Polyphenol oxidase, an amino acid transporter, and a transgenic cell, tissue, and organism comprising same

Abstract

The present invention provides isolated DNA molecule(s), isolated polypeptide(s), artificial vector(s), and cell(s) including same. Further provided are methods for increasing the amount of an amino acid, as well as for synthesizing L-DOPA including a derivative thereof.

Description

POLYPHENOL OXIDASE, AN AMINO ACID TRANSPORTER, AND A TRANSGENIC CELL, TISSUE, AND ORGANISM COMPRISING SAMEREFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0001] The contents of the electronic sequence listing (YEDA-P-055-PCT; size: 56,586 bytes; and date of creation: August 20, 2025) is herein incorporated by reference in its entirety.CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of priority of Israel Patent Application No. 315530, entitled “POLYPHENOL OXIDASE, AN AMINO ACID TRANSPORTER, AND A TRANSGENIC CELL, TISSUE, AND ORGANISM COMPRISING SAME” filed September 8, 2024, the contents of which are incorporated herein by reference in their entirety.FIELD OF INVENTION

[0003] The present invention relates to, inter alia, enzymes related to amino acid synthesis, including polynucleotides encoding same, and use of same in a method for synthesizing tyrosine, L-DOPA, and derivatives thereof.BACKGROUND

[0004] The chemical structure of melanin in plants is complex and varies, but it is generally composed of polymers derived from indole-5, 6-quinone or dihydroxyindole carboxylic acids. These polymers are formed through oxidative polymerization of phenolic compounds, primarily involving L-DOPA and related catechols, resulting in a heterogeneous, amorphous structure. Melanin performs a protective role in plant tissues defending them against UV radiation and other environmental stressors. It also plays a role in pathogen defense by acting as a barrier.

[0005] The causal gene for the black pigment accumulation was mapped by few research groups to the Blp (Black lemma and pericarp) locus with a physical size of 0.8 Mb in the “white” reference genomes MorexV3 and Barke.

[0006] An amino acid transporter BlpTr protein was recently linked to the process of melanin production in barley, although the subcellular localization of the transporter remainsdebatable and needs to be validated. The BlpTr homolog from rice was earlier suggested to be related to melanin formation (Fukuda et al., 2012; Zhu et al., 2011).

[0007] There is still a great need for enzymes, transporters, or their combination, and use of same, such as in a method for synthesizing tyrosine, L-DOPA, and / or derivatives thereof.SUMMARY

[0008] 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.

[0009] 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

[0010] Figs. 1A-1D include vertical bar graphs showing L-DOPA content and Hordeum vulgare polyphenol oxidase (HvPPOb) expression in barley seed tissues. (1A) L-DOPA content in hull / pericarp tissues of Barke (“white”) and melanin-containing HEB-06-137 barley at different stages of seed maturation. The measurements were performed on a LC- TripleQuad MS / MS with the calibration curve of L-DOPA for the absolute quantification. (IB) HvPPOb expression in different stages / organs of HEB-06-137 seeds (from TranSeq output). RT-qPCR analysis of HvPPOb expression in hull+pericarp tissue of different stages of Barke and HEB-06-137 seed maturation (1C) and in hull+pericarp of the different barley genotypes and pigment accumulation at late dough stage (ID).

[0011] Figs. 2A-2B include a phylogenetic tree and a multiple sequence alignment showing that phylogenetic analysis reveals a unique HvPPOb subclade among the BOP clade Poaceae grass species. (2A) A phylogenetic tree of PPOs from the BOP clade with focus on the unique HvPPOb subclade. The phylogenetic tree was generated by the neighbor joining(NJ) method with bootstrap analysis (1,000 replicates) from the amino acid sequence alignment of PPO proteins using MEGA 11 program. The tree was displayed with FigTree vl.4.0. Amino acid conservation of the N-termini from HvPPOb-closest homologues. (2B) Multiple sequence alignment and conservation analysis was performed in Praline sequence alignment program. In 2B the provided amino acid sequences from top to bottom are set forth in SEQ ID Nos: 2, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, and 26.

[0012] Figs. 3A-3F includes vertical bar graphs showing metabolites content changes in N. benthamiana leaves after transient overexpression. Tyrosine (3A), phenylalanine (3B), tryptophane (3C), L-DOPA (3D), dopamine (3E) and tyramine (3F) content in N. benthamiana leaves after the 4 days of the infiltration with Agrobacterium tumefasciens carrying the overexpression vectors. EV - empty vector, HvPPOb - melanin-associated polyphenol oxidase (PPO) from barley, BlpTr - melanin-associated amino acid transporter, LwPPO - PPO from Lophophora williamsii (peyote cactus), MpPPO - PPO from Mucuna pruriens, CYP76AD6 - CYP450 oxidase from beet, DOPA4 - CYP76AD6 co-expressed with AroG and PpAAH. Analysis was performed on LC-qTOF-MS.

[0013] Figs. 4A-4G include horizontal bar graphs showing the amount of the following amino acids or derivatives thereof: L-tyrosine (4A); L-DOPA (4B); dopamine (4C); tyramine (4D); 3 -methoxy -tyramine (4E); phenylalanine (4F); and tryptophan (4G), determined in N. benthamiana transduced to express: (1) PPOb (SEQ ID NO: 1) + BlpTr_1284 (SEQ ID NO: 3); (2) BlpTr_1284 (SEQ ID NO: 3); (3) BlpTr_1008; or (4) negative control (empty vector, EV).

[0014] Figs. 5A-5C include fluorescent micrographs showing the subcellular localization of HvPPOb-RFP and BlpTR_1284 in N benthamiana leaves. (5 A) Shown is the signal of HvPPOb-RFP in epidermal / mesophyll layer of infiltrated leaf disc. (5B) Shown is the same leaf disc as in (5A) after plasmolysis. (5C) Shown is the signal of BlpTR_1284 in pavement cells. The images were taken from the Nikon Al LFOV confocal microscope camera with magnification *20 with channel 1 mCherry (excitation 561.5 nm, emission 595 nm) for the proteins fused with mRFP (artificial red and green colors) and another Cy5 channel for chloroplast autofluorescence detection (artificial magenta color).

[0015] Figs. 6A-6F include vertical bar graphs showing production levels of tyrosine (6A), L-DOPA (6B), dopamine (6C), tyramine (6D), phenylalanine (6E), and tryptophan (6F) in ‘early dough’ (ED) and Tate dough’ (LD) stages of stable transgenic lines of common wheat (Triticum aestivum, cv. Fielder). The regenerative genes GROWTH-REGULATING FACTOR (GRP and GRF-INTERACTING FACTOR (GIF were used together withHvPPOb alone GRF -GIF -HvPPOb lines #1-3) or with a double gene construct including HvPPOb and BlpTR (GRF-GIF-D24 lines #1-4).

[0016] Figs. 7A-7B include photographs showing visual phenotype of mature GRF-GIF (control, 7A) and GRF-GIF-D24#3 (7B) spikelet.DETAILED DESCRIPTIONDNA molecules and vectors

[0017] According to some embodiments, there is provided a polynucleotide or an isolated DNA molecule comprising a nucleic acid sequence comprising SEQ ID Nos: 1, 3, or any combination thereof (“polynucleotide of the invention”).

[0018] In some embodiments, the isolated DNA molecule comprises a first isolated DNA molecule, a second isolated DNA molecule, or both.

[0019] In some embodiments, a first isolated DNA molecule comprises a nucleic acid sequence having at least 86%, 90%, 95%, or 99% homology or identity to SEQ ID NO: 1, or any value and range therebetween. In some embodiments, a first isolated DNA molecule comprises a nucleic acid sequence having 86-100%, 90-100%, 95-100%, or 99-100 % homology or identity to SEQ ID NO: 1. Each possibility represents a separate embodiment of the invention.

[0020] In some embodiments, the first isolated DNA molecule comprises a nucleic acid sequence encoding a polypeptide being a polyphenol oxidase (PPO), having a PPO activity, or both.

[0021] As used herein, the terms “polyphenol oxidase” and “PPO” are interchangeable, and refer to any peptide, polypeptide, or a protein, capable catalyzing one- and two- electron oxidation of phenols to quinones at the expense of O2.

[0022] PPOs are copper metalloproteins catalyzing the o- hydroxylation of a monophenol, followed by its oxidation to the o-diquinone, or the oxidation of an o-dihydroxyphenol to the o-diquinone. PPO-generated products, e.g., quinones are highly reactive, electrophilic molecules which covalently modify and crosslink a variety of cellular constituents.

[0023] In some embodiments, PPO activity comprises o- hydroxylation of a monophenol. In some embodiments, PPO activity comprises oxidation of a monophenol to an o- diquinone. In some embodiments, PPO activity comprises oxidation of an o- dihydroxyphenol to an o-diquinone.

[0024] A second isolated DNA molecule comprising a nucleic acid sequence having at least 94%, 96%, 98%, or 99% homology or identity to SEQ ID NO: 3, or any value and rangetherebetween. In some embodiments, a second isolated DNA molecule comprises a nucleic acid sequence having 94-100%, 96-100%, 98-100%, or 99-100 % homology or identity to SEQ ID NO: 3. Each possibility represents a separate embodiment of the invention.

[0025] In some embodiments, the second isolated DNA molecule comprises nucleic acid sequence encoding a polypeptide being an amino acid transporter.

[0026] As used herein, the term “amino acid transporter” refers to any peptide, polypeptide, or a protein, capable of transporting or mediating transport of an amino acid or a plurality thereof across a membrane. In some embodiments, a membrane comprises a cell membrane. In some embodiments, a membrane comprises an organelle membrane. In some embodiments, a membrane comprises a cell membrane and an organelle membrane. In some embodiments, an organelle is an organelle of a cell (e.g., the cell of the invention). In some embodiments, transport is into, out of, or both, a cell, an organelle, or both.

[0027] As used herein, the term “amino acid” refers to a class of organic compounds that contain at least one amino group and one carboxyl group. Amino acids include leucine, isoleucine, valine, arginine, lysine, asparagine, serine, glycine, glutamine, tryptophan, methionine, threonine, cysteine, tyrosine, phenylalanine, glutamic acid, aspartic acid, alanine, histidine, and proline.

[0028] 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. In some embodiments, the isolated DNA molecule is a polynucleotide.

[0029] 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 by primer linkers multiple nucleic acid molecules together.

[0030] 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).

[0031] 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.

[0032] In some embodiments, the first isolated DNA molecule comprises 1,600 to 1,900 nucleotides. In some embodiments, the first isolated DNA molecule is 1,650 to 1,850 nucleotides long. In some embodiments, the first isolated DNA molecule comprises 1,700 to 1,850 nucleotides. In some embodiments, the first isolated DNA molecule is 1,700 to 1,800 nucleotides long.

[0033] In some embodiments, the second isolated DNA molecule comprises 900 to 1,100 nucleotides. In some embodiments, the second isolated DNA molecule is 950 to 1,050 nucleotides long. In some embodiments, the second isolated DNA molecule comprises 970 to 1,020 nucleotides. In some embodiments, the second isolated DNA molecule is 990 to 1,010 nucleotides long.

[0034] According to some embodiments, there is provided an artificial vector comprising the isolated DNA molecule or polynucleotide of the invention (such as disclosed herein), or combination thereof.

[0035] In some embodiments, the artificial vector comprises the first isolated DNA molecule of the invention; the second isolated DNA molecule of the invention; or both.

[0036] In some embodiments, the artificial vector comprises a plasmid. In some embodiments, the artificial vector comprises or is an Agrobacterium or an agrob acterium- based vector comprising the isolated DNA molecule(s) of the invention. 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 PPO, an amino acid transport, or both, encoding nucleic acid sequence as disclosed herein. In some embodiments, the artificial vector is for use in heterologous expression of a PPO, an amino acid transport, or both, encoding nucleic acid sequence asdisclosed herein in a cell, a tissue, or an organism. In some embodiments, the artificial vector is for use in producing / synthesizing or the production / synthesis of L-DOPA, or a derivative thereof in a cell, a tissue, or an organism. In some embodiments, the artificial vector is for use in producing / synthesizing or the production / synthesis of dopamine in a cell, a tissue, or an organism. In some embodiments, L-DOPA derivative is a benzylisoquinoline alkaloids (BIA). In some embodiments, L-DOPA derivative is selected from: dopamine, melanin, betalain, mescaline, morphine, codeine, epinephrine, norepinephrine, papaverine, or any combination thereof.

[0037] In some embodiments, the artificial vector is for use in increasing amounts or accumulation of an amino acid in a cell, a tissue, or an organism. In some embodiments, the amino acid is selected from: tyrosine, phenylalanine, tryptophan, or any combination thereof.

[0038] 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.

[0039] 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.

[0040] 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 of interest 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 transcript! on / translati on 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.

[0041] 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 kilobases.

[0042] 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.

[0043] 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.

[0044] 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.

[0045] 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.

[0046] 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.

[0047] 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.

[0048] 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.

[0049] In some embodiments, the artificial vector comprises a polynucleotide encoding a protein comprising an amino acid sequence as described herein.

[0050] As used herein, the term “genetic mutation” refers to a change or multiple changes in a nucleotide sequence of a gene or related regulatory region that alters the nucleotide sequence as compared to its native or wild-type sequence. Mutations include, for example, substitutions, insertions, and deletions, in whole or in part, within the wild-type sequence. Such substitutions, insertions, or deletions can be single nucleotide changes (e.g., one or more point mutations), or can be two or more nucleotide changes, which may result in substantial changes to the sequence. Mutations can occur within the coding region of the gene as well as within the non-coding and regulatory sequence of the gene. The term “genetic mutation” is intended to include silent and conservative mutations within a coding region as well as changes which alter the amino acid sequence of the polypeptide encoded by the gene. A genetic mutation in a gene coding sequence may, for example, increase, decrease, or otherwise alter the activity (e.g., import activity) of the polypeptide product encoded by the gene. A genetic mutation in a regulatory sequence may increase, decrease, or otherwise alter the expression of sequences operably linked to the altered regulatory sequence.

[0051] It is routine for one of ordinary skill in the art to make mutations in a gene of interest. Mutations include substitutions, insertions, deletions, and / or truncations of one or more specific amino acid residues or of one or more specific nucleotides or codons in the polypeptide or polynucleotide of interest. Mutagenesis and directed evolution methods are well known in the art for creating variants. See, e.g., U.S. Pat. No. 7,783,428; 6,586,182; 6,117,679; and Ling, et al., 1999, “Approaches to DNA mutagenesis: an overview,” Anal. Biochem., 254(2): 157-78; Smith, 1985, “In vitro mutagenesis,” Ann. Rev. Genet., 19:423- 462; Carter, 1986, “Site-directed mutagenesis,” Biochem. J., 237 : l-7; and Minshull, et al., 1999, “Protein evolution by molecular breeding,” Current Opinion in Chemical Biology, 3:284-290. For example, the lambda red system can be used to knock-out genes in A. coli (see, e.g., Datta et al., Gene, 379:109-115 (2006)).

[0052] As used herein, “heterologous” as used in the context of a nucleic acid or polypeptide sequence, “heterologous gene”, or “heterologous sequence”, refers to a nucleotide or polypeptide sequence that is not normally found in a given cell in nature. As used herein, a heterologous sequence encompasses a nucleic acid sequence that is exogenously introduced into a given cell. “Heterologous gene” includes a native gene, or fragment thereof, that has been introduced into the host cell in a form that is different from the corresponding native gene. For example, a heterologous gene may include a native coding sequence that is a portion of a chimeric gene to include a native coding sequence that is a portion of a chimericgene to include non-native regulatory regions that is reintroduced into the host cell. A heterologous gene may also include a native gene, or fragment thereof, introduced into a non-native host cell. Thus, a heterologous gene may be foreign or native to the recipient cell; a nucleic acid sequence that is naturally found in a given cell but expresses an unnatural amount of the nucleic acid and / or the polypeptide which it encodes; and / or two or more nucleic acid sequences that are not found in the same relationship to each other in nature. As used herein, the term “endogenous gene” refers to a native gene in its natural location in the genome of an organism. As used herein, the term “transgene” refers to a gene that has been introduced into the host organism, e.g., host bacterial cell, genome.

[0053] In some embodiments, the first isolated DNA molecule, the second isolated DNA molecule, or both, are codon optimized for expression in a cell of the invention.

[0054] As used herein the term “codon-optimized” refers to the modification of codons in a gene or a coding region of a nucleic acid molecule to improve translation in a host cell or organism of a transcript RNA molecule transcribed from the coding sequence, or to improve transcription of a coding sequence. Codon optimization includes, but is not limited to, processes including selecting codons for the coding sequence to suit the codon preference of the expression host organism. Such optimization includes replacing at least one, or more than one, or a significant number, of codons with one or more codons that are more frequently used in the genes of the host organism.Polypeptides

[0055] According to some embodiments, there is provided a polypeptide encoded by: (a) the first isolated DNA molecule of the invention; (b) the second isolated DNA molecule of the invention; (c) the artificial vector of the invention; or (d) any combination of (a) to (c).

[0056] In some embodiments, the polypeptide is encoded by a nucleic acid sequence comprising or consisting of SEQ ID Nos: 1, 3, or a combination thereof.

[0057] In some embodiments, the polypeptide is an isolated polypeptide.

[0058] 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 acidpolymers. 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.

[0059] As used herein, the terms "isolated polypeptide" refers to a polypeptide 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 polypeptide contains the polypeptide 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 polypeptide is a synthesized polypeptide. Synthesis of polypeptides is well known in the art and may be performed, for example, by heterologous expression in a transformed cell, such as exemplified herein.

[0060] In some embodiments, the polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 2.

[0061] In some embodiments, the polypeptide comprises an amino acid sequence with at least 82%, at least 85%, at least 90%, at least 95%, or at least 97% homology or identity to SEQ ID NO: 2, or any value and range therebetween. In some embodiments, the polypeptide comprises an amino acid sequence with 82% to 100%, 85% 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.

[0062] In some embodiments, the polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2, or a functional analog having at least 82% homology thereto, is characterized by having an activity of a PPO, a phenolic compound, a phenol-containing compound oxidation, or any combination thereof.

[0063] In some embodiments, the phenolic compound, phenol-containing compound, or both, comprises or is tyrosine.

[0064] In some embodiments, the polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 4.

[0065] In some embodiments, the polypeptide comprises an amino acid sequence with at least 93%, at least 95%, at least 97%, or at least 99% homology or identity to SEQ ID NO: 4, or any value and range therebetween. In some embodiments, the polypeptide comprises an amino acid sequence with 93% to 100%, 95% to 100%, 97% to 100%, or 99% to 100% homology or identity to SEQ ID NO: 4. Each possibility represents a separate embodiment of the invention.

[0066] In some embodiments, the polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 4, or a functional analog having at least 93% homology thereto, is characterized by having an activity of an amino acid transporter.

[0067] In some embodiments, the amino acid is selected from: tyrosine, phenylalanine, tryptophan, or any combination thereof.

[0068] As used herein, the term “functional analog” refers to any peptide / polypeptide / protein having an amino acid sequence substantially identical to one of the sequences specifically shown herein in which one or more residues have been conservatively substituted with a functionally similar residue and which displays the abilities as described herein, PPO activity and / or amino acid transport activity. Examples of conservative substitutions include the substitution of one non-polar (hydrophobic) residue such as isoleucine, valine, leucine or methionine for another, the substitution of one polar (hydrophilic) residue for another such as between arginine and lysine, between glutamine and asparagine, between glycine and serine, the substitution of one basic residue such as lysine, arginine or histidine for another, or the substitution of one acidic residue, such as aspartic acid or glutamic acid for another. Each possibility represents a separate embodiment of the present invention.

[0069] In some embodiments, a functional analog to the isolated polypeptide of the invention comprises an amino acid sequence with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% homology to the amino acid sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 4, or any value and range therebetween. Each possibility represents a separate embodiment of the invention.

[0070] In some embodiments, the isolated polypeptide comprises a plurality of polypeptides. In some embodiments, the plurality of polypeptides comprises a plurality of different polypeptides. In some embodiments, a plurality of different polypeptides comprises at least two polypeptides each of which having a different amino acid sequence, e.g., not sharing at least one amino acid (such as when aligned by their sequence in a multiple sequence alignment). In some embodiments, the plurality of polypeptides comprises a first amino acid sequence having at least 82% homology to SEQ ID NO: 2, and a second amino acid sequence having at least 93% homology to SEQ ID NO: 4.

[0071] 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 canbe anywhere from 0-100% identical, or any value there between. Identity can be determined by 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.

[0072] 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.

[0073] 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.Cells and Compositions

[0074] According to some embodiments, there is provided a transgenic, transduced, or transformed cell comprising any one of: (a) the first isolated DNA molecule of the invention; (b) the second isolated DNA molecule of the invention; (c) the artificial vector of the invention; (d) the isolated polypeptide of the invention; and (e) any combination of (a) to (d).

[0075] 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 hashad exogenous polynucleotide, such as an isolated DNA molecule of the invention, 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 or in its genome. In some embodiments, the exogenous polynucleotide (e.g., the isolated DNA molecule of the invention) or vector is stably integrated into the cell. In some embodiments, the transgenic cell expresses a polynucleotide / isolated DNA molecule of the invention. In some embodiments, the transgenic cell expresses an artificial vector of the invention. In some embodiments, the transgenic cell expresses a polypeptide of the invention. In some embodiments, the transgenic cell is a cell that is naturally devoid of an isolated DNA molecule of the invention that has been transformed or genetically modified to include the isolated DNA molecule of the invention. In some embodiments, CRISPR technology is used to modify the genome of the cell, as described herein. In some embodiments, any molecular tool known to a person of skill in the art for the purpose of introducing an exogenous DNA molecule, e.g., the isolated DNA molecule of the invention, is contemplated herein.

[0076] In some embodiments, the cell is a unicellular organism, a cell of a multicellular organism, or a cell in a culture. In some embodiments, the cell is a plant cell. In some embodiments, the cell is a cell of an alga. In some embodiments, the cell is a microalga. In some embodiments, the alga is a macroalga. In some embodiments, the cell is a cell of a microalga.

[0077] In some embodiments, a unicellular organism comprises a fungus or a bacterium. In some embodiments, a unicellular organism comprises a microalga.

[0078] In some embodiments, a fungus comprises a yeast cell.

[0079] In some embodiments, a cell is an insect cell. In some embodiments, the cell comprises an insect cell line.

[0080] Types of insect cell lines suitable for transformation and / or heterologous expression are common and would be apparent to one of ordinary skill in the art. Non-limiting examples of such insect cell lines include, but are not limited to, Sf-9 cells, SR+ Schneider cells, S2 cells, and others.

[0081] According to some embodiments, there is provided a homogenate, lysate, extract, derived from a transgenic cell of the invention, any combination thereof, or any fraction thereof.

[0082] 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), detergentbased lysis, solvent (e.g., polar or nonpolar solvent), liquid chromatography mass spectrometry, or others.

[0083] According to some embodiments, there is provided a transgenic plant, a transgenic plant tissue or a plant part. In some embodiments, there is provided a transgenic plant, or any portion, seed, tissue or organ thereof, comprising at least one transgenic plant cell of the invention. In some embodiments, the transgenic plant, transgenic plant tissue or plant part, comprises: transgenic plant, a part thereof, or a tissue derived therefrom, comprising: (a) the first isolated DNA molecule of the invention; (b) the second isolated DNA molecule of the invention; (c) the artificial vector of the invention; (d) the isolated polypeptide of the invention; (e) the transgenic, transduced, or transformed cell of the invention; and (f) any combination of (a) to (e).

[0084] According to some embodiments, there is provided a homogenate, lysate, extract, exudate, or any combination thereof, being derived from a transgenic plant of the invention, a part thereof, or any fraction thereof.

[0085] In some embodiments, an exudate comprises a root exudate. In some embodiments, an exudate comprises a whole plant exudate.

[0086] In some embodiments, a plant comprises a whole plant. In some embodiments, a plant or a part thereof comprises a suspension of plant cells. In some embodiments, a plant or a part thereof comprises a hairy root culture. In some embodiments, a plant comprises a water plant. In some embodiments, a water plant comprises or is duckweed, water lettuce, or both. In some embodiments, a plant part comprises a storage organ of a plant. In some embodiments, a storage organ of a plant comprises a tuber, a swollen root, or both. In some embodiments, a plant part is an edible organ of a plant. In some embodiments, an edible organ of a plant comprises or is a fruit, a flower, a leaf, a seed, a stem, or any combination thereof. In some embodiments, a plant is a tree. In some embodiments, a plant comprises or is an extracellular vesicle of a plant cell.

[0087] In some embodiments the transgenic plant, part thereof, or tissue derived therefrom comprises a plant selected from: tobacco, rice, wheat, barley, tomato, duckweed, beet (or beetroot), sugar cane, maize, legume, soybean, cassava, potato, sorghum, or any combination thereof.

[0088] 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. 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.

[0089] According to some embodiments, there is provided a composition comprising: (a) the first isolated DNA molecule of the invention; (b) the second isolated DNA molecule of the invention; (c) the artificial vector of the invention; (d) the isolated polypeptide of the invention; (e) the transgenic, transduced, or transformed cell of the invention; (f) the transgenic plant, part thereof, or tissue derived therefrom of the invention; or (g) any combination of (a) to (g); and an acceptable carrier.

[0090] 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, 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, magnesiumstearate, 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 nontoxic, 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.

[0091] The carrier may comprise, in total, from about 0.1% to about 99.99999% by weight of the pharmaceutical compositions presented herein.Methods of synthesis

[0092] According to some embodiments, there is provided a method for increasing amount or abundance of an amino acid in a cell. According to some embodiments, there is provided a method for increasing amount or abundance of an amino acid in a plant, such as a water plant.

[0093] In some embodiments, increasing or increased is compared to a control. In some embodiments, a control comprises a control cell. In some embodiments, a control cell is devoid of a nucleic acid sequence having at least 94% homology to SEQ ID NO: 3. In some embodiments, a control cell is devoid of an artificial vector comprising a nucleic acid sequence having at least 94% homology to SEQ ID NO: 3. In some embodiments, a control comprises a control plant, such as a water plant. In some embodiments, a control plant is devoid of a nucleic acid sequence having at least 94% homology to SEQ ID NO: 3. In some embodiments, a control plant, such as a water plant, is devoid of an artificial vector comprising a nucleic acid sequence having at least 94% homology to SEQ ID NO: 3. In some embodiments, a control cell comprises a nucleic acid sequence of SEQ ID NO: 27, or an artificial vector comprising thereof. In some embodiments, a control cell overexpresses a nucleic acid sequence of SEQ ID NO: 27. In some embodiments, a control cell is devoid of a nucleic acid sequence having at least 80%, 85%, 90%, 95%, 97% homology, or any value and range therebetween to SEQ ID NO: 29. In some embodiments, a control cell is devoid of polypeptide having at least 80%, 85%, 90%, 95%, 97% homology, or any value and range therebetween to SEQ ID NO: 30.

[0094] In some embodiments, the plant is a water plant. In some embodiments, the cell is a microalga cell. In some embodiments, the plant is selected from: tobacco, rice, wheat, barley, tomato, and duckweed.

[0095] In some embodiments, the method comprises: (a) providing a cell comprising an artificial vector comprising a nucleic acid sequence having at least 94% homology to SEQ ID NO: 3; and (b) culturing the cell from step (a) such that a polypeptide encoded by the artificial vector is expressed. In some embodiments, the method comprises: (a) providing a plant comprising an artificial vector comprising a nucleic acid sequence having at least 94% homology to SEQ ID NO: 3; and (b) culturing the plant from step (a) such that a polypeptide encoded by the artificial vector is expressed.

[0096] In some embodiments, the method further comprises a step before step (a), comprising introducing or transfecting the cell with the artificial vector. In someembodiments, the artificial vector comprises a plasmid or agrobacterium. In some embodiments, the method further comprises a step before step (a), comprising introducing or transfecting at least one cell of the plant with the artificial vector. In some embodiments, the artificial vector comprises a plasmid or agrobacterium.

[0097] In some embodiments, the method comprises culturing a cell comprising an artificial vector comprising a nucleic acid sequence having at least 94% homology to SEQ ID NO: 3 such that a polypeptide encoded by the artificial vector is expressed. In some embodiments, the method comprises culturing a plant comprising an artificial vector comprising a nucleic acid sequence having at least 94% homology to SEQ ID NO: 3 such that a polypeptide encoded by the artificial vector is expressed.

[0098] In some embodiments, the amino acid is selected from: tyrosine, phenylalanine, tryptophan, or any combination thereof.

[0099] According to some embodiments, there is provided a method for synthesizing L- DOPA or a derivative thereof in a cell. According to some embodiments, there is provided a method for synthesizing L-DOPA or a derivative thereof in a plant. According to some embodiments, there is provided a method for synthesizing L-DOPA. According to some embodiments, there is provided a method for synthesizing dopamine.

[0100] In some embodiments, the method comprises culturing a cell comprising an artificial vector comprising: (i) a first nucleic acid sequence having at least 86% homology to SEQ ID NO: 1; and / or (ii) a second nucleic acid sequence having at least 94% homology to SEQ ID NO: 3 such that a first polypeptide encoded by the first nucleic sequence of the artificial vector and / or a second polypeptide encoded by the second nucleic sequence of the artificial vector are expressed. In some embodiments, the method comprises culturing a plant comprising an artificial vector comprising: (i) a first nucleic acid sequence having at least 86% homology to SEQ ID NO: 1; and / or (ii) a second nucleic acid sequence having at least 94% homology to SEQ ID NO: 3 such that a first polypeptide encoded by the first nucleic sequence of the artificial vector and / or a second polypeptide encoded by the second nucleic sequence of the artificial vector are expressed.

[0101] In some embodiments, the method comprises: (a) providing a cell comprising an artificial vector comprising: (i) a first nucleic acid sequence having at least 86% homology to SEQ ID NO: 1; and / or (ii) a second nucleic acid sequence having at least 94% homology to SEQ ID NO: 3; and (b) culturing the cell from step (a) such that a first polypeptide encoded by the first nucleic sequence of the artificial vector and / or a second polypeptide encoded by the second nucleic sequence of the artificial vector are expressed. In some embodiments, the method comprises: (a) providing a plant comprising an artificial vector comprising: (i) a firstnucleic acid sequence having at least 86% homology to SEQ ID NO: 1; and / or (ii) a second nucleic acid sequence having at least 94% homology to SEQ ID NO: 3; and (b) culturing the plant from step (a) such that a first polypeptide encoded by the first nucleic sequence of the artificial vector and / or a second polypeptide encoded by the second nucleic sequence of the artificial vector are expressed.

[0102] In some embodiments, the method further comprises a step before the culturing comprising introducing or transfecting the cell with the artificial vector. In some embodiments, the method further comprises a step before the culturing comprising introducing or transfecting at least one cell of the plant with the artificial vector.

[0103] In some embodiments, synthesizing comprises increasing the amount of L-DOPA or a derivative thereof synthesized by the cell, the plant, or both (e.g., compared to a control cell or a control plant, respectively). In some embodiments, synthesizing comprises increasing the amount of dopamine synthesized by the cell (e.g., compared to a control cell).

[0104] In some embodiments, the cell further comprises at least one third nucleic acid sequence. In some embodiments, at least one cell of the plant further comprises at least one third nucleic acid sequence. In some embodiments, the at least one third nucleic acid sequence encodes at least one third polypeptide. In some embodiments, the at least one third polypeptide is capable of catalyzing conversion of L-DOPA to the derivative or an intermediate product therebetween. In some embodiments, the intermediate product is a metabolite or a transient metabolite being a part of a biosynthetic pathway for producing the derivative from L-DOPA.

[0105] In some embodiments, L-DOPA derivative is benzylisoquinoline alkaloid (BIA). In some embodiments, L-DOPA derivative is selected from: dopamine, melanin, betalain, mescaline, morphine, codeine, epinephrine, norepinephrine, papaverine, or any combination thereof.

[0106] 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.

[0107] In some embodiments, introducing or transfecting comprises transferring an artificial nucleic acid molecule or vector comprising the isolated DNA molecule of the invention into a cell; or modifying the genome of a cell to include the isolated DNA molecule of the invention. In some embodiments, transferring comprises transfection. In some embodiments, transferring comprises transformation. In some embodiments, transferring compriseslipofection. In some embodiments, transferring comprises nucleofection. In some embodiments, transferring comprises viral infection.

[0108] In some embodiments, artificial nucleic acid molecule or vector comprises: a plasmid, an agrobacterium, or both.

[0109] As used herein, the terms “transfecting” and “introducing” are interchangeable.

[0110] According to some embodiments, there is provided a method for obtaining an extract from a transgenic cell or a transfected cell of the invention.

[0111] In some embodiments, the method comprises culturing a transgenic cell or a transfected cell of the invention in a medium and extracting the transgenic cell or the transfected cell. In some embodiments, the method comprises culturing a plant comprising at least one transgenic cell or a transfected cell of the invention in a medium and isolating an amino acid, L-DOPA, a derivative thereof, or any combination thereof, from the medium.

[0112] In some embodiments, isolating comprises purifying. In some embodiments, isolating comprises exuding (e.g., exudation) at least one root of the plant.

[0113] In some embodiments, the method comprises the steps: (a) culturing a transgenic cell or a transfected cell of the invention 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.

[0114] In some embodiments, the method further comprises a step preceding or before step (b), comprising separating the cultured transgenic cell or the cultured transfected cell from the medium.

[0115] In some embodiments, the method further comprises a step proceeding or after the culturing, comprising separating the cultured plant from the medium.

[0116] Methods for separating cells 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.

[0117] 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 method of the invention. According to some embodiments, there is provided a secret of the plant, obtained according to the method of the invention. According to some embodiments, there is provided an exudate of the plant, obtained according to the method of the invention.

[0118] According to another aspect, there is provided a method for increasing synthesis of L-DOPA or a derivative thereof in a cell. In some embodiments, the cell is a cell of a plant.According to another aspect, there is provided a method for increasing synthesis of L-DOPA or a derivative thereof in a plant.

[0119] In some embodiments, the method comprises increasing the activity, abundance, or both, of a PPO in a plasma membrane of a cell or an extracellular space at least partially separated from the cell by the plasma membrane, thereby increasing the synthesis of L- DOPA or a derivative thereof in the cell.

[0120] In some embodiments, the method comprises increasing the activity, abundance, or both, of a PPO in a plasma membrane of a cell of a plant or an extracellular space at least partially separated from the cell of the plant by the plasma membrane, thereby increasing the synthesis of L-DOPA or a derivative thereof in the plant.

[0121] In some embodiments, the method comprises increasing the activity, abundance, or both, of an amino acid transporter in a plasma membrane of a cell of a plant or an extracellular space at least partially separated from the cell of the plant by the plasma membrane, thereby increasing the synthesis of L-DOPA or a derivative thereof in the plant.

[0122] In some embodiments, the method comprises increasing the activity, abundance, or both, of a PPO and of an amino acid transporter in a plasma membrane of a cell of a plant or an extracellular space at least partially separated from the cell of the plant by the plasma membrane, thereby increasing the synthesis of L-DOPA or a derivative thereof in the plant.

[0123] In some embodiments, increasing the activity, abundance or both, comprises increasing the expression level of a nucleic acid sequence encoding the PPO, the amino acid transporter, or both. In some embodiments, increasing the activity, abundance or both, comprises increasing the rate of translocation or localization of the PPO, the amino acid transporter, or both, to the plasma membrane of the cell, the extracellular space at least partially separated from the cell by the plasma membrane, or both.

[0124] In some embodiments, the cell is a transgenic, transformed, or transfected cell.

[0125] In some embodiments, the extracellular space is an apoplast of a plant cell.

[0126] In some embodiments, the PPO comprises a signal sequence targeting the PPO to the plasma membrane of the cell or the extracellular space at least partially separated from the cell by the plasma membrane. In some embodiments, the amino acid transporter comprises a signal sequence targeting the amino acid transporter to the plasma membrane of the cell or the extracellular space at least partially separated from the cell by the plasma membrane. In some embodiments, the signal sequence is an apoplast sorting signal.

[0127] In some embodiments, the method comprises increasing the activity, abundance, or both, of a PPO, of an amino acid transport, or both in a plasma membrane of a cell, thereby increasing the synthesis of L-DOPA or a derivative thereof in the cell. In some embodiments,the method comprises increasing the activity, abundance, or both, of a PPO, of an amino acid transporter, or both in the apoplast of the cell, thereby increasing the synthesis of L- DOPA or a derivative thereof in the cell. In some embodiments, the method comprises increasing the activity, abundance, or both, of a PPO, of an amino acid transport, or both in a plasma membrane of a cell, the apoplast of a cell, or both, thereby increasing the synthesis of L-DOPA or a derivative thereof in the cell.

[0128] As used herein, the term “apoplast sorting signal” refers to a specific sequence of amino acids within a protein that directs the protein to the apoplast, which is the extracellular space in plant tissues. This signal sequence ensures that the protein is transported across the plasma membrane and localized in the apoplast, where it can perform its intended function. The apoplast sorting signal is crucial for targeting proteins to the correct cellular compartment, thereby facilitating their role in processes such as cell wall modification, defense responses, and intercellular communication.

[0129] Exemplary references providing a detailed understanding of the role and importance of apoplast sorting signals in plant biology, highlighting their function in targeting proteins to the apoplast and ensuring their proper localization and activity, include, but are not limited to “Plant Cell Wall Proteins: Structure, Function, and Applications” by Z. A. Popper, G. Michel, C. Herve, D. A. Domozych, W. G. Willats, M. G. Tuohy, B. Kloareg, and D. B. Stengel, or “The Role of Signal Peptides in Protein Targeting to the Apoplast” by S. M. Smith and J. D. Raikhel.

[0130] In some embodiments, increasing the rate of translocation or localization of the PPO, the amino acid transporter, or both, to the plasma membrane of the cell, the extracellular space at least partially separated from the cell by the plasma membrane, or both, comprises expressing a nucleic acid sequence encoding the PPO, the amino acid transporter, or both, in conjunction (e.g., in-frame) with a nucleic acid sequence encoding a sorting signal to the plasma membrane of the cell, the extracellular space at least partially separated from the cell by the plasma membrane, or both. In some embodiments, the sorting signal is a plasma membrane sorting signal. In some embodiments, the sorting signal is an apoplast sorting signal.

[0131] In some embodiments, the PPO comprises the amino acid sequence set forth in SEQ ID NO: 2, or a homolog thereof having at least 83%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% homology thereto, or any value and range therebetween. Each possibility represents a separate embodiment of the invention.

[0132] In some embodiments, the cell comprises: (i) a first nucleic acid sequence having at least 86% homology to SEQ ID NO: 1; and (ii) a second nucleic acid sequence having atleast 94% homology to SEQ ID NO: 3. In some embodiments, the method further comprises introducing or transfecting the cell with an artificial vector comprising the first nucleic acid sequence and the second nucleic acid sequence.

[0133] In some embodiments, the method further comprises a step comprising extracting L- DOPA, a derivative thereof, or any combination thereof, from the apoplast of the cell.

[0134] In some embodiments, the PPO is a plant PPO. In some embodiments, the PPO is an animal PPO. In some embodiments, the PPO is a dicto or a dicotyledon plant PPO. In some embodiments, a plant comprises a dictos or a dicotyledon plant.General

[0135] 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.

[0136] 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.

[0137] 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.

[0138] 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 furtherunderstood 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."

[0139] 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.

[0140] 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.

[0141] 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

[0142] 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; Birrenet 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.EXAMPLE 1

[0143] In the current analysis, the inventors compared seeds of common “white” barley (cv Barke) which do not accumulate melanin with the melanin accumulating (‘melanistic’) HEB- 06-137 landrace backcrossed to Barke with the homozygous Blp locus. The inventors performed metabolomics analysis of hull / pericarp tissue from seeds at soft (SD), early (ED) and late dough (LD) maturation stages. The inventors found enrichment of L-DOPA (Fig. 1A), tyrosine and other phenols at ED and LD stages of the “black” barley genotype. These compounds are known precursors for melanin formation. The inventors performed de- novo genome sequencing of “black” HEB-06-137 accession as well as full-length transcript sequencing (IsoSec; Pacbio technology) of hull / pericarp tissues of HEB-06-137 and Barke. The list of transcripts (obtained by IsoSec) was used as a reference transcriptome for the TranSeq (3 ’end transcript sequencing) analysis of awns, hull / pericarp and endosperm tissues of HEB-06-137 and Barke at ED, SD andLD stages. De-novo sequencing revealed high level of polymorphism between the ‘melanistic’ HEB-06-137 and “white ” (MorexV3 and Barke) Blp loci. One of the major differences was the presence of at least 0.4 Mb insertion in HEB- 06-137 Blp region. Through the current IsoSeq sequencing analysis the inventors revealed 2 genes encoded in the HEB-06-137 Blp 0.4 Mb insertion that show expression in the lemmapericarp tissue. One of them was putative purple acid phosphatase gene (PAP27) with the 89% similarity to “white” homologue, located in Blp locus of MorexV3. But, as RNA seq results showed, this novel PAP had low expression that was not specified to any organ / stage. The second gene was a putative Polyphenol oxidase gene (termed herein ‘HvPPOlP SEQ ID Nos: 1-2), the expression of which correlates with the accumulation of L-DOPA (Figs. 1A-1C). This enzyme, like the majority of plant PPOs, has a tyrosinase domain and willlikely oxidase tyrosine to L-DOPA. RT-PCR analysis revealed that high HvPPOb expression in hull+pericarp tissues was associated with the late stages of seed development in melaninforming accessions only (Figs. 1C-1D).

[0144] Phylogenetic analysis of PPOs belonging to the BOP clade (i.e., those Poaceae C3 grasses species phylogenetically related to barley) showed that HvPPOb shares a separated, yet uncharacterized specific subclade with another polyphenol oxidase from barley and with PPOs from other cereals (Fig. 2A). Amino acid conservation analysis of proteins in this subclade (none of them besides HvPPOb ever associated with black pigment accumulation) revealed that HvPPOb possess a unique polymorphism in its N-terminal region encoding the first 100 amino acids (Fig. 2B). These differences may impact the subcellular localization of HvPPOb providing the enzyme access to its substrates.

[0145] Although being isolated from the barley cultivar of different origin, BlpTr (SEQ ID Nos: 3-4) was shown to be located also in the Blp locus suggesting that the identified herein BlpTr (SEQ ID Nos: 3-4) and HvPPOb (SE ID Nos: 1 -2) are part of a metabolic gene cluster. In view of the high level of L-tyrosine and L-DOPA in black barley, the inventors suggest to overexpress both HvPPOb and BlpTr to increase production of L-DOPA, and derivatives thereof, such as melanin or precursors thereof, in multiple species including tobacco, rice, wheat, barley, tomato, and duckweed. This overexpression may be achieved under the regulation of different promotors.

[0146] In preliminary experiments, the inventors performed transiently overexpressed HvPPOb and BlpTr in the tobacco species Nicotiana benthamiana (N. Benthamiana). To check the specificity of HvPPOb effects on L-DOPA accumulation, the inventors also overexpressed peyote cactus (L. williamsit) PPO (LwPPO; does not accumulate L-DOPA) and Mucuna Pruriens PPO (MpPPO; L-DOPA over accumulating legume plant) in N. benthamiana alone or in combination with BlpTr (SEQ ID NO: 3). Both enzymes were chosen based on the correlation between their expression and metabolite accumulation, e.g., L-DOPA in Mucuna, and L-DOPA derived mescaline in peyote, which are known from an internal transcriptomic / metabolomic data. As an additional combination that may generate L-DOPA accumulation, the inventors used CYP76AD6, an enzyme from beet eto vulgaris that can generate L-DOPA from tyrosine. This enzyme is from a different protein / enzyme family (i.e., Cytochrome P450). The inventors also generated the DOPA4 construct in which CYP76AD6 is co-expressed with 2 genes that increase tyrosine availability, e.g., the bacterial feedback-insensitive 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (AroG) (the first enzyme of the shikimate pathway), and an amino acid hydroxylase (AAH)from the moss Physcomitrella patens (catalyzing the hydroxylation of phenylalanine to tyrosine). Both the single CYP76AD6 and D0PA4 vectors were transiently overexpressed in N. benthamiana alone or in combination with BlpTR (SEQ ID NO: 3).

[0147] The inventors constructed the following GoldenBraid vectors using tomato UBIQUITIN 10 promoter region (pSIUBQlO') for driving the expression of the target genes: EV - empty a2 vector; BlpTr - a2-pSlUBQ10: BlpTr :tUBQ,' HvPPOb - a2- pSlUBQ10:HvPPOb:tUBQ, LwPPO - a2-pSlUBQ10:LwPPO:tUBQ, MpPPO - a2- pSlUBQ10:MpPPO:tUBQ, CYP76AD6 - a2-pSlUBQ10:CYP76AD6:tUBQ, and DOPA4 a2- pSIUBQ10:CYP76AD6:tUBQ-p35S:AroG:t35S-pNOS:PpAAH:tNOS.

[0148] For each promoter, a corresponding terminator (t) was used, e.g., tUBQ, t35S, and tNOS.

[0149] Individual expression of HvPPOb, MpPPO and CYP76AD6 oxidases led to the accumulation of L-DOPA in N. benthamiana leaves and the effect of MpPPO and CYP76AD6 was significantly higher than HvPPOb. The coexpression of MpPPO and CYP76AD6 with BlpTr caused significant but relatively slight effect (2.8-fold for MpPPO and 2-fold for CYP76AD6) compared to single gene overexpression. However, coexpression of BlpTr with HvPPOb induced a 100-fold elevation in DOPA levels compared to HvPPOb expressing leaves and 2-fold elevation compared to DOPA4 expressing leaves (Fig. 3D). Thus, BlpTr+HvPPOb was the most efficient combination for the induction of L- DOPA accumulation in the N benthamiana leaves substantially increasing it compared to the previously described mechanism.

[0150] Transient overexpression of BlpTR increased the levels of tyrosine (96-fold), phenylalanine (4-fold) and tryptophan (about 8-fold) in N. benthamiana leaves. The combination of transporter with the oxidases resulted in similar results (Fig. 3A-3C). A stronger elevation of the phenylalanine levels was observed in DOPA4 likely due to the enhancement of shikimate pathway by AroG (Fig. 3B). At the same time transporter expression led to slightly increased tyramine amounts (Fig. 3F) and had no significant effect on the L-DOPA and dopamine levels (Fig. 3D-3E).

[0011] As for dopamine and L-DOPA accumulation, no effect was observed for LwPPOb overexpression (Figs. 3D-3E). Overexpressing any one of HvPPOb, MpPPO and CYP76AD6 led to about 25-30-fold increase of dopamine levels in the leaves of N. benthamiana. Noteworthy, combination of the above-mentioned oxidases with BlpTr mostly leveled the increase of dopamine (Fig. 3E).

[0152] Further, the inventors compared the full length (e.g., 1284) BlpTr of the invention (SEQ ID Nos: 3-4) to Blpl008, a short isoform of BlpTr as described in Li et al., (Plant Biotechnology Journal (2024) 22, pp. 1282-1298). In a comparative assay, tyrosine accumulation in N. benthamiana expressing BlpTrl284 (SEQ ID NO: 3) was significantly increased compared to controls, inclusive of BlpTrl008 (SEQ ID NO: 27), in which tyrosine levels were negligible to non-detected (Fig. 4A). Further, levels of phenylalanine and tryptophan were also substantially greater in N benthamiana expressing BlpTr 1284 compared to BlpTrl008 (Figs. 4F-4G). Further, the co-expression of PPOb (SEQ ID NO: 1) and BlpTrl284 (SEQ ID NO: 3) resulted in increased amounts of tyrosine derivatives, including L-DOPA and dopamine (Figs. 4B-4C), this is in sharp contrast to the controls inclusive of BlpTrl284 (SEQ ID NO: 3) alone, BlpTrl008 (SEQ ID NO: 27), and empty vector, wherein these compounds were basically not detected. Further, co-expression of PPOb (SEQ ID NO: 1) and BlpTrl284 (SEQ ID NO: 3) resulted in increased amounts of 3- methoxy-tyramine (Fig. 4E), a precursor in mescaline biosynthesis.EXAMPLE 2

[0153] To investigate the putative subcellular localization of HvPPOb protein (SEQ ID NO: 2), its coding sequence was fused to mRFP (monomeric Red Fluorescent Protein) sequence and the fused HvPPOb-mRFP protein was transiently expressed under S1UBQ promotor in N benthamiana and an infiltrated leaf area was assessed by confocal microscopy. After 36 hours of infiltration the signal of HvPPOb-mRFP was observed at the plasma membrane region (Fig. 5A). Additional incubation of the infiltrated leaf disc in 0.1 M NaCl induced partial plasmolysis and the widening of the signal area (Fig. 5B) suggesting the possible apoplastic localization of the HvPPOb-mRFP. This preliminary data may suggest the non- canonical subcellular localization of HvPPOb. The plant polyphenol oxidases characterized thus far are known to have a plastidial localization that is conditioned by their plastidial signal peptide at the N-terminus, which is not present in HvPPOb (SEQ ID NO: 2).

[0154] According to Li et al. 2024, the short isoform of the transporter BlpTr 1008 (SEQ ID NO: 28) is localized to the plasma membrane. The localization of long BlpTrl284 transporter form (SEQ ID NO: 4), disclosed herein for the first time to the best of our understanding, was examined similarly to HvPPOb (described above). The signal of BlpTrl284-RFP was associated to plasma membrane (Fig. 5C). This suggests that the 92 amino acids of the N-terminus of the long transporter form do not alter the localization of the protein, but rather its substrate preference, likely resulting in higher fidelity of BlpTrl284to transport tyrosine (Fig. 4A). Taken together, the current data may suggest a mechanism of precursor transport as well as L-DOPA and melanin synthesis in plant cell that is different from Li et al., (2024).EXAMPLE 3Metabolic profiling of transgenic wheat hulls shows HvPPOb / BlpTr mediated L- DOPA / dopamine accumulation and melanin formation

[0155] To demonstrate the possible involvement of HvPPOb in L-DOPA / Dopamine biosynthesis and / or melanin formation in cereals, the stable transgenic lines of common wheat (Triticum aestivum, cv. Fielder) were generated. For the generation of the lines, regenerative genes GROWTH-REGULATING FACTOR (GRF) and GRF -INTERACTING FACTOR (GIF) were used together with candidate genes. For single HvPPOb overexpression (OX) GRF-GIF chimera (Prusty et al., 2024, BioRxiv) was joined with HvPPOb-OX cassette (under maize ZmUBQ promotor) resulting in GRF-GIF -HvPPOb lines. In the previous N. Benthamiana transient expression experiments HvBlpTr_1284 increased the tyrosine flux and thus facilitated the action of HvPPOb resulting in the increase of L-DOPA and dopamine. For double gene construct HvPPOb and BlpTR ORFs were joined through p2a peptide junction under double 35S promotor and included into cassette with GRF-GIF resulting in GRF-GIF-D24 lines. The expression of the expressed candidates in the obtained lines in TO were checked by qRT-PCR (in the leaves) and lines with the strongest expression were used for the metabolic analysis. For background metabolite level determination in wheat the GRF-GIF lines were used.

[0156] The metabolite profiling of wheat hulls was conducted at early (ED) and late dough (LD) stages providing clear evidence of altered aromatic amino acid metabolism in the transgenic lines. In the GRF / GIF baseline, the ED stage is characterized by low to zero levels of dopamine and L-DOPA with moderate pools of tyrosine, phenylalanine, and tryptophan. At LD, there is a strong natural increase in aromatic amino acids, particularly tyrosine, phenylalanine, and tryptophan, while dopamine and DOPA remain at trace levels (Figs. 6A- 6F)

[0157] Introduction of HvPPOb dramatically changes this profile. The levels of tyrosine during seed development did not differ from control in hull of GRF -GIF -HvPPOb lines (Fig. 6A). Nonetheless, at ED stage, the HvPPOb overexpressing lines accumulate L-DOPA and dopamine well above the GRF / GIF control (where their concentration was minor). AtLD, the levels of L-DOPA elevated 4 to 50 times compared to ED, but the level of dopamine decreased significantly, although, remained drastically higher than in control (Figs. 6B-6C).

[0158] The most striking changes are observed in the 1)24 lines, particularly in D24#3. At ED D24#3 lines show higher tyrosine and L-DOPA and comparable levels of dopamine than HvPPOb alone (Figs. 6A-6C). At LD it exhibits extreme metabolite accumulation. Tyrosine reaches 2-fold and L-DOPA levels reach more than 12-fold compared to the strongest HvPPOb overexpression line. Dopamine accumulated similarly to GRF -GIF -HvPPOb lines (Figs. 6A-6C)

[0159] Moreover, D24#3 hulls had blackish phenotype (Fig. 7B). This visual phenotype aligns directly with the metabolic data. Plant melanins are typically derived from the oxidation and polymerization of L-DOPA and dopamine by PPO enzymes. The D24#3 line combines both an abundant substrate pool, due to transporter-mediated supply of aromatic amino acids, particularly tyrosine, and high PPO activity to catalyze oxidation. The resulting accumulation of melanin-like polymers explains the blackish pigmentation of the hulls.

[0160] Tyramine content did not change significantly between the studied variants, demonstrating overall decrease in LD relative to ED stage.

[0161] The overexpression of both candidate genes in GRF-GIF-D24 also did not alter the accumulation of phenylalanine and tryptophan (Figs. 6E-6F) in the hull tissues. Earlier, the induction of these aromatic amino acids under transient expression of BlpTrl284 was shown in the leaves of N. Benthamiana (Figs. 3B-3C). In wheat hulls of D24 lines the accumulation of Phe and Trp was not observed (Figs. 6E-6F) possibly due to a limitation in their availability or / and quick metabolization.

[0162] In conclusion, the metabolic and phenotypic data strongly support that D24#3 undergoes melanin biosynthesis in the hulls. HvPPOb channels tyrosine into the L- DOPA / dopamine pathway, BlpTr enhances precursor availability, and the combination produces sufficient flux for visible pigment accumulation. This demonstrates that melanin formation can be engineered in wheat hulls by coordinating enzyme and transporter coexpression. It also highlights that the transporter is a critical factor for achieving the high substrate levels required for polymerization.

[0163] 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 increasing amount or abundance of an amino acid in a cell compared to a control cell, the method comprising: a. providing a cell comprising an artificial vector comprising a nucleic acid sequence having at least 94% homology to SEQ ID NO: 3; and b. culturing said cell from step (a) such that a polypeptide encoded by said artificial vector is expressed, thereby increasing the amount or abundance of the amino acid in the cell compared to a control cell.

2. The method of claim 1, further comprising a step before said step (a), comprising introducing or transfecting said cell with said artificial vector.

3. The method of claim 1 or 2, wherein said amino acid is selected from the group consisting of: tyrosine, phenylalanine, tryptophan, and any combination thereof.

4. A method for synthesizing L-DOPA or a derivative thereof in a cell, the method comprising: a. providing a cell comprising an artificial vector comprising: (i) a first nucleic acid sequence having at least 86% homology to SEQ ID NO: 1; and (ii) a second nucleic acid sequence having at least 94% homology to SEQ ID NO: 3; and b. culturing said cell from step (a) such that a first polypeptide encoded by said first nucleic sequence of said artificial vector and a second polypeptide encoded by said second nucleic sequence of said artificial vector are expressed, thereby synthesizing L-DOPA in the cell.

5. The method of claim 4, further comprising a step before said step (a), comprising introducing or transfecting said cell with said artificial vector.

6. The method of claim 4 or 5, wherein said synthesizing comprises increasing the amount of L-DOPA or a derivative thereof synthesized by said cell compared to a control cell.

7. The method of any one of claims 4 to 6, wherein said L-DOPA derivative comprises dopamine, melanin, betalain, mescaline, morphine, codeine, epinephrine, norepinephrine, papaverine, or any combination thereof.

8. The method of any one of claims 4 to 7, wherein said cell further comprises at least one third nucleic acid sequence encoding at least one third polypeptide being capable of catalyzing conversion of said L-DOPA to said derivative, or an intermediate product therebetween.

9. A method for increasing synthesis of L-DOPA or a derivative thereof in a cell, the method comprising increasing the activity, abundance, or both, of a PPO in a plasma membrane of said cell or an extracellular space at least partially separated from said cell by said plasma membrane, thereby increasing the synthesis of L-DOPA or a derivative thereof in the cell.

10. The method of claim 9, wherein said cell is a transgenic, transformed, or transfected cell.

11. The method of claim 9 or 10, wherein said cell is a plant cell.

12. The method of claim 11, wherein said extracellular space is an apoplast of said plant cell.

13. The method of any one of claim 9 to 12, wherein said PPO comprises a signal sequence targeting said PPO to said plasma membrane of said cell or said extracellular space at least partially separated from said cell by said plasma membrane.

14. The method of claim 13, wherein said PPO comprises the amino acid sequence set forth in SEQ ID NO: 2, or a homolog thereof having at least 83% homology thereto.

15. The method of any one of claims 9 to 14, wherein said cell comprises: (i) a first nucleic acid sequence having at least 86% homology to SEQ ID NO: 1; and (ii) a second nucleic acid sequence having at least 94% homology to SEQ ID NO: 3.

16. The method of claim 15, further comprising introducing or transfecting said cell with an artificial vector comprising said first nucleic acid sequence and said second nucleic acid sequence.

17. The method of any one of claims 9 to 16, wherein said increasing is compared to a control cell.

18. The method of any one of claims 9 to 17, wherein said L-DOPA derivative comprises dopamine, melanin, betalain, mescaline, morphine, codeine, epinephrine, norepinephrine, papaverine, or any combination thereof.

19. The method of any one of claims 9 to 18, wherein said cell further comprises at least one third nucleic acid sequence encoding at least one third polypeptide being capable of catalyzing conversion of said L-DOPA to said derivative, or an intermediate product therebetween.

20. A first isolated DNA molecule comprising a nucleic acid sequence having at least 87% homology or identity to SEQ ID NO: 1.

21. The first isolated DNA molecule of claim 20, wherein said nucleic acid sequence encodes a polypeptide being a polyphenol oxidase (PPO).

22. A second isolated DNA molecule comprising a nucleic acid sequence having at least 94% homology or identity to SEQ ID NO: 3.

23. The second isolated DNA molecule of claim 22, wherein said nucleic acid sequence encodes a polypeptide being an amino acid transporter.

24. An artificial vector comprising any one of: a. the first isolated DNA molecule of claim 20 or 21; b. the second isolated DNA molecule of claim 22 or 23; and c. both (a) and (b).

25. The artificial vector of claim 24, being a plasmid or an expression vector.

26. An isolated polypeptide encoded by any one of:a. the first isolated DNA molecule of claim 20 or 21; b. the second isolated DNA molecule of claim 22 or 23; c. the artificial vector of claim 24 or 25; and d. any combination of (a) to (c).

27. The isolated polypeptide of claim 26, comprising an amino acid sequence having at least 82% homology to SEQ ID NO: 2.

28. The isolated polypeptide of claim 27, characterized by having an activity of a phenol- containing compound oxidation.

29. The isolated polypeptide of claim 28, wherein said phenol-containing compound is tyrosine.

30. The isolated polypeptide of claim 26, comprising an amino acid sequence having at least 93% homology to SEQ ID NO: 4.

31. The isolated polypeptide of claim 30, characterized by having an activity of an amino acid transporter.

32. The isolated polypeptide of claim 31, wherein said amino acid is tyrosine.

33. The isolated protein of any one of claims 26 to 32, comprising a plurality of isolated polypeptides comprising a first amino acid sequence having at least 82% homology to SEQ ID NO: 2, and a second amino acid sequence having at least 93% homology to SEQ ID NO: 4.

34. A transgenic, transduced, or transformed cell comprising any one of: a. the first isolated DNA molecule of claim 20 or 21; b. the second isolated DNA molecule of claim 22 or 23; c. the artificial vector of claim 24 or 25; d. the isolated polypeptide of one of claims 26 to 33; and e. any combination of (a) to (d).

35. The transgenic, transduced, or transformed cell of claim 34, being any one of: a unicellular organism, a cell of a multicellular organism, and a cell in a culture.

36. The transgenic, transduced, or transformed cell of claim 35, wherein said unicellular organism comprises a fungus or a bacterium.

37. The transgenic, transduced, or transformed cell of claim 36, wherein said fungus is a yeast cell.

38. The transgenic, transduced, or transformed cell of any one of claims 35 to 37, wherein said multicellular organism is a plant or a part thereof.

39. A transgenic plant, a part thereof, or a tissue derived therefrom, comprising: a. the first isolated DNA molecule of claim 20 or 21; b. the second isolated DNA molecule of claim 22 or 23; c. the artificial vector of claim 24 or 25; d. the isolated polypeptide of one of claims 26 to 33; e. the transgenic, transduced, or transformed cell of any one of claims 34 to 38; and f. any combination of (a) to (e).

40. The transgenic plant, part thereof, or tissue derived therefrom of claim 39, being a plant selected from the group consisting of: tobacco, rice, wheat, barley, tomato, and duckweed.

41. A composition comprising any one of: a. the first isolated DNA molecule of claim 20 or 21; b. the second isolated DNA molecule of claim 22 or 23; c. the artificial vector of claim 24 or 25; d. the isolated polypeptide of one of claims 26 to 33;e. the transgenic, transduced, or transformed cell of any one of claims 34 to 38; f. the transgenic plant, part thereof, or tissue derived therefrom of claim 39 or 40; and g. any combination of (a) to (f); and an acceptable carrier.

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