CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority from U.S. Provisional Application Ser. No. 62/936,387 filed on Nov. 15, 2019, which is incorporated herein by reference in its entirety.
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT Not Applicable.
INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ON A COMPACT DISC The Sequence Listing, which is a part of the present disclosure, includes a computer readable form and a written sequence listing comprising nucleotide and/or amino acid sequences of the present invention. The sequence listing information recorded in computer readable form is identical to the written sequence listing. The ASCII text file, entitled “psilocybinseq.text”, was created on Nov. 15, 2019 using PatentIn version 3.5 and is incorporated herein by reference in its entirety. The subject matter of the Sequence Listing is incorporated herein by reference in its entirety.
FIELD The present invention generally relates to the production of psilocybin and its intermediates (e.g., tryptamine, 4-hydroxytryptamine, norbaeocystin, baeocystin, and psilocin) in a modified heterologous microorganism.
INTRODUCTION Mental health problems, which may also be referred to as mental illness or psychiatric disorder, are behavioral or mental patterns which impair the functioning of individuals across the world. Psilocybin has been increasingly evaluated for treating mental health problems. Such mental health disorders include: personality disorders, anxiety disorders, major depressions, and various addictions. In contrast to anxiolytic medicines, usage of psilocybin does not lead to physical dependence.
SUMMARY The present teachings include a recombinant host organism. The recombinant host organism can include: a plurality of cells transfected by a set of genes for synthesizing psilocybin in the recombinant host organism via at least a first pathway and a second pathway. The recombinant host organism can be a fungal species comprising: Schizosaccharomyces cerevisiae, Schizosaccharomyces japonicus, Schizosaccharomyces pombe, Schizosaccharomyces cryophilus, Saccharomyces cerevisiae, Kluyveromyces lactis, Kluyveromyces dobzhanskii, and Yarrowia lipolytica. The set of genes can include any combination of a gene selected from a group consisting of PsiD, PsiH, PsiK, and PsiM.
In accordance with a further aspect, PsiD can comprise codon optimized nucleic acid sequences SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3 that encode for isolated amino acid sequences SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16, respectively; PsiH can comprise codon optimized nucleic acid sequences SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6 that encode for isolated amino acid sequences SEQ ID NO: 17 SEQ ID NO: 18, and SEQ ID NO: 19, respectively; PsiK can comprise codon optimized nucleic acid sequences SEQ ID NO: 7 and SEQ ID NO: 8 that encode for isolated amino acid sequences SEQ ID NO: 20 and SEQ ID NO: 21, respectively; and PsiM can comprises codon optimized nucleic acid sequences SEQ ID NO: 9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO: 12, and SEQ ID NO: 13 that encode for isolated amino acid sequences SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24; SEQ ID NO: 25, and SEQ ID NO: 26, respectively.
In accordance with a further aspect, the set of genes can express amino acid sequences that increase titers of psilocybin in the plurality of cells.
In accordance with a further aspect, the set of genes can synthesize intermediates of psilocybin, wherein the intermediates comprise: tryptamine, 4-hydroxytryptamine, norbaeocystin, baeocystin, and psilocin.
In accordance with a further aspect, a protein can be heterologous to the plurality of cells and an exogenous substrate, wherein the protein is encoded by codon optimized SEQ ID NO: 36.
In accordance with a further aspect, the carbon source can include at least one of: glucose, galactose, sucrose, fructose, corn syrup, corn steep liquor, ethanol, and molasses.
In accordance with another aspect, the first pathway can be a shikimate-chorismate pathway and the second pathway can be a L-tryptophan pathway
In accordance with another aspect, the first pathway can be modified by codon optimized SEQ ID NO: 27, SEQ ID NO. 28, SEQ ID NO: 29, SEQ ID NO: 30, and SEQ ID NO: 31 and the second pathway is modified by codon optimized SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, and SEQ ID NO: 35.
The present teaching include a plurality of sequences containing nucleotides or amino acids for producing psilocybin in a recombinant host organism, wherein the plurality of sequences comprise SEQ ID NO: 1-SEQ ID NO: 36.
In accordance with a further aspect, an isolated amino acid sequence comprises SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16, wherein SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16 can be at least 50% similar to each other, and wherein SEQ ID NO: 14 is encoded by codon optimized SEQ ID NO: 1, SEQ ID NO: 15 is encoded by codon optimized SEQ ID NO: 2, and SEQ ID NO: 16 is encoded by codon optimized SEQ ID NO: 3.
In accordance with a further aspect, an isolated amino acid sequence comprises at least one of: SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19, wherein SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19 are at least 40% similar to each other, and wherein SEQ ID NO: 17 is encoded by codon optimized SEQ ID NO: 4, SEQ ID NO: 18 is encoded by codon optimized SEQ ID NO: 5, and SEQ ID NO: 19 is encoded by codon optimized SEQ ID NO: 6.
In accordance with a further aspect, an isolated amino acid sequence comprises at least one of: SEQ ID NO: 20 and SEQ ID NO: 21, wherein SEQ ID NO: 20 and SEQ ID NO: 21 are at least 85% similar to each other; and wherein SEQ ID NO: 21 is encoded by codon optimized SEQ ID NO: 7 and SEQ ID NO: 22 is encoded by codon optimized SEQ ID NO: 8.
In accordance with a further aspect, an isolated amino acid sequence comprises at least one of: SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, and SEQ ID NO: 26, wherein SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, and SEQ ID NO: 26 are at least 55% similar to each other, and wherein SEQ ID NO: 22 is encoded by codon optimized SEQ ID NO: 9, SEQ ID NO: 23 is encoded by codon optimized SEQ ID NO: 10, SEQ ID NO: 24 is encoded by SEQ ID NO: 11, SEQ ID NO: 25 is encoded by SEQ ID NO: 12, and SEQ ID NO: 26 is encoded by SEQ ID NO: 13.
The present teachings include a method. The method can include: transfecting a plurality of cells in a recombinant host organism a set of genes for synthesizing psilocybin via at least a first pathway and a second pathway; and increasing titers of psilocybin in the plurality of cells via the set of genes; and synthesizing intermediates of psilocybin via the set of genes. The recombinant host organism can be a fungal species comprising: Schizosaccharomyces cerevisiae, Schizosaccharomyces japonicus, Schizosaccharomyces pombe, Schizosaccharomyces cryophilus, Saccharomyces cerevisiae, Kluyveromyces lactis, Kluyveromyces dobzhanskii, and Yarrowia lipolytica. The The set of genes can include a gene from a group consisting of: PsiD, PsiH, PsiK, and PsiM.
In accordance with a further aspect, PsiD can comprise codon optimized nucleic acid sequences SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3 that encode for isolated amino acid sequences SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16, respectively; wherein PsiH can comprise codon optimized nucleic acid sequences SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6 that encode for isolated amino acid sequences SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19, respectively; wherein PsiK can comprise codon optimized nucleic acid sequences SEQ ID NO: 7 and SEQ ID NO: 8 that encode for isolated amino acid sequences SEQ ID NO: 20 and SEQ ID NO: 21, respectively; and wherein PsiM can comprise codon optimized nucleic acid sequences SEQ ID NO: 9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO: 12, and SEQ ID NO: 13 that encode for isolated amino acid sequences SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24; SEQ ID NO: 25, and SEQ ID NO: 26, respectively.
In accordance with a further aspect, the carbon source can include at least one of: glucose, galactose, sucrose, fructose, corn syrup, corn steep liquor, ethanol, and molasses.
In accordance with a further aspect, the method can also include an exogenous substrate and a transporter protein.
In accordance with a further aspect, the first pathway can be a shikimate-chorismate pathway modified by codon optimized SEQ ID NO: 27, SEQ ID NO. 28, SEQ ID NO: 29, SEQ ID NO: 30, and SEQ ID NO: 31 and the second pathway can be a L-tryptophan pathway modified by codon optimized SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, and SEQ ID NO: 35.
In accordance with a further aspect, the transporter protein can be encoded by codon optimized SEQ ID NO: 36.
In accordance with a further aspect, the intermediates can include: tryptamine, 4-hydroxytryptamine, norbaeocystin, baeocystin, and psilocin.
These and other features, aspects, and advantages of the present teachings will become better understood with reference to the following description, examples and appended claims.
DRAWINGS Those of skill in the art will understand that the drawings, described below, are for illustrative purposes only. The drawings are not intended to limit the scope of the present teachings in any way.
FIG. 1 depicts a table of amino acids and codon triplets.
FIG. 2 depicts a table of genes and enzymes inserted into a recombinant host organism.
FIG. 3 depicts the biosynthesis of psilocybin.
FIG. 4-7 depicts sequence alignments.
FIG. 8 depicts endogenous pathways in a host organism.
FIG. 9 depicts a scheme to increase metabolic flux through shikimate-chorismate and L-tryptophan pathways.
FIG. 10 depicts a heterologous recombinant host organism.
FIG. 11 depicts HPLC chromatograms and UV/Vis spectra.
DETAILED DESCRIPTION Abbreviations and Definitions To facilitate understanding of the invention, a number of terms and abbreviations as used herein are defined below as follows:
Amino acids: As used herein, the term “amino acids” refer to the molecular basis for constructing and assembling proteins, such as enzymes. (See FIG. 1 for a table of amino acids.). Peptide bonds (i.e., polypeptides) are formed between amino acids and assemble three-dimensionally (3-D). The 3-D assembly can influence the properties, function, and conformational dynamics of the protein. Within biological systems, the protein may: (i) catalyze reactions as enzymes; (ii) transport vesicles, molecules, and other entities within cells as transporter entities; (iii) provide structure to cells and organisms as protein filaments; (iv) replicate deoxyribonucleic acid (DNA); and (v) coordinate actions of cells as cell signalers.
Nucleotides: As used herein, the term “nucleotides” refers to the molecular basis for constructing and assembling nucleic acids, such as DNA and ribonucleic acid (RNA). There are two types of nucleotides—purines and pyrimidines. The specific purines are adenine (A) and guanine (G). The specific pyrimidines are cytosine (C), uracil (U), and thymine (T). T is found in DNA, whereas U is found in RNA. The genetic code defines the sequence of nucleotide triplets (i.e., codons) for specifying which amino acids are added during protein synthesis.
Genes: As used herein, the term “genes” refers to regions of DNA. Amino acid sequences in the proteins, as defined by the sequence of a gene, are encoded in the genetic code.
The present invention is directed to biosynthetic production of psilocybin and related intermediates in recombinant organisms. The syntheses of psilocybin and intermediates of psilocybin in a laboratory environment typically involve tedious techniques of organic chemistry. Often reproducibility is elusive and the solvents used during the syntheses of psilocybin and intermediates of psilocybin are environmentally toxic. Decarboxylations, selective methylations, and selective phosphorylations can be difficult to obtain via the techniques of organic chemistry. Further, the yields and purity of the intermediates for obtaining the target molecules can be low using the techniques of organic chemistry, where the starting molecule is L-tryptophan and the target molecule is psilocybin.
The systems and method herein disclose more environmentally benign processes which can have higher throughputs (i.e., more robust processes). The systems and methods herein include: (i) growing modified recombinant host cells and thereby yielding a recombinant host organism; (ii) expressing engineered psilocybin biosynthesis genes and enzymes in the recombinant host organism; (iii) producing or synthesizing psilocybin and/or intermediates of psilocybin in the recombinant host organism; (iv) fermenting the recombinant host organism; and (v) isolating the psilocybin and/or intermediates of psilocybin from the recombinant host organism. Endogenous pathways of the recombinant host can be modified by the systems and methods herein to produce high purity psilocybin and/or intermediates of psilocybin.
Reference is made to the figures to further describe the systems and methods disclosed herein.
Referring to FIG. 2, a table lists the enzymes involved in the direct biosynthesis of psilocybin and psilocybin intermediates in species of fungus (i.e., mushrooms). Gene source organisms provide a genetic starting source (i.e., raw gene sequences) which is codon optimized and engineered to function in the recombinant host organisms. The recombinant host organisms include but are not limited to: Schizosaccharomyces cerevisiae, Schizosaccharomyces japonicus, Schizosaccharomyces pombe, Schizosaccharomyces cryophilus, Saccharomyces cerevisiae, Kluyveromyces lactis, Kluyveromyces dobzhanskii, and Yarrowia lipolytica.
Further, the genes/enzymes that are inserted or engineered into the recombinant host are PsiD, PsiH, PsiK, and PsiM.
A PsiD enzyme, which is a decarboxylase (e.g., L-tryptophan decarboxylase) derives from a gene source organism herein—Psilocybe cubensis, Psilocybe cyanescens, and Gymnopilus junonius. The decarboxylase can catalyze the decarboxylation of an aliphatic carboxylic acid (i.e., release carbon dioxide) L-tryptophan to tryptamine and 4-hydroxy-L-tryptophan to 4-hydroxytryptamine, as depicted in FIG. 3.
A PsiH enzyme, which is a monooxygenase (e.g., Tryptamine 4-monooxygenase) derives from a gene source organism herein—Psilocybe cubensis, Psilocybe cyanescens, and Gymnopilus junonius. The monooxygenase can catalyze the oxidative hydroxylation of the phenyl ring of tryptamine to 4-hydroxytryptamine, as depicted in FIG. 3.
A PsiK enzyme, which is a kinase (e.g., 4-hydroxytryptamine kinase) derives from a gene source organism herein—Psilocybe cubensis and Psilocybe cyanescens. The kinase can catalyze the phosphorylation (i.e., adding O═P(OH)2) of the phenolic oxygen of 4-hydroxytryptamine to norbaeocystin, as depicted in FIG. 3. The kinase can also catalyze the phosphorylation of psilocin to psilocybin.
A PsiM enzyme, which is a methyl transferase (e.g., psilocybin synthase) derives from a gene source organism herein—Psilocybe cubensis, Psilocybe cyanescens, Panaeolus cynascens, Gymnopilus junonius, and Gymnopilus dilepis. The methyl transferase can catalyze the alkylation (i.e., adding a methyl (CH3) group) of the primary amine in norbaeocystin to baecystin, as depicted in FIG. 3. Another alkylation can take place where the methyl transferase when the secondary amine of baecystin becomes a tertiary amine of psilocybin, as depicted in FIG. 3.
As depicted in FIG. 3, the engineered PsiD, PsiH, PsiK, and PsiM enzymes act on substrates in the psilocybin biosynthetic pathway to produce intermediates of psilocybin and psilocybin itself. The initial substrate for psilocybin intermediates and psilocybin can be L-tryptophan and/or 4-hydroxy-L-tryptophan. These initial substrates can be produced endogenously in a recombinant host as described and/or provided exogenously to a fermentation involving a recombinant host, whereby the host uptakes the starting substrates to feed into the psilocybin biosynthetic pathway. The recombinant host herein described that is expressing all, one, or multiple combinations of the engineered PsiD, PsiH, PsiK, PsiM genes can produce tryptamine, 4-hydroxytryptamine, norbaeocystin, baeocystin, psilocybin, and psilocin. Psilocybin may be converted to psilocin due to spontaneous dephosphorylation. Psilocin is in turn an intermediate which can be acted on by the PsiK enzyme to produce psilocybin.
As depicted in FIG. 4, the amino acid alignments of recombinant PsiD enzymes are presented. Recombinant PsiD enzymes have been reengineered from various fungal species to function in heterologous recombinant host organisms herein. The gene used in the pair wise alignment is the PsiD gene from the fungal species—Psilocybe cubensis, Psilocybe cyanescens, and Gymnopilus junonius. The alignment is performed with EMBOSS Needle Pair wise Sequence Alignment statistic (EBLOSUM62) with Psilocybe cubensis (PsiD gene) as a reference. The identity percentage and similarity percentage of the amino acid sequences are presented.
For the PsiD gene, codon optimized nucleic acid sequences SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3 encode for isolated amino acid sequences SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16, respectively. SEQ ID NO: 14 is Psilocybe cubensis (PsiD gene); SEQ ID NO: 15 is Psilocybe cyanescens (PsiD gene); and SEQ ID NO: 16 is Gymnopilus junonius (PsiD gene).
As depicted in FIG. 5, the amino acid alignment of recombinant PsiH enzymes are presented. Recombinant PsiH enzymes have been reengineered from various fungal species to function in heterologous recombinant host organisms herein. The gene used in the pair wise alignment is the PsiH gene from the fungal species—Psilocybe cubensis, Psilocybe cyanescens, and Gymnopilus junonius. The alignment is performed with EMBOSS Needle Pair wise Sequence Alignment statistic (EBLOSUM62) with Psilocybe cubensis (PsiH gene) as a reference. The identity percentage and similarity percentage of the amino acid sequences are presented.
For the PsiH gene, codon optimized nucleic acid sequences SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6 encode for isolated amino acid sequences SEQ ID NO: 17 SEQ ID NO: 18, and SEQ ID NO: 19, respectively. SEQ ID NO: 17 is Psilocybe cubensis (PsiH gene); SEQ ID NO: 18 is Psilocybe cyanescens (PsiH gene); and SEQ ID NO: 19 is Gymnopilus junonius (PsiH gene).
As depicted in FIG. 6, the amino acid alignment of recombinant PsiK enzymes are presented. Recombinant PsiK enzymes have been reengineered from various fungal species to function in heterologous recombinant host organisms herein. The gene used in the pair wise alignment is the PsiK gene from the fungal species—Psilocybe cubensis and Psilocybe cyanescens. The alignment is performed with EMBOSS Needle Pair wise Sequence Alignment statistic (EBLOSUM62) with Psilocybe cubensis (PsiK gene) as a reference. The identity percentage and similarity percentage of the amino acid sequences are presented.
For the PsiK gene, codon optimized nucleic acid sequences SEQ ID NO: 7 and SEQ ID NO: 8 encode for isolated amino acid sequences SEQ ID NO: 20 and SEQ ID NO: 21, respectively. SEQ ID NO: 20 is Psilocybe cubensis (PsiK gene) and SEQ ID NO: 21 is Psilocybe cyanescens (PsiK gene).
As depicted in FIG. 7, the amino acid alignment of recombinant PsiM enzymes are presented. Recombinant PsiM enzymes have been reengineered from various fungal species to function in heterologous recombinant host organisms herein. The gene used in the pair wise alignment is the PsiM gene from the fungal species—Psilocybe cubensis, Psilocybe cyanescens, Panaeolus cynascens, Gymnopilus junonius, and Gymnopilus dilepis. The alignment is performed with EMBOSS Needle Pair wise Sequence Alignment statistic (EBLOSUM62) with Psilocybe cubensis (PsiM gene) as a reference. The identity percentage and similarity percentage of the amino acid sequences are presented.
For the PsiM gene, codon optimized nucleic acid sequences SEQ ID NO: 9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO: 12, and SEQ ID NO: 13 encode for isolated amino acid sequences SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24; SEQ ID NO: 25, and SEQ ID NO: 26, respectively. SEQ ID NO: 22 is Psilocybe cubensis (PsiM gene); SEQ ID NO: 23 is Psilocybe cyanescens (PsiM gene); SEQ ID NO: 24 is Panaeolus cynascens (PsiM gene); SEQ ID NO: 25 is Gymnopilus junonius (PsiM gene), and SEQ ID NO: 26 is Gymnopilus dilepis (PsiM gene).
As depicted in FIG. 8, the endogenous pathways of a recombinant host organism produce precursors for the engineered PsiD, PsiH, PsiK, PsiM genes. Pathways relating to chorismate, L-glutamine, and L-serine, feed into the endogenous pathway for L-tryptophan production, which a recombinant host organism expressing the psilocybin biosynthetic pathway herein described can use to create tryptamine, 4-hydroxytryptamine, norbaeocystin, baeocystin, psilocin, and psilocybin. The enzymes in the endogenous pathways of the recombinant host organism are encircled in FIG. 8. Glycolysis and gluconeogenesis in combination with ARO3, ARO4, ARO1, and ARO2 enzymes can be subjected to the depicted precursors at the specified point in the pathway to selectively yield chrorismate. The glutamate biosynthesis pathway in combination with a GLN1 enzyme can be subjected to the depicted precursor at the specified point in the pathway to selectively yield L-glutamine. Glycolysis in combination with SER3, SER33, SER1, and SER2 enzymes can be subjected to the depicted precursors at the specified points in the pathway to selectively yield L-serine. Chorismate and L-glutamine in combination with TRP1, TRP2, TRP3, and TRP4 enzymes can be subjected to the depicted precursors at the specified point to selectively yield (1S,2R)-1-C-indol-3-yl)glycerol 3-phosphate. The addition of L-serine to (1S,2R)-1-C-indol-3-yl)glycerol 3-phosphate in the presence of the TRP1 enzyme can yield L-tryptophan.
As depicted in FIG. 9, a scheme to increase metabolic flux through the shikimate-chorismate and L-tryptophan pathways is disclosed. The increased metabolic flux through the shikimate-chorismate and L-tryptophan pathways increases the production of L-tryptophan, a key precursor compound for the production of psilocybin and intermediates of psilocybin. Specific enzymes in the described native pathways are overexpressed. Enzymes subject to allosteric inhibition are mutated and overexpressed to render the enzymes insensitive to feedback mechanisms. Enzymes that consume pathway intermediates for off-pathway compound production are hereby deleted.
L-tryptophan production is improved herein by overexpressing a series of enzymes that first increase production of the aromatic compound intermediate, chorismate in a series of enzymatic reactions known as the shikimate pathway. As described in FIG. 5, the shikimate-chorismate pathway initial precursors, PEP and E4P are converted into 3-deoxy-D-arabinoheptulosonate 7-phosphate (DAHP), catalyzed by ARO3 and ARO4 enzymes.
Overexpression of the genes encoding ARO3 enzyme (as encoded by codon optimized SEQ ID NO: 29), and a feedback-resistant mutant ARO4 K229L enzyme (as encoded by codon optimized SEQ ID NO: 30) are described herein and can increase metabolic flux through the pathway. In addition, genes that encode key enzymes, ARO1 enzyme (as encoded by codon optimized SEQ ID NO: 27) and ARO2 (as encoded by codon optimized SEQ ID NO: 28) are overexpressed as part of a series of enzymes that can convert DAHP to chorismate. In addition, the gene that encodes the Escherichia coli shikimate kinase II (AROL enzyme) can be overexpressed to increase pathway flux from DHAP to chorismate via codon optimized SEQ ID NO: 31.
Chorismate as a general precursor compound can be converted specifically to L-tryptophan by overexpressing a series of enzymes in the L-tryptophan pathway. As described in FIG. 9, flux through the L-tryptophan pathway can be increased by overexpressing the genes that encode specific enzymes, TRP1 enzyme (as encoded by codon optimized by SEQ ID NO: 32), TRP3 enzyme (as encoded by codon optimized by SEQ ID NO: 34), and TRP4 enzyme (as encoded by codon optimized by SEQ ID NO: 35). Furthermore, overexpression of the gene that encodes the feedback-resistant mutant of TRP2 S76L enzyme (as encoded by SEQ ID NO: 33) is described herein.
Chorismate is a precursor that feeds into the metabolic pathways that produce a variety of aromatic alcohols and aromatic amino acids. The mechanism made operable by systems and methods herein reduce pathway flux into pathways that produce off-pathway targets. As described in FIG. 9, genes that encode native enzymes—PDC5 enzyme and ARO10 enzyme—have been deleted to reduce pathway flux through the pathways that produce aromatic alcohols. The gene that encodes the native enzyme, ARO7 enzyme has been deleted to reduce production of tyrosine and phenylalanine. Genes that encode PDZ1 and PDZ2 enzymes have been deleted to reduce pathway flux through the pABA production pathway.
As depicted in FIG. 10, a modified heterologous recombinant host organism is: (i) expressing endogenous pathways for L-tryptophan; (ii) expressing a recombinant version of the TAT2 L-tryptophan importer protein; and (iii) selectively expressing recombinant psilocybin biosynthetic pathway genes. Such a recombinant host can produce tryptamine, 4-hydroxytryptamine, norbaeocystin, baeocystin, psilocin, and psilocybin from L-tryptophan. L-tryptophan can be created by the host through endogenous pathways (FIG. 8) or engineered pathways (FIG. 9). L-tryptophan may also be fed to the recombinant host organism by media supplementation and up taken by the host expressing the recombinant TAT2 importer protein. Accordingly, contact with the L-tryptophan and the recombinant host organism in the media can selectively direct flux towards psilocybin. Other carbon sources can make contact with the recombinant host organism in the media, wherein the other carbon sources include at least one of: glucose, galactose, sucrose, corn steep liquor, ethanol, fructose, and molasses.
Besides the recombinant TAT2 importer protein, which is encoded by a codon optimized L-tryptophan importer (SEQ ID NO: 36), the nucleotide and amino acid sequences provided are in the order of the psilocybin pathway: PsiD, PsiH, PsiK, and PsiM genes which encode for the respective enzymes. In the systems and methods herein, PsiD enzyme selectively and cleanly catalyzes decarboxylation; the PsiH enzyme catalyzes selective hydroxylation at the 4-position of an indole; the PsiK enzyme catalyzes selective phosphorylation at the hydroxylated 4-position of an indole; and the PsiM enzyme catalyzes selective and stepwise methylations of an amine group, respectively.
By expressing the PsiD gene in the recombinant host organism, codon optimized nucleic acid sequences SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3 encode for isolated amino acid sequences SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16, respectively. Using the techniques of organic chemistry, decarboxylations would require harsh and toxic tin hydrides (e.g., Barton Decarboxylation), as opposed to the selective and clean decarboxylation by the PsiD enzyme in the recombinant host.
By expressing the PsiH gene in the recombinant host organism, codon optimized nucleic acid sequences SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6 encode for isolated amino acid sequences SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19, respectively. Phenyl group functionalization is often done at high temperatures and pressures, while leading to a mixture of products (e.g., hydroxylations at the 5, 6, and 7 positions of the indole). The regioisomers of the hydroxylated products at the 5, 6, and 7 positions of the indole are structurally distinct from each other, but also structurally similar to each other. Separation of such regioisomers can be very challenging and requires cumbersome separation techniques (e.g., slow column chromatography with poor separation (i.e., the regiosiomers have similar Rf values to each other) and low accompanying yields). In contrast, the PsiH enzyme catalyzes selective hydroxylation of indole at the 4-position in the recombinant host organism herein at standard room conditions (˜25 degrees Celsius at ˜1 atm of atmospheric pressure). The systems and methods herein can produce and increase the titers of the hydroxylated indole at the 4-position within the recombinant organism. Using the purification techniques, as described in more detail with respect to the Examples, a sample can be obtained, which exclusively contains the hydroxylated indole at the 4-position. This is indicative of a more facile procedure for obtaining the hydroxylated indole at the 4-position, in comparison to the techniques of organic chemistry.
By expressing the PsiK gene in the recombinant host organism, codon optimized nucleic acid sequences SEQ ID NO: 7 and SEQ ID NO: 8 encode for isolated amino acid sequences SEQ ID NO: 20 and SEQ ID NO: 21, respectively. Primary amines and indole nitrogen are nucleophilic groups than can compete with phenolic oxygen for phosphorylation. In contrast, the recombinant host supports the PsiK enzyme catalysis of selective phosphorylation of the phenolic oxygen. The recombinant host and the PsiK enzyme can also catalyze the undoing of de-phosphorylations that yield psilocin. Stated another way, the recombinant host organism and the expressed PsiK gene for encoding the PsiK enzyme can convert psilocin back to the target molecule psilocybin. Stated yet another way, the recombinant host organism and the expressed PsiK gene for encoding the PsiK enzyme can provide a corrective mechanism to obtain the target molecule psilocybin.
By expressing the PsiM gene in the recombinant host organism, codon optimized nucleic acid sequences SEQ ID NO: 9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO: 12, and SEQ ID NO: 13 encode for isolated amino acid sequences SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, and SEQ ID NO: 26, respectively. The primary amine when subjected to methyl iodide may get over alkylated to the quaternary amine. Further, the reaction is not selective as monoalklyated and dialkylated products may also be obtained. To further complicate the alkylation, the nitrogen of the indole is sufficiently nucleophilic to perform alkylations. In contrast, the PsiM enzyme catalyzes selective methylation at the primary amine in the recombinant host organism, which is also stepwise. The first methylation yields norbaeocystin and the second methylation yields psilocybin. The indole nitrogen does not get methylated.
SEQ ID NO: 1-SEQ ID NO: 36 of the systems and methods herein aid in increasing titers of psilocybin in the recombinant host organism in comparison to the titers of psilocybin in natural state of the host organism. As described above, the mutations at specific points of the pathways above direct flux toward yielding psilocybin in the recombinant host organism.
EXAMPLES Aspects of the present teachings may be further understood in light of the following examples, which should not be construed as limiting the scope of the present teachings in any way.
The following examples are provided to illustrate various aspects of the present invention. They are not intended to limit the invention, which is defined by the accompanying claims.
In the examples below, genetically engineered host cells may be any species of yeast herein, including but not limited to any species of Saccharomyces, Candida, Schizosaccharomyces, Yarrowia, etc., which have been genetically altered to produce precursor molecules, intermediate molecules, and psilocybin molecules. Additionally, genetically engineered host cells may be any species of filamentous fungus, including but not limited to any species of Aspergillus, which have been genetically altered to produce precursor molecules, intermediate molecules, and psilocybin molecules. Some of the species of yeast herein for the recombinant host organism include but are not limited to: Schizosaccharomyces cerevisiae, Schizosaccharomyces japonicus, Schizosaccharomyces pombe, Schizosaccharomyces cryophilus, Saccharomyces cerevisiae, Kluyveromyces lactis, Kluyveromyces dobzhanskii, and Yarrowia lipolytica.
The gene sequences from gene source organisms are codon optimized to improve expression using techniques disclosed in U.S. patent application Ser. No. 15/719,430, filed Sep. 28, 2017, entitled “An Isolated Codon Optimized Nucleic Acid”. The gene source organisms can include, but are not limited to: Psilocybe cubensis, Psilocybe cyanescens, Panaeolus cynascens, Gymnopilus junonius, and Gymnopilus dilepis. DNA sequences are synthesized and cloned using techniques known in the art. Gene expression can be controlled by inducible or constitutive promoter systems using the appropriate expression vectors. Genes are transformed into an organism using standard yeast or fungus transformation methods to generate modified host strains (i.e., the recombinant host organism). The modified strains express genes for: (i) producing L-tryptophan and precursor molecules to L-tryptophan; (ii) increasing an output of L-tryptophan molecules and precursor molecules to L-tryptophan molecules; (iii) increasing the import of exogenous L-tryptophan into the host strain; and (iv) the genes for the psilocybin biosynthetic pathway. In the presence or absence of exogenous L-tryptophan, fermentations are run to determine if the cell will convert the L-tryptophan into psilocybin. The L-tryptophan and psilocybin pathway genes herein can be integrated into the genome of the cell or maintained as an episomal plasmid. Samples are: (i) prepared and extracted using a combination of fermentation, dissolution, and purification steps; and (ii) analyzed by HPLC for the presence of precursor molecules, intermediate molecules, and psilocybin molecules.
Using the systems and methods herein, the genes which can be expressed to encode for a corresponding enzyme or other type of proteins include but are not limited to: PsiM, PsiH, PsiD, PsiK, TRP1, TRP2 S76L, TRP3, TRP4, ARO1, ARO2, ARO3, ARO4 K229L, and AROL. For example, the PsiM gene is expressed or (overexpressed) to encode for the PsiM enzyme; the PsiH gene is overexpressed to encode for the PsiH enzyme; and so forth. These PsiM, PsiH, PsiD, and PsiK genes can derive from: Psilocybe cubensis, Psilocybe cyanescens, Panaeolus cynascens, Gymnopilus junonius, and Gymnopilus dilepis. These TRP1, TRP2 S76L, TRP3, TRP4, AR01, ARO2, ARO3, and ARO4 K229L genes can derive from Saccharomyces cerevisiae. These AROL genes can derive from Escherichia coli. Further, these genes are transformed into Schizosaccharomyces cerevisiae, Schizosaccharomyces japonicus, Schizosaccharomyces pombe, Schizosaccharomyces cryophilus, Saccharomyces cerevisiae, Kluyveromyces lactis, Kluyveromyces dobzhanskii, and Yarrowia lipolytica. The PsiM, PsiH, PsiD, PsiK, TRP1, TRP2 S76L, TRP3, TRP4, ARO1, ARO2, ARO3, ARO4 K229L, and AROL genes which derive from at least one of: Psilocybe cubensis, Psilocybe cyanescens, Panaeolus cynascens, Gymnopilus junonius, Gymnopilus dilepis, Saccharomyces cerevisiae, and Escherichia coli can be expressed at the same time. Gene sequences can be determined using the techniques disclosed in U.S. Nonprovisional patent application Ser. No. 16/558,909 filed on Sep. 3, 2019, entitled “Automated Pipeline”.
Example 1—Construction of Saccharomyces cerevisiae Platform Strains with Elevated Metabolic Flux Towards L-Tryptophan Via Overexpression of the Feedback Resistant Mutant, ARO4 K229L The optimized ARO4 K229L gene is synthesized using DNA synthesis techniques known in the art. The optimized gene can be cloned into vectors with the proper regulatory elements for gene expression (e.g. promoter, terminator) and the derived plasmid can be confirmed by DNA sequencing. As an alternative to expression from an episomal plasmid, the optimized ARO4 K229L gene is inserted into the recombinant host genome. Integration is achieved by a single cross-over insertion event of the plasmid. Strains with the integrated gene can be screened by rescue of auxotrophy and genome sequencing.
Example 2—Construction of Saccharomyces cerevisiae Platform Strains with Elevated Metabolic Flux Towards L-Tryptophan Via Deletion of PDC5 Deletion of PDC5 is performed by replacement of the PDC5 gene with the URA3 cassette in the recombinant host. The PDC5 URA3 knockout fragment, carrying the marker cassette, URA3, and homologous sequence to the targeted gene, PDC5, can be generated by bipartite PCR amplification. The PCR product is transformed into a recombinant host and transformants can be selected on synthetic URA drop-out media. Further verification of the modification in said strain can be carried out by genome sequencing, and analyzed by the techniques disclosed in U.S. Nonprovisional patent application Ser. No. 16/558,909 filed on Sep. 3, 2019, entitled “Automated Pipeline”.
Example 3—Method of Growth Modified host cells that yield recombinant host cells, such as the psilocybin-producing strain herein, express engineered psilocybin biosynthesis genes and enzymes. More specifically, the psilocybin-producing strain herein is grown in rich culture media containing yeast extract, peptone and a carbon source of glucose, galactose, sucrose, fructose, corn syrup, corn steep liquor, ethanol, and/or molasses. The recombinant host cells are grown in either shake flasks or fed-batch bioreactors. Fermentation temperatures can range from 25 degrees Celsius to 37 degrees Celsius at a pH range from pH 4 to pH 7.5. Exogenous L-tryptophan can be added to media to supplement the precursor pool for psilocybin production, which can be up taken by strains expressing the TAT2 L-tryptophan importer protein. The strains herein can be harvested during a fermentation period ranging from 12 hours onward from the start of fermentation.
Example 4—Detection of Isolated Product To identify fermentation derived psilocybin produced by a recombinant host expressing the engineered psilocybin biosynthetic pathway, an Agilent 1100 series liquid chromatography (LC) system equipped with a HILIC column (Obelisc N, SIELC, Wheeling, Ill. USA) is used. A gradient is used of mobile phase A (ultraviolet (UV) grade H2O+0.1% Formic Acid) and mobile phase B (UV grade acetonitrile+0.1% Formic Acid). Column temperature is set at 40 degree Celsius. Compound absorbance is measured at 220 nanometers (nm) and 270 nm wavelength using a diode array detector (DAD) and spectral analysis from 200 nm to 400 nm wavelengths. A 0.1 milligram (mg)/milliliter (mL) analytical standard is made from psilocybin certified reference material (Cayman Chemical Company, USA). Each sample is prepared by diluting fermentation biomass from a recombinant host expressing the engineered psilocybin biosynthesis pathway 1:1 in 100% ethanol and filtered in 0.2 um nanofilter vials. Samples are compared to the psilocybin analytical standard retention time and UV-visible spectra for identification. As depicted in inset A of FIG. 11, a fermentation derived product is obtained which has absorption of 300 au at 220 nm with a retention time of 4.55 minutes in a HPLC chromatogram. As depicted in inset B of FIG. 11, the fermentation derived product obtained matches the retention time of the psilocybin analytical standard in the overlaid HPLC chromatograms. This indicates that the fermentation derived product is psilocybin. As depicted in inset C of FIG. 11, the UV-visible spectra of the fermentation derived product and the psilocybin analytical standard are identical. This further corroborates that the fermentation derived product is psilocybin.
OTHER EMBODIMENTS The detailed description set-forth above is provided to aid those skilled in the art in practicing the present invention. However, the invention described and claimed herein is not to be limited in scope by the specific embodiments herein disclosed because these embodiments are intended as illustration of several aspects of the invention. Any equivalent embodiments are intended to be within the scope of this invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description which does not depart from the spirit or scope of the present inventive discovery. Such modifications are also intended to fall within the scope of the appended claims.
REFERENCES CITED All publications, patents, patent applications and other references cited in this application are incorporated herein by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application or other reference was specifically and individually indicated to be incorporated by reference in its entirety for all purposes. Citation of a reference herein shall not be construed as an admission that such is prior art to the present invention.
SEQUENCE LISTINGS
(Psilocybe cubensis (PSID gene))
SEQ ID NO: 1
ATGCAAGTCATCCCCGCGTGCAACAGCGCAGCTAT
AAGGTCACTTTGTCCGACCCCCGAGAGCTTTAGAA
ATATGGGCTGGCTTTCCGTGAGCGATGCCGTCTAT
AGCGAATTTATAGGTGAACTTGCGACGAGAGCATC
TAATAGAAACTACAGCAATGAGTTCGGTTTAATGC
AACCAATACAAGAATTTAAAGCGTTCATCGAGAGT
GATCCCGTTGTACACCAAGAGTTTATCGACATGTT
TGAAGGCATCCAAGATTCTCCGAGGAACTACCAAG
AACTATGTAACATGTTCAATGATATTTTTAGGAAG
GCTCCCGTATACGGAGATTTGGGCCCTCCGGTCTA
CATGATTATGGCGAAGTTGATGAATACAAGGGCGG
GTTTCAGTGCGTTCACAAGACAACGTCTGAACCTG
CATTTTAAAAAGCTGTTCGATACCTGGGGTTTATT
TCTTTCATCCAAAGACAGCAGGAATGTCCTGGTAG
CTGACCAGTTTGATGATAGGCACTGCGGCTGGCTG
AACGAGAGGGCATTATCTGCGATGGTGAAACACTA
TAATGGGCGTGCATTTGATGAAGTATTTCTATGTG
ACAAAAATGCACCCTATTACGGCTTTAATTCATAC
GACGATTTCTTCAATAGGAGGTTCCGTAATAGAGA
CATTGATAGACCCGTTGTCGGCGGCGTGAACAACA
CGACGCTTATATCAGCAGCCTGTGAGTCTCTGTCT
TATAACGTCAGCTATGACGTGCAATCCTTAGATAC
TTTAGTTTTCAAAGGTGAGACGTACTCATTAAAAC
ATCTTTTGAATAATGATCCATTTACGCCACAATTC
GAGCACGGTTCCATATTGCAAGGATTCCTAAACGT
GACAGCATATCATCGTTGGCACGCGCCGGTTAACG
GAACTATCGTCAAGATAATCAACGTTCCTGGTACT
TATTTCGCACAAGCGCCGTCTACCATCGGTGATCC
GATCCCAGATAATGACTATGATCCACCGCCATATC
TAAAGAGTCTTGTGTACTTCAGTAACATTGCAGCG
AGACAGATTATGTTCATAGAAGCTGATAACAAGGA
GATAGGCCTAATTTTCCTGGTTTTTATAGGCATGA
CAGAAATTTCAACGTGTGAAGCAACGGTATCCGAG
GGGCAACATGTCAATAGAGGGGACGACCTGGGTAT
GTTTCATTTCGGGGGCTCTTCTTTTGCCCTTGGCC
TGCGTAAAGACTGCCGTGCCGAAATTGTTGAGAAG
TTCACGGAGCCCGGGACAGTTATAAGGATTAACGA
AGTCGTCGCCGCCTTGAAGGCTTAA
(Psilocybe cyanescens (PSID gene))
SEQ ID NO: 2
ATGCAAGTGCTTCCTGCTTGCCAAAGCTCTGCCCT
TAAAACCCTGTGTCCGAGCCCCGAGGCTTTTAGAA
AGCTGGGATGGCTACCTACGTCTGACGAAGTGTAC
AACGAGTTCATAGATGATCTGACTGGCAGGACTTG
CAATGAGAAGTATAGCAGCCAAGTAACCCTGTTAA
AGCCAATCCAAGACTTCAAGACTTTCATAGAGAAT
GACCCGATAGTATATCAAGAGTTCATTAGCATGTT
TGAGGGCATAGAACAGAGCCCTACTAACTATCATG
AGCTATGTAACATGTTCAACGATATTTTTCGTAAG
GCACCCCTATACGGAGACTTAGGACCACCTGTCTA
CATGATAATGGCACGTATTATGAATACGCAGGCGG
GTTTTTCAGCGTTCACCAAAGAATCTCTGAACTTC
CATTTTAAGAAGCTATTCGACACGTGGGGTCTATT
CCTAAGCTCTAAAAATTCCAGAAACGTACTTGTCG
CCGATCAGTTTGACGACAAACATTACGGATGGTTT
TCTGAGAGAGCAAAGACTGCGATGATGATCAACTA
TCCAGGACGTACATTCGAGAAGGTCTTCATCTGTG
ACGAGCATGTGCCTTATCACGGATTTACTTCCTAT
GACGACTTCTTTAACAGGAGATTTCGTGACAAGGA
TACAGACCGTCCCGTCGTCGGTGGCGTCACCGACA
CGACGTTGATAGGCGCGGCATGTGAAAGTTTATCT
TATAACGTTTCTCACAACGTCCAATCACTGGACAC
CCTTGTCATAAAAGGCGAGGCGTACTCTTTAAAAC
ACCTTCTGCATAATGACCCATTTACGCCACAGTTT
GAACATGGATCTATCATCCAAGGATTCTTGAACGT
TACAGCCTATCACAGATGGCACTCTCCAGTTAACG
GCACTATTGTGAAGATTGTAAACGTACCAGGGACA
TACTTTGCCCAGGCGCCCTATACCATAGGTAGCCC
AATCCCTGATAATGACCGGGACCCGCCGCCCTACT
TGAAGAGCCTTGTTTATTTTAGCAACATTGCTGCC
AGACAGATTATGTTTATTGAGGCTGACAATAAAGA
TATTGGCCTTATCTTTCTTGTGTTCATTGGCATGA
CTGAAATTAGCACATGTGAAGCGACGGTATGCGAA
GGACAGCACGTTAACAGAGGCGATGACCTTGGGAT
GTTTCATTTTGGGGGATCGAGTTTTGCATTGGGGC
TTAGAAAAGATAGCAAAGCAAAAATACTAGAAAAA
TTTGCAAAGCCGGGAACAGTAATAAGGATTAACGA
GCTGGTGGCATCCGTCAGAAAATAA
(Gymnopilus junonius (PSID gene))
SEQ ID NO: 3
ATGTCATCTCCTCGTATCGTGCTGCACAGGGTTGG
TGGCTGGCTGCCTAAAGACCAAAACGTGCTAGAAG
CATGGCTGAGCAAGAAGATTGCTAAAGCAAAAACT
AGAAATAGGGCTCCAAAAGATTGGGCTCCTGTGAT
TCAAGACTTCCAGAGACTGATAGAGACCGATGCCG
AGATCTACATGGGTTTCCATCAGATGTTCGAGCAG
GTCCCCAAGAAAACTCCGTACGATAAAGACCCCAC
CAATGAGCAATGGCAAGTAAGAAATTATATGCACA
TGTTAGATCTGTTCGACCTAATTATAACCGAGGCA
CCGGATTTCGAACAAAATGATCTTGTTGGATTTCC
AATAAATGCAATCCTGGATTGGCCCATGGGGACCC
CCGGTGGGCTTACTGCATTTATTAACCCTAAAGTA
AATATTATGTTTCATAAAATGTTTGACGTTTGGGC
AGTATTTCTGTCATCTCCAGCATCATGCTACGTCC
TAAATACAAGCGATAGCGGTTGGTTCGGTCCCGCT
GCAACCGCAGCTATACCCAACTTCAAAGAGACCTT
CATCTGCGACCCAAGTCTGCCATACCTAGGGTACA
CTAGCTGGGATAATTTCTTCACCAGGCTGTTTAGG
CCGGGGGTGCGTCCTGTCGAGTTCCCGAACAATGA
TGCCATTGTTAACAGTGCGTGTGAATCCACGGTTT
ATAATATAGCTCCAAACATTAAACCACTAGATAAA
TTTTGGATTAAGGGAGAGCCGTATTCCCTAAATCA
CATACTTAATAACGACCCGTACGCGAGCCAGTTCG
TAGGTGGAACCATATCCCAAGCATTCTTATCTGCG
CTGAACTATCACCGTTGGGCGAGTCCGGTTAACGG
CAACATTGTCAAGGTCGTCAATGTTCCGGGTACAT
ACTACGCGGAGTCCCCAGTTACCGGTTTTGGGAAT
CCAGAAGGGCCAGATCCAGCGGCGCCCAATCTATC
TCAAGGTTTCATTACTGCTGTGGCTGCGAGAGCCC
TGATTTTCATAGAGGCCGATAACCCTAACATCGGA
TTAATGTGTTTTGTGGGGGTTGGCATGGCAGAGGT
CTCAACATGTGAAGTTACCGTGAGTGTAGGCGATG
TTGTCAAGAAAGGAGATGAGATTGGAATGTTCCAT
TTCGGGGGAAGCACTCACTGCTTGATATTTAGGCC
ACAAACAAAAATTACGTTCAATCCCGACTATCCTG
TGTCAACCGCCGTACCCTTGAATGCTGCAGTGGCA
ACCGTCGTATAA
(Psilocybe cubensis (PSIH gene))
SEQ ID NO: 4
ATGATTGCCGTCTTATTCTCTTTTGTCATAGCTGG
CTGCATCTATTATATAGTATCCCGTCGTGTGCGTC
GTTCAAGACTTCCGCCCGGACCACCAGGCATCCCT
ATCCCCTTTATCGGCAATATGTTTGACATGCCCGA
AGAATCACCCTGGTTGACGTTTCTGCAATGGGGCA
GAGATTATAATACAGACATTTTGTATGTAGATGCA
GGCGGAACTGAGATGGTAATATTGAATACCCTTGA
GACAATCACTGATTTGTTAGAAAAGAGGGGGTCTA
TATATTCTGGCAGGCTAGAAAGTACCATGGTTAAT
GAGTTGATGGGGTGGGAGTTTGATCTAGGATTCAT
CACCTACGGTGATCGTTGGAGAGAGGAGAGAAGGA
TGTTCGCGAAAGAGTTCAGCGAAAAGGGAATCAAA
CAATTCAGGCACGCCCAAGTAAAGGCGGCGCATCA
ACTTGTCCAACAGCTGACAAAAACACCGGATCGTT
GGGCTCAACACATACGTCATCAGATAGCCGCCATG
TCTTTAGACATCGGCTATGGCATAGACTTAGCGGA
GGATGATCCATGGTTAGAAGCAACACACTTAGCTA
ACGAAGGACTGGCGATAGCTTCCGTCCCAGGAAAA
TTTTGGGTAGACTCATTTCCGTCTCTGAAATACCT
ACCAGCCTGGTTTCCTGGAGCTGTCTTCAAACGTA
AGGCAAAAGTATGGAGGGAGGCAGCAGACCATATG
GTGGACATGCCATATGAGACTATGAGGAAATTGGC
GCCACAGGGCTTGACTAGACCATCCTATGCATCTG
CAAGACTACAGGCCATGGACCTAAACGGTGATTTG
GAGCACCAAGAGCACGTAATTAAAAACACAGCAGC
CGAAGTGAACGTCGGAGGGGGAGATACAACCGTCT
CTGCGATGAGTGCGTTCATACTAGCGATGGTCAAG
TATCCGGAAGTACAGCGTAAAGTCCAGGCCGAGCT
AGACGCACTTACTAACAACGGCCAGATTCCCGATT
ACGACGAGGAAGACGATAGTCTACCTTACTTGACC
GCATGTATTAAAGAGTTATTTAGATGGAATCAAAT
TGCGCCCCTAGCGATTCCTCACAAGTTAATGAAAG
ACGATGTATATAGGGGTTATCTAATACCTAAGAAT
ACGCTAGTTTTTGCAAACACATGGGCGGTCCTGAA
CGACCCTGAAGTCTACCCAGACCCTAGCGTATTTA
GGCCGGAGCGTTATTTAGGACCCGACGGTAAGCCC
GATAATACTGTCAGGGACCCCAGGAAGGCTGCGTT
CGGGTATGGGAGGAGGAACTGTCCAGGAATACACT
TAGCCCAATCAACCGTCTGGATAGCCGGAGCGACC
TTACTTAGTGCGTTTAATATCGAGAGGCCAGTTGA
CCAGAATGGGAAACCCATCGATATTCCAGCAGACT
TCACAACCGGGTTTTTCAGGCATCCTGTTCCTTTT
CAGTGCCGTTTCGTGCCTAGGACTGAACAGGTCTC
CCAATCAGTCAGTGGGCCGTAA
(Psilocybe cyanescens (PSIH gene))
SEQ ID NO: 5
ATGGCGCCTTTGACAACCATGATTCCGATCGTTCT
ATCTCTTCTAATAGCGGGGTGTATATATTATATCA
ACGCAAGGAGAATTAAAAGGTCCAGGTTGCCACCA
GGACCGCCGGGTATTCCTATTCCATTCATCGGGAA
CATGTTCGACATGCCAAGCGAAAGTCCCTGGCTAA
TCTTCCTACAATGGGGACAAGAGTACCAGACCGAT
ATAATTTACGTTGACGCGGGAGGAACTGATATGAT
AATACTTAATTCCCTAGAGGCAATTACAGATCTGT
TAGAGAAAAGGGGCTCATTGTATAGCGGGAGGTTG
GAATCCACGATGGTAAACGAGCTAATGGGTTGGGA
GTTTGATTTCGGTTTCATACCTTACGGTGAAAGAT
GGAGGGAAGAACGTCGTATGTTCGCCAAAGAGTTT
TCTGAGAAGAACATAAGGCAGTTTAGACACGCCCA
AGTAAAGGCTGCCAATCAGCTAGTGCGTCAACTAA
CCGATAAACCGGACAGGTGGTCACACCACATAAGG
CATCAAATCGCGTCCATGGCCCTGGACATCGGTTA
CGGAATCGATCTTGCTGAAGACGATCCGTGGATCG
CAGCTTCCGAACTGGCGAATGAAGGCTTGGCTGTA
GCCTCAGTGCCAGGATCTTTTTGGGTAGATACGTT
CCCGTTTCTTAAATATTTGCCAAGTTGGTTACCTG
GCGCGGAGTTCAAAAGAAACGCAAAGATGTGGAAG
GAAGGAGCAGATCATATGGTCAATATGCCTTACGA
AACGATGAAAAAGCTAAGCGCACAAGGACTGACTA
GACCATCATATGCAAGTGCGAGGCTACAGGCTATG
GACCCGAACGGGGATCTTGAACATCAAGAAAGAGT
GATCAAAAATACGGCCACGCAGGTAAATGTTGGTG
GTGGGGATACTACAGTCGGGGCAGTAAGTGCGTTT
ATCCTTGCGATGGTAAAATACCCGGAAGTTCAAAG
GAAAGTACAAGCCGAGCTGGACGAGTTCACGAGCA
AGGGGAGGATACCGGATTACGATGAAGATAACGAT
TCTCTTCCCTATCTATCGGCTTGCTTCAAAGAGCT
GTTCAGGTGGGGCCAGATTGCGCCTTTGGCGATTG
CTCATAGGCTGATAAAGGACGATGTCTATAGGGAA
TATACTATCCCAAAGAATGCTCTGGTCTTTGCGAA
CAATTGGTATGGGCGTACTGTATTGAATGACCCTT
CTGAGTATCCCAATCCTTCAGAATTTAGACCTGAA
AGGTACTTGGGGCCCGATGGTAAGCCAGATGACAC
CGTCAGGGACCCAAGAAAGGCAGCGTTTGGGTACG
GACGTAGAGTGTGTCCAGGGATACACCTGGCGCAG
AGCACGGTCTGGATTGCTGGTGTCGCGTTGGTATC
TGCCTTCAACATTGAGCTGCCCGTGGACAAAGACG
GGAAATGTATAGATATTCCGGCGGCCTTCACGACG
GGATTCTTTAGATAA
(Gymnopilus junonius (PSIH gene))
SEQ ID NO: 6
ATGATGTCCGAGATGAATGGGATGGATAAATTGGC
GCTATTGACGACGTTATTAGCTGCCGGTTTTCTAT
ACTTCAAGAATAAGCGTCGTTCCGCGTTGCCGTTC
CCGCCAGGGCCGAAAAAGCATCCCCTTTTAGGTAA
CTTGCTGGACCTTCCGAAGAAGCTGGAGTGGGAGA
CGTACAGAAGATGGGGAAAAGAATACAATTCAGAT
GTAATACATGTTAGCGCGGGGAGTGTAAACTTAAT
TATCGTTAATTCCTTTGAAGCTGCGACAGACCTGT
TTGATAAGAGATCAGCCAATTATTCAAGTAGGCCA
CAATTCACGATGGTGAGAGAACTGATGGGATGGAA
TTGGTTGATGTCTGCATTAATATACGGTGACAAGT
GGAGAGAGCAACGTAGGTTGTTTCAGAAACATTTC
AGTACAACGAATGCCGAACTTTACCAAAATACACA
ATTAGAATATGTTCGTAAAGCCCTGCAGCATCTGC
TAGAAGAGCCTTCAGATTTTATGGGAATAACACGT
CACATGGCTGGGGGCGTCAGCATGTCCCTGGCATA
TGGCTTAAACATTCAGAAGAAAAACGACCCTTTTG
TTGACCTTGCACAAAGGGCAGTGCACAGCATAACA
GAGGCCTCAGTTCCTGGGACATTTTGGGTAGACGT
AATGCCTTGGCTAAAGTATATTCCAGAATGGGTGC
CGGGTGCTGGCTTTCAGAAGAAGGCTAGAGTGTGG
AGGAAATTACAGCAAGATTTTCGTCAGGTCCCATA
TCAGGCAGCTCTGAAAGACATGGCTTCAGGGAAAG
CTAAACCATCATTTGCAAGTGAGTGTTTGGAGACG
ATAGACGACAATGAGGATGCACAAAGGCAAAGGGA
GGTGATAAAAGACACAGCTGCCATTGTATTCGCAG
CCGGTGCGGATACAAGCCTTAGTGGAATCCATACA
TTATTCGCCGCAATGTTGTGTTACCCAGAGGTCCA
GAAGAAAGCACAAGAAGAACTGGATCGTGTCTTGG
GTGGGAGACGTCTACCGGAATTTACCGATGAGCCC
AACATGCCCTACATCTCTGCGTTAGTGAAGGAAAT
ATTGAGGTGGAAACCGGCTACTCCGATTGGCGTAC
CCCACTTAGCCAGCGAGGATGACGTTTACAACGGA
TATTACATACCAAAACGTGCGGTTGTCATAGGCAA
CAGCTGGGCTATGCTTCATGATGAGGAAACTTATC
CGGACCCAAGCACCTTTAACCCTGACAGATTTTTG
ACCACAAATAAAAGCACTGGAAAATTGGAATTAGA
TCCCACAGTGAGAGATCCCGCTTTAATGGCCTTCG
GATTTGGTAGACGTATGTGTCCAGGACGTGATGTA
GCTCTTTCTGTCATATGGCTGACTATCGCAAGCGT
TTTAGCAACGTTTAATATTACCAAGGCGATAGACG
AAAACGGGAAGGAACTGGAACCGGATGTACAGTAC
TGGAGCGGTCTAATCGTCCACCCGCTGCCATTCAA
ATGTACGATCAAGCCAAGATCAAAGGCAGCGGAAG
AACTTGTGAAATCTGGCGCAGACGCCTATTAA
(Psilocybe cubensis (PSIK gene))
SEQ ID NO: 7
ATGGCATTCGACTTGAAAACTGAAGACGGGCTAAT
AACTTACCTAACGAAACACCTTTCTTTGGATGTGG
ATACATCAGGTGTGAAAAGGTTAAGCGGTGGCTTC
GTTAACGTGACCTGGAGAATAAAACTAAACGCACC
CTATCAGGGTCACACATCAATAATTCTAAAGCACG
CACAGCCGCATATGTCAACCGACGAAGACTTCAAA
ATTGGCGTGGAGCGTTCCGTCTATGAGTACCAGGC
TATCAAACTTATGATGGCCAATAGGGAGGTGCTAG
GGGGTGTTGACGGGATCGTGTCTGTGCCAGAGGGG
TTGAACTACGACCTTGAAAATAATGCATTGATCAT
GCAGGACGTAGGTAAGATGAAGACCCTATTAGACT
ACGTAACGGCAAAACCCCCGCTTGCGACTGATATA
GCACGTTTGGTAGGTACAGAGATTGGGGGTTTCGT
GGCTAGACTGCATAACATAGGGAGGGAGAGGAGAG
ACGACCCGGAGTTCAAGTTTTTCTCTGGAAATATA
GTCGGCAGGACAACAAGCGATCAACTATACCAAAC
AATTATCCCTAACGCAGCTAAGTACGGGGTAGATG
ACCCTCTACTGCCTACCGTTGTAAAAGATCTGGTC
GATGATGTCATGCACAGTGAGGAGACTCTTGTAAT
GGCGGATTTATGGAGCGGCAATATACTTCTACAGT
TGGAGGAGGGGAATCCTTCAAAGTTACAGAAAATC
TACATTTTAGATTGGGAATTGTGTAAATACGGCCC
AGCTTCACTAGACCTTGGGTATTTCTTGGGTGATT
GCTACCTGATTTCTCGTTTCCAAGATGAGCAGGTC
GGCACAACTATGAGACAAGCCTACTTACAAAGCTA
CGCTCGTACCTCTAAACATTCCATAAACTACGCCA
AGGTCACTGCGGGAATTGCAGCACATATAGTGATG
TGGACAGACTTTATGCAGTGGGGGAGTGAGGAAGA
GAGAATTAACTTCGTCAAGAAAGGCGTGGCCGCCT
TCCATGACGCAAGAGGGAACAATGATAATGGTGAA
ATCACCTCTACTCTGTTGAAGGAGAGTTCAACTGC
CTAA
(Psilocybe cyanescens (PSIK gene))
SEQ ID NO: 8
ATGACTTTCGATCTAAAAACGGAGGAGGGCTTATT
ATCTTATCTTACCAAGCATTTAAGTTTAGACGTAG
CACCGAATGGTGTCAAAAGATTATCTGGTGGATTC
GTCAATGTGACTTGGAGGGTAGGGTTAAATGCACC
GTACCATGGGCACACGTCTATAATCCTTAAACACG
CTCAACCACATTTAAGCTCCGATATTGACTTCAAA
ATAGGGGTGGAAAGAAGTGCGTATGAGTACCAGGC
TTTGAAGATTGTCTCTGCCAACAGCAGCCTACTTG
GTTCTTCTGATATCCGTGTCTCAGTTCCAGAAGGT
TTGCACTATGATGTTGTGAATAACGCCCTAATCAT
GCAGGACGTGGGTACAATGAAGACCTTGCTGGACT
ATGTTACAGCGAAACCCCCTATATCTGCTGAAATT
GCCAGCCTAGTAGGTAGTCAGATTGGCGCTTTCAT
AGCAAGATTACACAATTTGGGCAGAGAAAATAAAG
ATAAGGACGACTTTAAATTTTTCTCCGGAAATATA
GTTGGGAGGACGACGGCAGACCAACTGTATCAGAC
CATAATTCCTAATGCGGCAAAATATGGAATCGATG
ACCCAATTCTTCCAATAGTTGTCAAAGAACTTGTT
GAAGAAGTCATGAACTCAGAGGAAACCCTGATTAT
GGCGGACCTATGGAGCGGTAATATCTTGCTACAGT
TCGACGAGAACAGTACGGAACTAACCCGTATTTGG
CTGGTAGACTGGGAGCTATGCAAGTACGGGCCGCC
GTCACTGGATATGGGTTACTTCTTGGGCGACTGCT
TTTTGGTAGCTAGATTCCAAGACCAACTTGTAGGC
ACATCTATGAGACAAGCATACCTTAAAAGCTACGC
ACGTAACGTAAAAGAGCCGATCAACTATGCTAAGG
CCACAGCAGGCATCGGCGCTCATTTGGTAATGTGG
ACTGACTTCATGAAGTGGGGTAACGATGAAGAAAG
GGAGGAGTTCGTGAAAAAGGGGGTCGAAGCATTCC
ACGAGGCCAACGAAGACAATAGGAACGGAGAGATA
ACGAGCATATTGGTGAAAGAGGCATCACGTACGTA
A
(Psilocybe cubensis (PSIM gene))
SEQ ID NO: 9
ATGCACATCAGAAACCCCTATAGAACCCCCATAGA
TTACCAGGCGCTGAGTGAGGCCTTTCCACCATTGA
AGCCCTTTGTATCCGTAAACGCTGATGGTACGAGT
TCCGTAGATCTAACGATCCCGGAGGCGCAACGTGC
GTTCACTGCCGCATTGTTACATAGAGATTTCGGGC
TAACCATGACTATACCGGAAGATAGACTGTGCCCT
ACTGTCCCTAACAGGTTAAATTATGTACTGTGGAT
TGAAGATATTTTCAACTACACGAATAAGACCCTGG
GGCTGAGCGATGACAGACCGATAAAGGGGGTGGAT
ATTGGCACAGGCGCCAGCGCAATATACCCTATGCT
TGCTTGCGCCAGGTTTAAGGCATGGTCCATGGTAG
GGACAGAGGTAGAACGTAAATGTATTGATACGGCT
AGACTAAATGTCGTCGCCAATAATCTACAGGATAG
ATTGAGTATATTAGAGACATCCATCGACGGTCCCA
TTCTTGTTCCAATCTTCGAGGCCACAGAAGAATAT
GAGTATGAGTTCACCATGTGTAATCCGCCATTCTA
CGATGGTGCGGCCGACATGCAGACCTCTGACGCGG
CCAAAGGATTCGGCTTTGGAGTGGGGGCCCCTCAC
TCTGGAACAGTTATCGAAATGTCCACTGAAGGAGG
GGAGTCCGCATTCGTAGCCCAGATGGTGAGAGAGA
GCTTGAAACTGCGTACCAGATGCAGATGGTATACG
TCTAATCTTGGGAAATTAAAAAGCCTAAAGGAGAT
TGTGGGTCTTTTAAAAGAGCTGGAGATTTCCAACT
ACGCCATAAACGAGTACGTCCAAGGGTCTACCAGA
AGATACGCCGTCGCGTGGTCTTTTACTGACATTCA
GCTTCCAGAGGAGCTATCTCGTCCCAGTAACCCGG
AATTGTCCTCCTTGTTTTAA
(Psilocybe cyanescens (PSIM gene))
SEQ ID NO: 10
ATGCATATCAGGAATCCGTACCGTGACGGCGTGGA
CTACCAGGCATTAGCCGAGGCTTTCCCGGCGCTAA
AGCCACACGTCACTGTCAATTCAGACAATACAACT
TCTATAGATTTCGCGGTACCCGAGGCCCAGAGACT
TTACACCGCAGCATTACTTCATAGGGACTTTGGTT
TAACCATAACCTTACCCGAGGATAGACTATGTCCT
ACGGTCCCGAATAGATTGAACTATGTGTTGTGGGT
GGAAGATATACTGAAGGTTACGTCAGACGCATTGG
GATTACCGGATAATAGACAAGTGAAAGGTATTGAT
ATTGGAACAGGAGCAAGCGCAATTTATCCCATGTT
AGCTTGTTCCAGGTTTAAGACTTGGTCCATGGTAG
CTACAGAGGTGGATCAAAAATGCATAGATACCGCA
AGGCTAAACGTAATAGCTAATAACCTTCAGGAGAG
ATTGGCAATCATAGCCACTTCCGTGGACGGGCCTA
TTCTTGTTCCTCTGTTGCAGGCTAATTCCGACTTT
GAATATGACTTCACCATGTGCAATCCGCCCTTTTA
CGACGGCGCCTCTGATATGCAGACAAGTGATGCCG
CTAAAGGCTTTGGCTTCGGAGTAAACGCACCTCAC
ACTGGGACAGTACTTGAAATGGCGACAGAAGGAGG
GGAAAGTGCGTTCGTTGCCCAAATGGTTCGTGAGT
CCTTGAACCTGCAGACTAGATGCAGGTGGTTCACA
TCTAATTTGGGTAAACTAAAATCACTGTACGAGAT
TGTGGGTCTATTAAGAGAACACCAGATTTCTAACT
ACGCCATAAATGAGTATGTACAAGGCGCAACTCGT
AGGTATGCAATTGCGTGGAGTTTCATAGATGTAAG
ACTGCCCGACCATTTGTCCAGACCATCTAATCCCG
ATCTATCCAGTTTGTTTTAA
(Panaeolus cyanescens (PSIM gene))
SEQ ID NO: 11
ATGCATAACCGTAACCCGTATAGGGACGTGATTGA
TTACCAAGCACTTGCGGAAGCCTACCCGCCCCTAA
AACCCCACGTCACGGTGAACGCGGATAACACGGCA
TCCATAGATCTTACGATCCCCGAGGTCCAGAGGCA
ATACACAGCAGCTCTTTTACATCGTGATTTCGGAT
TAACTATCACACTACCAGAAGATAGGCTGTGCCCG
ACAGTACCGAACCGTTTAAACTATGTATTGTGGAT
AGAGGATATATTTCAGTGTACGAATAAGGCTCTGG
GATTGTCAGATGACAGACCCGTTAAGGGGGTAGAT
ATAGGGACCGGCGCCTCCGCCATCTATCCAATGCT
TGCTTGCGCGAGGTTTAAGCAGTGGTCCATGATTG
CCACAGAAGTGGAGCGTAAGTGCATAGATACAGCG
AGATTGAATGTCCTGGCGAATAACTTACAGGACCG
TTTGTCAATTCTTGAGGTTTCAGTAGACGGCCCGA
TTTTGGTACCCATCTTTGATACCTTCGAGCGTGCG
ACAAGCGATTACGAATTTGAGTTCACGATGTGTAA
CCCTCCATTTTACGACGGGGCCGCGGATATGCAAA
CATCAGATGCAGCTAAGGGTTTCGGTTTTGGAGTT
AACGCTCCACACTCCGGTACCGTGATAGAGATGGC
TACTGAAGGAGGTGAGGCTGCTTTTGTGGCGCAAA
TGGTCCGTGAGAGCATGAAGTTACAGACAAGGTGT
CGTTGGTTTACAAGCAACTTAGGCAAGCTAAAATC
ACTGCATGAAATTGTTGCTTTGTTGAGAGAATCCC
AGATCACAAACTATGCCATAAATGAGTACGTTCAG
GGGACGACGAGAAGGTACGCTCTTGCTTGGTCCTT
CACAGACATAAAACTTACTGAGGAACTTTACAGGC
CCTCCAATCCAGAATTAGGACCTCTTTGCAGCACA
TTTGTCTAA
(Gymnopilus dilepis (PSIM gene))
SEQ ID NO: 12
ATGCACATTAGAAACCCTTACTTAACACCTCCGGA
CTACGAGGCCCTTGCGGAGGCCTTCCCCGCACTAA
AGCCTTATGTTACAGTTAACCCCGATAAGACTACT
ACAATTGACTTTGCCATACCGGAGGCTCAGAGATT
ATACACGGCTGCTCTACTTTACAGGGACTTTGGAC
TGACAATAACATTGCCGCCGGATAGGTTATGCCCA
ACCGTGCCCAATAGGCTTAATTATGTTTTGTGGAT
TCAGGACATTCTGCAGATTACCTCCGCTGCCTTGG
GCTTGCCAGAGGCTAGACAAGTAAAGGGAGTAGAC
ATAGGTACCGGAGCGGCAGCGATATACCCTATTCT
TGGTTGCAGCCTTGCAAAGAATTGGTCTATGGTGG
GGACAGAGGTCGAACAAAAATGTATCGACATAGCG
CGTCAAAACGTGATTTCAAATGGATTGCAGGATAG
GATAACCATAACTGCTAATACCATAGACGCTCCCA
TTCTGCTGCCCTTATTTGAAGGAGACAGTAACTTC
GAATGGGAGTTCACCATGTGTAACCCGCCATTTTA
CGACGGCGCTGCGGACATGGAGACAAGCCAGGACG
CTAAAGGCTTCGGGTTCGGCGTCAACGCCCCGCAT
ACAGGAACAGTGGTGGAAATGGCCACGGACGGTGG
TGAGGCTGCATTCGTCAGCCAAATGGTGAGAGAGT
CCTTGCACCTAAAGACACGTTGTAGATGGTTCACG
TCCAATCTAGGTAAATTGAAGAGTCTACATGAAAT
TGTGGGATTGTTGCGTGAACACCAAATTACCAACT
ACGCGATAAATGAATATGTTCAGGGAACGACACGT
AGATACGCGATTGCATGGTCATTTACTGACCTACG
TCTATCAGACCACCTGCCACGTCCTCCGAACCCCG
ATCTATCAGCCCTATTTTAA
(Gymnopilus junonius (PSIM gene))
SEQ ID NO: 13
ATGCACTCTCGTAACCCTTATAGATCCCCTCCTGA
TTTCGCGGCATTAAGTGCGGCTTATCCTCCGCTGT
CACCATACATAACTACCGATCTAAGCAGCGGTCGT
AAAACAATTGACTTTAGAAATGAGGAAGCGCAACG
TCGTCTAACTGAGGCTATCATGTTGCGTGACTTCG
GCGTTGTGTTAAACATACCATCTAACAGGCTGTGC
CCGCCTGTGCCGAATCGTATGAACTATGTACTTTG
GATACAAGATATAGTTTACGCGCACCAGACAATAC
TGGGAGTGAGTTCTCGTCGTATCAGAGGTCTTGAT
ATTGGTACTGGTGCTACCGCTATATATCCTATACT
GGCATGCAAGAAAGAGCAGAGCTGGGAGATGGTTG
CAACTGAATTGGACGACTACTCCTATGAGTGTGCA
TGTGATAACGTGTCATCCAACAATATGCAGACTTC
CATTAAAGTAAAGAAGGCTTCGGTAGATGGGCCCA
TCCTGTTCCCAGTGGAAAACCAAAATTTCGACTTT
AGCATGTGCAACCCGCCTTTCTACGGCTCTAAGGA
GGAGGTGGCGCAATCCGCAGAGTCAAAAGAACTGC
CGCCCAATGCTGTTTGCACGGGTGCAGAGATCGAG
ATGATATTTAGTCAAGGAGGAGAAGAGGGTTTCGT
AGGTAGAATGGTAGAGGAATCAGAGAGGTTGCAAA
CGAGATGCAAATGGTACACTTCAATGCTTGGTAAG
ATGTCTAGTGTAAGCACTATAGTTCAGGCTCTGCG
TGCGAGATCAATTATGAATTATGCTTTGACAGAAT
TTGTACAAGGACAAACCCGTAGGTGGGCGATAGCT
TGGTCTTTCTCCGACACTCACTTACCGGATGCCGT
CAGTAGAATCTCTAGTTAA
(Psilocybe cubensis (PSID gene))
SEQ ID NO: 14
MQVIPACNSAAIRSLCPTPESFRNMGWLSVSDAVY
SEFIGELATRASNRNYSNEFGLMQPIQEFKAFIES
DPVVHQEFIDMFEGIQDSPRNYQELCNMFNDIFRK
APVYGDLGPPVYMIMAKLMNTRAGFSAFTRQRLNL
HFKKLFDTWGLFLSSKDSRNVLVADQFDDRHCGWL
NERALSAMVKHYNGRAFDEVFLCDKNAPYYGFNSY
DDFFNRRFRNRDIDRPVVGGVNNTTLISAACESLS
YNVSYDVQSLDTLVFKGETYSLKHLLNNDPFTPQF
EHGSILQGFLNVTAYHRWHAPVNGTIVKIINVPGT
YFAQAPSTIGDPIPDNDYDPPPYLKSLVYFSNIAA
RQIMFIEADNKEIGLIFLVFIGMTEISTCEATVSE
GQHVNRGDDLGMFHFGGSSFALGLRKDCRAEIVEK
FTEPGTVIRINEVVAALKA
(Psilocybe cyanescens (PSID gene))
SEQ ID NO: 15
MQVLPACQSSALKTLCPSPEAFRKLGWLPTSDEVY
NEFIDDLTGRTCNEKYSSQVTLLKPIQDFKTFIEN
DPIVYQEFISMFEGIEQSPTNYHELCNMFNDIFRK
APLYGDLGPPVYMIMARIMNTQAGFSAFTKESLNF
HFKKLFDTWGLFLSSKNSRNVLVADQFDDKHYGWF
SERAKTAMMINYPGRTFEKVFICDEHVPYHGFTSY
DDFFNRRFRDKDTDRPVVGGVTDTTLIGAACESLS
YNVSHNVQSLDTLVIKGEAYSLKHLLHNDPFTPQF
EHGSIIQGFLNVTAYHRWHSPVNGTIVKIVNVPGT
YFAQAPYTIGSPIPDNDRDPPPYLKSLVYFSNIAA
RQIMFIEADNKDIGLIFLVFIGMTEISTCEATVCE
GQHVNRGDDLGMFHFGGSSFALGLRKDSKAKILEK
FAKPGTVIRINELVASVRK
(Gymnopilus junonius (PSID gene))
SEQ ID NO: 16
MSSPRIVLHRVGGWLPKDQNVLEAWLSKKIAKAKT
RNRAPKDWAPVIQDFQRLIETDAEIYMGFHQMFEQ
VPKKTPYDKDPTNEQWQVRNYMHMLDLFDLIITEA
PDFEQNDLVGFPINAILDWPMGTPGGLTAFINPKV
NIMFHKMFDVWAVFLSSPASCYVLNTSDSGWFGPA
ATAAIPNFKETFICDPSLPYLGYTSWDNFFTRLFR
PGVRPVEFPNNDAIVNSACESTVYNIAPNIKPLDK
FWIKGEPYSLNHILNNDPYASQFVGGTISQAFLSA
LNYHRWASPVNGNIVKVVNVPGTYYAESPVTGFGN
PEGPDPAAPNLSQGFITAVAARALIFIEADNPNIG
LMCFVGVGMAEVSTCEVTVSVGDVVKKGDEIGMFH
FGGSTHCLIFRPQTKITFNPDYPVSTAVPLNAAVA
TVV
(Psilocybe cubensis (PSIH gene))
SEQ ID NO: 17
MIAVLFSFVIAGCIYYIVSRRVRRSRLPPGPPGIP
IPFIGNMFDMPEESPWLTFLQWGRDYNTDILYVDA
GGTEMVILNTLETITDLLEKRGSIYSGRLESTMVN
ELMGWEFDLGFITYGDRWREERRMFAKEFSEKGIK
QFRHAQVKAAHQLVQQLTKTPDRWAQHIRHQIAAM
SLDIGYGIDLAEDDPWLEATHLANEGLAIASVPGK
FWVDSFPSLKYLPAWFPGAVFKRKAKVWREAADHM
VDMPYETMRKLAPQGLTRPSYASARLQAMDLNGDL
EHQEHVnCNTAAEVNVGGGDTTVSAMSAFILAMVK
YPEVQRKVQAELDALTNNGQIPDYDEEDDSLPYLT
ACIKELFRWNQIAPLAIPHKLMKDDVYRGYLIPKN
TLVFANTWAVLNDPEVYPDPSVFRPERYLGPDGKP
DNTVRDPRKAAFGYGRRNCPGIHLAQSTVWIAGAT
LLSAFNIERPVDQNGKPIDIPADFTTGFFRHPVPF
QCRFVPRTEQVSQSVSGP
(Psilocybe cyanescens (PSIH gene))
SEQ ID NO: 18
MAPLTTMIPIVLSLLIAGCIYYINARRIKRSRLPP
GPPGIPIPFIGNMFDMPSESPWLIFLQWGQEYQTD
IIYVDAGGTDMIILNSLEAITDLLEKRGSLYSGRL
ESTMVNELMGWEFDFGFIPYGERWREERRMFAKEF
SEKNIRQFRHAQVKAANQLVRQLTDKPDRWSHHIR
HQIASMALDIGYGIDLAEDDPWIAASELANEGLAV
ASVPGSFWVDTFPFLKYLPSWLPGAEFKRNAKMWK
EGADHMVNMPYETMKKLSAQGLTRPSYASARLQAM
DPNGDLEHQERVIKNTATQVNVGGGDTTVGAVSAF
ILAMVKYPEVQRKVQAELDEFTSKGRIPDYDEDND
SLPYLSACFKELFRWGQIAPLAIAHRLIKDDVYRE
YTIPKNALVFANNWYGRTVLNDPSEYPNPSEFRPE
RYLGPDGKPDDTVRDPRKAAFGYGRRVCPGIHLAQ
STVWIAGVALVSAFNIELPVDKDGKCIDIPAAFTT
GFFR
(Gymnopilus junonius (PSIH gene))
SEQ ID NO: 19
MMSEMNGMDKLALLTTLLAAGFLYFKNKRRSALPF
PPGPKKHPLLGNLLDLPKKLEWETYRRWGKEYNSD
VIHVSAGSVNLIIVNSFEAATDLFDKRSANYSSRP
QFTMVRELMGWNWLMSALIYGDKWREQRRLFQKHF
STTNAELYQNTQLEYVRKALQHLLEEPSDFMGITR
HMAGGVSMSLAYGLNIQKKNDPFVDLAQRAVHSIT
EASVPGTFWVDVMPWLKYIPEWVPGAGFQKKARVW
RKLQQDFRQVPYQAALKDMASGKAKPSFASECLET
IDDNEDAQRQREVIKDTAAIVFAAGADTSLSGIHT
LFAAMLCYPEVQKKAQEELDRVLGGRRLPEFTDEP
NMPYISALVKEILRWKPATPIGVPHLASEDDVYNG
YYIPKRAVVIGNSWAMLHDEETYPDPSTFNPDRFL
TTNKSTGKLELDPTVRDPALMAFGFGRRMCPGRDV
ALSVIWLTIASVLATFNITKAIDENGKELEPDVQY
WSGLIVHPLPFKCTIKPRSKAAEELVKSGADAY
(Psilocybe cubensis (PSIK gene))
SEQ ID NO: 20
MAFDLKTEDGLITYLTKHLSLDVDTSGVKRLSGGF
VNVTWRIKLNAPYQGHTSIILKHAQPHMSTDEDFK
IGVERSVYEYQAIKLMMANREVLGGVDGIVSVPEG
LNYDLENNALIMQDVGKMKTLLDYVTAKPPLATDI
ARLVGTEIGGFVARLHNIGRERRDDPEFKFFSGNI
VGRTTSDQLYQTIIPNAAKYGVDDPLLPTVVKDLV
DDVMHSEETLVMADLWSGNILLQLEEGNPSKLQKI
YILDWELCKYGPASLDLGYFLGDCYLISRFQDEQV
GTTMRQAYLQSYARTSKHSINYAKVTAGIAAHIVM
WTDFMQWGSEEERINFVKKGVAAFHD ARGNNDNG
EITSTLLKESSTA
(Psilocybe cyanescens (PSIK gene))
SEQ ID NO: 21
MTFDLKTEEGLLSYLTKHLSLDVAPNGVKRLSGGF
VNVTWRVGLNAPYHGHTSIILKHAQPHLSSDIDFK
IGVERSAYEYQALKIVSANSSLLGSSDIRVSVPEG
LHYDVVNNALIMQDVGTMKTLLDYVTAKPPISAEI
ASLVGSQIGAFIARLHNLGRENKDKDDFKFFSGNI
VGRTTADQLYQTIIPNAAKYGIDDPILPIVVKELV
EEVMNSEETLIMADLWSGNILLQFDENSTELTRIW
LVDWELCKYGPPSLDMGYFLGDCFLVARFQDQLVG
TSMRQAYLKSYARNVKEPINYAKATAGIGAHLVMW
TDFMKWGNDEEREEFVKKGVEAFHEANEDNRNGEI
TSILVKEASRT
(Psilocybe cyanescens (PSIM gene))
SEQ ID NO: 22
MHIRNPYRDGVDYQALAEAFPALKPHVTVNSDNTT
SIDFAVPEAQRLYTAALLHRDFGLTITLPEDRLCP
TVPNRLNYVLWVEDILKVTSDALGLPDNRQVKGID
IGTGASAIYPMLACSRFKTWSMVATEVDQKCIDTA
RLNVIANNLQERLAIIATSVDGPILVPLLQANSDF
EYDFTMCNPPFYDGASDMQTSDAAKGFGFGVNAPH
TGTVLEMATEGGESAFVAQMVRESLNLQTRCRWFT
SNLGKLKSLYEIVGLLREHQISNYAINEYVQGATR
RYAIAWSFIDVRLPDHLSRPSNPDLSSLF
(Psilocybe cubensis (PSIM gene))
SEQ ID NO: 23
MHIRNPYRTPIDYQALSEAFPPLKPFVSVNADGTS
SVDLTIPEAQRAFTAALLHRDFGLTMTIPEDRLCP
TVPNRLNYVLWIEDIFNYTNKTLGLSDDRPIKGVD
IGTGASAIYPMLACARFKAWSMVGTEVERKCIDTA
RLNVVANNLQDRLSILETSIDGPILVPIFEATEEY
EYEFTMCNPPFYDGAADMQTSDAAKGFGFGVGAPH
SGTVIEMSTEGGESAFVAQMVRESLKLRTRCRWYT
SNLGKLKSLKEIVGLLKELEISNYAINEYVQGSTR
RYAVAWSFTDIQLPEELSRPSNPELSSLF
(Panaeolus cyanescens (PSIM gene))
SEQ ID NO: 24
MHNRNPYRDVIDYQALAEAYPPLKPHVTVNADNTA
SIDLTIPEVQRQYTAALLHRDFGLTITLPEDRLCP
TVPNRLNYVLWIEDIFQCTNKALGLSDDRPVKGVD
IGTGASAIYPMLACARFKQWSMIATEVERKCIDTA
RLNVLANNLQDRLSILEVSVDGPILVPIFDTFERA
TSDYEFEFTMCNPPFYDGAADMQTSDAAKGFGFGV
NAPHSGTVIEMATEGGEAAFVAQMVRESMKLQTRC
RWFTSNLGKLKSLHEIVALLRESQITNYAINEYVQ
GTTRRYALAWSFTDIKLTEELYRPSNPELGPLCST
FV
(Gymnopilus junonius (PSIM gene))
SEQ ID NO: 25
MHSRNPYRSPPDFAALSAAYPPLSPYITTDLSSGR
KTIDFRNEEAQRRLTEAIMLRDFGVVLNIPSNRLC
PPVPNRMNYVLWIQDIVYAHQTILGVSSRRIRGLD
IGTGATAIYPILACKKEQSWEMVATELDDYSYECA
CDNVSSNNMQTSIKVKKASVDGPILFPVENQNFDF
SMCNPPFYGSKEEVAQSAESKELPPNAVCTGAEIE
MIFSQGGEEGFVGRMVEESERLQTRCKWYTSMLGK
MSSVSTIVQALRARSIMNYALTEFVQGQTRRWAIA
WSFSDTHLPDAVSRISS
(Gymnopilus dilepis (PSIM gene))
SEQ ID NO: 26
MHIRNPYLTPPDYEALAEAFPALKPYVTVNPDKTT
TIDFAIPEAQRLYTAALLYRDFGLTITLPPDRLCP
TVPNRLNYVLWIQDILQITSAALGLPEARQVKGVD
IGTGAAAIYPILGCSLAKNWSMVGTEVEQKCIDIA
RQNVISNGLQDRITITANTIDAPILLPLFEGDSNF
EWEFTMCNPPFYDGAADMETSQDAKGFGFGVNAPH
TGTVVEMATDGGEAAFVSQMVRESLHLKTRCRWFT
SNLGKLKSLHEIVGLLREHQITNYAINEYVQGTTR
RYAIAWSFTDLRLSDHLPRPPNPDLSALF
(Saccharmyces cerevisiae (ARO1 gene))
SEQ ID NO: 27
ATGGTTCAACTAGCCAAGGTTCCAATACTAGGAAA
CGATATAATACACGTTGGATATAATATACACGATC
ATCTTGTAGAGACAATTATTAAACACTGTCCTTCT
TCTACTTACGTCATCTGTAACGATACTAACCTTAG
CAAGGTACCTTATTACCAGCAACTGGTTCTGGAGT
TCAAAGCAAGTCTTCCCGAAGGCTCCAGACTACTA
ACCTACGTGGTCAAACCGGGCGAGACGTCTAAGAG
TAGGGAGACGAAGGCGCAGTTAGAGGATTATCTTT
TAGTAGAAGGGTGCACTCGTGATACGGTCATGGTA
GCCATCGGCGGAGGTGTCATCGGTGACATGATCGG
TTTCGTAGCCTCCACGTTCATGAGAGGTGTGAGGG
TAGTACAGGTTCCGACGTCTCTTTTAGCAATGGTA
GACTCATCCATAGGCGGTAAAACGGCGATCGATAC
TCCGCTAGGAAAGAACTTCATTGGAGCCTTTTGGC
AGCCAAAATTTGTTCTTGTGGATATCAAGTGGCTT
GAAACACTAGCTAAACGTGAATTTATCAACGGCAT
GGCAGAAGTGATCAAGACAGCGTGCATCTGGAACG
CTGATGAATTTACTCGTCTCGAATCCAACGCGTCA
CTGTTCCTAAACGTAGTAAATGGTGCGAAAAATGT
AAAGGTGACTAACCAGCTGACGAACGAGATAGATG
AGATCAGCAACACGGATATTGAAGCCATGTTGGAC
CATACTTATAAACTGGTATTAGAGAGTATTAAGGT
TAAAGCGGAGGTGGTAAGCAGCGATGAAAGGGAGA
GCAGTCTTAGGAACCTTTTAAACTTCGGGCATAGC
ATAGGTCACGCGTATGAAGCCATACTGACACCCCA
GGCTTTACATGGAGAGTGCGTATCCATCGGCATGG
TAAAAGAAGCAGAACTATCAAGGTATTTTGGGATA
CTTTCTCCGACCCAGGTGGCGCGTCTAAGCAAAAT
TCTAGTTGCGTACGGATTGCCCGTTAGCCCCGATG
AGAAATGGTTTAAAGAGCTTACACTTCATAAGAAG
ACACCCTTGGACATACTGCTAAAGAAGATGAGCAT
CGACAAGAAAAATGAAGGAAGCAAGAAGAAGGTCG
TAATCCTAGAGTCTATCGGCAAATGTTACGGAGAC
TCAGCTCAGTTTGTTTCAGACGAAGACTTACGTTT
TATATTGACAGATGAAACACTAGTATATCCTTTTA
AGGATATTCCCGCTGATCAGCAGAAAGTCGTGATT
CCACCCGGAAGTAAATCAATAAGCAATCGTGCTTT
AATCTTAGCAGCTCTGGGGGAGGGACAGTGCAAGA
TCAAGAACCTATTACACTCCGACGACACCAAACAT
ATGCTGACCGCAGTCCACGAGTTAAAAGGTGCTAC
CATCAGTTGGGAGGATAACGGAGAAACAGTGGTCG
TAGAGGGCCATGGCGGGAGCACTCTATCGGCTTGT
GCTGATCCCTTATACTTAGGCAACGCGGGGACGGC
GAGTAGATTCTTAACATCACTGGCGGCACTAGTGA
ACAGTACATCCTCCCAAAAGTATATCGTACTAACA
GGCAACGCAAGGATGCAGCAACGTCCGATAGCGCC
CCTTGTTGACAGCTTACGTGCTAACGGGACAAAGA
TCGAGTACTTGAACAACGAAGGTTCTTTGCCGATC
AAAGTGTACACTGATTCTGTATTTAAAGGCGGCCG
TATTGAGTTGGCTGCGACAGTTAGTTCCCAATACG
TGAGCAGTATCCTGATGTGTGCGCCTTACGCAGAA
GAGCCCGTGACTTTAGCTTTGGTAGGTGGGAAACC
GATCAGTAAACTATACGTTGATATGACAATTAAGA
TGATGGAAAAGTTCGGCATCAATGTGGAGACCTCA
ACCACGGAACCCTACACATACTACATTCCGAAGGG
GCATTACATTAATCCAAGTGAGTACGTAATCGAGA
GCGACGCTTCATCCGCTACCTATCCGTTAGCATTC
GCCGCAATGACCGGTACCACCGTAACAGTCCCCAA
CATCGGCTTTGAATCTCTGCAGGGCGACGCTAGAT
TCGCAAGAGACGTCCTAAAGCCGATGGGGTGTAAA
ATCACCCAAACGGCTACGTCTACAACCGTCAGTGG
ACCACCCGTCGGTACGCTAAAGCCATTAAAACACG
TTGATATGGAACCAATGACAGACGCCTTCTTAACC
GCATGCGTTGTAGCCGCAATCAGTCATGACTCCGA
CCCCAATTCAGCGAACACTACTACTATCGAGGGGA
TCGCAAACCAAAGGGTTAAAGAATGCAACAGAATC
TTAGCGATGGCTACCGAGCTGGCAAAGTTTGGAGT
AAAGACAACAGAACTTCCCGATGGCATACAGGTCC
ATGGGCTAAATTCCATCAAGGACCTTAAAGTCCCA
TCTGACAGCTCAGGACCCGTCGGAGTCTGTACTTA
TGATGACCATAGGGTTGCCATGTCATTTTCCCTTT
TGGCTGGCATGGTAAACAGTCAGAATGAGAGAGAT
GAAGTGGCAAACCCAGTTAGGATCTTAGAGAGGCA
CTGCACCGGAAAGACGTGGCCAGGCTGGTGGGACG
TTCTGCACAGCGAACTTGGAGCGAAGCTGGATGGT
GCCGAGCCGCTAGAATGCACATCCAAAAAGAACTC
TAAGAAGAGCGTAGTCATAATAGGCATGAGAGCTG
CGGGCAAAACTACTATCTCTAAGTGGTGCGCAAGT
GCGCTGGGTTACAAGTTGGTAGATTTAGATGAATT
GTTCGAGCAGCAGCATAATAACCAATCAGTAAAAC
AATTTGTAGTCGAGAATGGTTGGGAGAAATTCAGA
GAGGAAGAGACCAGGATATTCAAGGAGGTTATTCA
AAATTACGGCGACGACGGGTATGTCTTTAGCACTG
GGGGAGGGATCGTCGAATCCGCGGAGAGCAGGAAA
GCACTAAAGGACTTCGCCAGTTCCGGTGGGTATGT
GCTTCACTTACATCGTGATATAGAGGAGACGATAG
TCTTCCTACAAAGTGATCCATCCAGGCCGGCGTAT
GTTGAGGAGATTAGGGAGGTCTGGAACCGTAGAGA
AGGCTGGTATAAAGAATGTAGTAATTTTAGCTTTT
TCGCACCTCACTGTAGCGCAGAGGCGGAGTTTCAA
GCACTTAGACGTTCATTCAGTAAGTATATAGCTAC
GATCACGGGGGTCCGTGAAATAGAGATTCCTAGTG
GGAGGAGTGCGTTTGTATGCTTAACTTTTGACGAT
CTAACTGAGCAAACGGAGAATCTGACGCCTATATG
CTACGGGTGTGAAGCCGTAGAGGTGCGTGTTGATC
ATCTTGCCAATTATTCCGCAGACTTCGTTAGCAAG
CAATTAAGCATACTGAGAAAAGCGACCGACAGTAT
ACCCATTATCTTCACCGTCCGTACTATGAAACAAG
GCGGTAATTTTCCCGATGAAGAGTTCAAGACATTG
CGTGAGTTGTACGACATAGCTCTTAAAAACGGAGT
GGAGTTCCTTGATTTGGAACTTACTCTGCCTACAG
ATATACAGTACGAAGTCATCAACAAGAGAGGTAAT
ACGAAGATCATTGGGTCTCATCATGACTTCCAGGG
TTTGTACAGCTGGGACGATGCTGAATGGGAAAACA
GATTCAATCAGGCACTGACTCTTGACGTAGATGTG
GTGAAATTTGTGGGTACCGCGGTGAATTTCGAGGA
CAACTTACGTTTGGAACATTTTCGTGACACGCACA
AAAATAAACCACTAATAGCAGTTAACATGACGTCT
AAGGGCTCAATCAGTAGGGTACTAAATAATGTATT
GACTCCGGTTACTTCAGACCTTTTACCGAACAGCG
CAGCGCCTGGTCAATTGACGGTTGCACAGATTAAT
AAAATGTATACATCTATGGGAGGAATTGAGCCTAA
AGAGCTATTTGTGGTGGGGAAGCCAATCGGCCACT
CAAGATCACCTATACTACACAATACTGGGTATGAG
ATTTTGGGTCTACCTCACAAATTCGATAAATTTGA
GACGGAAAGCGCACAATTAGTGAAGGAGAAATTGT
TAGACGGGAACAAGAATTTCGGTGGTGCAGCGGTG
ACCATCCCTTTAAAGCTAGACATAATGCAGTACAT
GGATGAACTTACGGACGCTGCGAAGGTGATTGGGG
CGGTAAACACAGTAATCCCTTTGGGTAACAAGAAA
TTCAAGGGTGATAATACGGACTGGTTAGGGATAAG
GAACGCACTTATAAATAATGGTGTGCCCGAGTACG
TGGGGCATACTGCCGGACTTGTAATAGGTGCTGGT
GGTACCAGTAGGGCGGCACTGTACGCTTTGCATAG
CTTAGGTTGCAAGAAGATCTTTATCATCAATAGAA
CAACTAGTAAACTGAAGCCACTGATAGAATCACTA
CCCTCCGAGTTTAACATCATTGGAATAGAGTCTAC
GAAATCCATCGAGGAGATTAAAGAACACGTCGGAG
TCGCTGTTAGCTGCGTGCCTGCCGATAAGCCCTTA
GATGACGAGCTACTGAGTAAGTTAGAACGTTTCCT
TGTCAAGGGTGCACATGCGGCTTTCGTCCCAACAC
TGCTAGAGGCTGCCTATAAACCCAGCGTAACACCT
GTTATGACCATAAGTCAGGACAAGTATCAATGGCA
CGTGGTGCCGGGTTCCCAGATGCTGGTCCATCAAG
GTGTTGCACAATTTGAAAAATGGACTGGTTTCAAG
GGGCCCTTCAAAGCCATATTTGACGCCGTGACTAA
AGAGTAA
(Saccharomyces cerevisiae (ARO2 gene))
SEQ ID NO: 28
ATGTCCACATTCGGTAAACTTTTCCGTGTCACTAC
ATACGGCGAGTCACACTGCAAATCTGTGGGGTGCA
TAGTAGACGGCGTTCCGCCGGGCATGAGTTTAACC
GAAGCGGACATTCAACCTCAGCTTACCCGTAGGAG
GCCCGGTCAGAGCAAGTTATCCACCCCGAGGGACG
AAAAGGACCGTGTAGAGATCCAAAGCGGAACGGAA
TTTGGGAAGACACTTGGTACGCCTATCGCTATGAT
GATTAAAAACGAGGATCAACGTCCGCACGATTACT
CCGACATGGACAAGTTCCCTAGGCCGAGTCACGCC
GATTTTACGTACTCAGAGAAATACGGAATAAAAGC
CTCCAGCGGTGGGGGCCGTGCTTCCGCGAGAGAAA
CCATTGGAAGAGTAGCATCCGGTGCAATAGCAGAG
AAGTTCCTAGCACAGAACTCAAATGTTGAAATTGT
CGCTTTCGTCACGCAAATAGGTGAGATCAAGATGA
ACCGTGACAGTTTCGACCCAGAATTTCAACACCTT
CTAAATACAATTACGAGGGAGAAGGTAGATAGCAT
GGGTCCAATAAGATGCCCCGACGCTTCCGTCGCGG
GATTGATGGTGAAGGAAATTGAAAAATATCGTGGG
AACAAGGATTCTATTGGGGGTGTAGTAACTTGCGT
AGTCAGAAATCTACCTACAGGGTTGGGTGAACCGT
GTTTTGACAAACTGGAGGCGATGCTGGCACATGCC
ATGTTATCCATACCAGCAAGTAAAGGATTTGAAAT
AGGATCTGGCTTCCAGGGTGTAAGCGTACCAGGAA
GCAAACACAATGATCCCTTTTACTTTGAAAAAGAG
ACTAACCGTCTTCGTACAAAGACAAACAACTCCGG
TGGGGTGCAAGGGGGCATCTCTAATGGTGAGAACA
TTTACTTTTCCGTACCATTTAAGAGCGTGGCTACA
ATAAGCCAAGAGCAAAAGACCGCAACTTACGATGG
AGAAGAAGGAATCCTCGCAGCTAAGGGTAGGCACG
ATCCTGCGGTCACACCGCGTGCAATTCCCATAGTG
GAAGCTATGACCGCCCTAGTACTAGCAGATGCGTT
ACTAATACAGAAAGCCAGGGATTTTTCTAGGTCAG
TCGTACATTAA
(Saccharomyces cerevisiae (ARO3 gene))
SEQ ID NO: 29
ATGTTCATCAAGAATGACCATGCTGGTGATAGAAA
GAGACTAGAGGACTGGCGTATAAAGGGTTATGACC
CTCTAACTCCGCCTGATTTGCTACAGCACGAGTTT
CCTATATCAGCAAAAGGGGAAGAAAATATCATCAA
GGCTCGTGATAGTGTATGTGATATACTGAACGGAA
AGGATGACAGACTTGTGATAGTAATTGGACCCTGT
TCTCTGCATGATCCGAAGGCGGCCTACGACTATGC
CGACAGATTAGCCAAAATATCCGAAAAGCTGTCAA
AAGATCTTTTAATTATCATGCGTGCATACCTAGAG
AAGCCTCGTACAACCGTTGGATGGAAAGGGTTGAT
AAACGACCCGGATATGAACAATAGTTTTCAGATTA
ATAAAGGCCTTCGTATAAGCCGTGAGATGTTTATA
AAACTAGTTGAGAAATTACCTATTGCAGGAGAAAT
GCTTGACACGATTTCCCCTCAGTTCTTATCTGACT
GTTTCTCACTAGGTGCAATTGGTGCTAGGACTACC
GAGTCACAGTTACATCGTGAACTGGCCAGCGGTCT
GTCTTTCCCCATTGGCTTTAAAAATGGTACCGATG
GTGGCCTTCAAGTAGCAATTGATGCTATGAGAGCT
GCGGCCCACGAACACTACTTTTTGTCTGTGACCAA
ACCTGGCGTAACAGCGATTGTGGGAACTGAAGGGA
ACAAGGACACCTTCCTAATCCTGAGAGGGGGCAAG
AACGGGACTAATTTTGACAAGGAGTCAGTTCAAAA
CACTAAGAAGCAATTGGAGAAGGCGGGCCTTACTG
ACGATTCTCAGAAGAGAATCATGATAGACTGCAGC
CATGGCAACTCAAATAAAGATTTCAAAAATCAACC
CAAAGTCGCCAAGTGTATCTACGATCAACTAACCG
AAGGAGAAAATAGTTTATGCGGGGTGATGATAGAG
AGTAATATAAACGAAGGAAGACAGGATATTCCTAA
GGAAGGCGGAAGAGAGGGTCTGAAGTACGGGTGTT
CTGTGACAGACGCTTGCATAGGATGGGAGAGCACG
GAACAGGTTTTGGAGCTGCTGGCAGAAGGGGTGCG
TAATAGAAGGAAAGCCTTAAAGAAGTAA
(Saccharomyces cerevisiae (ARO4 K2229L gene))
SEQ ID NO: 30
ATGAGCGAATCTCCGATGTTCGCCGCAAACGGCAT
GCCTAAGGTAAATCAAGGGGCCGAGGAGGACGTGA
GAATATTAGGTTATGACCCGCTTGCCAGTCCTGCA
TTGCTTCAGGTACAGATTCCAGCAACGCCAACGTC
CTTAGAAACAGCAAAAAGGGGACGTCGTGAAGCTA
TAGACATCATCACTGGCAAGGACGACCGTGTCCTA
GTAATAGTTGGTCCGTGCTCTATCCATGACCTTGA
GGCTGCACAGGAGTATGCACTAAGGTTGAAGAAAT
TGTCTGATGAACTGAAAGGTGATCTTAGTATAATC
ATGCGTGCATATTTAGAGAAACCGCGTACGACGGT
AGGCTGGAAAGGGCTAATTAACGATCCGGATGTGA
ATAATACCTTTAACATCAACAAGGGTCTACAGAGT
GCGCGTCAGTTATTCGTGAACTTAACAAATATCGG
ACTGCCGATAGGCTCCGAGATGCTGGACACGATAT
CTCCCCAGTATTTGGCTGACCTTGTTTCTTTTGGA
GCTATAGGTGCAAGGACTACTGAGAGTCAGTTACA
TAGAGAGTTGGCATCAGGACTTAGCTTCCCTGTAG
GATTTAAGAACGGTACAGACGGCACTCTTAATGTC
GCGGTCGATGCCTGCCAGGCAGCCGCCCATTCACA
TCATTTTATGGGAGTGACATTACACGGGGTGGCCG
CTATCACAACGACTAAAGGGAATGAGCACTGTTTT
GTTATCCTTAGAGGAGGAAAGAAAGGTACGAATTA
TGATGCGAAAAGTGTAGCAGAGGCCAAAGCGCAAC
TTCCTGCCGGTTCAAACGGACTTATGATTGACTAT
TCCCATGGAAACTCAAATAAGGACTTTAGGAATCA
GCCAAAAGTTAACGATGTGGTATGCGAACAGATCG
CGAACGGTGAAAATGCGATTACGGGTGTTATGATC
GAGTCAAATATAAATGAAGGTAACCAAGGTATCCC
GGCAGAGGGCAAAGCGGGCCTGAAGTACGGTGTAT
CTATTACGGATGCCTGTATAGGTTGGGAGACAACC
GAAGACGTCCTAAGGAAACTTGCCGCCGCGGTTAG
ACAGAGACGTGAAGTCAATAAGAAGTAA
(Escherichia coli (AROL gene))
SEQ ID NO: 31
ATGACCCAGCCATTATTTCTGATCGGTCCTCGTGG
GTGCGGGAAAACGACGGTTGGCATGGCCTTAGCTG
ACAGTTTGAATCGTAGATTCGTGGACACCGACCAG
TGGCTACAGTCTCAGCTTAACATGACGGTGGCCGA
AATTGTAGAACGTGAAGAATGGGCTGGTTTTCGTG
CAAGAGAAACAGCCGCATTGGAAGCTGTGACGGCG
CCTTCAACGGTGATAGCTACGGGAGGTGGTATTAT
TTTGACCGAATTTAATAGGCACTTCATGCAGAATA
ATGGCATAGTGGTTTACCTATGCGCTCCTGTGTCT
GTCTTGGTAAACCGTTTGCAAGCCGCACCAGAAGA
AGACTTGCGTCCAACCCTGACGGGGAAGCCACTGT
CTGAGGAAGTGCAAGAGGTACTGGAGGAAAGGGAC
GCTCTATACCGTGAGGTGGCTCACATCATAATTGA
CGCTACGAATGAGCCATCACAGGTAATTTCTGAGA
TCCGTTCAGCGTTGGCCCAAACCATCAATTGTTAA
(Saccharomyces cerevisiae (TRP1 gene))
SEQ ID NO: 32
ATGTCAGTGATTAACTTTACAGGCTCCTCAGGTCC
CTTGGTCAAGGTCTGCGGCTTGCAATCAACAGAGG
CCGCTGAATGCGCCCTAGACTCAGATGCAGACCTT
TTAGGCATCATCTGTGTCCCCAACAGAAAGCGTAC
TATTGATCCTGTTATTGCGCGTAAGATCAGTTCTT
TGGTCAAGGCGTATAAGAACTCCTCAGGAACCCCC
AAGTATCTGGTAGGGGTATTCAGGAATCAACCTAA
AGAAGACGTCTTGGCCCTAGTTAATGACTACGGCA
TAGACATAGTCCAGTTGCACGGAGACGAAAGCTGG
CAAGAATATCAGGAATTTTTGGGGCTGCCGGTTAT
AAAAAGGCTGGTTTTCCCTAAGGACTGTAACATAC
TGTTATCAGCCGCATCACAGAAGCCGCATTCCTTT
ATACCTCTTTTCGACTCCGAGGCCGGAGGCACTGG
TGAATTACTGGACTGGAACAGCATTTCAGATTGGG
TAGGGAGGCAGGAGAGCCCAGAATCTCTTCATTTT
ATGTTGGCAGGGGGCCTTACGCCGGAAAATGTTGG
AGATGCATTGAGGTTGAACGGAGTTATAGGTGTGG
ATGTCAGTGGTGGGGTTGAAACGAATGGTGTTAAA
GACAGCAACAAAATAGCAAATTTTGTCAAGAATGC
CAAAAAGTAA
(Saccharomyces cerevisiae (TRP2 S76L gene))
SEQ ID NO: 33
ATGACGGCGAGCATTAAAATTCAGCCAGACATTGA
CAGTTTAAAGCAGTTGCAGCAACAGAATGACGACT
CTTCCATTAACATGTATCCCGTGTATGCGTATCTG
CCTTCTTTGGATTTGACACCTCACGTTGCTTACTT
AAAGTTAGCTCAACTTAATAATCCAGATAGAAAGG
AGTCTTTCTTACTTGAAAGTGCTAAGACCAATAAT
GAGCTGGACAGATATCTTTTCATAGGGATCAGTCC
AAGGAAGACCATTAAGACCGGGCCCACTGAAGGCA
TTGAGACTGACCCATTAGAAATCCTTGAAAAAGAA
ATGTCTACTTTCAAAGTCGCCGAAAACGTCCCAGG
CCTTCCCAAATTAAGCGGCGGGGCGATAGGTTACA
TATCATACGACTGTGTACGTTACTTCGAACCCAAG
ACTAGGCGTCCCTTGAAAGATGTGCTTAGGTTACC
AGAGGCGTACTTGATGCTTTGTGACACGATAATCG
CATTTGACAATGTCTTCCAAAGGTTTCAAATTATT
CACAATATTAACACAAACGAAACGTCTTTGGAGGA
AGGATACCAGGCGGCTGCGCAGATAATCACGGATA
TTGTATCTAAGTTGACAGACGACAGCTCCCCCATT
CCGTACCCGGAGCAACCCCCTATCAAACTAAACCA
AACCTTTGAATCCAACGTAGGCAAAGAGGGGTATG
AAAATCACGTCTCCACTCTCAAAAAGCACATAAAG
AAAGGTGACATAATCCAAGGTGTGCCCAGCCAGAG
AGTGGCGAGGCCTACATCTTTACATCCATTCAACA
TATATAGGCATCTTAGAACCGTGAACCCATCACCT
TATCTATTTTACATAGACTGCCTAGATTTCCAGAT
AATAGGGGCTAGTCCCGAATTGCTGTGTAAATCAG
ATTCAAAGAATCGTGTTATTACACACCCCATAGCT
GGCACAGTCAAGAGGGGTGCTACCACTGAGGAAGA
TGACGCTCTGGCAGATCAGCTACGTGGTTCTTTGA
AAGATAGGGCTGAGCATGTTATGCTGGTTGACTTA
GCAAGAAACGACATCAATCGTATATGCGATCCCCT
AACGACTTCCGTTGACAAACTTTTGACCATTCAGA
AGTTCAGCCACGTACAGCACTTAGTCTCTCAGGTC
TCTGGCGTCCTAAGGCCTGAGAAAACTCGTTTCGA
TGCATTCAGAAGCATATTTCCCGCGGGTACAGTGA
GTGGGGCCCCAAAGGTGCGTGCAATGGAGCTTATA
GCCGAGCTAGAAGGCGAGCGTAGGGGAGTGTACGC
AGGGGCCGTAGGCCATTGGTCTTATGACGGCAAGA
CCATGGATAATTGTATTGCACTAAGGACCATGGTC
TATAAAGATGGGATTGCATACTTGCAGGCAGGAGG
TGGGATTGTCTATGACAGCGATGAGTACGATGAGT
ATGTAGAAACAATGAATAAAATGATGGCGAATCAT
TCCACGATAGTGCAGGCGGAGGAGTTATGGGCGGA
TATTGTGGGTAGTGCATAA
(Saccharomyces cerevisiae (TRP3 gene))
SEQ ID NO: 34
ATGTCTGTCCACGCAGCCACCAACCCGATAAATAA
GCATGTCGTTCTGATTGATAATTACGACTCCTTCA
CGTGGAATGTTTATGAGTATCTTTGCCAGGAGGGA
GCGAAGGTTAGCGTTTACCGTAATGACGCTATCAC
GGTCCCAGAAATTGCAGCACTGAATCCCGATACCC
TTCTGATATCACCAGGCCCGGGCCATCCCAAGACA
GATTCTGGTATTAGCAGAGATTGCATCAGATACTT
CACTGGAAAAATTCCAGTTTTTGGGATATGTATGG
GGCAGCAATGCATGTTTGACGTGTTTGGCGGGGAA
GTGGCTTATGCGGGTGAAATAGTGCACGGAAAGAC
TAGTCCCATATCCCATGATAACTGCGGTATCTTTA
AGAATGTCCCCCAGGGTATTGCAGTTACAAGATAT
CATAGCTTGGCTGGCACTGAAAGTAGTCTGCCTAG
CTGCCTAAAGGTGACTGCCTCTACTGAAAACGGGA
TAATCATGGGGGTAAGGCACAAGAAGTACACCGTC
GAGGGGGTGCAATTCCACCCAGAGAGTATTTTAAC
CGAAGAAGGACATCTAATGATCCGTAATATTCTTA
ATGTTTCTGGCGGAACGTGGGAGGAAAATAAATCA
AGCCCATCCAATTCCATCCTAGATAGGATATACGC
CAGGCGTAAAATTGACGTAAACGAACAGTCAAAGA
TTCCCGGTTTCACCTTTCAGGACTTACAATCTAAC
TATGATCTTGGCCTTGCCCCGCCTCTGCAAGATTT
TTATACCGTGCTGAGCAGTAGTCATAAGAGGGCTG
TGGTCCTAGCGGAGGTGAAGCGTGCCTCCCCTAGC
AAAGGTCCAATCTGCCTGAAGGCCGTTGCTGCTGA
ACAAGCCCTTAAATATGCTGAGGCTGGGGCGAGTG
CAATTAGCGTTCTAACAGAACCCCACTGGTTCCAC
GGGAGCCTTCAAGACCTTGTGAATGTAAGAAAGAT
CTTGGATCTAAAATTTCCGCCAAAAGAGAGACCCT
GCGTGCTTAGGAAAGAGTTTATATTTTCCAAATAC
CAAATATTGGAGGCACGTCTAGCTGGTGCAGATAC
TGTCCTTTTGATTGTAAAGATGTTGTCCCAACCAT
TACTGAAAGAGCTATATAGTTACTCAAAGGATTTA
AACATGGAGCCGTTAGTGGAAGTAAATAGCAAGGA
GGAGCTACAACGTGCCCTGGAAATTGGTGCCAAGG
TTGTTGGAGTTAACAATCGTGACTTGCATTCCTTC
AACGTAGACTTGAATACAACAAGTAATTTGGTCGA
ATCTATCCCAAAAGATGTGCTGTTGATTGCACTTT
CCGGTATCACAACACGTGATGACGCCGAAAAGTAT
AAAAAGGAGGGGGTGCACGGGTTTTTGGTGGGTGA
GGCGTTAATGAAATCTACAGATGTAAAGAAGTTTA
TTCATGAGCTGTGCGAATAA
(Saccharomyces cerevisiae (TRP4 gene))
SEQ ID NO: 35
ATGAGCGAAGCTACTCTATTAAGTTATACCAAAAA
GCTACTAGCAAGCCCACCTCAGCTTAGTTCCACCG
ACCTACACGATGCACTACTTGTCATCCTAAGTCTA
CTTCAGAAGTGCGACACCAATTCTGATGAGTCCTT
GTCTATTTATACGAAGGTGTCTTCCTTTTTAACAG
CCCTAAGGGTGACTAAGTTAGATCATAAGGCGGAA
TATATTGCCGAGGCTGCAAAAGCAGTTTTGCGTCA
CTCAGATCTGGTCGATCTACCTTTACCTAAAAAGG
ATGAGCTGCATCCTGAAGATGGTCCTGTTATCTTG
GACATTGTGGGTACTGGGGGTGATGGACAGAATAC
CTTTAACGTGTCAACGTCAGCCGCTATTGTGGCCT
CAGGTATTCAGGGACTGAAGATTTGCAAACACGGA
GGTAAAGCATCTACCTCAAACAGCGGAGCTGGAGA
TCTGATTGGGACATTGGGATGCGATATGTTCAAAG
TGAATAGTAGCACAGTCCCCAAATTGTGGCCAGAC
AATACATTTATGTTCTTATTGGCTCCATTCTTTCA
TCATGGGATGGGTCATGTAAGCAAGATTCGTAAGT
TTCTTGGAATACCTACGGTATTTAACGTATTGGGG
CCGCTGTTACACCCCGTATCCCATGTGAATAAGAG
GATACTTGGAGTGTATTCAAAAGAGTTGGCGCCAG
AATATGCGAAGGCAGCAGCCTTGGTCTATCCAGGG
TCAGAAACGTTTATTGTGTGGGGCCATGTTGGGCT
TGACGAGGTGAGCCCCATAGGAAAGACTACCGTGT
GGCACATCGATCCGACAAGCTCAGAACTAAAGTTG
AAGACCTTCCAGCTGGAGCCATCTATGTTCGGTCT
GGAGGAGCACGAGCTGAGTAAATGCGCCTCATATG
GACCTAAGGAGAATGCTCGTATATTAAAGGAGGAA
GTCCTTTCCGGCAAATACCACCTAGGCGACAATAA
TCCAATATATGATTACATTCTGATGAATACTGCAG
TATTATACTGCCTGTCCCAAGGGCACCAAAACTGG
AAGGAAGGTATTATCAAAGCCGAGGAGTCAATTCA
CAGCGGGAATGCCTTGAGATCGCTAGAACATTTCA
TTGATTCAGTATCTTCCCTTTAA
(Saccharomyces cerevisiae (TAT2 gene))
SEQ ID NO: 36
ATGACCGAAGATTTCATCAGTAGCGTCAAAAGGTC
AAATGAAGAGCTTAAAGAGAGAAAATCTAATTTTG
GGTTTGTAGAGTACAAGTCAAAACAACTTACCTCC
AGTAGCTCACACAACTCCAACTCTTCACACCATGA
TGACGACAACCAGCACGGTAAAAGAAACATCTTTC
AGCGTTGTGTGGATTCTTTTAAATCCCCTCTGGAT
GGGTCTTTCGACACCTCCAATCTGAAAAGAACACT
GAAACCTCGTCATTTAATAATGATCGCAATAGGAG
GTAGTATAGGTACTGGTCTTTTCGTGGGTTCAGGG
AAGGCTATAGCGGAAGGCGGACCACTTGGCGTTGT
GATCGGATGGGCCATTGCGGGTAGCCAAATAATAG
GTACTATACATGGGTTAGGAGAGATCACGGTAAGA
TTTCCAGTAGTCGGTGCGTTTGCCAACTACGGCAC
CCGTTTCTTGGACCCGAGCATTAGTTTTGTAGTCT
CCACTATATACGTGCTACAGTGGTTCTTTGTCCTA
CCCCTAGAGATTATTGCTGCGGCGATGACCGTGCA
ATACTGGAACAGTTCTATCGATCCGGTAATATGGG
TCGCAATTTTCTATGCCGTCATCGTCTCAATCAAT
TTGTTTGGAGTTAGGGGTTTCGGAGAAGCTGAATT
CGCCTTCTCAACTATTAAGGCAATCACTGTCTGTG
GCTTCATAATCTTATGTGTAGTCTTGATCTGCGGC
GGAGGACCCGATCACGAATTCATTGGTGCTAAATA
CTGGCATGATCCTGGCTGCCTGGCAAACGGGTTTC
CTGGAGTCTTGAGTGTCCTTGTCGTTGCGTCATAC
AGCCTAGGAGGCATAGAAATGACTTGCTTAGCCTC
TGGGGAAACGGACCCAAAGGGACTTCCCTCAGCTA
TAAAACAGGTTTTCTGGCGTATTTTGTTTTTCTTC
TTAATTTCTTTAACTCTAGTGGGATTTTTAGTTCC
TTACACCAACCAAAATCTACTAGGTGGCTCCTCTG
TCGATAATAGTCCCTTCGTTATCGCGATTAAGCTA
CACCATATCAAAGCTCTTCCGTCTATTGTTAACGC
AGTTATCCTTATTTCCGTGCTATCCGTGGGTAACA
GTTGCATCTTTGCCAGCTCCAGAACTCTGTGTAGC
ATGGCACATCAAGGACTGATACCGTGGTGGTTCGG
CTATATTGACAGAGCTGGCAGACCCCTGGTTGGGA
TTATGGCCAATTCTCTTTTCGGCTTATTGGCGTTC
CTTGTTAAATCTGGCTCCATGAGTGAGGTGTTTAA
TTGGCTGATGGCTATAGCCGGACTGGCGACATGTA
TTGTGTGGTTATCTATAAATCTTTCCCATATAAGA
TTCCGTCTTGCAATGAAGGCCCAAGGAAAGTCCCT
GGATGAACTTGAATTCGTAAGCGCGGTTGGTATAT
GGGGATCTGCTTATTCCGCACTTATCAATTGCTTA
ATACTTATTGCTCAATTTTATTGCTCTTTATGGCC
AATCGGGGGTTGGACATCCGGAAAAGAGAGGGCAA
AGATTTTCTTTCAGAATTATCTTTGCGCCCTGATT
ATGTTATTTATATTCATCGTCCATAAGATCTATTA
TAAATGTCAAACGGGAAAGTGGTGGGGTGTTAAAG
CTCTGAAGGACATCGACCTAGAGACCGACAGGAAG
GACATAGACATCGAAATAGTTAAACAAGAAATCGC
TGAAAAGAAGATGTATTTGGACTCCAGACCTTGGT
ACGTGAGGCAGTTTCATTTTTGGTGCTAA
