytic cultures of Psilocybe cubensis, Psilocybe cyanescens, and Paneolus campanulatus. J. M. Neal, R. G. Benedict, L. R. Brady. J. Pharm. Sci. 57: 1661 (1968)
72. Pharmacotheon. J. Ott. Natural Products Co., Kennewick, WA (1993)
73. Transformation of E. coli using homopolymer-linked plasmid chimeras. S. L. Peacock, C. M. McIver, J. J. Monahan. Biochim. Biophys. Acta 655: 243 (1981)
74. Determination of psilocybin in Psilocybe semilanceata by capillary zone electrophoresis. S. Pedersen-Bjergaard, E. Sannes, K. E. Rasmussen, F. Tønnesen. J. Chrom. 694: 375 (1997)
75. Psilocybian mycetismus with special reference to Paneolus. S. H. Pollock. J. Psyched. Drugs 8: 43 (1976)
76. GLC-mass spectral analysis of psilocin and psilocybin. D. B. Repke, D. T. Leslie, D. M. Mandell, N. G. Kish. J. Pharm. Sci. 66: 743 (1977)
77. Baeocystin in Psilocybe semilanceata. D. B. Repke. J. Pharm. Sci. 66: 113 (1977)
78. Baeocystin in Psilocybe, Conocybe and Paneolus. D. B. Repke. Lloydia 40: 566 (1977)
79. Psilocin analogs. III. Synthesis of 5-methoxy- and 5-hydroxy-1,2,3,4-tetrahydro-9H-pyrido[3,4-b]indoles. D. B. Repke, W. J. Ferguson. J. Het. Chem. 19: 845 (1982)
80. Site of action of growth inhibitory tryptophan analogues in Catharanthus roseus cell suspension cultures. F. Sasse, M. Buchholz, J. Berlin. Z. Naturforsch. 38c: 910, 916 (1983)
81. Pihkal. A. Shulgin, A. Shulgin. Transform Press, Berkeley, CA (1991)
82. Tihkal. A. Shulgin, A. Shulgin. Transform Press, Berkeley, CA (1997)
83. Psilocybin in Fruchtkörpern von Inocybe aeruginascens. M. Smerdzieva, M. Wurst, T. Koza, J. Gartz. Planta Med. 83 (1986)
84. A rapid and inexpensive method for isolation of shuttle vector DNA from yeast for the transformation of E. coli. R. Soni, J. A. H. Murray. Nucl. Acid. Res. 20: 5852 (1992)
85. A highly efficient directional cDNA cloning method utilizing an asymmetrically tailed linker-primer plasmid. N. Spickofsky, R. F. Margolskee. Nucl. Acid Res. 19: 7105 (1991)
86. The mushroom cultivator. P. Stamets, J. S. Chilton. Agarikon Press, Olympia, WA (1983)
87. Occurence of 5-hydroxylated indole derivatives in Paneolina foenescii (Fries) Kuehner from various origin. T. Stijve, C. Hischenhuber, D. Ashley. Z. Mycol. 50: 361 (1984)
88. Psilocin, psilocybin, serotonin and urea in Paneolus cyanescens from various origin. T. Stijve. Persoonia 15: 117 (1992)
89. Convenient and effective methods for in vitro cultivation of mycelium and fruiting bodies of Lentinus edodes. Y. H. Tan, D. Moore. Mycol. Res. 96: 14077 (1992)
90. A simple method for rescuing autonomous plasmids from fission yeast. A. Topal, S. Karaer, G. Temizkan. Elsevier Techn. Tips Online (97)
91. Basic yeast methods. J. H. Toyn. Meth. Mol. Cell. Biol. 5: 249 (1995)
92. Influence of plant hormones on a wood-rotting fungus, Coriolus versicolor. S. I. Tsujiyama, J. I. Azuma, K. Okamura. Transact. Mycol. Soc. Japan 34: 369
93. Occurence of serotonin in a hallucinogenic mushroom. V. E. Tyler, JR. Science. 128: 718 (1958)
94. Exogenous regulators in the mycelium of Pleurotus ostreatus after exogenous application. K. Vinklarkova, Z. Sladky. Folia Microbiol. Praha 23: 55 (1978)
95. Mushrooms, Russia and history. Volumes 1 and 2. V. P. Wasson, R. G. Wasson. Pantheon Books, New York (1957)
96. A new psilocybian species of Copelandia. R. A. Weeks. J. Nat. Prod. 42: 469 (1979)
97. Analysis of psychotropic compounds in fungi of the genus Psilocybe by reversed phase high-performance liquid chromatography. M. Wurst, M. Semerdzieva, J. Vokoun. J. Chrom. 286: 229 (1984)
98. Analysis and isolation of indole alkaloids of fungi by high-performance liquid chromatography. M. Wurst, R. Kysilka, T. Koza. J. Chrom. 593: 201 (1992)
Quicksilver:
Bioboy,
Thanks for the ref! What do you think the odds of this enzyme working on a more simple compound (tryptamine) compared to the one the authors used (desacetoxyvindoline) ??
Don't suppose anybody has molecular ligand-binding software they want to share? (Windows or MacOS)
(e.g. Sybyl, Autodock, Insight ...but those are all unix i think??)
-quicksilver-
stormwind:
Man Oh Man - when you guys get these yeasts bred and multiplied, please let J***F or someone market some - I'm kinda short on realchem practical knowledge, but I could handle some sourdough starter without blowing the house up I think...
"I held my nose I closed my eyes
I took a drink
I didn't know if it was day or night..."
ZyGoat:
This is probably a stupid question, but why go to all the trouble of creating a transgenic psilocin producing yeast/bacteria when there already exists a load of fungal organisms capable of this?
Navigation
[0] Message Index
[#] Next page
[*] Previous page
Go to full version