Method for synthesizing dopamine

Abstract

The present invention provides a method for synthesizing dopamine or a precursor thereof.

Description

METHOD FOR SYNTHESIZING DOPAMINEREFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0001] The contents of the electronic sequence listing (YEDA-P-041-PCT.xml; size: 72,170 bytes; and date of creation: December 3, 2024) 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 / 608,317, titled “METHOD FOR SYNTHESIZING DOPAMINE”, filed 11 December 2023, the contents of which are incorporated herein by reference in their entirety.FIELD OF INVENTION

[0003] The present invention relates to, inter alia, the use of enzymes including polynucleotides encoding the same, in a method for synthesizing dopamine.BACKGROUND

[0004] In recent years, there has been a resurgence of interest in psychedelics due to their reported efficacy in producing long-lasting symptom reduction in phase II clinical trials, offering potential transformative effects on neuroscience and psychiatry as currently understood. The historical use of psychedelic cacti can be traced back nearly 6,000 years. Mescaline, the primary hallucinogenic compound found in these cacti, was first isolated and identified by Arthur Heffter in 1897. This 5-HT2A serotonin receptor agonist exerts 1,000- 3,000 times less potent effects than LSD and 30 times less potent than psilocybin due to much lower binding affinity. Mescaline is classified as a Schedule I controlled substance and is prohibited in most countries. Nevertheless, Lophophora williamsii (L. williamsii, Peyote) which serves as the primary natural reservoir of mescaline, has obtained legal sanction for ceremonial utilization by the Native American Church as a medium for spiritual connection with the Great Spirit (God).

[0005] The biosynthetic pathway of mescaline in Peyote has been postulated for over fifty years, primarily based on radioisotope labeling studies and extensive metabolic profiling. Despite this, the specific enzymes responsible for each step have yet to be elucidated.Additionally, this prediction suggests that some metabolites may be biosynthesized through multiple routes.

[0006] Further, to the best of our knowledge there has yet been identified a hydroxylating enzyme in Peyote which accepts tyramine as a substrate for the production of dopamine.SUMMARY

[0007] The present invention, in some embodiments, is based, at least in part, on the identification of a group of hydroxylating enzymes isolated from cacti, which shown to synthesize dopamine from a precursor thereof in Nicotiana benthamiana (N. benthamiana) plants.

[0008] According to the first aspect, there is provided a method for synthesizing dopamine comprising the steps: (a) providing a cell comprising a first isolated DNA molecule comprising a nucleic acid sequence having at least 80% homology to SEQ ID NO: 12; and (b) culturing the cell from step (a) such that a polypeptide encoded by the first isolated DNA molecule is expressed, thereby synthesizing dopamine.

[0009] According to another aspect, there is provided a method for synthesizing dopamine comprising contacting tyramine with an effective amount of an isolated polypeptide comprising an amino acid sequence having at least 80% homology or identity to SEQ ID NO: 28, thereby synthesizing dopamine.

[0010] According to another aspect, there is provided an extract obtained according to the method of the invention.

[0011] According to another aspect, there is provided a dopamine synthesized according to the method of the invention.

[0012] According to another aspect, there is provided a composition comprising the dopamine of the invention, and an acceptable carrier.

[0013] According to another aspect, there is provided a method for synthesizing dopamine comprising the steps: (a) providing a cell comprising an isolated DNA molecule comprising a nucleic acid sequence having at least 80% homology to SEQ ID NO: 33; and (b) culturing the cell from step (a) such that a polypeptide encoded by the isolated DNA molecule is expressed, thereby synthesizing dopamine.

[0014] In some embodiments, the polypeptide is characterized by having an activity comprising introducing a hydroxyl group into a substrate.

[0015] In some embodiments, the culturing comprises supplementing the cell with an effective amount of tyramine.

[0016] In some embodiments, the cell is a unicellular organism, a cell of a multicellular organism, and a cell in a culture.

[0017] In some embodiments, the unicellular organism comprises a fungus or a bacterium.

[0018] In some embodiments, the fungus is a yeast cell.

[0019] In some embodiments, the cell further comprises a second isolated DNA molecule comprising a nucleic acid sequence having at least 80% homology to SEQ ID NO: 33.

[0020] In some embodiments, any one of the first isolated DNA molecule, the second isolated DNA molecule, or both, is incorporated in a plasmid or an agrobacterium.

[0021] In some embodiments, the cell is a transgenic cell or a cell transfected with the first isolated DNA molecule or the plasmid or agrobacterium.

[0022] In some embodiments, the method further comprises supplementing the cell with an effective amount of L-tyrosine, L-DOPA, or both.

[0023] In some embodiments, the method further comprises a step comprising extracting the cell, thereby obtaining an extract from the cell.

[0024] In some embodiments, the extract of the invention comprises dopamine.

[0025] In some embodiments, the acceptable carrier is a pharmaceutically acceptable carrier.

[0026] In some embodiments, the composition is a pharmaceutical composition.

[0027] In some embodiments, the polypeptide is characterized by having an activity comprising releasing a carboxyl group from a substrate.

[0028] In some embodiments, the culturing comprises supplementing the cell with an effective amount of L-DOPA.

[0029] 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.

[0030] 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

[0031] Figs. 1A-1D include graphs and a synthesis scheme showing that LwCYP12 and LwTyDC2 catalyze the production of dopamine. (1A) Ion abundance peak areas (mean ± s.d.; n = 4 biological replicates) of dopamine following transient expression of two CYP candidates in N. benthamiana leaves. CYP11 and CYP12 (LwCYP12) exhibit high similarities (51% and 62%, respectively) to BvCYP76AD6, a betalain-related enzyme previously identified in beetroot, which has been shown to catalyze the hydroxylation of L- tyrosine into L-DOPA. LwCYP12 was found to catalyze the hydroxylation of tyramine to dopamine. Metabolites were identified by exact mass, retention time and MS / MS spectra. EV, empty vector. (IB) Ion abundance peak areas (mean ± s.d.; n = 3-4 biological replicates) of dopamine following in vitro assay using a lysate from E. coli cells transformed with TyDC candidates, utilizing L-DOPA as substrates in the presence of pyridoxal 5-phosphate. (1C) A scheme showing the reaction catalyzed by LwCYP12, and LwTyDC2 for the production of dopamine from tyramine, and L-DOPA, respectively. (ID) Ion abundance peak areas (mean ± s.d.; n = 4 biological replicates) display the production of dopamine following the stepwise transient expression of LwTyDC2 and LwCYP12 in N. benthamiana leaves. EV, empty vector.DETAILED DESCRIPTIONMethods of synthesis

[0032] According to the first aspect, there is provided a method for synthesizing dopamine.

[0033] In some embodiments, the method comprises providing a cell comprising: (i) a first isolated DNA molecule comprising a nucleic acid sequence having at least 75%, 80%, 90%, 95%, or 99% homology or identity to a nucleic acid sequence as set forth in SEQ ID Nos: 1- 16, or any combination thereof; (ii) a plasmid or an agrobacterium comprising a nucleic acid sequence set forth in SEQ ID Nos: 1-16, or any combination thereof; or (iii) both (i) and (ii). In some embodiments, the method comprises culturing the cell such that at least onepolypeptide encoded by the first isolated DNA molecule, the plasmid or agrobacterium, or both, is expressed.

[0034] In some embodiments, the first isolated DNA molecule, the plasmid, the agrobacterium, or any combination thereof, comprises SEQ ID Nos: 2, 3, 12, or any combination thereof.

[0035] In some embodiments, the first isolated DNA molecule, the plasmid, the agrobacterium, or any combination thereof, comprises SEQ ID Nos: 2, 3, and 12.

[0036] In some embodiments, the first isolated DNA molecule, the plasmid, the agrobacterium, or any combination thereof, comprises SEQ ID NO: 12.

[0037] In some embodiments, the at least one polypeptide is characterized by having an activity of introducing a hydroxyl group into a substrate.

[0038] In some embodiments, the substrate comprises a substrate molecule. In some embodiments, the substrate comprises an organic compound or molecule.

[0039] In some embodiments, culturing comprises supplementing the cell with an effective amount of a dopamine precursor.

[0040] In some embodiments, a dopamine precursor comprises any compound known to a person of skill in the art to be involved and / or being a member of the dopamine biosynthetic pathway.

[0041] In some embodiments, a dopamine precursor is or comprises tyramine.

[0042] In some embodiments, the method further comprises producing tyramine.

[0043] In some embodiments, there is provided a method for producing tyramine.

[0044] In some embodiments, the method (such as for producing tyramine) comprises providing a cell comprising: (i) an isolated DNA molecule comprising a nucleic acid sequence having at least 75%, 80%, 90%, 95%, or 99% homology or identity to a nucleic acid sequence as set forth in SEQ ID Nos: 33-35, or any combination thereof; (ii) a plasmid or an agrobacterium comprising a nucleic acid sequence set forth in SEQ ID Nos: 33-35, or any combination thereof; or (iii) both (i) and (ii). In some embodiments, the method comprises culturing the cell such that at least one polypeptide encoded by the isolated DNA molecule, the plasmid or agrobacterium, or both, is expressed.

[0045] In some embodiments, the method further comprises supplementing the cell with an effective amount of L-tyrosine, L-DOPA, or both.[046 J In some embodiments, an effective amount refers to supplementing the cell with a dopamine precursor, such as selected from: L-tyrosine, L-DOPA, tyramine, or any combination thereof, in a concentration ranging between: 0.1-100 mM, 0.5-50 mM, 1-40 mM, 0.3-30 mM, 0.7-20 mM, 1-25 mM, 0.6-10 mM, 1-5 mM, or 1-3 mM. Each possibility represents a separate embodiment of the invention.

[0047] In some embodiments, the cell comprises or is a transgenic cell or a cell transfected with the first isolated DNA molecule of the invention or the plasmid or agrobacterium of the invention.

[0048] In some embodiments, the cell further comprises: (i) a second isolated DNA molecule comprising a nucleic acid sequence having at least 75%, 80%, 90%, 95%, or 99% homology or identity to a nucleic acid sequence as set forth in SEQ ID Nos: 33-35, or any combination thereof; (ii) a plasmid or an agrobacterium comprising a nucleic acid sequence set forth in SEQ ID Nos: 33-35, or any combination thereof; or (iii) both (i) and (ii)80% homology to SEQ ID NO: 33.

[0049] In some embodiments, the second isolated DNA molecule, the plasmid, the agrobacterium, or any combination thereof, comprises SEQ ID NO: 33.

[0050] In some embodiments, the at least one polypeptide is characterized by having an activity of releasing a carboxyl group from a substrate.

[0051] In some embodiments, the method further comprises a step before step (a), comprising introducing or transfecting the cell with the plasmid or agrobacterium.

[0052] 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.

[0053] In some embodiments, introducing or transfecting comprises transferring an artificial nucleic acid molecule or vector comprising the polynucleotide disclosed herein into a cell; or modifying the genome of a cell to include the polynucleotide disclosed herein. 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.

[0054] In some embodiments, artificial nucleic acid molecule or vector comprises: a plasmid, an agrobacterium, or both.

[0055] As used herein, the terms “transfecting” and “introducing” are interchangeable.

[0056] According to another aspect, there is provided a method comprising contacting a dopamine precursor with an effective amount of at least one isolated polypeptide comprising an amino acid sequence selected from SEQ ID Nos: 17-32, or any combination thereof. In some embodiments, the method is for producing dopamine.

[0057] According to another aspect, there is provided a method comprising contacting a dopamine precursor with an effective amount of at least one isolated polypeptide comprising an amino acid sequence set forth in SEQ ID NO: 36. In some embodiments, the method is for producing tyramine, dopamine, or both.

[0058] In some embodiments, the contacting is with isolated proteins. In some embodiments, each isolated protein of the different isolated proteins comprises a different amino acid. In some embodiments, the different isolated protein comprises an amino acid sequence having at least 75%, 80%, 90%, 95%, or 99% homology or identity to the amino acid sequences set forth in SEQ ID Nos: 18, 19, 28, or any combination thereof. Each possibility represents a separate embodiment of the invention.

[0059] In some embodiments, the different isolated protein comprises an amino acid sequence having at least 75%, 80%, 90%, 95%, or 99% homology or identity to the amino acid sequences set forth in SEQ ID Nos: 18, 19, and 28. Each possibility represents a separate embodiment of the invention.

[0060] In some embodiments, the contacting is with an isolated protein comprising an amino acid sequence having at least 75%, 80%, 90%, 95%, or 99% homology or identity to the amino acid sequences set forth in SEQ ID NO: 36. Each possibility represents a separate embodiment of the invention.

[0061] In some embodiments, the method comprises providing a cell comprising: (i) an isolated DNA molecule comprising a nucleic acid sequence having at least 80% homology to SEQ ID NO: 33; (ii) a plasmid or agrobacterium comprising the isolated DNA molecule of (i); or both (i) and (ii). In some embodiments, the method comprises culturing the cell such that a polypeptide encoded by any one of the isolated DNA molecule, the plasmid or agrobacterium, and both, is expressed.

[0062] In some embodiments, the polypeptide is characterized by having an activity comprising releasing a carboxyl group from a substrate.

[0063] In some embodiments, the culturing comprises supplementing the cell with an effective amount of L-DOPA.

[0064] In some embodiments, contacting is in a cell-free system.

[0065] According to another aspect, there is provided a dopamine synthesized according to the method of the invention.

[0066] According to some embodiments, there is provided a method for obtaining an extract from a transgenic cell or a transfected cell.

[0067] In some embodiments, the method comprises culturing a transgenic cell or a transfected cell in a medium and extracting the transgenic cell or the transfected cell.

[0068] In some embodiments, the method comprises the steps: (a) culturing a transgenic cell or a transfected cell in a medium; and (b) extracting the transgenic cell or the transfected cell, thereby obtaining an extract from the transgenic cell or the transfected cell.

[0069] In some embodiments, the transgenic cell or the transfected cell is the transgenic cell disclosed herein.

[0070] In some embodiments, the transgenic cell or the transfected cell comprises an isolated DNA molecule comprising a nucleic acid sequence set forth in SEQ ID Nos: 1-16, or any combination thereof. In some embodiments, the transgenic cell or the transfected cell comprises an isolated DNA molecule comprising a nucleic acid sequence set forth in SEQ ID Nos: 2, 3, 12, or any combination thereof.

[0071] In some embodiments, the transgenic cell or the transfected cell comprises the polynucleotide of the invention or a plurality thereof, as disclosed herein.

[0072] In some embodiments, the transgenic cell or the transfected cell comprises the plasmid or agrobacterium as disclosed herein.

[0073] In some embodiments, the cell is a transgenic cell, or a cell transfected with a polynucleotide as disclosed herein.

[0074] In some embodiments, the method further comprises a step proceeding or after step (b), comprising separating the cultured transgenic cell or the cultured transfected cell from the medium.

[0075] Method for separating cell from a medium are common and may include, but not limited to, centrifugation, ultracentrifugation, or other, as would be apparent to one of ordinary skill in the art.

[0076] According to some embodiments, there is provided an extract of a transgenic cell or a transfected cell obtained according to the herein disclosed method.

[0077] According to some embodiments, there is provided a medium or a portion thereof separated from a cultured transgenic cell or a cultured transfected cell, obtained according to the herein disclosed method.

[0078] According to some embodiments, there is provided a composition comprising: (a) the extract disclosed herein; (b) the medium disclosed herein or a portion thereof; or (c) any combination of (a) and (b), and an acceptable carrier, as described herein.

[0079] In some embodiments, a portion comprises a fraction or a plurality thereof.Polynucleotides, proteins, cells, and compositions

[0080] The present invention, in some embodiments, is directed to polynucleotides including isolated protein encodes by the same, that are involved in production / or synthesis of dopamine, and methods of using same in the production or synthesis of dopamine.

[0081] In some embodiments, the polynucleotide is an isolated polynucleotide. In some embodiments, the polynucleotide is a DNA molecule. In some embodiments, the polynucleotide is an isolated DNA molecule. In some embodiments, the DNA molecule is an isolated DNA molecule. In some embodiments, the DNA molecule is a complementary DNA (cDNA) molecule.

[0082] As used herein, the terms "isolated polynucleotide" and "isolated DNA molecule" refers to a nucleic acid molecule that is essentially free from contaminating cellular components, such as carbohydrate, lipid, or other proteinaceous impurities associated with the nucleic acid in nature. Typically, a preparation of isolated DNA or RNA contains the nucleic acid in a highly purified form, e.g., at least about 80% pure, at least about 90% pure, at least about 95% pure, greater than 95% pure, or greater than 99% pure. In some embodiments, the isolated polynucleotide is any one of DNA, RNA, and cDNA. In some embodiments, the isolated polynucleotide is a synthesized polynucleotide. Synthesis of polynucleotides is well known in the art and may be performed, for example, by ligating or covalently linking multiple nucleic acid molecules together by primer linkers.

[0083] In some embodiments, the isolated DNA molecule is or comprises the first isolated DNA molecule, the second isolated DNA molecule, or both.

[0084] In some embodiments, the hydroxylating enzyme is encoded by the first isolated DNA molecule.[085 J In some embodiments, the decarboxylating enzyme is encoded by the second isolated DNA molecule.

[0086] 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. The nitrogenous 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).

[0087] 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.

[0088] In some embodiments, the polynucleotide comprises or consists of the nucleic acid sequence set forth in SEQ ID NO: 1.

[0089] In some embodiments, the polynucleotide comprises a nucleic acid sequence with at least 83%, at least 85%, at least 87%, at least 95%, or at least 99% homology or identity to SEQ ID NO: 1, or any value and range therebetween. Each possibility represents a separate embodiment of the invention. In some embodiments, the polynucleotide comprises a nucleic acid sequence with 83% to 100%, 88% to 100%, 90% to 100%, or 95% to 100% homology or identity to SEQ ID NO: 1. Each possibility represents a separate embodiment of the invention. In some embodiments, CYP1, as disclosed herein, comprises or consists of the nucleic acid sequence set forth in SEQ ID NO: 1.

[0090] In some embodiments, the polynucleotide comprises or consists of the nucleic acid sequence set forth in SEQ ID NO: 2.

[0091] In some embodiments, the polynucleotide comprises a nucleic acid sequence with at least 83%, at least 85%, at least 87%, at least 95%, or at least 99% homology or identity to SEQ ID NO: 2, or any value and range therebetween. Each possibility represents a separate embodiment of the invention. In some embodiments, the polynucleotide comprises a nucleic acid sequence with 83% to 100%, 88% to 100%, 90% to 100%, or 95% to 100% homology or identity to SEQ ID NO: 2. Each possibility represents a separate embodiment of the invention. In some embodiments, LwCYP2, as disclosed herein, comprises or consists of the nucleic acid sequence set forth in SEQ ID NO: 2.

[0092] In some embodiments, the polynucleotide comprises or consists of the nucleic acid sequence set forth in SEQ ID NO: 3.

[0093] In some embodiments, the polynucleotide comprises a nucleic acid sequence with at least 83%, at least 85%, at least 87%, at least 95%, or at least 99% homology or identity to SEQ ID NO: 3, or any value and range therebetween. Each possibility represents a separate embodiment of the invention. In some embodiments, the polynucleotide comprises a nucleic acid sequence with 83% to 100%, 88% to 100%, 90% to 100%, or 95% to 100% homology or identity to SEQ ID NO: 3. Each possibility represents a separate embodiment of the invention. In some embodiments, LwCYP3, as disclosed herein, comprises or consists of the nucleic acid sequence set forth in SEQ ID NO: 3.

[0094] In some embodiments, the polynucleotide comprises or consists of the nucleic acid sequence set forth in SEQ ID NO: 4.

[0095] In some embodiments, the polynucleotide comprises a nucleic acid sequence with at least 83%, at least 85%, at least 87%, at least 95%, or at least 99% homology or identity to SEQ ID NO: 4, or any value and range therebetween. Each possibility represents a separate embodiment of the invention. In some embodiments, the polynucleotide comprises a nucleic acid sequence with 83% to 100%, 88% to 100%, 90% to 100%, or 95% to 100% homology or identity to SEQ ID NO: 4. Each possibility represents a separate embodiment of the invention. In some embodiments, CYP4, as disclosed herein, comprises or consists of the nucleic acid sequence set forth in SEQ ID NO: 4.

[0096] In some embodiments, the polynucleotide comprises or consists of the nucleic acid sequence set forth in SEQ ID NO: 5.

[0097] In some embodiments, the polynucleotide comprises a nucleic acid sequence with at least 83%, at least 85%, at least 87%, at least 95%, or at least 99% homology or identity to SEQ ID NO: 5, or any value and range therebetween. Each possibility represents a separate embodiment of the invention. In some embodiments, the polynucleotide comprises a nucleic acid sequence with 83% to 100%, 88% to 100%, 90% to 100%, or 95% to 100% homology or identity to SEQ ID NO: 5. Each possibility represents a separate embodiment of the invention. In some embodiments, CYP5, as disclosed herein, comprises or consists of the nucleic acid sequence set forth in SEQ ID NO: 5.

[0098] In some embodiments, the polynucleotide comprises or consists of the nucleic acid sequence set forth in SEQ ID NO: 6.

[0099] In some embodiments, the polynucleotide comprises a nucleic acid sequence with at least 83%, at least 85%, at least 87%, at least 95%, or at least 99% homology or identity to SEQ ID NO: 6, or any value and range therebetween. Each possibility represents a separate embodiment of the invention. In some embodiments, the polynucleotide comprises a nucleic acid sequence with 83% to 100%, 88% to 100%, 90% to 100%, or 95% to 100% homology or identity to SEQ ID NO: 6. Each possibility represents a separate embodiment of the invention. In some embodiments, CYP6, as disclosed herein, comprises or consists of the nucleic acid sequence set forth in SEQ ID NO: 6.

[0100] In some embodiments, the polynucleotide comprises or consists of the nucleic acid sequence set forth in SEQ ID NO: 7.

[0101] In some embodiments, the polynucleotide comprises a nucleic acid sequence with at least 83%, at least 85%, at least 87%, at least 95%, or at least 99% homology or identity to SEQ ID NO: 7, or any value and range therebetween. Each possibility represents a separate embodiment of the invention. In some embodiments, the polynucleotide comprises a nucleic acid sequence with 83% to 100%, 88% to 100%, 90% to 100%, or 95% to 100% homology or identity to SEQ ID NO: 7. Each possibility represents a separate embodiment of the invention. In some embodiments, CYP7, as disclosed herein, comprises or consists of the nucleic acid sequence set forth in SEQ ID NO: 7.

[0102] In some embodiments, the polynucleotide comprises or consists of the nucleic acid sequence set forth in SEQ ID NO: 8.

[0103] In some embodiments, the polynucleotide comprises a nucleic acid sequence with at least 83%, at least 85%, at least 87%, at least 95%, or at least 99% homology or identity to SEQ ID NO: 8, or any value and range therebetween. Each possibility represents a separate embodiment of the invention. In some embodiments, the polynucleotide comprises a nucleic acid sequence with 83% to 100%, 88% to 100%, 90% to 100%, or 95% to 100% homology or identity to SEQ ID NO: 8. Each possibility represents a separate embodiment of the invention. In some embodiments, CYP8, as disclosed herein, comprises or consists of the nucleic acid sequence set forth in SEQ ID NO: 8.

[0104] In some embodiments, the polynucleotide comprises or consists of the nucleic acid sequence set forth in SEQ ID NO: 9.

[0105] In some embodiments, the polynucleotide comprises a nucleic acid sequence with at least 83%, at least 85%, at least 87%, at least 95%, or at least 99% homology or identity to SEQ ID NO: 9, or any value and range therebetween. Each possibility represents a separateembodiment of the invention. In some embodiments, the polynucleotide comprises a nucleic acid sequence with 83% to 100%, 88% to 100%, 90% to 100%, or 95% to 100% homology or identity to SEQ ID NO: 9. Each possibility represents a separate embodiment of the invention. In some embodiments, CYP9, as disclosed herein, comprises or consists of the nucleic acid sequence set forth in SEQ ID NO: 9.

[0106] In some embodiments, the polynucleotide comprises or consists of the nucleic acid sequence set forth in SEQ ID NO: 10.

[0107] In some embodiments, the polynucleotide comprises a nucleic acid sequence with at least 83%, at least 85%, at least 87%, at least 95%, or at least 99% homology or identity to SEQ ID NO: 10, or any value and range therebetween. Each possibility represents a separate embodiment of the invention. In some embodiments, the polynucleotide comprises a nucleic acid sequence with 83% to 100%, 88% to 100%, 90% to 100%, or 95% to 100% homology or identity to SEQ ID NO: 10. Each possibility represents a separate embodiment of the invention. In some embodiments, CYP10, as disclosed herein, comprises or consists of the nucleic acid sequence set forth in SEQ ID NO: 10.

[0108] In some embodiments, the polynucleotide comprises or consists of the nucleic acid sequence set forth in SEQ ID NO: 11.

[0109] In some embodiments, the polynucleotide comprises a nucleic acid sequence with at least 83%, at least 85%, at least 87%, at least 95%, or at least 99% homology or identity to SEQ ID NO: 11, or any value and range therebetween. Each possibility represents a separate embodiment of the invention. In some embodiments, the polynucleotide comprises a nucleic acid sequence with 83% to 100%, 88% to 100%, 90% to 100%, or 95% to 100% homology or identity to SEQ ID NO: 11. Each possibility represents a separate embodiment of the invention. In some embodiments, CYP11, as disclosed herein, comprises or consists of the nucleic acid sequence set forth in SEQ ID NO: 11.

[0110] In some embodiments, the polynucleotide comprises or consists of the nucleic acid sequence set forth in SEQ ID NO: 12.

[0111] In some embodiments, the polynucleotide comprises a nucleic acid sequence with at least 83%, at least 85%, at least 87%, at least 95%, or at least 99% homology or identity to SEQ ID NO: 12, or any value and range therebetween. Each possibility represents a separate embodiment of the invention. In some embodiments, the polynucleotide comprises a nucleic acid sequence with 83% to 100%, 88% to 100%, 90% to 100%, or 95% to 100% homology or identity to SEQ ID NO: 12. Each possibility represents a separate embodiment of theinvention. In some embodiments, LwCYP12, as disclosed herein, comprises or consists of the nucleic acid sequence set forth in SEQ ID NO: 12.

[0112] In some embodiments, the polynucleotide comprises or consists of the nucleic acid sequence set forth in SEQ ID NO: 13.

[0113] In some embodiments, the polynucleotide comprises a nucleic acid sequence with at least 83%, at least 85%, at least 87%, at least 95%, or at least 99% homology or identity to SEQ ID NO: 13, or any value and range therebetween. Each possibility represents a separate embodiment of the invention. In some embodiments, the polynucleotide comprises a nucleic acid sequence with 83% to 100%, 88% to 100%, 90% to 100%, or 95% to 100% homology or identity to SEQ ID NO: 13. Each possibility represents a separate embodiment of the invention.

[0114] In some embodiments, the polynucleotide comprises or consists of the nucleic acid sequence set forth in SEQ ID NO: 14.

[0115] In some embodiments, the polynucleotide comprises a nucleic acid sequence with at least 83%, at least 85%, at least 87%, at least 95%, or at least 99% homology or identity to SEQ ID NO: 14, or any value and range therebetween. Each possibility represents a separate embodiment of the invention. In some embodiments, the polynucleotide comprises a nucleic acid sequence with 83% to 100%, 88% to 100%, 90% to 100%, or 95% to 100% homology or identity to SEQ ID NO: 14. Each possibility represents a separate embodiment of the invention.

[0116] In some embodiments, the polynucleotide comprises or consists of the nucleic acid sequence set forth in SEQ ID NO: 15.

[0117] In some embodiments, the polynucleotide comprises a nucleic acid sequence with at least 83%, at least 85%, at least 87%, at least 95%, or at least 99% homology or identity to SEQ ID NO: 15, or any value and range therebetween. Each possibility represents a separate embodiment of the invention. In some embodiments, the polynucleotide comprises a nucleic acid sequence with 83% to 100%, 88% to 100%, 90% to 100%, or 95% to 100% homology or identity to SEQ ID NO: 15. Each possibility represents a separate embodiment of the invention.

[0118] In some embodiments, the polynucleotide comprises or consists of the nucleic acid sequence set forth in SEQ ID NO: 16.

[0119] In some embodiments, the polynucleotide comprises a nucleic acid sequence with at least 83%, at least 85%, at least 87%, at least 95%, or at least 99% homology or identity to SEQ ID NO: 16, or any value and range therebetween. Each possibility represents a separate embodiment of the invention. In some embodiments, the polynucleotide comprises a nucleic acid sequence with 83% to 100%, 88% to 100%, 90% to 100%, or 95% to 100% homology or identity to SEQ ID NO: 16. Each possibility represents a separate embodiment of the invention.

[0120] In some embodiments, the polynucleotide of the invention comprises 1,400 to 1,700 nucleotides. In some embodiments, the polynucleotide of the invention is 1,400 to 1,700 nucleotides long.

[0121] In some embodiments, 1,400 to 1,700 nucleotides comprises: at least 1,400 nucleotides, at least 1,500 nucleotides, at least 1,600 nucleotides, at least 1,650 nucleotides, at least 1,450 nucleotides, at least 1,550 nucleotides, or at least 1,490 nucleotides, or any value and range therebetween. Each possibility represents a separate embodiment of the invention. In some embodiments, 1,400 to 1,700 nucleotides comprises: 1,400 to 1,450 nucleotides, 1,500 to 1,575 nucleotides, 1,400 to 1,650 nucleotides, 1,600 to 1,700 nucleotides, 1,450 to 1,650 nucleotides, 1,420 to 1,590 nucleotides, or 1,410 to 1,690 nucleotides. Each possibility represents a separate embodiment of the invention.

[0122] In some embodiments, the polynucleotide comprises a plurality of polynucleotides. In some embodiments, the polynucleotide comprises a plurality of types of polynucleotides. As used herein, the term “plurality” comprises any integer equal to or greater than 2. In some embodiments, the polynucleotide comprises at least 2, at least 3, at least 5, at least 7, at least 10, or at least 11 different nucleic acid sequences, or any value and range therebetween, wherein each of the different nucleic acid sequences is selected from SEQ ID Nos: 1-16. Each possibility represents a separate embodiment of the invention. In some embodiments, the polynucleotide comprises 1-12, 2-12, 4-12, 6-12, 8-12, 10-12, 2-6, 1-3, or 2-3 different nucleic acid sequences, wherein each of the different nucleic acid sequences is selected from SEQ ID Nos: 1-16.

[0123] In some embodiments, the polynucleotide comprises a plurality of polynucleotide molecules, wherein each of the plurality of the polynucleotide molecules comprises a different nucleic acid sequence, and wherein the different nucleic acid sequences are selected from SEQ ID Nos.: 1-16.

[0124] In some embodiments, the polynucleotide encodes a protein characterized by hydroxylating activity. In some embodiments, the polynucleotide encodes a protein characterized by having an activity of introducing a hydroxyl group to a substrate. In some embodiments, the polynucleotide encodes a protein being a hydroxylase. In some embodiments, the encoded protein belongs to the Cytochrome P450 (CYP) family. In some embodiments, hydroxylase is a hydroxylase derived from a cactus. In some embodiments, the cactus is a peyote. In some embodiments, the polynucleotide encoding a hydroxylating protein is selected from SEQ ID Nos: 1-16, or an analog thereof having at least 70%, 80%, 90%, or 95% homology or identity thereto.

[0125] In some embodiments, the polynucleotide comprises or consists of the nucleic acid sequence set forth in SEQ ID NO: 33.

[0126] In some embodiments, the polynucleotide comprises a nucleic acid sequence with at least 83%, at least 85%, at least 87%, at least 95%, or at least 99% homology or identity to SEQ ID NO: 33, or any value and range therebetween. Each possibility represents a separate embodiment of the invention. In some embodiments, the polynucleotide comprises a nucleic acid sequence with 83% to 100%, 88% to 100%, 90% to 100%, or 95% to 100% homology or identity to SEQ ID NO: 33. Each possibility represents a separate embodiment of the invention. In some embodiments, LwTyDC2, as disclosed herein, comprises or consists of the nucleic acid sequence set forth in SEQ ID NO: 33.

[0127] In some embodiments, the polynucleotide comprises or consists of the nucleic acid sequence set forth in SEQ ID NO: 34.

[0128] In some embodiments, the polynucleotide comprises a nucleic acid sequence with at least 83%, at least 85%, at least 87%, at least 95%, or at least 99% homology or identity to SEQ ID NO: 34, or any value and range therebetween. Each possibility represents a separate embodiment of the invention. In some embodiments, the polynucleotide comprises a nucleic acid sequence with 83% to 100%, 88% to 100%, 90% to 100%, or 95% to 100% homology or identity to SEQ ID NO: 34. Each possibility represents a separate embodiment of the invention. In some embodiments, TyDC4, as disclosed herein, comprises or consists of the nucleic acid sequence set forth in SEQ ID NO: 34.

[0129] In some embodiments, the polynucleotide comprises or consists of the nucleic acid sequence set forth in SEQ ID NO: 35.

[0130] In some embodiments, the polynucleotide comprises a nucleic acid sequence with at least 83%, at least 85%, at least 87%, at least 95%, or at least 99% homology or identity toSEQ ID NO: 35, or any value and range therebetween. Each possibility represents a separate embodiment of the invention. In some embodiments, the polynucleotide comprises a nucleic acid sequence with 83% to 100%, 88% to 100%, 90% to 100%, or 95% to 100% homology or identity to SEQ ID NO: 35. Each possibility represents a separate embodiment of the invention. In some embodiments, TyDC5, as disclosed herein, comprises or consists of the nucleic acid sequence set forth in SEQ ID NO: 35.

[0131] In some embodiments, the polynucleotide of the invention comprises 1,400 to 1,700 nucleotides. In some embodiments, the polynucleotide of the invention is 1,400 to 1,700 nucleotides long.

[0132] In some embodiments, 1,400 to 1,700 nucleotides comprises: at least 1,400 nucleotides, at least 1,500 nucleotides, at least 1,600 nucleotides, at least 1,650 nucleotides, at least 1,450 nucleotides, at least 1,550 nucleotides, or at least 1,490 nucleotides, or any value and range therebetween. Each possibility represents a separate embodiment of the invention. In some embodiments, 1,400 to 1,700 nucleotides comprises: 1,400 to 1,450 nucleotides, 1,500 to 1,575 nucleotides, 1,400 to 1,650 nucleotides, 1,600 to 1,700 nucleotides, 1,450 to 1,650 nucleotides, 1,420 to 1,590 nucleotides, or 1,410 to 1,690 nucleotides. Each possibility represents a separate embodiment of the invention.

[0133] In some embodiments, the polynucleotide comprises a plurality of polynucleotides. In some embodiments, the polynucleotide comprises a plurality of types of polynucleotides. As used herein, the term “plurality” comprises any integer equal to or greater than 2. In some embodiments, the polynucleotide comprises at least 2, or at least 3 different nucleic acid sequences, or any value and range therebetween, wherein each of the different nucleic acid sequences is selected from SEQ ID Nos: 33-35. Each possibility represents a separate embodiment of the invention. In some embodiments, the polynucleotide comprises 1-2, 1-3, or 2-3 different nucleic acid sequences, wherein each of the different nucleic acid sequences is selected from SEQ ID Nos: 33-35.

[0134] In some embodiments, the polynucleotide comprises a plurality of polynucleotide molecules, wherein each of the plurality of the polynucleotide molecules comprises a different nucleic acid sequence, and wherein the different nucleic acid sequences are selected from SEQ ID Nos.: 33-35.

[0135] In some embodiments, the polynucleotide encodes a protein characterized by decarboxylating activity. In some embodiments, the polynucleotide encodes a protein characterized by having an activity of releasing a carboxyl group from a substrate, e.g.,releasing CO2. In some embodiments, the polynucleotide encodes a protein being a decarboxylase. In some embodiments, the decarboxylase is a decarboxylase derived from a cactus. In some embodiments, the cactus is a peyote. In some embodiments, the polynucleotide encoding a decarboxylating protein is selected from SEQ ID Nos: 33-35, or an analog thereof having at least 70%, 80%, 90%, or 95% homology or identity thereto.

[0136] According to some embodiments, there is provided an artificial nucleic acid molecule comprising the polynucleotide disclosed herein.

[0137] In some embodiments, the artificial vector comprises a plasmid. In some embodiments, the artificial vector comprises or is an agrobacterium comprising the artificial nucleic acid molecule. In some embodiments, the artificial vector is an expression vector. In some embodiments, the artificial vector is a plant expression vector. In some embodiments, the artificial vector is for use in expressing a decarboxylase, hydroxylase, methyl transferase, or any combination thereof, encoding nucleic acid sequence as disclosed herein. In some embodiments, the artificial vector is for use in heterologous expression of a decarboxylase, hydroxylase, methyl transferase, or any combination thereof, encoding nucleic acid sequence as disclosed herein in a cell, a tissue, or an organism. In some embodiments, the artificial vector is for use in producing or the production of dopamine in a cell, a tissue, or an organism.

[0138] Expressing a polynucleotide within a cell is well known to one skilled in the art. It can be carried out by, among many methods, transfection, viral infection, or direct alteration of the cell's genome. In some embodiments, the polynucleotide is in an expression vector such as plasmid or viral vector. A vector nucleic acid sequence generally contains at least an origin of replication for propagation in a cell and optionally additional elements, such as a heterologous polynucleotide sequence, expression control element (e.g., a promoter, enhancer), selectable marker (e.g., antibiotic resistance), poly- Adenine sequence.

[0139] The vector may be a DNA plasmid delivered via non-viral methods or via viral methods. The viral vector may be a retroviral vector, a herpesviral vector, an adenoviral vector, an adeno- associated viral vector, a virgaviridae viral vector, or a poxviral vector. The barley stripe mosaic virus (BSMV), the tobacco rattle virus and the cabbage leaf curl geminivirus (CbLCV) may also be used. The promoters may be active in plant cells. The promoters may be a viral promoter.

[0140] In some embodiments, the polynucleotide as disclosed herein is operably linked to a promoter. The term "operably linked" is intended to mean that the nucleotide sequence ofinterest is linked to the regulatory element or elements in a manner that allows for expression of the nucleotide sequence (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 polynucleotide of the invention. In some embodiments, the promoter is a heterologous promoter. In some embodiments, the promoter is the endogenous promoter.

[0141] 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.

[0142] 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.

[0143] 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.

[0144] 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 Vlll, 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.

[0145] 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, metallothionein promoter, murine mammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrin promoter, or other promoters shown effective for expression in eukaryotic cells.

[0146] 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.

[0147] In some embodiments, plant viral vectors are used. In some embodiments, a wildtype 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.

[0148] 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 transienttransfection, 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.

[0149] 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.

[0150] In some embodiments, the artificial vector comprises a polynucleotide encoding a protein comprising an amino acid sequence as described herein.

[0151] In some embodiments, the protein comprises or consists of the amino acid sequence set forth in SEQ ID NO: 17.

[0152] In some embodiments, the protein comprises an amino acid sequence having at least 87%, at least 92%, at least 95%, or at least 99% homology or identity to SEQ ID NO: 17, or any value and range therebetween. Each possibility represents a separate embodiment of the invention. In some embodiments, the protein comprises an amino acid sequence with 87% to 100%, 90% to 100%, or 95% to 100% homology or identity to SEQ ID NO: 17. Each possibility represents a separate embodiment of the invention. In some embodiments, CYP1, as disclosed herein, comprises or consists of the amino acid sequence set forth in SEQ ID NO: 17.

[0153] In some embodiments, the protein comprises or consists of the amino acid sequence set forth in SEQ ID NO: 18.

[0154] In some embodiments, the protein comprises an amino acid sequence having at least 87%, at least 92%, at least 95%, or at least 99% homology or identity to SEQ ID NO: 18, or any value and range therebetween. Each possibility represents a separate embodiment of the invention. In some embodiments, the protein comprises an amino acid sequence with 87% to 100%, 90% to 100%, or 95% to 100% homology or identity to SEQ ID NO: 18. Each possibility represents a separate embodiment of the invention. In some embodiments, LwCYP2, as disclosed herein, comprises or consists of the amino acid sequence set forth in SEQ ID NO: 18.

[0155] In some embodiments, the protein comprises or consists of the amino acid sequence set forth in SEQ ID NO: 19.

[0156] In some embodiments, the protein comprises an amino acid sequence having at least 87%, at least 92%, at least 95%, or at least 99% homology or identity to SEQ ID NO: 19, or any value and range therebetween. Each possibility represents a separate embodiment of the invention. In some embodiments, the protein comprises an amino acid sequence with 87% to 100%, 90% to 100%, or 95% to 100% homology or identity to SEQ ID NO: 19. Each possibility represents a separate embodiment of the invention. In some embodiments, LwCYP3, as disclosed herein, comprises or consists of the amino acid sequence set forth in SEQ ID NO: 19.

[0157] In some embodiments, the protein comprises or consists of the amino acid sequence set forth in SEQ ID NO: 20.

[0158] In some embodiments, the protein comprises an amino acid sequence having at least 87%, at least 92%, at least 95%, or at least 99% homology or identity to SEQ ID NO: 20, or any value and range therebetween. Each possibility represents a separate embodiment of the invention. In some embodiments, the protein comprises an amino acid sequence with 87% to 100%, 90% to 100%, or 95% to 100% homology or identity to SEQ ID NO: 20. Each possibility represents a separate embodiment of the invention. In some embodiments, CYP4, as disclosed herein, comprises or consists of the amino acid sequence set forth in SEQ ID NO: 20.

[0159] In some embodiments, the protein comprises or consists of the amino acid sequence set forth in SEQ ID NO: 21.

[0160] In some embodiments, the protein comprises an amino acid sequence having at least 87%, at least 92%, at least 95%, or at least 99% homology or identity to SEQ ID NO: 21, or any value and range therebetween. Each possibility represents a separate embodiment of the invention. In some embodiments, the protein comprises an amino acid sequence with 87% to 100%, 90% to 100%, or 95% to 100% homology or identity to SEQ ID NO: 21. Each possibility represents a separate embodiment of the invention. In some embodiments, CYP5, as disclosed herein, comprises or consists of the amino acid sequence set forth in SEQ ID NO: 21.

[0161] In some embodiments, the protein comprises or consists of the amino acid sequence set forth in SEQ ID NO: 22.

[0162] In some embodiments, the protein comprises an amino acid sequence having at least 87%, at least 92%, at least 95%, or at least 99% homology or identity to SEQ ID NO: 22, or any value and range therebetween. Each possibility represents a separate embodiment of theinvention. In some embodiments, the protein comprises an amino acid sequence with 87% to 100%, 90% to 100%, or 95% to 100% homology or identity to SEQ ID NO: 22. Each possibility represents a separate embodiment of the invention. In some embodiments, CYP6, as disclosed herein, comprises or consists of the amino acid sequence set forth in SEQ ID NO: 22.

[0163] In some embodiments, the protein comprises or consists of the amino acid sequence set forth in SEQ ID NO: 23.

[0164] In some embodiments, the protein comprises an amino acid sequence having at least 87%, at least 92%, at least 95%, or at least 99% homology or identity to SEQ ID NO: 23, or any value and range therebetween. Each possibility represents a separate embodiment of the invention. In some embodiments, the protein comprises an amino acid sequence with 87% to 100%, 90% to 100%, or 95% to 100% homology or identity to SEQ ID NO: 23. Each possibility represents a separate embodiment of the invention. In some embodiments, CYP7, as disclosed herein, comprises or consists of the amino acid sequence set forth in SEQ ID NO: 23.

[0165] In some embodiments, the protein comprises or consists of the amino acid sequence set forth in SEQ ID NO: 24.

[0166] In some embodiments, the protein comprises an amino acid sequence having at least 87%, at least 92%, at least 95%, or at least 99% homology or identity to SEQ ID NO: 24, or any value and range therebetween. Each possibility represents a separate embodiment of the invention. In some embodiments, the protein comprises an amino acid sequence with 87% to 100%, 90% to 100%, or 95% to 100% homology or identity to SEQ ID NO: 24. Each possibility represents a separate embodiment of the invention. In some embodiments, CYP8, as disclosed herein, comprises or consists of the amino acid sequence set forth in SEQ ID NO: 24.

[0167] In some embodiments, the protein comprises or consists of the amino acid sequence set forth in SEQ ID NO: 25.

[0168] In some embodiments, the protein comprises an amino acid sequence having at least 87%, at least 92%, at least 95%, or at least 99% homology or identity to SEQ ID NO: 25, or any value and range therebetween. Each possibility represents a separate embodiment of the invention. In some embodiments, the protein comprises an amino acid sequence with 87% to 100%, 90% to 100%, or 95% to 100% homology or identity to SEQ ID NO: 25. Each possibility represents a separate embodiment of the invention. In some embodiments, CYP9,as disclosed herein, comprises or consists of the amino acid sequence set forth in SEQ ID NO: 25.

[0169] In some embodiments, the protein comprises or consists of the amino acid sequence set forth in SEQ ID NO: 26.

[0170] In some embodiments, the protein comprises an amino acid sequence having at least 87%, at least 92%, at least 95%, or at least 99% homology or identity to SEQ ID NO: 26, or any value and range therebetween. Each possibility represents a separate embodiment of the invention. In some embodiments, the protein comprises an amino acid sequence with 87% to 100%, 90% to 100%, or 95% to 100% homology or identity to SEQ ID NO: 26. Each possibility represents a separate embodiment of the invention. In some embodiments, CYP10, as disclosed herein, comprises or consists of the amino acid sequence set forth in SEQ ID NO: 26.

[0171] In some embodiments, the protein comprises or consists of the amino acid sequence set forth in SEQ ID NO: 27.

[0172] In some embodiments, the protein comprises an amino acid sequence having at least 87%, at least 92%, at least 95%, or at least 99% homology or identity to SEQ ID NO: 27, or any value and range therebetween. Each possibility represents a separate embodiment of the invention. In some embodiments, the protein comprises an amino acid sequence with 87% to 100%, 90% to 100%, or 95% to 100% homology or identity to SEQ ID NO: 27. Each possibility represents a separate embodiment of the invention. In some embodiments, CYP11, as disclosed herein, comprises or consists of the amino acid sequence set forth in SEQ ID NO: 27.

[0173] In some embodiments, the protein comprises or consists of the amino acid sequence set forth in SEQ ID NO: 28.

[0174] In some embodiments, the protein comprises an amino acid sequence having at least 87%, at least 92%, at least 95%, or at least 99% homology or identity to SEQ ID NO: 28, or any value and range therebetween. Each possibility represents a separate embodiment of the invention. In some embodiments, the protein comprises an amino acid sequence with 87% to 100%, 90% to 100%, or 95% to 100% homology or identity to SEQ ID NO: 28. Each possibility represents a separate embodiment of the invention. In some embodiments, LwCYP12, as disclosed herein, comprises or consists of the amino acid sequence set forth in SEQ ID NO: 28.

[0175] In some embodiments, the protein comprises or consists of the amino acid sequence set forth in SEQ ID NO: 29.

[0176] In some embodiments, the protein comprises an amino acid sequence having at least 87%, at least 92%, at least 95%, or at least 99% homology or identity to SEQ ID NO: 29, or any value and range therebetween. Each possibility represents a separate embodiment of the invention. In some embodiments, the protein comprises an amino acid sequence with 87% to 100%, 90% to 100%, or 95% to 100% homology or identity to SEQ ID NO: 29. Each possibility represents a separate embodiment of the invention.

[0177] In some embodiments, the protein comprises or consists of the amino acid sequence set forth in SEQ ID NO: 30.

[0178] In some embodiments, the protein comprises an amino acid sequence having at least 87%, at least 92%, at least 95%, or at least 99% homology or identity to SEQ ID NO: 30, or any value and range therebetween. Each possibility represents a separate embodiment of the invention. In some embodiments, the protein comprises an amino acid sequence with 87% to 100%, 90% to 100%, or 95% to 100% homology or identity to SEQ ID NO: 30. Each possibility represents a separate embodiment of the invention.

[0179] In some embodiments, the protein comprises or consists of the amino acid sequence set forth in SEQ ID NO: 31.

[0180] In some embodiments, the protein comprises an amino acid sequence having at least 87%, at least 92%, at least 95%, or at least 99% homology or identity to SEQ ID NO: 31, or any value and range therebetween. Each possibility represents a separate embodiment of the invention. In some embodiments, the protein comprises an amino acid sequence with 87% to 100%, 90% to 100%, or 95% to 100% homology or identity to SEQ ID NO: 31. Each possibility represents a separate embodiment of the invention.

[0181] In some embodiments, the protein comprises or consists of the amino acid sequence set forth in SEQ ID NO: 32.

[0182] In some embodiments, the protein comprises an amino acid sequence having at least 87%, at least 92%, at least 95%, or at least 99% homology or identity to SEQ ID NO: 32, or any value and range therebetween. Each possibility represents a separate embodiment of the invention. In some embodiments, the protein comprises an amino acid sequence with 87% to 100%, 90% to 100%, or 95% to 100% homology or identity to SEQ ID NO: 32. Each possibility represents a separate embodiment of the invention.

[0183] In some embodiments, the protein comprising an amino acid sequence selected from SEQ ID Nos: 17-32, or an analog thereof comprising 80-100% homology or identity thereto is characterized by hydroxylating activity, as described herein. In some embodiments, the protein is characterized by having an activity of introducing a hydroxyl group into a substrate.

[0184] In some embodiments, the protein comprises or consists of the amino acid sequence set forth in SEQ ID NO: 36.

[0185] In some embodiments, the protein comprises an amino acid sequence having at least 87%, at least 92%, at least 95%, or at least 99% homology or identity to SEQ ID NO: 36, or any value and range therebetween. Each possibility represents a separate embodiment of the invention. In some embodiments, the protein comprises an amino acid sequence with 87% to 100%, 90% to 100%, or 95% to 100% homology or identity to SEQ ID NO: 36. Each possibility represents a separate embodiment of the invention. In some embodiments, LwTyCD2, as disclosed herein, comprises or consists of the amino acid sequence set forth in SEQ ID NO: 36.

[0186] In some embodiments, the protein comprises or consists of the amino acid sequence set forth in SEQ ID NO: 37.

[0187] In some embodiments, the protein comprises an amino acid sequence having at least 87%, at least 92%, at least 95%, or at least 99% homology or identity to SEQ ID NO: 37, or any value and range therebetween. Each possibility represents a separate embodiment of the invention. In some embodiments, the protein comprises an amino acid sequence with 87% to 100%, 90% to 100%, or 95% to 100% homology or identity to SEQ ID NO: 37. Each possibility represents a separate embodiment of the invention. In some embodiments, TyCD4, as disclosed herein, comprises or consists of the amino acid sequence set forth in SEQ ID NO: 37.

[0188] In some embodiments, the protein comprises or consists of the amino acid sequence set forth in SEQ ID NO: 38.

[0189] In some embodiments, the protein comprises an amino acid sequence having at least 87%, at least 92%, at least 95%, or at least 99% homology or identity to SEQ ID NO: 38, or any value and range therebetween. Each possibility represents a separate embodiment of the invention. In some embodiments, the protein comprises an amino acid sequence with 87% to 100%, 90% to 100%, or 95% to 100% homology or identity to SEQ ID NO: 38. Each possibility represents a separate embodiment of the invention. In some embodiments,TyCD5, as disclosed herein, comprises or consists of the amino acid sequence set forth in SEQ ID NO: 38.

[0190] In some embodiments, the protein comprising an amino acid sequence selected from SEQ ID Nos: 36-38, or an analog thereof comprising 80-100% homology or identity thereto is characterized by decarboxylating activity, as described herein. In some embodiments, the protein is characterized by having an activity of releasing a carboxyl group from a substrate (such as resulting with the release of CO2).

[0191] In some embodiments, the protein is an isolated protein.

[0192] As used herein, the terms "peptide", "polypeptide" and "protein" are interchangeable and refer to a polymer of amino acid residues. In another embodiment, the terms "peptide", "polypeptide" and "protein" as used herein encompass native peptides, peptidomimetics (typically including non-peptide bonds or other synthetic modifications) and the peptide analogues peptoids and semipeptoids or any combination thereof. In another embodiment, the peptides, polypeptides, and proteins described have modifications rendering them more stable while in the organism or more capable of penetrating into cells. In one embodiment, the terms "peptide", "polypeptide" and "protein" apply to naturally occurring amino acid polymers. In another embodiment, the terms "peptide", "polypeptide" and "protein" apply to amino acid polymers in which one or more amino acid residue is an artificial chemical analogue of a corresponding naturally occurring amino acid.

[0193] As used herein, the terms "isolated protein" refers to a protein that is essentially free from contaminating cellular components, such as carbohydrate, lipid, or other proteinaceous impurities associated with the nucleic acid in nature. Typically, a preparation of an isolated protein contains the protein in a highly purified form, e.g., at least about 80% pure, at least about 90% pure, at least about 95% pure, greater than 95% pure, or greater than 99% pure. In some embodiments, the isolated protein is a synthesized protein. Synthesis of protein is well known in the art and may be performed, for example, by heterologous expression in a transformed cell, such as exemplified herein.

[0194] The terms “homology” or “identity”, as used interchangeably herein, refer to sequence identity between two amino acid sequences or two nucleic acid sequences, with identity being a stricter comparison. The phrases “percent identity or homology” and “% identity or homology” refer to the percentage of sequence identity found in a comparison of two or more amino acid sequences or nucleic acid sequences. Two or more sequences can be anywhere from 0-100% identical, or any value there between. Identity can be determinedby comparing a position in each sequence that can be aligned for purposes of comparison to a reference sequence. When a position in the compared sequence is occupied by the same nucleotide base or amino acid, then the molecules are identical at that position. A degree of identity of amino acid sequences is a function of the number of identical amino acids at positions shared by the amino acid sequences. A degree of identity between nucleic acid sequences is a function of the number of identical or matching nucleotides at positions shared by the nucleic acid sequences. A degree of homology of amino acid sequences is a function of the number of amino acids at positions shared by the polypeptide sequences.

[0195] The following is a non-limiting example for calculating homology or sequence identity between two sequences (the terms are used interchangeably herein). The sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non- homologous sequences can be disregarded for comparison purposes). The optimal alignment is determined as the best score using the GAP program in the GCG software package with a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frame shift gap penalty of 5. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences.

[0196] In some embodiments, % homology or identity as described herein are calculated or determined using the basic local alignment search tool (BLAST). In some embodiments, % homology or identity as described herein are calculated or determined using Blossum 62 scoring matrix.

[0197] According to some embodiments, there is provided a transgenic cell comprising: (a) the polynucleotide disclosed herein; (b) the artificial nucleic acid molecule disclosed herein; (c) the plasmid or agrobacterium disclosed herein; (d) the protein disclosed herein; or any combination thereof.

[0198] 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 anartificial 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.

[0199] In some embodiments, the cell is a unicellular organism, a cell of a multicellular organism, and a cell in a culture.

[0200] In some embodiments, a unicellular organism comprises a fungus or a bacterium.

[0201] In some embodiments, the fungus is a yeast cell.

[0202] In some embodiments, the cell is a prokaryote or a eukaryote cell.

[0203] According to some embodiments, there is provided an extract derived from a transgenic cell disclosed herein, or any fraction thereof.

[0204] In some embodiments, the extract comprises the polynucleotide of the invention, an isolated DNA molecule as disclosed herein, an isolated protein as disclosed herein, or any combination thereof.

[0205] According to some embodiments, there is provided a homogenate, lysate, extract, derived from a transgenic cell disclosed herein, any combination thereof, or any fraction thereof.

[0206] In some embodiments, the extract comprises dopamine.

[0207] 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.

[0208] In some embodiments, the transgenic plant, transgenic plant tissue or plant part, comprises: (a) the polynucleotide disclosed herein; (b) the plasmid or agrobacterium disclosed herein; (c) the isolated protein of the invention; (d) the transgenic cell disclosed herein; or any combination thereof.

[0209] In some embodiments, the transgenic plant comprises: a transgenic plant tissue or a plant part, any portion thereof, seed, tissue, organ thereof, or any combination thereof. In some embodiments, the transgenic plant comprises at least one transgenic plant cell as disclosed herein.

[0210] In some embodiments, the transgenic plant, transgenic plant tissue, or plant part consists of transgenic plant cells of the invention. In some embodiments, the transgenic plant, transgenic plant tissue, or plant part comprises at least: 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 99% transgenic cells of the invention, or any value and range therebetween. Each possibility represents a separate embodiment of the invention. In some embodiments, the transgenic plant, transgenic plant tissue, or plant part comprises 20%-50%, 20%-60%, 20%-70%, 20%-80%, 20%-90%, or 20%-100% transgenic cells of the invention. Each possibility represents a separate embodiment of the invention.

[0211] In some embodiments, the composition comprises: (a) a polynucleotide as disclosed herein (for example, an isolated DNA molecule); (b) a plasmid or agrobacterium as disclosed herein; (c) an isolated protein as disclosed herein; (d) a transgenic cell as disclosed herein; (e) an extract as disclosed herein; (f) a transgenic plant tissue or plant part as disclosed herein; or (g) any combination of (a) to (f), and an acceptable carrier.

[0212] 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; oilssuch as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, com 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 nonlimiting 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.

[0213] The carrier may comprise, in total, from about 0.1% to about 99.99999% by weight of the pharmaceutical compositions presented herein.General

[0214] 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.

[0215] 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.

[0216] 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.

[0217] 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."

[0218] 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 specifically embraced by the present invention and are disclosed herein just as if each and every combination was individually and explicitly disclosed. In addition, all subcombinations 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.

[0219] 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.

[0220] 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

[0221] 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 LIII 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 LIII 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.EXAMPLE 1De novo sequencing and annotation of the Peyote genome

[0222] The inventors obtained an l lx Pacbio HiFi based-assembly. Approximately 37 K contigs were generated (l lx 3.2 Gb genome size, N50 127 Kb), and genes were annotated using Pacbio Iso-Seq and Illumina paired-end-based transcriptomes from different tissues. The BUSCO completeness values were 94.1% for the primary genome assembly, 97.6% for Illumina-based transcriptome, and 88.9% for Pacbio Isoseq.

[0223] The inventors focused on two enzyme families, CYP and PPG. Approximately 270 CYP and 3 PPO genes were annotated in the Peyote genome. Out of these genes, the inventors selected candidates based on relative expression levels in Peyote tissues, favoring those highly expressed in the skin and button, and lowly expressed in thin or thick roots. The inventors also considered the protein size, the presence of protein family (Pfam) motifs, and the similarity to functional enzymes involved in secondary metabolite biosynthesis reported in literature.Functional characterization of CYPs and DC from Peyote

[0224] The inventors selected ten CYP candidates based on their relative expression levels in Peyote tissues and similarity to previously reported CYPs with known functions. The inventors also selected a single PPO homolog that was highly expressed in the areal parts of the cactus. The inventors then opted to evaluate the activities of these potential candidates by transiently expressing them individually in Nicotiana benthamiana (N. benthamiana).

[0225] The inventors did not observe a pronounced increase in dopamine.

[0226] The inventors further identified two more CYP candidates CYP11, and CYP12 that exhibit high similarities (51% and 62%, respectively) to BvCYP76AD6, a betalain-related enzyme previously identified in beetroot, which has been shown to catalyze the hydroxylation of L-tyrosine into L-DOPA. Further, the inventors have found that CYP12 (LwCYP12; (nucleic acid sequence and amino acid sequence as set forth in SEQ ID NO: 12 and 24, respectively)) led to a decrease in tyramine, and a significant increase in dopamine (Fig. 1A). Further, TyDC2 (LwTyDC2; nucleic acid sequence and amino acid sequence as set forth in SEQ ID NO: 25 and 28, respectively) was active showing high catalytic activity for the production of dopamine (Fig. IB). Therefore, it was concluded that LwCYP12 and LwTyDC2 catalyze production of dopamine from tyramine, and L-DOPA, respectively (Fig. 1C). The inventors also showed in N. benthamiana that LwTyDC2 catalyzed the production of tyramine from L-tyrosine, and that a combination of LwTyDC2 and LwCYP12 lead to an increase in dopamine (Fig. ID).

[0227] Therefore, the inventors suggest that LwCYP12, LwTyDC2, or both, are suitable for use in a method for synthesizing dopamine, a precursor thereof, such as tyramine, or both.

[0228] 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 method for synthesizing dopamine comprising the steps: a. providing a cell comprising a first isolated DNA molecule comprising a nucleic acid sequence having at least 80% homology to SEQ ID NO: 12; and b. culturing said cell from step (a) such that a polypeptide encoded by said first isolated DNA molecule is expressed, thereby synthesizing dopamine.

2. The method of claim 1, wherein said polypeptide is characterized by having an activity comprising introducing a hydroxyl group into a substrate.

3. The method of claim 1 or claim 2, wherein said culturing comprises supplementing said cell with an effective amount of tyramine.

4. The method of any one of claims 1 to 3, wherein said cell is a unicellular organism, a cell of a multicellular organism, and a cell in a culture.

5. The method of claim 4, wherein said unicellular organism comprises a fungus or a bacterium.

6. The method of claim 5, wherein said fungus is a yeast cell.

7. The method of any one of claims 1 to 6, wherein any one of said first isolated DNA molecule, said second isolated DNA molecule, or both, is incorporated in a plasmid or an agrobacterium.

8. The method of claim 7, wherein said cell is a transgenic cell or a cell transfected with said first isolated DNA molecule or said plasmid or agrobacterium.

9. The method of any one of claims 1 to 8, wherein said cell further comprises a second isolated DNA molecule comprising a nucleic acid sequence having at least 80% homology to SEQ ID NO: 33.

10. The method of claim 9, further comprising supplementing said cell with an effective amount of L-tyrosine, L-DOPA, or both.

11. A method for synthesizing dopamine comprising contacting tyramine with an effective amount of an isolated polypeptide comprising an amino acid sequence having at least 80% homology or identity to SEQ ID NO: 28, thereby synthesizing dopamine.

12. The method of any one of claims 1 to 11, further comprising a step comprising extracting said cell, thereby obtaining an extract from said cell.

13. An extract obtained according to the method of claim 12.

14. The extract of claim 13, comprising dopamine.

15. Dopamine synthesized according to the method of any one of claims 1 to 14.

16. A composition comprising the dopamine of claim 15, and an acceptable carrier.

17. The composition of claim 16, wherein said acceptable carrier is a pharmaceutically acceptable carrier.

18. The composition of claim 16 or 17, being a pharmaceutical composition.

19. A method for synthesizing dopamine comprising the steps: a. providing a cell comprising an isolated DNA molecule comprising a nucleic acid sequence having at least 80% homology to SEQ ID NO: 33; and b. culturing said cell from step (a) such that a polypeptide encoded by said isolated DNA molecule is expressed, thereby synthesizing dopamine.

20. The method of claim 19, wherein said polypeptide is characterized by having an activity comprising releasing a carboxyl group from a substrate.

21. The method of claim 19 or 20, wherein said isolated DNA molecule is incorporated in a plasmid or an agrobacterium.

22. The method of any one of claims 19 to 21, wherein said culturing comprises supplementing said cell with an effective amount of L-DOPA.

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