BIOSYNTHETIC PRODUCTION OF PSILOCYBIN AND RELATED INTERMEDIATES

IN RECOMBINANT ORGANISMS

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority from U.S. Provisional Application Serial No. 62/936,387 filed on November 15, 2019, which is incorporated herein by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR

DEVELOPMENT

[0002] Not Applicable.

INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ON A COMPACT

DISC

[0003] 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 November 15, 2019 using Patentln 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

[0004] 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

[0005] 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

[0006] 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.

[0007] 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.

[0008] 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.

[0009] 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.

[0010] 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.

[0011] In accordance with a further aspect, the carbon source can include at least one of: glucose, galactose, sucrose, fructose, com syrup, corn steep liquor, ethanol, and molasses.

[0012] In accordance with another aspect, the first pathway can be a shikimate- chorismate pathway and the second pathway can be a L-tryptophan pathway

[0013] 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.

[0014] 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.

[0015] 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.

[0016] 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.

[0017] 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.

[0018] 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.

[0019] 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.

[0020] 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.

[0021] 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.

[0022] In accordance with a further aspect, the method can also include an exogenous substrate and a transporter protein.

[0023] 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.

[0024] In accordance with a further aspect, the transporter protein can be encoded by codon optimized SEQ ID NO: 36.

[0025] In accordance with a further aspect, the intermediates can include: tryptamine, 4-hydroxytryptamine, norbaeocystin, baeocystin, and psilocin.

[0026] 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

[0027] 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.

[0028] Fig. 1 depicts a table of amino acids and codon triplets.

[0029] Fig. 2 depicts a table of genes and enzymes inserted into a recombinant host organism

[0030] Fig. 3 depicts the biosynthesis of psilocybin.

[0031] Fig. 4-7 depicts sequence alignments.

[0032] Fig. 8 depicts endogenous pathways in a host organism.

[0033] Fig. 9 depicts a scheme to increase metabolic flux through shikimate- chorismate and L-tryptophan pathways.

[0034] Fig. 10 depicts a heterologous recombinant host organism.

[0035] Fig. 11 depicts HPLC chromatograms and UV/Vis spectra.

DETAILED DESCRIPTION

[0036] Abbreviations and Definitions

[0037] To facilitate understanding of the invention, a number of terms and abbreviations as used herein are defined below as follows:

[0038] Amino acids: As used herein, the term “amino acids” refer to the molecular basis for constructing and assembling proteins, such as enzymes. (See Figure 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.

[0039] 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.

[0040] 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.

[0041] 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.

[0042] 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.

[0043] Reference is made to the figures to further describe the systems and methods disclosed herein.

[0044] 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.

[0045] Further, the genes/enzymes that are inserted or engineered into the recombinant host are PsiD, PsiH, PsiK, and PsiM.

[0046] 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.

[0047] 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.

[0048] 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 phosphorylatation (i.e., adding 0=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.

[0049] 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 (C¾) 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.

[0050] 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.

[0051] 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.

[0052] 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).

[0053] 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.

[0054] 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).

[0055] 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.

[0056] 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).

[0057] 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.

[0058] 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).

[0059] 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 AR03, AR04, AROl, and AR02 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, SERI, 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 TRPl, TRP2, TRP3, and TRP4 enzymes can be subjected to the depicted precursors at the specified point to selectively yield (lS,2R)-l-C-indol-3-yl)glycerol 3-phosphate. The addition of L-serine to (lS,2R)-l-C-indol-3-yl)glycerol 3-phosphate in the presence of the TRPl enzyme can yield L-tryptophan.

[0060] 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.

[0061] 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 AR03 and AR04 enzymes.

[0062] Overexpression of the genes encoding AR03 enzyme (as encoded by codon optimized SEQ ID NO: 29), and a feedback-resistant mutant AR04 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, AROl enzyme (as encoded by codon optimized SEQ ID NO: 27) and AR02 (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.

[0063] 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 TRIM 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.

[0064] 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 AROIO enzyme - have been deleted to reduce pathway flux through the pathways that produce aromatic alcohols. The gene that encodes the native enzyme, AR07 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 pAB A production pathway.

[0065] 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.

[0066] 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.

[0067] 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.

[0068] 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 R _f 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 procedue for obtaining the hydroxylated indole at the 4- position, in comparison to the techniques of organic chemistry.

[0069] 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.

[0070] 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.

[0071] 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.

[0072] EXAMPLES

[0073] 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. [0074] 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.

[0075] 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.

[0076] The gene sequences from gene source organisms are codon optimized to improve expression using techniques disclosed in U.S. Patent Application No. 15/719430, filed September 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. [0077] 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, AROl, AR02, AR03, AR04 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, AROl, AR02, AR03, and AR04 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, TRPl, TRP2 S76L, TRP3, TRP4, AROl, AR02, AR03, AR04 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 No. 16/558,909 filed on September 3, 2019, entitled “Automated Pipeline”.

[0078] Example 1 - Construction of Saccharomyces cerevisiae platform strains with elevated metabolic flux towards L-tryptophan via overexpression of the feedback resistant mutant, AR04 K229L

[0079] The optimized AR04 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 AR04 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.

[0080] Example 2 - Construction of Saccharomyces cerevisiae platform strains with elevated metabolic flux towards L-tryptophan via deletion of PDC5 [0081] 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 No. 16/558,909 filed on September 3, 2019, entitled “Automated Pipeline”.

[0082] Example 3 - Method of Growth

[0083] 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, com 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.

[0084] Example 4 - Detection of Isolated Product

[0085] 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 (Obelise N, SIELC, Wheeling, IL USA) is used. A gradient is used of mobile phase A (ultraviolet (UV) grade H ₂ O+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.

[0086] Other Embodiments

[0087] 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.

[0088] References Cited

[0089] 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

SEQ ID NO: 1 (Psilocybe cubensis (PSID gene))

ATGCAAGTCATCCCCGCGTGCAACAGCGCAGCTATAAGGTCACTTTGTCCGACCC

CCGAGAGCTTTAGAAATATGGGCTGGCTTTCCGTGAGCGATGCCGTCTATAGCGA

ATTTATAGGTGAACTTGCGACGAGAGCATCTAATAGAAACTACAGCAATGAGTT

CGGTTTAATGCAACCAATACAAGAATTTAAAGCGTTCATCGAGAGTGATCCCGTT

GTACACCAAGAGTTTATCGACATGTTTGAAGGCATCCAAGATTCTCCGAGGAACT

ACCAAGAACTATGTAACATGTTCAATGATATTTTTAGGAAGGCTCCCGTATACGG

AGATTTGGGCCCTCCGGTCTACATGATTATGGCGAAGTTGATGAATACAAGGGCG

GGTTTCAGTGCGTTCACAAGACAACGTCTGAACCTGCATTTTAAAAAGCTGTTCG

ATACCTGGGGTTTATTTCTTTCATCCAAAGACAGCAGGAATGTCCTGGTAGCTGA

CCAGTTTGATGATAGGCACTGCGGCTGGCTGAACGAGAGGGCATTATCTGCGAT

GGTGAAACACTATAATGGGCGTGCATTTGATGAAGTATTTCTATGTGACAAAAAT

GCACCCTATTACGGCTTTAATTCATACGACGATTTCTTCAATAGGAGGTTCCGTA

ATAGAGACATTGATAGACCCGTTGTCGGCGGCGTGAACAACACGACGCTTATAT

CAGCAGCCTGTGAGTCTCTGTCTTATAACGTCAGCTATGACGTGCAATCCTTAGA

TACTTTAGTTTTCAAAGGTGAGACGTACTCATTAAAACATCTTTTGAATAATGAT

CCATTTACGCCACAATTCGAGCACGGTTCCATATTGCAAGGATTCCTAAACGTGA

CAGCATATCATCGTTGGCACGCGCCGGTTAACGGAACTATCGTCAAGATAATCAA

CGTTCCTGGTACTTATTTCGCACAAGCGCCGTCTACCATCGGTGATCCGATCCCA

GATAATGACTATGATCCACCGCCATATCTAAAGAGTCTTGTGTACTTCAGTAACA

TTGCAGCGAGACAGATTATGTTCATAGAAGCTGATAACAAGGAGATAGGCCTAA

TTTTCCTGGTTTTTATAGGCATGACAGAAATTTCAACGTGTGAAGCAACGGTATC

CGAGGGGCAACATGTCAATAGAGGGGACGACCTGGGTATGTTTCATTTCGGGGG

CTCTTCTTTTGCCCTTGGCCTGCGTAAAGACTGCCGTGCCGAAATTGTTGAGAAG

TTCACGGAGCCCGGGACAGTTATAAGGATTAACGAAGTCGTCGCCGCCTTGAAG

GCTTAA

SEQ ID NO: 2 (Psilocybe cyanescens (PSID gene))

ATGCAAGTGCTTCCTGCTTGCCAAAGCTCTGCCCTTAAAACCCTGTGTCCGAGCC

CCGAGGCTTTTAGAAAGCTGGGATGGCTACCTACGTCTGACGAAGTGTACAACG

AGTTCATAGATGATCTGACTGGCAGGACTTGCAATGAGAAGTATAGCAGCCAAG

TAACCCTGTTAAAGCCAATCCAAGACTTCAAGACTTTCATAGAGAATGACCCGAT

AGT AT AT C AAGAGTT C ATT AGC AT GTTTGAGGGC AT AGAAC AGAGCCCT ACT AAC

TATCATGAGCTATGTAACATGTTCAACGATATTTTTCGTAAGGCACCCCTATACG

GAGACTTAGGACCACCTGTCTACATGATAATGGCACGTATTATGAATACGCAGGC

GGGTTTTTCAGCGTTCACCAAAGAATCTCTGAACTTCCATTTTAAGAAGCTATTC

GACACGTGGGGTCTATTCCTAAGCTCTAAAAATTCCAGAAACGTACTTGTCGCCG

ATCAGTTTGACGACAAACATTACGGATGGTTTTCTGAGAGAGCAAAGACTGCGA

TGATGATCAACTATCCAGGACGTACATTCGAGAAGGTCTTCATCTGTGACGAGCA

TGTGCCTTATCACGGATTTACTTCCTATGACGACTTCTTTAACAGGAGATTTCGTG

ACAAGGATACAGACCGTCCCGTCGTCGGTGGCGTCACCGACACGACGTTGATAG

GCGCGGCATGTGAAAGTTTATCTTATAACGTTTCTCACAACGTCCAATCACTGGA

CACCCTTGTCATAAAAGGCGAGGCGTACTCTTTAAAACACCTTCTGCATAATGAC

CCATTTACGCCACAGTTTGAACATGGATCTATCATCCAAGGATTCTTGAACGTTA

CAGCCTATCACAGATGGCACTCTCCAGTTAACGGCACTATTGTGAAGATTGTAAA

34 CGT ACC AGGGACATACTTTGCCC AGGC GCCCTATACCATAGGTAGCCCAATCCCT

GATAATGACCGGGACCCGCCGCCCTACTTGAAGAGCCTTGTTTATTTTAGCAACA

TTGCTGCCAGACAGATTATGTTTATTGAGGCTGACAATAAAGATATTGGCCTTAT

CTTTCTTGTGTTCATTGGCATGACTGAAATTAGCACATGTGAAGCGACGGTATGC

GAAGGACAGCACGTTAACAGAGGCGATGACCTTGGGATGTTTCATTTTGGGGGA

TCGAGTTTTGCATTGGGGCTTAGAAAAGATAGCAAAGCAAAAATACTAGAAAAA

TTTGCAAAGCCGGGAACAGTAATAAGGATTAACGAGCTGGTGGCATCCGTCAGA

A A AT A A

SEQ ID NO: 3 (Gymnopilus junonius (PSID gene))

ATGTCATCTCCTCGTATCGTGCTGCACAGGGTTGGTGGCTGGCTGCCTAAAGACC

AAAACGTGCTAGAAGCATGGCTGAGCAAGAAGATTGCTAAAGCAAAAACTAGA

AATAGGGCTCCAAAAGATTGGGCTCCTGTGATTCAAGACTTCCAGAGACTGATA

GAGACCGATGCCGAGATCTACATGGGTTTCCATCAGATGTTCGAGCAGGTCCCCA

AGAAAACTCCGTACGATAAAGACCCCACCAATGAGCAATGGCAAGTAAGAAATT

ATATGCACATGTTAGATCTGTTCGACCTAATTATAACCGAGGCACCGGATTTCGA

ACAAAATGATCTTGTTGGATTTCCAATAAATGCAATCCTGGATTGGCCCATGGGG

ACCCCCGGTGGGCTTACTGCATTTATTAACCCTAAAGTAAATATTATGTTTCATA

AAATGTTTGACGTTTGGGCAGTATTTCTGTCATCTCCAGCATCATGCTACGTCCTA

AATACAAGCGATAGCGGTTGGTTCGGTCCCGCTGCAACCGCAGCTATACCCAACT

TCAAAGAGACCTTCATCTGCGACCCAAGTCTGCCATACCTAGGGTACACTAGCTG

GGATAATTTCTTCACCAGGCTGTTTAGGCCGGGGGTGCGTCCTGTCGAGTTCCCG

AACAATGATGCCATTGTTAACAGTGCGTGTGAATCCACGGTTTATAATATAGCTC

CAAACATTAAACCACTAGATAAATTTTGGATTAAGGGAGAGCCGTATTCCCTAAA

TCACATACTTAATAACGACCCGTACGCGAGCCAGTTCGTAGGTGGAACCATATCC

CAAGCATTCTTATCTGCGCTGAACTATCACCGTTGGGCGAGTCCGGTTAACGGCA

ACATTGTCAAGGTCGTCAATGTTCCGGGTACATACTACGCGGAGTCCCCAGTTAC

CGGTTTTGGGAATCCAGAAGGGCCAGATCCAGCGGCGCCCAATCTATCTCAAGG

TTTCATTACTGCTGTGGCTGCGAGAGCCCTGATTTTCATAGAGGCCGATAACCCT

AACATCGGATTAATGTGTTTTGTGGGGGTTGGCATGGCAGAGGTCTCAACATGTG

A AGT T AC C GT G AGT GT AGGC GAT GTT GT C A AG A A AGG AG AT GAG AT T GG A AT GT

TCCATTTCGGGGGAAGCACTCACTGCTTGATATTTAGGCCACAAACAAAAATTAC

GTTCAATCCCGACTATCCTGTGTCAACCGCCGTACCCTTGAATGCTGCAGTGGCA

ACCGTCGTATAA

SEQ ID NO: 4 (Psilocybe cubensis (PSIH gene))

ATGATTGCCGTCTTATTCTCTTTTGTCATAGCTGGCTGCATCTATTATATAGTATC

CCGTCGTGTGCGTCGTTCAAGACTTCCGCCCGGACCACCAGGCATCCCTATCCCC

TTTATCGGCAATATGTTTGACATGCCCGAAGAATCACCCTGGTTGACGTTTCTGC

A AT GGGGC AGAGATT AT A AT AC AGAC ATTTT GT AT GT AG AT GC AGGC GGA AC T G

AG AT GGT A AT ATTGA AT AC CC TT GAGAC A AT C ACTG ATTTGTT AGA A A AGAGGG

GGTCT AT AT ATTCTGGC AGGCT AGAAAGT ACC ATGGTT AATGAGTT GAT GGGGT G

GGAGTTT GATCT AGGATTC ATC ACCT ACGGT GATCGTTGGAGAGAGGAGAGAAG

GATGTTCGCGAAAGAGTTCAGCGAAAAGGGAATCAAACAATTCAGGCACGCCCA

AGTAAAGGCGGCGCATCAACTTGTCCAACAGCTGACAAAAACACCGGATCGTTG

GGCTCAACACATACGTCATCAGATAGCCGCCATGTCTTTAGACATCGGCTATGGC

ATAGACTTAGCGGAGGATGATCCATGGTTAGAAGCAACACACTTAGCTAACGAA

35 GGACTGGCGATAGCTTCCGTCCCAGGAAAATTTTGGGTAGACTCATTTCCGTCTC

TGAAATACCTACCAGCCTGGTTTCCTGGAGCTGTCTTCAAACGTAAGGCAAAAGT

AT GGAGGGAGGC AGC AGACC AT AT GGT GGAC AT GCC AT AT GAGACT AT GAGGAA

ATTGGCGCCACAGGGCTTGACTAGACCATCCTATGCATCTGCAAGACTACAGGCC

ATGGACCTAAACGGTGATTTGGAGCACCAAGAGCACGTAATTAAAAACACAGCA

GCCGAAGTGAACGTCGGAGGGGGAGATACAACCGTCTCTGCGATGAGTGCGTTC

ATACTAGCGATGGTCAAGTATCCGGAAGTACAGCGTAAAGTCCAGGCCGAGCTA

GACGCACTTACTAACAACGGCCAGATTCCCGATTACGACGAGGAAGACGATAGT

CTACCTTACTTGACCGCATGTATTAAAGAGTTATTTAGATGGAATCAAATTGCGC

CCCTAGCGATTCCTCACAAGTTAATGAAAGACGATGTATATAGGGGTTATCTAAT

ACCTAAGAATACGCTAGTTTTTGCAAACACATGGGCGGTCCTGAACGACCCTGAA

GTCTACCCAGACCCTAGCGTATTTAGGCCGGAGCGTTATTTAGGACCCGACGGTA

AGCCCGATAATACTGTCAGGGACCCCAGGAAGGCTGCGTTCGGGTATGGGAGGA

GGAACTGTCCAGGAATACACTTAGCCCAATCAACCGTCTGGATAGCCGGAGCGA

CCTTACTTAGTGCGTTTAATATCGAGAGGCCAGTTGACCAGAATGGGAAACCCAT

CGATATTCCAGCAGACTTCACAACCGGGTTTTTCAGGCATCCTGTTCCTTTTCAGT

GCCGTTTCGTGCCTAGGACTGAACAGGTCTCCCAATCAGTCAGTGGGCCGTAA

SEQ ID NO: 5 (Psilocybe cyanescens (PSIH gene))

ATGGCGCCTTTGACAACCATGATTCCGATCGTTCTATCTCTTCTAATAGCGGGGT

GTATATATTATATCAACGCAAGGAGAATTAAAAGGTCCAGGTTGCCACCAGGAC

CGCCGGGTATTCCTATTCCATTCATCGGGAACATGTTCGACATGCCAAGCGAAAG

TCCCTGGCTAATCTTCCTACAATGGGGACAAGAGTACCAGACCGATATAATTTAC

GTTGACGCGGGAGGAACTGATATGATAATACTTAATTCCCTAGAGGCAATTACA

GATCTGTT AGAGAAA AGGGGCTC ATTGT AT AGCGGGAGGTTGGAATCC ACGAT G

GT AAACGAGCT A ATGGGTT GGGAGTTT GATTTCGGTTT CAT ACCTT AC GGT GAAA

GAT GGAGGGAAGAACGTCGT AT GTTCGCC AAAGAGTTTTCTGAGAAGAAC AT AA

GGCAGTTTAGACACGCCCAAGTAAAGGCTGCCAATCAGCTAGTGCGTCAACTAA

CCGATAAACCGGACAGGTGGTCACACCACATAAGGCATCAAATCGCGTCCATGG

CCCTGGACATCGGTTACGGAATCGATCTTGCTGAAGACGATCCGTGGATCGCAGC

TTCCGAACTGGCGAATGAAGGCTTGGCTGTAGCCTCAGTGCCAGGATCTTTTTGG

GTAGATACGTTCCCGTTTCTTAAATATTTGCCAAGTTGGTTACCTGGCGCGGAGTT

C A A A AG A A AC GC A A AGAT GT GGA AGGA AGGAGC AG ATC AT AT GGT C A AT AT GC

CTTACGAAACGATGAAAAAGCTAAGCGCACAAGGACTGACTAGACCATCATATG

CAAGTGCGAGGCTACAGGCTATGGACCCGAACGGGGATCTTGAACATCAAGAAA

GAGT GAT C AAAAAT ACGGCC ACGC AGGT AAAT GTTGGT GGT GGGGAT ACT AC AG

TCGGGGCAGTAAGTGCGTTTATCCTTGCGATGGTAAAATACCCGGAAGTTCAAAG

GAAAGTACAAGCCGAGCTGGACGAGTTCACGAGCAAGGGGAGGATACCGGATT

ACGATGAAGATAACGATTCTCTTCCCTATCTATCGGCTTGCTTCAAAGAGCTGTT

CAGGTGGGGCCAGATTGCGCCTTTGGCGATTGCTCATAGGCTGATAAAGGACGA

TGTCTATAGGGAATATACTATCCCAAAGAATGCTCTGGTCTTTGCGAACAATTGG

TATGGGCGTACTGTATTGAATGACCCTTCTGAGTATCCCAATCCTTCAGAATTTA

GACCTGAAAGGTACTTGGGGCCCGATGGTAAGCCAGATGACACCGTCAGGGACC

CAAGAAAGGCAGCGTTTGGGTACGGACGTAGAGTGTGTCCAGGGATACACCTGG

CGCAGAGCACGGTCTGGATTGCTGGTGTCGCGTTGGTATCTGCCTTCAACATTGA

GCTGCCCGTGGACAAAGACGGGAAATGTATAGATATTCCGGCGGCCTTCACGAC

GGGATTCTTTAGATAA

36 SEQ ID NO: 6 (Gymnopilus junonius (PSIH gene))

AT GAT GT C C GAG AT G A AT GGG AT GG AT A A ATT GGC GC T AT T G AC G AC GTT ATT AG

CTGCCGGTTTTCTATACTTCAAGAATAAGCGTCGTTCCGCGTTGCCGTTCCCGCCA

GGGCCGAAAAAGCATCCCCTTTTAGGTAACTTGCTGGACCTTCCGAAGAAGCTGG

AGT GGG AG AC GT AC AG A AG AT GGGGA A A AG A AT AC A AT T C AG AT GT AAT AC ATG

TTAGCGCGGGGAGTGTAAACTTAATTATCGTTAATTCCTTTGAAGCTGCGACAGA

CCTGTTTGATAAGAGATCAGCCAATTATTCAAGTAGGCCACAATTCACGATGGTG

AGAGA ACTG AT GGG AT GGA ATT GGTT GAT GT C T GC ATT AAT AT AC GGT GAC A AG

TGGAGAGAGCAACGTAGGTTGTTTCAGAAACATTTCAGTACAACGAATGCCGAA

CTTTACCAAAATACACAATTAGAATATGTTCGTAAAGCCCTGCAGCATCTGCTAG

AAGAGCCTTCAGATTTTATGGGAATAACACGTCACATGGCTGGGGGCGTCAGCA

TGTCCCTGGCATATGGCTTAAACATTCAGAAGAAAAACGACCCTTTTGTTGACCT

TGCACAAAGGGCAGTGCACAGCATAACAGAGGCCTCAGTTCCTGGGACATTTTG

GGTAGACGTAATGCCTTGGCTAAAGTATATTCCAGAATGGGTGCCGGGTGCTGGC

TTTC AGAAGA AGGCT AGAGT GTGGAGGAAATT AC AGC AAGATTTTCGT C AGGTC

CCATATCAGGCAGCTCTGAAAGACATGGCTTCAGGGAAAGCTAAACCATCATTT

GCA AGT GAGT GTTT GGAGAC GAT AGACGAC A AT G AGGAT GC AC A A AGGC A A AG

GGAGGTGATAAAAGACACAGCTGCCATTGTATTCGCAGCCGGTGCGGATACAAG

CCTTAGTGGAATCCATACATTATTCGCCGCAATGTTGTGTTACCCAGAGGTCCAG

AAGAAAGCACAAGAAGAACTGGATCGTGTCTTGGGTGGGAGACGTCTACCGGAA

TTTACCGATGAGCCCAACATGCCCTACATCTCTGCGTTAGTGAAGGAAATATTGA

GGTGGAAACCGGCTACTCCGATTGGCGTACCCCACTTAGCCAGCGAGGATGACG

TTT AC AACGGAT ATT AC AT ACC AAAACGT GCGGTT GTC AT AGGC AAC AGCTGGGC

TATGCTTCATGATGAGGAAACTTATCCGGACCCAAGCACCTTTAACCCTGACAGA

TTTTT GACC AC AAAT AAAAGC ACTGGAAAATTGGAATT AGATCCC AC AGTGAGA

GATCCCGCTTTAATGGCCTTCGGATTTGGTAGACGTATGTGTCCAGGACGTGATG

TAGCTCTTTCTGTCATATGGCTGACTATCGCAAGCGTTTTAGCAACGTTTAATATT

ACC AAGGCGATAGACGAAAACGGGAAGGAACTGGAACCGGATGTAC AGT ACTG

GAGCGGTCTAATCGTCCACCCGCTGCCATTCAAATGTACGATCAAGCCAAGATCA

AAGGCAGCGGAAGAACTTGTGAAATCTGGCGCAGACGCCTATTAA

SEQ ID NO: 7 (Psilocybe cubensis (PSIK gene))

ATGGCATTCGACTTGAAAACTGAAGACGGGCTAATAACTTACCTAACGAAACAC

CTTTCTTTGGATGTGGATACATCAGGTGTGAAAAGGTTAAGCGGTGGCTTCGTTA

ACGTGACCTGGAGAATAAAACTAAACGCACCCTATCAGGGTCACACATCAATAA

TTCTAAAGCACGCACAGCCGCATATGTCAACCGACGAAGACTTCAAAATTGGCG

TGGAGCGTTCCGTCTATGAGTACCAGGCTATCAAACTTATGATGGCCAATAGGGA

GGTGCTAGGGGGTGTTGACGGGATCGTGTCTGTGCCAGAGGGGTTGAACTACGA

CCTT GAAAAT AAT GC ATTGAT CAT GC AGGACGT AGGT AAGAT GAAGACCCT ATT

AGACTACGTAACGGCAAAACCCCCGCTTGCGACTGATATAGCACGTTTGGTAGGT

AC AGAGATT GGGGGTTTC GT GGC T AGACTGC AT A AC AT AGGGAGGGAGAGGAGA

GACGACCCGGAGTTCAAGTTTTTCTCTGGAAATATAGTCGGCAGGACAACAAGC

GATCAACTATACCAAACAATTATCCCTAACGCAGCTAAGTACGGGGTAGATGAC

CCTCTACTGCCTACCGTTGTAAAAGATCTGGTCGATGATGTCATGCACAGTGAGG

AGACTCTTGTAATGGCGGATTTATGGAGCGGCAATATACTTCTACAGTTGGAGGA

GGGGA ATCC TT C A A AGTT AC AGA A A AT C T AC ATTTT AGATTGGGA ATT GT GT AAA

TACGGCCCAGCTTCACTAGACCTTGGGTATTTCTTGGGTGATTGCTACCTGATTTC

37 TCGTTTCCAAGATGAGCAGGTCGGCACAACTATGAGACAAGCCTACTTACAAAG CTACGCTCGTACCTCTAAACATTCCATAAACTACGCCAAGGTCACTGCGGGAATT GC AGC AC AT AT AGT GAT GT GGAC AGACTTT AT GC AGT GGGGGAGT GAGGA AGAG AGAATTAACTTCGTCAAGAAAGGCGTGGCCGCCTTCCATGACGCAAGAGGGAAC AATGATAATGGTGAAATCACCTCTACTCTGTTGAAGGAGAGTTCAACTGCCTAA

SEQ ID NO: 8 (Psilocybe cyanescens (PSIK gene))

ATGACTTTCGATCTAAAAACGGAGGAGGGCTTATTATCTTATCTTACCAAGCATT

TAAGTTTAGACGTAGCACCGAATGGTGTCAAAAGATTATCTGGTGGATTCGTCAA

TGTGACTTGGAGGGTAGGGTTAAATGCACCGTACCATGGGCACACGTCTATAATC

CTTAAACACGCTCAACCACATTTAAGCTCCGATATTGACTTCAAAATAGGGGTGG

AAAGAAGTGCGTATGAGTACCAGGCTTTGAAGATTGTCTCTGCCAACAGCAGCCT

ACTTGGTTCTTCTGATATCCGTGTCTCAGTTCCAGAAGGTTTGCACTATGATGTTG

TGAATAACGCCCTAATCATGCAGGACGTGGGTACAATGAAGACCTTGCTGGACT

ATGTTACAGCGAAACCCCCTATATCTGCTGAAATTGCCAGCCTAGTAGGTAGTCA

GATTGGCGCTTT CAT AGC A AG ATT AC AC AATTT GGGC AGAGAAAAT AAAGAT AA

GGACGACTTTAAATTTTTCTCCGGAAATATAGTTGGGAGGACGACGGCAGACCA

ACTGTATCAGACCATAATTCCTAATGCGGCAAAATATGGAATCGATGACCCAATT

CTTCCAATAGTTGTCAAAGAACTTGTTGAAGAAGTCATGAACTCAGAGGAAACC

CTGATTATGGCGGACCTATGGAGCGGTAATATCTTGCTACAGTTCGACGAGAACA

GTACGGAACTAACCCGTATTTGGCTGGTAGACTGGGAGCTATGCAAGTACGGGC

CGCCGTCACTGGATATGGGTTACTTCTTGGGCGACTGCTTTTTGGTAGCTAGATTC

CAAGACCAACTTGTAGGCACATCTATGAGACAAGCATACCTTAAAAGCTACGCA

CGTAACGTAAAAGAGCCGATCAACTATGCTAAGGCCACAGCAGGCATCGGCGCT

C ATTT GGT A AT GT GGACTGACTT CAT GA AGT GGGGT AAC GAT GA AGA A AGGGAG

GAGTTCGTGAAAAAGGGGGTCGAAGCATTCCACGAGGCCAACGAAGACAATAG

GAACGGAGAGAT AACGAGC AT ATT GGT GAAAGAGGC AT C ACGT ACGT AA

SEQ ID NO: 9 (Psilocybe cubensis (PSIM gene))

ATGCACATCAGAAACCCCTATAGAACCCCCATAGATTACCAGGCGCTGAGTGAG

GCCTTTCCACCATTGAAGCCCTTTGTATCCGTAAACGCTGATGGTACGAGTTCCG

TAGATCTAACGATCCCGGAGGCGCAACGTGCGTTCACTGCCGCATTGTTACATAG

AGATTTCGGGCTAACCATGACTATACCGGAAGATAGACTGTGCCCTACTGTCCCT

AAC AGGTT AAATT AT GT ACTGTGGATT GAAGAT ATTTT C AACT AC ACGA AT AAGA

CCCTGGGGCTGAGCGATGACAGACCGATAAAGGGGGTGGATATTGGCACAGGCG

CCAGCGCAATATACCCTATGCTTGCTTGCGCCAGGTTTAAGGCATGGTCCATGGT

AGGG AC AG AGGT AG A AC GT A A AT GT ATT GAT AC GGC T AG AC T A A AT GTC GTC GC

CAATAATCTACAGGATAGATTGAGTATATTAGAGACATCCATCGACGGTCCCATT

CTTGTTCCAATCTTCGAGGCCACAGAAGAATATGAGTATGAGTTCACCATGTGTA

ATCCGCCATTCTACGATGGTGCGGCCGACATGCAGACCTCTGACGCGGCCAAAG

GATTCGGCTTTGGAGTGGGGGCCCCTCACTCTGGAACAGTTATCGAAATGTCCAC

T G A AGGAGGGG AGT C CGC ATTCGT AGCC C AGAT GGT GAGAGAGAGC TT GA A AC T

GCGTACCAGATGCAGATGGTATACGTCTAATCTTGGGAAATTAAAAAGCCTAAA

GGAGATTGTGGGTCTTTTAAAAGAGCTGGAGATTTCCAACTACGCCATAAACGA

GTACGTCCAAGGGTCTACCAGAAGATACGCCGTCGCGTGGTCTTTTACTGACATT

CAGCTTCCAGAGGAGCTATCTCGTCCCAGTAACCCGGAATTGTCCTCCTTGTTTTA

A

38 SEQ ID NO: 10 (Psilocybe cyanescens (PSIM gene))

ATGCATATCAGGAATCCGTACCGTGACGGCGTGGACTACCAGGCATTAGCCGAG

GCTTTCCCGGCGCTAAAGCCACACGTCACTGTCAATTCAGACAATACAACTTCTA

TAGATTTCGCGGTACCCGAGGCCCAGAGACTTTACACCGCAGCATTACTTCATAG

GGACTTTGGTTTAACCATAACCTTACCCGAGGATAGACTATGTCCTACGGTCCCG

A AT AGATTGA AC T AT GT GTT GT GGGT GG A AGAT AT AC T GA AGGTT ACGT C AGAC

GC ATT GGGATT AC CGGAT A AT AGAC A AGT GA A AGGT ATTG AT ATTGG A AC AGGA

GCAAGCGCAATTTATCCCATGTTAGCTTGTTCCAGGTTTAAGACTTGGTCCATGG

T AGC T AC AG AGGT GG AT C A A A A AT GC AT AGAT ACC GC A AGGC T A A AC GT A AT AG

CTAATAACCTTCAGGAGAGATTGGCAATCATAGCCACTTCCGTGGACGGGCCTAT

TCTTGTTCCTCTGTTGCAGGCTAATTCCGACTTTGAATATGACTTCACCATGTGCA

ATCCGCCCTTTTACGACGGCGCCTCTGATATGCAGACAAGTGATGCCGCTAAAGG

CTTTGGCTTCGGAGTAAACGCACCTCACACTGGGACAGTACTTGAAATGGCGACA

GAAGGAGGGGAAAGTGCGTTCGTTGCCCAAATGGTTCGTGAGTCCTTGAACCTG

CAGACTAGATGCAGGTGGTTCACATCTAATTTGGGTAAACTAAAATCACTGTACG

AGATTGT GGGTCT ATT AAGAGA AC ACC AGATTTCT AACT ACGCC AT AAAT GAGT A

TGTACAAGGCGCAACTCGTAGGTATGCAATTGCGTGGAGTTTCATAGATGTAAGA

CTGCCCGACCATTTGTCCAGACCATCTAATCCCGATCTATCCAGTTTGTTTTAA

SEQ ID NO: 11 (Panaeolus cyanescens (PSIM gene))

ATGCATAACCGTAACCCGTATAGGGACGTGATTGATTACCAAGCACTTGCGGAA

GCCTACCCGCCCCTAAAACCCCACGTCACGGTGAACGCGGATAACACGGCATCC

ATAGATCTTACGATCCCCGAGGTCCAGAGGCAATACACAGCAGCTCTTTTACATC

GTGATTTCGGATTAACTATCACACTACCAGAAGATAGGCTGTGCCCGACAGTACC

GAACCGTTTAAACTATGTATTGTGGATAGAGGATATATTTCAGTGTACGAATAAG

GCTCTGGGATTGTCAGATGACAGACCCGTTAAGGGGGTAGATATAGGGACCGGC

GCCTCCGCCATCTATCCAATGCTTGCTTGCGCGAGGTTTAAGCAGTGGTCCATGA

TT GCC AC AGAAGTGGAGCGT AAGT GC AT AGAT AC AGCGAGATTGAAT GTCCTGG

CGAATAACTTACAGGACCGTTTGTCAATTCTTGAGGTTTCAGTAGACGGCCCGAT

TTTGGTACCCATCTTTGATACCTTCGAGCGTGCGACAAGCGATTACGAATTTGAG

TTCACGATGTGTAACCCTCCATTTTACGACGGGGCCGCGGATATGCAAACATCAG

ATGCAGCTAAGGGTTTCGGTTTTGGAGTTAACGCTCCACACTCCGGTACCGTGAT

AGAGATGGCTACTGAAGGAGGTGAGGCTGCTTTTGTGGCGCAAATGGTCCGTGA

GAGC ATGAAGTT AC AGAC AAGGT GTCGTTGGTTT AC AAGC AACTT AGGC AAGCT

AAAATCACTGCATGAAATTGTTGCTTTGTTGAGAGAATCCCAGATCACAAACTAT

GCCATAAATGAGTACGTTCAGGGGACGACGAGAAGGTACGCTCTTGCTTGGTCCT

TCACAGACATAAAACTTACTGAGGAACTTTACAGGCCCTCCAATCCAGAATTAGG

ACCTCTTTGCAGCACATTTGTCTAA

SEQ ID NO: 12 (Gymnopilus dilepis (PSIM gene))

ATGCACATTAGAAACCCTTACTTAACACCTCCGGACTACGAGGCCCTTGCGGAGG

CCTTCCCCGCACTAAAGCCTTATGTTACAGTTAACCCCGATAAGACTACTACAAT

TGACTTTGCCATACCGGAGGCTCAGAGATTATACACGGCTGCTCTACTTTACAGG

GACTTTGGACTGACAATAACATTGCCGCCGGATAGGTTATGCCCAACCGTGCCCA

ATAGGCTTAATTATGTTTTGTGGATTCAGGACATTCTGCAGATTACCTCCGCTGCC

39 TTGGGCTTGCCAGAGGCTAGACAAGTAAAGGGAGTAGACATAGGTACCGGAGCG

GCAGCGATATACCCTATTCTTGGTTGCAGCCTTGCAAAGAATTGGTCTATGGTGG

GGACAGAGGTCGAACAAAAATGTATCGACATAGCGCGTCAAAACGTGATTTCAA

ATGGATTGCAGGATAGGATAACCATAACTGCTAATACCATAGACGCTCCCATTCT

GCTGCCCTTATTTGAAGGAGACAGTAACTTCGAATGGGAGTTCACCATGTGTAAC

CCGCCATTTTACGACGGCGCTGCGGACATGGAGACAAGCCAGGACGCTAAAGGC

TTCGGGTTCGGCGTC AACGCCCCGC AT AC AGGAAC AGT GGT GGAAAT GGCC ACG

GACGGTGGTGAGGCTGCATTCGTCAGCCAAATGGTGAGAGAGTCCTTGCACCTA

AAGACACGTTGTAGATGGTTCACGTCCAATCTAGGTAAATTGAAGAGTCTACATG

AAATTGT GGGATTGTT GCGT GAAC ACC AAATT ACC A ACT ACGCGAT AAATGAAT

AT GTT C AGGGAACGAC ACGT AG AT AC GC GATT GC AT GGT C ATTT ACTGACCT ACG

TCTATCAGACCACCTGCCACGTCCTCCGAACCCCGATCTATCAGCCCTATTTTAA

SEQ ID NO: 13 (Gymnopilus junonius (PSIM gene))

ATGCACTCTCGTAACCCTTATAGATCCCCTCCTGATTTCGCGGCATTAAGTGCGG

CTTATCCTCCGCTGTCACCATACATAACTACCGATCTAAGCAGCGGTCGTAAAAC

AATTGACTTTAGAAATGAGGAAGCGCAACGTCGTCTAACTGAGGCTATCATGTTG

CGTGACTTCGGCGTTGTGTTAAACATACCATCTAACAGGCTGTGCCCGCCTGTGC

CGAATCGTATGAACTATGTACTTTGGATACAAGATATAGTTTACGCGCACCAGAC

AATACTGGGAGTGAGTTCTCGTCGTATCAGAGGTCTTGATATTGGTACTGGTGCT

ACCGCT AT AT ATCCT AT ACTGGC AT GC AAGAAAGAGC AGAGCTGGGAGATGGTT

GCAACTGAATTGGACGACTACTCCTATGAGTGTGCATGTGATAACGTGTCATCCA

ACAATATGCAGACTTCCATTAAAGTAAAGAAGGCTTCGGTAGATGGGCCCATCCT

GTTCCCAGTGGAAAACCAAAATTTCGACTTTAGCATGTGCAACCCGCCTTTCTAC

GGCTCTAAGGAGGAGGTGGCGCAATCCGCAGAGTCAAAAGAACTGCCGCCCAAT

GCTGTTT GC AC GGGT GC AGAGATCGAGAT GAT ATTT AGT C A AGGAGGAGA AGAG

GGTTTCGT AGGT AGAAT GGT AGAGGAAT C AGAGAGGTT GC AAACGAGAT GC AA A

TGGTACACTTCAATGCTTGGTAAGATGTCTAGTGTAAGCACTATAGTTCAGGCTC

T GCGT GCGAGAT C A ATT AT GA ATT AT GC TTTG AC AGA ATTT GT AC A AGG AC A A AC

CCGTAGGTGGGCGATAGCTTGGTCTTTCTCCGACACTCACTTACCGGATGCCGTC

AGT AGAATCTCT AGTT AA

SEQ ID NO: 14 (Psilocybe cubensis (PSID gene))

MQ VIP ACN S A AIRSLCPTPESFRNMGWLS V SD AVY SEFIGEL ATRASNRNY SNEF GL MQPIQEFKAFIESDPVVHQEFIDMFEGIQDSPRNYQELCNMFNDIFRKAPVYGDLGPP VYMIM AKLMNTRAGF S AF TRQRLNLHFKKLFDTW GLFL S SKD SRNVL V AD QFDDR HCGWLNERAL S AMVKHYNGRAFDEVFLCDKNAP YY GFN S YDDFFNRRFRNRDIDR P VV GGVNNTTLIS AACESL S YNV S YD V Q SLDTL VFKGET Y SLKHLLNNDPFTPQFEH GSILQGFLN VT A YHRWH AP VN GTI VKIINVPGT YF AQ AP S TIGDPIPDND YDPPP YLK S LVYF SNIAARQIMFIEADNKEIGLIFLVFIGMTEISTCEAT V SEGQHVNRGDDLGMFHF GGS SF ALGLRKDCRAEIVEKFTEPGT VIRINEVVAALKA

SEQ ID NO: 15 (Psilocybe cyanescens (PSID gene))

MQVLPACQSSALKTLCPSPEAFRKLGWLPTSDEVYNEFIDDLTGRTCNEKYSSQVTL LKPIQDFKTFIENDPIVYQEFISMFEGIEQSPTNYHELCNMFNDIFRKAPLYGDLGPPV YMIMARIMNTQAGF S AF TKE SLNFHFKKLFDTW GLFL S SKN SRN VL V AD QFDDKH Y

40 GWF SERAKT AMMINYPGRTFEKVFICDEHVP YHGFT S YDDFFNRRFRDKDTDRP VV GGVTDTTLIGAACESLSYNVSHNVQSLDTLVIKGEAYSLKHLLHNDPFTPQFEHGSII QGFLNVTAYHRWHSPVNGTIVKIVNVPGTYFAQAPYTIGSPIPDNDRDPPPYLKSLVY FSNIAARQIMFIEADNKDIGLIFLVFIGMTEISTCEATVCEGQHVNRGDDLGMFHFGGS SF ALGLRKD SKAKILEKF AKPGT VIRINEL VAS VRK

SEQ ID NO: 16 (Gymnopilus junonius (PSID gene))

MS SPRIVLHRVGGWLPKDQNVLEAWLSKKIAKAKTRNRAPKDWAP VIQDF QRLIET DAEIYMGFHQMFEQVPKKTPYDKDPTNEQWQVRNYMHMLDLFDLIITEAPDFEQND L V GFPINAILDWPMGTPGGLT AFINPKVNIMFHKMFD VW AVFL S SP ASC YVLNT SD S GWF GP AAT AAIPNFKETFICDP SLP YLGYT S WDNFFTRLFRPGVRPVEFPNND AIVN S ACE S T VYNIAPNIKPLDKF WIKGEP Y SLNHILNNDP Y AS QF VGGTIS Q AFL S ALN YHR W ASP VN GNI VK VVN VPGT Y Y AE SP VT GF GNPEGPDP A APNL S QGFIT A V A ARALIFIE ADNPNIGLMCF VGV GMAE VSTCE VT V S VGD VVKKGDEIGMFHF GGSTHCLIFRPQT KITFNPD YP V S T A VPLN A A V AT V V

SEQ ID NO: 17 (Psilocybe cubensis (PSIH gene))

MIAVLFSFVIAGCIYYIVSRRVRRSRLPPGPPGIPIPFIGNMFDMPEESPWLTFLQW GRD

YNTDILYVDAGGTEMVILNTLETITDLLEKRGSIYSGRLESTMVNELMGWEFDLGFI T

YGDRWREERRMFAKEFSEKGIKQFRHAQVKAAHQLVQQLTKTPDRWAQHIRHQIA

AMSLDIGYGIDLAEDDPWLEATHLANEGLAIASVPGKFWVDSFPSLKYLPAWFPGAV

FKRKAKVWREAADHMVDMPYETMRKLAPQGLTRPSYASARLQAMDLNGDLEHQE

HVIKNTAAEVNVGGGDTTVSAMSAFILAMVKYPEVQRKVQAELDALTNNGQIPDYD

EEDD SLP YLT ACIKELFRWN QI APL AIPHKLMKDD V YRGYLIPKNTL VF ANTW A VLN

DPEV YPDPS VFRPERYLGPDGKPDNT VRDPRK AAF GY GRRN CPGIHL AQ ST VWI AGA

TLLSAFNIERPVDQNGKPIDIPADFTTGFFRHPVPFQCRFVPRTEQVSQSVSGP

SEQ ID NO: 18 (Psilocybe cyanescens (PSIH gene))

MAPLTTMIPIVLSLLIAGCIYYINARRIKRSRLPPGPPGIPIPFIGNMFDMPSESPW LIFLQ WGQEY QTDIIYVD AGGTDMIILN SLEAITDLLEKRGSL Y SGRLESTMVNELMGWEFD F GFIP Y GERWREERRMF AKEF SEKNIRQFRHAQ VK AANQL VRQLTDKPDRW SHHIR HQIASMALDIGYGIDLAEDDPWIAASELANEGLAVASVPGSFWVDTFPFLKYLPSWL PGAEFKRNAKMWKEGADHMVNMPYETMKKLSAQGLTRPSYASARLQAMDPNGDL EHQER VIKNT AT Q VN V GGGDTT V GA V S AFIL AM VK YPE V QRK V Q AELDEF T SKGRI PD YDEDND SLP YL S ACFKELFRW GQIAPL AIAHRLIKDD V YRE YTIPKNAL VF ANNW Y GRTVLNDP SEYPNP SEFRPERYLGPDGKPDDT VRDPRK AAF GY GRRV CPGIHL AQ S TVWIAGVALVSAFNIELPVDKDGKCIDIPAAFTTGFFR

SEQ ID NO: 19 (Gymnopilus junonius (PSIH gene))

MMSEMNGMDKLALLTTLLAAGFLYFKNKRRSALPFPPGPKKHPLLGNLLDLPKKLE WETYRRW GKEYN SD VIHV S AGS VNLIIVN SFEAATDLFDKRS ANY S SRPQFTMVREL MGWNWLMS ALI Y GDKWREQRRLF QKHF STTNAEL Y QNT QLEYVRKALQHLLEEP S DFMGITRHM AGGV SMSL AY GLNIQKKNDPF VDL AQRAVHSITE AS VPGTF WVD VMP WLKYIPEWVPGAGFQKKARVWRKLQQDFRQVPYQAALKDMASGKAKPSFASECLE TIDDNED AQRQRE VIKDT A AI VF A AGADT SL S GIHTLF AAMLC YPE V QKK AQEELDR

41 VLGGRRLPEFTDEPNMPYISALVKEILRWKPATPIGVPHLASEDDVYNGYYIPKRAVV IGN SWAMLHDEETYPDPSTFNPDRFLTTNKSTGKLELDPTVRDP ALMAF GF GRRMCP GRD VALS VIWLTIAS VLATFNITKAIDENGKELEPD V QYW SGLIVHPLPFKCTIKPRSK A AEEL VK S GAD AY

SEQ ID NO: 20 (Psilocybe cubensis (PSIK gene))

MAFDLKTEDGLITYLTKHLSLDVDTSGVKRLSGGFVNVTWRIKLNAPYQGHTSIILK HAQPHMSTDEDFKIGVERS VYEY Q AIKLMMANREVLGGVDGIV SVPEGLNYDLENN ALIMQDVGKMKTLLDYVTAKPPLATDIARLVGTEIGGFVARLHNIGRERRDDPEFKF F SGNIV GRTTSDQL Y QTIIPNAAKY GVDDPLLPT VVKDLVDDVMHSEETLVMADLW SGNILLQLEEGNPSKLQKIYILDWELCKYGPASLDLGYFLGDCYLISRFQDEQVGTTM RQ A YLQ S YARTSKHSINY AK VT AGI AAHIVMWTDFMQW GSEEERINF VKKGV AAFH DARGNNDNGEITSTLLKESSTA

SEQ ID NO: 21 (Psilocybe cyanescens (PSIK gene))

MTFDLKTEEGLLSYLTKHLSLDVAPNGVKRLSGGFVNVTWRVGLNAPYHGHTSIILK HAQPHL S SDIDFKIGVERS AYEYQ ALKI VS ANS SLLGS SDIRVS VPEGLHYD VVNNALI MQDVGTMKTLLDYVTAKPPISAEIASLVGSQIGAFIARLHNLGRENKDKDDFKFFSG NIVGRTTADQLY QTIIPNAAKY GIDDPILPIVVKELVEEVMN SEETLIMADLW SGNILL QFDEN STELTRIWLVDWELCKY GPPSLDMGYFLGDCFLVARFQDQL VGTSMRQ AYL KS YARNVKEPINY AK AT AGIGAHL VMWTDFMKW GNDEEREEF VKKGVEAFHE ANE DNRNGEITSILVKEASRT

SEQ ID NO: 22 (Psilocybe cyanescens (PSIM gene))

MHIRNP YRDGVD Y Q AL AE AFP ALKPHVT VN SDNTTSIDF AVPEAQRL YT AALLHRDF GLTITLPEDRLCPT VPNRLNYVLWVEDILK VT SD ALGLPDNRQ VKGIDIGT GAS AIYP MLACSRFKTW SMVATEVDQKCIDTARLNVIANNLQERLAIIATSVDGPILVPLLQAN S DFEYDFTMCNPPFYDGASDMQTSDAAKGFGFGVNAPHTGTVLEMATEGGESAFVA QM VRE SLNL Q TRCRWF T SNLGKLK SL YEI V GLLREHQI SN Y AINE Y V Q G ATRR Y AI A WSFIDVRLPDHLSRPSNPDLSSLF

SEQ ID NO: 23 (Psilocybe cubensis (PSIM gene))

MHIRNP YRTPIDY Q AL SEAFPPLKPF V S VNADGT S SVDLTIPEAQRAFT AALLHRDF G LTMTIPEDRLCPTVPNRLNYVLWIEDIFNYTNKTLGLSDDRPIKGVDIGTGASAIYPML ACARFKAWSMVGTEVERKCIDTARLNVVANNLQDRLSILETSIDGPILVPIFEATEEY EYEFTMCNPPFYDGAADMQTSDAAKGFGFGVGAPHSGTVIEMSTEGGESAFVAQM VRE SLKLRTRCRW YT SNLGKLK SLKEI V GLLKELEISN Y AINE Y VQGS TRRY A V AW S F TDIQLPEEL SRP SNPEL S SLF

SEQ ID NO: 24 (Panaeolus cyanescens (PSIM gene))

MHNRNP YRD VID Y Q AL AE AYPPLKPHVT VNADNT ASIDLTIPE V QRQ YT AALLHRDF GLTITLPEDRLCPTVPNRLNYVLWIEDIFQCTNKALGLSDDRPVKGVDIGTGASAIYP MLACARFKQWSMIATEVERKCIDTARLNVLANNLQDRLSILEVSVDGPILVPIFDTFE RATSDYEFEFTMCNPPFYDGAADMQTSDAAKGFGFGVNAPHSGTVIEMATEGGEAA

42 FVAQMVRESMKLQTRCRWFTSNLGKLKSLHEIVALLRESQITNYAINEYVQGTTRRY ALAW SFTDIKLTEELYRPSNPELGPLCSTF V

SEQ ID NO: 25 (Gymnopilus junonius (PSIM gene))

MH SRNP YRSPPDF A AL S A A YPPL SP YITTDL S S GRKTIDFRNEE AQRRLTE AIMLRDF G

VVLNIP SNRLCPP VPNRMNYVLWIQDIV Y AHQTILGV S SRRIRGLDIGTGAT AIYPIL A

CKKEQSWEMVATELDDYSYECACDNVSSNNMQTSIKVKKASVDGPILFPVENQNFD

FSMCNPPFYGSKEEVAQSAESKELPPNAVCTGAEIEMIFSQGGEEGFVGRMVEESER L

QTRCKWYTSMLGKMSSVSTIVQALRARSIMNYALTEFVQGQTRRWAIAWSFSDTHL

PDAVSRISS

SEQ ID NO: 26 (Gymnopilus dilepis (PSIM gene))

MfflRNP YLTPPD YE AL AEAFP ALKP YVTVNPDKTTTIDF AIPE AQRL YT AALL YRDF G LTITLPPDRLCPTVPNRLNYVLWIQDILQITSAALGLPEARQVKGVDIGTGAAAIYPIL GC SL AKNW SM V GTE VEQKCIDI ARQN VI SN GLQDRITIT ANTID APILLPLFEGD SNFE WEFTMCNPPF YDGAADMETSQD AKGF GF GVNAPHTGTVVEMATDGGEAAF VSQM VRE SLHLKTRCRWF T SNLGKLK SLHEI V GLLREHQITN Y AINE Y V QGTTRR Y AI AW SF TDLRL SDHLPRPPNPDL S ALF

SEQ ID NO: 27 (Saccharmyces cerevisiae (AROl gene))

ATGGTTCAACTAGCCAAGGTTCCAATACTAGGAAACGATATAATACACGTTGGAT

ATAATATACACGATCATCTTGTAGAGACAATTATTAAACACTGTCCTTCTTCTACT

TACGTCATCTGTAACGATACTAACCTTAGCAAGGTACCTTATTACCAGCAACTGG

TTCTGGAGTTCAAAGCAAGTCTTCCCGAAGGCTCCAGACTACTAACCTACGTGGT

CAAACCGGGCGAGACGTCTAAGAGTAGGGAGACGAAGGCGCAGTTAGAGGATT

ATCTTTTAGTAGAAGGGTGCACTCGTGATACGGTCATGGTAGCCATCGGCGGAGG

TGTCATCGGTGACATGATCGGTTTCGTAGCCTCCACGTTCATGAGAGGTGTGAGG

GTAGTACAGGTTCCGACGTCTCTTTTAGCAATGGTAGACTCATCCATAGGCGGTA

AAACGGCGATCGATACTCCGCTAGGAAAGAACTTCATTGGAGCCTTTTGGCAGCC

AAAATTTGTTCTTGTGGATATCAAGTGGCTTGAAACACTAGCTAAACGTGAATTT

AT C A AC GGC AT GGC AG A AGT GAT C A AGAC AGC GT GC ATCTGGA ACGC T GAT GA A

TTTACTCGTCTCGAATCCAACGCGTCACTGTTCCTAAACGTAGTAAATGGTGCGA

AAAAT GT AAAGGT GACT AACC AGCTGACGA ACGAGAT AG AT GAG AT C AGC AAC A

CGGATATTGAAGCCATGTTGGACCATACTTATAAACTGGTATTAGAGAGTATTAA

GGTTAAAGCGGAGGTGGTAAGCAGCGATGAAAGGGAGAGCAGTCTTAGGAACCT

TTTAAACTTCGGGCATAGCATAGGTCACGCGTATGAAGCCATACTGACACCCCAG

GCTTT AC AT GGAG AGT GC GT ATCC AT C GGC AT GGT A A A AG A AGC AG A AC T AT C A

AGGTATTTTGGGATACTTTCTCCGACCCAGGTGGCGCGTCTAAGCAAAATTCTAG

TTGCGTACGGATTGCCCGTTAGCCCCGATGAGAAATGGTTTAAAGAGCTTACACT

TCATAAGAAGACACCCTTGGACATACTGCTAAAGAAGATGAGCATCGACAAGAA

AAATGAAGGAAGCAAGAAGAAGGTCGTAATCCTAGAGTCTATCGGCAAATGTTA

CGGAGACTCAGCTCAGTTTGTTTCAGACGAAGACTTACGTTTTATATTGACAGAT

GAAACACTAGTATATCCTTTTAAGGATATTCCCGCTGATCAGCAGAAAGTCGTGA

TTCCACCCGGAAGTAAATCAATAAGCAATCGTGCTTTAATCTTAGCAGCTCTGGG

GGAGGGACAGTGCAAGATCAAGAACCTATTACACTCCGACGACACCAAACATAT

GCTGACCGCAGTCCACGAGTTAAAAGGTGCTACCATCAGTTGGGAGGATAACGG

43 AGAAACAGTGGTCGTAGAGGGCCATGGCGGGAGCACTCTATCGGCTTGTGCTGA

TCCCTTATACTTAGGCAACGCGGGGACGGCGAGTAGATTCTTAACATCACTGGCG

GCACTAGTGAACAGTACATCCTCCCAAAAGTATATCGTACTAACAGGCAACGCA

AGGATGCAGCAACGTCCGATAGCGCCCCTTGTTGACAGCTTACGTGCTAACGGG

ACAAAGATCGAGTACTTGAACAACGAAGGTTCTTTGCCGATCAAAGTGTACACT

GATTCTGTATTTAAAGGCGGCCGTATTGAGTTGGCTGCGACAGTTAGTTCCCAAT

ACGTGAGCAGTATCCTGATGTGTGCGCCTTACGCAGAAGAGCCCGTGACTTTAGC

TTTGGTAGGTGGGAAACCGATCAGTAAACTATACGTTGATATGACAATTAAGATG

ATGGAAAAGTTCGGCATCAATGTGGAGACCTCAACCACGGAACCCTACACATAC

TACATTCCGAAGGGGCATTACATTAATCCAAGTGAGTACGTAATCGAGAGCGAC

GCTTCATCCGCTACCTATCCGTTAGCATTCGCCGCAATGACCGGTACCACCGTAA

CAGTCCCCAACATCGGCTTTGAATCTCTGCAGGGCGACGCTAGATTCGCAAGAGA

CGTCCTAAAGCCGATGGGGTGTAAAATCACCCAAACGGCTACGTCTACAACCGT

CAGTGGACCACCCGTCGGTACGCTAAAGCCATTAAAACACGTTGATATGGAACC

AATGACAGACGCCTTCTTAACCGCATGCGTTGTAGCCGCAATCAGTCATGACTCC

GACCCCAATTCAGCGAACACTACTACTATCGAGGGGATCGCAAACCAAAGGGTT

AAAGA ATGC AAC AGAATCTT AGCGAT GGCT ACCGAGCTGGC AAAGTTT GGAGT A

AAGACAACAGAACTTCCCGATGGCATACAGGTCCATGGGCTAAATTCCATCAAG

GACCTTAAAGTCCCATCTGACAGCTCAGGACCCGTCGGAGTCTGTACTTATGATG

ACCATAGGGTTGCCATGTCATTTTCCCTTTTGGCTGGCATGGTAAACAGTCAGAA

TGAGAGAGATGAAGTGGCAAACCCAGTTAGGATCTTAGAGAGGCACTGCACCGG

AAAGACGTGGCCAGGCTGGTGGGACGTTCTGCACAGCGAACTTGGAGCGAAGCT

GGATGGTGCCGAGCCGCTAGAATGCACATCCAAAAAGAACTCTAAGAAGAGCGT

AGT CAT A AT AGGC AT GAGAGC T GC GGGC A A A ACT ACT AT C TC T A AGT GGT GCGC

AAGTGCGCTGGGTTACAAGTTGGTAGATTTAGATGAATTGTTCGAGCAGCAGCAT

AATAACCAATCAGTAAAACAATTTGTAGTCGAGAATGGTTGGGAGAAATTCAGA

GAGGAAGAGACCAGGATATTCAAGGAGGTTATTCAAAATTACGGCGACGACGGG

TATGTCTTTAGCACTGGGGGAGGGATCGTCGAATCCGCGGAGAGCAGGAAAGCA

CTAAAGGACTTCGCCAGTTCCGGTGGGTATGTGCTTCACTTACATCGTGATATAG

AGGAGACGATAGTCTTCCTACAAAGTGATCCATCCAGGCCGGCGTATGTTGAGG

AG AT T AGGG AGGT C T GG A AC C GT AG AG A AGGC T GGT AT A A AG A AT GT AGT A AT T

TTAGCTTTTTCGCACCTCACTGTAGCGCAGAGGCGGAGTTTCAAGCACTTAGACG

TTCATTCAGTAAGTATATAGCTACGATCACGGGGGTCCGTGAAATAGAGATTCCT

AGTGGGAGGAGTGCGTTTGTATGCTTAACTTTTGACGATCTAACTGAGCAAACGG

AGAATCTGACGCCTATATGCTACGGGTGTGAAGCCGTAGAGGTGCGTGTTGATCA

TCTTGCCAATTATTCCGCAGACTTCGTTAGCAAGCAATTAAGCATACTGAGAAAA

GCGACCGACAGTATACCCATTATCTTCACCGTCCGTACTATGAAACAAGGCGGTA

ATTTTCCCGATGAAGAGTTCAAGACATTGCGTGAGTTGTACGACATAGCTCTTAA

AAACGGAGTGGAGTTCCTTGATTTGGAACTTACTCTGCCTACAGATATACAGTAC

GAAGT CAT C AAC AAGAGAGGT AAT ACGAAGAT C ATTGGGTCTC AT CAT GACTTC

CAGGGTTTGTACAGCTGGGACGATGCTGAATGGGAAAACAGATTCAATCAGGCA

CTGACTCTTGACGTAGATGTGGTGAAATTTGTGGGTACCGCGGTGAATTTCGAGG

ACAACTTACGTTTGGAACATTTTCGTGACACGCACAAAAATAAACCACTAATAGC

AGTTAACATGACGTCTAAGGGCTCAATCAGTAGGGTACTAAATAATGTATTGACT

CCGGTTACTTCAGACCTTTTACCGAACAGCGCAGCGCCTGGTCAATTGACGGTTG

CACAGATTAATAAAATGTATACATCTATGGGAGGAATTGAGCCTAAAGAGCTAT

TTGTGGTGGGGAAGCCAATCGGCCACTCAAGATCACCTATACTACACAATACTGG

GTATGAGATTTTGGGTCTACCTCACAAATTCGATAAATTTGAGACGGAAAGCGCA

CAATTAGTGAAGGAGAAATTGTTAGACGGGAACAAGAATTTCGGTGGTGCAGCG

44 GTGACCATCCCTTTAAAGCTAGACATAATGCAGTACATGGATGAACTTACGGACG

CTGCGAAGGTGATTGGGGCGGTAAACACAGTAATCCCTTTGGGTAACAAGAAAT

TCAAGGGTGATAATACGGACTGGTTAGGGATAAGGAACGCACTTATAAATAATG

GTGTGCCCGAGTACGTGGGGCATACTGCCGGACTTGTAATAGGTGCTGGTGGTAC

CAGTAGGGCGGCACTGTACGCTTTGCATAGCTTAGGTTGCAAGAAGATCTTTATC

ATCAATAGAACAACTAGTAAACTGAAGCCACTGATAGAATCACTACCCTCCGAG

TTTAACATCATTGGAATAGAGTCTACGAAATCCATCGAGGAGATTAAAGAACAC

GTCGGAGTCGCTGTTAGCTGCGTGCCTGCCGATAAGCCCTTAGATGACGAGCTAC

TGAGTAAGTTAGAACGTTTCCTTGTCAAGGGTGCACATGCGGCTTTCGTCCCAAC

ACTGCTAGAGGCTGCCTATAAACCCAGCGTAACACCTGTTATGACCATAAGTCAG

GAC AAGT AT C AATGGC ACGT GGT GCCGGGTTCCC AGAT GCTGGTCC AT C AAGGT

GTTGCACAATTTGAAAAATGGACTGGTTTCAAGGGGCCCTTCAAAGCCATATTTG

ACGCCGT GACT AAAGAGT AA

SEQ ID NO: 28 (Saccharomyces cerevisiae (AR02 gene))

ATGTCCACATTCGGTAAACTTTTCCGTGTCACTACATACGGCGAGTCACACTGCA

AATCTGTGGGGTGCATAGTAGACGGCGTTCCGCCGGGCATGAGTTTAACCGAAG

CGGACATTCAACCTCAGCTTACCCGTAGGAGGCCCGGTCAGAGCAAGTTATCCAC

CCCGAGGGACGAAAAGGACCGTGTAGAGATCCAAAGCGGAACGGAATTTGGGA

AGACACTTGGTACGCCTATCGCTATGATGATTAAAAACGAGGATCAACGTCCGC

ACGATTACTCCGACATGGACAAGTTCCCTAGGCCGAGTCACGCCGATTTTACGTA

CTCAGAGAAATACGGAATAAAAGCCTCCAGCGGTGGGGGCCGTGCTTCCGCGAG

AGAAACCATTGGAAGAGTAGCATCCGGTGCAATAGCAGAGAAGTTCCTAGCACA

GAACTCAAATGTTGAAATTGTCGCTTTCGTCACGCAAATAGGTGAGATCAAGATG

AACCGTGACAGTTTCGACCCAGAATTTCAACACCTTCTAAATACAATTACGAGGG

AGAAGGTAGATAGCATGGGTCCAATAAGATGCCCCGACGCTTCCGTCGCGGGAT

T GAT GGT G A AGG A A ATT G A A A A AT AT C GT GGG A AC A AGG AT TC T AT T GGGGGT G

TAGTAACTTGCGTAGTCAGAAATCTACCTACAGGGTTGGGTGAACCGTGTTTTGA

CAAACTGGAGGCGATGCTGGCACATGCCATGTTATCCATACCAGCAAGTAAAGG

ATTTGAAATAGGATCTGGCTTCCAGGGTGTAAGCGTACCAGGAAGCAAACACAA

TGATCCCTTTTACTTTGAAAAAGAGACTAACCGTCTTCGTACAAAGACAAACAAC

TCCGGT GGGGT GC AAGGGGGC ATCTCT AAT GGTGAGAAC ATTT ACTTTTCCGT AC

CATTTAAGAGCGTGGCTACAATAAGCCAAGAGCAAAAGACCGCAACTTACGATG

GAGAAGAAGGAATCCTCGCAGCTAAGGGTAGGCACGATCCTGCGGTCACACCGC

GTGCAATTCCCATAGTGGAAGCTATGACCGCCCTAGTACTAGCAGATGCGTTACT

AATACAGAAAGCCAGGGATTTTTCTAGGTCAGTCGTACATTAA

SEQ ID NO: 29 (Saccharomyces cerevisiae (AR03 gene))

AT GTT CAT C A AG A AT GAC CAT GCTGGT GAT AGA A AGAGAC T AGAGGACTGGCGT ATAAAGGGTTATGACCCTCTAACTCCGCCTGATTTGCTACAGCACGAGTTTCCTA TAT C AGC A A A AGGGGA AGA A A AT AT CAT C A AGGCTCGT GAT AGT GT AT GT GAT A TACTGAACGGAAAGGATGACAGACTTGTGATAGTAATTGGACCCTGTTCTCTGCA TGATCCGAAGGCGGCCTACGACTATGCCGACAGATTAGCCAAAATATCCGAAAA

45 GCTGTCAAAAGATCTTTTAATTATCATGCGTGCATACCTAGAGAAGCCTCGTACA

ACCGTTGGATGGAAAGGGTTGATAAACGACCCGGATATGAACAATAGTTTTCAG

ATTAATAAAGGCCTTCGTATAAGCCGTGAGATGTTTATAAAACTAGTTGAGAAAT

TACCTATTGCAGGAGAAATGCTTGACACGATTTCCCCTCAGTTCTTATCTGACTGT

TTCTC ACT AGGTGC AATT GGT GCT AGGACT ACCGAGT C AC AGTT AC ATCGT GAAC

TGGCCAGCGGTCTGTCTTTCCCCATTGGCTTTAAAAATGGTACCGATGGTGGCCT

TCAAGTAGCAATTGATGCTATGAGAGCTGCGGCCCACGAACACTACTTTTTGTCT

GTGACCAAACCTGGCGTAACAGCGATTGTGGGAACTGAAGGGAACAAGGACACC

TTCCTAATCCTGAGAGGGGGCAAGAACGGGACTAATTTTGACAAGGAGTCAGTT

CAAAACACTAAGAAGCAATTGGAGAAGGCGGGCCTTACTGACGATTCTCAGAAG

AGAAT CAT GAT AGACTGC AGCC AT GGC A ACTC AAAT AAAGATTT C AAA AATC AA

CCCAAAGTCGCCAAGTGTATCTACGATCAACTAACCGAAGGAGAAAATAGTTTA

TGC GGGGT GAT GAT AG AG AGT A AT AT A A AC G A AGG A AG AC AGG AT AT TC C T A AG

GAAGGCGGAAGAGAGGGTCTGAAGTACGGGTGTTCTGTGACAGACGCTTGCATA

GGAT GGGAGAGC ACGGAAC AGGTTTT GGAGCTGCTGGC AGAAGGGGT GCGT AAT

AG A AGG A A AGC C TT A A AG A AGT A A

SEQ ID NO: 30 (Saccharomyces cerevisiae (AR04 K2229L gene))

ATGAGCGAATCTCCGATGTTCGCCGCAAACGGCATGCCTAAGGTAAATCAAGGG

GCCGAGGAGGACGTGAGAATATTAGGTTATGACCCGCTTGCCAGTCCTGCATTGC

TTCAGGTACAGATTCCAGCAACGCCAACGTCCTTAGAAACAGCAAAAAGGGGAC

GTCGTGAAGCTATAGACATCATCACTGGCAAGGACGACCGTGTCCTAGTAATAGT

TGGTCCGTGCTCTATCCATGACCTTGAGGCTGCACAGGAGTATGCACTAAGGTTG

A AG A A ATT GT C T GAT GA AC T GA A AGGT GATCTT AGT AT A AT CAT GCGT GC AT ATT

TAGAGAAACCGCGTACGACGGTAGGCTGGAAAGGGCTAATTAACGATCCGGATG

TGAATAATACCTTTAACATCAACAAGGGTCTACAGAGTGCGCGTCAGTTATTCGT

GAACTTAACAAATATCGGACTGCCGATAGGCTCCGAGATGCTGGACACGATATC

TCCCCAGTATTTGGCTGACCTTGTTTCTTTTGGAGCTATAGGTGCAAGGACTACTG

AGAGTCAGTTACATAGAGAGTTGGCATCAGGACTTAGCTTCCCTGTAGGATTTAA

GAACGGTACAGACGGCACTCTTAATGTCGCGGTCGATGCCTGCCAGGCAGCCGC

CCATTCACATCATTTTATGGGAGTGACATTACACGGGGTGGCCGCTATCACAACG

ACTAAAGGGAATGAGCACTGTTTTGTTATCCTTAGAGGAGGAAAGAAAGGTACG

AATTATGATGCGAAAAGTGTAGCAGAGGCCAAAGCGCAACTTCCTGCCGGTTCA

AACGGACTTATGATTGACTATTCCCATGGAAACTCAAATAAGGACTTTAGGAATC

AGCCAAAAGTTAACGATGTGGTATGCGAACAGATCGCGAACGGTGAAAATGCGA

TTACGGGTGTTATGATCGAGTCAAATATAAATGAAGGTAACCAAGGTATCCCGG

CAGAGGGCAAAGCGGGCCTGAAGTACGGTGTATCTATTACGGATGCCTGTATAG

GTTGGGAGACAACCGAAGACGTCCTAAGGAAACTTGCCGCCGCGGTTAGACAGA

GAC GT GA AGT C A AT A AG A AGT A A

SEQ ID NO: 31 (Escherichia coli (AROL gene))

ATGACCCAGCCATTATTTCTGATCGGTCCTCGTGGGTGCGGGAAAACGACGGTTG

GCATGGCCTTAGCTGACAGTTTGAATCGTAGATTCGTGGACACCGACCAGTGGCT

ACAGTCTCAGCTTAACATGACGGTGGCCGAAATTGTAGAACGTGAAGAATGGGC

46 TGGTTTTCGTGCAAGAGAAACAGCCGCATTGGAAGCTGTGACGGCGCCTTCAAC

GGTGATAGCTACGGGAGGTGGTATTATTTTGACCGAATTTAATAGGCACTTCATG

CAGAATAATGGCATAGTGGTTTACCTATGCGCTCCTGTGTCTGTCTTGGTAAACC

GTTTGCAAGCCGCACCAGAAGAAGACTTGCGTCCAACCCTGACGGGGAAGCCAC

TGTCTGAGGAAGTGCAAGAGGTACTGGAGGAAAGGGACGCTCTATACCGTGAGG

TGGCTCACATCATAATTGACGCTACGAATGAGCCATCACAGGTAATTTCTGAGAT

CC GTT C AGC GTT GGCC C A A ACC AT C A ATTGTT A A

SEQ ID NO: 32 (Saccharomyces cerevisiae (TRP1 gene))

ATGTCAGTGATTAACTTTACAGGCTCCTCAGGTCCCTTGGTCAAGGTCTGCGGCT

TGCAATCAACAGAGGCCGCTGAATGCGCCCTAGACTCAGATGCAGACCTTTTAG

GCATCATCTGTGTCCCCAACAGAAAGCGTACTATTGATCCTGTTATTGCGCGTAA

GATCAGTTCTTTGGTCAAGGCGTATAAGAACTCCTCAGGAACCCCCAAGTATCTG

GTAGGGGTATTCAGGAATCAACCTAAAGAAGACGTCTTGGCCCTAGTTAATGACT

ACGGC AT AG AC AT AGTCC AGTT GC ACGGAGACGAAAGCTGGC AAGAAT ATC AGG

AATTTTTGGGGCTGCCGGTTATAAAAAGGCTGGTTTTCCCTAAGGACTGTAACAT

ACTGTTATCAGCCGCATCACAGAAGCCGCATTCCTTTATACCTCTTTTCGACTCCG

AGGCCGGAGGC ACTGGT GAATT ACTGGACTGGA AC AGC ATTTC AGATT GGGT AG

GGAGGCAGGAGAGCCCAGAATCTCTTCATTTTATGTTGGCAGGGGGCCTTACGCC

GGAAAATGTTGGAGATGCATTGAGGTTGAACGGAGTTATAGGTGTGGATGTCAG

T GGT GGGGTT GA A AC GA AT GGT GTT A A AGAC AGC A AC A A A AT AGC A A ATTTTGT

C AAGAAT GC C A A A A AGT A A

SEQ ID NO: 33 (Saccharomyces cerevisiae (TRP2 S76L gene))

ATGACGGCGAGCATTAAAATTCAGCCAGACATTGACAGTTTAAAGCAGTTGCAG

CAACAGAATGACGACTCTTCCATTAACATGTATCCCGTGTATGCGTATCTGCCTT

CTTTGGATTTGACACCTCACGTTGCTTACTTAAAGTTAGCTCAACTTAATAATCCA

GATAGAAAGGAGTCTTTCTTACTTGAAAGTGCTAAGACCAATAATGAGCTGGAC

AGATATCTTTTCATAGGGATCAGTCCAAGGAAGACCATTAAGACCGGGCCCACT

GAAGGC ATT GAGACTGACCC ATT AGAAATCCTT GAAAAAGAA ATGTCT ACTTT C A

AAGTCGCCGAAAACGTCCCAGGCCTTCCCAAATTAAGCGGCGGGGCGATAGGTT

ACATATCATACGACTGTGTACGTTACTTCGAACCCAAGACTAGGCGTCCCTTGAA

AGATGTGCTTAGGTTACCAGAGGCGTACTTGATGCTTTGTGACACGATAATCGCA

TTTGACAATGTCTTCCAAAGGTTTCAAATTATTCACAATATTAACACAAACGAAA

CGTCTTTGGAGGAAGGATACCAGGCGGCTGCGCAGATAATCACGGATATTGTAT

CTAAGTTGACAGACGACAGCTCCCCCATTCCGTACCCGGAGCAACCCCCTATCAA

ACTAAACCAAACCTTTGAATCCAACGTAGGCAAAGAGGGGTATGAAAATCACGT

CTCC ACTCTC AAAAAGC AC AT AAAGAA AGGTGAC AT AATCC AAGGT GT GCCC AG

CCAGAGAGTGGCGAGGCCTACATCTTTACATCCATTCAACATATATAGGCATCTT

AGAACCGTGAACCCATCACCTTATCTATTTTACATAGACTGCCTAGATTTCCAGA

TAATAGGGGCTAGTCCCGAATTGCTGTGTAAATCAGATTCAAAGAATCGTGTTAT

TACACACCCCATAGCTGGCACAGTCAAGAGGGGTGCTACCACTGAGGAAGATGA

47 CGCTCTGGCAGATCAGCTACGTGGTTCTTTGAAAGATAGGGCTGAGCATGTTATG

CTGGTTGACTTAGCAAGAAACGACATCAATCGTATATGCGATCCCCTAACGACTT

CCGTTGACAAACTTTTGACCATTCAGAAGTTCAGCCACGTACAGCACTTAGTCTC

TCAGGTCTCTGGCGTCCTAAGGCCTGAGAAAACTCGTTTCGATGCATTCAGAAGC

ATATTTCCCGCGGGTACAGTGAGTGGGGCCCCAAAGGTGCGTGCAATGGAGCTT

AT AGCCGAGCT AGAAGGCGAGCGT AGGGGAGTGT ACGC AGGGGCCGT AGGCC AT

TGGTCTTATGACGGCAAGACCATGGATAATTGTATTGCACTAAGGACCATGGTCT

AT A A AG AT GGGATTGC AT AC TT GC AGGC AGGAGGT GGGATT GT C T AT GAC AGCG

ATGAGTACGATGAGTATGTAGAAACAATGAATAAAATGATGGCGAATCATTCCA

CGATAGTGCAGGCGGAGGAGTTATGGGCGGATATTGTGGGTAGTGCATAA

SEQ ID NO: 34 (Saccharomyces cerevisiae (TRIG gene))

ATGTCTGTCCACGCAGCCACCAACCCGATAAATAAGCATGTCGTTCTGATTGATA

ATTACGACTCCTTCACGTGGAATGTTTATGAGTATCTTTGCCAGGAGGGAGCGAA

GGTTAGCGTTTACCGTAATGACGCTATCACGGTCCCAGAAATTGCAGCACTGAAT

CCCGATACCCTTCTGATATCACCAGGCCCGGGCCATCCCAAGACAGATTCTGGTA

TTAGCAGAGATTGCATCAGATACTTCACTGGAAAAATTCCAGTTTTTGGGATATG

TATGGGGCAGCAATGCATGTTTGACGTGTTTGGCGGGGAAGTGGCTTATGCGGGT

GAAATAGTGCACGGAAAGACTAGTCCCATATCCCATGATAACTGCGGTATCTTTA

AGAATGTCCCCCAGGGTATTGCAGTTACAAGATATCATAGCTTGGCTGGCACTGA

AAGTAGTCTGCCTAGCTGCCTAAAGGTGACTGCCTCTACTGAAAACGGGATAATC

ATGGGGGTAAGGCACAAGAAGTACACCGTCGAGGGGGTGCAATTCCACCCAGAG

AGTATTTTAACCGAAGAAGGACATCTAATGATCCGTAATATTCTTAATGTTTCTG

GCGGAACGTGGGAGGAAAATAAATCAAGCCCATCCAATTCCATCCTAGATAGGA

TATACGCCAGGCGTAAAATTGACGTAAACGAACAGTCAAAGATTCCCGGTTTCA

CCTTTCAGGACTTACAATCTAACTATGATCTTGGCCTTGCCCCGCCTCTGCAAGAT

TTTTATACCGTGCTGAGCAGTAGTCATAAGAGGGCTGTGGTCCTAGCGGAGGTGA

AGCGTGCCTCCCCTAGCAAAGGTCCAATCTGCCTGAAGGCCGTTGCTGCTGAACA

AGCCCTTAAATATGCTGAGGCTGGGGCGAGTGCAATTAGCGTTCTAACAGAACC

CCACTGGTTCCACGGGAGCCTTCAAGACCTTGTGAATGTAAGAAAGATCTTGGAT

CTAAAATTTCCGCCAAAAGAGAGACCCTGCGTGCTTAGGAAAGAGTTTATATTTT

CCAAATACCAAATATTGGAGGCACGTCTAGCTGGTGCAGATACTGTCCTTTTGAT

TGTAAAGATGTTGTCCCAACCATTACTGAAAGAGCTATATAGTTACTCAAAGGAT

TTAAACATGGAGCCGTTAGTGGAAGTAAATAGCAAGGAGGAGCTACAACGTGCC

CTGGAAATTGGTGCCAAGGTTGTTGGAGTTAACAATCGTGACTTGCATTCCTTCA

ACGTAGACTTGAATACAACAAGTAATTTGGTCGAATCTATCCCAAAAGATGTGCT

GTTGATTGCACTTTCCGGTATCACAACACGTGATGACGCCGAAAAGTATAAAAA

GGAGGGGGTGC ACGGGTTTTTGGT GGGT GAGGCGTT AAT GAAATCT AC AGAT GT

A A AGA AGTTT ATTC AT GAGCTGT GC GA AT A A

SEQ ID NO: 35(Saccharomyces cerevisiae (TRP4 gene))

48 ATGAGCGAAGCTACTCTATTAAGTTATACCAAAAAGCTACTAGCAAGCCCACCTC

AGCTTAGTTCCACCGACCTACACGATGCACTACTTGTCATCCTAAGTCTACTTCA

GAAGTGCGACACCAATTCTGATGAGTCCTTGTCTATTTATACGAAGGTGTCTTCC

TTTTTAACAGCCCTAAGGGTGACTAAGTTAGATCATAAGGCGGAATATATTGCCG

AGGCTGCAAAAGCAGTTTTGCGTCACTCAGATCTGGTCGATCTACCTTTACCTAA

AAAGGATGAGCTGCATCCTGAAGATGGTCCTGTTATCTTGGACATTGTGGGTACT

GGGGGTGATGGACAGAATACCTTTAACGTGTCAACGTCAGCCGCTATTGTGGCCT

CAGGTATTCAGGGACTGAAGATTTGCAAACACGGAGGTAAAGCATCTACCTCAA

ACAGCGGAGCTGGAGATCTGATTGGGACATTGGGATGCGATATGTTCAAAGTGA

ATAGTAGCACAGTCCCCAAATTGTGGCCAGACAATACATTTATGTTCTTATTGGC

TCCATTCTTTCATCATGGGATGGGTCATGTAAGCAAGATTCGTAAGTTTCTTGGA

ATACCTACGGTATTTAACGTATTGGGGCCGCTGTTACACCCCGTATCCCATGTGA

ATAAGAGGATACTTGGAGTGTATTCAAAAGAGTTGGCGCCAGAATATGCGAAGG

CAGCAGCCTTGGTCTATCCAGGGTCAGAAACGTTTATTGTGTGGGGCCATGTTGG

GCTTGACGAGGTGAGCCCCATAGGAAAGACTACCGTGTGGCACATCGATCCGAC

AAGCTCAGAACTAAAGTTGAAGACCTTCCAGCTGGAGCCATCTATGTTCGGTCTG

GAGGAGCACGAGCTGAGTAAATGCGCCTCATATGGACCTAAGGAGAATGCTCGT

ATATTAAAGGAGGAAGTCCTTTCCGGCAAATACCACCTAGGCGACAATAATCCA

ATATATGATTACATTCTGATGAATACTGCAGTATTATACTGCCTGTCCCAAGGGC

ACCAAAACTGGAAGGAAGGTATTATCAAAGCCGAGGAGTCAATTCACAGCGGGA

ATGCCTTGAGATCGCTAGAACATTTCATTGATTCAGTATCTTCCCTTTAA

SEQ ID NO: 36 (Saccharomyces cerevisiae (TAT2 gene))

AT GACCGAAGATTT CAT C AGT AGCGT C AA AAGGTC AAAT GAAGAGCTT AAAGAG AGAAAATCTAATTTTGGGTTTGTAGAGTACAAGTCAAAACAACTTACCTCCAGTA GCTCACACAACTCCAACTCTTCACACCATGATGACGACAACCAGCACGGTAAAA GAAACATCTTTCAGCGTTGTGTGGATTCTTTTAAATCCCCTCTGGATGGGTCTTTC GACACCTCCAATCTGAAAAGAACACTGAAACCTCGTCATTTAATAATGATCGCAA TAGGAGGTAGTATAGGTACTGGTCTTTTCGTGGGTTCAGGGAAGGCTATAGCGGA AGGCGGACCACTTGGCGTTGTGATCGGATGGGCCATTGCGGGTAGCCAAATAAT AGGTACTATACATGGGTTAGGAGAGATCACGGTAAGATTTCCAGTAGTCGGTGC GTTTGCCAACTACGGCACCCGTTTCTTGGACCCGAGCATTAGTTTTGTAGTCTCCA CTATATACGTGCTACAGTGGTTCTTTGTCCTACCCCTAGAGATTATTGCTGCGGCG ATGACCGTGCAATACTGGAACAGTTCTATCGATCCGGTAATATGGGTCGCAATTT TCTATGCCGTCATCGTCTCAATCAATTTGTTTGGAGTTAGGGGTTTCGGAGAAGC TGAATTCGCCTTCTCAACTATTAAGGCAATCACTGTCTGTGGCTTCATAATCTTAT GTGTAGTCTTGATCTGCGGCGGAGGACCCGATCACGAATTCATTGGTGCTAAATA CTGGCATGATCCTGGCTGCCTGGCAAACGGGTTTCCTGGAGTCTTGAGTGTCCTT GTCGTTGCGTCATACAGCCTAGGAGGCATAGAAATGACTTGCTTAGCCTCTGGGG AAACGGACCCAAAGGGACTTCCCTCAGCTATAAAACAGGTTTTCTGGCGTATTTT _{GTTTTTCTTCTTAATTTCTTTAACTCTAGTGGGATTTTTAGTTCCTTACACCAAC CA} AAATCTACTAGGTGGCTCCTCTGTCGATAATAGTCCCTTCGTTATCGCGATTAAG

49 CTACACCATATCAAAGCTCTTCCGTCTATTGTTAACGCAGTTATCCTTATTTCCGT

GCTATCCGTGGGTAACAGTTGCATCTTTGCCAGCTCCAGAACTCTGTGTAGCATG

GCACATCAAGGACTGATACCGTGGTGGTTCGGCTATATTGACAGAGCTGGCAGA

CCCCTGGTTGGGATTATGGCCAATTCTCTTTTCGGCTTATTGGCGTTCCTTGTTAA

ATCTGGCTCCATGAGTGAGGTGTTTAATTGGCTGATGGCTATAGCCGGACTGGCG

ACATGTATTGTGTGGTTATCTATAAATCTTTCCCATATAAGATTCCGTCTTGCAAT

GAAGGCCC AAGGAAAGTCCCTGGATGA ACTTGAATTCGT AAGCGCGGTT GGT AT

ATGGGGATCTGCTTATTCCGCACTTATCAATTGCTTAATACTTATTGCTCAATTTT

ATTGCTCTTTATGGCCAATCGGGGGTTGGACATCCGGAAAAGAGAGGGCAAAGA

TTTTCTTTCAGAATTATCTTTGCGCCCTGATTATGTTATTTATATTCATCGTCCATA

AGATCTATTATAAATGTCAAACGGGAAAGTGGTGGGGTGTTAAAGCTCTGAAGG

ACATCGACCTAGAGACCGACAGGAAGGACATAGACATCGAAATAGTTAAACAAG

AAATCGCTGAAAAGAAGATGTATTTGGACTCCAGACCTTGGTACGTGAGGCAGT

TTCATTTTTGGTGCTAA

50