Modified yeast cell and a method of using same

Abstract

The present invention provides a transformed or transgenic yeast cell including at least a partially inactive acetyltransferase encoding gene. Further provided is a method for increasing production levels of at least one arylalkyl amine in a yeast cell heterologously expressing at least one enzyme having an arylalkyl amine substrate, synthesizing an arylalkyl amine product, or both.

Description

MODIFIED YEAST CELL AND A METHOD OF USING SAMEREFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0001] The contents of the electronic sequence listing (YEDA-P-052-PCT.xml; size: 2,091 bytes; and date of creation: May 5, 2025) is herein incorporated by reference in its entirety.CROSS REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of priority of U.S. Provisional Patent Application No. 63 / 653,241, entitled “MODIFIED YEAST CELL AND A METHOD OF USING SAME”, filed 30 May 2024, the contents of which are incorporated herein by reference in their entirety.FIELD OF INVENTION

[0003] The present invention relates to a modified yeast cell, having at least partially reduced acetyl transferase activity, and methods of using same, such as for increasing production efficiency of a compound from an arylalkyl amine precursor thereof, in a yeast cell.BACKGROUND

[0004] The biosynthetic pathway of arylalkyl amines, such as in the production of mescaline in Peyote cacti has recently been elucidated. This pathway includes four groups of enzymes responsible for the six steps required for mescaline biosynthesis. While reconstructing the pathway in the yeast Saccharomyces cerevisiae, all arylalkyl amine intermediates were found to be Wacetylated, which likely reduces the production yield of each subsequent intermediate in the pathway, let alone, the desired product, e.g., mescaline.

[0005] An arylalkyl amine / V-acctyl transferase from S. cerevisiae (scAANAT, GenBank accession number S49826) has been reported to be functional on several arylalkyl amines, including mescaline, dopamine, tyramine, 3-methoxy-tyramine, and others.

[0006] There is still a great need for a modified yeast characterized by at least partially inactive acetyl transferring activity, such as harboring a knocked-down scAANAT, and methods of using same for increasing the production of arylalkyl amines which is of utmost importance for the industry relying on fermentation.SUMMARY

[0007] According to the first aspect, there is provided a transformed or transgenic yeast cell comprising at least a partially inactive acetyltransferase encoding gene.

[0008] According to another aspect, there is provided an extract derived from the transformed or transgenic cell of the invention, or any fraction thereof.

[0009] According to another aspect, there is provided a composition comprising: (a) the transformed or transgenic cell of the invention; (b) the extract of the invention; or (c) a combination of (a) and (b), and an acceptable carrier.

[0010] According to another aspect, there is provided a method for increasing production levels of at least one arylalkyl amine in a yeast cell heterologously expressing at least one enzyme having an arylalkyl amine substrate, synthesizing an arylalkyl amine product, or both, the method comprising contacting the yeast cell with an effective amount of an acetyltransferase inhibitor, thereby increasing the production levels of the at least one arylalkyl amine in the yeast cell.

[0011] According to another aspect, there is provided a composition comprising at least one arylalkyl amine synthesized according to the method of the invention, and a pharmaceutically acceptable carrier.

[0012] In some embodiments, the acetyltransferase encoding gene is fully inactive.

[0013] In some embodiments, the acetyltransferase encoding gene is constitutively inactive or conditionally inactive.

[0014] In some embodiments, the acetyltransferase is an arylalkyl amine A / - acetyltransferase (AANAT).

[0015] In some embodiments, the transformed or transgenic yeast cell is a Saccharomyces cerevisiae cell.

[0016] In some embodiments, the transformed or transgenic yeast cell further comprises at least one artificial DNA molecule comprising a nucleic acid sequence encoding at least one enzyme having an arylalkyl amine substrate, synthesizing an arylalkyl amine product, or both.

[0017] In some embodiments, the arylalkyl amine is phenethylamine.

[0018] In some embodiments, the arylalkyl amine is a psychoactive compound.

[0019] In some embodiments, the psychoactive compound is a psychedelic drug.

[0020] In some embodiments, the inactive gene is knocked down.

[0021] In some embodiments, knocked down is by any one of: antisense RNA, short interfering RNA (siRNA), short hairpin RNA (shRNA), double stranded RNA (dsRNA), and any combination thereof.

[0022] In some embodiments, the antisense RNA, siRNA, shRNA, dsRNA, and any combination thereof, is complementary to a transcript of the acetyltransferase encoding gene.

[0023] In some embodiments, the antisense RNA, siRNA, shRNA, dsRNA is transcribed from a polynucleotide sequence being integrated in a genome of the transformed or transgenic yeast cell, integrated in an artificial vector introduced into the transformed or transgenic yeast cell, or both.

[0024] In some embodiments, the nucleic acid sequence encoding an enzyme having a substrate, synthesizing a product, or both, being an arylalkyl amine, and any one of the antisense RNA, siRNA, shRNA, dsRNA, and any combination thereof, are operably linked.

[0025] In some embodiments, the extract comprises a psychoactive compound.

[0026] In some embodiments, the inhibitor inhibits: transcription of a gene encoding the acetyltransferase, translation of a transcript of the gene encoding the acetyltransferase, acetylation activity of a protein product of the gene encoding the acetyltransferase, or any combination thereof.

[0027] In some embodiments, the inhibitor induces or promotes degradation of a transcript of the gene encoding the acetyltransferase.

[0028] In some embodiments, the inhibitor is an RNA interfering (RNAi) oligonucleotide or a polynucleotide.

[0029] In some embodiments, the RNAi oligonucleotide or polynucleotide is selected from the group consisting of: antisense RNA, short interfering RNA (siRNA), short hairpin RNA (shRNA), double stranded RNA (dsRNA), and any combination thereof.

[0030] In some embodiments, the method further comprises a step before the contacting comprising introducing to the yeast cell an artificial DNA molecule encoding the at least one enzyme having an arylalkyl amine substrate, synthesizing an arylalkyl amine product, or both.

[0031] In some embodiments, the method further comprises a step comprising extracting the yeast cell so as to obtain the at least one arylalkyl amine.

[0032] In some embodiments, the at least one arylalkyl amine is a psychoactive compound.

[0033] Unless otherwise defined, all technical and / or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention, exemplary methods and / or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting.

[0034] Further embodiments and the full scope of applicability of the present invention will become apparent from the detailed description given hereinafter. However, it should be understood that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.BRIEF DESCRIPTION OF THE FIGURES

[0035] Figs. 1A-1B include a predicted biosynthetic pathway of mescaline 1 and related phenethylamines in Peyote as well as structures of tetrahydroisoquinoline (THIQ) alkaloids. (1A) The active enzymes identified in this study are marked by their names. TyDC, L-tyrosine / L- DOPA decarboxylase; CYP, cytochrome P450; PPO, polyphenol oxidase; OMT, O- methyltransferase; NMT, A-methyltransferase. Putative types of enzymes appear in black. According to the predicted pathway, some metabolites may be biosynthesized in more than one route as depicted by the main molecules marked in yellow, orange, and blue according to modules I-III, respectively. Decarboxylation, hydroxylation, (9-methylation, and A-methylation reactions are marked in pink, red, blue, and green, respectively. (IB) Chemical structures of several known tetrahydroisoquinolines (TIQs) from Peyote putatively assigned according to LC -MS / MS fragmentation.

[0036] Figs. 2A-2C include chromatograms showing functional characterization of CYP proteins. (2A) Extracted ion chromatograms of tyramine 4 and dopamine 3 and C, MS / MS spectra of A-acetyl-tyramine 32 and A-acetyl-dopamine 33 obtained from the supernatant collected after co-expression of LwTyDC2 and LwCYP76AD131 in yeast (n =4 biological replicates). Metabolites were analyzed using the acquired data-dependent MS / MS (dd-MS2) data. Chromatograms were normalized to the highest value. Identification of A-acetyl- phenethylamines was conducted by comparing the accurate mass, retention time, and MS / MSspectra of the observed peaks with those obtained from yeast transformed with the EV after supplementation of each intermediate, as well as MS / MS spectra from analytical standards (2B). Full details including typical fragmentation patterns of the V-acctylatcd metabolites appear in Fig. 6. (2C) Extracted ion chromatograms of mass corresponding to L-DOPA 5 ( 198.076— > 152.071) obtained from the supernatant following expression of either LwCYP76AD131 alone or co-expression of LwTyDC2 and LwCYP76AD131 in yeast (n =4 biological replicates) demonstrate the absence of L-DOPA 5 production by this enzyme.

[0037] Fig. 3 includes chromatograms showing characterization of Peyote cytochrome P450 (CYP) enzymes. Extracted ion chromatograms and chemical structures of A- acetyl tyramine 32 and A-acetyl dopamine 33 obtained after co-expression of LwTyDC2 and CYP76AD131 in yeast (n =4 biological replicates). Metabolites were analyzed using the acquired data-dependent MS / MS (dd-MS2) data. Chromatograms were normalized to the highest value. A-Acetylation occurs in yeast by an endogenous acetylating enzyme as shown for samples transformed with the empty vector (EV) and supplemented with each intermediate (blue chromatograms).

[0038] Figs. 4A-4E include chemical structures and chromatograms showing that reconstruction of the mescaline pathway in yeast shows de novo production of phenethylamines and V-acctyl- phenethylamines from the mescaline pathway. (4A) Schematic representation of experimental approach and extracted ion chromatograms of products observed in yeast following co- expression of LwTyDC2, LwCYP76AD131, LwOMTl and LwOMTl l. Metabolites were analyzed using the acquired data-dependent MS / MS (dd-MS2) data. Chromatograms were normalized to the highest value. Identification of A-acetyl-phenethylamines was by comparing the accurate mass, retention time, and MS / MS spectra of the observed peaks with those obtained from yeast transformed with the EV after supplementation of each intermediate, as well as MS / MS spectra from analytical standards (Fig. 2C). (4B) Schematic representation of experimental approach and MS / MS spectral matching of A-acetylated products observed in yeast transformed with the EV following incubation with each intermediate. Putative identification of AAiccty I -phenethyl a ines was by accurate mass and by comparing MS / MS spectra with the non- acetylated phenethylamine as annotated on the fragmentation structures. Full details including typical fragmentation patterns of the A-acetylated metabolites appear in Fig. 6. scAANAT, yeast A-acetyltransferase. (4C) Extracted ion chromatograms of phenethylamines and 4D, MS / MS spectra of A-acetylated products observed following co-expression of LwTyDC2, LwCYP76AD131, LwOMTl and LwOMTl l and incubation with each intermediate from the mescaline 1 pathway individually. Metabolites were analyzed using the acquired data-dependentMS / MS (dd-MS2) data. Chromatograms were normalized to the highest value. Products in each assay were identified according to analytical standards. 3-Methoxy-tyramine 7 and 3,4- dihidroxy-5-methoxy-phenethylamine 6 products were not observed following incubation with dopamine 3 and 3-methoxy-tyramine 7, respectively. (4D) Putative identification of N- acetyl- phenethylamines was by comparing the accurate mass, retention time, and MS / MS spectra of the observed peaks with those obtained from yeast transformed with the EV after supplementation of each intermediate. V-Acetyl-3-hydroxy-4,5-dimethoxy-phenethylamine 37 and V-acctyl- mescaline 38 were putatively identified by accurate mass and by comparing MS / MS spectra with the non- acetylated phenethylamine as annotated on the fragmentation structures in (4E). Full details including typical fragmentation patterns of V-acctylatcd metabolites appear in Fig. 6. EV, empty vector.

[0039] Figs. 5A-5B include a non-limiting scheme, chromatograms, and chemical structures showing in vivo reconstruction of the mescaline pathway in yeast (S. cerevisiae). (5A) A schematic representation of the experimental approach and observed products in yeast following co-expression of LwTyDC2, LwCYP76AD131, LwOMTl and LwOMTl l, and incubation with each intermediate from the mescaline 1 pathway individually (n =2 biological replicates). N- Acetylation occurs in yeast by an endogenous Y-acctylating enzyme (scAANAT) as presented for samples transformed with the empty vector (EV) and supplemented with each intermediate (Fig. 4B). Detected phenethylamines and A-acetyl-phenethylamines in each sample appear in green and red, respectively. The Peyote enzymes that exhibit activity with the supplemented intermediate and the respective products are highlighted in blue. (5B) Extracted ion chromatograms and chemical structures of A- acetylated products from (5A). Identification of N- acetylated phenethylamines was according to yeast transformed with the EV and following incubation with each intermediate as standard (blue chromatogram, full details appear in Figs. 4D-4E). Metabolites were analyzed using the acquired data-dependent MS / MS (dd-MS2) data. Chromatograms were normalized to the highest value.

[0040] Fig. 6 includes a table summarizing the full description of the identified Y-acctylatcd phenethylamines in Saccharomyces cerevisiae following feeding with mescaline pathway intermediates and expression of Peyote enzymes.DETAILED DESCRIPTION

[0041] According to the first aspect, there is provided a transformed or transgenic yeast cell comprising at least a partially inactive acetyltransferase encoding gene.

[0042] In some embodiments, the transgenic cell is devoid of an acetyl transferring enzyme, e.g., acetyltransferase. In some embodiments, the transgenic cell comprises a knocked-out acetyl transferring enzyme encoding gene or a transcript thereof. In some embodiments, the transgenic cell comprises a knocked-down (or silenced) acetyl transferring enzyme encoding gene or a transcript thereof. In some embodiments, the transgenic cell comprises an RNA interfering (RNAi) oligonucleotide or polynucleotide capable of hybridizing to, inducing degradation of, or both, an acetyl transferring enzyme encoding gene or a transcript thereof. In some embodiments, the transgenic cell synthesizes RNAi oligonucleotide(s) or polynucleotide(s) hybridizing to, inducing degradation of, or both, an acetyl transferring enzyme encoding gene or a transcript thereof. In some embodiments, the transgenic cell further comprises RNAi oligonucleotide(s) or polynucleotide(s) capable of hybridizing to, inducing degradation of, or both, an acetyl transferring enzyme encoding gene or a transcript thereof.

[0043] As used herein, the term “at least partially” comprises 99% at most, 95% at most, 90% at most, 80% at most, 70% at most, 60% at most, 50% at most, 40% at most, 30% at most, 20% at most, 10%, or any value and range therebetween. Each possibility represents a separate embodiment of the invention.

[0044] In some embodiments, the transformed or transgenic yeast cell comprises an acetyltransferase encoding gene being fully inactive. In some embodiments, fully inactive comprises non-active, deactivated, or both. In some embodiments, fully inactive is 100% inactive.

[0045] In some embodiments, the acetyltransferase encoding gene is constitutively inactive or conditionally inactive.

[0046] In some embodiments, the transformed or transgenic yeast cell comprises or is a Saccharomyces cerevisiae cell.

[0047] In some embodiments, the acetyltransferase is an arylalkyl amine A-acetyltransferase (AANAT). In some embodiments, S. cerevisiae acetyltransferase an arylalkyl amine N- acetyltransferase (scAANAT). In some embodiments, the scAANAT comprises the amino acid sequence as set forth in the Genbank under accession no. Q12447.1.

[0048] In some embodiments, scAANAT comprises the amino acid sequence: MASSSSTLPLHMYIRPLIIEDLKQILNLESQGFPPNERASEEIISFRLINCPELCSGLFIREIE GKEVKKETEIGHIMGTKIPHEYITIESMGKEQVESSNHIGIHSVVIKPEYQKKNEATEEET DYIQKLSNQEIGNKIVLIAHEPLIPFYERVGFKIIAENTNVAKDKNFAEQKWIDMERELI KEEYDN (SEQ ID NO: 1).

[0049] In some embodiments, the transformed or transgenic yeast cell further comprises at least one artificial DNA molecule. In some embodiments, the artificial DNA molecule comprises a nucleic acid sequence encoding at least one enzyme having an arylalkyl amine substrate, synthesizing an arylalkyl amine product, or both. In some embodiments, the artificial DNA molecule is integrated in the genome of the transformed or transgenic cell. In some embodiments, the artificial DNA is integrated in an artificial vector or a plasmid. In some embodiments, the artificial DNA is an artificial vector or a plasmid. In some embodiments, a vector is an expression vector.

[0050] In some embodiments, an arylalkyl amine comprises phenethylamine.

[0051] In some embodiments, an arylalkyl amine comprises a psychoactive compound.

[0052] In some embodiments, a psychoactive compound comprises a psychedelic drug.

[0053] Non-limiting examples of arylalkyl amines include but are not limited to mescaline, dopamine, tyramine, 3-methoxy-tyramine, or others.

[0054] In some embodiments, an arylalkyl amine comprises mescaline, dopamine, tyramine, 3- methoxy-tyramine, or any combination thereof.

[0055] In some embodiments, an inactive gene is knocked down.

[0056] As used herein, the term “knocked down”, or “knockdown”, refers to a condition in which the expression of the gene is reduced.

[0057] In some embodiments, the transcription of the gene is reduced. In some embodiments, the translation of the gene is reduced. In some embodiments, the expression of the gene is reduced by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or by at least 99%. Each embodiment refers to a separate embodiment of the invention. In some embodiments, the reduction can occur either through a genetic modification or by treatment with a reagent such as a short DNA or RNA oligonucleotide that has a nucleic acid sequence complementary to either gene or a mRNA transcript.

[0058] In some embodiments, the knocked down gene is achieved by a genetic modification or mutation, leading to gene silencing. In some embodiments, the knocked down gene is by a stable knockdown, referring to constituent reduced gene expression. In some embodiments, the gene reduced expression is caused by an oligonucleotide binding to mRNA or temporarily binding to a gene. In some embodiments, the knocked down gene is a transient knockdown referring to a temporary change in gene expression that does not modify the chromosomal DNA. In some embodiments, the transient knockdown, is achieved by at least one of: blocking or reducing transcription, degradation of the mRNA transcript, blocking or reducing mRNA translation, blocking or reducing the binding of pre-mRNA splicing sites, or nuclease cleavage sites used formaturation of other functional RNAs, including miRNA. In some embodiments, blocking or reducing transcription can be achieved by small interfering RNA (siRNA) or RNase-H dependent antisense. Methods for generation of knockdown genes are known in the art, comprising RNA interference, CRISPRs, and TALENs.

[0059] In some embodiments, knocked down is by any one of: antisense RNA, short interfering RNA (siRNA), short hairpin RNA (shRNA), double stranded RNA (dsRNA), and any combination thereof.

[0060] In some embodiments, antisense RNA, siRNA, shRNA, dsRNA, or any combination thereof, is complementary to a transcript of the acetyltransferase encoding gene.

[0061] In some embodiments, antisense RNA, siRNA, shRNA, dsRNA, or any combination thereof, is transcribed from a polynucleotide sequence being integrated in a genome of the transformed or transgenic yeast cell, integrated in an artificial vector introduced into the transformed or transgenic yeast cell, or both.

[0062] In some embodiments, the nucleic acid sequence encoding an enzyme having an arylalkyl amine substrate, synthesizing an arylalkyl amine product, or both, and the antisense RNA, siRNA, shRNA, dsRNA, or any combination thereof, are operably linked.

[0063] Typically, RNA interference (RNAi) refers to the process of sequence- specific post transcriptional gene silencing mediated by small interfering RNAs (siRNA). Long double stranded RNA (dsRNA) in cells typically stimulates the activity of a ribonuclease III enzyme referred to as dicer. Dicer is involved in the processing of the long dsRNA into short pieces of siRNA. siRNAs derived from dicer activity are typically about 21-23 nucleotides in length and include duplexes of about 19 base pairs.

[0064] The RNAi response also features an endonuclease complex containing a siRNA, commonly referred to as an RNA-induced silencing complex (RISC), which mediates cleavage of single stranded RNA having sequence complementary to the antisense strand of the siRNA duplex. Cleavage of the target RNA takes place in the middle of the region complementary to the antisense strand of the siRNA duplex. Without being bound to any mechanism of processing or action, the present invention relates to double stranded.

[0065] RNAs, whether processed or not, is a tool for down regulating gene expression.

[0066] Gene expression can also be down regulated by microRNAs, single- stranded RNA molecules of about 21-23 nucleotides in length, encoded by genes that are transcribed from DNA but not translated into protein. MicroRNAs base pair with their complementary mRNA molecules and induce mRNA degradation in the RISC complex. Gene expression can further be down regulated by an antisense oligonucleotide complementary to a region of an mRNA wherein theantisense oligonucleotide is capable of specifically hybridizing with the region of the mRNA, thereby inhibiting the expression of a gene. Thus, the present invention encompasses double stranded RNAs, micro RNAs, antisense oligonucleotides, short hairpin RNAs (shRNAs), each capable of down regulating the yeast acetyl transferase, particularly of S. cerevisiae.

[0067] The term "operably linked" is intended to mean that the nucleotide sequence(s) of interest is linked to the regulatory element or elements in a manner that allows for expression of the nucleotide sequence(s) (e.g., in an in vitro transcription / translation system or in a host cell when the vector is introduced into the host cell). In some embodiments, the promoter is operably linked to the artificial DNA molecule disclosed herein, the RNAi oligonucleotide or polynucleotide as disclosed herein, or both. In some embodiments, the promoter is a heterologous promoter. In some embodiments, the promoter is the endogenous promoter. In some embodiments, expression of the artificial DNA molecule is operably linked to the expression of the RNAi oligonucleotide or polynucleotide. In some embodiments, transcription of the artificial DNA molecule is operably linked to the transcription of the RNAi oligonucleotide or polynucleotide. In some embodiments, the artificial DNA molecule and the RNAi oligonucleotide or polynucleotide are expressed or transcribed together or at the same time. In some embodiments, when the artificial DNA molecule is expressed or transcribed, the RNAi oligonucleotide or polynucleotide is also expressed or transcribed.

[0068] In some embodiments, the vector is introduced into the cell by standard methods including electroporation (e.g., as described in From et al., Proc. Natl. Acad. Sci. USA 82, 5824 (1985)), heat shock, infection by viral vectors, high velocity ballistic penetration by small particles with the nucleic acid either within the matrix of small beads or particles, or on the surface (Klein et al., Nature 327. 70-73 (1987)), such as biolistic use of coated particles, and needle-like particles, Agrobacterium Ti plasmids and / or the like.

[0096] The term "promoter" as used herein refers to a group of transcriptional control modules that are clustered around the initiation site for an RNA polymerase i.e., RNA polymerase II. Promoters are composed of discrete functional modules, each consisting of approximately 7-20 bp of DNA, and containing one or more recognition sites for transcriptional activator or repressor proteins. The promoter may extend upstream or downstream of the transcriptional start site and may be any size ranging from a few base pairs to several kilo-bases.

[0069] In some embodiments, the polynucleotide is transcribed by RNA polymerase II (RNAP II and Pol II). RNAP II is an enzyme found in eukaryotic cells, known to catalyze the transcription of DNA to synthesize precursors of mRNA and most snRNA and microRNA.

[0070] In some embodiments, a plant expression vector is used. In one embodiment, the expression of a polypeptide coding sequence is driven by a number of promoters. In some embodiments, viral promoters such as the 35S RNA and 19S RNA promoters of CaMV [Brisson et al., Nature 310:511-514 (1984)], or the coat protein promoter to TMV [Takamatsu et al., EMBO J. 6:307-311 (1987)] are used. In another embodiment, plant promoters are used such as, for example, the small subunit of RUBISCO [Coruzzi et al., EMBO J. 3: 1671-1680 (1984); and Brogli et al., Science 224:838- 843 (1984)] or heat shock promoters, e.g., soybean hspl7.5-E or hspl7.3-B [Gurley et al., Mol. Cell. Biol. 6:559-565 (1986)]. In one embodiment, constructs are introduced into plant cells using Ti plasmid, Ri plasmid, plant viral vectors, direct DNA transformation, microinjection, electroporation and other techniques well known to the skilled artisan. See, for example, Weissbach & Weissbach [Methods for Plant Molecular Biology, Academic Press, NY, Section VIII, pp 421-463 (1988)]. Other expression systems such as insects and mammalian host cell systems, which are well known in the art, can also be used by the present invention.

[0071] In some embodiments, expression vectors containing regulatory elements from eukaryotic viruses such as retroviruses are used by the present invention. SV40 vectors include pSVT7 and pMT2. In some embodiments, vectors derived from bovine papilloma virus include pBV-lMTHA, and vectors derived from Epstein Bar virus include pHEBO, and p205. Other exemplary vectors include pMSG, pAV009 / A+, pMTO10 / A+, pMAMneo-5, baculovirus pDSVE, and any other vector allowing expression of proteins under the direction of the SV-40 early promoter, SV-40 later promoter, metallo thionein promoter, murine mammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrin promoter, or other promoters shown effective for expression in eukaryotic cells.

[0072] In some embodiments, recombinant viral vectors, which offer advantages such as systemic infection and targeting specificity, are used for in vivo expression. In one embodiment, systemic infection is inherent in the life cycle of, for example, the retrovirus and is the process by which a single infected cell produces many progeny virions that infect neighboring cells. In one embodiment, the result is that a large area becomes rapidly infected, most of which was not initially infected by the original viral particles. In one embodiment, viral vectors are produced that are unable to spread systemically. In one embodiment, this characteristic can be useful if the desired purpose is to introduce a specified gene into only a localized number of targeted cells.

[0073] In some embodiments, plant viral vectors are used. In some embodiments, a wild-type virus is used. In some embodiments, a deconstructed virus such as are known in the art is used. In some embodiments, Agrobacterium is used to introduce the vector of the invention into a virus.

[0074] Various methods can be used to introduce the expression vector of the present invention into cells. Such methods are generally described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Springs Harbor Laboratory, New York (1989, 1992), in Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Md. (1989), Chang et al., Somatic Gene Therapy, CRC Press, Ann Arbor, Mich. (1995), Vega et al., Gene Targeting, CRC Press, Ann Arbor Mich. (1995), Vectors: A Survey of Molecular Cloning Vectors and Their Uses, Butterworths, Boston Mass. (1988) and Gilboa et at. [Biotechniques 4 (6): 504-512, 1986] and include, for example, stable or transient transfection, lipofection, electroporation, agrobacterium Ti plasmids and infection with recombinant viral vectors. In addition, see U.S. Pat. Nos. 5,464,764 and 5,487,992 for positive-negative selection methods.

[0075] It will be appreciated that other than containing the necessary elements for the transcription and translation of the inserted coding sequence (encoding the polypeptide), the expression construct of the present invention can also include sequences engineered to optimize stability, production, purification, yield, or activity of the expressed polypeptide.

[0076] According to another aspect, there is provided an extract derived from the transformed or transgenic cell of the invention, or any fraction thereof.

[0077] In some embodiments, the extract comprises a psychoactive compound.

[0078] According to another aspect, there is provided a composition comprising: (a) the transformed or transgenic cell of the invention; (b) the extract of the invention; or (c) a combination of (a) and (b), and an acceptable carrier. In some embodiments, the carrier comprises a biologically acceptable carrier. In some embodiments, the carrier comprises a pharmaceutically acceptable carrier. In some embodiments, the carrier comprises a nutraceutically acceptable carrier.

[0079] According to another aspect, there is provided a method for increasing production levels of at least one arylalkyl amine in a yeast cell heterologously expressing at least one enzyme having an arylalkyl amine substrate, synthesizing an arylalkyl amine product, or both.

[0080] In some embodiments, the method comprises contacting a yeast cell with an effective amount of an acetyltransferase inhibitor, thereby increasing the production levels of the at least one arylalkyl amine in the yeast cell.

[0081] In some embodiments, the contacting comprise contacting with an effective amount of an inducer activating, promoting, pushing, driving, or any combination thereof, the expression of the acetyltransferase inhibitor being an RNAi oligonucleotide or polynucleotide.

[0082] In some embodiments, the acetyltransferase inhibitor inhibits: transcription of a gene encoding an acetyltransferase, translation of a transcript of a gene encoding an acetyltransferase, acetylation activity of a protein product of a gene encoding an acetyltransferase, or any combination thereof.

[0083] In some embodiments, an acetyltransferase inhibitor induces or promotes degradation of a transcript of a gene encoding an acetyltransferase.

[0084] In some embodiments, an acetyltransferase inhibitor is an RNA interfering (RNAi) oligonucleotide or a polynucleotide.

[0085] In some embodiments, an RNAi oligonucleotide or polynucleotide is selected from: antisense RNA, short interfering RNA (siRNA), short hairpin RNA (shRNA), double stranded RNA (dsRNA), or any combination thereof.

[0086] In some embodiments, acetyltransferase is AANAT as described herein.

[0087] In some embodiments, the yeast cell is a transformed or transgenic yeast cell.

[0088] In some embodiments, the method further comprises a step comprising introducing to the yeast cell an artificial DNA molecule encoding the at least one enzyme having an arylalkyl amine substrate, synthesizing an arylalkyl amine product, or both, thereby producing a transformed or transgenic yeast cell.

[0089] In some embodiments, the method further comprises a step before the contacting comprising introducing to the yeast cell an artificial DNA molecule encoding the at least one enzyme having an arylalkyl amine substrate, synthesizing an arylalkyl amine product, or both, thereby producing a transformed or transgenic yeast cell.

[0090] In some embodiments, the method further comprises a step comprising extracting the yeast cell. In some embodiments, the extracting comprises extracting at least one arylalkyl amine from the yeast cell.

[0091] According to another aspect, there is provided at least one arylalkyl amine synthesized according to the method of the invention.

[0092] According to another aspect, there is provided a composition comprising at least one arylalkyl amine, and a pharmaceutically acceptable carrier.

[0093] The term "nucleic acid" is well known in the art. A "nucleic acid" as used herein will generally refer to any molecule (e.g., a strand) of DNA, RNA or a derivative or analog thereof, comprising nucleotides. Nucleotides are comprised of nucleosides and phosphate groups. Thenitrogenous bases of nucleosides include, for example, naturally occurring purine or pyrimidine nucleosides as found in DNA (e.g., an adenine "A," a guanine "G," a thymine "T" or a cytosine "C") or RNA (e.g., an A, a G, an uracil "U" or a C).

[0094] The term "nucleic acid molecule" includes but is not limited to single- stranded RNA (ssRNA), double- stranded RNA (dsRNA), single-stranded DNA (ssDNA), double- stranded DNA (dsDNA), small RNAs, circular nucleic acids, fragments of genomic DNA or RNA, degraded nucleic acids, amplification products, modified nucleic acids, plasmid or organellar nucleic acids, and artificial nucleic acids such as oligonucleotides.

[0095] As used herein, the term "transgenic cell" refers to any cell that has undergone human manipulation on the genomic or gene level. In some embodiments, the transgenic cell has had exogenous polynucleotide, such as an isolated DNA molecule as disclosed herein, introduced into it. In some embodiments, a transgenic cell comprises a cell that has an artificial vector introduced into it. In some embodiments, a transgenic cell is a cell which has undergone genome mutation or modification. In some embodiments, a transgenic cell is a cell that has undergone CRISPR genome editing. In some embodiments, a transgenic cell is a cell that has undergone targeted mutation of at least one base pair of its genome. In some embodiments, the exogenous polynucleotide (e.g., the isolated DNA molecule disclosed herein) or vector is stably integrated into the cell. In some embodiments, the transgenic cell expresses a polynucleotide of the invention. In some embodiments, the transgenic cell expresses a vector of the invention. In some embodiments, the transgenic cell expresses a protein of the invention. In some embodiments, the transgenic cell, is a cell that is devoid of a polynucleotide of the invention that has been transformed or genetically modified to include the polynucleotide of the invention. In some embodiments, CRISPR technology is used to modify the genome of the cell, as described herein.

[0096] In some embodiments, the cell is a unicellular organism, a cell of a multicellular organism, and a cell in a culture. In some embodiments, a unicellular organism comprises a fungus or a bacterium. In some embodiments, the fungus is a yeast cell. In some embodiments, the cell is a prokaryote or a eukaryote cell. In some embodiments, the yeast cell is or comprises a Saccharomyces cerevisiae cell.

[0097] Methods and / or means for extracting, lysing, homogenizing, fractionating, or any combination thereof, a cell or a culture of same, are common and would be apparent to one of ordinary skill in the art of cell biology and biochemistry. Non-limiting examples include, but are not limited to, pressure lysis (e.g., such as using a French press), enzymatic lysis, soluble-insoluble phase separation (such for obtaining a supernatant and a pellet), detergent-based lysis, solvent (e.g., polar, or nonpolar solvent), liquid chromatography mass spectrometry, or others.

[0098] As used herein, the term “carrier”, “excipient”, or “adjuvant” refers to any component of a composition, e.g., pharmaceutical or nutraceutical, that is not the active agent. As used herein, the term “pharmaceutically acceptable carrier” refers to non-toxic, inert solid, semi-solid liquid filler, diluent, encapsulating material, formulation auxiliary of any type, or simply a sterile aqueous medium, such as saline. Some examples of the materials that can serve as pharmaceutically acceptable carriers are sugars, such as lactose, glucose and sucrose, starches such as corn starch and potato starch, cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt, gelatin, talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol, polyols such as glycerin, sorbitol, mannitol and polyethylene glycol; esters such as ethyl oleate and ethyl laurate, agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline, Ringer's solution; ethyl alcohol and phosphate buffer solutions, as well as other non-toxic compatible substances used in pharmaceutical formulations. Some non-limiting examples of substances which can serve as a carrier herein include sugar, starch, cellulose and its derivatives, powered tragacanth, malt, gelatin, talc, stearic acid, magnesium stearate, calcium sulfate, vegetable oils, polyols, alginic acid, pyrogen-free water, isotonic saline, phosphate buffer solutions, cocoa butter (suppository base), emulsifier (e.g. carbomer, hydroxypropyl cellulose, sodium lauryl sulfate) as well as other non-toxic pharmaceutically compatible substances used in other pharmaceutical formulations. Wetting agents and lubricants such as sodium lauryl sulfate, as well as coloring agents, flavoring agents, excipients, stabilizers, antioxidants, and preservatives may also be present. Any non-toxic, inert, and effective carrier may be used to formulate the compositions contemplated herein. Suitable pharmaceutically acceptable carriers, excipients, and diluents in this regard are well known to those of skill in the art, such as those described in The Merck Index, Thirteenth Edition, Budavari et al., Eds., Merck & Co., Inc., Rahway, N.J. (2001); the CTFA (Cosmetic, Toiletry, and Fragrance Association) International Cosmetic Ingredient Dictionary and Handbook, Tenth Edition (2004); and the “Inactive Ingredient Guide,” U.S. Food and Drug Administration (FDA) Center for Drug Evaluation and Research (CDER) Office of Management, the contents of all of which are hereby incorporated by reference in their entirety. Examples of pharmaceutically acceptable excipients, carriers, and diluents useful in the present compositions include distilled water, physiological saline, Ringer's solution, dextrose solution, Hank's solution, and DMSO.These additional inactive components, as well as effective formulations and administration procedures, are well known in the art and are described in standard textbooks, such as Goodman and Gillman’s: The Pharmacological Bases of Therapeutics, 8th Ed., Gilman et al. Eds. Pergamon Press (1990); Remington’s Pharmaceutical Sciences, 18th Ed., Mack Publishing Co., Easton, Pa. (1990); and Remington: The Science and Practice of Pharmacy, 21st Ed., Lippincott Williams & Wilkins, Philadelphia, Pa., (2005), each of which is incorporated by reference herein in its entirety. The presently described composition may also be contained in artificially created structures such as liposomes, ISCOMS, slow -releasing particles, and other vehicles which increase the half-life of the peptides or polypeptides in serum. Liposomes include emulsions, foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions, lamellar layers, and the like. Liposomes for use with the presently described peptides are formed from standard vesicle-forming lipids which generally include neutral and negatively charged phospholipids and a sterol, such as cholesterol. The selection of lipids is generally determined by considerations such as liposome size and stability in the blood. A variety of methods are available for preparing liposomes as reviewed, for example, by Coligan, J. E. et al, Current Protocols in Protein Science, 1999, John Wiley & Sons, Inc., New York, and see also U.S. Pat. Nos. 4,235,871, 4,501,728, 4,837,028, and 5,019,369.

[0099] The carrier may comprise, in total, from about 0.1% to about 99.99999% by weight of the pharmaceutical compositions presented herein.

[0100] Method for introducing or transfecting a cell with an artificial nucleic acid molecule or vector (e.g., plasmid and / or agrobacterium) are common and would be apparent to one of ordinary skill in the art.

[0101] In some embodiments, introducing or transfecting comprises transferring an artificial nucleic acid molecule or vector comprising a polynucleotide into a cell; or modifying the genome of a cell to include a polynucleotide. In some embodiments, the transferring comprises transfection. In some embodiments, the transferring comprises transformation. In some embodiments, the transferring comprises lipofection. In some embodiments, the transferring comprises nucleofection. In some embodiments, the transferring comprises viral infection.

[0102] In some embodiments, artificial nucleic acid molecule or vector comprises: a plasmid, an agrobacterium, or both.

[0103] As used herein, the terms “transfecting” and “introducing” are interchangeable.General

[0104] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0105] As used herein, the term "about" when combined with a value refers to plus and minus 10% of the reference value. For example, a length of about 1,000 nanometers (nm) refers to a length of 1,000 nm ± 100 nm.

[0106] It is noted that as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a polynucleotide" includes a plurality of such polynucleotides and reference to "the polypeptide" includes reference to one or more polypeptides and equivalents thereof known to those skilled in the art, and so forth. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as "solely," "only" and the like in connection with the recitation of claim elements or use of a "negative" limitation.

[0107] In those instances where a convention analogous to "at least one of A, B, and C, etc." is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e.g., "a system having at least one of A, B, and C" would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). It will be further understood by those within the art that virtually any disjunctive word and / or phrase presenting two or more alternative terms, whether in the description, claims, or drawings, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase "A or B" will be understood to include the possibilities of "A" or "B" or "A and B."

[0108] It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub- combination. All combinations of the embodiments pertaining to the invention are specificallyembraced by the present invention and are disclosed herein just as if each and every combination was individually and explicitly disclosed. In addition, all sub-combinations of the various embodiments and elements thereof are also specifically embraced by the present invention and are disclosed herein just as if each and every such sub -combination was individually and explicitly disclosed herein.

[0109] Additional objects, advantages, and novel features of the present invention will become apparent to one ordinarily skilled in the art upon examination of the following examples, which are not intended to be limiting. Additionally, each of the various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below finds experimental support in the following examples.

[0110] Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples.EXAMPLES

[0111] Generally, the nomenclature used herein, and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological, and recombinant DNA techniques. Such techniques are thoroughly explained in the literature. See, for example, "Molecular Cloning: A laboratory Manual" Sambrook et al., (1989); "Current Protocols in Molecular Biology" Volumes I-III Ausubel, R. M., ed. (1994); Ausubel et al., "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Maryland (1989); Perbal, "A Practical Guide to Molecular Cloning", John Wiley & Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific American Books, New York; Birren et al. (eds) "Genome Analysis: A Laboratory Manual Series", Vols. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); methodologies as set forth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; "Cell Biology: A Laboratory Handbook", Volumes I-III Cellis, J. E., ed. (1994); "Culture of Animal Cells - A Manual of Basic Technique" by Freshney, Wiley-Liss, N. Y. (1994), Third Edition; "Current Protocols in Immunology" Volumes I-III Coligan J. E., ed. (1994); Stites et al. (eds), "Basic and Clinical Immunology" (8th Edition), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (eds), "Strategies for Protein Purification and Characterization - A Laboratory Course Manual" CSHL Press (1996); all of which are incorporated by reference. Other general references are provided throughout this document.Materials and MethodsMaterials

[0112] Unless otherwise stated, all the analytical metabolites were >95% pure. Mescaline (hydrochloride) 1, dopamine (hydrochloride) 3, 3-MeO-tyramine (hydrochloride) 7 and hordenine 13 were purchased from Cayman Chemical (Ann Arbor, MI, USA). L-DOPA 5 and L-tyrosine 2 were purchased from Sigma-Aldrich (Rehovot, Israel), 3,4-diOH-5-MeO- phenethylamine (hydrochloride) 6 was purchased from Rare Chemicals GmbH (Kiel, Germany), 4-OH-3,5-diMeO-phenethylamine (hydrochloride) 9 was purchased from Ambinter (Orleans, France), and tyramine 4 was purchased from Holland Moran (Yehud, Israel). N-p-trans- Coumaroyltyramine, A-trans-caffeoyltyramine, A-trans-feruloyltyramine and A-Zrans-feruloyl- 3 -methoxy tyramine were purchased from BioBioPha (Yunnan, China).LC-MS chemical analysis

[0113] Unless otherwise stated, 100 mg frozen powdered plant tissue were extracted with 300 pl 80% methanol and 0.1% formic acid, sonicated for 15 min, agitated for 30 min, and centrifuged at 14,000 g for 10 min. The supernatant was filtered through a 0.22 pm syringe filter. Samples were analyzed using an ultrahigh-performance liquid chromatography (UPLC, Waters Acquity) with a diode array detector connected either to a quadrupole time-of-flight (XEVO G2-S or Synapt HDMS qTOF systems, Waters), an Orbitrap IQ-X Tribrid MS, or an Obitrap Exploris 240 MS (Thermo Scientific, Bremen, Germany). The chromatographic separation was performed on a 100 mm x 2.1 mm i.d. (internal diameter), 1.7 pm UPLC BEH-T3 C18 column (Waters Acquity). The mobile phase consisted of 0.1% formic acid in acetonitrile:water (1:99, v / v; phase A) and 0.1% formic acid in acetonitrile (phase B). The multistep method was as follows: Initial conditions were 0% B for 4 min, raised to 75% B until 12 min, raised to 100% B in 0.2 min, held at 100% B until 15 min, decreased to 0% B in 0.2 min, and held at 0% B until 18 min for re- equilibration of the system. The flow rate was 0.3 ml min-1, and the column temperature was kept at 35 °C.

[0114] Electrospray ionization (ESI) was used in positive ionization mode at an m / z range of 50- 1,000 Da. With the qTOF system masses were detected with the following settings: capillary 1 kV, source temperature 140 °C, desolvation temperature 450 °C, and desolvation gas flow 800 1 h-1. MS / MS experiments were performed according to the specific protonated masses with following settings: capillary spray of 1 kV; cone voltage of 30 eV; the collision energy ramp was 10-45 eV. Argon was used as the collision gas. On the Orbitrap MS systems the source parameters were: sheath gas flow rate, auxiliary gas flow rate and sweep gas flow rate: 45, 10 and 1 arbitrary units, respectively; vaporizer temperature: 275 °C; ion transfer tube temperature: 275 °C; spray voltage: 2.3 kV. The instrument was operated in full MS1with data dependentMS / MS (MS-dd-MS2). Data acquisition in full MS1mode was 60,000 resolution, normalized automatic gain control target of 25% and a maximum injection time of 50 ms. Data acquisition in dd-MS2mode was with 15,000 resolution, a normalized AGC target of 20%, maximum IT of 150 ms, isolation window of 1.5 m / z and normalized collision energy of 40. Identification of metabolites 1-7 and 9 was performed according to analytical standards. All the other metabolites were putatively identified according to LC-MS / MS.Heterologous expression in yeast

[0115] CYP76AD131, TyDC2, CYP84A22O, 0MT1 and 0MT11 coding sequences were individually cloned into pESC plasmids allowing simultaneous expression of up to two genes in one plasmid. LwCYP76AD131 was cloned into pESC-Trp; LwCYP84A22O and LwOMTH were cloned into pESC-Leu; and LwTyDC2 and LwOMTl were cloned into pESC-His. The cloning was performed by in-fusion and the pESC vectors were linearized by PCR. The pESC constructs were sequenced and transformed into S. cerevisiae WAT21 using the Yeastmaker yeast transformation system (Clontech) as follows: (a) LwTyDC2-pESC-His and LwCYP76AD131- pESC-Trp; (b) LwCYP76AD131-pESC-Trp; (c) LwCYP84A220-pESC-Leu; and (d) LwTyDC2- LwOMTl -pESC-His, LwCYP76AD131 -pESC-Trp and LwOMTl -pESC-Leu. Each strain was additionally transformed with the appropriate EVs as control. Transformed yeast were grown on SD minimal media supplemented with appropriate amino acids and 2% glucose. Colonies were screened and the presence of the transgene was confirmed by colony PCR. For induction of gene expression, transformed cells were grown in 5 mL SD minimal medium with 2% glucose and after 24 h transferred to an SD minimal medium with 2% galactose without additional supplementation or containing 1 mM of either tyramine 4, dopamine 3, 3-MeO-tyramine 7, 3,4- diOH-5-MeO-phenethylamine 6 or 4-OEL3, 5-diMeO-phenethylamine 9, and grown for additional 48 h at 30 °C.

[0116] Cultures were centrifuged at 8,000 g for 1 min and the pellet and supernatant were collected separately. The supernatant was lyophilized, 1 mL of methanol with 0.1% formic acid was added, sonicated for 30 min, centrifuged at 4,300 r.p.m for 10 min and then the clear supernatant was collected and dried under a stream of N2. Dry residues were dissolved in 200 pL of methanol, sonicated for 20 min, centrifuged at 15,000 g for 10 min and filtered through a 0.22 pm filter for LC-MS analysis. The cell pellet was weighed, double the amount of glass beads (diameter 500 pm) and 500 pL of methanol with 0.1% formic acid was added and vortexed at maximum speed for 10 min. Lysed cells were centrifuged at 21,000 g for 5 min and clear supernatant was collected and dried under a stream of N2. Dry residues were dissolved in 100 pLof methanol, sonicated for 20 min, centrifuged at 15,000 g for 10 min and filtered through a 0.22 pm filter for LC-MS analysis. Samples were analyzed using the acquired dd-MS2data according to the following transitions: L-tyrosine 2: 182.081 ->165.055; dopamine 3: 154.086^137.056; tyramine 4: 138.091^121.065; 3,4-diOH-5-MeO-phenethylamine 6: 184.097^ 167.070; 3-MeO-tyramine 7: 168.102^151.075; 4-OH-3,5-diMeO-phenethylamine 9 and 3-OH-4,5-diMeO-phenethylamine 10: 198.113 181.086; mescaline 1: 212.128^195.101; V-acetyl-tyramine 32: 180.102— > 121.065; V-acetyl -dopamine 33: 196.097^137.056; V-acetyl- 3-MeO-tyramine 34: 210.113 ->151.075; 7V-acetyl-3,4-diOH-5-MeO-phenethylamine 35: 226.107-> 167.070; 7V-acetyl-4-OH-3,5-diMeO-phenethylamine 36 and 7V-acetyl-3-OH-4,5- diMeO-phenethylamine 37: 240.123~> 181 .086; V-acetyl-mescaline 1: 254.139^195.101.

[0117] Peyote enzymes are disclosed in Berman et al., “Discovery of the Mescaline Hallucinogen Biosynthetic Pathway in the Entheogenic Peyote (Lophophora williamsii) Cactus”; Molecular Plant, 2024.EXAMPLE 1Acetylation of phenethylamine intermediates reduces heterologous mescaline production yield in recombinant yeast

[0118] The inventors co-expressed LwTyDC2 and LwCYP76AD131 in S. cerevisiae yeast and noted the production of tyramine 4 and a minor peak of dopamine 3 (Fig. 2A), accompanied by their putative A- acetylated forms (Figs. 2B and 3). The inventors did not observe peaks of L- DOPA 5 in these samples or following the expression of only LwCYP76AD131 (Fig. 2C). Incubation of empty vector (EV) transformed yeast with either tyramine 4 or dopamine 3 resulted in the appearance of peaks with similar mass and retention time as A-acetyl-tyramine 32 and N- acetyl-dopamine 33 (Fig. 3). Identification of A- acetylated metabolites was further confirmed using analytical standards (Fig. 2A). The detection of dopamine 3 and A- acetyl -dopamine 33 confirms the tyramine 4 hydroxylating activity of LwCYP76AD131 and suggests competition between the expressed Peyote enzymes and an endogenous A-acetylating enzyme in yeast for the newly formed phenethylamines.

[0119] The inventors attempted to reconstruct the mescaline 1 pathway by co-expressing LwTyDC2, LwCYP76AD131, LwOMTl and LwOMTl l enzymes in yeast. Following two days of galactose induction, the inventors observed peaks of tyramine 4 and dopamine 3, in addition to their respective A- acetylated forms both in the pellet and the supernatant of the transformed yeast (Fig. 4A). However, no other intermediates from the pathway were detected. Following thedetection of iV-acetylated metabolites the inventors incubated EV transformed yeast with individual intermediates from the pathway to account for potential A-acctylation by a yeast N- acetyltransferase. Following two days of protein induction the inventors could detect all N- acetylated intermediates from the pathway (metabolites 32-36, Figs. 4B and 6). Consistent with these findings, an arylalkyl amine A-accty I transferase from S. cerevisiae (scAANAT, GenBank accession number S49826) has been reported to be functional on several phenethylamines, including mescaline 1, dopamine 3, tyramine 4 and 3-MeO-tyramine 7, suggesting it may be responsible for the observed activity.

[0120] To confirm the in vivo activity of the enzymes, the inventors further supplemented yeast co-expressing the four enzymes from Peyote with each pathway intermediate individually (Fig. 5A). Subsequently, depending on the supplemented substrate, the inventors detected the phenethyl products tyramine 4, dopamine 3, 4-OH-3,5-diMeO-phenethylamine 9, 3-OH-4,5- diMeO-phenethylamine 10, and mescaline 1 (Figs. 4C and 5A). Additionally, the inventors observed the appearance of A-acctylatcd products, including A- acetyl tyramine 32, A-acctyl- dopamine 33, A-acetyl-3-MeO-tyramine 34, A-acetyl-4-OH-3,5-diMeO-phenethylamine 36, and two newly observed metabolites putatively identified as A-acctyl-3-OH-4,5-diMcO- phenethylamine 37 and A-acctyl- mescaline 38 (Figs. 4D-4E, 5A-5B, and 6). Following incubation with 3-MeO-tyramine 7, only a low abundance peak of A-acctyl-3,4-diOH-5-McO- phenethylamine 35 was detected (this could not be verified via MS / MS; Fig. 5B). Overall, the production of the phenethylamine intermediates and their A-acctylatcd forms by yeast co- expressing the Peyote enzymes confirms the enzymes’ activity. However, the presence of N- acetylation competition possibly by scAANAT and potentially low catalytic activity of specific enzymes prevented the inventors from achieving in vivo synthesis of mescaline 1. This highlights the complexity in future engineering efforts targeting the mescaline 1 pathway in yeast, suggesting that knocking down scAANAT may be necessary to enable its production.

[0121] In view of the foregoing, it is concluded that in order to increase biosynthetic production yield of a compound, such as a psychedelic or a hallucinating compound having a phenethylamine precursor, in a yeast cell, the activity of acetyl transferring enzyme(s), inclusive of AANAT is to be reduced or inhibited.

[0122] Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications, and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims.

Claims

CLAIMSWhat is claimed is:

1. A transformed or transgenic yeast cell comprising at least a partially inactive acetyltransferase encoding gene.

2. The transformed or transgenic yeast cell of claim 1, wherein said acetyltransferase encoding gene is fully inactive.

3. The transformed or transgenic yeast cell of claim 1 or 2, wherein said acetyltransferase encoding gene is constitutively inactive or conditionally inactive.

4. The transformed or transgenic yeast cell of any one of claims 1 to 3, wherein said acetyltransferase is an arylalkyl amine A-accty I transferase (AANAT).

5. The transformed or transgenic yeast cell of any one of claims 1 to 4, being a Saccharomyces cerevisiae cell.

6. The transformed or transgenic yeast cell of any one of claims 1 to 5, further comprising at least one artificial DNA molecule comprising a nucleic acid sequence encoding at least one enzyme having an arylalkyl amine substrate, synthesizing an arylalkyl amine product, or both.

7. The transformed or transgenic yeast cell of claim 6, wherein said arylalkyl amine is phenethylamine .

8. The transformed or transgenic yeast cell of claim 6 or 7, wherein said arylalkyl amine is a psychoactive compound.

9. The transformed or transgenic yeast cell of claim 8, wherein said psychoactive compound is a psychedelic drug.

10. The transformed or transgenic yeast cell of any one of claims 1 to 9, wherein said inactive gene is knocked down.

11. The transformed or transgenic yeast cell of claim 10, wherein said knocked down is by any one of: antisense RNA, short interfering RNA (siRNA), short hairpin RNA (shRNA), double stranded RNA (dsRNA), and any combination thereof.

12. The transformed or transgenic yeast cell of claim 11, wherein any one of said antisense RNA, siRNA, shRNA, dsRNA, and any combination thereof, is complementary to a transcript of said acetyltransferase encoding gene.

13. The transformed or transgenic yeast cell of claim 11 or 12, wherein any one of said antisense RNA, siRNA, shRNA, dsRNA is transcribed from a polynucleotide sequence being integrated in a genome of said transformed or transgenic yeast cell, integrated in an artificial vector introduced into said transformed or transgenic yeast cell, or both.

14. The transformed or transgenic yeast cell of any one of claims 11 to 13, wherein said nucleic acid sequence encoding an enzyme having a substrate, synthesizing a product, or both, being an arylalkyl amine, and any one of said antisense RNA, siRNA, shRNA, dsRNA, and any combination thereof, are operably linked.

15. An extract derived from the transformed or transgenic cell of any one of claims 1 to 14, or any fraction thereof.

16. The extract of claim 15, comprising a psychoactive compound.

17. A composition comprising: a. the transformed or transgenic cell of any one of claims 1 to 14; b. the extract of claim 15 or 16; or c. a combination of (a) and (b), and an acceptable carrier.

18. A method for increasing production levels of at least one arylalkyl amine in a yeast cell heterologously expressing at least one enzyme having an arylalkyl amine substrate, synthesizing an arylalkyl amine product, or both, the method comprising contacting said yeast cell with an effective amount of an acetyltransferase inhibitor, thereby increasing the production levels of the at least one arylalkyl amine in the yeast cell.

19. The method of claim 18, wherein said inhibitor inhibits: transcription of a gene encoding said acetyltransferase, translation of a transcript of said gene encoding said acetyltransferase,acetylation activity of a protein product of said gene encoding said acetyltransferase, or any combination thereof.

20. The method of claim 18 or 19, wherein said inhibitor induces or promotes degradation of a transcript of said gene encoding said acetyltransferase.

21. The method of any one of claims 18 to 20, wherein said inhibitor is an RNA interfering (RNAi) oligonucleotide or a polynucleotide.

22. The method of claim 21, wherein said RNAi oligonucleotide or polynucleotide is selected from the group consisting of: antisense RNA, short interfering RNA (siRNA), short hairpin RNA (shRNA), double stranded RNA (dsRNA), and any combination thereof.

23. The method of any one of claims 18 to 22, wherein said acetyltransferase is AANAT.

24. The method of any one of claims 18 to 23, wherein said yeast cell is a transformed or transgenic yeast cell.

25. The method of any one of claims 18 to 24, wherein said yeast cell is S. cerevisiae cell.

26. The method of any one of claims 18 to 25, further comprising a step before said contacting comprising introducing to said yeast cell an artificial DNA molecule encoding said at least one enzyme having an arylalkyl amine substrate, synthesizing an arylalkyl amine product, or both.

27. The method of any one of claims 18 to 26, further comprising a step comprising extracting said yeast cell so as to obtain said at least one arylalkyl amine.

28. The method of any one of claims 18 to 27, wherein said at least one arylalkyl amine is a psychoactive compound.

29. The method of claim 28, wherein said psychoactive compound is a psychedelic compound.

30. A composition comprising at least one arylalkyl amine synthesized according to the method of any one of claims 18 to 29, and a pharmaceutically acceptable carrier.

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