Compositions including CYP76AD1-β clade peptides and their uses

Abstract

This invention provides recombinant polynucleotides comprising nucleic acids encoding CYP76AD6 or related genes, and their use for the production of L-DOPA from tyrosine and for the treatment of dopamine-responsive disorders such as Parkinson's disease. This invention also provides recombinant polynucleotides comprising nucleic acids encoding CYP76AD1 and / or CYP76AD6, nucleic acids encoding DOPA 4,5-dioxygenase (DOD) such as beet DODA1, and in some cases, nucleic acids encoding betaine-related glucosyltransferases such as the four o'clock gene cyclic-DOPA 5-O-glucosyltransferase (cDOPA5GT), and their use for the production of betaine. Finally, this invention provides chimeric polypeptides, expression vectors, cells, compositions, and organisms (including plants), and their use in the various methods of this invention.

Description

Technical Field

[0001] This invention provides recombinant polynucleotides comprising nucleic acids encoding CYP76AD6 or related genes, and their use for the production of L-DOPA from tyrosine and for the treatment of dopamine-responsive disorders such as Parkinson's disease. This invention also provides recombinant polynucleotides comprising nucleic acids encoding CYP76AD1 and / or CYP76AD6, nucleic acids encoding DOPA 4,5-dioxygenase (DOD) such as beet DODA1, and in some cases, nucleic acids encoding betaine-related glucosyltransferases such as the four o'clock gene cyclic-DOPA 5-O-glucosyltransferase (cDOPA5GT), and their use for the production of betaine. Finally, this invention provides chimeric polypeptides, expression vectors, cells, compositions, and organisms (including plants), and their use in the various methods of this invention. Background Technology

[0002] Betaines are tyrosine-derived, reddish-purple and yellow plant pigments found only in a group of angiosperms (Caryophyllales), where they exist in a manner that is chemically distinct from and widely distributed anthocyanin pigments. The betaine class contains a large number of compounds, which are generally divided into two groups: the reddish-purple betaine red and the yellow betaine xanthophyll.

[0003] The key enzyme in betaine biosynthesis, DOPA 4,5-dioxygenase (DOD), converts L-DOPA into betaine aldehydes, which form the basic backbone of all betaine pigments. Figure 1 The spontaneous binding of betaine with amines or with L-DOPA derivatives leads to the formation of yellow betaine xanthophyll or reddish-purple betaine red, respectively. Betaine pigment-related glucosyltransferases have also been characterized in several Caryophyllales species, catalyzing the 5-O glucosylation of cyclic DOPA or, alternatively, the 5-O or 6-O glucosylation of betaine. The enzyme catalyzing the formation of L-DOPA from tyrosine is unknown.

[0004] Due to their high stability, pH independence, and antioxidant properties, beet pigments can be used as natural food colorings and dietary supplements.

[0005] While there are many potential edible plant sources of anthocyanins, betaine pigments are rarely found in edible plants, with red beet being the only major source of betaine pigment extracts currently used commercially. Despite its high betaine content, red beet extract has several drawbacks as a food coloring source; it primarily produces betaine glycosides, thus limiting color variability; it carries an unpleasant earthy taste due to the presence of geosmin and various pyrazines; and there is a risk of soil-borne microbial residues. Currently, there is no large-scale use of naturally derived betaine xanthophyll as a food dye. For example, yellow beet is unlikely to be used due to the presence of phenolic compounds that are easily oxidized and mask the yellow hue of betaine xanthophyll. Clearly, it is beneficial to develop alternative sources of betaine pigments, particularly betaine xanthophyll, as there are currently no commercially available natural yellow water-soluble pigments for the food industry. Heterogeneous production of betaine pigments could provide many novel viable sources for these pigments, such as plants, plant cell cultures, algae, and yeast.

[0006] L-3,4-dihydroxyphenylalanine (L-DOPA), an intermediate in the betaine pathway, is also a commercially valuable metabolite widely used in the treatment of Parkinson's disease.

[0007] Parkinson's disease is a progressive neurological disorder that primarily affects the motor system. It is the second most common neurodegenerative disease and the most common motor disorder, affecting an estimated 5 million people worldwide. A key characteristic of Parkinson's disease is low dopamine levels, an important signaling molecule in the nervous system. The most effective treatment for Parkinson's disease is the administration of L-DOPA (3,4-dihydroxyphenylalanine), which is converted into dopamine in the brain. Because dopamine cannot cross the blood-brain barrier, L-DOPA is the most effective medication for treating Parkinson's disease.

[0008] L-DOPA is also widely marketed as a dietary supplement and is a precursor to other high-value metabolites, including, for example, catecholamines (such as dopamine and adrenaline), benzyl isoquinoline alkaloids (such as morphine and other opioids), betaine, and melanin.

[0009] Although L-DOPA is produced in many plant and animal species, it rarely accumulates in large quantities. This is partly due to the fact that L-DOPA is generally formed by tyrosinases, which catalyze the hydroxylation of tyrosine to L-DOPA, but also immediately convert L-DOPA to its oxidized form, dopaquinone.

[0010] L-DOPA for the pharmaceutical industry is currently prepared using one of several methods, all of which have significant limitations because chemical synthesis can only be achieved in expensive processes involving numerous chemical reactions and requiring costly substrates and demanding production conditions. The production of L-DOPA using tyrosinase (also known as polyphenol oxidase-PPO) biotechnology has also been explored. The dual reaction of the aforementioned tyrosinase is problematic for the commercial production of L-DOPA because the product of interest is directly metabolized to commercially unusable dopaquinone, thus becoming a major bottleneck for its enzymatic biosynthesis to produce L-DOPA on a large scale from tyrosine. Therefore, a more efficient and cheaper method for preparing L-DOPA is needed, and this need remains unmet.

[0011] Compared to other major categories of plant pigments (i.e., anthocyanins and carotenoids), the biosynthesis of betaine is still not well understood, especially regarding the enzymes that catalyze the conversion of tyrosine to L-DOPA in the betaine synthesis pathway of the Caryophyllales order. Summary of the Invention

[0012] In one embodiment, the present invention provides a recombinant polynucleotide comprising a nucleic acid encoding the CYP76AD6 gene, a nucleic acid encoding the CYP76AD15 gene, or a combination thereof, under the control of a promoter. In one embodiment, the polynucleotide further comprises a nucleic acid encoding DOPA 4,5-dioxygenase (DOD).

[0013] In another embodiment, the present invention provides a recombinant polynucleotide comprising a nucleic acid encoding CYP76AD1, a nucleic acid encoding DOPA 4,5-dioxygenase (DOD), and a nucleic acid encoding a betaine-associated glucosyltransferase, wherein the nucleic acids are in-frame inserted into the polynucleotide. In one embodiment, the polynucleotide further comprises a nucleic acid encoding CYP76AD6.

[0014] In another embodiment, the present invention provides a composition comprising the recombinant polynucleotide as described above.

[0015] In another embodiment, the present invention provides an expression vector comprising the recombinant polynucleotide as described above.

[0016] In another embodiment, the present invention provides a cell comprising the expression vector described above.

[0017] In another embodiment, the present invention provides a chimeric polypeptide encoded by the recombinant polynucleotide as described above.

[0018] In another embodiment, the present invention provides a method for generating L-DOPA in cells, comprising the steps of: contacting the cells with a recombinant polynucleotide including a nucleic acid encoding a CYP76AD6 gene, a nucleic acid encoding a CYP76AD15 gene, or a combination thereof, under conditions sufficient to generate L-DOPA, thereby generating L-DOPA.

[0019] In another embodiment, the present invention provides a method for producing L-DOPA from tyrosine, comprising the steps of: combining CYP76AD6, CYP76AD15 or a combination thereof with tyrosine under conditions sufficient to produce L-DOPA, thereby producing L-DOPA.

[0020] In another embodiment, the present invention provides the use of the CYP76AD1-β clade gene or one or more cells from an organism comprising high levels of the CYP76AD1-β clade gene in the preparation of a composition for treating or inhibiting a patient’s dopamine-responsive disorder.

[0021] In another embodiment, the present invention provides a method for producing beet pigment, the method comprising the steps of: contacting, under conditions sufficient to produce beet pigment, a recombinant polynucleotide comprising a nucleic acid encoding a CYP76AD6 gene, a nucleic acid encoding a CYP76AD15 gene, or a combination thereof, under the control of a promoter, a nucleic acid encoding DOPA 4,5-dioxygenase (DOD), and a nucleic acid encoding CYP76AD1 with one or more cells, wherein the nucleic acid is in-frame inserted into the polynucleotide, thereby producing beet pigment.

[0022] In another embodiment, the present invention provides a method for producing betaine, the method comprising the steps of: contacting, under conditions sufficient to produce betaine, a recombinant polynucleotide comprising a nucleic acid encoding a CYP76AD6 gene, a nucleic acid encoding a CYP76AD15 gene, or a combination thereof, under the control of a promoter, and a nucleic acid encoding DOPA 4,5-dioxygenase (DOD), with one or more cells, wherein the nucleic acid is in-frame inserted into the polynucleotide, thereby producing betaine.

[0023] In another embodiment, the present invention provides a method for increasing the resistance of a plant or plant part to one or more biotic or abiotic stressors, comprising the steps of: contacting one or more cells of the plant or plant part with a nucleic acid encoding CYP76AD6, a nucleic acid encoding CYP76AD15, a nucleic acid encoding CYP76AD1, or a combination thereof, a nucleic acid encoding DOPA 4,5-dioxygenase (DOD), and optionally a nucleic acid encoding a betaine-associated glucosyltransferase, under conditions sufficient to produce betaine, thereby producing betaine and increasing the resistance of the plant or plant part to the one or more stressors.

[0024] In another embodiment, the present invention provides a method for increasing the resistance of a plant or plant part to one or more fungal diseases, comprising the steps of: contacting one or more cells of a plant or plant part with a nucleic acid encoding CYP76AD6, a nucleic acid encoding CYP76AD15, a nucleic acid encoding CYP76AD1, or a combination thereof, a nucleic acid encoding DOPA 4,5-dioxygenase (DOD), and optionally a nucleic acid encoding a betaine-associated glucosyltransferase, under conditions sufficient to produce betaine, thereby producing betaine and increasing the resistance of the plant or plant part to the one or more fungal diseases.

[0025] In another embodiment, the present invention provides a method for increasing the level of one or more betaine pigments in an organism or a portion of an organism, comprising inducing or permitting the expression of DOPA 4,5-dioxygenase (DOD) and CYP76AD6, CYP76AD1, or combinations thereof in at least a portion of the organism, wherein if the organism expresses CYP76AD1 and DOD instead of CYP76AD6 or CYP76AD15, the organism also expresses a betaine-associated glucosyltransferase, thereby increasing the level of one or more betaine pigments in the organism or the portion of the organism.

[0026] In another embodiment, the present invention provides an organism or a portion thereof comprising a nucleic acid sequence encoding a CYP76AD1-α clade gene, a nucleic acid sequence encoding a CYP76AD1-β clade gene, or a combination thereof, and a recombinant nucleic acid sequence encoding a nucleic acid sequence encoding DOPA 4,5-dioxygenase (DOD).

[0027] In another embodiment, the present invention provides a method for producing betaine from tyrosine, comprising the steps of: contacting a composition comprising CYP76AD1, DOPA 4,5-dioxygenase (DOD), and CYP76AD6, CYP76AD15, or combinations thereof with tyrosine under conditions sufficient to produce betaine, thereby producing betaine.

[0028] In another embodiment, the present invention provides a method for producing betaine from tyrosine, comprising the steps of: contacting a composition comprising CYP76AD6, CYP76AD15 or a combination thereof, and DOPA 4,5-dioxygenase (DOD) with tyrosine under conditions sufficient to produce betaine, thereby producing betaine. Attached Figure Description

[0029] The subject matter considered to be the invention is specifically pointed out and clearly claimed in the concluding section of the specification. However, regarding the organization and operation methods, as well as its purpose, features, and advantages, the invention can be understood by referring to the following detailed description and accompanying drawings. Figure 1 For best reading comprehension, see the attached image:

[0030] Figure 1 Betaine biosynthetic pathway. Genes involved in the pathway are shown in magenta. Enzymatic reactions: EI, hydroxylation of tyrosine; EII, DOPA-4,5 dioxygenase; EIII, oxidation of DOPA; EIV, glucosylation of cyclic DOPA; EV, glucosylation of betaine; EVI, additional glucosylation of betaine; EVII, acylation of betaine. Predicted spontaneous reaction: SI, condensation (formation of aldehyde imine). Spontaneous cyclization reactions follow enzymatic reactions EII and EIII and are therefore marked with an asterisk. EV can alternatively be catalyzed by betaine-6-O-glucosyltransferase, leading to the formation of betaine instead of betaine glycoside. Dashed lines indicate reactions of the alternative pathway, shown in four o'clocks, where cyclic DOPA is first glycosylated and then condensed with betaine aldehydes to form betaine glycosides.

[0031] Figure 2A-2G MjCYP76 (SEQ ID NO: 1) was identified as a candidate gene associated with betaine pigments. Gene expression in Mirabilis jalapa was analyzed in transcriptome datasets from 24 tissues, including four floral organ types across five developmental stages, red or green stem epidermis, and red or green leaves. Figure 2A :petal. Figure 2B Stamens and filaments. Figure 2C : Stem epidermis from nodes or internodes. Figure 2D :stigma. Figure 2E :anther. Figure 2F Red young or mature green leaves. Figure 2GThe beet pigment-related genes cyclic-DOPA-5-O-glucosyltransferase (cDOPA5GT; SEQ ID NO: 2), DOPA 4,5-dioxygenase (MjDOD; SEQ ID NO: 3), and cytochrome P450 CYP76AD3 (gene bank accession number HQ656026.1; SEQ ID NO: 4) exhibited a parallel pigment accumulation expression pattern. The cytochrome P450 gene MjCYP76 (SEQ ID NO: 1) was highly co-expressed with cDOPA5GT (Pearson correlation r = 0.90) and less co-expressed with MjDOD (r = 0.67).

[0032] Figure 3 Gene expression patterns of polyphenol oxidase encoding genes MjPPO1 (SEQ ID NO: 5); MjPPO2 (SEQ ID NO: 6); MjPPO3 (SEQ ID NO: 7); and MjPPO4 (SEQ ID NO: 8) as deduced from the Mirabilis jalapa RNA sequence dataset.

[0033] Figure 4A Following infection with the pTRV2:CYP76AD1 or pTRV2:CYP76AD1-CYP76AD6 vector, qRT-PCR analysis of the four CYP76AD paralogs showed partial downregulation, indicating off-target silencing. Upregulation of Bv20048 was observed in tissues infected with pTRV2:CYP76AD1-CYP76AD6. Relative quantification (RQ) values ​​are expressed as the mean ± SE of three biological replicates. An asterisk indicates statistical significance of differential expression compared to unsilenced tissues. *, P < 0.05; **, P < 0.01.

[0034] Figure 4B Phylogenetic tree of CYP76AD paralogs in sugar beets. CYP76AD6 (Genome Accession No. KT962274; SEQ ID NO: 30); CYP76AD1 (Genome Accession No. AET43289.1; SEQ ID NO: 14); BvCYP76new (Genome Accession No. KU041699; SEQ ID NO: 9); Bv20048 (Genome Accession No. KU041700); Bv10427 (Genome Accession No. KU041701); Bv31911 (Genome Accession No. KU041702); Bv15899 (Genome Accession No. KU041703); Bv15885 (Genome Accession No. KU041704).

[0035] Figures 5A-5CCo-silencing of CYP76AD1 and CYP76AD6 inhibited betalain production. Virus-induced gene silencing (VIGS) assays in red beets were used to silence either BvDODA1 or CYP76AD1. CYP76AD1 was also co-silenced with each of two novel candidates, BvCYP76new (SEQ ID NO: 9) and CYP76AD6. While silencing CYP76AD1 alone or co-silencing with BvCYP76new prevented the formation of betalains but not betalains, co-silencing of CYP76AD1 and CYP76AD6 prevented the production of both types of pigments, resulting in a phenotype similar to that obtained by silencing BvDODA1. Reduced betalain levels were observed through a visible decrease in yellow color and a lack of fluorescence under blue light (a typical characteristic of betalains). Figure 5A The image presents a whole-plant image 3.5 weeks after infiltration; Figure 5B The image shows a single leaf, bright field, and... Figure 5C The image shows a single leaf in blue light.

[0036] Figure 6A Relative quantification of betalains in VIGS-silenced beet leaves. Relative betalain content was assessed spectrophotometrically by absorbance measurements at 475 nm and 600 nm. Values ​​represent the mean ± SEM of four biological replicates, each replicate consisting of gene-silencing tissues from two to three plants. An asterisk indicates a statistically significant difference from pTRV2:CYP76AD1. **, P < 0.01. RQ, relative quantification.

[0037] Figure 6B Quantitative real-time PCR (qRT-PCR) analysis of CYP76AD1 and CYP76AD6. CYP76AD1 was downregulated in tissues infected with pTRV2:CYP76AD1 or pTRV2:CYP76AD1-CYP76AD6. CYP76AD6 was significantly downregulated after infection with pTRV2:CYP76AD1-CYP76AD6. Relative quantitation (RQ) values ​​are expressed as the mean ± SE of three biological replicates. An asterisk indicates statistical significance of differential expression compared to uninfected plants. **, P < 0.01.

[0038] Figures 7A-7F Recombinant expression of CYP76AD1 or CYP76AD6 in tobacco leaves enables the production of L-DOPA and betaine. Figure 7A Co-infiltration of Agrobacterium containing plasmids used to express CYP76AD1 in tobacco leaves with cDOPA5GT (pAD1-GT) and BvDODA1 (pDODA) resulted in red pigment deposition. Figure 7BLC-MS analysis of the red pigment tissue revealed the presence of betaine and isobetaine. Figure 7C Co-infiltration of the CYP76AD6 expression vector (pAD6) and pDODA in tobacco leaves led to yellow pigment deposition. Figure 7D The infiltration of pAD1-GT into tobacco leaves enables the production of L-DOPA. L-DOPA was not detected in the control experiment expressing YFP (pYFP). Figure 7E Analysis of tissues containing yellow pigment deposits allowed for the identification of several betaine compounds, including pomegranate cactus flavonoids. Figure 7F The infiltration of pAD6 into *Nicotiana benthamiana* leaves enables the production of L-DOPA. L-DOPA (pDODA + pYFP) was not detected in the control experiment. XIC, extracted ion chromatogram.

[0039] Figures 8A-8C Cytochrome P450 CYP76AD1 and CYP76AD6 differ in activity and phylogenetics.

[0040] Figure 8A Recombinant expression of CYP76AD1, CYP76AD6, and BvDODA1 in yeast cells. The images presented are of yeast grown in medium after overnight galactose induction. Each yeast clone was transformed with three vectors to express CYP76AD1, CYP76AD6, BvDODA1, and β-glucuronidase (GUS) in different combinations and grown in standard SD medium without L-DOPA supplementation. Combined expression of BvDODA1 and CYP76AD1 resulted in reddish-purple pigment deposition in the medium, while expression of BvDODA1 with CYP76AD6 resulted in yellow pigment deposition, indicating a difference in catalytic activity between CYP76AD1 and CYP76AD6. Expression of BvDODA1 with both cytochrome P450s resulted in orange-red pigment deposition in the medium. No pigment deposition was observed when each cytochrome P450 or BvDODA1 was expressed alone.

[0041] Figure 8BMultiple sequence alignment of CYP76AD1-like protein sequences from caryophyllales plants that produce betaine pigments was processed into a phylogenetic tree of maximum probability, resulting in the formation of two separate clades, named the α and β clades (Brockington et al., 2015). CYP76AD6 belongs to the β clade, while CYP76AD1 belongs to the α clade. Within the CYP76AD1-β clade: B. vulgaris, beet (Beta vulgaris) (CYP76AD6; SEQ ID NO: 30); Bv. maritima, beet subspecies maritima (accession number AKI33834.1; SEQ ID NO: 10); F. latifolia, Froelichia latifolia (accession number AKI33838.1; SEQ ID NO: 11); A. caracasana, prickly amaranth (Alternanthera caracasana) (accession number AKI33835.1; SEQ ID NO: 12); A. ficoidea, five-colored amaranth (Alternanthera ficoidea) (accession number AKI33831.1; SEQ ID NO: 13). Within the CYP76AD1-α clade: B. vulgaris, beetroot (CYP76AD1; accession number AET43289.1; SEQ ID NO: 14); A. cruentus, amaranth (Amaranthus cruentus) (CYP76AD2; accession number AET43291.1; SEQ ID NO: 15); M. jalapa, four o'clock flower (Mirabilis jalapa) (CYP76AD3; accession number AET43292.1; SEQ ID NO: 16); C. cristata, cockscomb flower (Celosia cristata) (CYP76AD4; accession number AGI78466.1; SEQ ID NO: 17).

[0042] Figure 8CLiquid chromatography-mass spectrometry (LC-MS) analysis of the pigment yeast culture medium showed that, in addition to several betaine chromatids (including glutamine-betaine and valine betaine (Table 2; not shown in the chromatograms)) were observed when CYP76AD1, CYP76AD6, or both were expressed with BvDODA1. Betaine red betaine and isobetaine were observed only with CYP76AD1 expression. No peaks corresponding to betaine pigment compounds were found without BvDODA1 expression. The y-axis (peak intensity) of all six chromatograms were correlated. XIC, extracted ion chromatogram corresponding to the mass of betaine aldehyde [M + H = 212.05] and betaine isomer [M + H = 389.10]. Time, retention time (minutes).

[0043] Figure 9 Alignment of the protein sequences of CYP76AD1 (SEQ ID NO: 14) and CYP76AD6 (SEQ ID NO: 18) showed 72% identity between them.

[0044] Figure 10 Schematic diagram of the pX11 overexpression vector (SEQ ID NO: 19). KanR, nptII gene conferring kanamycin resistance; DODA1, beet DOPA 4,5-dioxygenase; CYP76AD1, beet cytochrome P450; cDOPA5GT, Mirabilis ring-DOPA-5-O-glucosyltransferase; nos, carmine synthase promoter / terminator; 35S, CaMV 35S promoter / terminator; Act2, Arabidopsis actin 2 terminator; Ub10, Arabidopsis ubiquitin 10 promoter;

[0045] Figure 11A-1 1B. Betaine is produced in a variety of natural, non-productive plant species.

[0046] Figure 11AAgrobacterium-mediated transformation of the pX11 vector overexpressing cytochrome P450 CYP76AD1, DOPA 4,5-dioxygenase (BvDODA1), and cyclic DOPA5-O-glucosyltransferase (cDOPA5GT) induces the development of reddish-purple callus or germination in various Solanaceae species, typically observed within 1–2 weeks in the following tissue cultures: Solanum lycopersicum (tomato), Solanum tuberosum (potato), Solanum melongena (eggplant), Nicotiana tabacum (tobacco), Nicotiana glauca (tree tobacco), Solanum nigrum (European black nightshade), Petunia x hybrida (petunia), and Nicotiana benthamiana.

[0047] Figures 11B1-11B2 Liquid chromatography-mass spectrometry (LC-MS) was used to analyze the properties of tobacco trees (Tobacco monotrees). Figure 11B1 ) and Benedict's tobacco ( Figure 11B2 Callus tissue was sampled and analyzed to allow for the identification of betaine betaine glycosides and isobetaine from both species, in addition to the identification of a novel, unidentified betaine compound (betaine III; Table 2) found only in tobacco tree tissue. XIC, extraction ion chromatograms corresponding to the masses of the novel betaine [M + H = 593.10] and the betaine glycoside isomer [M + H = 551.10]. Time, retention time (minutes).

[0048] Figures 12A-12C. Identification and quantification of betalains in engineered and naturally produced species.

[0049] Figure 12A: LC-MS analysis of *Nicotiana benthamiana* leaves infiltrated with *Agrobacterium tumefaciens* vector pX11 ( Figure 12A2 It can recognize betaine, which exhibits similarity to extracts from red beet (sweet beet) leaves ( Figure 12A1 It has the same precise mass, retention time, fragmentation mode, and UV-VIS absorption spectrum as betaine.

[0050] Figure 12B Introducing Agrobacterium containing the pX11 vector into Tobacco Benzoenta leaves resulted in strong red pigment deposition in the introduced area, which was usually observed within 2-3 days.

[0051] Figure 12CThe betaine content of pX11-Agrobacterium-infiltrated and pX11-transformed tobacco was evaluated compared with several betaine-producing species. Betaine content was assessed spectrophotometrically by measuring absorbance at 535 nm and 600 nm. Values ​​represent the mean ± SE of four biological replicates, each containing 100 mg of tissue (fresh weight).

[0052] Figures 13A-13E Pigmentation phenotypes observed in transgenic tobacco plants engineered to produce beet pigments. Three beet pathway genes, namely CYP76AD1, BvDODA1, and cDOPA5GT (in the pX11 binary vector), are heterologously expressed in tobacco, resulting in red pigment deposition in various plant organs, including leaves, stems, roots, and flowers. Figure 13A Left: Wild-type tobacco; Right: PX11-converted tobacco. Figure 13B Top row: wild-type tobacco flowers; bottom row: pX11 flowers. On pX11 roots ( Figure 13C ), stem epidermis, magnified 20 times ( Figure 13D ), and leaf glandular hairs, magnified 20 times ( Figure 13E Pigment accumulation in the ) is also obvious.

[0053] Figures 14A-14B It produces betaine pigments in food crops such as tomatoes, potatoes, and eggplants.

[0054] Figure 14A Transformation of the pX11 vector resulted in the formation of red pigment plants in potatoes (Solanum tuberosum var. Désirée - top left), eggplants (Solanum melongena line DR2 - bottom left), and tomatoes (Solanum lycopersicum var. MicroTom - right).

[0055] Figure 14B Betaine and isobetaine were identified as the main betalains in potato tubers, tomato fruits, and eggplant fruits by LC-MS analysis. Extracted ion chromatograms (XIC) of the corresponding betaine / isobetaine masses [M + H = 551.1] and the betaine peak UV-VIS absorption for all three tissues are shown.

[0056] Figure 15Schematic diagram of overexpression vectors for pX11 (SEQ ID NO: 19), pX11 (E8) (SEQ ID NO: 20), pX11 (CHS) (SEQ ID NO: 21), pX12 (SEQ ID NO: 22), and pX13 (SEQ ID NO: 23). KanR, nptII gene conferring kanamycin resistance; DODA1, beet DOPA 4,5-dioxygenase; CYP76AD1 / CYP76AD6, beet cytochrome P450; cDOPA5GT, four o'clock ring-DOPA-5-O-glucosyltransferase; nos, carmine synthase promoter / terminator; 35S, CaMV 35S promoter / terminator; Act2, Arabidopsis actin 2 terminator; Ub10, Arabidopsis ubiquitin 10 promoter; E8, tomato E8 promoter; CHS, petunia chalcon synthase promoter.

[0057] Figures 16A-16B Fruit-specific accumulation of betaine in tomatoes.

[0058] Figure 16A Introducing the pX11(E8) vector into tomato (Solanum lycopersicum var. M-82) resulted in beet red pigment deposition in mid- to mature fruits.

[0059] Figure 16B : Whole fruit and cross section of wild-type and pX11 (E8) tomato fruit.

[0060] Figures 17A-17B The color changes in genetically modified tobacco plants that produce betalains were determined by altering the betalain / betaflavin ratio.

[0061] Figure 17A Introducing pX11, pX12, or pX13 carriers into tobacco results in flowers of different colors. When viewed from the top (top row) or magnified and observed under bright field (middle row) or blue light (bottom row), betaine typically exhibits fluorescence.

[0062] Figure 17B LC-MS analysis of tobacco petals of pX11, pX12, and pX13. Extracted ion chromatograms (XICs) of masses [M + H = 309.1, 331.1, 340.1, 551.1] correspond to proline-betaine, tyramine-betaine, glutamine-betaine, and betaine / isobetaine, respectively. Vertical axis correlation.

[0063] Figures 18A-18B Betaine is produced in tobacco BY2 cells.

[0064] Figure 18AIntroducing pX11 or pX13 into tobacco BY2 cells resulted in red-purple or yellow-orange pigment deposition, respectively.

[0065] Figure 18B LC-MS analysis of pX11 and pX13 BY2 cells. Extracted ion chromatograms (XIC) of mass [M + H = 283.1, 340.1, 551.1], corresponding to alanine-betaine, glutamine-betaine, and betaine / isobetaine, respectively. Vertical axis correlation.

[0066] Figure 19 Schematic diagram of the pDOPA1-pDOPA4 (SEQ ID NO: 24-27) overexpression vector. nptII, neomycin phosphotransferase II, a gene conferring kanamycin resistance; CYP76AD6, beet cytochrome P450; AroG, Escherichia coli AroG175 (DAHPS) mutant; AAH, sphagnum moss aromatic amino acid hydroxylase; 35S, CaMV 35S promoter / terminator; Ub10, tomato ubiquitin 10 promoter.

[0067] Figures 20A-20C L-DOPA is produced in tobacco plants and BY2 cells. Figure 20A L-DOPA in leaf extracts of tobacco plants expressing pDOPA1 and pDOPA3 was identified by LC-MS analysis. Figure 20B Expression of pDOPA2 and pDOPA4 in BY2 cells caused varying degrees of melanization of callus tissue after several weeks of culture. Figure 20C The relative quantification of L-DOPA in pDOPA2, pDOPA4 and wild-type BY2 cells was performed by LC-MS analysis.

[0068] Figures 21A-21B Seed germination determination of tobacco that produces betaine under osmotic and salinity stress conditions. Figure 21A Wild-type and pX11 tobacco seedlings grown under high salinity stress conditions (150 mM NaCl) and control conditions (MS) were photographed and seed germination rates were recorded. Figure 21B Wild-type and pX11 tobacco seedlings grown under osmotic stress conditions (400 mM mannitol) and control conditions (MS) were photographed and seed germination rates were recorded.

[0069] Figures 22A-22C Analysis of botrytis cinerea resistance in tobacco plants that produce betaine. Figure 22A Leaves of wild-type and pX11 tobacco plants were infected with droplets of Botrytis cinerea suspension and photographed 5 days after infection. Figure 22BLesion size was recorded for wild-type and pX11 plants infected with a total of approximately 500 Botrytis cinerea species per plant. The average lesion area is shown for approximately 30 plants per genotype. Figure 22C Compared to the leaves of pX11 plants, infected leaves of wild-type tobacco plants showed increased signs of necrosis.

[0070] Figures 23A-23D Recombinant expression of CYP76AD15 in Nicotiana benthamiana enables the production of betaine and L-DOPA. Figure 23A Co-infiltration of Agrobacterium containing plasmids used to express CYP76AD15 and BvDODA1 in tobacco leaves resulted in yellow pigment deposition in the infiltrated area. Figure 23B LC-MS analysis of the yellow pigment tissue revealed the presence of several betaine flavonoids, including dopamine-betaine [M + H = 347.1] and valine-betaine [M + H = 311.1]. XIC, extraction ion chromatogram. Figure 23C : UV-VIS absorption of peaks representing dopamine-betaine and valine-betaine in the analyzed tissue. Figure 23D Mass spectrometry of the L-DOPA peak identified in leaf tissue of Nicotiana benthamiana expressing CYP76AD15 showed typical L-DOPA fragments [M + H = 181.05, 152.07] and molecular ions [M + H = 198.07]. Detailed Implementation

[0071] In the following detailed description, numerous specific details are set forth in order to provide a thorough understanding of the invention. However, those skilled in the art will understand that the invention can be practiced without these specific details. In other instances, well-known methods, procedures, and components have not been described in detail so as not to obscure the invention.

[0072] In one embodiment, the present invention provides a composition comprising an enzyme that catalyzes the first step of betaine synthesis in the Caryophyllales order, and the use of the enzyme in plant genetic engineering and for the production of L-DOPA from tyrosine. Typically, tyrosinases are substrate-mixed oxygenases that exhibit undesirable catechol oxidase activity. Previous studies have shown that plant cytochrome P450 CYP76AD1 exhibits this characteristic mixing, and that undesirable byproducts are still produced despite screening strategies identifying mutations that increase enzyme substrate specificity and activity (DeLoache et al., 2015; Nat Chem Biol. 2015 July; 11(7): 465-71, the entire contents of which are incorporated herein by reference). Therefore, it is not expected that different plant cytochrome P450s will be specific to tyrosine and catalyze only the reaction for L-DOPA, thus avoiding the accumulation of undesirable end products.

[0073] CYP76AD6

[0074] This invention provides a novel gene (belonging to the cytochrome P450 gene family) that catalyzes the hydroxylation of tyrosine to form L-DOPA. This enzyme potentially offers significant advantages over current tyrosinases used to produce L-DOPA; while tyrosinases catalyze the formation of L-DOPA from tyrosine and immediately oxidize it to the unusable metabolite dopaquinone, the novel P450 enzyme catalyzes only the formation of L-DOPA without oxidizing it. Therefore, this P450 enzyme can be used for a more efficient method of L-DOPA production than using tyrosinases.

[0075] Therefore, in one embodiment, the present invention provides a novel enzyme that can be used for the biosynthesis of L-DOPA, a drug for treating Parkinson's disease, and a precursor to economically important drug benzylisoquinoline alkaloids (e.g., morphine). This enzyme can potentially be used for the commercial production of L-DOPA in several ways; its encoding gene can be expressed in plants or microorganisms to induce in vivo production of L-DOPA, or alternatively, it can be recombinantly expressed and used for in vitro enzymatic catalysis.

[0076] L-DOPA and betaine production methods

[0077] Produced from proteins

[0078] In one embodiment, the method of the present invention is carried out in vitro, such that the tyrosine and enzymes required for the production of L-DOPA or beet pigment are provided in vitro.

[0079] In one embodiment, the present invention provides a method for producing L-DOPA from tyrosine, comprising the steps of: combining a CYP76AD1-β clade protein with tyrosine under conditions sufficient to produce L-DOPA, thereby producing L-DOPA.

[0080] In one embodiment, the present invention provides a method for producing L-DOPA from tyrosine, comprising the steps of: combining CYP76AD6, CYP76AD15 or a combination thereof with tyrosine under conditions sufficient to produce L-DOPA, thereby producing L-DOPA.

[0081] In another embodiment, the present invention provides a method for producing L-DOPA from tyrosine in vitro, comprising the steps of: combining CYP76AD6, CYP76AD15 or a combination thereof with tyrosine under conditions sufficient to produce L-DOPA, thereby producing L-DOPA.

[0082] In another embodiment, the present invention provides a method for producing betaine from tyrosine, comprising the step of contacting tyrosine with CYP76AD6, CYP76AD1, or a combination thereof under conditions sufficient to produce betaine, thereby producing betaine. In one embodiment, the method further comprises the step of contacting tyrosine with CYP76AD6, CYP76AD15, or a combination thereof, and CYP76AD1 or a combination thereof with DOPA 4,5-dioxygenase (DOD).

[0083] In another embodiment, the present invention provides a method for producing betaine from tyrosine, comprising the steps of: contacting tyrosine with a CYP76AD1-β clade protein and DOPA 4,5-dioxygenase (DOD) and optionally CYP76AD1 under conditions sufficient to produce betaine, thereby producing betaine.

[0084] In another embodiment, the present invention provides a method for producing betaine from tyrosine, comprising the steps of: contacting tyrosine with CYP76AD6, CYP76AD15 or a combination thereof and DOPA 4,5-dioxygenase (DOD) and optionally CYP76AD1 under conditions sufficient to produce betaine, thereby producing betaine.

[0085] In another embodiment, the present invention provides a method for producing a beet pigment mixture, comprising the steps of: contacting tyrosine with CYP76AD6, CYP76AD15 or a combination thereof, CYP76AD1, DOPA 4,5-dioxygenase (DOD) and beet pigment-associated glucosyltransferase under conditions sufficient to produce a combination of beet red and beet yellow pigments, thereby producing a beet pigment mixture.

[0086] In one embodiment, the production of beet pigment described in this invention is carried out without the provision of L-DOPA, which has never been done before. In one embodiment, the beet pigment comprises betalain and betalain. In one embodiment, the beet pigment, betalain, and betalain can be used as food coloring agents or other dyes.

[0087] In one embodiment, betalain produces a reddish-purple color. In one embodiment, betalain produces a yellow color. In one embodiment, a combination of betalain and betalain produces an orange color. In one embodiment, a combination of betalain and betalain produces an orange-red color.

[0088] In another embodiment, the present invention provides a method for producing an orange dye, comprising the steps of: contacting tyrosine with CYP76AD6, CYP76AD15 or a combination thereof, CYP76AD1, DOPA 4,5-dioxygenase (DOD) and betaine-associated glucosyltransferase under conditions sufficient to produce a combination of betaine red and betaine yellow, thereby producing an orange dye.

[0089] In another embodiment, the present invention provides a method for producing betaine from tyrosine, comprising the steps of: contacting tyrosine with CYP76AD6, CYP76AD15 or a combination thereof and DOPA4,5-dioxygenase (DOD) under conditions sufficient to produce betaine, thereby producing betaine.

[0090] In another embodiment, the present invention provides a method for producing a yellow dye, comprising the steps of: contacting tyrosine with CYP76AD6, CYP76AD15, or a combination thereof, and DOPA 4,5-dioxygenase (DOD) under conditions sufficient to produce betaine, thereby producing a yellow dye. In one embodiment, the yellow dye is a food coloring agent.

[0091] In one embodiment, the method of the present invention produces one or more betaine compounds. In one embodiment, the betaine comprises prickly pear cactus flavin I (glutamine-betaine). In another embodiment, the betaine comprises proline-betaine. In another embodiment, the betaine comprises dopaflavin-hexoside. In another embodiment, as described in Table 2 below, the betaine comprises betaine I (unknown). In another embodiment, the betaine comprises histamine-betaine. In another embodiment, the betaine comprises alanine-betaine. In another embodiment, the betaine comprises dopamine-betaine. In yet another embodiment, the betaine comprises valine-betaine.

[0092] In another embodiment, the present invention provides a method for producing betalain from tyrosine, comprising the steps of: contacting tyrosine with CYP76AD1, DOPA 4,5-dioxygenase (DOD) and betalain-associated glucosyltransferase under conditions sufficient for betalain production, thereby producing betalain.

[0093] In another embodiment, the present invention provides a method for producing a reddish-purple dye, comprising the steps of: contacting tyrosine with CYP76AD1, DOPA 4,5-dioxygenase (DOD), and a betaine-associated glucosyltransferase under conditions sufficient to produce betaine, thereby producing a reddish-purple dye. In one embodiment, the reddish-purple dye is a food coloring agent.

[0094] In one embodiment, the method described herein provides a glycosylated betaine pigment (betaine) instead of a glycoside aglycone (betaine). In one embodiment, betaine is more stable than betaine, thus offering significant advantages. Betaine is a highly unstable compound and ascorbic acid must be added to prevent its degradation. Betaine is more readily degraded by enzymatic and non-enzymatic oxidative processes. Betaine pigment extracts used for food coloring contain betaine. Due to its instability, betaine is not suitable for food coloring.

[0095] In another embodiment, the method of the present invention produces one or more betalains. In one embodiment, the betalain comprises betaine. In another embodiment, the betalain comprises isobetaine. In another embodiment, as described in Table 2 below, the betalain comprises betalain I (unknown) or betalain II (unknown). In another embodiment, the method of the present invention produces one or more betalain fragments, wherein in one embodiment, the betalain fragment is betaine.

[0096] In another embodiment, the present invention provides a method for producing a pigment or dye, comprising the steps of: contacting tyrosine with CYP76AD6, CYP76AD15 or a combination thereof and DOPA 4,5-dioxygenase (DOD) and optionally CYP76AD1 under conditions sufficient to produce beet pigment, thereby producing a pigment or dye.

[0097] In another embodiment, the present invention provides a method for increasing the level of one or more betaine pigments in an organism, plant, or plant part, comprising inducing or permitting the expression of DOPA 4,5-dioxygenase (DOD), betaine-associated glucosyltransferase, and CYP76AD6, CYP76AD15, or combinations thereof, CYP76AD1, or combinations thereof, in the organism, plant, or plant part, thereby increasing the level of one or more betaine pigments in the organism, plant, or plant part. In one embodiment, the plant is an ornamental plant. In another embodiment, the plant is a food crop. In one embodiment, the organism or plant does not naturally produce detectable levels of betaine pigments.

[0098] From nucleic acid production

[0099] In another embodiment, the method of the present invention is carried out in vivo. In one embodiment, tyrosine is endogenous to the cells. In another embodiment, tyrosine is provided to the cells. In another embodiment, the cells are transformed with a gene that enhances tyrosine availability. In one embodiment, the gene is AroG175 (Tzin et al., 2012 NewPhytologist 194:430-439; Genebank Registry No. JC233128.1, SEQ ID NO: 28), aromatic amino acid hydroxylase (AAH) (Pribat et al., 2010 Plant Cell 22:3410-3422; Genebank Registry No. HQ003815.1, SEQ ID NO: 29), or a combination thereof. In one embodiment, the enzyme required for the production of betaine is provided to the cells by transferring a polynucleotide encoding the enzyme.

[0100] In one embodiment, the present invention provides a method for generating L-DOPA, comprising the steps of: contacting an organism with nucleic acid encoding a gene of the CYP76AD1-β clade under conditions sufficient to generate L-DOPA, thereby generating L-DOPA.

[0101] In another embodiment, the present invention provides a method for generating L-DOPA, comprising the steps of: contacting an organism with a nucleic acid encoding CYP76AD6, CYP76AD15, or a combination thereof under conditions sufficient to generate L-DOPA, thereby generating L-DOPA.

[0102] In another embodiment, the present invention provides a method for generating L-DOPA, comprising the steps of: contacting an organism with a nucleic acid encoding CYP76AD6, CYP76AD15, or a combination thereof under conditions sufficient to generate L-DOPA, thereby generating L-DOPA.

[0103] In one implementation, the organism is a plant. In one implementation, the plant is a tobacco plant.

[0104] In another embodiment, the present invention provides a method for producing betaine, comprising the steps of: contacting an organism with nucleic acids encoding the CYP76AD1-β clade gene, nucleic acids encoding the CYP76AD1-α clade gene, or a combination thereof, under conditions sufficient to produce betaine, thereby producing betaine.

[0105] In another embodiment, the present invention provides a method for producing beet pigment, comprising the steps of: contacting an organism with a nucleic acid encoding CYP76AD6, CYP76AD15 or a combination thereof, a nucleic acid encoding CYP76AD1 or a combination thereof under conditions sufficient to produce beet pigment, thereby producing beet.

[0106] In one embodiment, the method further includes the step of contacting a tyrosine-containing organism with a nucleic acid encoding DOPA 4,5-dioxygenase (DOD).

[0107] In one embodiment, the nucleic acids encoding genes disclosed in this invention specifically include CYP76AD6, CYP76AD15 or combinations thereof, CYP76AD1, and DOD, the nucleic acids being present together on a single recombinant polynucleotide, as described in more detail below.

[0108] In another embodiment, the present invention provides a method for producing betaine, comprising the steps of: contacting an organism with nucleic acids encoding CYP76AD6, CYP76AD15 or a combination thereof, nucleic acids encoding CYP76AD1, nucleic acids encoding DOPA 4,5-dioxygenase (DOD), and nucleic acids encoding betaine-associated glucosyltransferases under conditions sufficient for betaine production, thereby producing betaine.

[0109] In another embodiment, the present invention provides a method for producing an orange dye, comprising the steps of: contacting an organism containing tyrosine with a nucleic acid encoding CYP76AD6, CYP76AD15 or a combination thereof, a nucleic acid encoding CYP76AD1, a nucleic acid encoding DOPA 4,5-dioxygenase (DOD), and a nucleic acid encoding a betaine-associated glucosyltransferase under conditions sufficient to produce betaine pigment, thereby producing an orange dye.

[0110] In another embodiment, the generation of beet pigments described in this invention is carried out without a substrate feed.

[0111] In another embodiment, the present invention provides a method for producing betaine, comprising the steps of: contacting an organism containing tyrosine with a nucleic acid encoding CYP76AD6, CYP76AD15 or a combination thereof, a nucleic acid encoding DOPA 4,5-dioxygenase (DOD), and a nucleic acid encoding a betaine-associated glucosyltransferase under conditions sufficient for betaine production, thereby producing betaine.

[0112] In another embodiment, the present invention provides a method for producing a yellow dye, comprising the steps of: contacting an organism containing tyrosine with a nucleic acid encoding CYP76AD6, CYP76AD15 or a combination thereof, or a nucleic acid encoding DOPA 4,5-dioxygenase (DOD), under conditions sufficient to produce betaine, thereby producing a yellow dye.

[0113] In another embodiment, the present invention provides a method for producing betaine, comprising the steps of: contacting an organism containing tyrosine with nucleic acids encoding CYP76AD1, DOPA 4,5-dioxygenase (DOD), and betaine-associated glucosyltransferase under conditions sufficient for betaine production, thereby producing betaine. In one embodiment, the method for producing betaine is primarily a method for producing betaine.

[0114] In another embodiment, the present invention provides a method for producing a reddish-purple dye, comprising the steps of: contacting an organism containing tyrosine with nucleic acids encoding CYP76AD1, DOPA 4,5-dioxygenase (DOD), and betaine-associated glucosyltransferase under conditions sufficient to produce betaine, thereby producing a reddish-purple dye.

[0115] In one embodiment, the method described above provides a method for producing a natural reddish-purple dye. In another embodiment, the method described above provides a method for producing a natural yellow dye. In yet another embodiment, the method described above provides a method for producing a natural orange dye. In one embodiment, the dye can be used on textiles.

[0116] In one embodiment, the organism is a plant. In another embodiment, the organism is yeast. In yet another embodiment, the organism is bacteria. In still another embodiment, the organism is algae.

[0117] In one embodiment, the method of the present invention is carried out in a plant. In one embodiment, the pigment color in the plant depends on other natural or engineered pigments expressed in the plant. Thus, in one embodiment, the method of the present invention produces red, yellow, orange, and brown pigments, or any pigment color known in the art.

[0118] It should also be understood that pigment color can also be altered by changing the expression ratio of CYP76AD6, CYP76AD15 or combinations thereof and / or CYP76AD1.

[0119] In another embodiment, the present invention provides a method for producing betaine from tyrosine, comprising the steps of: contacting tyrosine with a nucleic acid encoding CYP76AD6, CYP76AD15 or a combination thereof, encoding DOPA 4,5-dioxygenase (DOD) and optionally encoding CYP76AD1 under conditions sufficient to produce betaine, thereby producing betaine.

[0120] In another embodiment, the present invention provides a method for producing an orange dye, comprising the steps of: contacting tyrosine with a nucleic acid encoding CYP76AD1, a nucleic acid encoding CYP76AD6, CYP76AD15 or a combination thereof, and a nucleic acid encoding DOPA 4,5-dioxygenase (DOD) under conditions sufficient to produce beet pigment, thereby producing an orange dye.

[0121] In another embodiment, the present invention provides a method for producing betaine from tyrosine, comprising the steps of: contacting tyrosine with a nucleic acid encoding CYP76AD6, CYP76AD15 or a combination thereof, and a nucleic acid encoding DOPA 4,5-dioxygenase (DOD) under conditions sufficient to produce betaine, thereby producing betaine.

[0122] In another embodiment, the present invention provides a method for producing a yellow dye, comprising the steps of: contacting tyrosine with a nucleic acid encoding CYP76AD6, CYP76AD15 or a combination thereof, and encoding DOPA 4,5-dioxygenase (DOD) under conditions sufficient to produce betaine, thereby producing a yellow dye.

[0123] In another embodiment, the present invention provides a method for producing betaine from tyrosine, comprising the steps of: contacting tyrosine with a nucleic acid encoding CYP76AD1 and a nucleic acid encoding DOPA 4,5-dioxygenase (DOD) under conditions sufficient to produce betaine, thereby producing betaine.

[0124] In another embodiment, the present invention provides a method for producing a reddish-purple dye, comprising the steps of: contacting tyrosine with nucleic acids encoding CYP76AD1, DOPA 4,5-dioxygenase (DOD), and betaine-associated glucosyltransferase under conditions sufficient to produce betaine, thereby producing a reddish-purple dye.

[0125] In one embodiment, the dye of the present invention is a food coloring agent. In one embodiment, the method of the present invention for producing the dye can be applied to the production of pigments, etc.

[0126] In one embodiment, the present invention provides food colorings, dyes, or pigments produced by the method of the present invention or comprising compositions of the present invention.

[0127] In another embodiment, the present invention provides a method for generating L-DOPA in cells, comprising the steps of: contacting the cells with a polynucleotide comprising a nucleic acid encoding CYP76AD6, CYP76AD15 or a combination thereof under conditions sufficient to generate L-DOPA, thereby generating L-DOPA.

[0128] In another embodiment, the present invention provides a method for producing betaine, orange dye or combinations thereof in cells, comprising the steps of: contacting the cells with nucleic acids encoding CYP76AD6, CYP76AD15 or combinations thereof, and nucleic acids encoding CYP76AD1 or combinations thereof under conditions sufficient to produce betaine, thereby producing betaine, orange dye or combinations thereof.

[0129] In another embodiment, the present invention provides a method for producing betaine, a yellow dye, or a combination thereof in cells, comprising the steps of: contacting the cells with nucleic acids encoding CYP76AD6, CYP76AD15, or a combination thereof, and nucleic acids encoding DOPA 4,5-dioxygenase (DOD) under conditions sufficient for the production of betaine, thereby producing betaine, a yellow dye, or a combination thereof.

[0130] In another embodiment, the present invention provides a method for producing betalains, a reddish-purple dye, or a combination thereof in cells, comprising the steps of: contacting the cells with nucleic acids encoding CYP76AD1, DOPA 4,5-dioxygenase (DOD), and betalain-associated glucosyltransferase under conditions sufficient for betalain production, thereby producing betalains, a reddish-purple dye, or a combination thereof.

[0131] In one implementation, the cells described above are part of a cell line.

[0132] In one embodiment, beet pigments (including betaine, betaine red, or combinations thereof) obtained by the method of the present invention are produced in a cell line. In one embodiment, the cell line is a plant cell line. In one embodiment, the plant cell line is tobacco BY2 or Arabidopsis T87.

[0133] In another embodiment, the L-DOPA obtained by the method of the present invention is produced in a cell line. In one embodiment, the cell line is a tobacco cell line. In another embodiment, the tobacco cell line is BY2.

[0134] In another embodiment, the present invention provides a method for producing a metabolite of L-DOPA, comprising the steps of: contacting cells with nucleic acids encoding CYP76AD6, CYP76AD15, or a combination thereof under conditions sufficient for L-DOPA production to produce L-DOPA, and allowing the formation of L-DOPA metabolites, or further contacting said cells with nucleic acids encoding an enzyme that metabolizes L-DOPA, thereby producing L-DOPA metabolites. In one embodiment, the L-DOPA metabolite is a catecholamine, a benzylisoquinoline alkaloid, betaine, melanin, or a combination thereof. In one embodiment, the catecholamine is dopamine, norepinephrine, or a combination thereof. In one embodiment, the benzylisoquinoline alkaloid is an opioid, which in one embodiment is morphine.

[0135] In one embodiment, the enzyme metabolizing L-DOPA is DOPA 4,5-dioxygenase (DOD). In another embodiment, the enzyme is CYP76AD1. In yet another embodiment, the enzyme is a glucosyltransferase. In one embodiment, the glucosyltransferase is cyclic-DOPA 5-O-glucosyltransferase or betaine-5-O-glucosyltransferase. In one embodiment, the cyclic-DOPA 5-O-glucosyltransferase is the Mirabilis jalapa gene cyclic-DOPA 5-O-glucosyltransferase (cDOPA5GT).

[0136] In another embodiment, the enzyme that metabolizes L-DOPA is DOPA decarboxylase. In another embodiment, the enzyme that metabolizes L-DOPA is dopamine β-hydroxylase. In another embodiment, the enzyme that metabolizes L-DOPA is aromatic L-amino acid decarboxylase. In another embodiment, the enzyme that metabolizes L-DOPA is catechol-O-methyltransferase. In another embodiment, the enzyme that metabolizes L-DOPA is phenylethanolamine N-methyltransferase.

[0137] In another embodiment, the enzyme that metabolizes L-DOPA is norcoclaurine synthase (NCS). In another embodiment, the enzyme that metabolizes L-DOPA is norcoclaurine 6-O-methyltransferase (6OMT). In another embodiment, the enzyme that metabolizes L-DOPA is coclaurine-N-methyltransferase (CNMT). In yet another embodiment, the enzyme that metabolizes L-DOPA is 3'-hydroxy-N-methylcoclaurine 4'-O-methyltransferase (4'OMT).

[0138] Gene transformation methods

[0139] In one embodiment, the present invention provides a method including the step of “contacting” a cell with a polynucleotide or expression vector described herein.

[0140] In one embodiment, contact includes transforming cells with the nucleic acid molecules or constructs of the present invention. Methods of transforming plant cells with nucleic acid sequences are known in the art. As used herein, the terms “transformation” or “transforming” can refer to the process by which exogenous DNA (e.g., DNA constructs, including expression vectors) enters and alters recipient cells into transformed, genetically modified, or transgenic cells. Transformation can be stable, in which the nucleic acid sequence is integrated into the plant genome and thus presents stable and heritable traits; or transient, in which the nucleic acid sequence is expressed by the transformed cells but not integrated into the genome and thus presents transient traits.

[0141] In one embodiment, the present invention provides a transgenic plant. In one embodiment, the transgenic plant of the present invention is genetically modified using exogenous or heterologous genes. In one embodiment, the transgenic plant of the present invention is used for biofuels. In another embodiment, the transgenic plant of the present invention is a food crop.

[0142] In another embodiment, the present invention provides cis-gene plants. In one embodiment, the cis-gene plants of the present invention are genetically modified but do not contain exogenous or heterologous genes. According to this aspect and in one embodiment, betaine enzyme can be overexpressed in plants already containing betaine enzyme, thereby altering the ratio between betaine red and betaine xanthophyll. In one embodiment, the food crop of the present invention is cis-gene.

[0143] Any method or delivery system can be used to deliver and / or transform (plant cells) / transfect (algal cells) nucleic acid vectors encoding CYP76AD6 and its homologs, parahomologs, etc., into host cells (e.g., plant protoplasts). The vector can be delivered to the host cell alone or in combination with other reagents. Transient expression systems can also be used. Homologous recombination can also be used.

[0144] In one embodiment, polynucleotides as described herein are provided to the cells of the present invention via transformation. As is known to those skilled in the art, transformation can be accomplished by a variety of means. Such methods include, but are not limited to, particle bombardment-mediated transformation (e.g., Finer et al., 1999, Curr. Top. Microbiol. Immunol., 240:59), protoplast electroporation (e.g., Bates, 1999, 111:359), viral infection (e.g., Porta and Lomonossoff, 1996, Mol. Biotechnol. 5:209), microinjection, and liposome injection. Other exemplary delivery systems that can be used to facilitate cellular uptake of nucleic acids include calcium phosphate and other chemical mediators for intracellular transport, microinjection compositions, and homologous recombination compositions (e.g., for integrating genes into preselected sites within the cell chromosome). Alternative methods may involve, for example, the use of liposomes, electroporation, or chemicals that increase the uptake of free (or “naked”) DNA, transformation using viruses or pollen, and the use of microprojection. Standard molecular biology techniques are common in the field (e.g., Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, New York).

[0145] Plant transformation

[0146] Various methods exist for introducing exogenous genes into both monocots and dicots (see Potrykus I 1991. Annu Rev Plant Physiol Plant Mol Biol 42, 205-225; Shimamoto K. et al., 1989. Nature 338, 274-276). Transformation methods may include, for example, but not limited to, the use of liposomes, electroporation, chemicals that increase the uptake of cell-free DNA, direct injection of DNA into plants, particle gun bombardment, transformation using viruses, and transformation using microscopy.

[0147] In one embodiment, in another embodiment, in agrobacterium-mediated transformation (e.g., Komari et al., 1998, Curr. Opin. Plant Biol., 1:161), including introgression transformation in flowers, the polynucleotides described herein are provided to cells. In one embodiment, introgression induces transient expression of genes in the plant to produce the desired protein by injecting a suspension of Agrobacterium tumefaciens containing one or more desired genes into plant leaves. In one embodiment, transformation can be carried out via Agrobacterium-mediated gene transfer. Agrobacterium-mediated systems include the use of plasmid vectors containing defined DNA fragments integrated into the plant's genomic DNA. The inoculation method of plant tissues varies depending on the plant species and the Agrobacterium delivery system. Transformation can be carried out with any suitable tissue explant, which provides a good source for initiating whole-plant differentiation (see Horsch et al., 1988. Plant Molecular Biology Manual A5, 1-9, Kluwer Academic Publishers, Dordrecht).

[0148] Plant transformation methods are disclosed in US patent applications US 20110209247; US 20110113514; US20100199371; US ​​20070079396; US 20080307541; US ​​20030028913; and US20030196219; and US patents US 5,015,580; US 5,550,318; US 5,538,880; US 6,160,208; US 6,399,861; US ​​6,403,865; US 5,635,055; US 5,824,877; US 5,591,616; US 5,981,840 and US It is fully described in 6,384,301, the entire contents of which are incorporated herein by reference.

[0149] In one implementation of a plant transformation system based on Agrobacterium tumefaciens, other portions of the transformation construct will include left and right boundary sequences of T-DNA to facilitate the incorporation of recombinant polynucleotides into the plant genome.

[0150] In one implementation, transformation can be carried out via direct DNA uptake. Several methods exist for the direct transfer of DNA into plant cells. In electroporation, protoplasts are briefly exposed to a strong electric field, opening micropores to allow DNA entry. In microinjection, DNA is directly and mechanically injected into cells using a micropipette. In particle bombardment, DNA is adsorbed onto microspheres such as magnesium sulfate crystals or tungsten particles, and these microspheres are physically accelerated into cells or plant tissues.

[0151] In another embodiment, using gene gun technology or electroporation, suitable plant viruses are used to deliver polynucleotides, as described herein, to cells via viral transformation (transduction).

[0152] In one embodiment, the heterologous gene of the polynucleotide of the present invention is integrated into a plant chromosome. In another embodiment, the heterologous gene of the polynucleotide of the present invention is retained outside the chromosome. In one embodiment, the heterologous gene is located in a plasmid within the cell. Plasmids suitable for either application are known to those skilled in the art.

[0153] In one implementation, the DNA is randomly inserted, i.e., inserted into the genome of the target plant line at a non-specific location. In another implementation, the DNA is targeted to achieve site-specific integration, such as replacing an existing gene in the genome, using a promoter already present in the plant genome, or inserting the recombinant polynucleotide into a predetermined site known to be active in gene expression. Several site-specific recombination systems are known to function in plants, including cre-lox and FLP-FRT.

[0154] In one embodiment, the transformation method of the present invention is practiced on tissue cultures in a culture medium and a control environment. “Culture medium” refers to a mixture of various nutrients used for the in vitro growth of cells, i.e., outside a complete living organism. Recipient cell targets include, but are not limited to, meristematic cells, callus, immature embryos, and gamete cells such as microspores, pollen, sperm, and egg cells. Any cell intended to regenerate a fertile plant can be used as a recipient cell. Callus can be generated from tissue sources, including, but not limited to, immature embryos, apical meristems of young shoots, and microspores. Cells capable of proliferating as callus are also recipient cells for gene transformation. Practical transformation methods and materials for creating the genetically modified plants of the present invention, such as various culture media and recipient target cells, transformation of immature embryos, and subsequent regeneration of fertile genetically modified plants, are known in the art.

[0155] In another embodiment, the introduction of polynucleotides into plant cells allows betalains to be expressed or detected in any plant organ, including leaves, stems, roots, flowers, seeds, tubers, or fruits, or combinations thereof.

[0156] In another embodiment, the method of the present invention can be carried out throughout the plant, such that the entire plant expresses betaine as described herein. In yet another embodiment, the compositions of the present invention can describe a whole plant that has been genetically modified to express the polynucleotides or polypeptides of the present invention.

[0157] Algal transformation

[0158] In one embodiment, the method for transforming algae includes any of the methods described above. In one embodiment, the transformation of algae is accomplished using glass bead-assisted transformation, particle gun-mediated (biological projectile) transformation, treatment with cellulase to weaken its cell wall, or homologous recombination.

[0159] On the other hand, the nucleic acids of the present invention can be transformed into algae. In one embodiment, the algae are unicellular algae. In another embodiment, the algae are multicellular algae. In one embodiment, the algae are cyanobacteria, diatoms, Chlamydomonas, Dunaliella, or hemocytes. Genes can be overexpressed in the algae. Betaine, DOPA, or combinations thereof can be produced through such transgenic algae. Methods for algal transformation are well known in the art and are fully described in U.S. Patent Application Publications US20150011008; US 20150004704; US 20130130389; US 20120094385; US 20120034698; US20110300633; and US 20040133937, which are incorporated herein by reference in their entirety.

[0160] In one embodiment, the genetically modified algae of the present invention can be used for biofuel production.

[0161] yeast conversion

[0162] On the other hand, the nucleic acids of the present invention can be transformed into yeast. The gene can be overexpressed in yeast. Betaine, DOPA, or combinations thereof can be produced through this transgenic yeast. Methods for yeast transformation are described below and are well known in the art, and are fully described in U.S. Patent Application Publications US 20090264320; US 20010031724; US20030049785; US 20050158861; US ​​20070264716; US 20090325247; and US 20100190223, the entire contents of which are incorporated herein by reference.

[0163] Viral transformation

[0164] In another embodiment, the nucleic acid of the present invention can be transformed into a virus. In yet another embodiment, the nucleic acid can be overexpressed in a virus. Betaine, dyes, pigments, DOPA, or combinations thereof can be produced through such viral overexpression.

[0165] Gene transformation markers

[0166] In one implementation, in any given experiment, DNA is introduced into only a small percentage of the target cells.

[0167] In one embodiment, cells containing transferred exogenous DNA can be identified by color, as described for yeast in Example 6. In one embodiment, cells expressing CYP76AD1 and DODA are reddish-purple. In one embodiment, cells expressing CYP76AD6, CYP76AD15 or a combination thereof, and DODA are yellow. In one embodiment, cells expressing CYP76AD1, CYP76AD6, CYP76AD15 or a combination thereof, and DODA are orange or orange-red.

[0168] In another implementation, biomarker genes are used to provide an effective system for identifying those cells that have been stably transformed by receiving genetically modified DNA constructs and integrating them into their genome.

[0169] In another implementation, the selected gene provides a selective marker confers resistance to a selective agent, such as an antibiotic or herbicide. Potentially transformed cells are exposed to the selective agent. Within a population of surviving cells, typically those cells where the resistance-conferring gene has been integrated and expressed at sufficient levels to allow cell survival, the cells can be further tested to confirm stable integration of the exogenous DNA.

[0170] Useful selective marker genes include those that confer resistance to antibiotics (e.g., kanamycin (nptII), hygromycin B (aph IV), nptII, hpt, aadA, and gentamicin (aac3 and aacC4)) or to herbicides (e.g., glufosinate (bar or pat) and glyphosate (EPSPS)). In another embodiment, the selective gene is an antimetabolite. In one embodiment, the antimetabolite is dhf.

[0171] Screenable markers that provide the ability to visually identify transformants can also be used, such as genes expressing colored or fluorescent proteins (e.g., cat, lacZ, uidA, luciferase, or green fluorescent protein (GFP)), or genes expressing β-glucuronidase or the uidA gene (GUS) known for various chromogenic substrates. It is also anticipated that combinations of screenable and selectable markers will be useful for identifying transformed cells.

[0172] In one embodiment, the selection gene is a positive selection marker gene conditioned by a non-toxic agent, which may be a substrate for growth or an induction of growth and differentiation of the transformed tissue.

[0173] In one implementation, cells exposed to a selective reagent and surviving, or cells scoring positive in a screening assay, can be cultured in a regeneration medium and allowed to mature into plants. Before being transferred to a greenhouse or growth chamber for maturation, developing seedlings can be transferred to a plant growth mixture with less soil and, for example, at approximately 85% relative humidity, 600 ppm CO2, and 25-250 micrograms of Einstein m3. -2 s -1 The plants are hardened in an environmentally controlled chamber. They are preferably matured in a growth chamber or greenhouse. Depending on the initial tissue, the plants regenerate approximately 6 weeks to 10 months after the transformant is identified. During regeneration, the cells are grown into plants on a solid culture medium at approximately 19 to 28°C. Once the regenerated plants reach the bud and root development stages, they can be transferred to a greenhouse for further growth and testing. The plants can be pollinated using conventional plant breeding methods known to those skilled in the art and the seeds produced.

[0174] Progeny plants can be used to recover and test the expression of exogenous recombinant polynucleotides. Useful assays include, for example, "molecular biology" assays such as Southern Blot and Northern Blot and PCR; "biochemical" assays such as detecting the presence of RNA (e.g., double-stranded RNA) or protein products, for example by immunological means (ELISA and Northern Blot) or by enzymatic function; plant part assays such as leaf or root assays; and also by analyzing the phenotype of the entire regenerated plant.

[0175] Those skilled in the art will be able to select a suitable vector for introducing the coding nucleic acid sequence in a relatively intact state. Therefore, any vector that will produce a host cell (e.g., a plant protoplast) carrying the introduced coding nucleic acid should be sufficient. The choice of vector, or whether to use a vector at all, is typically guided by the chosen transformation method.

[0176] promoter

[0177] On the other hand, cell, tissue, or organ-specific promoters can be used to transform the genes of the present invention and express them in specific cells, tissues, or organs. For example, fruit-specific promoters can be used to transform the genes of the present invention into plants (e.g., tomatoes) or plant cells to produce betaine in fruits (e.g., tomato fruits).

[0178] In one embodiment, the recombinant polynucleotide of the present invention includes at least one promoter. In one embodiment, the recombinant polynucleotide of the present invention includes a single promoter controlling the expression of tandem nucleic acids within the same reading frame. In another embodiment, each nucleic acid in the recombinant polynucleotide of the present invention is controlled by a separate promoter.

[0179] In one embodiment, the promoter is a constitutive promoter. In another embodiment, the constitutive promoter is the CaMV 35S promoter. In yet another embodiment, the constitutive promoter is the crown gall alkaloid promoter. In yet another embodiment, the constitutive promoter is a monocotyledonous promoter. In yet another embodiment, the constitutive promoter is the plant ubiquitin promoter (Ubi). In yet another embodiment, the constitutive promoter is the Arabidopsis ubiquitin-10 promoter. In yet another embodiment, the constitutive promoter is the tomato ubiquitin-10 promoter (SlUb10). In yet another embodiment, the constitutive promoter is the rice actin-1 promoter (Act-1). In yet another embodiment, the constitutive promoter is the zeatol dehydrogenase-1 promoter (Adh-1).

[0180] In another embodiment, the promoter is an inducible promoter. In one embodiment, the inducible promoter is a galactose-inducible promoter. In another embodiment, the inducible promoter is selected from AlcR / AlcA (ethanol-inducible); GR fusion, GVG, and pOp / LhGR (dexamethasone-inducible); XVE / OlexA (β-estradiol-inducible); and heat shock-inducible. In another embodiment, the inducible promoter is selected from tetracycline, dexamethasone, copper, salicylic acid, and herbicide-inducible promoters. In another embodiment, mGal4:VP16 / UAS or pOp / LhG4 can be used for trans-activation, and alc-switch, GVE / VGE, and XVE systems can be used for chemical induction. These methods are known in the art.

[0181] In another embodiment, the promoter is a tissue-specific or developmental stage-specific promoter. In yet another embodiment, the promoter is a synthetic promoter, which is produced by bringing together major portions of promoter regions from different sources.

[0182] In some embodiments, a fruit-specific promoter can be used to transform the gene of the present invention in combination with one or more other genes (e.g., anthocyanin genes) into a plant (e.g., tomato) or plant cell to produce betaine in a fruit (e.g., tomato fruit). In one embodiment, the gene of the present invention can be transformed into a transgenic plant (e.g., a transgenic tomato plant already transformed with a recombinant anthocyanin gene) or plant cell to produce betaine in a fruit (e.g., tomato fruit).

[0183] Therefore, in one embodiment, the promoter of any polynucleotide described herein may be a fruit-specific promoter. In one embodiment, the fruit-specific promoter is the E8 promoter. In another embodiment, the promoter may be a flower-specific promoter. In one embodiment, the flower-specific promoter is the chalcone synthase (CHS) promoter. In one embodiment, the CHS promoter is the petunia CHS promoter. In another embodiment, the promoter may be a root-specific promoter. In another embodiment, the promoter may be a stem-specific promoter. In another embodiment, the promoter may be a leaf-specific promoter. In another embodiment, the promoter may be a seed-specific promoter. In another embodiment, the promoter may be a tuber-specific promoter.

[0184] In one implementation, the promoter may be specific to a portion of the tissue. For example, in one implementation, the promoter may be specific to petals, stamens, anthers, stigmas, or combinations thereof.

[0185] In another embodiment, the same nucleic acid can be expressed under different nonconstitutive promoter sequences to engineer organisms exhibiting two colors of pigmentation and / or expressing betalains and betalains in different organs or different parts of the same organ. For example, in one embodiment, CYP76AD6 or CYP76AD15 or their homologs can be expressed under a flower-specific promoter, and CYP76AD1 can be expressed under a fruit-specific promoter, such that the fruit is reddish-purple due to the presence of both betalains and betalains, and the flower is yellow due to the presence of only betalains. Furthermore, inducible promoters can be used to engineer organisms exhibiting two colors of pigmentation and / or expressing betalains and betalains in different organs or different parts of the same organ.

[0186] In another embodiment, the promoter may be specific to the developmental stage. For example, in one embodiment, the promoter may be specific to stage 1, which is when the flower is approximately 1 cm long. In another embodiment, the developmental stage is stage 2, which is when the flower is approximately 2 cm long. In another embodiment, the developmental stage is stage 3, which is when the flower is approximately 3 cm long. In another embodiment, the developmental stage is stage 4, which is when the flower is approximately 4 cm long. In yet another embodiment, the developmental stage is stage 5, which is when the flower is approximately 5-6 cm long.

[0187] In one embodiment, the promoter may be tissue- and developmental stage-specific. In one embodiment, the fruit-specific E8 promoter is expressed in maturing and mature fruits. In another embodiment, the flower-specific CHS promoter is expressed in petals during flower maturation.

[0188] Gene silencing applications

[0189] In one embodiment, the present invention provides a method for treating or inhibiting dopamine-responsive disorders in a subject, comprising the step of administering a food crop, cell, or cell line containing high levels of CYP76AD1-β clade polypeptide, thereby providing the subject with L-DOPA to treat or inhibit the dopamine-responsive disorder in the subject. In one embodiment, the food crop, cell, or cell line endogenously produces betaine. In one embodiment, the expression of DOPA 4,5-dioxygenase, cyclic DOPA 5-O-glucosyltransferase, and the CYP76AD1-α clade gene in the food crop, cell, or cell line has been inhibited.

[0190] In one embodiment, the present invention provides a method for producing yellow plant parts in plants containing betaine, comprising silencing CYP76AD1 gene expression.

[0191] In one embodiment, the present invention provides a method for producing green plant parts in plants containing betaine, comprising silencing gene expression of CYP76AD1 and CYP76AD6, CYP76AD15 or combinations thereof.

[0192] In one embodiment, the plant part includes leaves, stems, roots, flowers, tubers, fruits, seeds, or combinations thereof.

[0193] In one implementation, gene silencing is gene knockout; in another, it is a reduction in gene expression. In one implementation, virus-induced gene silencing is used to silence genes encoding CYP76AD1 and / or CYP76AD6. In one implementation, virus-induced gene silencing (VIGS) is a technique utilizing an RNA-mediated antiviral defense mechanism. In plants infected with unmodified viruses, this mechanism specifically targets the viral genome. However, in cases where the viral vector carries an insert derived from a host gene, the process can be additionally targeted at the corresponding mRNA. This approach is illustrated in Examples 1 and 3 below.

[0194] Other gene silencing methods that can be used in this invention include gene silencing of the three major untranslated regions / microRNAs using antisense oligonucleotides, ribozymes, or RNA interference. These methods are known in the art and are described in Example 1 below.

[0195] In another embodiment, the gene encoding CYP76AD1 and / or CYP76AD6, CYP76AD15 or a combination thereof is knocked out using methods known in the art.

[0196] In one embodiment, the plant contains betalains, which are betalains in one embodiment and betalains in another embodiment.

[0197] In one embodiment, the gene silencing method is carried out in plants containing betaine. In one embodiment, the plants containing betaine are from the order Caryophyllales.

[0198] In one embodiment, the gene silencing method is carried out in the order Caryophyllales, which includes cacti, carnations, amaranth, ice plants, beets, and many carnivorous plants. In another embodiment, the plant belongs to the suborder Caryophyllaceae. In yet another embodiment, the plant belongs to the suborder Polygonaceae. In another embodiment, the plant is selected from one of the following families in the order Caryophyllales: Hairy Fruit Family; Aizoaceae; Amaranthaceae; Amaranthus Family; Rhizophoraceae; Theaceae Family; Bryaceae; Basellaceae; Cactaceae; Caryophyllaceae; Draconis Family; Draconis Family; Droseraceae; Droseraceae; Droseraceae; Pleurotace ...

[0199] In some embodiments, the polynucleotides of the present invention are prepared using PCR technology with procedures and methods known to those skilled in the art. In some embodiments, the procedure involves the merging of two different DNA sequences (see, for example, "Current Protocols in Molecular Biology", eds. Ausubel et al., John Wiley & Sons, 1992).

[0200] Polynucleotides

[0201] In one embodiment, the present invention provides a recombinant polynucleotide comprising a nucleic acid encoding CYP76AD6. In one embodiment, the nucleic acid sequence encoding CYP76AD6 includes:

[0202]

[0203] In one implementation, the nucleic acid sequence encoding CYP76AD6 includes the following sequence:

[0204]

[0205] In one implementation, the nucleic acid encoding CYP76AD6 is driven by the CaMV 35S promoter (pDOPA1); Figure 19 In another embodiment, the nucleic acid encoding CYP76AD6 is driven by the tomato ubiquitin 10 promoter (S1Ub10) (pDOPA2; Figure 19 ).

[0206] In one implementation, nucleic acids encoding CYP76AD6, CYP76AD15, or a combination thereof are expressed together with AroG175 and aromatic amino acid hydroxylase (AAH).

[0207] In one embodiment, the polynucleotide comprising a nucleic acid encoding CYP76AD6, CYP76AD15, or a combination thereof further comprises a neomycin phosphotransferase II (nptII) gene, which in one embodiment confers kanamycin resistance.

[0208] In another embodiment, the present invention provides a recombinant polynucleotide comprising a nucleic acid encoding a CYP76AD1-α clade gene. In one embodiment, the CYP76AD1-α clade gene comprises CYP76AD1. In one embodiment, the nucleic acid sequence encoding CYP76AD1 comprises:

[0209]

[0210] In another embodiment, the CYP76AD1-α clade gene includes A. Crocuentus, amaranth (CYP76AD2, accession number AET43291.1; SEQ ID NO: 15); M. jalapa, four o'clock flower (CYP76AD3, accession number AET43292.1; SEQ ID NO: 16); C. crispata, cockscomb flower (CYP76AD4, accession number AGI78466.1; SEQ ID NO: 17); or combinations thereof.

[0211] In one embodiment, the present invention provides a recombinant polynucleotide comprising a nucleic acid encoding DOPA 4,5-dioxygenase (DOD). In one embodiment, DOD is beet DODA1 (BvDODA1). In one embodiment, the nucleic acid sequence encoding BvDODA1 comprises:

[0212]

[0213] In one embodiment, the present invention provides a recombinant polynucleotide comprising a nucleic acid encoding a betaine-associated glucosyltransferase. In one embodiment, the nucleic acid sequence encoding the betaine-associated glucosyltransferase is a nucleic acid sequence encoding a circular-DOPA 5-O-glucosyltransferase (cDOPA5GT). In one embodiment, cDOPA5GT is derived from Mirabilis jalapa. In one embodiment, the nucleic acid sequence encoding cDOPA5GT comprises:

[0214]

[0215] In one embodiment, several nucleic acids are combined into a single recombinant polynucleotide as described herein. In one embodiment, the present invention provides a recombinant polynucleotide comprising a nucleic acid encoding CYP76AD6, CYP76AD15, or a combination thereof, and a nucleic acid encoding DOD. In another embodiment, the present invention provides a recombinant polynucleotide comprising a nucleic acid encoding CYP76AD1 and a nucleic acid encoding DOD. In another embodiment, the present invention provides a recombinant polynucleotide comprising a nucleic acid encoding CYP76AD1 and a nucleic acid encoding CYP76AD6. In yet another embodiment, the present invention provides a recombinant polynucleotide comprising a nucleic acid encoding CYP76AD1, a nucleic acid encoding CYP76AD6, CYP76AD15, or a combination thereof, and a nucleic acid encoding DOD.

[0216] In another embodiment, several nucleic acids are incorporated into the cell, but each nucleic acid is a separate polynucleotide as described herein.

[0217] In one embodiment, the polynucleotide of the present invention comprises a nucleic acid sequence encoding CYP76AD6, CYP76AD15, or a combination thereof, and a nucleic acid sequence encoding CYP76AD1. In another embodiment, the polynucleotide of the present invention comprises a nucleic acid sequence encoding CYP76AD6, CYP76AD15, or a combination thereof, and a nucleic acid sequence encoding a DOD enzyme. In another embodiment, the polynucleotide of the present invention comprises a nucleic acid sequence encoding CYP76AD1 and a nucleic acid sequence encoding a DOD enzyme. In another embodiment, the polynucleotide of the present invention comprises a nucleic acid sequence encoding CYP76AD6, CYP76AD15, or a combination thereof, a nucleic acid sequence encoding CYP76AD1, and a nucleic acid sequence encoding a DOD enzyme. In yet another embodiment, the polynucleotide of the present invention comprises a nucleic acid sequence encoding CYP76AD6, CYP76AD15, or a combination thereof, a nucleic acid sequence encoding CYP76AD1, and a nucleic acid sequence encoding a betaine-associated glucosyltransferase. In another embodiment, the polynucleotide of the present invention comprises a nucleic acid sequence encoding CYP76AD6, CYP76AD15, or a combination thereof, a nucleic acid sequence encoding a DOD enzyme, and a nucleic acid sequence encoding a betaine-associated glucosyltransferase. In another embodiment, the polynucleotide of the present invention comprises a nucleic acid sequence encoding CYP76AD1, a nucleic acid sequence encoding a DOD enzyme, and a nucleic acid sequence encoding a betaine-associated glucosyltransferase. In yet another embodiment, the polynucleotide of the present invention comprises a nucleic acid sequence encoding CYP76AD6, CYP76AD15, or a combination thereof, a nucleic acid sequence encoding CYP76AD1, a nucleic acid sequence encoding a DOD enzyme, and a nucleic acid sequence encoding a betaine-associated glucosyltransferase.

[0218] In another embodiment, any recombinant polynucleotide described above also includes a betaine-associated glucosyltransferase. In one embodiment, the betaine-associated glucosyltransferase is a cyclic-DOPA 5-O-glucosyltransferase or a betaine-5-O-glucosyltransferase. In one embodiment, the cyclic-DOPA 5-O-glucosyltransferase is the four o'clock gene cyclic-DOPA 5-O-glucosyltransferase (cDOPA5GT) (SEQ ID NO: 2).

[0219] In another embodiment, the present invention provides a recombinant polynucleotide comprising a nucleic acid encoding CYP76AD1, a nucleic acid encoding CYP76AD6, CYP76AD15 or a combination thereof, a nucleic acid encoding DOPA 4,5-dioxygenase (DOD), and a nucleic acid sequence encoding a betaine-associated glucosyltransferase, wherein said nucleic acids are in the same reading frame.

[0220] In another embodiment, the present invention provides a recombinant polynucleotide comprising a nucleic acid encoding CYP76AD1, a nucleic acid encoding DOPA 4,5-dioxygenase (DOD), and a nucleic acid sequence encoding a betaine-associated glucosyltransferase, wherein the nucleic acids are in the same reading frame.

[0221] In another embodiment, the present invention provides recombinant polynucleotides comprising nucleic acids tandemly expressed encoding CYP76AD6, CYP76AD15 or a combination thereof, and nucleic acids encoding DOPA 4,5-dioxygenase (DOD).

[0222] In another embodiment, the present invention provides a recombinant polynucleotide comprising a nucleic acid sequence encoding CYP76AD1, CYP76AD6, CYP76AD15 or a combination thereof expressed in tandem, a nucleic acid encoding DOPA 4,5-dioxygenase (DOD), and a nucleic acid sequence encoding a betaine-associated glucosyltransferase.

[0223] In one embodiment, the polynucleotide as described herein comprises multiple nucleic acid sequences within the same reading frame. In another embodiment, the polynucleotide as described herein comprises multiple nucleic acid sequences expressed in tandem.

[0224] In one embodiment, the polynucleotide of the present invention comprises a nucleic acid encoding CYP76AD1, a nucleic acid encoding DOPA 4,5-dioxygenase (DOD), and a nucleic acid sequence encoding a betaine-associated glucosyltransferase, wherein said nucleic acids are in the same reading frame. In one embodiment, the DOD gene is under the CaMV 35S promoter. In one embodiment, the CYP76AD1 gene is under the CaMV 35S promoter. In one embodiment, the betaine-associated glucosyltransferase gene is under the Arabidopsis ubiquitin-10 promoter. In one embodiment, the polynucleotide further comprises a kanamycin resistance gene. In one embodiment, the polynucleotide is a pX11 vector, as described in Example 8 below. In one embodiment, the nucleic acid sequence of the pX11 vector comprises (SEQ ID NO: 19).

[0225] In another embodiment, the CYP76AD1 gene is in the E8 promoter (pX11 (E8), SEQ ID NO: 20; Figure 15 In another embodiment, the CYP76AD1 gene is located under the CHS promoter (pX11(CHS)SEQ ID NO: 21; Figure 15 )Down.

[0226] In another embodiment, the polynucleotide of the present invention comprises a nucleic acid encoding CYP76AD6, CYP76AD15, or a combination thereof, a nucleic acid encoding DOPA 4,5-dioxygenase (DOD), and a nucleic acid encoding betaine-associated glucosyltransferase, wherein said nucleic acids are in the same reading frame. In one embodiment, the DOD gene is under the CaMV 35S promoter. In one embodiment, the CYP76AD6, CYP76AD15, or a combination thereof gene is under the CaMV 35S promoter. In one embodiment, the betaine-associated glucosyltransferase gene is under the Arabidopsis ubiquitin-10 promoter. In one embodiment, the polynucleotide further comprises a kanamycin resistance gene. In one embodiment, the polynucleotide is a pX13 vector, as described in Example 14 below.

[0227] In another embodiment, CYP76AD6, CYP76AD15, or a combination thereof are under the E8 promoter. In yet another embodiment, CYP76AD6, CYP76AD15, or a combination thereof are under the CHS promoter.

[0228] In another embodiment, the polynucleotide of the present invention comprises a nucleic acid encoding CYP76AD6, CYP76AD15, or a combination thereof, a nucleic acid encoding CYP76AD1, a nucleic acid encoding DOPA 4,5-dioxygenase (DOD), and a nucleic acid sequence encoding a betaine-associated glucosyltransferase, wherein said nucleic acids are in the same reading frame. In one embodiment, the DOD gene is under the CaMV 35S promoter. In one embodiment, the CYP76AD6, CYP76AD15, or a combination thereof gene is under the CaMV 35S promoter. In one embodiment, the CYP76AD1 gene is under the CaMV 35S promoter. In one embodiment, the betaine-associated glucosyltransferase gene is under the Arabidopsis ubiquitin-10 promoter. In one embodiment, the polynucleotide further comprises a kanamycin resistance gene. In one embodiment, the polynucleotide is a pX12 vector, as described in Example 14 below.

[0229] In another embodiment, the CYP76AD6, CYP76AD15, or a combination thereof are under the E8 promoter. In another embodiment, the CYP76AD6, CYP76AD15, or a combination thereof are under the CHS promoter. In another embodiment, the CYP76AD1 gene is under the E8 promoter. In another embodiment, the CYP76AD1 gene is under the CHS promoter.

[0230] In one embodiment, the present invention provides a nucleic acid comprising encoding a CYP76AD1-β clade gene under the control of a promoter. In one embodiment, the CYP76AD1-β clade gene is CYP76AD6. In another embodiment, the CYP76AD1-β clade gene is CYP76AD15.

[0231] In one embodiment, the present invention provides a recombinant nucleic acid comprising a CYP76AD1-β clade gene from red beet. In one embodiment, the present invention provides a recombinant nucleic acid comprising a CYP76AD1-β clade gene not derived from beet. In one embodiment, the present invention provides a recombinant nucleic acid comprising the CYP76AD1-β clade gene but excluding nucleic acid sequences from CYP76AD1 paralogs of beet (DeLoache et al., 2015), which does not produce betaine when transformed into yeast with DOD. In one embodiment, the present invention provides a recombinant nucleic acid comprising the CYP76AD1-β clade gene but excluding the nucleic acid encoding SEQ ID NO: 34.

[0232] In another embodiment, the CYP76AD1-β clade gene is the CYP76AD1-β clade gene as described by Brockington et al. in 2015 (New Phytol. Sep 2015; 207(4): 1170-80, the entire contents of which are incorporated herein by reference). In one embodiment, the CYP76AD1-β clade gene is the CYP76AD1-β clade gene described in Supplementary Figure 2 by Brockington et al. in 2015.

[0233] In one embodiment, the present invention provides a recombinant polynucleotide comprising a nucleic acid encoding the CYP76AD6 gene, the CYP76AD15 gene, or a combination thereof under the control of a promoter.

[0234] In one embodiment, the present invention provides a recombinant polynucleotide comprising a nucleic acid encoding a CYP76AD1-β clade gene and a nucleic acid encoding DOPA 4,5-dioxygenase (DOD), wherein the nucleic acid is in-frame inserted into the polynucleotide. In one embodiment, the polynucleotide further comprises a nucleic acid encoding a betaine-associated glucosyltransferase.

[0235] In one embodiment, the nucleic acid encoding the CYP76AD1-β clade gene is BV. maritima, subspecies of beet maritima (Genus Bank Accession No. AKI33834.1; SEQ ID NO: 10); F. latifolia, Froelichialatifolia (Genus Accession No. AKI33838.1; SEQ ID NO: 11); A. caracasana, spiny amaranth (Genus Accession No. AKI33835.1; SEQ ID NO: 12); A. ficoidea, five-colored amaranth (Genus Accession No. AKI33831.1; SEQ ID NO: 13) or a combination thereof.

[0236] In one embodiment, the present invention provides a recombinant polynucleotide comprising a nucleic acid encoding the CYP76AD6 gene, the CYP76AD15 gene, or a combination thereof, and a nucleic acid encoding DOPA 4,5-dioxygenase (DOD), wherein the nucleic acid is in-frame inserted into the polynucleotide. In one embodiment, the polynucleotide further comprises a nucleic acid encoding a betaine-associated glucosyltransferase.

[0237] In another embodiment, the invention includes additional nucleic acids encoding polypeptides that modify the structure of beet pigments. In one embodiment, the polypeptide is a glycosylation enzyme. In another embodiment, the polypeptide is an acylase. In another embodiment, the additional nucleic acid encodes a polypeptide involved in subcellular transport. In another embodiment, the additional nucleic acid encodes a polypeptide involved in detoxification.

[0238] Homologous and variant

[0239] In another embodiment, the recombinant polynucleotide or polypeptide of the present invention is homologous to the sequence described herein, either explicitly or by reference to gene library entries. When referring to any amino acid or nucleic acid sequence, the terms “homology,” “homologous,” etc., in one embodiment refer respectively to the percentage of amino acids or nucleotides in the candidate sequence that are identical to the residues of the corresponding native polypeptide or polynucleotide, achieving the maximum percentage of homology if desired after sequence alignment and nick introduction, without considering any conserved substitutions as part of sequence identity. Methods and computer programs for alignment are known in the art, such as BLAST, DOMAIN, BEAUTY (BLAST-enhanced alignment utility), GENPEPT, and TREMBL packages.

[0240] In one embodiment, according to the compositions and methods of the present invention, homologs of CYP76AD1 can be used instead of CYP76AD1.

[0241] In another embodiment, according to the compositions and methods of the present invention, homologs of CYP76AD6 can be used instead of CYP76AD6.

[0242] In another embodiment, according to the compositions and methods of the present invention, homologs of DOPA 4,5-dioxygenase (DOD) can be used instead of DOPA 4,5-dioxygenase (DOD).

[0243] Homology can be at the nucleotide sequence and / or encoded amino acid sequence level. In one embodiment, the nucleic acid and / or amino acid sequences share at least about 50%, or 60%, or 70%, or 80% homology. In another embodiment, the nucleic acid and / or amino acid sequences share at least about 90%, 95%, 96%, 97%, 98%, or 99% homology with the sequences of the present invention. Homology can be at the nucleotide sequence and / or encoded amino acid sequence level. In one embodiment, the nucleic acid and / or amino acid sequences share at least about 72% homology. In another embodiment, the nucleic acid and / or amino acid sequences share at least about 75% homology.

[0244] In another embodiment, homology is determined by identifying candidate sequence hybridization, the methods of which are well described in the art (see, for example, “Nucleic Acid Hybridization” Hames, BD, and Higgins SJ, Eds. (1985); Sambrook et al., 2001, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, NY; and Ausubel et al., 1989, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, NY).

[0245] In one embodiment, the homolog is isolated from another Caryophyllales plant. In one embodiment, the homolog is isolated from cacti, carnations, amaranth, ice plants, beets, carnivorous plants, or combinations thereof. In one embodiment, the carnivorous plant is a Diplophyllaceae family (e.g., *Spicata*), a sundew family (e.g., *Sundew*, *Venus*, *Sundew*), a Dewy Pine family (e.g., *Dewy Pine* or *Portugese Sundew*), or a Nepenthes family (e.g., *Nepenthes tropicalis* or *Monkey Cup*).

[0246] In one embodiment, homologs are isolated based on time expression. In another embodiment, homologs are isolated based on developmental stage. In one embodiment, the developmental stage is stage 1, which is when the flower is approximately 1 cm long. In another embodiment, the developmental stage is stage 2, which is when the flower is approximately 2 cm long. In yet another embodiment, the developmental stage is stage 3, which is when the flower is approximately 3 cm long. In yet another embodiment, the developmental stage is stage 4, which is when the flower is approximately 4 cm long. In yet another embodiment, the developmental stage is stage 5, which is when the flower is approximately 5-6 cm long.

[0247] In another embodiment, homologs are isolated based on tissue expression. In one embodiment, tissue expression of CYP76AD1, CYP76AD6, CYP76AD15, or combinations thereof, or DOD homologs, is performed in floral tissue. In one embodiment, the floral tissue is a petal. In another embodiment, the floral tissue is a stamen. In another embodiment, the floral tissue is an anther. In yet another embodiment, the floral tissue is a stigma.

[0248] In another embodiment, the recombinant polynucleotide or polypeptide of the present invention is a variant of the sequence described herein. In another embodiment, the recombinant polynucleotide or polypeptide of the present invention is an isotype of the sequence described herein. In another embodiment, the recombinant polynucleotide or polypeptide of the present invention is a fragment of the sequence described herein. In one embodiment, the recombinant polynucleotide or polypeptide of the present invention is a functional variant of the sequence described herein. In another embodiment, the recombinant polynucleotide or polypeptide of the present invention is a functional isotype of the sequence described herein. In another embodiment, the recombinant polynucleotide or polypeptide of the present invention is a functional fragment of the sequence described herein. In another embodiment, the recombinant polynucleotide or polypeptide of the present invention is a functional homolog of the sequence described herein.

[0249] In another embodiment, the present invention provides recombinant polynucleotides comprising nucleic acids encoding polypeptides as described herein.

[0250] In one embodiment, the nucleic acid sequence encoding CYP76AD6 is as shown in SEQ ID NO: 30. In another embodiment, the polynucleotide of the present invention optionally includes a nucleic acid sequence encoding CYP76AD6. In one embodiment, the nucleic acid sequence of CYP76AD6 is as shown in SEQ ID NO: 30. In another embodiment, CYP76AD6 in the methods and compositions of the present invention is a homolog of SEQ ID NO: 30. In another embodiment, CYP76AD6 in the methods and compositions of the present invention is a variant of SEQ ID NO: 30. In another embodiment, CYP76AD6 in the methods and compositions of the present invention is a fragment of SEQ ID NO: 30. In another embodiment, CYP76AD6 in the methods and compositions of the present invention is an isotype of SEQ ID NO: 30. In another embodiment, CYP76AD6 in the methods and compositions of the present invention is a functional homolog, functional variant, functional fragment, or functional isotype of SEQ ID NO: 30.

[0251] As demonstrated in Example 19, CYP76AD15, an ortholog of CYP76AD6 in four o'clock flower, exhibits the same activity as CYP76AD6 during transient expression in tobacco Benzoinus. In another embodiment, CYP76AD15 or its homologs, isotypes, or variants may be used in place of CYP76AD6 in the compositions and methods of the present invention. In one embodiment, the CYP76AD15 gene product from four o'clock flower has a function similar to that of the CYP76AD6 gene product from sugar beet. In one embodiment, CYP76AD15 from four o'clock flower is a gene encoding an enzyme involved in the conversion of tyrosine to L-DOPA.

[0252] In one embodiment, the present invention provides a recombinant polynucleotide comprising a nucleic acid encoding CYP76AD15. In one embodiment, the nucleic acid sequence encoding CYP76AD15 comprises:

[0253]

[0254] In another embodiment, the polynucleotide of the present invention comprises a nucleic acid sequence encoding BvCYP76new, as described in Example 3. In another embodiment, the polynucleotide of the present invention comprises a nucleic acid sequence encoding MjCYP76 from Mirabilis jalapa (SEQ ID NO: 1). In one embodiment, CYP76AD15, BvCYP76new, or MjCYP76 may be used in the compositions and methods of the present invention. In another embodiment, other cytochrome P450 encoding genes co-expressed with one or more known beet pigment-related genes used as bait may be used in the compositions and methods of the present invention. In one embodiment, CYP82G1-like (SEQ ID NO: 36), CYP78A9-like (SEQ ID NO: 37), CYP86B1-like (SEQ ID NO: 38), or combinations thereof may be used in the compositions and methods of the present invention. In one embodiment, CYP76AD6 may be used instead.

[0255] In one embodiment, the polynucleotide of the present invention comprises a nucleic acid sequence encoding CYP76AD1. In one embodiment, the polynucleotide of the present invention optionally comprises a nucleic acid sequence encoding CYP76AD1. In one embodiment, the nucleic acid sequence of CYP76AD1 is as shown in SEQ ID NO: 32. In another embodiment, CYP76AD1 in the methods and compositions of the present invention is a homolog of SEQ ID NO: 32. In another embodiment, CYP76AD1 in the methods and compositions of the present invention is a variant of SEQ ID NO: 32. In another embodiment, CYP76AD1 in the methods and compositions of the present invention is a fragment of SEQ ID NO: 32. In another embodiment, CYP76AD1 in the methods and compositions of the present invention is an isotype of SEQ ID NO: 32. In another embodiment, CYP76AD1 in the methods and compositions of the present invention is a functional homolog, functional variant, functional fragment, or functional isotype of SEQ ID NO: 32.

[0256] In another embodiment, the polynucleotide of the present invention comprises a nucleic acid sequence encoding CYP76AD3 (SEQ ID NO: 4), as described in Example 9. In one embodiment, CYP76AD3 may be used instead of CYP76AD1 in the compositions and methods of the present invention.

[0257] In one embodiment, the polynucleotide of the present invention comprises a nucleic acid sequence encoding beet DODA1 (BvDODA1). In one embodiment, the polynucleotide of the present invention optionally comprises a nucleic acid sequence encoding BvDODA1. In one embodiment, the nucleic acid sequence of BvDODA1 is as shown in SEQ ID NO: 33. In another embodiment, BvDODA1 used in the methods and compositions of the present invention is a homolog of SEQ ID NO: 33. In another embodiment, BvDODA1 used in the methods and compositions of the present invention is a variant of SEQ ID NO: 33. In another embodiment, BvDODA1 used in the methods and compositions of the present invention is a fragment of SEQ ID NO: 33. In another embodiment, BvDODA1 used in the methods and compositions of the present invention is an isotype of SEQ ID NO: 33. In another embodiment, BvDODA1 used in the methods and compositions of the present invention is a functional homolog, functional variant, functional fragment, or functional isotype of SEQ ID NO: 33.

[0258] In another embodiment, the DOD enzyme is PgDOD from Portulaca grandiflora (accession number AJ580598; SEQ ID NO: 39), MjDOD from Mirabilis jalapa (accession number AB435372; SEQ ID NO: 3), BgDOD from Bougainvillea glabra (accession number AB435373; SEQ ID NO: 40), or AmDOD from Venus flytrap (accession number P87064; SEQ ID NO: 41).

[0259] In one embodiment, the polynucleotide of the present invention comprises a nucleic acid sequence encoding a circular DOPA 5-O-glucosyltransferase (cDOPA5GT). In one embodiment, the polynucleotide of the present invention optionally comprises a nucleic acid sequence including cDOPA5GT. In one embodiment, the nucleic acid sequence of cDOPA5GT is as shown in SEQ ID NO: 2. In another embodiment, cDOPA5GT in the methods and compositions of the present invention is a homolog of SEQ ID NO: 2. In another embodiment, cDOPA5GT in the methods and compositions of the present invention is a variant of SEQ ID NO: 2. In another embodiment, cDOPA5GT in the methods and compositions of the present invention is a fragment of SEQ ID NO: 2. In another embodiment, cDOPA5GT in the methods and compositions of the present invention is an isotype of SEQ ID NO: 2. In another embodiment, cDOPA5GT in the methods and compositions of the present invention is a functional homolog, functional variant, functional fragment, or functional isotype of SEQ ID NO: 2.

[0260] In one embodiment, a “variant” refers to an amino acid or nucleic acid sequence (or, in other embodiments, an organism or tissue) that differs from the majority population but is still sufficiently similar to a common pattern to be considered one of them, such as a splicing variant. In one embodiment, a variant may be a sequence-conserved variant, while in another embodiment, a variant may be a functionally conserved variant. In one embodiment, a variant may comprise the addition, deletion, or substitution of one amino acid. In one embodiment, a variant may comprise the addition, deletion, or substitution of two amino acids, or a combination thereof. In one embodiment, a variant may comprise the addition, deletion, or substitution of three amino acids, or a combination thereof. In one embodiment, a variant may comprise the addition, deletion, or substitution of four amino acids, or a combination thereof. In one embodiment, a variant may comprise the addition, deletion, or substitution of five amino acids, or a combination thereof. In one embodiment, a variant may comprise the addition, deletion, or substitution of seven amino acids, or a combination thereof. In one embodiment, a variant may comprise the addition, deletion, or substitution of ten amino acids, or a combination thereof. In one embodiment, a variant may comprise the addition, deletion, or substitution of two to fifteen amino acids, or a combination thereof. In one embodiment, a variant may comprise the addition, deletion, or substitution of three to twenty amino acids, or a combination thereof. In one implementation, the variant may include the addition, deletion, or substitution of 4-25 amino acids, or a combination thereof.

[0261] In one embodiment, the term "fragment" is used herein to refer to a protein or polypeptide that is shorter or contains fewer amino acids than a full-length protein or polypeptide. In another embodiment, a fragment refers to a nucleic acid that is shorter or contains fewer nucleotides than a full-length nucleic acid. In another embodiment, a fragment is an N-terminal fragment. In another embodiment, a fragment is a C-terminal fragment. In one embodiment, a fragment is an intra-sequence portion of a protein, peptide, or nucleic acid. In another embodiment, a fragment is a functional portion within a protein, peptide, or nucleic acid.

[0262] In one embodiment, "isotype" refers to a protein isotype or a gene isotype. In one embodiment, a protein isotype is a protein encoded by the same gene, or a different form of a protein from different genes having similar amino acid sequences and functions. In one embodiment, a gene isotype is mRNA generated from the same locus but with different transcription start sites (TSS), protein-coding DNA sequences (CDS), and / or untranslated regions (UTRs). In one embodiment, a gene isotype has altered activity, while in another embodiment, a gene isotype does not have altered activity. Thus, in one embodiment, an isotype is an alternative form of a molecule (e.g., a protein) that has the same function as the first molecule but may have minor differences in structure or sequence. In one embodiment, an isotype can be generated by different but related genes, or in another embodiment, it can be generated by the same gene through alternative splicing. In another embodiment, an isotype is caused by a single nucleotide polymorphism.

[0263] In one embodiment, the term "function" as used herein refers to the innate ability of a protein, peptide, nucleic acid, fragment, or variant thereof to exhibit biological activity or function. In one embodiment, such biological function is its binding property with an interacting partner (e.g., a membrane-associated receptor), and in another embodiment, it is its trimerizing property. In the case of functional fragments and functional variants of the present invention, these biological functions can be modified, for example, for their specificity or selectivity, while retaining the underlying biological function.

[0264] Many methods for measuring the biological activity of proteins, peptides, or molecules are known in the relevant fields, such as protein assays using labeled substrates, substrate analysis by chromatographic methods (e.g., HPLC or thin-layer chromatography), spectrophotometry, etc. (see, for example, Maniatis et al. (2001) Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Laboratory Press, ColdSpring Harbor, NY).

[0265] expression carrier

[0266] In another embodiment, the present invention provides an expression vector comprising any polynucleotide of the present invention as described herein.

[0267] In another embodiment, the present invention provides an expression vector comprising a recombinant polynucleotide comprising a nucleic acid encoding CYP76AD1, a nucleic acid encoding DOPA 4,5-dioxygenase (DOD), and optionally a nucleic acid sequence encoding betaine-associated glucosyltransferase, wherein the nucleic acids are in the same reading frame.

[0268] In another embodiment, the present invention provides an expression vector comprising recombinant polynucleotides, the recombinant polynucleotides comprising tandemly expressed nucleic acids encoding CYP76AD6, CYP76AD15 or a combination thereof and nucleic acids encoding DOPA 4,5-dioxygenase (DOD).

[0269] In another embodiment, the present invention provides an expression vector comprising a recombinant polynucleotide comprising a nucleic acid tandemly expressed encoding CYP76AD1, CYP76AD6, CYP76AD15 or a combination thereof, a nucleic acid encoding DOPA 4,5-dioxygenase (DOD), and optionally a nucleic acid sequence encoding a betaine-associated glucosyltransferase.

[0270] In one embodiment, the polynucleotide of the present invention is inserted into an expression vector (i.e., a nucleic acid construct) to enable the recombinant polypeptide to be expressed. In one embodiment, the expression vector of the present invention includes additional sequences that adapt the vector for replication and integration in prokaryotes. In one embodiment, the expression vector of the present invention includes additional sequences that adapt the vector for replication and integration in eukaryotes. In one embodiment, the expression vector of the present invention includes a shuttle vector that adapts the vector for replication and integration in both prokaryotes and eukaryotes. In some embodiments, the cloning vector contains transcription and translation initiation sequences (e.g., promoters, enhancers) and transcription and translation terminators (e.g., polyadenylation signals).

[0271] In one embodiment, various prokaryotic or eukaryotic cells can be used as host expression systems to express the peptides of the present invention. In some embodiments, these include, but are not limited to, microorganisms, such as bacteria transformed with recombinant phage DNA, plasmid DNA, or copious DNA expression vectors containing peptide coding sequences; yeast transformed with recombinant yeast expression vectors containing peptide coding sequences; and plant cell systems infected with recombinant viral expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors containing peptide coding sequences (e.g., Ti plasmid).

[0272] In one embodiment, the viral vector of the present invention overexpresses the pathway gene described herein. In another embodiment, viral infection of cells expressing the pathway gene described herein induces overexpression of the pathway gene in the cells.

[0273] In some embodiments, non-bacterial expression systems (e.g., mammalian expression systems such as CHO cells) are used to express the polypeptides of the present invention. In one embodiment, the expression vector used to express the polynucleotides of the present invention in mammalian cells is a pCI-DHFR vector containing a CMV promoter and a neomycin resistance gene.

[0274] In some embodiments, within the bacterial system of the present invention, a number of expression vectors may be advantageously selected depending on the intended use for expressing the polypeptide. In one embodiment, a large quantity of polypeptide is desired. In another embodiment, a vector that directs the expression of a high level of protein product is desired, which may be fused with a hydrophobic signal sequence to direct the expression product into the bacterial periplasm or a culture medium in which the protein product is easily purified. In one embodiment, a fusion protein is engineered to have a specific cleavage site to aid in the recovery of the polypeptide. In one embodiment, vectors suitable for this operation include, but are not limited to, the pET series of E. coli expression vectors (Studier et al., Methods in Enzymol. 185:60-891990).

[0275] In one embodiment, a yeast expression system is used. In one embodiment, as disclosed in U.S. Patent No. 5,932,447, a variety of vectors containing constitutive or inducible promoters can be used in yeast. In another embodiment, a vector that promotes the integration of exogenous DNA sequences into the yeast chromosome is used.

[0276] In one embodiment, the expression vector of the present invention may further include additional polynucleotide sequences that allow translation of several proteins, such as sequences from a single mRNA (e.g., an internal ribosome entry site (IRES)) and sequences for genomic integration of promoter chimeric polypeptides.

[0277] In some embodiments, mammalian expression vectors include, but are not limited to, pcDNA3, pcDNA3.1 (+ / -), pGL3, pZeoSV2 (+ / -), pSecTag2, pDisplay, pEF / myc / cyto, pCMV / myc / cyto, pCR3.1, pSinRep5, DH26S, DHBB, pNMT1, pNMT41, pNMT81 (available from Invitrogen), pCI (available from Promega), pMbac, pPbac, pBK-RSV and pBK-CMV (available from Strategene), pTRES (available from Clontech), and derivatives thereof.

[0278] In some embodiments, the present invention uses expression vectors containing regulatory portions derived from eukaryotic viruses such as retroviruses. SV40 vectors include pSVT7 and pMT2. In some embodiments, vectors derived from bovine papillomavirus include pBV-1MTHA, and vectors derived from Epstein-Barr virus include pHEBO and p2O5. Other exemplary vectors include pMSG, pAV009 / A+, pMTO10 / A+, pMAMneo-5, baculovirus pDSVE, and any other vectors that allow expression of proteins guided by the SV-40 early promoter, SV-40 late promoter, metallothionein promoter, murine mammary tumor virus promoter, Raoult's sarcoma virus promoter, polyhedrosis protein promoter, or other promoters that are effectively expressed in eukaryotic cells.

[0279] In some embodiments, recombinant viral vectors can be used to express the peptides of the present invention in vivo because they offer advantages such as lateral spread and target specificity. In one embodiment, lateral spread is inherent in the life cycle of, for example, retroviruses and is a process in which a single infected cell produces numerous budding progeny viral particles that infect neighboring cells. In one embodiment, the result is rapid, large-area infection, with most of the virus initially uninfected by the original viral particles. In one embodiment, a viral vector that cannot spread laterally is produced. In one embodiment, this feature may be useful if the desired objective is to introduce a specific gene into only a local number of target cells.

[0280] In one embodiment, various methods can be used to introduce the expression vector of the present invention into cells. These methods include, for example, stable or transient transfection, lipid transfection, electroporation, and infection with a recombinant viral vector.

[0281] In some implementations, introducing nucleic acids via viral infection offers several advantages over other methods such as lipid transfection and electroporation, because the infectious nature of the virus allows for higher transfection efficiency.

[0282] In one embodiment, it should be understood that the polypeptides of the present invention can also be expressed by nucleic acid constructs administered to an individual using any suitable administration modality described above (i.e., in vivo gene therapy). In one embodiment, the nucleic acid construct is introduced into suitable cells as needed using a suitable gene delivery vector / method (transfection, transduction, homologous recombination, etc.) and expression system, and then the modified cells are amplified in a culture and returned to the individual (i.e., ex vivo gene therapy).

[0283] In one embodiment, a plant expression vector is used. In another embodiment, expression of the polypeptide coding sequence is driven by a variety of promoters. In some embodiments, viral promoters are used, such as the 35S RNA and 19S RNA promoters of CaMV [Brisson et al., Nature 310: 511-514 (1984)] or the capsid protein promoter of TMV [Takamatsu et al., EMBO J. 6: 307-311 (1987)]. In another embodiment, plant promoters are used, such as 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, such as soybean hsp17.5-E or hsp17.3-B [Gurley et al., Mol. Cell. Biol. 6: 559-565 (1986)]. In one embodiment, the construct is introduced into plant cells using Ti plasmids, Ri plasmids, plant viral vectors, direct DNA transformation, microinjection, electroporation, and other techniques well known to those skilled in the art. See, for example, Weissbach & Weissbach [Methods for Plant Molecular Biology, Academic Press, NY, Section VIII, pp 421-463 (1988)]. Other expression systems known in the art, such as insect and mammalian host cell systems, may also be used in this invention.

[0284] It should be understood that, in addition to the necessary portions of transcription and translation containing the coding sequence (coding polypeptide) for insertion, the expression constructs of the present invention may also include sequences engineered to optimize the stability, production, purification, yield, or activity of the expressed polypeptide.

[0285] In some embodiments, various methods may be used to introduce the expression vector of the present invention into a host cell system. In some embodiments, such methods are generally described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Springs Harbor Laboratory, New York (1989, 1992), 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 al. [Biotechniques 4(6): 504-512, 1986], and include, for example, stable or transient transfection, lipid transfection, electroporation, and infection with recombinant viral vectors. See also the positive and negative selection methods in U.S. Patent Nos. 5,464,764 and 5,487,992.

[0286] In some embodiments, the transformed cells are cultured under effective conditions, which allow for the expression of large quantities of recombinant peptides. In some embodiments, effective culture conditions include, but are not limited to, effective culture media, bioreactors, temperature, pH, and oxygen conditions that allow for protein production. In one embodiment, an effective culture medium refers to any culture medium in which cells are cultured to produce the recombinant peptides of the present invention. In some embodiments, the culture medium typically comprises an aqueous solution having assimilated carbon, nitrogen, and phosphorus sources, as well as suitable salts, minerals, metals, and other nutrients, such as vitamins. In some embodiments, the cells of the present invention can be cultured in conventional fermentation bioreactors, shake flasks, test tubes, microtiter plates, and culture dishes. In some embodiments, the culture is carried out at temperatures, pH, and oxygen levels suitable for the recombinant cells. In some embodiments, the culture conditions are within the scope of the expertise of those skilled in the art.

[0287] In some embodiments, depending on the vector and host system used for production, the polypeptides obtained by the present invention may be retained in recombinant cells, secreted into fermentation medium, secreted into the space between two cell membranes (e.g., the periplasmic space in Escherichia coli); or retained on the outer surface of a cell or viral membrane.

[0288] Composition

[0289] In another embodiment, the present invention provides a composition comprising one or more recombinant polynucleotides as described herein. In yet another embodiment, the present invention provides a composition comprising recombinant nucleic acids as described herein.

[0290] In one embodiment, the present invention provides a recombinant polynucleotide comprising a nucleic acid encoding CYP76AD1, a nucleic acid encoding DOPA 4,5-dioxygenase (DOD), and optionally a nucleic acid sequence encoding a betaine-associated glucosyltransferase, wherein said nucleic acids are in the same reading frame.

[0291] In another embodiment, the present invention provides a composition comprising a recombinant polynucleotide comprising a nucleic acid tandemly expressed encoding CYP76AD6, CYP76AD15 or a combination thereof, and a nucleic acid encoding DOPA 4,5-dioxygenase (DOD).

[0292] In another embodiment, the present invention provides an expression vector comprising recombinant polynucleotides as described herein.

[0293] In another embodiment, the present invention provides a composition comprising an expression vector comprising a recombinant polynucleotide, the recombinant polynucleotide comprising a nucleic acid encoding CYP76AD1, a nucleic acid encoding DOPA 4,5-dioxygenase (DOD), and optionally a nucleic acid sequence encoding a betaine-associated glucosyltransferase, wherein said nucleic acids are in the same reading frame.

[0294] In another embodiment, the present invention provides a composition comprising an expression vector comprising a recombinant polynucleotide, the recombinant polynucleotide comprising a nucleic acid tandemly expressed encoding CYP76AD6, CYP76AD15 or a combination thereof, and a nucleic acid encoding DOPA 4,5-dioxygenase (DOD).

[0295] In one embodiment, a composition comprising the recombinant polynucleotide or chimeric polypeptide of the present invention is provided. In one embodiment, the polypeptide and polynucleotide of the present invention may be provided to an individual.

[0296] In another embodiment, the present invention provides cells comprising an expression vector or recombinant polynucleotide as described herein. In yet another embodiment, the present invention provides compositions comprising cells as described herein.

[0297] cell

[0298] In another embodiment, the present invention provides cells comprising recombinant polynucleotides as described herein. In yet another embodiment, the present invention provides cells comprising an expression vector as described herein.

[0299] In another embodiment, the present invention provides a cell comprising a recombinant polynucleotide comprising a nucleic acid encoding CYP76AD1, a nucleic acid encoding DOPA 4,5-dioxygenase (DOD), and optionally a nucleic acid sequence encoding a betaine-associated glucosyltransferase, wherein the nucleic acids are in the same reading frame.

[0300] In another embodiment, the present invention provides a cell comprising recombinant polynucleotides, the recombinant polynucleotides comprising tandemly expressed nucleic acids encoding CYP76AD6, CYP76AD15 or a combination thereof, and nucleic acids encoding DOPA 4,5-dioxygenase (DOD).

[0301] In another embodiment, the present invention provides a cell comprising a recombinant polynucleotide comprising a nucleic acid tandemly expressed encoding CYP76AD1, CYP76AD6, CYP76AD15 or a combination thereof, a nucleic acid encoding DOPA 4,5-dioxygenase (DOD), and optionally a nucleic acid sequence encoding a betaine-associated glucosyltransferase.

[0302] In another embodiment, the present invention provides a cell comprising an expression vector comprising a recombinant polynucleotide, the recombinant polynucleotide comprising a nucleic acid encoding CYP76AD1, a nucleic acid encoding DOPA 4,5-dioxygenase (DOD), and optionally a nucleic acid sequence encoding a betaine-associated glucosyltransferase, wherein said nucleic acids are in the same reading frame.

[0303] In another embodiment, the present invention provides a cell comprising an expression vector comprising a recombinant polynucleotide, the recombinant polynucleotide comprising a nucleic acid tandemly expressed encoding CYP76AD6, CYP76AD15 or a combination thereof and a nucleic acid encoding DOPA4,5-dioxygenase (DOD).

[0304] In another embodiment, the present invention provides a cell comprising an expression vector comprising a recombinant polynucleotide comprising a nucleic acid tandemly expressed encoding CYP76AD1, CYP76AD6, CYP76AD15 or a combination thereof, a nucleic acid encoding DOPA 4,5-dioxygenase (DOD), and optionally a nucleic acid sequence encoding a betaine-associated glucosyltransferase.

[0305] In one embodiment, a plant cell suspension culture can be produced when the gene of the present invention can be transformed into plant cells. In one embodiment, the plant cells are plant stem cells. Betaine, L-DOPA, or combinations thereof can be isolated from the plant suspension culture. Methods for preparing plant cell suspension cultures are well known in the art and are fully described in U.S. Patent Application Publications US2014 / 0051135; US 2011 / 0251408; US 2010 / 0159545; and US 2007 / 0026506, the entire contents of which are incorporated herein by reference.

[0306] In one embodiment, the plant cells are selected from tobacco (Nicotiana tabacum), tomato (Solanum lycopersicum), potato (Solanum tuberosum), eggplant (Solanum melongena), tree tobacco (Nicotiana glauca), European black nightshade (Solanum nigrum), petunia (Petunia x hybrida), and tobacco Benedict. In another embodiment, the plant cells are derived from any of the plants described below.

[0307] In one embodiment, a polynucleotide is introduced into a plant cell, which can be derived from any plant organ of interest, as described below. In one embodiment, the polynucleotide is introduced into a portion of a plant organ. In one embodiment, the polynucleotide is introduced into a portion of a flower. In one embodiment, a portion of the flower is a petal, stamen, anther, stigma, or a combination thereof.

[0308] The present invention also provides a method for manufacturing transgenic seeds, which can be used to produce transgenic plant crops with enhanced traits resulting from the expression of a stably integrated recombinant DNA construct.

[0309] In another embodiment, the cells of the present invention are cells derived from microorganisms. In another embodiment, the cells are yeast cells. In one embodiment, the yeast cells are *Saccharomyces cerevisiae* cells. In another embodiment, the cells of the present invention are bacterial cells. In one embodiment, the bacterial cells are *Escherichia coli* cells. In another embodiment, the cells are *Aquilaria sinensis*, *Aspergillus oryzae*, *Yersinia lipolytica*, *Bacillus* JPJ, *Brucella* SGJ, *Erwinia spp.*, *Citrobacter freundii*, commensal bacteria, or *Pseudomonas aeruginosa* cells. In another embodiment, the bacterial cells are derived from bacteria involved in dairy product fermentation. In one embodiment, the bacteria are *Streptococcus lactis*. In another embodiment, the bacteria are *Lactobacillus*. In one embodiment, the *Lactobacillus bulgaricus*. In another embodiment, the bacteria are *Lactococcus lactis* or *Leuconostoc mesenteroides*.

[0310] Plants and plant parts

[0311] In another embodiment, the present invention provides a plant or part thereof produced by a method of increasing the level of one or more betaine pigments or L-DOPA in the plant or plant part, the method comprising inducing or permitting the expression of DOPA 4,5-dioxygenase (DOD), CYP76AD6, CYP76AD15 or a combination thereof, CYP76AD1 or a combination thereof in the plant or plant part. In one embodiment, plants expressing DOD and CYP76AD1 but not CYP76AD6 or CYP76AD15 also express betaine-associated glucosyltransferases.

[0312] In one embodiment, the plant does not naturally produce betaine. In another embodiment, the plant produces low levels of betaine. In yet another embodiment, the plant does not naturally produce detectable levels of betaine.

[0313] In another embodiment, the plant or plant part described herein naturally produces betaine; however, the genetically modified plant or plant part has altered levels of betaine or a specific betaine, thereby altering the color and / or properties of the genetically modified plant. In one embodiment, the alteration of betaine level is an increase. In another embodiment, the alteration of betaine level is a decrease.

[0314] In another embodiment, the present invention provides a plant or a portion thereof comprising a nucleic acid sequence encoding CYP76AD1, a nucleic acid sequence encoding CYP76AD6, CYP76AD15 or a combination thereof, and a nucleic acid sequence encoding DOPA 4,5-dioxygenase (DOD) or a combination thereof.

[0315] In another embodiment, betaine or L-DOPA is produced throughout the plant as described herein. In one embodiment, the seed is genetically modified to produce betaine or L-DOPA throughout the plant. In another embodiment, betaine or L-DOPA is produced in a specific organ of the plant. In yet another embodiment, betaine or L-DOPA is produced in a portion of a plant organ. For example, betaine or L-DOPA can be produced in some plant cells in the leaves or other organs of the plant.

[0316] In another embodiment, the present invention provides an ornamental plant produced by a method of increasing the level of one or more betaine pigments in a plant or a plant part thereof, the method comprising inducing or permitting the expression of DOPA 4,5-dioxygenase (DOD) and CYP76AD6, CYP76AD15 or combinations thereof, CYP76AD1 or combinations thereof in said plant or plant part. In one embodiment, if the ornamental plant expresses CYP76AD1 and DOD but not CYP76AD6 or CYP76AD15, then betaine-associated glucosyltransferases are further expressed.

[0317] In another embodiment, the present invention provides an ornamental plant comprising a nucleic acid sequence encoding CYP76AD1, CYP76AD6, CYP76AD15 or a combination thereof, and a nucleic acid sequence encoding DOPA 4,5-dioxygenase (DOD).

[0318] In one embodiment, different parts or organs of the organism of the present invention contain different pigment deposits due to the expression of multiple cytochrome P450s (e.g., CYP76AD6, CYP76AD1) under different nonconstitutive promoter sequences (e.g., fruit-specific, flower-specific, or inducible promoters). In one embodiment, the organism is a plant and the different pigment deposits are in different parts of the plant. In one embodiment, the plant is an ornamental plant.

[0319] In another embodiment, the present invention provides a food crop produced by a method of increasing the level of one or more betaine pigments in a plant or part thereof used as a food crop, the method comprising inducing or permitting the expression of DOPA 4,5-dioxygenase (DOD) and CYP76AD6, CYP76AD15 or combinations thereof, CYP76AD1 or combinations thereof in said plant or part thereof. In one embodiment, if the food crop expresses CYP76AD1 and DOD but not CYP76AD6 or CYP76AD15, then betaine-associated glucosyltransferases are further expressed.

[0320] In another embodiment, the present invention provides a plant or part thereof used as a food crop, comprising a nucleic acid sequence encoding CYP76AD1, CYP76AD6, CYP76AD15 or a combination thereof, and a nucleic acid sequence encoding DOPA 4,5-dioxygenase (DOD).

[0321] In one embodiment, the compositions and methods of the present invention comprise one or more plant parts. In one embodiment, the plant part as described herein is a plant organ. In one embodiment, the plant part is a leaf, stem, root, flower, seed, tuber, or fruit. In one embodiment, the plant part is an edible portion of a plant. In one embodiment, the compositions and methods of the present invention comprise one or more portions of one or more plant parts. In one embodiment, a portion of the plant part is a portion of a flower. In one embodiment, a portion of the flower is a petal, stamen, anther, or stigma.

[0322] In one embodiment, CYP76AD6, CYP76AD15, or combinations thereof, as well as other genes of the invention described herein, can be transformed into plants or plant cells. As used herein, the term "plant" can refer to any monocotyledonous or dicotyledonous plant. Examples of monocotyledonous plants include, but are not limited to, maize, wheat, rice, sugarcane, and banana. Examples of dicotyledonous plants include, but are not limited to, soybeans, beans, peas, lentils, peanuts, tomatoes, potatoes, cotton, and perennial fruit trees (including grapes, apples, and oranges).

[0323] In one embodiment, the plant is tobacco (Nicotiana tabacum), tomato (Solanum lycopersicum), potato (Solanum tuberosum), eggplant (Solanum melongena), tree tobacco (Nicotiana glauca), European black nightshade (Solanum nigrum), petunia (Petunia x hybrida), or Benzoin's tobacco. In another embodiment, the plant is beet. In yet another embodiment, the plant is four o'clock flower (Mirabilis jalapa).

[0324] In one embodiment, the plant of the present invention is a crop plant. In one embodiment, the crop plant is *Solanum tuberosum* (potato). In another embodiment, the crop plant is *Zea mays* (corn). In another embodiment, the crop plant is *Oryza sativa* (rice). In another embodiment, the crop plant is *Manihot esculenta* (cassava). In another embodiment, the crop plant is *Hordeum vulgare* (barley). In another embodiment, the crop plant is *Triticum aestivum* (wheat). In another embodiment, the crop plant is *Sorghum bicolor* (sorghum). In another embodiment, the crop plant is *Brassica napus* (canola). In another embodiment, the crop plant is *Ricinus communis* (castor bean). In another embodiment, the crop plant is *Phaseolus vulgaris* (bean). In another embodiment, the crop plant is *Gossypium histrum* (cotton). In another embodiment, the crop plant is *Glycine max* (soybean). In another embodiment, the crop plant is *Beta vulgaris* (beetroot). In another embodiment, the crop plant is *Musa acuminate* (banana). In another embodiment, the crop is *Capsicum annuum* (bell pepper and chili pepper). In another embodiment, the crop is *Cicer arietinum* (chickpea). In another embodiment, the crop is *Solanumlycopersicum* (tomato). In another embodiment, the crop is *Elaeis guineensis* (African oil palm). In another embodiment, the crop is *Setaria italic* (millet).

[0325] In another embodiment, the plant of the invention is bamboo, specifically Arundinaria gigantea and Arundinaria tecta in one embodiment. In another embodiment, the plant is duckweed.

[0326] In another embodiment, the plant of the invention is a moss. In one embodiment, the moss is peat moss. In one embodiment, the species of peat moss is *Cristatum* or *Subnitens*. In one embodiment, the moss is used for peat. In one embodiment, peat is used as fuel, as a horticultural soil additive, and in the production of malted Scotch whisky. In another embodiment, the moss is used for decorative purposes, such as in gardens and the florist trade. In another embodiment, the moss is used as an insulator. In another embodiment, the moss is used as a liquid absorbent. In another embodiment, the moss is used in first-aid dressings, diapers, or napkins. In another embodiment, the moss is *Sphaerocarpus serrata*. In another embodiment, the moss is *Sphaerocarpus aquatilis*, which in one embodiment is used for fire suppression.

[0327] The plants included in this invention are any plants suitable for transformation technology, including gymnosperms and angiosperms (including both monocots and dicots).

[0328] Examples of monocotyledonous angiosperms include, but are not limited to, asparagus, waxy corn and sweet corn, barley, wheat, rice, sorghum, onion, pearl millet, rye and oats, and other grains.

[0329] Examples of dicotyledonous angiosperms include, but are not limited to, tomatoes, tobacco, cotton, rapeseed, broad beans, soybeans, peppers, lettuce, peas, alfalfa, clover, rapeseed crops or cabbage (e.g., cabbage, broccoli, cauliflower, Brussels sprouts), radishes, carrots, beets, eggplants, spinach, cucumbers, squash, melons, cantaloupes, sunflowers, and various ornamental plants.

[0330] In another embodiment, any species of woody, ornamental, or decorative, crop or cereal, fruit or vegetable plant, and algae (e.g., Chlamydomonas reinhardtii) may be used in the compositions and methods provided herein. Non-limiting examples of plants include plants from the genera Arabidopsis or rice. Other examples include plants from the following genera: sweet flag, goat's foot grass, onion, oilless camphor, snapdragon, celery, peanut, beet, birch, brassica, chili pepper, water fern, citrus, Japanese cedar, cycad, shepherd's purse, California poppy, eucalyptus, soybean, cotton, ear grass, sunflower, barley, sweet potato, lettuce, flax, tulip tree, lotus, lupin, tomato, alfalfa, pine needle chrysanthemum, tobacco, yellow water lily, foxtail grass, avocado, common bean, small sedge, spruce, pine, trifoliate orange, poplar, plum, black locust, rosa, sugarcane, long inflorescence, rye, sesame, eggplant, sorghum, stevia, salt mustard, cocoa, straight-fruited grass, wheat, grape, corn, or zinnia.

[0331] Examples of woody species include poplar, pine, redwood, cedar, and oak.

[0332] In some embodiments, the plants of the present invention are crop plants (e.g., cereals and legumes, corn, wheat, potato, cassava, rice, sorghum, millet, cassaya, barley, peas, and other root, tuber, or seed crops). Exemplary cereal crops used in the compositions and methods of the present invention include, but are not limited to, any species of grass or cereal plant (e.g., barley, corn, oats, rice, wild rice, rye, wheat, millet, sorghum, triticale, etc.) and non-grass plants (e.g., buckwheat, flax, legumes, or soybeans, etc.). Cereal plants providing seeds of interest include oilseed plants and legumes. Other seeds of interest include cereal seeds such as corn, wheat, barley, rice, sorghum, rye, etc. Oilseed plants include cotton, soybeans, safflower, sunflower, brassica, corn, alfalfa, palm, coconut, etc. Other important seed crops are oilseed rape, sugar beet, corn, sunflower, soybean, and sorghum. Legumes include beans and peas. Legumes include guar beans, locust beans, fenugreek, soybeans, garden beans, cowpeas, mung beans, lima beans, broad beans, lentils, chickpeas, etc.

[0333] Horticultural plants to which this invention can be applied may include lettuce, chicory, and vegetable rapeseed (including cabbage, broccoli, and cauliflower), as well as carnations and geraniums. This invention can also be applied to tobacco, gourds, carrots, strawberries, sunflowers, tomatoes, peppers, chrysanthemums, poplars, eucalyptus, and pine trees.

[0334] This invention can be used for the transformation of other plant species, including but not limited to rapeseed (Brassica napus, Brassica rapa subspecies), alfalfa (Medicago sativa), rye (Secale cereale), sorghum (Sorghumbicolor), sunflower (Helianthus), wheat (Triticum aestivum), soybean (Glycine max), tobacco (Nicotiana tabacum, Nicotiana benthamiana), tree tobacco (Nicotiana glauca), peanut (Arachis hypogaea), cotton (Gossypium hirsutum), sweet potato (Ipomoea batatus), cassava (Manihot esculenta), coffee (Coffea genus), coconut (Cocos nucifera), pineapple (Ananas comosus), citrus tree (Citrus genus), cocoa (Theobroma cacao), tea (Camellia sinensis), banana (Musa genus), avocado (Persea americana), fig (Ficus casica), and guava (Psidium spp.). guajava), mango (Mangifera indica), olive oil (Olea europaea), papaya (Carica papaya), cashew (Anacardium occidentale), macadamia nut (Macadamia integrifolia), almond (Prunus amygdalus), beet (Beta vulgaris), tomato (Solanum lycopersicum), eggplant (Solanum melongena), black nightshade (Solanum nigrum), petunia (Petunia x hybrida), oats, barley, millet, fruits, vegetables, ornamental plants, turf, and conifers.

[0335] In addition to plants, the present invention provides any clone of such plants, seeds, self-pollinated or hybrid offspring and progeny, and any part of any of these (e.g., cuttings, seeds). The present invention provides any plant propagule, that is, any part that can be used for sexual or asexual reproduction or propagation, including cuttings, seeds, etc. The present invention also includes plants that are the sexual or asexual offspring, clones, or progeny of such plants, or any part or propagule of said plant, offspring, clones, or progeny. Plant extracts and derivatives are also provided.

[0336] In this application, the terms "plant, plant part, seed, or plant cell transformed with or by a construct or interchangeably with a construct, or transformed with or by a nucleic acid" should be understood to mean a plant, plant part, seed, or plant cell carrying the construct or the nucleic acid as a transgenic organism, i.e., as a result of the introduction of the construct or the nucleic acid by a biological means. The plant, plant part, seed, or plant cell thus contains the recombinant construct or the recombinant nucleic acid. Any plant, plant part, seed, or plant cell that no longer contains the recombinant construct or the recombinant nucleic acid after past introduction is referred to as a null isolate, null zygote, or null control, but is not considered, within the meaning of this application, a plant, plant part, seed, or plant cell transformed with the construct or the nucleic acid.

[0337] In one embodiment, tyrosine is endogenous to the organism or part of the organism of the present invention. In another embodiment, the organism or part of the organism of the present invention synthesizes tyrosine. In another embodiment, the organism or part of the organism of the present invention is genetically modified to produce tyrosine. In another embodiment, the organism or part of the organism of the present invention is provided with or supplied with tyrosine. In another embodiment, the organism or part of the organism containing endogenous tyrosine is genetically modified as described herein to produce a greater amount of tyrosine in order to increase the production of L-DOPA or betaine. In another embodiment, the organism or part of the organism containing endogenous tyrosine is supplied with tyrosine to have a greater amount of available tyrosine, thereby increasing the production of L-DOPA or betaine.

[0338] Methods for extracting L-DOPA and beet pigment

[0339] In one embodiment, the method of the present invention further provides a method for extracting L-DOPA or beet pigment as described in Example 1, as well as other L-DOPA or beet pigment extraction methods known in the art, such as Misra and Wagner (Indian J Biochem Biophys. Feb. 2007; 44(1): 56-60), which are incorporated herein by reference in their entirety.

[0340] In another embodiment, L-DOPA or beet pigment is extracted from algae, yeast, or bacterial cultures using methods known in the art.

[0341] In another embodiment, the present invention provides a method for harvesting L-DOPA or betaine, the method comprising the steps of producing L-DOPA or betaine as described herein, and further comprising the step of extracting said L-DOPA or said betaine from a plant, plant part, colony, organ, tissue, or cell to produce said L-DOPA or said betaine. In one embodiment, the plant part is a leaf, stem, root, flower, seed, tuber, or fruit.

[0342] In one embodiment, L-DOPA or betaine is extracted from the roots of a plant. In another embodiment, L-DOPA or betaine is secreted into a liquid or other medium by the plant roots and then harvested from the liquid or other medium. In yet another embodiment, L-DOPA or betaine is extracted from a culture medium containing a plant cell suspension (e.g., BY2 tobacco).

[0343] In another embodiment, L-DOPA or beet pigment is extracted from the aquatic plant. In one embodiment, the aquatic plant is duckweed. In one embodiment, the duckweed is *Lemna trisulca*; *Lemna japonica*; *Lemna obscura*; *Lemna tenera*; *Lemna turionifera*; *Lemna yungensis*; or combinations thereof.

[0344] polypeptide

[0345] In one embodiment, the present invention provides a chimeric polypeptide encoded by a recombinant polynucleotide encoding a nucleic acid encoding CYP76AD6. In one embodiment, the amino acid sequence encoding CYP76AD6 includes:

[0346] MDNATLAVILSILFVFYHIFKSFFTNSSSRRLPPGPKPVPIFGNIFDLGEKPHRSFANLSKIHGPLISLKLGSVTTIVVSSASVAEEMFLKNDQALANRTIPDSVRAGDHDKLSMSWLPVSQKWR NMRKISAVQLLSNQKLDASQPLRQAKVKQLLSYVQVCSEKMQPVDIGRAAFTTSLNLLSNTFFSIELASHESSASQEFKQLMWNIMEEIGRPNYADFFPILGYIDPFGIRRRLAGYFDKLIDVFQD IIRERQKLRSSNSSGAKQTNDILDTLLKLHEDNELSMPEINHLLVDIFDAGTDTTASTLEWAMAELVKNPEMMTKVQIEIEQALGKDCLDIQESDISKLPYLQAIIKETLRLHPPTVFLLPRKADN DVELYGYVVPKNAQVLVNLWAIGRDPKVWKNPEVFSPERFLDCNIDYKGRDFELLPFGAGRRICPGLTLAYRMLNLMLATLLQNYNWKLEDGINPKDLDMDEKFGITLQKVKPLQVIPVPRN (SEQ ID NO: 18).

[0347] In another embodiment, the present invention provides a chimeric polypeptide encoded by a recombinant polynucleotide comprising a nucleic acid encoding CYP76AD1. In one embodiment, the amino acid sequence encoding CYP76AD1 comprises:

[0348] MDHATLAMILAIWFISFHFIKLLFSQQTTKLLPPGPKPLPIIGNILEVGKKPHRSFANLAKIHGPLISLRLGSVTTIVVSSADVAKEMFLKKDHPLSNRTIPNSVTAGDHHKLTMSWLPVSPKWR NFRKITAVHLLSPQRLDACQTFRHAKVQQLYEYVQECAQKGQAVDIGKAAFTTSLNLLSKLFFSVELAHHKSHTSQEFKELIWNIMEDIGKPNYADYFPILGCVDPSGIRRRLACSFDKLIAVFQ GIICERLAPDSSTTTTTTTDDVLDVLLQLFKQNELTMGEINHLLVDIFDAGTDTTSTFEWVMTELIRNPEMMEKAQEEIKQVLGKDKQIQESDIINLPYLQAIIKETLRLHPPTVFLLPRKADT DVELYGYIVPKDAQILVNLWAIGRDPNAWQNADIFSPERFIGCEIDVKGRDFGLLPFGAGRRICPGMNLAIRMLTLMLATLLQFFNWKLEGDISPKDLDMDEKFGIALQKTKPLKLIPIPRY (SEQ ID NO: 14).

[0349] In another embodiment, the present invention provides a chimeric polypeptide encoded by a recombinant polynucleotide comprising a nucleic acid encoding DOPA 4,5-dioxygenase (DOD). In one embodiment, the DOD enzyme is beet DODA1 (BvDODA1). In one embodiment, the amino acid sequence encoding BvDODA1 comprises:

[0350] MKMMNGEDANDQMIKESFFITHGNPILTVEDTHPLRPFFETWREKIFSKKPKAILIISGHWETVKPTVNAVHINDTIHDFDDYPAAMYQFKYPAPGEPELARKVEEILKKSGFETAETDQKRGLDHGAWVPLMLMYPEA DIPVCQLSVQPHLDGTYHYNLGRALAPLKNDGVLIIGSGSATHPLDETPHYFDGVAPWAAAFDSWLRKALINGRFEEVNIYESKAPNWKLAHPFPEHFYPLHVVLGAAGEKWKAELIHSSWDHGTLCHGSYKFTSA (SEQ ID NO: 42).

[0351] In one embodiment, the present invention provides a chimeric polypeptide encoded by a recombinant polynucleotide comprising a nucleic acid encoding a circular-DOPA 5-O-glucosyltransferase (cDOPA5GT). In one embodiment, the amino acid sequence encoding cDOPA5GT comprises:

[0352] MTAIKMNTNGEGETQHILMIPFMAQGHLRPFLELAMFLYKRSHVIITLLTTPLNAGFLRHLLHHHSYSSSGIRIVELPFNSTNHGLPPGIENTDKLTLPLVVSLFHSTISLDPHLRDYISRHFSPA RPPLCVIHDVFLGWVDQVAKDVGSTGVVFTTGGAYGTSAYVSIWNDLPHQNYSDDQEFPLPGFPENHKFRRSQLHRFLRYADGSDDWSKYFQPQLRQSMKSFGWLCNSVEEIETLGFSILRNYTKL PIWGIGPLIASPVQHSSSDNNSTGAEFVQWLSLKEPDSVLYISFGSQNTISPTQMMELAAGLESSEKPFLWVIRAPFGFDINEEMRPEWLPEGFEERMKVKKQGKLVYKLGPQLEILNHESIGGFL THCGWNSILESLREGVPMLGWPLAAEQAYNLKYLEDEMGVAVELARGLEGEISKEKVKRIVEMILERNEGSKGWEMKNRAVEMGKKLKDAVNEEEKELKGSSVKAIDDFLDAVMQAKLEPSLQ (SEQ ID NO: 43).

[0353] In one embodiment, the polypeptide of the present invention comprises an amino acid sequence encoding CYP76AD6, CYP76AD15, or a combination thereof, and an amino acid sequence encoding CYP76AD1. In another embodiment, the polypeptide of the present invention comprises an amino acid sequence encoding CYP76AD6, CYP76AD15, or a combination thereof, and an amino acid sequence encoding a DOD enzyme. In another embodiment, the polypeptide of the present invention comprises an amino acid sequence encoding CYP76AD6, CYP76AD15, or a combination thereof, an amino acid sequence encoding CYP76AD1, and an amino acid sequence encoding a DOD enzyme. In yet another embodiment, the polypeptide of the present invention comprises an amino acid sequence encoding CYP76AD6, CYP76AD15, or a combination thereof, an amino acid sequence encoding CYP76AD1, and an amino acid sequence encoding a betaine-associated glucosyltransferase. In another embodiment, the polypeptide of the present invention comprises an amino acid sequence encoding CYP76AD6, CYP76AD15, or a combination thereof, an amino acid sequence encoding a DOD enzyme, and an amino acid sequence encoding a betaine-associated glucosyltransferase. In another embodiment, the polypeptide of the present invention comprises an amino acid sequence encoding CYP76AD1, an amino acid sequence encoding a DOD enzyme, and an amino acid sequence encoding a betaine-associated glucosyltransferase. In yet another embodiment, the polypeptide of the present invention comprises an amino acid sequence encoding CYP76AD6, CYP76AD15, or a combination thereof, an amino acid sequence encoding CYP76AD1, an amino acid sequence encoding a DOD enzyme, and an amino acid sequence encoding a betaine-associated glucosyltransferase.

[0354] In another embodiment, any of the polypeptides described above further includes a betaine-associated glucosyltransferase. In one embodiment, the betaine-associated glucosyltransferase is a cyclic-DOPA 5-O-glucosyltransferase or a betaine-5-O-glucosyltransferase. In one embodiment, the cyclic-DOPA 5-O-glucosyltransferase is the four o'clock gene cyclic-DOPA 5-O-glucosyltransferase (cDOPA5GT) (SEQ ID NO: 43).

[0355] In one embodiment, the present invention provides a chimeric polypeptide encoded by the recombinant polynucleotides described herein.

[0356] In another embodiment, the present invention provides a chimeric polypeptide comprising CYP76AD6, CYP76AD15 or a combination thereof, CYP76AD1, DOPA 4,5-dioxygenase (DOD), betaine-associated glucosyltransferase or a combination thereof, wherein the protein is expressed in tandem.

[0357] In another embodiment, the present invention provides a chimeric polypeptide comprising CYP76AD1, DOPA 4,5-dioxygenase (DOD), and optionally a betaine-associated glucosyltransferase, wherein the proteins are expressed in tandem.

[0358] In another embodiment, the present invention provides a chimeric polypeptide comprising CYP76AD6, CYP76AD15 or a combination thereof and DOPA 4,5-dioxygenase (DOD), wherein the protein is expressed in tandem.

[0359] In another embodiment, the present invention provides a chimeric polypeptide comprising CYP76AD6, CYP76AD15 or a combination thereof, CYP76AD1 or a combination thereof, DOPA 4,5-dioxygenase (DOD) and optionally a betaine-associated glucosyltransferase, wherein the protein is expressed in tandem.

[0360] In one implementation, a "chimeric" or "fusion" polypeptide or protein is a polypeptide or protein produced by linking two or more genes that originally encode a single protein.

[0361] It should be understood that, according to the present invention, CYP76AD6 and CYP76AD6-like polypeptides from plants other than sugar beets can be used in the compositions and methods described herein and can replace CYP76AD6 from sugar beets. In one embodiment, the CYP76AD6-like polypeptide is from the order Caryophyllales. In one embodiment, the CYP76AD6-like polypeptide is a homolog within the CYP76AD1-β clade, as described in Brockington et al. (2015) (New Phytol. Sep 2015; 207(4): 1170-80), which is incorporated herein by reference in its entirety.

[0362] In one embodiment, CYP76AD15 or its homologs, isotypes, or variants may be used in place of CYP76AD6 in the compositions and methods of the present invention. CYP76AD15 from Mirabilis jalapa has similar functions to CYP76AD6 from beets. In one embodiment, CYP76AD15 is an enzyme involved in the conversion of tyrosine to L-DOPA. In one embodiment, the amino acid sequence of CYP76AD15 includes:

[0363] MENTMLGVILATIFLTFHIMKMLFSPSKVKLPPGPRPLPIIGNILELGDKPHRSFANLAKIHGPLVTLKLGSVTTIVVSSSEVAKEMFLKNDQPLANRTIPDSVRAGNHDKLSMSWLPVSPKWRNLR KISAVQLLSTQRLDASQAHRQAKIKQLIEYVKKCSKIGQYVDIGQVAFTTSLNLLSNTFFSKELASFDSDNAQEFKQLMWCIMEEIGRPNYADYFPILGYVDPFGARRRLSRYFDQLIEVFQVIIRE RLTHDNNIVGNNNDVLATLLDLYKQNELTMDEINHLLVDIFDAGTDTTASTLEWAMSELIKNPHIMAKAQEEVRRATMSHGGATVAEIQESDINNLPYIQSIIKETLRLHPPTVFLLPRKADVDVQL FGYVVPKNAQVLVNLWAIGRDPNVWPDPEVFSPERFMDCEIDVKGRDFELLPFGAGRRICPGLSLAYRMLNLMLANMVHSFDWKLPGVENGSGSEMDSLDMDEKFGITLQKVQPLKVIPVSRK (SEQ ID NO: 44).

[0364] In one embodiment, the present invention provides isolated polypeptides comprising amino acid sequences encoding CYP76AD6, CYP76AD15, or combinations thereof, or another polypeptide described herein. In another embodiment, the present invention provides non-naturally occurring polypeptides comprising amino acid sequences encoding CYP76AD6, CYP76AD15, or combinations thereof, or another polypeptide described herein. In one embodiment, the CYP76AD6, CYP76AD15, or combinations thereof sequences are modified sequences. In one embodiment, it is an enhanced sequence. In one embodiment, it is an optimized sequence.

[0365] In one embodiment, the polypeptide of the present invention optionally includes CYP76AD6. In one embodiment, the amino acid sequence of CYP76AD6 is as shown in SEQ ID NO: 18. In another embodiment, CYP76AD6 in the methods and compositions of the present invention is a homolog of SEQ ID NO: 18. In another embodiment, CYP76AD6 in the methods and compositions of the present invention is a variant of SEQ ID NO: 18. In another embodiment, CYP76AD6 in the methods and compositions of the present invention is a fragment of SEQ ID NO: 18. In another embodiment, CYP76AD6 in the methods and compositions of the present invention is an isotype of SEQ ID NO: 18. In another embodiment, CYP76AD6 in the methods and compositions of the present invention is a functional homolog, functional variant, functional fragment, or functional isotype of SEQ ID NO: 18.

[0366] In one embodiment, the polypeptide of the present invention optionally includes CYP76AD15. In one embodiment, the amino acid sequence of CYP76AD15 is as shown in SEQ ID NO: 44. In another embodiment, CYP76AD15 in the methods and compositions of the present invention is a homolog of SEQ ID NO: 44. In another embodiment, CYP76AD15 in the methods and compositions of the present invention is a variant of SEQ ID NO: 44. In another embodiment, CYP76AD15 in the methods and compositions of the present invention is a fragment of SEQ ID NO: 44. In another embodiment, CYP76AD15 in the methods and compositions of the present invention is an isotype of SEQ ID NO: 44.

[0367] In another embodiment, CYP76AD15 used in the methods and compositions of the present invention is a functional homolog, functional variant, functional fragment, or functional isotype of SEQ ID NO: 44.

[0368] In one embodiment, the polypeptide of the present invention optionally includes CYP76AD1. In one embodiment, the amino acid sequence of CYP76AD1 is as shown in SEQ ID NO: 14. In another embodiment, CYP76AD1 used in the methods and compositions of the present invention is a homolog of SEQ ID NO: 14. In another embodiment, CYP76AD1 used in the methods and compositions of the present invention is a variant of SEQ ID NO: 14. In another embodiment, CYP76AD1 used in the methods and compositions of the present invention is a fragment of SEQ ID NO: 14. In another embodiment, CYP76AD1 used in the methods and compositions of the present invention is an isotype of SEQ ID NO: 14.

[0369] In another embodiment, CYP76AD1 used in the methods and compositions of the present invention is a functional homolog, functional variant, functional fragment, or functional isotype of SEQ ID NO: 14.

[0370] In one embodiment, the polypeptide of the present invention optionally includes BvDODA1. In one embodiment, the amino acid sequence of BvDODA1 is as shown in SEQ ID NO: 42. In another embodiment, BvDODA1 used in the methods and compositions of the present invention is a homolog of SEQ ID NO: 42. In another embodiment, BvDODA1 used in the methods and compositions of the present invention is a variant of SEQ ID NO: 42. In another embodiment, BvDODA1 used in the methods and compositions of the present invention is a fragment of SEQ ID NO: 42. In another embodiment, BvDODA1 used in the methods and compositions of the present invention is an isotype of SEQ ID NO: 42.

[0371] In another embodiment, BVDODA1 used in the methods and compositions of the present invention is a functional homolog, functional variant, functional fragment, or functional isotype of SEQ ID NO: 42.

[0372] In one embodiment, the polypeptide of the present invention optionally includes cDOPA5GT. In one embodiment, the amino acid sequence of cDOPA5GT is as shown in SEQ ID NO: 43. In another embodiment, cDOPA5GT used in the methods and compositions of the present invention is a homolog of SEQ ID NO: 43. In another embodiment, cDOPA5GT used in the methods and compositions of the present invention is a variant of SEQ ID NO: 43. In another embodiment, cDOPA5GT used in the methods and compositions of the present invention is a fragment of SEQ ID NO: 43. In another embodiment, cDOPA5GT used in the methods and compositions of the present invention is an isotype of SEQ ID NO: 43.

[0373] In another embodiment, cDOPA5GT used in the methods and compositions of the present invention is a functional homolog, functional variant, functional fragment, or functional isotype of SEQ ID NO: 43.

[0374] In some embodiments, the terms “peptide,” “engineered peptide,” or “protein” as used herein include native peptides (degradation products, synthetic peptides, or recombinant peptides) and peptide analogs (typically, synthetic peptides), as well as peptide-like peptides and hemipeptides that, in some embodiments, have modifications that make the peptide more stable in vivo or more capable of penetrating cells.

[0375] In some embodiments, modifications include, but are not limited to, C-terminal modifications, peptide bond modifications (including, but not limited to, CH2-NH, CH2-S, CH2-S=O, O=C-NH, CH2-O, CH2-CH2, S=C-NH, CH=CH, or CF=CH), backbone modifications, and residue modifications. Methods for preparing peptide-like compounds are known in the art and are described in detail, for example, in Quantitative Drug Design, CA Ramsden Gd., Chapter 17.2, F. Choplin Pergamon Press (1992), which is incorporated herein by reference as if fully described herein. Further details in this regard are provided below.

[0376] In some embodiments, the polypeptide bond (-CO-NH-) within the polypeptide is substituted. In some embodiments, the polypeptide bond is substituted with an N-methylation bond (-N(CH3)-CO-). In some embodiments, the polypeptide bond is substituted with an ester bond (-C(R)HCOOC(R)-N-). In some embodiments, the polypeptide bond is substituted with a ketomethylene bond (-CO-CH2-). In some embodiments, the polypeptide bond is substituted with an α-aza bond (-NH-N(R)-CO-) (where R is any alkyl group, such as methyl) or a carbamate bond (-CH2-NH-). In some embodiments, the polypeptide bond is substituted with a hydroxyethylene bond (-CH(OH)-CH2-). In some embodiments, the polypeptide bond is substituted with a thioamide bond (-CS-NH-). In some embodiments, the polypeptide bond is substituted with an alkene double bond (-CH=CH-). In some embodiments, the polypeptide bond is substituted with a retroamide bond (-NH-CO-). In some embodiments, the polypeptide bond is replaced by a polypeptide derivative (-N(R)-CH2-CO-), where R is a "normal" side chain naturally present on a carbon atom. In some embodiments, these modifications occur at any bond along the polypeptide chain, and in one embodiment, they occur simultaneously at several (2-3) bonds.

[0377] In some embodiments, the natural aromatic amino acids of the polypeptide (e.g., Trp, Tyr, and Phe) are replaced with synthetic non-natural acids (e.g., phenylglycine, TIC, naphthylalanine (Nol), cyclic methylated derivatives of Phe, halogenated derivatives of Phe, or o-methyl-Tyr). In some embodiments, the polypeptide of the present invention comprises one or more modified amino acids or one or more non-amino acid monomers (e.g., fatty acids, complex carbohydrates, etc.).

[0378] In one embodiment, "amino acid" or "amino acid sequence" is understood to include 20 naturally occurring amino acids; those that are frequently post-translational modified in vivo, including, for example, hydroxyproline, phosphoserine, and phosphothreonine; and other less common amino acids, including but not limited to 2-aminoadipic acid, hydroxylysine, isodesyl base, valine, leucine, and ornithine. In one embodiment, "amino acid" includes D- and L-amino acids.

[0379] In some embodiments, the polypeptides of the present invention are used in therapeutic agents that require the polypeptide to be in a soluble form. In some embodiments, the polypeptides of the present invention comprise one or more non-natural or natural polar amino acids (including, but not limited to, serine and threonine) that can increase polypeptide solubility due to the presence of hydroxyl side chains.

[0380] In some embodiments, the engineered peptides of the present invention are used in linear form, but those skilled in the art will understand that cyclic engineered peptides can also be used where cyclization does not significantly interfere with the characteristics of the engineered peptide.

[0381] In some embodiments, the engineered peptides of the present invention are biochemically synthesized, for example, using standard solid-phase techniques. In some embodiments, these biochemical methods include proprietary solid-phase synthesis, partially solid-phase synthesis, fragment condensation, or classical solution synthesis.

[0382] In some embodiments, recombinant protein technology is used to generate the engineered peptides of the present invention. In some embodiments, recombinant protein technology is used to generate relatively long peptides (e.g., longer than 18-25 amino acids). In some embodiments, recombinant protein technology is used to generate large quantities of the engineered peptides of the present invention. In some implementations, recombination techniques are described in Bitter et al., (1987) Methods in Enzymol. 153: 516-544; Studier et al., (1990) Methods in Enzymol. 185: 60-89; Brisson et al., (1984) Nature 310: 511-514; Takamatsu et al., (1987) EMBO J. 6: 307-311; Coruzzi et al., (1984) EMBO J. 3: 1671-1680; Brogli et al., (1984) Science 224: 838-843; Gurley et al., (1986) Mol. Cell. Biol. 6: 559-565; and Weissbach & Weissbach, 1988, Methods for Plant Molecular Biology, Academic Press, NY, Section VIII, pp. Pages 421-463, the entire contents of which are incorporated herein by reference.

[0383] In one embodiment, the present invention provides a chimeric polypeptide comprising a CYP76AD1-β clade polypeptide. In one embodiment, the CYP76AD1-β clade polypeptide is CYP76AD6. In another embodiment, the CYP76AD1-β clade polypeptide is CYP76AD15. In one embodiment, the present invention provides a chimeric polypeptide comprising a CYP76AD1-β clade polypeptide but excluding the amino acid sequence of a CYP76AD1 paralog of beet known as Bv9_228610_yqeq or Bv9_228860_ickx (which does not produce betaine when converted to yeast with DOD). In one embodiment, the present invention provides a chimeric polypeptide comprising a CYP76AD1-β clade polypeptide but excluding the following amino acid sequence:

[0384] MDNATLAVILSILFVFYHIFKSFFTNSSSRRLPPGPKPVPIFGNIFDLGEKPHRSFANLSKIHGPLISLKLGSVTTIVVSSASVAEEMFLKNDQALANRTIPDSVRAGDHDKLSMSWLPVSQKWR NMRKISAVQLLSNQKLDASQPLRQTKVKQLLSYVQDCSKKMQPVDIGRAAFTTSLNLLSNTFFSIELASHESSASQEFKQLMWNIMEEIGRPNYADFFPILGYIDPFGIRRRLAGYFDKLIDVFQD IIRERQKLRSSNSSGAKQTNDILDTLLKLHEDNELSMPEINHLLVDIFDAGTDTTASTLEWAMAELVKNPEMMTKVQIEIEQALGKDCLDIQESDISKLPYLQGIIKETLRLHPPTVFLLPRKADN DVELYGYVVPKNAQVLVNLWAIGRDPKVWKNPEVFSPERFLDCNIDYKGRDFELLPFGAGRRICPGLTLAYRMLNLMLATLLQNYNWKLEDGINPKDLDMDEKFGITLQKVKPLQVIPVPRN (SEQ ID NO: 34).

[0385] In one embodiment, the present invention provides a method comprising the step of “contacting” tyrosine with one or more polypeptides described herein. In one embodiment, the contacting step is performed in vitro under conditions that allow for the production of L-DOPA or betaine, as described herein.

[0386] peptide recovery

[0387] In one embodiment, the method of the present invention further provides a method for extracting betaine or L-DOPA from plants as described in Example 1, as well as other betaine extraction methods known in the art. In another embodiment, betaine is extracted from algae, yeast, or bacterial cultures using methods known in the art. In one embodiment, the algae, yeast, or bacterial cultures are genetically modified to express betaine or L-DOPA.

[0388] In one implementation, peptide recovery is affected after a predetermined culture time.

[0389] In one implementation, the phrase “recovered peptide” as used herein refers to the collection of the entire fermentation medium containing the peptide without the need for additional separation or purification steps.

[0390] In one embodiment, the polypeptides of the present invention are purified using a variety of standard protein purification techniques, including, but not limited to, affinity chromatography, ion exchange chromatography, filtration, electrophoresis, hydrophobic interaction chromatography, gel filtration chromatography, reversed-phase chromatography, concanavalin A chromatography, chromatographic focusing, and differential dissolution.

[0391] In one embodiment, to facilitate recovery, the expressed coding sequence can be engineered to encode the polypeptide of the present invention and the cleavable fusion moiety. In one embodiment, the fusion protein can be engineered such that the polypeptide can be easily separated by affinity chromatography; for example, by immobilization on a column specific to the cleavable moiety. In one embodiment, a cleavage site is engineered between the polypeptide and the cleavable moiety, and the polypeptide can be released from the multi-column by treatment with an appropriate enzyme or reagent that specifically cleaves the fusion protein at that site [see, for example, Booth et al., Immunol. Lett. 19:65-70 (1988); and Gardella et al., J. Biol. Chem. 265:15854-15859 (1990)].

[0392] In one embodiment, the polypeptide of the present invention is recovered in a "substantially pure" form.

[0393] In one implementation, the phrase "substantially pure" refers to the purity of the protein that allows for its effective use in the applications described herein.

[0394] In one embodiment, the polypeptide of the present invention can also be synthesized using an in vitro expression system. In one embodiment, the in vitro synthesis method is well known in the art, and the components of the system are commercially available.

[0395] The method for producing betaine flavonoids in vitro using DOD enzymes and their substrate L-DOPA, along with the addition of amino acids, is described by Sekiguchi et al. (2010) (Journal of Agricultural and Food Chemistry 58, 12504-12509), which is incorporated herein by reference in its entirety. In one embodiment, the in vitro synthesis method of betaine flavonoids of the present invention is as described in Sekiguchi et al., but with the addition of a step to synthesize L-DOPA from tyrosine, instead of providing L-DOPA as described by Sekiguchi et al.

[0396] In one embodiment, red betaine (betalain) is produced in vitro under the same or similar conditions as those used for betaine production, except that the CYP76AD1 enzyme is provided together with the DOD enzyme, and the substrate can be tyrosine or L-DOPA. In one embodiment, the cofactor NADPH may be added to activate the CYP76AD1 enzyme.

[0397] In some implementations, recombinant peptides are synthesized and purified; their therapeutic efficacy can be determined in vivo or in vitro.

[0398] Pharmaceutical Composition

[0399] In another embodiment, a pharmaceutical composition comprising the recombinant polynucleotide or chimeric polypeptide of the present invention is provided. In one embodiment, the polypeptide and polynucleotide of the present invention may be provided to an individual as part of a pharmaceutical composition, and mixed in the individual with a pharmaceutically acceptable carrier.

[0400] In one embodiment, a "pharmaceutical composition" refers to a formulation of one or more active ingredients described herein with other chemical components (e.g., physiologically suitable carriers and excipients). The purpose of a pharmaceutical composition is to facilitate the administration of the compound to a living organism.

[0401] In one implementation, "active ingredient" refers to a recombinant polynucleotide or chimeric polypeptide that causes biological or biochemical effects.

[0402] In other embodiments, the composition includes additional components. In one embodiment, such additional components may include a carrier or diluent, including but not limited to gums, starches (e.g., corn starch, pregelatinized starch), sugars (e.g., lactose, mannitol, sucrose, dextrose), cellulose materials (e.g., microcrystalline cellulose), acrylates (e.g., polymethyl acrylate), calcium carbonate, magnesium oxide, talc, or mixtures thereof.

[0403] In other embodiments, pharmaceutically acceptable carriers for liquid formulations are water or non-aqueous solutions, suspensions, emulsions, or oils. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, and injectable organic esters (such as ethyl oleate). Aqueous carriers include water, alcohol / aqueous solutions, emulsions, or suspensions, including saline solutions and buffer media. Examples of oils are oils of animal, plant, or synthetic origin, such as peanut oil, soybean oil, olive oil, sunflower oil, cod liver oil, another marine oil, or lipids derived from milk or eggs.

[0404] In another embodiment, parenteral carriers (for subcutaneous, intravenous, intra-arterial, or intramuscular injection) include sodium chloride solution, Ringer's dextran, dextran and sodium chloride, lactated Ringer's solution, and non-volatile oils. Intravenous carriers include fluids and nutritional supplements, such as electrolyte supplements based on Ringer's dextran. Examples are sterile liquids, such as water and oil, with or without surfactants and other pharmaceutically acceptable adjuvants. Generally, water, saline, aqueous dextran and related sugar solutions, and glycols (such as propylene glycol or polyethylene glycol) are preferred liquid carriers, particularly for injectable solutions. Examples of oils are oils of animal, plant, or synthetic origin, such as peanut oil, soybean oil, olive oil, sunflower oil, cod liver oil, another marine oil, or lipids derived from milk or eggs.

[0405] In other embodiments, the composition further includes binders (e.g., gum arabic, corn starch, gelatin, carbomer, ethyl cellulose, guar gum, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, povidone), disintegrants (e.g., corn starch, potato starch, alginic acid, silica, croscarmellose sodium, croscarmellose, guar gum, sodium glycolate starch), buffers of various pH and ionic strengths (e.g., Tris-HCl, acetate, phosphate), additives to prevent adsorption to surfaces (e.g., albumin or gelatin), and detergents (e.g., Tween 20, Tween 80, Pluronic). F68, bile salts), protease inhibitors, surfactants (e.g., sodium lauryl sulfate), penetration enhancers, solubilizers (e.g., glycerol, polyvinylglycerol), antioxidants (e.g., ascorbic acid, sodium metabisulfite, butylated hydroxyanisole), stabilizers (e.g., hydroxypropyl cellulose, hydroxypropyl methylcellulose), thickeners (e.g., carbomer, colloidal silica, ethyl cellulose, guar gum), sweeteners (e.g., aspartame, citric acid), preservatives (e.g., thimerosal, benzyl alcohol, parabens), lubricants (e.g., stearic acid, magnesium stearate, polyethylene glycol, sodium lauryl sulfate), flow aids (e.g., colloidal silica), plasticizers (e.g., diethyl phthalate, triethyl citrate), emulsifiers (e.g., carbomer, hydroxypropyl cellulose, sodium lauryl sulfate), polymer coatings (e.g., poloxamer or poloxamine), coating and film-forming agents (e.g., ethyl cellulose, acrylates, polymethyl methacrylate), and / or adjuvants. Each of the excipients described above represents a separate embodiment of the present invention.

[0406] In another embodiment, the pharmaceutical composition provided herein is a controlled-release composition, i.e., a composition that releases the antigen over a period of time after administration. Controlled-release or sustained-release compositions comprise formulations in lipophilic reservoirs (e.g., fatty acids, waxes, oils). In another embodiment, the composition is an immediate-release composition, i.e., a composition in which all the antigen is released immediately after administration.

[0407] In another embodiment, the pharmaceutical composition is delivered using a controlled-release system. In another embodiment, the agent is administered via intravenous infusion, an implantable osmotic pump, a transdermal patch, liposomes, or other administration methods. In another embodiment, a pump is used (see Langer, supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14:201 (1987); Buchwald et al., Surgery 88:507 (1980); Saudek et al., N. Engl. J. Med. 321:574 (1989)). In another embodiment, a polymeric material is used; for example, in microspheres or implants.

[0408] In another embodiment, the composition further includes incorporating the active material into or onto particulate formulations of polymeric compounds (such as polylactic acid, polyglycolic acid, hydrogels, etc.), or into liposomes, microemulsions, micelles, monolayer or multilayer vesicles, erythrocyte shadows, or protoplasts. This composition will affect the physical state, solubility, stability, in vivo release rate, and in vivo clearance rate.

[0409] The present invention also includes particulate compositions coated with a polymer (e.g., poloxamer or poloxamine) and a compound coupled to an antibody or a ligand coupled to a tissue-specific receptor, ligand, or antigen.

[0410] The present invention also includes compounds modified with covalently linked water-soluble polymers, such as polyethylene glycol, copolymers of polyethylene glycol and polypropylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, or polyproline. Modified compounds are known to exhibit significantly longer half-lives in the blood after intravenous injection compared to their corresponding unmodified counterparts (Abuchowski et al., 1981; Newmark et al., 1982; Katre et al., 1987). In another embodiment, such modification also increases the solubility of the compound in aqueous solution, eliminates aggregation, improves the physical and chemical stability of the compound, and significantly reduces the immunogenicity and reactivity of the compound. In another embodiment, the desired in vivo bioactivity compared to the unmodified compound is achieved by administering this polymer-compound adduct at a lower frequency or at a lower dose.

[0411] The preparation of pharmaceutical compositions containing active ingredients, for example by mixing, granulation, or tableting processes, is well understood in the art. In another embodiment, the active ingredient is formulated into the composition in the form of a neutral, pharmaceutically acceptable salt. Pharmaceutically acceptable salts include acid addition salts (formed from the free amino groups of polypeptide or antibody molecules) formed with inorganic acids (e.g., hydrochloric acid or phosphoric acid) or organic acids (e.g., acetic acid, oxalic acid, tartaric acid, and mandelic acid). Salts formed from free carboxyl groups can also be derived from inorganic bases (e.g., sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, or ferric hydroxide) and organic bases (e.g., isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine, and procaine).

[0412] Each of the above-described additives, excipients, formulations, and methods of application represents a separate embodiment of the present invention.

[0413] In one embodiment, the method of the present invention includes administering the polynucleotide or polypeptide of the present invention into a pharmaceutically acceptable carrier.

[0414] In another embodiment, the pharmaceutical composition containing the polypeptide can be administered to a subject by any method known to those skilled in the art, such as parenteral, transmucosal, transdermal, intramuscular, intravenous, intradermal, intranasal, subcutaneous, intraperitoneal, intravenous, intracranial, or intravaginal administration. In another embodiment, the composition of the present invention is administered by epidermal injection, in another embodiment by intramuscular injection, in another embodiment by subcutaneous injection, and in another embodiment by intramural injection into the respiratory tract.

[0415] In another embodiment of the method and composition of the present invention, the pharmaceutical composition is administered orally and thus formulated into a form suitable for oral administration, i.e., as a solid or liquid dosage form. Suitable solid oral dosage forms include tablets, capsules, pills, granules, and microgranules, etc. Suitable liquid oral dosage forms include solutions, suspensions, dispersants, emulsions, and oils, etc. In another embodiment of the invention, the composition is formulated into capsules. In another embodiment, the composition of the present invention comprises hard gel capsules.

[0416] In another embodiment, the pharmaceutical composition is administered via intravenous, intra-arterial, or intramuscular injection of a liquid formulation. Suitable liquid formulations include solutions, suspensions, dispersants, emulsions, and oils. In another embodiment, the pharmaceutical composition is administered intravenously and is therefore formulated to a form suitable for intravenous administration. In another embodiment, the pharmaceutical composition is administered intra-arterially and is therefore formulated to a form suitable for intra-arterial administration. In another embodiment, the pharmaceutical composition is administered intramuscularly and is therefore formulated to a form suitable for intramuscular administration.

[0417] In another embodiment, the pharmaceutical composition is applied topically to the body surface and is thus formulated into a form suitable for topical application. Suitable topical formulations include gels, ointments, creams, lotions, and drops.

[0418] In another embodiment, the pharmaceutical composition is administered in the form of a suppository, such as a rectal suppository or a urethral suppository. In another embodiment, the pharmaceutical composition is administered via subcutaneous implantation of microspheres. In yet another embodiment, the microspheres provide controlled release of the antigen over a period of time.

[0419] In another embodiment, the pharmaceutical composition is delivered in vesicles, such as liposomes.

[0420] Plant regeneration

[0421] In one embodiment, the transgenic plant of the present invention is grown under conditions suitable for expressing the recombinant DNA construct. The regeneration, development, and culture of plants from single-plant protoplast transformants or from various transformed explants are well known in the art (see Weissbach and Weissbach, In.: Methods for Plant Molecular Biology, (ed.), 1988 Academic Press, Inc., San Diego, Calif.). This regeneration and growth process typically involves selecting transformed cells, culturing those individualized cells through the typical stages of embryonic development to rooted embryos. Transgenic embryos and seeds are regenerated similarly. The resulting transgenic rooted cuttings are then planted in a suitable plant growth medium (e.g., soil).

[0422] Following transformation, plant cells transformed with plant expression vectors can be regenerated from single cells, callus tissue, or leaves, for example, according to standard plant tissue culture techniques. Using methods known in the art, virtually any plant can be completely regenerated from its cells, tissues, and organs.

[0423] The transformed plants can then be grown and pollinated with the same or different transformed lines, and the resulting hybrids will exhibit the expression of the desired phenotypic trait. Two or more generations can be grown to ensure that the expression of the desired phenotypic trait is stably maintained and inherited, and then the seeds are harvested to ensure that the expression of the desired phenotypic trait has been achieved.

[0424] In one implementation, plant cells are regenerated to obtain a whole plant from the transformation process. As used herein, the terms “growth” or “regeneration” refer to the growth of a whole plant from plant cells, plant cell groups, plant parts (including seeds), or plant slices (e.g., from protoplasts, callus, or tissue portions).

[0425] Protoplast regeneration varies by plant species, but typically begins with the preparation of a protoplast suspension. Embryogenesis can then be induced from the protoplast suspension in some species. In one implementation, the culture medium will contain various amino acids and hormones necessary for growth and regeneration. Examples of hormones used include auxin and cytokinin. Effective regeneration will depend on the culture medium, genotype, and culture process. If these variables are controlled, regeneration is reproducible. Regeneration can also occur from plant callus, explants, organs, or parts.

[0426] In asexually propagated crops, mature genetically modified plants are propagated using cuttings or tissue culture techniques to produce multiple identical plants. The desired genetically modified plants are selected to obtain new varieties, which are then propagated asexually for commercial use.

[0427] In seed-propagated crops, mature genetically modified plants can self-pollinate to produce homozygous inbred plants. The resulting inbred plants produce seeds containing gene mutations. These seeds can be grown to produce plants that exhibit selected phenotypes, such as increased lateral root growth, nutrient uptake, overall plant growth, and / or increased nutrient or reproductive yield.

[0428] Parts obtained from regenerated plants (e.g., flowers, seeds, leaves, branches, and fruits) are included in this invention, provided that the cells contained in these parts contain the isolated nucleic acids of this invention. Progeny and variants of the regenerated plants, as well as mutants, are also included within the scope of this invention, provided that these parts contain the introduced nucleic acid sequence. In one embodiment, genetically modified plants expressing selectable markers can be screened for delivery of the nucleic acids of this invention using, for example, standard immunoblotting and DNA detection techniques. In another embodiment, the genetically modified plant cells can be evaluated at the expression level of the genetically modified nucleic acids. Expression at the RNA level can be determined first to identify and quantify positive expression plants. Standard techniques for RNA analysis can be used and include PCR amplification assays using oligonucleotide primers (the oligonucleotide primers are designed to amplify only the genetically modified RNA template) and solution hybridization assays using specific probes for the genetically modified nucleic acids. Protein expression in RNA-positive plants can then be analyzed using Western immunoblotting analysis with the specific reactive antibodies of this invention. Additionally, expression sites within genetically modified tissues can be located using in situ hybridization and immunocytochemistry according to standard protocols, using specific polynucleotide probes and antibodies for the genetically modified nucleic acids. In one implementation, numerous genetically modified lines are screened for the incorporated nucleic acid in order to identify and select plants with the most suitable expression profile.

[0429] In one embodiment, the present invention provides a homozygous genetically modified plant containing two added nucleic acid sequences, one gene located at the same locus on each chromosome of a chromosome pair. Homozygous genetically modified plants can be obtained by sexually mating (self-pollinating) heterozygous genetically modified plants containing a single added genetically modified nucleic acid to germinate some of the produced seeds, and the resulting plants can be analyzed for polynucleotide expression changes relative to control plants (i.e., native, non-genetically modified). Backcrossing to parental plants and hybridization with non-genetically modified plants is also considered.

[0430] Transformed plant cells obtained through any of the above-described transformation techniques can be cultured to regenerate whole plants with the transformed genotype. In one embodiment, this regeneration technique relies on treatment with certain plant hormones in a tissue culture growth medium.

[0431] Then, as is well known in the art, the regenerated plant containing the foreign, exogenous gene encoding the protein of interest can be further propagated. The specific method of propagation will depend on the starting plant tissue and the specific plant species to be propagated.

[0432] In one embodiment, the resulting transformed plants are propagated asexually. In another embodiment, the resulting transformed plants are propagated using conventional breeding techniques. In one specific embodiment, the regenerated plants are self-pollinated to provide homozygous transgenic plants. Alternatively, pollen obtained from the regenerated plants is hybridized with seed-growing plants of agronomically important lines, or pollen from these important lines is used to pollinate the regenerated plants. The transgenic plants of the present invention containing the desired polypeptides are cultured using methods well known to those skilled in the art.

[0433] Products from genetically modified plants

[0434] In one embodiment, the present invention provides a food crop containing betaine pigment. In one embodiment, the food crop does not endogenously contain betaine pigment. In another embodiment, the food crop endogenously contains betaine pigment, and the method of the present invention is used to increase or enhance the amount of betaine pigment present in the food crop.

[0435] In one embodiment, food crops containing betaine or an increased amount of betaine are nutritionally enhanced. In one embodiment, food crops containing betaine or an increased amount of betaine possess antioxidant properties. In one embodiment, ingestion of food crops containing betaine or an increased amount of betaine protects subjects from degenerative diseases or conditions. In one embodiment, ingestion of betaine or betaine-containing food crops can protect subjects from cancers (e.g., skin, lung, cervical, ovarian, and bladder cancer), heart disease, or neurodegenerative diseases. Betaine can also inhibit lipid peroxidation and heme breakdown; prevent oxidative hemolysis of red blood cells; and bind to human low-density lipoprotein, increasing its antioxidant capacity.

[0436] In one embodiment, the present invention provides a fruit comprising the betaine pigment of the present invention. In another embodiment, the present invention provides a vegetable comprising the betaine pigment of the present invention. In yet another embodiment, the present invention provides an underground organ, such as a potato, comprising the betaine pigment of the present invention. In yet another embodiment, the present invention provides a juice derived from a fruit or vegetable comprising the betaine pigment of the present invention.

[0437] In one embodiment, in addition to the beet pigment of the present invention, the plant organ or its derivative (if juice) also contains natural or engineered anthocyanins, carotene or combinations thereof.

[0438] In another embodiment, the present invention provides a dietary supplement comprising beet pigments produced from plants or plant parts as described herein or as described herein.

[0439] In another embodiment, the present invention provides allelochemicals comprising L-DOPA produced by plants or plant parts as described herein or as described herein.

[0440] Other uses

[0441] Heterogeneous production of beet pigments enables the biofortification and enhancement of the nutritional quality of staple foods, as well as the development of new ornamental plant varieties. The simple biosynthetic pathways of these pigments begin with the ubiquitous precursor tyrosine and require the expression of 2 to 3 genes, allowing these pigments to be produced in many plant species or other species.

[0442] In another embodiment, the present invention provides a method for producing ornamental plants, comprising the steps of: contacting plant cells with nucleic acid sequences encoding CYP76AD1, CYP76AD6, CYP76AD15, or combinations thereof, and a nucleic acid sequence encoding DOPA 4,5-dioxygenase (DOD), under conditions sufficient to produce betaine, wherein if the cells are contacted with nucleic acid sequences encoding CYP76AD1 and DOD but not encoding CYP76AD6 or CYP76AD15, then the cells are contacted with a nucleic acid sequence encoding a betaine-associated glucosyltransferase, thereby producing an ornamental plant. In one embodiment, the plant is not a naturally betaine-expressing plant. In one embodiment, the plant is not a caryophyllales plant. In one implementation, the plant species are: Syngonium podophyllum, Boston fern, Canary Island date palm, Aglaonema, Bird's nest fern, Rhapis excelsa, Aspidistra elatior, Orange succulent, European maidenhair fern, Dieffenbachia grandiflora, European dwarf palm, Epipremnum aureum, Croton tigrinum, Alocasia macrorrhiza, Ficus lyrata, Philodendron simsii, Philodendron simsii, Dracaena sanderiana, Adiantum capillus-veneris, Dwarf coconut palm, Cocos nudiflora, Dracaena marginata, Chestnut palm, Maranta peacockii, Fern ferns, Maranta scoparia, Cycas revoluta, Bambusa textilis, Maranta scoparia, Ficus elastica, Cocos nudiflora, Spider plant, Venus flytrap, Peppermint, Silver-veined single-herb flower, Zamioculcas zamiifolia, Tradescantia pallida, Ficus weepingii, Maranta velutina, or cactus.

[0443] In one embodiment, altering the color of a plant, particularly the color of its flowers, will affect plant pollination. In one embodiment, the method of the present invention will increase or enhance plant pollination. In another embodiment, the method of the present invention will reduce plant pollination. In yet another embodiment, the method of the present invention will alter the organism attracted by the plant, thereby altering plant pollination. In one embodiment, the present invention provides a method for increasing plant pollination, comprising the step of inserting the recombinant polynucleotide or one or more nucleic acids of the present invention into at least one cell of the plant.

[0444] In another embodiment, the present invention provides a method for increasing plant pollination, the method comprising the steps of: contacting a plant with a nucleic acid sequence encoding CYP76AD1, CYP76AD6, CYP76AD15, or a combination thereof, and optionally a nucleic acid sequence encoding DOPA 4,5-dioxygenase (DOD), under conditions sufficient to produce betaine, thereby altering the plant's color and thus increasing pollination. In one embodiment, the plant is not a naturally betaine-expressing plant. In one embodiment, the plant is not a caryophyllales plant.

[0445] In another embodiment, the method of the present invention provides a method for changing the color of an organism. In one embodiment, the organism is a fish.

[0446] In another embodiment, the method of the present invention provides a method for producing an organism, wherein one or more portions of the organism are reddish-purple, the method comprising the steps of: contacting one or more cells of the organism with a recombinant polynucleotide, the recombinant polynucleotide comprising a nucleic acid encoding CYP76AD1, a nucleic acid encoding DOPA 4,5-dioxygenase (DOD), and a nucleic acid encoding a betaine-associated glucosyltransferase, wherein the nucleic acid is in-frame inserted into the polynucleotide.

[0447] In another embodiment, the method of the present invention provides a method for producing an organism, wherein one or more portions of the organism are yellow, the method comprising the steps of: contacting one or more cells of the organism with a recombinant polynucleotide, the recombinant polynucleotide comprising a nucleic acid encoding CYP76AD6 and a nucleic acid encoding DOPA 4,5-dioxygenase (DOD), wherein the nucleic acid is in-frame inserted into the polynucleotide.

[0448] In another embodiment, the method of the present invention provides a method for producing an organism, wherein one or more portions of the organism are orange, the method comprising the steps of: contacting one or more cells of the organism with a recombinant polynucleotide, the recombinant polynucleotide comprising a nucleic acid encoding CYP76AD6, a nucleic acid encoding CYP76AD1, a nucleic acid encoding DOPA4,5-dioxygenase (DOD), and a nucleic acid encoding a betaine-associated glucosyltransferase, wherein the nucleic acid is in-frame inserted into the polynucleotide.

[0449] In one implementation, the organism is a fish. In another implementation, the fish is an ornamental fish.

[0450] In one embodiment, the present invention provides a method for producing ornamental fish, the method comprising contacting fish cells with polynucleotides comprising nucleic acids encoding DOPA 4,5-dioxygenase (DOD) and nucleic acids encoding CYP76AD6, CYP76AD15, CYP76AD1, or combinations thereof, wherein if the cells are contacted with nucleic acids encoding CYP76AD1 and DOD but not CYP76AD6 or CYP76AD15, the cells are further contacted with nucleic acids encoding betaine-associated glucosyltransferases.

[0451] In another embodiment, the present invention provides a method for producing a plant expressing betaine, the method comprising the steps of: contacting one or more cells of the plant with a nucleic acid sequence encoding CYP76AD1, CYP76AD6, CYP76AD15 or a combination thereof, and a nucleic acid sequence encoding DOPA 4,5-dioxygenase (DOD); screening the one or more cells to identify cells containing the nucleic acid sequences; and regenerating a plant or plant part from the cells, thereby producing a plant expressing betaine.

[0452] In one embodiment, the plant does not naturally express betaine. In another embodiment, the plant expresses betaine, and the method of the present invention is used to increase the amount of one or more betaines expressed by the plant.

[0453] In another embodiment, the present invention provides a method for producing a food crop expressing betaine, comprising the steps of: contacting one or more cells of a food crop plant with a nucleic acid sequence encoding CYP76AD1, CYP76AD6, CYP76AD15 or a combination thereof, and a nucleic acid sequence encoding DOPA 4,5-dioxygenase (DOD); screening the one or more cells to identify cells containing the nucleic acid sequences; and regenerating a plant or plant part from the cells to produce a food crop expressing betaine.

[0454] In one embodiment, a "food crop" or "crop" is a plant intended to be grown primarily for human or animal consumption. In one embodiment, a food crop is a banana, yam, sorghum, sweet potato, soybean, cassava, rice, wheat, or corn. In another embodiment, a food crop is an eggplant, pepper, broccoli, broccoli, buckwheat, corn, barley, grapes, berries, tomatoes, cucumbers, artichokes, onions, rhubarb, blackberries, blueberries, zucchini, radishes, carrots, Brussels sprouts, lettuce, melon, beans, peas, grains, peanuts, sugarcane, watermelon, papaya, apple, pear, peach, cherry, strawberry, or pumpkin.

[0455] In one embodiment, the strong antioxidant activity of betaine is beneficial to human health.

[0456] Their potential health-promoting properties have been extensively studied, including anticancer, lipid-lowering, anti-inflammatory, hepatoprotective, and antidiabetic activities. Therefore, in one embodiment, the present invention provides a method for treating or inhibiting cancer, hyperlipidemia, inflammation, liver disease, and diabetes, the method comprising feeding a subject in need a plant or plant part of the present invention expressing betaine, betaine red, and / or betaine xanthophyll, thereby treating or inhibiting said subject's cancer, hyperlipidemia, inflammation, liver disease, or diabetes.

[0457] In one embodiment, the subject ingests food crops of the present invention containing betaine or an increased amount of betaine to protect the subject against inflammatory-immune damage (including but not limited to glomerulonephritis, vasculitis, autoimmune diseases, adult respiratory distress syndrome, rheumatoid arthritis, inflammatory bowel disease, pancreatitis); cancer (including but not limited to radiation-induced cancer, cervical cancer, hepatocellular carcinoma, carcinogenesis promoters, cancer in inflammatory bowel disease); ischemia / reoxygenation (including but not limited to stroke, myocardial infarction, organ transplantation (heart, lung, skin, cornea, kidney), organ preservation, reattachment of severed limbs, frostbite, Dupuy's palm contracture, hemorrhagic shock, endotoxic shock, crush injury); metal overload (including but not limited to hemochromatosis, thalassemia, quasi-Cholke's disease, chemotherapy for leukemia, fulminant hepatic failure, Wilson's disease, alcohol-induced iron overload, nickel-induced carcinogenesis, lead poisoning); toxins (including but not limited to hemolytic drugs, lead, halogenated hydrocarbons, ozone, nitrogen oxides). Asbestos, other mineral dust, sulfur dioxide, paraquat, aluminum, cigarette smoke, diabetic drugs, fava beans (hemolytic agents), anthracyclines (cardiotoxicity), heavy metals (nephrotoxicity), photosensitizing drugs, contact dermatitis; eye diseases (including but not limited to cataract development, worsening after ocular hemorrhage, photochemical retinal damage, retinopathy of prematurity (posterior fibrosis of the lens)); congenital diseases (including but not limited to porphyria, sickle cell anemia, Fanconi anemia, neuronal cerebrospinal lipofuscinitis, thalassemia); insufficient antioxidant protection (including but not limited to Keshan disease (severe selenium deficiency), hemolytic disease of prematurity, retinopathy of prematurity, bronchopulmonary dysplasia, intracranial hemorrhage, neurodegeneration caused by severe vitamin E deficiency (congenital defects affecting intestinal fat absorption), acquired immune dysfunction syndrome); brain and central nervous system disorders (including but not limited to stroke, trauma, neurotoxicity (e.g., aluminum), hyperbaric oxygen therapy, Parkinson's disease, exacerbation of traumatic injury, cerebral malaria).

[0458] In another embodiment, the present invention provides a novel method for producing betaine in non-natural betaine-expressing species, wherein the betaine can be used as a natural food coloring agent. Therefore, the present invention also provides natural food coloring agents comprising the betaine produced as described herein. In one embodiment, betaine is extracted from plants expressing betaine, as described herein. In another embodiment, betaine is produced in vitro in cell lines. Heterogeneous production of betaine enables biofortification and enhancement of the nutritional quality of basic foods.

[0459] In another embodiment, the method of the present invention can be used to express one or more beet pigments in genetically engineered plants to alter their color, thereby improving the aesthetic quality of food crops or products.

[0460] Therefore, in one embodiment, the present invention provides a method for treating or inhibiting the diseases, disorders, and symptoms mentioned above, the method comprising the step of providing a subject with a food crop that has been genetically modified to express betaine pigments as described herein. In another embodiment, the present invention provides the use of the polynucleotides, nucleic acids, and other compositions described herein in the preparation of compositions for treating or inhibiting the aforementioned diseases, disorders, and symptoms. In one embodiment, the composition is a pharmaceutical composition. In another embodiment, the present invention provides the use of the polynucleotides, nucleic acids, and other compositions described herein in the treatment or inhibition of the aforementioned diseases, disorders, and symptoms.

[0461] In another embodiment, food crops containing betaine or in increased amounts of betaine have an extended shelf life compared to the same food crops lacking betaine or having low levels of betaine. In another embodiment, food crops containing betaine or in increased amounts of betaine have increased resistance to fungal diseases compared to the same food crops lacking betaine or having low levels of betaine.

[0462] In another embodiment, the beet pigment produced by the method described herein can be used for food preservation.

[0463] Therefore, in one embodiment, the present invention provides a method for increasing the shelf life of food crops, the method comprising the steps of: contacting one or more cells of a food crop plant containing tyrosine with nucleic acids encoding CYP76AD6, CYP76AD15 or combinations thereof, nucleic acids encoding CYP76AD1 or combinations thereof, under conditions sufficient to produce betaine, thereby producing betaine and increasing the shelf life of the food crop.

[0464] Within the plant kingdom, betaines are found only in one group of angiosperms, the Caryophyllales order. Within this order, betaines and anthocyanins appear in a mutually exclusive manner, meaning no plant species produces both types of pigments. One of the most prominent features of the Caryophyllales order is its dominance in arid and semi-arid regions, as well as in saline-alkali soil habitats. While some families within the Caryophyllales order are distributed across a variety of habitats worldwide, members of several families are particularly adapted to arid or saline regions, including Aizoaceae (Oryza sativa family), Portulaca oleracea (Portulaca oleracea family), and most notably, Cacti (Cactaceae family; www.britannica.com / plant / Caryophyllales). The following section uses heterologous betaine production in plants to investigate the role of these pigments in conferring tolerance to various abiotic stress cues associated with arid regions (such as drought, high UV radiation, excessive light, and high salinity) and as plant defense compounds against pathogenic fungi.

[0465] In another embodiment, the present invention provides a method for increasing the resistance of an organism or part of an organism to one or more stress factors, the method comprising the step of: contacting one or more cells of the organism with a nucleic acid encoding CYP76AD6, CYP76AD15, or a combination thereof, a nucleic acid encoding CYP76AD1, or a combination thereof, under conditions sufficient to produce betaine, thereby producing betaine and increasing the resistance of the organism or part of the organism to the one or more stress factors. In one embodiment, the organism is a plant. In one embodiment, the organism contains tyrosine. In one embodiment, the stress factor is an abiotic stress factor. In another embodiment, the stress factor is high osmotic pressure, high salinity conditions, or a combination thereof. In another embodiment, the stress factor is drought. In another embodiment, the stress factor is excessive light. In one embodiment, the plant part is a seed. In one embodiment, the plant is a food crop.

[0466] In one embodiment, the method includes the step of contacting one or more cells of a plant or a plant part with nucleic acids encoding CYP76AD1, nucleic acids encoding DOPA 4,5-dioxygenase (DOD), and nucleic acids encoding betaine-associated glucosyltransferase.

[0467] In another embodiment, the present invention provides a method for increasing the resistance of a plant or plant part to high osmotic pressure, the method comprising the steps of: contacting one or more cells of a plant or plant part containing tyrosine with a nucleic acid encoding CYP76AD6, CYP76AD15 or a combination thereof, a nucleic acid encoding CYP76AD1 or a combination thereof, under conditions sufficient to produce betaine, thereby producing betaine and increasing the resistance of said plant or plant part to high osmotic pressure.

[0468] In another embodiment, the present invention provides a method for increasing the resistance of a plant or plant part to high salinity conditions, the method comprising the steps of: contacting one or more cells of a plant or plant part containing tyrosine with a nucleic acid encoding CYP76AD6, CYP76AD15 or a combination thereof, a nucleic acid encoding CYP76AD1 or a combination thereof, under conditions sufficient to produce betaine, thereby producing betaine and increasing the resistance of said plant or plant part to high salinity conditions.

[0469] In another embodiment, the present invention provides a method for increasing the seed germination rate of a plant, the method comprising the steps of: contacting one or more cells of a plant or a plant part with a polynucleotide including a nucleic acid encoding CYP76AD1, a nucleic acid encoding DODA1, and a nucleic acid encoding cDOPA5GT under conditions sufficient to produce betaine, thereby increasing the seed germination rate of said plant.

[0470] In another embodiment, the present invention provides a method for increasing the resistance of an organism or part of an organism to one or more fungal diseases, the method comprising the steps of: contacting one or more cells of the organism with nucleic acids encoding CYP76AD6, CYP76AD15, or combinations thereof, a nucleic acid encoding CYP76AD1, or combinations thereof, under conditions sufficient to produce betaine, thereby producing betaine and increasing the resistance of the organism or part of the organism to the one or more fungal diseases. In one embodiment, the organism contains tyrosine. In one embodiment, the organism is a plant. In one embodiment, the plant is a food crop.

[0471] In one embodiment, the fungus causing the fungal disease is a plant pathogenic fungus. In one embodiment, the fungus causing the fungal disease is *Botrytis cinerea*. In one embodiment, the plant is a tobacco plant. In one embodiment, the plant part is a leaf.

[0472] In another embodiment, the present invention provides a method for increasing the resistance of a plant or plant part to one or more fungal diseases, the method comprising the steps of: contacting one or more cells of a plant or plant part with nucleic acids encoding CYP76AD1, DOPA 4,5-dioxygenase (DOD), and DOPA-associated glucosyltransferase, under conditions sufficient to produce betaine, thereby producing betaine and increasing the resistance of said plant or plant part to one or more fungal diseases. In one embodiment, the plant is a food crop.

[0473] In another embodiment, the present invention provides a method for reducing the area of ​​lesions caused by fungal diseases in a plant or plant part, the method comprising the steps of: contacting one or more cells of the plant or plant part with nucleic acids encoding CYP76AD1, DOPA 4,5-dioxygenase (DOD), and β-betaine-associated glucosyltransferase, under conditions sufficient to produce betaine, thereby producing betaine and thus reducing the area of ​​lesions caused by fungal diseases in the plant or plant part.

[0474] In another embodiment, the present invention provides a method for reducing necrosis caused by fungal disease in a plant or plant part, the method comprising the steps of: contacting one or more cells of the plant or plant part with nucleic acids encoding CYP76AD1, DOPA 4,5-dioxygenase (DOD), and β-betaine-associated glucosyltransferase, under conditions sufficient to produce betaine pigment, thereby producing betaine pigment and thus reducing necrosis caused by fungal disease in the plant or plant part.

[0475] In another embodiment, the present invention provides a method for inhibiting the growth of a plant species near a first plant or plant part, the method comprising the steps of: contacting one or more cells of the first plant or plant part with nucleic acids encoding a CYP76AD1-β clade gene under conditions sufficient to produce and secrete L-DOPA, thereby inhibiting the growth of a plant species near the first plant or plant part. In one embodiment, the plant species is a heterologous plant species of the first plant.

[0476] In another embodiment, the present invention provides an allelopathic growth method for an organism, the method comprising the steps of: contacting one or more cells of the organism or a portion thereof with nucleic acid encoding a gene encoding the CYP76AD1-β clade under conditions sufficient to cause the organism or a portion thereof to produce and secrete L-DOPA, thereby allowing allelopathic growth of the organism.

[0477] In one implementation, allelopathy is a biological phenomenon in which an organism produces one or more biochemical substances that affect the germination, growth, survival, and reproduction of other organisms.

[0478] In another embodiment, the present invention provides a method for weed control, the method comprising the steps of: contacting one or more cells of the plant or plant part with a nucleic acid encoding a gene encoding the CYP76AD1-β clade under conditions sufficient to cause the plant or plant part to produce and secrete L-DOPA, thereby inhibiting weed growth near the plant or plant part.

[0479] In one embodiment, the step of contacting one or more cells of a plant or plant part with nucleic acids encoding CYP76AD6, CYP76AD15, or combinations thereof, nucleic acids encoding CYP76AD1, or combinations thereof, under conditions sufficient to produce betaine, includes contacting said one or more cells with nucleic acids encoding CYP76AD1, nucleic acids encoding DOPA 4,5-dioxygenase (DOD), and nucleic acids encoding betaine-associated glucosyltransferases. In one embodiment, DOD is beet DODA1. In one embodiment, the betaine-associated glucosyltransferase is cyclic DOPA 5-O-glucosyltransferase or betaine-5-O-glucosyltransferase. In one embodiment, the cyclic DOPA 5-O-glucosyltransferase is the four o'clock gene cyclic DOPA 5-O-glucosyltransferase (cDOPA5GT).

[0480] In one embodiment, the polynucleotide includes a nucleic acid. In one embodiment, the polynucleotide is pX11. In another embodiment, the polynucleotide is pX12. In yet another embodiment, the polynucleotide is pX13.

[0481] In one implementation, species expressing pX11 exhibit a predominantly reddish-purple color due to the production of higher amounts of betaine than betaine.

[0482] The invention described herein has many applications in a variety of industries, including, but not limited to, agriculture, food and beverage, pharmaceuticals, cosmetics, textiles, and consumer products.

[0483] In one embodiment, the application of the invention includes the production of betaine pigments and / or L-DOPA for use in fish feed. In another embodiment, the production of betaine pigments and / or L-DOPA is for inclusion in a food supplement. In one embodiment, the food supplement is for human use. In another embodiment, the food supplement is for non-human animals. In another embodiment, the food supplement is for veterinary use. In another embodiment, the food supplement is for aquaculture. In another embodiment, the food supplement is in the form of a vitamin. In another embodiment, the food supplement is added to milk or other dairy products.

[0484] In another embodiment, the present invention provides a method for increasing one or more beet pigments in a dairy product, the method comprising the steps of: a) contacting bacterial cells with a polynucleotide comprising a nucleic acid encoding DOPA 4,5-dioxygenase (DOD) and nucleic acids encoding CYP76AD6, CYP76AD15, CYP76AD1, or combinations thereof, wherein if the cells are contacted with nucleic acids encoding CYP76AD1 and DOD but not CYP76AD6 or CYP76AD15, the cells are also contacted with a nucleic acid encoding a beet pigment-associated glucosyltransferase; and b) fermenting the dairy product with bacteria grown from the cells, thereby increasing one or more beet pigments in the dairy product. In one embodiment, the bacteria are bacteria used in the fermentation process for preparing the dairy product. In one embodiment, the bacteria are lactobacilli.

[0485] On the other hand, the genes of the present invention can be transformed into plants or plant cells to protect them from one or more biotic stresses, such as, but not limited to, fungi, bacteria and viruses, and / or one or more abiotic stresses, such as, but not limited to, heat, cold, salt, drought and ultraviolet exposure.

[0486] In one embodiment, plant organs expressing the polynucleotides or polypeptides of the present invention can be used as food colorings, dyes, or pigments. In another embodiment, derivatives of plant organs expressing the polynucleotides or polypeptides of the present invention can be used as food colorings, dyes, or pigments.

[0487] In another embodiment, juice from the transformed fruit can be extracted from the fruit to manufacture beverages. In yet another embodiment, juice or other extracts from plant organs expressing the polynucleotides or polypeptides of the present invention can be used as food colorings, dyes, or pigments. In one embodiment, such juice can be used to dye yogurt, candy, chewing gum, and ice cream, etc. Such dyes or colorings can also be used to dye clothing in the textile industry.

[0488] In another embodiment, as described herein, extracts of transgenic plants or plant cells, as described herein, can be sprayed onto vulnerable plants to protect them from biotic and abiotic stresses, as described above. Therefore, the present invention includes extracts of plants or food crops as described herein, and methods for protecting plants from biotic stresses, abiotic stresses, or combinations thereof, comprising contacting the plant with a plant or plant extract produced by the method of the present invention as described herein.

[0489] In another embodiment, the production of betaine pigment and / or L-DOPA is for cosmetic use. In one embodiment, the production of betaine pigment and / or L-DOPA is for inclusion in sunscreens. In another embodiment, betaine pigment and / or L-DOPA for use in sunscreens are produced in yeast.

[0490] In another embodiment, the present invention provides a method for treating, inhibiting, suppressing, or preventing dopamine-responsive disorders in a subject, the method comprising the step of administering a food crop, cell, or cell line containing a high level of the CYP76AD1-β clade gene to the subject thereby providing L-DOPA to the subject, thereby treating, inhibiting, suppressing, or preventing dopamine-responsive disorders in the subject.

[0491] In another embodiment, the present invention provides the use of the CYP76AD1-β clade gene or a food crop, cell or cell line containing a high level of the CYP76AD1-β clade gene in the preparation of a composition for treating or inhibiting dopamine-responsive disorders in a subject.

[0492] In another embodiment, the present invention provides the use of the CYP76AD1-β clade gene or food crops, cells or cell lines containing high levels of the CYP76AD1-β clade gene in the treatment or inhibition of dopamine-responsive disorders in subjects.

[0493] In one embodiment, the CYP76AD1-β clade polypeptide is CYP76AD6, CYP76AD15, or a combination thereof. In one embodiment, the dopamine-responsive disorder is Parkinson's disease, dopamine-sensitive dystonia, or a combination thereof.

[0494] In another embodiment, the present invention provides a method for treating, inhibiting, suppressing, or preventing Parkinson's disease or dopamine-sensitive dystonia in a subject, the method comprising providing the subject with a genetically modified tyrosine-expressing plant or plant part expressing CYP76AD6, CYP76AD15, or a combination thereof, thereby providing the subject with L-DOPA, thereby treating, inhibiting, suppressing, or preventing Parkinson's disease or dopamine-sensitive dystonia in the subject.

[0495] In another embodiment, the present invention provides a method for treating, inhibiting, suppressing, or preventing Parkinson's disease or dopamine-sensitive dystonia in a subject, the method comprising administering to the subject CYP76AD6, CYP76AD15, or a combination thereof and an enzyme, optionally together with phenylalanine or tyrosine, thereby generating L-DOPA from tyrosine, thereby treating, inhibiting, suppressing, or preventing Parkinson's disease or dopamine-sensitive dystonia in the subject.

[0496] In another embodiment, the present invention provides a method for suppressing symptoms, stabilizing symptoms, delaying onset, and / or slowing the progression of Parkinson's disease or dopamine-sensitive dystonia in a subject, the method comprising providing the subject with a genetically modified tyrosine-expressing plant or plant part expressing CYP76AD6, CYP76AD15, or a combination thereof, thereby providing the subject with L-DOPA, thereby suppressing symptoms, stabilizing symptoms, delaying onset, and / or slowing the progression of Parkinson's disease or dopamine-responsive dystonia in the subject.

[0497] In another embodiment, the present invention provides a method for suppressing symptoms, stabilizing symptoms, delaying onset, and / or slowing the progression of Parkinson's disease or dopamine-sensitive dystonia in a subject, the method comprising administering to the subject CYP76AD6, CYP76AD15, or a combination thereof and an enzyme, optionally together with phenylalanine or tyrosine, thereby generating L-DOPA from tyrosine, thereby suppressing symptoms, stabilizing symptoms, delaying onset, and / or slowing the progression of Parkinson's disease or dopamine-sensitive dystonia in the subject.

[0498] In one implementation, Parkinson's disease is idiopathic Parkinson's disease, post-encephalitis Parkinson's syndrome, or symptomatic Parkinson's syndrome.

[0499] In one embodiment, a peripheral DOPA decarboxylase inhibitor (DDCI) is additionally administered to the subject. In one embodiment, the DDCI is carbidopa, benserazide, or a combination thereof.

[0500] In another embodiment, pyridoxine is additionally administered to the subject.

[0501] In one embodiment, CYP76AD6, CYP76AD15, or combinations thereof, enzymes produced from CYP76AD6, CYP76AD15, or combinations thereof, and / or L-DOPA, as described herein, are administered orally. In another embodiment, they are administered via a catheter. In yet another embodiment, the enzymes are administered epidurally, intracerebrally, or intraventricularly. Other suitable methods of administration are described below.

[0502] According to any method of the present invention, and in one embodiment, the subject is a human. In another embodiment, the subject is a rodent, specifically a mouse in one embodiment and a rat in another. In yet another embodiment, the subject is a dog, cat, cow, sheep, or pig. In yet another embodiment, the subject is a mammal. In yet another embodiment, the subject is any organism susceptible to Parkinson's disease.

[0503] In one embodiment, the method of the present invention is used in veterinary medicine. In one embodiment, the present invention provides treatment for domesticated mammals that are kept as human companions (e.g., dogs, cats, horses), have significant commercial value (e.g., dairy cows, beef cattle, sport animals), have significant scientific value (e.g., captive or free specimens of endangered species), or other values.

[0504] In one embodiment, the method of the present invention includes using the composition to treat and / or prevent a disease, disorder, or condition. In one embodiment, the method of the present invention includes therapeutic treatment or preventative or protective measures, wherein the aim is to prevent or alleviate a targeted pathological condition or disorder as described above. Therefore, in one embodiment, the method of the present invention may include treatment, inhibition, suppression, prevention, reduction of severity, delay of onset, reduction of associated symptoms, or combinations thereof, directly affecting or curing a disease, disorder, or condition. Therefore, in one embodiment, "treatment" specifically refers to delaying progression, accelerating remission, inducing remission, increasing remission, accelerating recovery, improving the efficacy of alternative therapies, or reducing resistance to alternative therapies, or combinations thereof. In one embodiment, "prevention" specifically refers to delaying symptom onset, preventing disease recurrence, reducing the number or frequency of recurrence events, increasing the latency period between symptom onsets, or combinations thereof. In one embodiment, "inhibition" or "suppression" specifically refers to reducing the severity of symptoms, reducing the severity of acute attacks, reducing the number of symptoms, reducing the incidence of disease-related symptoms, reducing the latency period of symptoms, improving symptoms, reducing secondary symptoms, reducing secondary infections, prolonging patient survival, or combinations thereof.

[0505] In one embodiment, the symptoms are primary, while in another, the symptoms are secondary. In one embodiment, "primary" refers to symptoms that are a direct result of a disease, disorder, or condition, while in another embodiment, "secondary" refers to symptoms that originate from or are caused by a primary cause. In one embodiment, the polynucleotides, polypeptides, compositions, cells, and their uses in this invention treat or inhibit primary or secondary symptoms or secondary complications associated with Parkinson's disease.

[0506] In another implementation, “symptoms” can be any manifestation of Parkinson’s disease, including tremor, bradykinesia, rigid muscles, impaired posture and balance, loss of automatic movement, speech changes, writing changes, or a combination thereof.

[0507] In another embodiment, the present invention provides a method for marking gene transformation, the method comprising the steps of: contacting cells with a polynucleotide under conditions sufficient to produce betaine in the cells, the polynucleotide comprising: a) a nucleic acid sequence encoding CYP76AD1, CYP76AD6, CYP76AD15 or a combination thereof; b) a nucleic acid sequence encoding DOPA 4,5-dioxygenase (DOD); and c) a nucleic acid sequence of additional interest, wherein the gene transformation in the cells is marked by a color produced by the production of betaine in the cells. In one embodiment, the contacting step further includes contact with a betaine-associated glucosyltransferase.

[0508] In one embodiment, the method further includes contacting the growth medium with ascorbic acid as a reducing agent to prevent spontaneous betaine oxidation, which would cause the pigment to polymerize and lose its purple color.

[0509] In another embodiment, the present invention provides a method for identifying betaine-related genes in an organism, the method comprising the steps of: searching a plant genome database for genes having an expression pattern highly correlated with the expression pattern of betaine-related genes described herein. In one embodiment, the gene is MjDOD, cDOPA5GT, CYP76AD3, or a combination thereof. In another embodiment, the gene is CYP76AD1, CYP76AD6, CYP76AD15, or a combination thereof.

[0510] combination

[0511] In one embodiment, the betaine pigment produced using the compositions and methods of the present invention can be applied or provided alone. In another embodiment, the betaine pigment produced using the compositions and methods of the present invention can be applied or provided together with an additional dietary supplement. In another embodiment, the betaine pigment produced using the compositions and methods of the present invention can be applied or provided together with a terpene, in one embodiment being astaxanthin. In yet another embodiment, the betaine pigment produced using the compositions and methods of the present invention can be applied or provided together with anthocyanins, carotenoids, or combinations thereof.

[0512] In another embodiment, the betaine pigment produced using the compositions and methods of the present invention can be produced together with additional components, which in one embodiment can be used as a dietary supplement. In another embodiment, the betaine pigment produced using the compositions and methods of the present invention can be produced together with terpenes, in one embodiment being astaxanthin. In yet another embodiment, the betaine pigment produced using the compositions and methods of the present invention can be produced together with anthocyanins, carotenoids, or combinations thereof.

[0513] In another embodiment, the present invention provides a food crop comprising the polynucleotides of the present invention. In one embodiment, the food crop comprising the polynucleotides of the present invention further comprises anthocyanins, carotene, or combinations thereof. In one embodiment, the food crop comprising the polynucleotides of the present invention is genetically modified to comprise anthocyanins, carotene, or combinations thereof. In one embodiment, the food crop is a purple anthocyanin tomato.

[0514] Any references cited in this article (including patents, patent applications, or scientific publications) are incorporated in their entirety through citation.

[0515] The following embodiments are presented to illustrate preferred embodiments of the invention more fully. However, they should not in any way be construed as limiting the broad scope of the invention.

[0516] Example

[0517] Example 1. Materials and Methods (1)

[0518] Plant materials and growing conditions

[0519] Four o'clock plants were grown in soil in a greenhouse with long-day conditions (25°C). Beetroot and Nicotiana benthamiana plants were grown in soil in a climate chamber (22°C; 70% humidity; 18 / 6 hours light / dark). Plant material for Four o'clock RNA sequencing was collected from the epidermis of red flowers at five developmental stages (stage 1 – flower length approximately 1 cm; stage 2 – 2 cm; stage 3 – 3 cm; stage 4 – 4 cm; stage 5 – 5-6 cm), red young leaves or green mature leaves, and stem nodes (red) or internodes (green). Plant material for beet RNA sequencing was collected from the hypocotyls of 10-day-old seedlings of three varieties (red beet, Bobbikin beet, and Kioja 'Striped' beet). Collected tissues were immediately frozen in liquid nitrogen and maintained at -80°C until processing.

[0520] Transcriptome sequencing, assembly and analysis

[0521] The Mirabilis jalapa and Beetroot libraries for Illumina high-throughput chain-specific RNA sequences were prepared as follows: Total RNA was extracted from sampled tissues using the TRIzol method based on the TRI Reagent User Manual (Sigma-Aldrich). Using methods known in the art with minor modifications, 5 μg of total RNA from each sample was used to prepare the RNA sequence library. In brief: poly(A) RNA was isolated from the total RNA using Dynabeads Oligo(dT)25 (Invitrogen), fragmented at 94 °C for 5 min, and eluted. First-strand cDNA was synthesized using reverse transcriptase SuperScript III (Invitrogen) with random primers and dNTPs, and second-strand cDNA was generated using DNA polymerase I (Enzymatics) and dUTPs. After end repair (Enzymatics), dA end extension (tailing) was performed using Klenow 3'-5' (Enzymatics) and adaptor ligation (Quick T4 DNA Ligase, NEB), and the second strand containing dUTPs was digested with uracil DNA glycosylase (Enzymatics). The cDNA ligated by the first-strand adaptor was used for PCR enrichment using NEBNext High-Fidelity PCR Master Mix (NEB) for 13–15 cycles. The collected libraries were merged and sequenced using an Illumina HiSeq2000 instrument. De novo assembly and calculation of normalized counts for the Mirabilis jalapa dataset were performed using Trinity software, Trinityrnaseq_r version 2013-02-25, according to standard procedures. Contiguous group annotations were assigned using Blast2GO. De novo genome-guided assembly of the B. vulgaris (beet) dataset was performed using the B. vulgaris genome resource (http: / / bvseq.molgen.mpg.de) and Trinity software, Trinityrnaseq_r version 2014-07-17, as a reference. Gene annotations were obtained using the comprehensive Trinotate module.

[0522] Generation of DNA constructs

[0523] The gene sequences used in this study are: cDOPA5GT (accession number AB182643.1; SEQ ID NO: 2), DODA1 (accession number HQ656027.1; SEQ ID NO: 33), and CYP76AD1 (accession number HQ656023.1; SEQ ID NO: 32). Other sequences are provided in Materials and Methods. CYP76AD6 was named by Dr. David R. Nelson of the University of Tennessee, USA, to maintain nomenclature consistency. Transcripts of four o'clock flower cDOPA5GT and beetroot CYP76AD1, BvDODA1, BvCYP76new, and CYP76AD6 were amplified from cDNA libraries of four o'clock flower red petals and beetroot red hypocotyls, prepared using a high-capacity cDNA reverse transcription kit (Life Technologies). PCR amplification was performed using 'Phusion' DNA polymerase (Finnzyme) and the oligonucleotides specified in Table 1. For VIGS assays, all gene fragments were cloned into the pTRV2 vector using XhoI and SacI restriction. The CYP76AD1 fragment was cloned into pTRV2:BvCYP76new and pTRV2:CYP76AD6 using EcoRI and BamHI restriction to construct vectors for co-silencing. DNA constructs for Agrobacterium-mediated infiltration and Agrobacterium-mediated plant transformation were constructed using Goldenbraid cloning. CYP76AD1, BvDODA1, CYP76AD6, and cDOPA5GT were cloned into the pUPD vector using the oligonucleotides specified in Table 1. pDODA, pAD1-GT, pYFP, ​​pAD6, and pX11 were 2α2, 2Ω2, 2α1, 2α1, and 3α1 vectors, respectively, all based on the pCAMBIA backbone.

[0524] Table 1. Oligonucleotides used in this study

[0525]

[0526] Virus-induced gene silencing in beets

[0527] Fragments of BvDODA1 (407 bp), CYP76AD1 (418 bp), BvCYP76new (471 bp), and CYP76AD6 (420 bp) were cloned into the pTRV2 vector, transformed into Agrobacterium line GV3101, and introduced into 10-day-old seedlings of the 'Blood' species of beet 'Bull' using the previously described vacuum infiltration method. Agrobacterium was brought to OD600 2.0 in VIGS infiltration buffer (10 mM MES, 10 mM MgCl2, 200 μM acetylsyleugenol) and incubated at room temperature for 3 hours, then mixed with Agrobacterium carrying pTRV1 at a 1:1 ratio before infiltration. For each experiment, 48 seedlings were infected. In experiments yielding visible phenotypes, depigmented spots were typically observed in more than half of the infected seedlings within 2–3 weeks. Tissue samples for spectrophotometric analysis were collected 3.5 weeks post-infection.

[0528] Quantitative real-time PCR (qRT-PCR) analysis

[0529] qRT-PCR analysis was performed on VIGS-infected red beet plants. Analysis was performed from three biological replicates of each experiment, each consisting of leaf tissue from two to three plants, sampled 3.5 weeks post-infection. RNA was extracted using the TRIzol method (according to the Sigma-Aldrich user manual for TRI reagents) according to the manufacturer's instructions, DNase-treated, and reverse transcribed to cDNA using a high-capacity cDNA reverse transcription kit (Applied Biosystems, Foster City, CA, USA). All oligonucleotides were designed using PRIMEREXPRESS software (Applied Biosystems) (Table 1). qRT-PCR was performed using Fast SYBR Green reagents and a StepOnePlus instrument (Applied Biosystems) under the following conditions: initial step in a thermal cycler at 95°C for 20 s, followed by 40 cycles of PCR amplification at 95°C for 3 s and 59°C for 30 s, followed by dissociation analysis to confirm the specificity of the PCR products. Each reaction consisted of a total volume of 10 μl containing 2.5 μM of each primer. The Ct method (Livak & Schmittgen, 2001) was used to calculate relative transcription levels, with the 'housekeeping' gene GAPDH as a reference.

[0530] Transient expression in Benzoic tobacco

[0531] Transient gene expression assays in *Nicotiana benthamiana* containing pDODA, pAD1-GT, pYFP, ​​pAD6, and pX11 vectors were performed using the previously described Agrobacterium introgression method. All constructs were transformed into *Agrobacterium tumefaciens* strain GV3101, excluding pX11 transformed into *Agrobacterium tumefaciens* strain EHA105. In all cases, *Agrobacterium* was grown overnight in LB medium and reached a final OD600 of 0.2 in introgression buffer. When co-introgression was performed, *Agrobacterium* carrying individual constructs reached an OD600 of 0.2 individually and were mixed in a 1:1 ratio prior to introgression. Tissues used for subsequent LC-MS analysis were sampled from leaves on day 7 post-introgression. For LC-MS analysis of L-DOPA and betaine, 3–4 biological replicates were sampled for each experiment, with each replicate consisting of introgressed tissue from 2–3 different leaves.

[0532] Metabolite extraction and analysis

[0533] For beet pigment analysis, the extraction solution (DDW containing 80% ethanol and 0.1% formic acid) was prepared in 200 μL at a concentration of 0.1 g. -1The proportion of tissue added to cryo-milled plant tissue (0.1–0.25 g) was determined. Samples were incubated at room temperature for 30 min, followed by sonication for 5 min, centrifugation for 5 min, and filtration through a 0.22 μm PVDF (Millipore) filter. Samples diluted 3-fold in DDW were analyzed using a high-resolution UPLC / PDA-qTOF system consisting of a UPLC (Waters Acquity) connected online to an Acquity PDA detector (200–700 nm) and a qTOF detector (tandem quadrupole / time-of-flight mass spectrometer, XEVO, Waters) equipped with an electrospray ionization (ESI) source. ESI was used in positive ionization mode in the m / z range of 50–1600 Da. The following settings were used: capillary – 1 kV, cone – 27 V, collision energy – 6 eV. For MS / MS or MSE runs, collision energy ramps from 15 to 40 eV were used. The compounds were separated on a UPLC HSS T3 column (Waters Acquity, 1.8 μm, 2.1 x 100 mm) using eluents A (1% acetonitrile, 0.1% formic acid) and B (100% acetonitrile, 0.1% formic acid) as follows: initial 3 min - isocratic elution in 100% A; 3.0–22.0 min - linear gradient to 75% A; 22.0–22.5 min - linear gradient to 100% B; 22.5–25.5 min - wash in 100% B; 25.5–26.0 min - return to 100% A; and 26.0–28.0 min - equilibration in 100% A. The column temperature was set at 35 °C and the flow rate at 0.3 mL / min. Betaine and isobetaine were partitioned using red beet leaf extract as a reference. Other beet pigment compounds were speculatively identified based on precise mass, UV-Vis spectroscopy, and MS / MS or MSE fragments (see Table 2 for details).

[0534] Table 2. Beetroot pigment compounds identified by LC-MS analysis.

[0535]

[0536] exist a pAD1-GT+pDODA1、 b pAD6+pDODA1 and c pX11, d Benedict's tobacco callus, e Tobacco callus tissue, f Infiltration experiment of *Agrobacterium tumefaciens* into tobacco transformed with pX11, and gCYP76AD1+DODA1, h CYP76AD6+DODA1 and i In the yeast expression experiment of CYP76AD1 / AD6+DODA1, betaine compounds were identified by LC-MS analysis. All fragments were obtained in MS / MS mode, except for the fragments of betaine and isobetaine, which were obtained by MS. （E） Mode determined. Rt, retention time; MS fragment, fragment quality obtained in positive ionization mode; UV / Vis, maximum absorption in the UV / Vis range; Hex, hexose.

[0537] For L-DOPA analysis, samples were extracted as described above and analyzed using a UPLC / PDA-MSMS instrument, where the UPLC separation module (Waters Acquity) was connected to a photodiode array detector (Waters Acquity 2996, 190-800 nm) and (in-line) to a triple quadrupole MS detector (Waters Xevo TQ MS) equipped with an ESI source. Compounds were separated using a Phenomenex Luna column (2 × 150 mm, 3 μm) with the following gradients: initial 1 min – linear gradient from 100% A to 90% A, 1–4 min – 90–75% A, 4–5 min – 75–0% A, 5–9.5 min – wash in 100% B, 9.5–10 min – return to 100% A, and 10–12 min – equilibration in 100% A, where A is water containing 0.1% formic acid and B is acetonitrile containing 0.1% formic acid. The column temperature was set to 35ºC, and the flow rate was 0.25 ml / min. Positive ionization mode analysis of the samples was performed using the following settings: capillary - 2 kV, cone - 26 eV, with two transitions for compound partition: 198.1 > 152.1 (collision - 15 eV) and 198.1 > 181.1 (collision - 10 eV). L-DOPA was identified by comparison with a commercial standard (Sigma-Aldrich) of L-DOPA dissolved in DDW (5 μM) and injected under the same LC-MS conditions.

[0538] To relative quantify betalains in VIGS-silenced beet leaves by spectrophotometry, samples were extracted using the same procedure detailed above, except that 80% methanol was used instead of 80% ethanol. The extracts were diluted 4-fold in the extraction solution, placed in 96-well plates, and analyzed using a Biotek Synergy HT microplate reader. The relative betalain content of each sample was assessed by measuring the absorbance at 475 nm and subtracting the absorbance at 600 nm.

[0539] To assess betalain content by spectrophotometry, the same extraction method was used as described above. Samples were diluted in DDW to obtain solutions with OD535 < 2.0. Betalain quantification was based on absorbance measurements at 535 nm and 600 nm and calculated using the previously described method.

[0540] Recombinant expression in yeast

[0541] According to the manufacturer's manual, the coding sequences for CYP76AD1, CYP76AD6, and BvDODA1 were PCR amplified and cloned into the BP site of the pDONR207 vector using Gateway Cloning (Life Technologies, Invitrogen), and then recombined into the LR site of the pAG423GAL-ccdB (for CYP76AD6), pAG425GAL (for BvDODA1), and pYES-DEST52 (for CYP76AD1, Invitrogen) vectors. Additionally, the coding sequence for β-glucuronidase (GUS) was introduced into these vectors for control assays. The target vectors were transformed into *Saccharomyces cerevisiae* strain BY4742 using the PEG / LiAc method, and the transformations were performed sequentially. Each yeast clone was ultimately transformed with three target vectors, expressing different combinations of CYP76AD1, CYP76AD6, BvDODA1, and GUS. The yeast strains were grown overnight in standard SD medium containing 2% glucose and lacking histidine, leucine, and uracil. Yeast was granulated and resuspended in 2 ml SD medium containing 2 mM ascorbic acid, 1% raffinose, and 2% galactose, but lacking histidine, leucine, and uracil, to an OD600 of 1.0, and grown overnight. The image in Figure 8 shows the culture medium for yeast growth after overnight galactose induction. For LC-MS analysis, 1 ml of liquid medium was dried in an evaporator and resuspended in 100 μl DDW.

[0542] Bioinformatics Analysis

[0543] For co-expression analysis, contigs corresponding to DOPA 4,5-dioxygenase (MjDOD), cyclic DOPA-5-O-glucosyltransferase (cDOPA5GT), and CYP76AD3 were used as 'bait'. Based on the normalized count of each gene in all 24 libraries, Pearson correlation values ​​were calculated by using R scripts to identify genes co-expressed with any of the 'bait'.

[0544] To generate a phylogenetic tree for CYP76AD1-like proteins, the CYP76AD6 protein sequence was first used for a BLASTp query in the NCBI nr database. The top four BLASTp matches with the highest sequence identity and complete protein sequence were selected and used for multiple sequence alignment along with the CYP76AD1 protein and its previously described orthologs CYP76AD2, CYP76AD3, and CYP76AD4. Multiple sequence alignment was performed using Clustal Omega, and the resulting phylogenetic tree was processed using MEGA software version 6.06. Validation was performed using a bootstrapping process of 100 iterations. Alignment between CYP76AD6 and CYP76AD1 was completed using Clustal Omega with default parameters and edited using GeneDoc software.

[0545] Plant transformation and regeneration

[0546] Agrobacterium-mediated plant transformation and regeneration were performed on tobacco leaves, eggplant, Nicotiana benthamiana, tomato, potato, petunia, and Nicotiana tree according to methods known in the art. A protocol for the transformation of Solanum nigrum is provided below. All plant species were transformed using Agrobacterium GV3101. Plant tissue cultures were performed in a climate chamber (22°C; 70% humidity; 16 / 8 hours light / dark, 2500 Lux light intensity).

[0547] The gene sequences used in this study

[0548] The provided sequences are contiguous sequences obtained from de novo assembly of beet and four o'clock transcriptome (RNA sequence) data. All sequences are presented in the 5'-3' orientation.

[0549] >MjCYP76 from Mirabilis jalapa

[0550]

[0551] >BvCYP76new from Beetroot

[0552]

[0553] Sample CYP82G1 from Mirabilis jalapa

[0554]

[0555] >Sample CYP78A9 from Mirabilis jalapa

[0556]

[0557] >CYP86B1 sample from Mirabilis jalapa

[0558]

[0559] Solanum nigrum hypocotyl transformation

[0560] culture medium

[0561] Jones liquid (pH 5.2)

[0562] Basic culture medium (adjusted to pH 5.8 with NaOH before adding agar)

[0563] Vitamin-containing MS (cat M0222) = 4.41 g / L

[0564] 3% sucrose = 30g / L

[0565] Agarose (plant agar) 0.6% = 6g / L

[0566] Table 3: IND1 (First Callus Induction Medium)

[0567]

[0568] Table 4: IND2 (Second Callus Induction Medium)

[0569]

[0570] *Prepare a culture medium without Kan for use as a control.

[0571] Table 5: SHOOT (Sprout Induction Medium)

[0572]

[0573] *Prepare a culture medium without Kan for use as a control.

[0574] Table 6: MAT (Maturation Culture Medium)

[0575]

[0576] *Prepare a culture medium without Kan for use as a control.

[0577] Table 7: ROOT (magenta rooting medium)

[0578]

[0579] *Prepare a culture medium without Kan for use as a control.

[0580] program

[0581] Always work in a flow fume hood, near a flame.

[0582] *Use forceps and scalpels sterilized with EtOH and flame.

[0583] * Seal with paraffin wax before removing from the flow fume hood.

[0584] Preparation of Agrobacterium:

[0585] Pick colonies newly transformed into Agrobacterium GV3101 and inoculate them into 7.5 ml LB buffer in a 50 ml Falcon tube.

[0586] Oscillating growth at 28℃o / n

[0587] Add Jones' liquid (pH 5.2) to a total volume of 25 ml.

[0588] Treatment of explants:

[0589] Use seedlings with enlarged cotyledons but no true leaves - remove individual seedlings using sterile tweezers and place them in sterile culture dishes (approximately 30 hypocotyls per plate).

[0590] The embryonic axis was cut using a sterile scalpel, which was then immersed in an Agrobacterium mixture before each cut.

[0591] For control purposes, the scalpel was dipped into Jones liquid before each cut.

[0592] All explants were placed on IND1 plates and placed in a growth chamber in the dark for 3 days (wrapped in foil).

[0593] Subsequent subculture:

[0594] Transfer the explants onto IND2 plates (spaced more than 20 per plate) and place them in a growth chamber for light / dark cycles.

[0595] If contamination is visible, check the plates for contamination every few days and transfer healthy explants to fresh culture medium more quickly.

[0596] Two to three weeks later, callus will form at the ends of the explants (where they were cut). The callus is then removed from the rest of the tissue and transferred to SHOOT medium.

[0597] If callus has not yet formed, transfer to fresh IND2 medium.

[0598] After 1-2 weeks, green shoots will begin to develop from the callus tissue. Transfer these callus tissues to MAT medium.

[0599] If no buds form, transfer to fresh SHOOT medium.

[0600] After 1-2 weeks, the buds should be large enough to be cut from the callus and transferred to ROOT medium (magenta).

[0601] If the bud is still very small, transfer it to fresh MAT medium.

[0602] Transplant seedlings to fresh ROOT medium every 2-3 weeks until they have enough roots to be transplanted into the soil.

[0603] Example 2. Transcriptome and co-expression analyses in Mirabilis jalapa revealed the involvement of members of the CYP76AD1 subfamily. Role in betaine biosynthesis

[0604] The primary objective of this study is to elucidate the first committed step in the biosynthesis of betaine in plants, which begins with the aromatic amino acid tyrosine. Achieving this goal will close gaps in our current understanding of the roles of plant enzymes and genes in the core betaine pathway. Figure 1 Since genomic and transcriptomic sequence data from plants that produce betaine are currently very limited, we generated a comprehensive transcriptomic dataset from betaine-colored Mirabilis jalapa (i.e., four o'clock) plants. Libraries of 24 Mirabilis jalapa tissues were sequenced, including both betaine-producing and non-betaine-producing tissues. A selected set of tissues provides a temporal and spatial representation of betaine synthesis, as samples were taken from four different floral parts (i.e., petals, stamens, anthers, and stigmas) across five developmental stages, each exhibiting increased pigmentation during development. Figures 2A to 2F ).

[0605] Transcriptome data were first analyzed by examining the gene expression patterns of betaine-related genes previously identified in Mirabilis jalapa (i.e., DOPA 4,5-dioxygenase (MjDOD), cyclic DOPA-5-O-glucosyltransferase (cDOPA5GT), and cytochrome P450 (CYP76AD3)). These three genes were found to exhibit a parallel pigment accumulation pattern (i.e., increased expression during flower development, with higher expression in red tissues than in green tissues). Figure 2G Therefore, co-expression analysis can be used to discover other betalain-related genes and to identify genes with expression patterns highly correlated with MjDOD, cDOPA5GT, or CYP76AD3.

[0606] It is generally believed that tyrosine hydroxylation in plants that produce betaine is catalyzed by tyrosinase (polyphenol oxidase). Therefore, we first searched for tyrosinase-encoding genes co-expressed with MjDOD, cDOPA5GT, or CYP76AD3, but none were found. Polyphenol oxidase expressed in a parallel betaine accumulation pattern could not be detected by manually examining genes annotated as polyphenol oxidase in the Mirabilis jalapa dataset. Figure 3Alternatively, L-DOPA formation could theoretically be catalyzed in Mirabilis jalapa by cytochrome P450 enzymes. Four cytochrome P450-encoding genes were found to be co-expressed with one or more known betaine-related genes used as bait: CYP82G1-like (SEQ ID NO: 36), CYP78A9-like (SEQ ID NO: 37), CYP86B1-like (SEQ ID NO: 38), and CYP76AD1-like (SEQ ID NO: 1). The latter, referred to below as MjCYP76, stood out as a promising candidate, exhibiting high expression values ​​across the dataset and being highly correlated with betaine accumulation. Figure 2G ).

[0607] Example 3. Co-silencing of CYP76AD1 and CYP76AD6 to inhibit the production of betaine in beets.

[0608] To verify whether MjCYP76 is indeed involved in betaine biosynthesis, a gene silencing assay was required. Since no betaine-producing plant has a robust and stable transformation program, we decided to apply Virus-Induced Gene Silencing (VIGS), a widely used method for transient gene silencing in plants and well-suited for tracking pigmentation phenotypes. However, gene silencing using the VIGS method has previously demonstrated particularly challenging results in Mirabilis jalapa (most likely due to the inhibitory activity of the Mirabilis jalapa antiviral protein (MAP),) and is only achievable when the MAP gene is co-silenced with the gene of interest. Attempts to perform gene silencing in Mirabilis jalapa using VIGS were unsuccessful in our case, including attempts to silence cDOPA5GT, which should have resulted in a partial loss of betaine pigmentation in the plant. Therefore, we continued our search for orthologs of MjCYP76 in different betaine-producing plant species where gene silencing assays could be efficiently performed. A highly efficient VIGS method using vacuum infiltration was previously described in Red Sugar beet, which was successfully used to silence betaine-related genes. Identifying orthologs of MjCYP76 in red beets can help assess the gene’s relevance to betalain biosynthesis.

[0609] To this end, we sequenced the transcriptomes of hypocotyl tissues from three beet varieties: the "Golden Beet" variety, which produces only yellow betaine, and the Red Beet and "Striped Beet" varieties, both with hypocotyls containing red pigment deposition. The MjCYP76 ortholog in beets is expected to be highly expressed in all three tissues, speculatively playing a key role in beet pigment biosynthesis. Homologs were identified by querying the beet transcriptome dataset and the MjCYP76 coding sequence using tBLASTx. A contiguous group of eight distinct cytochrome P450 genes representing high sequence homology (over 50% identity at the protein level) with MjCYP76 was identified, including the previously characterized CYP76AD1 (…). Figure 4A The gene with the highest score, BvCYP76new, was selected as a potential candidate to become a functional ortholog of MjCYP76 in sugar beets. Another gene in this group, referred to below as CYP76AD6, which shares 60% nucleotide identity and 53% amino acid identity with MjCYP76, exhibited significantly higher expression levels compared to other paralogs and was therefore also selected for subsequent analysis.

[0610] Fragments from BvCYP76new and CYP76AD6 were cloned into the VIGS vector pTRV2 and introduced into seedlings of the 'Blood' red beet variety 'Bull', which produces plants with highly pigmented dark red leaves, making it particularly suitable for gene silencing assays of beet pigment-related genes. Since the tyrosine hydroxylase reaction for L-DOPA formation is essential for the formation of all beet pigment compounds, including red betaine and yellow betaine xanthophyll, silencing BvCYP76new or CYP76AD6 was expected to produce plants displaying green patches due to the lack of beet pigment deposition. pTRV2:BvDODA1 and pTRV2:CYP76AD1 were also introduced into beet seedlings as positive controls for the assay. As previously reported, silencing BvCYP76new or CYP76AD6 did not result in any visible phenotype (data not shown), while silencing BvDODA1 and CYP76AD1 produced green or yellow patches, respectively. One possible explanation for the lack of a visible phenotype when the gene presumed to encode the enzyme that produces L-DOPA is silenced is that the enzyme may be duplicated by one or more other enzymes that also catalyze the same reaction. One such candidate is CYP76AD1, which, in addition to its experimentally demonstrated activity in converting L-DOPA to cyclic DOPA, may also catalyze the tyrosine hydroxylation step. Since silencing CYP76AD1 does not prevent the formation of betaine, and L-DOPA is an essential precursor to betaine (…),… Figure 1 Therefore, CYP76AD1 will have to repeat its action with another enzyme. This hypothesis can be tested by co-silencing BvCYP76new or CYP76AD6 together with CYP76AD1.

[0611] Therefore, we constructed a pTRV2 vector carrying a CYP76AD1 fragment tandemly with one of the BvCYP76new or CYP76AD6 candidates. In addition to co-silencing CYP76AD1 with each CYP76 candidate, subsequent experiments were conducted to silence BvDODA1, CYP76AD1, BvCYP76new, or CYP76AD6, as described in the aforementioned VIGS experiments. Plants co-silenced with both CYP76AD1 and BvCYP76new produced yellow patches in their leaves, similar to plants silenced only with CYP76AD1. However, co-silencing both CYP76AD1 and CYP76AD6 elicited a distinct green patch phenotype lacking betaine and betaine xanthophyll, similar to the phenotype obtained through BvDODA1 silencing. Figure 5A B). Differences in betalain accumulation levels can also be observed using blue light imaging. Figure 5C Betaine typically exhibits strong fluorescence under blue light. Spectrophotometric analysis (…) Figure 6A This further confirms that betaine accumulation is reduced in CYP76AD1-CYP76AD6 co-silenced tissues compared to CYP76AD1-silenced tissues. These findings support the concept of repetition in the tyrosine hydroxylation step of betaine biosynthesis in red beets, where either CYP76AD1 or CYP76AD6 can catalyze this step, as described above. Furthermore, the fact that CYP76AD1 silencing alone inhibits the production of betaine reds, rather than betaine xanthophyll, indicates that CYP76AD6 does not catalyze the conversion of L-DOPA to cyclic DOPA, but only catalyzes the formation of L-DOPA from tyrosine.

[0612] qRT-PCR analysis was performed to detect the expression of CYP76AD1 and CYP76AD6 in plants infected with either pTRV2:CYP76AD1 or pTRV2:CYP76AD1-CYP76AD6 vectors. It was found that CYP76AD1 was downregulated after infection with both vectors, while CYP76AD6 was downregulated only after infection with pTRV2:CYP76AD1-CYP76AD6. Figure 6B The expression of four CYP76AD1 and CYP76AD6 paralogs was also examined. Three of the four paralogs examined showed downregulation in tissues infected with at least one of the pTRV2:CYP76AD1 or pTRV2:CYP76AD1-CYP76AD6 vectors. Figure 4ATherefore, the possibility that one or more of these paralogs are involved in betaine biosynthesis cannot be ruled out at present. However, the lack of correlation between one of the three paralogs, BvCYP76new, and betaine biosynthesis can be inferred from the results of the silencing experiments, since silencing BvCYP76new alone does not lead to visible changes in phenotype, and silencing this gene together with CYP76AD1 produces the same phenotype observed when CYP76AD1 is silenced alone.

[0613] Example 4. Transient expression of CYP76AD1 in Nicotiana benthamiana enables the production of L-Dopa and betalains.

[0614] If CYP76AD1 does indeed catalyze the formation of L-DOPA and its conversion to cyclic DOPA, then heterologous expression of CYP76AD1, along with DOD enzymes and betaine-related glucosyltransferases (e.g., cyclic DOPA 5-O-glucosyltransferase or betaine-5-O-glucosyltransferase), should be sufficient for the biosynthesis of widely available glucosylated betaine and betaine glycosides. To test this, we generated overexpression constructs for the beet BvDODA1 and CYP76AD1 genes, as well as the four o'clock gene cyclic DOPA5-O-glucosyltransferase (cDOPA5GT), for transient, Agrobacterium infiltration-mediated overexpression in Nicotiana benthamiana leaves. Two vectors were prepared for this purpose: one for overexpressing BvDODA1 under the CaMV 35S promoter (pDODA), and the other for tandem expression of CYP76AD1 and cDOPA5GT driven by the CaMV 35S and Arabidopsis ubiquitin-10 promoters (pAD1-GT), respectively. Agrobacterium (Agrobacterium rhizogenes) carrying both pDODA and pAD1-GT vectors co-infiltrated into Tobacco Benzoinii leaves, resulting in dark red pigment deposition in the infiltrated area within 2-3 days after infiltration. Figure 7A Liquid chromatography-mass spectrometry (LC-MS) analysis revealed high levels of betaine in the pigmented leaf tissues, as well as the betaine isomer isobetaine (…). Figure 7B (Table 2). This result provides the first proof of concept for the possibility of engineering beet pigment production in-plant without substrate feed. Furthermore, the formation of beet red without L-DOPA feed or overexpression of tyrosine hydroxylases (e.g., tyrosinase) suggests that CYP76AD1 catalyzes L-DOPA formation in addition to its previously known activity of converting L-DOPA to cyclic-DOPA.

[0615] However, given the presence of L-DOPA in *N. benthamiana* leaves, it may be possible to produce betaine due to the activity of tyrosine hydroxylases via endogenous enzymes. To determine whether L-DOPA production is due to CYP76AD1 expression, pAD1-GT was infiltrated into *N. benthamiana* leaves lacking pDODA. The introduction of a vector overexpressing YFP protein (pYFP) was used as a control in this experiment. LC-MS analysis confirmed the presence of L-DOPA in tissues infiltrated with pAD1-GT, but not in tissues infiltrated with pYFP. Figure 7C ).

[0616] Example 5. Transient expression of CYP76AD6 in *Nicotiana benthamiana* enables the production of L-DOPA and betaine.

[0617] Tyrosine hydroxylase activity of CYP76AD6 was also assessed by transient overexpression in *Nicotiana benthamiana* leaves. An overexpression vector of CYP76AD6, constructed under the CaMV 35S promoter (pAD6), was inserted into *Agrobacterium*. Co-infiltration of pAD6 and pDODA resulted in yellow pigment deposition in the infiltrated area, visible within several days post-infiltration. Figure 7D LC-MS analysis of the tissue containing yellow pigment deposition revealed the presence of a major betaine compound, identified as pomegranate cactus flavin (proline-betaine flavin). Figure 7E Based on accurate quality and fragment data (Table 2), two other compounds were found to exhibit typical cytoflavin light absorption spectra, one of which was presumed to be dopaflavin-hexoside. Notably, to our knowledge, glycosylated betaine is not present in naturally occurring betaine-producing species. The fact that co-introduction of CYP76AD6 and BvDODA1 induces the production of betaine but not betaine red is consistent with the data obtained from the gene silencing assays described above, confirming that CYP76AD6 catalyzes only the single step of L-DOPA formation, without catalyzing its subsequent conversion to cyclic-DOPA. LC-MS validation showed that Agrobacterium introduction of the pAD6 vector alone resulted in the production of L-DOPA in *Nicotiana benthamiana* leaves. Figure 7F ).

[0618] Example 6. Recombinant expression of CYP76AD1 and CYP76AD6 allows for the synthesis of betaine in yeast.

[0619] The activities of CYP76AD1 and CYP76AD6 were further evaluated in conjunction with BvDODA1 through recombinant expression of each of the two enzymes in yeast. For this purpose, the complete coding sequences of BvDODA1, CYP76AD1, and CYP76AD6 were cloned into a galactose-inducible yeast overexpression vector and transformed into *Saccharomyces cerevisiae* cells. The yeast was then grown in standard SD medium without L-DOPA supplementation. Similar to the transient expression assay in *Nicotiana benthamiana* discussed above, reddish-purple pigment deposition was observed in yeast medium expressing CYP76AD1 and BvDODA1, while significant yellow pigment deposition was observed in yeast medium expressing CYP76AD6 and BvDODA1. Expression of each cytochrome P450 enzyme without BvDODA1 or expressing BvDODA1 alone did not result in the formation of red or yellow pigment. Furthermore, co-expression of CYP76AD1, CYP76AD6, and BvDODA1 resulted in orange pigment deposition in the medium. Figure 8A LC-MS analysis was used to verify the presence of betaine in media stained with yellow, reddish-purple, and orange pigments. Figure 7C (Table 2).

[0620] Example 7. CYP76AD6 belongs to a phylogenetic branch that was previously not associated with beet pigment biosynthesis.

[0621] A recently published study, involving a large-scale phylogenetic analysis of Caryophyllales plants, revealed several gene duplication events at the CYP76AD1 locus early in the evolutionary history of these plants. These events resulted in three clades within the CYP76AD1 lineage, named CYP76AD1-α, CYP76AD1-β, and CYP76AD1-γ. The inventors infer that the CYP76AD1-α clade is directly related to betaine biosynthesis because it includes the functionally characteristic beet CYP76AD1 gene and its ortholog in Mirabilis jalapa, CYP76AD3 (which is suggested to play a similar role in betaine biosynthesis). However, the functions of the genes belonging to the CYP76AD1-β and CYP76AD1-γ clades are currently unknown. Interestingly, the CYP76AD6 gene reported here [accession number 'Bv9_228610_yqeq.t1' in the beet genome] is located in the CYP76AD1-β clade, as demonstrated in the phylogenetic analysis provided by Brockington et al., and is further presented here in the most probable phylogenetic tree. Figure 8B The paired sequences of CYP76AD6 and CYP76AD1 proteins showed 72% identity. Figure 9 Their corresponding genes show 70% sequence identity at the nucleotide level.

[0622] Example 8. Red beet pigment in metabolically engineered transgenic plants requiring three biosynthetic genes Heterologous expression

[0623] As described above, we were able to produce red betaine and yellow betaine xanthophyll through transient gene overexpression in tobacco leaves. We then attempted to engineer betaine production in naturally non-producing plant species via stable transformation. To this end, a four-gene construct (hereinafter referred to as pX11) was generated using a Goldenbraid clone, in which CYP76AD1, BvDODA1, and cDOPA5GT were expressed in tandem and driven by a constitutive promoter, in addition to the kanamycin resistance gene used for transgenic selection. Figure 10 The pX11 vector was initially tested by infiltrating *Agrobacterium* into *Nicotiana benthamiana*. Similar to the co-infiltration of pDODA and pAD1-GT, the introduction of pX11 resulted in red pigment deposition in the infiltrated tissue. Figure 12B LC-MS analysis of pX11 infiltration into tissues was performed to determine the betaine pigment composition. Peaks corresponding to betaine and isobetaine were observed, as previously observed with co-infiltration of pDODA and pAD1-GT overexpression vectors. Figure 11A Also identified as a betaine compound is pomegranate cactus flavin (proline-betaine flavin), indicating a certain level of change towards betaine formation. Based on light absorption spectra and fragments, it includes the almost ubiquitous betaine red fragment betaine (m / z = 389; Table 2), and two other metabolites were identified as betaine flavins. pX11-infiltrated leaf tissues showed strong red pigmentation, seemingly producing betaine red in high quantities ( Figure 12B Therefore, the total betaine content in the infiltrated tissue was assessed by spectrophotometric analysis, revealing a content of 330 mg / kg. -1 The assessed value was higher than that of the red bracts of Bougainvillea glabra and the red petals of Mirabilis jalapa (containing 120 mg kg each). -1 and 250mg kg -1 It contains betalains, and is 2.3 times lower than that of red beetroot (containing 760 mg / kg). -1 () Figure 12C ).

[0624] Next, the pX11 vector was used for stable transformation in several plant species, including tobacco (Nicotiana tabacum), tomato (Solanum lycopersicum), potato (Solanum tuberosum), eggplant (Solanum melongena), tree tobacco (Nicotiana glauca), European black nightshade (Solanum nigrum), petunia (Petunia x hybrida), and *Nicotiana benthamiana*. Explants from different species were co-cultured with *Agrobacterium* on selective media. Within two days of co-culturing with the *Agrobacterium* of this invention, small patches of reddish-purple pigmentation were visible on the surface of the explants in *Nicotiana benthamiana*, tree tobacco, petunia, and tomato. In all transformed species, the introduction of pX11 resulted in the formation of reddish-purple callus, which typically appeared within 1-2 weeks of culture. Figure 11A LC-MS revealed that stained callus tissues of *Nicotiana benthamiana* and *Nicotiana treeensis* contained betaine and isobetaine. Furthermore, novel, unidentified betaine compounds were found in *Nicotiana treeensis* callus tissues (Figure 11B, Table 2).

[0625] Tissue cultures of pX11-transformed tobacco (Nicotiana tabacum L., Samsun-NN) ultimately resulted in mature plants with intense red pigmentation, exhibiting betaine accumulation in all major plant organs, including stems, leaves, roots, and flowers (Figure 13). LC-MS analysis of transgenic pX11 tobacco leaves confirmed the presence of betaine, betaine glycosides, isobetaine, and the betaine compound pachycarboxin I (glutamine-betaine) (Table 2). The betaine content in pX11 tobacco leaves was further measured spectrophotometrically to be 135 mg / kg. -1 ( Figure 12C ).

[0626] Example 9. discuss

[0627] Repeated catalysis of tyrosine hydroxylation by CYP76AD1 and CYP76AD6 is the first key step in the biosynthesis of betaine in plants.

[0628] In this study, in addition to its previously reported role in catalyzing subsequent reactions leading to the formation of cyclic DOPA, we have demonstrated that CYP76AD1 is involved in the hydroxylation of tyrosine to L-DOPA in red beet. Furthermore, we identified another beet cytochrome P450 enzyme, CYP76AD6, which repeats the catalytic first hydroxylation step with CYP76AD1. Figure 1The ortholog of CYP76AD6, MjCYP76, is primarily identified in four o'clock flower (Mirabilis jalapa) and exhibits a parallel betalain accumulation expression pattern. MjCYP76 and the previously identified CYP76AD3 likely perform the same functions as CYP76AD6 in four o'clock flower and as CYP76AD1 in red beet, respectively, although this still requires conclusive verification through functional analysis (primarily gene silencing assays in four o'clock flower).

[0629] CYP76AD6, initially identified as involved in L-DOPA formation, was detected by gene silencing assays. While silencing CYP76AD1 itself inhibits betalain formation rather than betalain formation, parallel silencing of CYP76AD1 and CYP76AD6 inhibits the formation of both betaine groups. Silencing CYP76AD6 alone does not produce a visible phenotype because the formation of L-DOPA and cyclic-DOPA continues to be catalyzed by CYP76AD1. This lack of a visible phenotype may partially explain why CYP76AD6 and the tyrosine hydroxylation step are the last to be found in the core betaine biosynthetic pathway. Although DOD and CYP76AD1-like genes can be identified in betaine-producing species through spontaneous or artificially induced mutations leading to a clearly visible phenotype, CYP76AD6 is not recognized due to its functional duplication with CYP76AD1. This indicates that the roles of CYP76AD1 and CYP76AD6 in betaine biosynthesis are further confirmed through recombinant expression in *Nicotiana benthamiana* and yeast cells, leading to the formation of betaine red or betaine xanthophyll, respectively, when combined with BvDODA1 expression. Furthermore, L-DOPA formation was detected in *Nicotiana benthamiana* when CYP76AD1 or CYP76AD6 was overexpressed in the absence of BvDODA1.

[0630] CYP76AD1 and CYP76AD6 belong to two separate clades within the CYP76AD1 subfamily, designated CYP76AD1-α and CYP76AD1-β, respectively. It is conceivable that these two cytochrome P450 enzymes would share some degree of structural correlation, as they repeatedly catalyze the same enzymatic reactions. Furthermore, cytochrome P450s belonging to a single subfamily have previously been reported to catalyze subsequent steps in the same biosynthetic pathway. It seems reasonable that, while the CYP76AD1-α clade could consist of enzymes with dual activities in L-DOPA and cyclic-DOPA formation, CYP76AD1-β could include enzymes that catalyze only L-DOPA formation. However, functional analysis of the corresponding genes from other beet pigment-producing species is needed to support this hypothesis.

[0631] Tyrosinase catalyzes the ortho-hydroxylation of monophenols and the oxidation of ortho-diphenols to ortho-quinones, thus directly converting tyrosine to dopaquinone, the oxidized form of L-DOPA. In a currently unknown reaction mechanism, CYP76AD1 also catalyzes the hydroxylation of tyrosine and the conversion of L-DOPA to cyclic-DOPA. Our experimental results indicate that CYP76AD6 is unique in that it catalyzes the ortho-hydroxylation of tyrosine but does not catalyze the conversion of L-DOPA to dopaquinone or cyclic-DOPA. The unique activity of the CYP76AD6 enzyme, compared to current production methods, is highly advantageous and unexpected for high-scale L-DOPA production. CYP76AD6 directly forms L-DOPA from the substrate tyrosine. The ubiquitous nature of tyrosine makes the expression of CYP76AD6 possible in various biological platforms, including plants and microorganisms.

[0632] A novel class of tyrosine hydroxylases has been discovered that can also promote the recognition of other cytochrome P450 genes and catalyze L-DOPA formation in the biosynthesis of other L-DOPA-derived metabolites. For example, in another well-defined pathway, the gene leading to tyrosine hydroxylation in the biosynthesis of benzylisoquinoline alkaloids (BIA) remains unknown. It is possible that in BIA-producing plants, L-DOPA could also be catalyzed by cytochrome P450s similar to CYP76AD1 and CYP76AD6.

[0633] Revealing the roles of CYP76AD1 and CYP76AD6 in L-DOPA formation has enhanced our understanding of betaine biosynthesis in plants and essentially completed the identification of genes and enzymes involved in the formation of core betaine structures. However, the betaine pathway remains relatively poorly understood regarding structural modifications of betaines via “decorative” enzymes (e.g., glycosylation and acylases), subcellular transport, and detoxification. Further characterization of the genes and regulators involved in these pigment biosyntheses is expected to be facilitated by elucidating core pathways and by increasing the availability of sequence data from betaine-producing plants.

[0634] Metabolic engineering for the production of betaine in plants

[0635] While anthocyanins and carotenoids are ubiquitous in the plant kingdom, betaine pigments are specific to the order Caryophyllales, where they are produced in a phylogenetic sequence that is mutually exclusive with anthocyanins. Their enigmatic evolutionary history, along with their nutritional benefits and economic value as natural food colorings, has recently fueled increased interest in these pigments, including the prospect of synthesizing them in microorganisms and plants.

[0636] In this study, we demonstrated for the first time the engineering of betaine production in plants without substrate feed through both transient gene expression and stable transformation. This was made possible by recognizing the additional tyrosine hydroxylase activity of CYP76AD1. Thus, it was found that expression of CYP76AD1 in combination with BvDODA1 and cDOPA5GT was sufficient for the biosynthesis of betaine without the need for exogenous L-DOPA supply. The use of the pX11 vector, including the CYP76AD1, BvDODA1, and cDOPA5GT genes, was first demonstrated through transient Agrobacterium infiltration-mediated expression in *Nicotiana benthamiana*, which resulted in high yields of betaine visible within two days of infiltration. Attempts to stably transform pX11 into plants and subsequently cultured them in tissue cultures resulted in pigmentation of the explants and the formation of reddish-purple callus and buds within days in multiple plant species, indicating the potential use of betaine as a visible marker for gene transformation. Stable transformation of tobacco plants with pX11 ultimately resulted in fully pigmented mature plants. LC-MS analysis of tobacco plants that produce betaine confirmed that betaine was the main betaine produced.

[0637] Therefore, the findings of this study pave the way for heterologous production of betaine in additional plant species, potentially leading to the development of nutritionally enhanced food crops and new ornamental varieties. In addition to engineered red-colored betaine-producing plants, betaine production can be primarily modified to be limited to betaine xanthophyll by expressing CYP76AD6 instead of CYP76AD1, as in transient expression experiments in *Nicotiana benthamiana*. The engineering of plants exhibiting both red and yellow pigmentation can hypothetically be achieved by expressing two cytochrome P450s under different nonconstitutive promoter sequences (e.g., fruit-specific, flower-specific, or inducible promoters). Engineered betaine-producing plants will also serve as a unique genetic resource for investigating pigment-based attractiveness of flowers to pollinators and fruits to fruit predators, as well as the role of pigments in assisting plant organs to resist abiotic and biotic cues.

[0638] Example 10. Materials and Methods (2)

[0639] Generation of DNA constructs

[0640] The gene sequences used in this study are provided below; the sequences of cDOPA5GT (accession number AB182643.1), BvDODA1 (accession number HQ656027.1), CYP76AD1 (HQ656023.1), CYP76AD6 (KT962274), AroG175 (JC233128.1), AAH (HQ003815.1), and CYP76AD15 were amplified from cDNA libraries of red petals of Mirabilis jalapa and red hypocotyls of Beetroot. DNA constructs for Agrobacterium benthamianum infiltration and Agrobacterium-mediated plant transformation were constructed using Goldenbraid cloning (Sarrion-Perdigones et al., 2013; GoldenBraid 2.0: A Comprehensive DNA Assembly Framework for Plant Synthetic Biology. Plant Physiology 162: 1618-1631). First, BvDODA1, CYP76AD1, CYP76AD6, CYP76AD15, cDOPA5GT, AroG175, and AAH were cloned into the pUPD vector. pX11, pX11(E8), pX11(CHS), pX13, pDOPA3, and pDOPA4 were 3... 1. Vectors. pX12, pDOPA1, and pDOPA2 are 3Ω1 vectors. All vectors are based on the pCAMBIA backbone (Roberts et al., 1997; A Comprehensive Set of Modular Vectors for Advanced Manipulations and Efficient Transformation of Plants. In pCAMBIA Vector Release Manual Rockefeller Foundation Meeting of the International Program on Rice Biotechnology, September 15-19, Malacca, Malaysia).

[0641] Plant transformation and regeneration

[0642] Agrobacterium-mediated plant transformation and regeneration were performed using the following methods: tobacco leaves (Horsch et al., 1985; Science 227: 1229-1231), eggplant (Van Eck and Snyder, 2006; Eggplant (Solanum melongena L.). In K Wang (ed.), Agrobacterium Protocols. Springer, pp 439-448), tomato (McCormick, 1997; Transformation of tomato with Agrobacterium tumefaciens. In K Lindsey (ed.), Plant tissue culture manual. Springer, pp 311-319), potato (Perl et al., 1992; Plant Molecular Biology 19: 815-823), and petunia (Conner et al., 2009; Transformation and regeneration of Petunia. In T Gerats, J Strommer (ed.), Petunia. Springer, pp 395-409). Transformation and culture of BY2 were performed according to An, 1985 (Plant Physiology 79:568-570). All plant species were transformed using Agrobacterium GV3101 strain. Plant tissue cultures were performed in a climate chamber (22°C; 70% humidity; 16 / 8 hours light / dark, 2500 Lux light intensity).

[0643] Transient expression of Tobacco Benzoviae

[0644] As described above, transient gene expression assays of BvDODA1 and CYP76AD15 genes in Nicotiana benthamiana and subsequent LC-MS analysis were performed.

[0645] Metabolite extraction and analysis

[0646] For LC-MS analysis, plant tissues were extracted from tomato fruit (pulp and peel), eggplant fruit (pulp), potato tuber (pulp), tobacco petals, and Nicotiana benthamiana leaves, as previously described (Polturak et al., 2016). For BY2 cell extraction, callus tissue (approximately 500 mg) sampled from culture dishes was thawed by two liquid nitrogen freeze-thaw cycles and then disrupted with metal beads. The cell extract was centrifuged to remove cell debris, and the supernatant was collected and filtered through a 0.22 μm PVDF (Millipore) filter prior to analysis. LC-MS analysis of L-DOPA and betaine pigments was performed as previously described (Polturak et al., 2016). The relative quantification of L-DOPA in BY2 cells was determined by the peak area based on the 198 > 152 transition.

[0647] Seed germination test

[0648] Seeds of pX11 and wild-type (WT) tobacco plants were sown in petri dishes containing intact Murashige-Skoog (MS) medium (4.4 g / L) and 1% agar. For stress condition determination, the MS medium additionally contained 150 mM NaCl or 400 mM mannitol. Seeds were sown in three petri dishes, with 50 seeds per genotype. The sealed plates were kept in the dark for one week and then transferred to a climate chamber (22°C; 70% humidity; light / dark 16 / 8 hours, 2500 Lux light intensity). Seed germination rate was assessed over 26 days.

[0649] Plant materials and growing conditions

[0650] Four o'clocks, tobacco, potatoes, melons, and tomatoes were grown in soil in a greenhouse with long-day conditions (25°C). Beetroot and Nicotiana benthamiana plants were grown in soil in a climate chamber (22°C; 70% humidity; 18 / 6 hours light / dark).

[0651] Example 11. pX11 expression in tomatoes, potatoes, and eggplants leads to red pigmentation in plants.

[0652] To examine the feasibility of heterologous betaine production in food crops, the pX11 vector was introduced into tomato (Solanum lycopersicum v. MicroTom), potato (Solanum tuberosum v. Désirée), and eggplant (Solanum melongena line DR2) via Agrobacterium-mediated stable transformation. Signs of reddish-purple pigment deposition were observed in the transformed explants of all three species within days of co-culturing with Agrobacterium. As previously observed with tobacco plants expressing pX11, the transformed explants eventually evolved into fully red-pigmented plants. Tomato and eggplant fruits, as well as potato tubers, exhibited intense reddish-purple pigmentation (Fig. 14). LC-MS analysis of tomato fruits, eggplant fruits, and potato tubers confirmed the presence of betaine, with betaine glycosides and isobetaine glycosides identified as the predominant betaine pigments (Fig. 14).

[0653] Example 12. Fruit-specific accumulation of betaine in tomato plants

[0654] In all plant species expressing pX11, beet pigment accumulation was observed in almost all plant tissues and organs, attributed to constitutive expression promoter sequences used for gene expression (i.e., CaMV 35S of the BvDODA1 and CYP76AD1 genes and the Arabidopsis ubiquitin-10 promoter of cDOPA5GT). We initially hypothesized that constitutive production of nitrogenous compounds could lead to a significant metabolic burden in transgenic plants. Therefore, we constructed a binary vector for expression in tomato that would result in pigment accumulation restricted to mid- and mature fruits. For this purpose, pX11 was modified to have a CYP76AD1 promoter driven by a fruit-specific E8 promoter instead of CaMV 35S. Figure 15 Transformation of the pX11 (E8) vector into tomato variety M-82 did indeed result in plants with betaine pigmentation limited to mature fruit (Fig. 16), although the betaine concentration was lower than that of MicroTom tomatoes expressing pX11. Notably, tomato, potato, and eggplant plants expressing pX11 ultimately did not exhibit any obvious developmental phenotypes or growth retardation.

[0655] Example 13. Flower-specific accumulation of betaine in petunias

[0656] Using a similar approach, another form of the pX11 vector was constructed, in which CYP76AD1 was driven by the flower-specific petunia chalcon synthase (CHS) promoter instead of CaMV 35S. Figure 15 It is expected that the conversion of pX11 (CHS) into petunia plants will lead to the production of plants that accumulate betaine pigment only in the petals during flower maturation.

[0657] Example 14. Expression of pX11, pX12, and pX13 in tobacco leads to flowers of different colors.

[0658] Due to the significant accumulation of betalains relative to betalains, plants expressing pX11 exhibit a predominantly reddish-purple hue. pX11 integrates the expression of CYP76AD1, which encodes an enzyme with dual activity in betalain biosynthesis, catalyzing the hydroxylation of tyrosine to L-DOPA and the conversion of L-DOPA to cyclic DOPA. As described above and in Polturak et al. 2016 (New Phytol. 2016 Apr; 210(1): 269-83, the entire contents of which are incorporated herein by reference), the associated enzyme CYP76AD6 in red beet uniquely exhibits tyrosine 3-hydroxylation activity to form L-DOPA. Since the cyclic DOPA derivative is essential for the formation of betalains, the intraplantational expression of CYP76AD6 instead of CYP76AD1 results in the formation of betalains rather than betalains, as previously observed with transient expression in Nicotiana benthamiana. To explore the possibility of generating transgenic plants that accumulate only beta-carotene-type betaine, a binary vector, pX13, was constructed to express BvDODA1 and CYP76AD6. Another vector, pX12, was designed to express the cytochrome P450 genes CYP76AD1 and CYP76AD6, as well as BvDODA1 and cDOPA5GT. Figure 15 Next, pX12 and pX13 were introduced into tobacco through stable transformation. While tobacco plants expressing pX13 produced only betalains, resulting in yellow-colored flowers, pX12 expression produced plants with orange-pink flowers (Fig. 17). LC-MS analysis of petals from pX11, pX12, and pX13 plants confirmed that the different colors observed in the flowers of the three strains were a result of different betalain / betalain ratios; pX11 flower extracts mainly contained betalains, pX13 extracts contained only betalains, while pX12 extracts contained both betalain pigments, with a lower betalain / betalain ratio than pX11 (Fig. 17). The difference in betalain accumulation relative to betalain accumulation was also observed by blue light imaging, with betalain exhibiting typical strong fluorescence (Gandia-Herrero et al., 2005). Therefore, changes toward the biosynthesis of red-purple betalains or yellow betalains can be manipulated by expressing CYP76AD1, CYP76AD6, or a combination of both.

[0659] Example 15. Expression of pX11 and pX13 in BY2 cells

[0660] The biotechnological production of beet pigments could provide a new viable source for their use as natural colorants in the food, pharmaceutical, and cosmetic industries. To date, most research has focused primarily on the cultivation of beet hairy root cultures or cell cultures for beet pigment production.

[0661] Genetic engineering for heterologous beet pigment production can develop many new sources of these pigments. One feasible source could be the culture of plant cell lines under investigation, such as the tobacco BY2 or Arabidopsis T87 lines. Previous attempts at beet pigment production using these lines have involved the expression of the MjDOD gene from Mirabilis jalapa and the tyrosinase gene from Lentinus edodes. Betalain production was demonstrated in both BY2 and T87 cells. However, within weeks, the cultured cells turned brown and became maldeveloped, attributable to the activity of the exogenous tyrosinase, most likely due to the toxic accumulation of dopaquinone and its derivatives (Nakatsuka et al., 2013).

[0662] To explore the potential applications of plant cell cultures producing betaine through the expression of only betaine-related genes, we expressed the pX11 and pX13 vectors in the tobacco cell line BY2, resulting in reddish-purple or yellow cells, respectively (Fig. 18). LC-MS analysis of cell extracts from cells expressing pX11 showed that betaine and isobetaine were the major betaine pigments. Several betaine flavonoids, including glutamine-betaine flavonoids and alanine-betaine flavonoids, were identified in both the pX11 and pX13 cell lines (Fig. 18). Notably, the pX11 and pX13 cell lines did not exhibit signs of browning or dysplasia in callus tissue expressing tyrosinase, as previously reported (Nakatsuka et al., 2013).

[0663] Example 16. L-DOPA production in tobacco and BY2 cells by expressing CYP76AD6

[0664] L-DOPA is an economically important compound, used in the treatment of Parkinson's disease and a precursor to high-value metabolites, including catecholamines, benzisoquinoline alkaloids, and betaine. Current methods for industrial-scale L-DOPA production exhibit key limitations. Compared to current methods, methods utilizing the tyrosine 3-hydroxylase activity of the CYP76AD6 enzyme offer significant advantages, as current methods directly generate L-DOPA from tyrosine. We investigated L-DOPA production via CYP76AD6 expression in tobacco plants and the tobacco cell line BY2. To this end, four different vectors expressing CYP76AD6 were constructed (…). Figure 19In one vector, CYP76AD6 is driven by the CaMV 35S promoter (hereinafter referred to as pDOPA1). In another vector, pDOPA2, CYP76AD6 is driven by the tomato ubiquitin 10 promoter (SlUb10). In vector pDOPA3, CYP76AD6 is expressed under the 35S promoter along with two other genes, AroG175 (Tzin et al., 2012) and aromatic amino acid hydroxylase (AAH) (Pribat et al., 2010), both of which are used to increase tyrosine availability. In vector pDOPA4, CYP76AD6 is expressed under the SlUb10 promoter along with AroG175 and AAH. All four vectors additionally express the neomycin phosphotransferase II (nptII) gene to confer selective kanamycin resistance in transformants. pDOPA1, pDOPA2, pDOPA3, and pDOPA4 were introduced into tobacco plants via Agrobacterium-mediated stable transformation. LC-MS analysis of first-generation (t0) plants confirmed the production of L-DOPA in lines expressing four different vectors. Analysis of pDOPA1 and pDOPA3 tobacco plants is shown in Figure 20. The four pDOPA vectors were also introduced into the BY2 cell line. BY2 cells expressing pDOPA2 or pDOPA4 showed varying degrees of gray to melanin deposition after several weeks of culture, likely due to the formation of L-DOPA derivatives, possibly caused by enzymatic or non-enzymatic oxidation of L-DOPA. LC-MS analysis of extracts obtained from callus expressing pDOPA2 and pDOPA4 showed the presence of different concentrations of L-DOPA (Figure 20).

[0665] Example 17. Beetroot pigments confer resistance to high osmotic and high salinity stress in plants.

[0666] Given the demonstrated role of betaine in conferring resistance to various abiotic stressors such as drought, high salinity, and excessive light in plants, the production of betaine in naturally non-producing plants could be used to increase their tolerance to abiotic stressors. Therefore, we proceeded to test this hypothesis by conducting seed germination assays on betaine-producing tobacco plants. Seeds of pX11 and wild-type (WT) tobacco were placed in Petri dishes containing Murashige-Skoog (MS) medium supplemented with 400 mM mannitol or 150 mM NaCl to simulate high osmotic stress or high salinity stress, respectively. Petri dishes containing MS medium without additional supplements served as controls. Germination rates of seeds sown on all plates were determined over a 26-day period. Throughout the experimental period, while pX11 and WT tobacco seeds showed similar germination rates in the control MS medium, pX11 seeds showed a significantly higher germination rate compared to WT seeds in plates containing 400 mM mannitol. In plates containing 150 mM NaCl, pX11 seeds showed a significantly higher germination rate after one week of incubation, but WT seeds achieved similar germination rates within two weeks and thereafter (Fig. 21). Therefore, compared with wild-type plants, betaine-producing tobacco plants exhibit increased tolerance to high osmotic and high salinity conditions, with higher germination rates under high osmotic stress and faster germination rates under high salinity conditions.

[0667] Example 18. Beetroot pigment confers resistance to botrytis cinerea infection in tobacco leaves.

[0668] Previously, betaine has been proposed to play a role in defense against pathogenic fungi, but evidence of its antifungal activity in the scientific literature is scarce. The production of heterologous betaine in plants provides an excellent platform for studying the antifungal activity of betaine within plants and can also be used to confer resistance against plant pathogenic fungi in target crop species. To investigate the potential antifungal effects of betaine, wild-type and pX11 tobacco plants were foliar infected with *Botrytiscinerea*, a lethal plant pathogen that causes significant crop losses both pre- and post-harvest. Droplets of different concentrations of *Botrytiscinerea* spore suspensions were applied to plant leaves, totaling approximately 100, 250, or 500 spores per plant. The extent of *Botrytiscinerea* infection was then assessed by measuring the size of lesions around the infection site daily. Compared to wild-type tobacco leaves, pX11 tobacco leaves exhibited significantly smaller lesion areas after infection (Figure 22). Within days of infection, infected wild-type tobacco leaves also showed signs of necrosis compared to pX11 leaves (Fig. 22). In summary, these results indicate that plants producing betaine have increased resistance to Botrytis cinerea infection.

[0669] Example 19. CYP76AD15 is a functional ortholog of CYP76AD6 in Mirabilis jalapa.

[0670] Given the natural existence of other plant species producing betaine pigments, consisting of red or yellow varieties, it seems reasonable that these species possess molecular mechanisms similar to those observed in red beets, where one cytochrome P450 enzyme catalyzes only tyrosine hydroxylation, while another catalyzes both tyrosine hydroxylation and cyclic DOPA formation. Functional orthologs of CYP76AD1 and CYP76AD6 may also exist in CYP76AD1- It was found in the CYP76AD1-β subclade. However, functional analysis of the corresponding genes in other species that produce betaine is needed to support this hypothesis.

[0671] Functional orthologs of CYP76AD1 have previously been identified in several plants, including Mirabilis jalapa, where CYP76AD3 was identified based on sequence similarity to CYP76AD1 (Hatlestad et al., 2012). However, the ortholog of CYP76AD6 has not been previously described. To assess the presence of CYP76AD6 orthologs in Mirabilis jalapa, a tBLASTx query was performed using the CYP76AD1 nucleotide sequence in a previously obtained Mirabilis jalapa transcriptome dataset (Polturak et al., 2016). This query led to the identification of several relevant genes, including one with the highest sequence similarity to CYP76AD6, hereinafter referred to as CYP76AD15. Interestingly, based on data obtained from Mirabilis jalapa transcriptome analysis, CYP76AD15 did not exhibit a parallel betaine accumulation expression pattern. Instead, the usual constitutive expression pattern of this gene was observed. CYP76AD15 activity was tested in Nicotiana benthamiana by Agrobacterium infiltration assay. Similar to CYP76AD6, CYP76AD15, when co-infiltrated with BvDODA1, allowed the production of betaine in *Nicotiana benthamiana* leaves, while when infiltrated alone, it produced L-DOPA (Figure 23). LC-MS analysis confirmed the presence of several betaine compounds, including dopamine-betaine and valine-betaine, identified based on the usual absorption pattern and MS / MS fragments. Therefore, CYP76AD15 was identified as a functional ortholog of CYP76AD6, catalyzing the formation of L-DOPA from tyrosine. Sequence analysis revealed that CYP76AD15, along with CYP76AD6, belongs to the CYP76AD1-β subclade, while CYP76AD3 and CYP76AD1 belong to the CYP76AD1-α clade, thus confirming the association between each subclade and specific catalytic activity.

[0672] While certain features of the invention have been described and illustrated herein, many modifications, substitutions, alterations, and equivalents will now occur to those skilled in the art. Therefore, it should be understood that the appended claims are intended to cover all such modifications and variations that fall within the true spirit of the invention.

Claims

1. A method of producing L-DOPA from tyrosine, comprising the steps of: Under conditions sufficient to produce L-DOPA, CYP76AD6, consisting of the amino acid sequence of SEQ ID NO: 18, is combined with tyrosine to produce L-DOPA.

2. The method of claim 1, wherein, The method is performed in vitro.

3. A method of producing a natural yellow dye comprising the steps of: Under conditions sufficient to produce betaine, cells are contacted with a. a nucleic acid encoding CYP76AD6, wherein the nucleic acid sequence encoding said CYP76AD6 consists of SEQ ID NO: 31, and b. a nucleic acid encoding DOPA 4,5-dioxygenase (DOD), thereby producing a natural yellow dye.

4. A method of producing a natural orange dye comprising the steps of: Under conditions sufficient to produce a combination of betaine and betaine xanthocyanin, cells are contacted with a nucleic acid encoding a. CYP76AD1, wherein the nucleic acid sequence encoding said CYP76AD1 consists of SEQ ID NO: 32, b. a nucleic acid encoding DOPA 4,5-dioxygenase (DOD), c. a nucleic acid encoding CYP76AD6, wherein the nucleic acid sequence encoding said CYP76AD6 consists of SEQ ID NO: 31, and d. a nucleic acid encoding a betaine-associated glucosyltransferase, thereby producing a natural orange dye.

5. A method for producing catecholamines, benzylisoquinoline alkaloids, betaine, melanin, or combinations thereof, wherein the method includes preparing an L-DOPA precursor, the preparation comprising contacting cells with a nucleic acid encoding CYP76AD6, wherein the nucleic acid sequence encoding said CYP76AD6 comprises SEQ ID NO:

31.

6. The method according to any one of claims 3 to 4, wherein the dye is a textile dye.

7. The method according to any one of claims 3 to 4, wherein the dye is an edible pigment.

8. The method according to any one of claims 1-5, wherein the method is carried out in bacterial or yeast cells.

9. The method according to any one of claims 3-5, wherein the cell is a plant cell that does not produce the dye.

10. The method according to any one of claims 3-5, wherein the cell is a bacterial or yeast cell.

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