Mogroside compositions and methods of producing same

Abstract

The present disclosure provides compositions and methods for producing transgenic plants and other organisms that exhibit improved production of mogroside compounds, particularly mogroside V, as well as mogroside compounds, plants, and plant parts produced thereby.

Description

[0001] Cross - Reference to Related Applications

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 376,194, filed on September 19, 2022, and U.S. Provisional Application No. 63 / 491,721, filed on March 22, 2023, under 35 U.S.C.§119(e), the entire contents of each of which are incorporated herein by reference in their entirety.

[0003] Incorporation of Sequence Listing

[0004] The sequence listing contains a file named "ELSS002WO_ST26.xml", which is 540 kilobytes (measured in MS - ), created on September 14, 2023, and contains 305 sequences, the entire contents of which are incorporated herein by reference in their entirety. Technical Field

[0005] This disclosure relates to the field of genetically engineered plants and other organisms, and more particularly to methods and compositions for producing plants and other organisms that exhibit increased yields of mogroside compounds, particularly mogroside V. This disclosure also relates to the use of such plants and other organisms in the production of new ingredients for food and beverages (such as plant extracts containing mogrosides, purified and partially purified components), and the resulting new foods and beverages (as well as other material compositions). Background Art

[0006] In addition to other industries, low - calorie or calorie - free sweeteners, particularly natural low - calorie or calorie - free sweeteners, are becoming increasingly important for the food and beverage industry as alternatives to traditional high - calorie sweeteners and artificial sweeteners. These alternative sweeteners are used as substitutes for artificial sweeteners or high - calorie sweeteners containing sucrose, fructose, and glucose. Like some artificial sweeteners, some of these alternative sweeteners provide a higher sweetening effect than an equivalent amount of caloric sweeteners, and thus require a smaller amount of these alternative sweeteners to achieve a sweetness equivalent to sugar. However, some low - calorie sweeteners may be expensive to produce and / or have adverse flavor characteristics and / or off - flavors, including but not limited to lingering sweetness, delayed onset of sweetness, negative mouthfeel and bitterness, metallic taste, cooling taste, astringency, and licorice - like taste.

[0007] Some natural plants produce low-calorie or calorie-free sweeteners. For example, mogrosides, an important class of natural sweeteners, are chemically a class of triterpene glycosides or mogrol glycosides naturally produced by Siraitia grosvenorii (also known as luohan guo; scientific name: Siraitia grosvenorii). Mogrosides contain "zero" calories (less than 5 calories per 8-ounce serving) and are 100 - 400 times sweeter than sucrose. Mogrosides have also been reported to have a variety of important pharmacological effects. However, although plants like Siraitia grosvenorii produce mogrosides, the production of mogrosides from these plants is limited and expensive due to their limited natural or agricultural production. In addition, Siraitia grosvenorii prefers to grow in subtropical mountainous areas and requires laborious pollination to bear fruit. Furthermore, attempts have been made to produce mogrosides in vitro or in microorganisms, but due to extensive processing and other problems, it has not been proven economically viable.

[0008] Accordingly, there is a need for new compositions and methods for the efficient production of mogrosides. Summary of the Invention

[0010] The present disclosure addresses these and other problems in the art by providing new compositions and methods for the efficient and cost-effective production of mogrosides, particularly sweet mogrosides, such as those containing more than three glucose residues in the molecule, including but not limited to mogroside V, isomogroside V, semenoside I, α-semenoside I, mogroside IV, mogroside IVA, mogroside III, mogroside IIIE, mogroside IIIA1, and 11-oxo-mogroside V, in plants and other organisms.

[0011] The present disclosure provides a transgenic plant, plant part or seed comprising: a) a first polynucleotide sequence encoding a cytochrome P450 polypeptide having at least 91% sequence identity with SEQ ID NO:2 or at least 95% sequence identity with SEQ ID NO:86; b) a second polynucleotide sequence encoding a cucurbitadienol synthase polypeptide having at least 90% sequence identity with SEQ ID NO:5; c) a third polynucleotide sequence encoding a cytochrome P450 polypeptide having at least 90% sequence identity with SEQ ID NO:7, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31 or SEQ ID NO:33; d) a fourth polynucleotide sequence encoding a uridine phosphorylase-dependent glycosyltransferase polypeptide having at least 90% sequence identity with SEQ ID NO:9; e) a fifth polynucleotide sequence encoding a uridine phosphorylase-dependent glycosyltransferase polypeptide having at least 90% sequence identity with SEQ ID NO:11; f) a sixth polynucleotide sequence encoding a squalene epoxidase polypeptide having at least 90% sequence identity with SEQ ID NO:13; g) a seventh polynucleotide sequence encoding an epoxide hydrolase polypeptide having at least 90% sequence identity with SEQ ID NO:15; or h) an eighth polynucleotide sequence encoding a truncated 3-hydroxy-3-methylglutaryl-CoA reductase polypeptide having at least 90% sequence identity with SEQ ID NO:17, SEQ ID NO:274, SEQ ID NO:276 or SEQ ID NO:278; wherein the first, second, third, fourth, fifth, sixth, seventh or eighth polynucleotide sequence is operably linked to a heterologous promoter, and wherein the transgenic plant, plant part or seed produces at least a first mogroside compound. In some embodiments, the first, second, third, fourth, fifth, sixth, seventh or eighth polynucleotide sequence is operably linked to a different heterologous promoter. In some embodiments, at least two of the first, second, third, fourth, fifth, sixth, seventh or eighth polynucleotide sequences are operably linked to a single heterologous promoter. In other embodiments, one or more of the first, second, third, fourth, fifth, sixth, seventh or eighth polynucleotide sequences are present in multiple copies and are operably linked to the same or different endogenous or heterologous promoters.

[0012] In various embodiments, the plant is a plant of the Cucurbitaceae, Solanaceae or Compositae. In some embodiments, the plant is a plant of the genus Cucurbita, Citrullus, Cucumis, Momordica, Solanum or Lettuce. In further embodiments, the plant is a watermelon, cantaloupe, honeydew melon, winter melon, Casaba melon, Persian melon, citron melon, musk melon, honeydew melon, crenshaw melon, Christmas melon, elf melon, caravelle melon, hamimelon, rock melon, golden Langkawi melon, Korean melon, saticoy melon, Galia melon, yulu melon, gold medal melon, ten melon, new century melon, banana melon, yubari king melon, sugar melon, tiger melon, climbing melon, horned melon, cucamelon, casabanana melon, melon), ginseng fruit, pineapple melon, camouflage melon, golden sponge melon, bitter melon, charentaismelon, crane melon, arrow melon, honey melon, rambutan melon, autumn melon, quick melon, lettuce, spinach, rice, oats, corn, sorghum, bitter apple (Citrullus colocynthis), pumpkin, beet, tobacco, miscanthus, tomato, cucumber, potato, amaranth or Nicotiana benthamiana plants. In specific embodiments, the plant is a watermelon, tomato, lettuce or cucumber plant, or other leafy large biomass green plants or other food crop plants that can be used as the base ingredient of processed foods. In certain embodiments, the plant is a monocot or a dicot. In some embodiments, the plant part is a fruit, leaf, root, flower, branch, cell, endosperm, ovule or pollen. In other embodiments, the plant is a species that can be used to produce mogroside for processing into food, wherein mogroside can replace or partially replace the need for added sugar.

[0013] In certain embodiments, the heterologous promoter is an inducible, plant, bacterial, viral, synthetic, constitutive, tissue-specific, developmentally regulated, cell cycle-regulated, temporally regulated, spatially regulated, and / or spatio-temporally regulated promoter. In other embodiments, the heterologous promoter is FSgt / PFLt (SEQ ID NO:62), FMVSgt (SEQ ID NO:69), CsVMV (SEQ ID NO:68), dMMV (SEQ ID NO:63), HLVH12 (SEQ ID NO:60), NOS (SEQ ID NO:66), ScBV (SEQ ID NO:67), DCMV (SEQ ID NO:61), CmYLCV (SEQ ID NO:64), FS1_1 (SEQ ID NO:70), FE_3 (SEQ ID NO:71), e35S (SEQ ID NO:65), AtUBQ10 (SEQ ID NO:259), PCLSV (SEQ ID NO:260), FS4 (SEQ ID NO:261), AtACT2 (SEQ ID NO:262), enhanced AtEf-1A (SEQ ID NO:263), FuasFScp (SEQ ID NO:264), FE4 (SEQ ID NO:269), cucumisin (SEQ ID NO:270), or SgCDS (SEQ ID NO:271) promoter. In further embodiments, the first, second, third, fourth, fifth, sixth, seventh, or eighth polynucleotide sequence is operably linked to a heterologous terminator. In still further embodiments, the first, second, third, fourth, fifth, sixth, seventh, or eighth heterologous terminator sequence is GmaxMYB2 (SEQ ID NO:74), 35S_T (SEQ ID NO:), ATHSP18.2 (SEQ ID NO:77), AtRBCS2b (SEQ ID NO:75), AtUBQ3 (SEQ ID NO:73), pea E9 (SEQ ID NO:76), pea 3A (SEQ ID NO:72), potato Ubi3 (SEQ ID NO:78), AtTubB9 (SEQ ID NO:79), AtFAD2 (SEQ ID NO:265), AtNDUFA8 (SEQ ID NO:266), CsHSP17.3 (SEQ ID NO:267), or CsHSP22 (SEQ ID NO:268) terminator sequence. In additional embodiments, the transgenic plant, plant part, or seed further comprises a selectable marker sequence. In some embodiments, the selectable marker sequence is a β-glucuronidase, green fluorescent protein, or antibiotic resistance sequence.In certain embodiments, the selectable marker sequence is a hygromycin B phosphotransferase (HygR) or neomycin phosphotransferase II (nptII) selectable marker sequence.

[0014] In some embodiments, the transgenic plant, plant part or cell further comprises i) a ninth polynucleotide sequence encoding a NADPH:cytochrome P450 reductase polypeptide having at least 90% sequence identity to SEQ ID NO:19; or j) a tenth polynucleotide sequence encoding a uridine phosphorylase-dependent glycosyltransferase polypeptide having at least 90% sequence identity to SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:120 or SEQ ID NO:126; k) an eleventh polynucleotide sequence encoding a 3-hydroxy-3-methylglutaryl-CoA synthase polypeptide having at least 90% sequence identity to SEQ ID NO:227; or l) a twelfth polynucleotide sequence encoding a geranyl diphosphate synthase polypeptide having at least 90% sequence identity to SEQ ID NO:256; wherein the ninth, tenth, eleventh or twelfth polynucleotide sequence is operably linked to a heterologous promoter. In other embodiments, the transgenic plant, plant part or cell further comprises a 2A linker, insulator, selectable marker or filler sequence.

[0015] In certain embodiments, at least the first mogroside compound is a non-natural mogrol precursor, mogrol, mogroside, or a metabolite or derivative thereof. In various embodiments, the mogroside is mogroside II A, mogroside IIA1, mogroside II A2, mogroside II E, 11-oxo-mogroside II, mogroside III, mogroside III A1, mogroside III A2, mogroside III E, 11-oxo-mogroside III, mogroside IV, mogroside IV A, 11-oxo-mogroside IV, esmogroside I, mogroside V, 11-oxo-mogroside V, or mogroside VI, or an isomer thereof. In additional embodiments, the transgenic plant, plant part, or seed produces at least 10 ng / g to 30 mg / g dry weight of at least the first mogroside compound. In some embodiments, the transgenic plant, plant part, or seed produces at least 10 ng / g, at least 25 ng / g, at least 50 ng / g, at least 75 ng / g, at least 100 ng / g, at least 250 ng / g, at least 500 ng / g, at least 750 ng / g, at least 1 mg / g, at least 2.5 mg / g, at least 5 mg / g, at least 7.5 mg / g, at least 10 mg / g, at least 12.5 mg / g, at least 15 mg / g, at least 17.5 mg / g, at least 20 mg / g, at least 22.5 mg / g, at least 25 mg / g, at least 27.5 mg / g, or at least 30 mg / g of at least the first mogroside compound. In further embodiments, the amount of at least the first mogroside compound is higher than the level in non-transgenic plants, plant parts, or seeds of the same species. In other embodiments, the proportion of individual mogroside compounds is different from the proportion of mogroside compounds in natural plant tissues or fruits or other plants or organisms that produce mogroside compounds.For example, the ratio of mogroside compounds produced by the plants or organisms of the present disclosure to mogroside compounds produced by natural plant tissues or fruits or other plants or organisms can be 1.1:1, 1.2:1, 1.3:1, 1.4:1, 1.5:1, 1.6:1, 1.7:1, 1.8:1, 1.9:1, 2:1, 2.5:1, 3:1, 4:1, 5:1, 6:1, 7:1, 7.5:1, 8:1, 9:1, 10:1, 12.5:1, 15:1, 20:1, 25:1, 30:1, 40:1, 50:1, 60:1, 70:1, 75:1, 80:1, 90:1, 100:1, 250:1, 500:1, 750:1, 1000:1 or more, or 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2, 1:2.5, 1:3, 1:4, 1:5, 1:6, 1:7, 1:7.5, 1:8, 1:9, 1:10, 1:12.5, 1:15, 1:20, 1:25, 1:30, 1:40, 1:50, 1:60, 1:70, 1:75, 1:80, 1:90, 1:100, 1:250, 1:500, 1:750, 1:1000 or lower. These ratios can apply to one or more of the following mogroside compounds: mogroside II A, mogroside II A1, mogroside II A2, mogroside II E, 11-oxo-mogroside II, mogroside III, mogroside III A1, mogroside III A2, mogroside III E, 11-oxo-mogroside III, mogroside IV, mogroside IV A, 11-oxo-mogroside IV, semenicoside I, mogroside V, 11-oxo-mogroside V or mogroside VI, or their isomers. Generally, a higher ratio of sweet mogroside compounds (such as those containing more than three glucose residues in the molecule, including but not limited to mogroside V, isomogroside V, semenicoside I, α-semenicoside I, mogroside IV, mogroside IV A, mogroside III, mogroside III E, mogroside III A1 and 11-oxo-mogroside V) to other mogroside compounds is used. In other words, this applies to the ratio of one or more mogroside compounds (such as mogroside V) to one or more other mogroside compounds.

[0016] The present disclosure also provides a recombinant host cell comprising: a) a first polynucleotide sequence encoding a cytochrome P450 polypeptide having at least 91% sequence identity with SEQ ID NO: 2 or at least 95% sequence identity with SEQ ID NO: 86; b) a second polynucleotide sequence encoding a cucurbitadienol synthase polypeptide having at least 90% sequence identity with SEQ ID NO: 5; c) a third polynucleotide sequence encoding a cytochrome P450 polypeptide having at least 90% sequence identity with SEQ ID NO: 7, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, or SEQ ID NO: 33; d) a fourth polynucleotide sequence encoding a uridine phosphorylase-dependent glycosyltransferase polypeptide having at least 90% sequence identity with SEQ ID NO: 9; e) a fifth polynucleotide sequence encoding a uridine phosphorylase-dependent glycosyltransferase polypeptide having at least 90% sequence identity with SEQ ID NO: 11; f) a sixth polynucleotide sequence encoding a squalene epoxidase polypeptide having at least 90% sequence identity with SEQ ID NO: 13; g) a seventh polynucleotide sequence encoding an epoxide hydrolase polypeptide having at least 90% sequence identity with SEQ ID NO: 15; or h) an eighth polynucleotide sequence encoding a truncated 3-hydroxy-3-methylglutaryl-CoA reductase polypeptide having at least 90% sequence identity with SEQ ID NO: 17, SEQ ID NO: 274, SEQ ID NO: 276, or SEQ ID NO: 278; wherein the first, second, third, fourth, fifth, sixth, seventh, or eighth polynucleotide sequence is operably linked to a heterologous promoter. In certain embodiments, the recombinant host cell produces at least a first mogroside compound.

[0017] The present disclosure also provides a processed low-calorie food or beverage product produced from a genetically modified plant, plant part, or seed, wherein the genetically modified plant, plant part, or seed comprises: a) a first polynucleotide sequence encoding a cytochrome P450 polypeptide having at least 91% sequence identity with SEQ ID NO: 2 or at least 95% sequence identity with SEQ ID NO: 86; b) a second polynucleotide sequence encoding a cucurbitadienol synthase polypeptide having at least 90% sequence identity with SEQ ID NO: 5; c) a third polynucleotide sequence encoding a cytochrome P450 polypeptide having at least 90% sequence identity with SEQ ID NO: 7, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, or SEQ ID NO: 33; d) a fourth polynucleotide sequence encoding a uridine phosphorylase-dependent glycosyltransferase polypeptide having at least 90% sequence identity with SEQ ID NO: 9; e) a fifth polynucleotide sequence encoding a uridine phosphorylase-dependent glycosyltransferase polypeptide having at least 90% sequence identity with SEQ ID NO: 11; f) a sixth polynucleotide sequence encoding a squalene epoxidase polypeptide having at least 90% sequence identity with SEQ ID NO: 13; g) a seventh polynucleotide sequence encoding an epoxide hydrolase polypeptide having at least 90% sequence identity with SEQ ID NO: 15; or h) an eighth polynucleotide sequence encoding a truncated 3-hydroxy-3-methylglutaryl-CoA reductase polypeptide having at least 90% sequence identity with SEQ ID NO: 17, SEQ ID NO: 274, SEQ ID NO: 276, or SEQ ID NO: 278; wherein the first, second, third, fourth, fifth, sixth, seventh, or eighth polynucleotide sequence is operably linked to a heterologous promoter, and wherein the genetically modified plant, plant part, or seed produces at least a first mogroside compound. In certain embodiments, the product is produced from the juice, extract, powder, fruit, or peel of the fruit, vegetable, legume, tuber, or grain of the genetically modified plant, plant part, or seed.

[0018] The present disclosure further provides a juice, powder or extract produced from a genetically modified plant, plant part or seed, the genetically modified plant, plant part or seed comprising: a) a first polynucleotide sequence encoding a cytochrome P450 polypeptide having at least 91% sequence identity with SEQ ID NO:2 or at least 95% sequence identity with SEQ ID NO:86; b) a second polynucleotide sequence encoding a cucurbitadienol synthase polypeptide having at least 90% sequence identity with SEQ ID NO:5; c) a third polynucleotide sequence encoding a cytochrome P450 polypeptide having at least 90% sequence identity with SEQ ID NO:7, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31 or SEQ ID NO:33; d) a fourth polynucleotide sequence encoding a uridine phosphorylase-dependent glycosyltransferase polypeptide having at least 90% sequence identity with SEQ ID NO:9; e) a fifth polynucleotide sequence encoding a uridine phosphorylase-dependent glycosyltransferase polypeptide having at least 90% sequence identity with SEQ ID NO:11; f) a sixth polynucleotide sequence encoding a squalene epoxidase polypeptide having at least 90% sequence identity with SEQ ID NO:13; g) a seventh polynucleotide sequence encoding an epoxide hydrolase polypeptide having at least 90% sequence identity with SEQ ID NO:15; or h) an eighth polynucleotide sequence encoding a truncated 3-hydroxy-3-methylglutaryl-CoA reductase polypeptide having at least 90% sequence identity with SEQ ID NO:17, SEQ ID NO:274, SEQ ID NO:276 or SEQ ID NO:278; wherein the first, second, third, fourth, fifth, sixth, seventh or eighth polynucleotide sequence is operably linked to a heterologous promoter, and wherein the genetically modified plant, plant part or seed produces at least a first mogroside compound.

[0019] The present disclosure also provides recombinant DNA molecules, comprising: a) a first polynucleotide sequence encoding a cytochrome P450 polypeptide having at least 91% sequence identity with SEQ ID NO:2 or at least 95% sequence identity with SEQ ID NO:86; b) a second polynucleotide sequence encoding a cucurbitadienol synthase polypeptide having at least 90% sequence identity with SEQ ID NO:5; c) a third polynucleotide sequence encoding a cytochrome P450 polypeptide having at least 90% sequence identity with SEQ ID NO:7, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31 or SEQ ID NO:33; d) a fourth polynucleotide sequence encoding a uridine phosphorylase-dependent glycosyltransferase polypeptide having at least 90% sequence identity with SEQ ID NO:9; e) a fifth polynucleotide sequence encoding a uridine phosphorylase-dependent glycosyltransferase polypeptide having at least 90% sequence identity with SEQ ID NO:11; f) a sixth polynucleotide sequence encoding a squalene epoxidase polypeptide having at least 90% sequence identity with SEQ ID NO:13; g) a seventh polynucleotide sequence encoding an epoxide hydrolase polypeptide having at least 90% sequence identity with SEQ ID NO:15; or h) an eighth polynucleotide sequence encoding a truncated 3-hydroxy-3-methylglutaryl-CoA reductase polypeptide having at least 90% sequence identity with SEQ ID NO:17, SEQ ID NO:274, SEQ ID NO:276 or SEQ ID NO:278, wherein the first, second, third, fourth, fifth, sixth, seventh or eighth polynucleotide sequence is operably linked to a heterologous promoter. In certain embodiments, the first, second, third, fourth, fifth, sixth, seventh or eighth polynucleotide sequence is operably linked to a different heterologous promoter. In other embodiments, at least two of the first, second, third, fourth, fifth, sixth, seventh or eighth polynucleotide sequences are operably linked to a single heterologous promoter. In some embodiments, one or more of the first, second, third, fourth, fifth, sixth, seventh or eighth polynucleotide sequences are present in multiple copies and are operably linked to the same or different endogenous or heterologous promoters.

[0020] The present disclosure also provides DNA molecules comprising polynucleotide sequences encoding cytochrome P450 polypeptides having at least 91% sequence identity to SEQ ID NO:2 or at least 95% sequence identity to SEQ ID NO:86. In certain embodiments, the DNA molecule is operably linked to a heterologous promoter. The present disclosure also provides DNA molecules exhibiting gene regulatory functional activity, comprising polynucleotide sequences selected from: a) sequences having at least 90% or 95% sequence identity to SEQ ID NO:70 or 71 and exhibiting promoter activity; b) sequences comprising SEQ ID NO:70 or 71; and c) fragments of SEQ ID NO:70 or 71, wherein the fragment exhibits promoter activity; wherein the DNA molecule is operably linked to a heterologous transcribable polynucleotide molecule. In certain embodiments, SEQ ID NO:70 and SEQ ID NO:71 result in increased expression in plant fruits.

[0021] The present disclosure also provides a method for producing at least a first mogroside compound, which comprises cultivating a transgenic plant or organism, the transgenic plant or organism comprising: a) a first polynucleotide sequence encoding a cytochrome P450 polypeptide having at least 91% sequence identity with SEQ ID NO: 2 or at least 95% sequence identity with SEQ ID NO: 86; b) a second polynucleotide sequence encoding a cucurbitadienol synthase polypeptide having at least 90% sequence identity with SEQ ID NO: 5; c) a third polynucleotide sequence encoding a cytochrome P450 polypeptide having at least 90% sequence identity with SEQ ID NO: 7, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31 or SEQ ID NO: 33; d) a fourth polynucleotide sequence encoding a uridine phosphorylase-dependent glycosyltransferase polypeptide having at least 90% sequence identity with SEQ ID NO: 9; e) a fifth polynucleotide sequence encoding a uridine phosphorylase-dependent glycosyltransferase polypeptide having at least 90% sequence identity with SEQ ID NO: 11; f) a sixth polynucleotide sequence encoding a squalene epoxidase polypeptide having at least 90% sequence identity with SEQ ID NO: 13; g) a seventh polynucleotide sequence encoding an epoxide hydrolase polypeptide having at least 90% sequence identity with SEQ ID NO: 15; or h) an eighth polynucleotide sequence encoding a truncated 3-hydroxy-3-methylglutaryl-CoA reductase polypeptide having at least 90% sequence identity with SEQ ID NO: 17, SEQ ID NO: 274, SEQ ID NO: 276 or SEQ ID NO: 278, wherein the first, second, third, fourth, fifth, sixth, seventh or eighth polynucleotide sequence is operably linked to a heterologous promoter, and wherein the transgenic plant, plant part or seed produces at least a first mogroside compound. In certain embodiments, the transgenic plant produces at least a first mogroside compound in a plant part or seed of the plant. In some embodiments, the transgenic plant produces at least a first mogroside compound in the fruit or leaf of the plant. In further embodiments, the method further comprises the step of isolating at least a first mogroside compound from the transgenic plant. In other embodiments, at least a first mogroside compound is isolated, purified or partially purified from a plant part or seed of the transgenic plant. In other embodiments, at least a first mogroside compound is isolated from the fruit, leaf, vegetable, bean, tuber or grain of the transgenic plant. In further embodiments, a combination of mogroside compounds is purified or partially purified from the transgenic plant, plant part or seed.

[0022] The present disclosure also provides a composition comprising: a) about 80% mogroside V, about 15% 11-oxo-mogroside V, and about 5% mogroside III-A1; or b) about 40% esgoside I, about 40% mogroside V, and about 20% 11-oxo-mogroside V. In certain embodiments, the composition comprises about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, or about 85% mogroside V, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, or about 20% 11-oxo-mogroside V, and about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, or about 10% mogroside III A1. In some embodiments, the composition comprises about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, or about 45% esgoside I, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, or about 45% mogroside V, and about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, or about 25% 11-oxo-mogroside V. In other embodiments, the composition is a liquid. In other embodiments, the composition is a dry powder. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] The following drawings form a part of this specification and are included to further illustrate certain aspects of the present disclosure. The present disclosure can be better understood by referring to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0024] Figure 1 . Biosynthetic pathway of mogrosides from Siraitia grosvenorii.

[0025] Figure 2 . Mogroside V produced by transient expression in watermelon fruits with various different expression constructs.

[0026] Figure 3 . Map of expression construct SP3139.

[0027] Figure 4 . Mogrosides and esgosides produced by transient expression in watermelon fruits using various constructs.

[0028] Figure 5 . Mogroside V produced by transient expression in watermelon fruits using various constructs.

[0029] Figure 6 . Siamenoside produced by transient expression in watermelon fruits using various constructs.

[0030] Figure 7 . Mogroside and siamenoside produced by transient expression in watermelon fruits using combinations of constructs SP2015, SP4332, SP5029, SP5030, SP5031, SP5032, and SP5033 (controls) and combinations excluding one of said constructs.

[0031] Figure 8 . Mogroside V produced by transient expression in watermelon fruits using combinations of constructs SP2015, SP4332, SP5029, SP5030, SP5031, SP5032, and SP5033 (controls) and combinations excluding one of said constructs.

[0032] Figure 9 . Mogroside V produced by transient expression in lettuce leaves infiltrated with construct SP1463, compared to a negative lettuce control and a mogroside standard mixture.

[0033] Figure 10 . Mass spectrometric fingerprinting of mogroside V produced by transient expression in lettuce leaves infiltrated with construct SP1463, compared to a negative lettuce control and a mogroside V reference standard.

[0034] Figure 11 . Map of expression construct SP1463.

[0035] Figure 12 . Mogroside and siamenoside produced by transient expression in lettuce leaves using various constructs.

[0036] Figure 13 . Mogroside V produced by transient expression in lettuce leaves using various constructs.

[0037] Figure 14 . Siamenoside produced by transient expression in lettuce leaves using various constructs.

[0038] Figure 15 . Mogroside and siamenoside produced by transient expression in lettuce leaves using combinations of constructs SP2015, SP4332, SP5029, SP5030, SP5031, SP5032, and SP5033 (controls) and combinations excluding one of said constructs.

[0039] Figure 16. Mogroside V produced by transient expression in lettuce leaves using combinations of constructs SP2015, SP4332, SP5029, SP5030, SP5031, SP5032, and SP5033 (controls) and combinations excluding one of the constructs.

[0040] Figure 17 . Production of mogroside V in transgenic watermelon using expression construct SP1463, compared to mogroside V reference standard.

[0041] Figure 18 . Mass spectrometry fingerprint analysis of mogroside V in transgenic watermelon using expression construct SP1463, compared to mogroside V reference standard.

[0042] Figure 19 . Production of mogrosides in various transgenic watermelon lines using expression constructs SP0336, SP1463, SP1908, SP3488, and SP3190.

[0043] Figure 20 . Map of expression construct SP0336.

[0044] Figure 21 . Map of expression construct SP1908.

[0045] Figure 22 . Production of various mogrosides and saponins in transgenic watermelon using expression constructs SP0565, SP0641, SP1463, SP1908, SP3015, SP3016, SP3029, SP3190, SP3308, and SP3488.

[0046] Figure 23 . Production of mogrosides and saponins in transgenic watermelon cell suspensions.

[0047] Figure 24 . Concentrations of mogrosides and saponins in transgenic watermelon cell suspensions.

[0048] Figure 25 . Production of various mogrosides and saponins in transgenic tomato using expression constructs SP0641, SP1463, SP1908, SP3016, SP3029, SP3190, and SP3684.

[0049] Figure 26 . Production of various mogrosides and saponins in transgenic potato using expression constructs SP0641, SP1463, SP3016, SP3029, SP3190, and SP3684.

[0050] Figure 27Mogroside V and an unknown mogroside V isomer are produced in transient watermelon fruits co-infiltrated with construct SP3684 and t72143 or green fluorescent protein (GFP).

[0051] Figure 28 Mass spectrometric fingerprint of mogroside V and an unknown mogroside V isomer.

[0052] Figure 29 Mogroside IV and an unknown mogroside IV isomer are produced in transient watermelon fruits co-infiltrated with construct SP3684 and t72143 or green fluorescent protein (GFP).

[0053] Figure 30 Mass spectrometric fingerprint of mogroside IV and an unknown mogroside IV isomer.

[0054] Figure 31 Mogroside III and an unknown mogroside III isomer are produced in transient watermelon fruits co-infiltrated with construct SP3684 and t72143 or green fluorescent protein (GFP).

[0055] Figure 32 Mass spectrometric fingerprint of mogroside III and an unknown mogroside III isomer.

[0056] Figure 33 Mogroside II and an unknown mogroside II isomer are produced in transient watermelon fruits co-infiltrated with construct SP3684 and t72143 or green fluorescent protein (GFP).

[0057] Figure 34 Mass spectrometric fingerprint of mogroside II and an unknown mogroside II isomer.

[0058] Figure 35 11-oxo-mogroside III and an unknown 11-oxo-mogroside III isomer are produced in transient watermelon fruits co-infiltrated with construct SP3684 and t72143 or green fluorescent protein (GFP).

[0059] Figure 36 Mass spectrometric fingerprinting of 11-oxo-mogroside III and an unknown 11-oxo-mogroside III isomer.

[0060] Figure 37 11-oxo-mogroside IV and an unknown 11-oxo-mogroside IV isomer are produced in transient watermelon fruits co-infiltrated with construct SP3684 and t72143 or green fluorescent protein (GFP).

[0061] Figure 38. Mass spectrometric fingerprint analysis of 11-oxo-mogroside IV and unknown 11-oxo-mogroside IV isomers.

[0062] Figure 39 . Production of 11-oxo-mogroside V and unknown 11-oxo-mogroside V isomers in transient watermelon fruits co-infiltrated with constructs SP3684 and t72143 or green fluorescent protein (GFP).

[0063] Figure 40 . Mass spectrometric fingerprint of 11-oxo-mogroside V and unknown 11-oxo-mogroside V isomers.

[0064] Figure 41 . Concentration-response curve of mogroside blend 80 / 15 / 5.

[0065] Figure 42 . Concentration-response curves of mogroside-V and mogroside blend 80 / 15 / 5.

[0066] Figure 43 . Time to onset and reach maximum sweetness of mogroside-V, mogroside blend 80 / 15 / 5, rebaudioside M, rebaudioside A, and monk fruit 50 (MF50).

[0067] Figure 44 . Sweetness decay profiles over time of mogroside-V, mogroside blend 80 / 15 / 5, rebaudioside M, rebaudioside A, and MF50.

[0068] Figure 45 . Descriptive analysis scores of the sweetener properties of mogroside-V, mogroside blend 80 / 15 / 5, rebaudioside M, rebaudioside A, and MF50.

[0069] Figure 46 . Descriptive analysis of the sweetener properties of mogroside-V, mogroside blend 80 / 15 / 5, rebaudioside M, rebaudioside A, and MF50.

[0070] Figure 47 . Concentration-response curve of V90.

[0071] Figure 48 . Concentration-response curves of mogroside V, blend 80 / 15 / 5, and V90.

[0072] Figure 49 . Time to onset and reach maximum sweetness of mogroside V and V90.

[0073] Figure 50 . Sweetness decay of mogroside V and V90.

[0074] Figure 51 . Descriptive analysis scores of mogroside V and V90.

[0075] Figure 52 . Concentration-response curve of 11-oxomogroside-V.

[0076] Figure 53 . Concentration-response curve of mogroside III A-1.

[0077] Figure 54 . Time to onset and reach maximum sweetness of mogroside V, 11-oxomogroside V, and mogroside III A-1.

[0078] Figure 55 . Decay of residual aftertaste of mogroside V, 11-oxomogroside V, and mogroside III A-1 over time.

[0079] Figure 56 . Descriptive analysis scores of sweetener properties of mogroside V, 11-oxomogroside V, and mogroside III A-1.

[0080] Figure 57 . Descriptive analysis of sweetener properties of mogroside V, 11-oxomogroside V, and mogroside III A-1.

[0081] Figure 58 . Concentrations of mogrosides and saponins in transgenic potatoes of the transgenic event transformed with vector SP1463 and subsequently re-transformed with S1UGT (SP5027).

[0082] Figure 59 . Concentrations of mogrosides and saponins in transgenic sugar beet calli transformed with vector SP3684.

[0083] Brief description of sequences

[0084] SEQ ID NO:1: Nucleic acid sequence of cytochrome P450-72 (CYP72Zm) based on maize codon usage.

[0085] SEQ ID NO:2: Amino acid sequence of cytochrome P450-72 (CYP72).

[0086] SEQ ID NO:3: Nucleic acid sequence of cytochrome P450-72 (CYP72GC) with a higher GC content than SEQ ID NO:1.

[0087] SEQ ID NO:4: Nucleic acid sequence of cucurbitadienol synthase (CDS).

[0088] SEQ ID NO:5: Amino acid sequence of cucurbitadienol synthase (CDS).

[0089] SEQ ID NO:6: Nucleic acid sequence of cytochrome P450-87 (CYP87).

[0090] SEQ ID NO:7: Amino acid sequence of cytochrome P450-87 (CYP87).

[0091] SEQ ID NO:8: Nucleic acid sequence of uridine phosphorylase-dependent glycosyltransferase-720 (UGT720).

[0092] SEQ ID NO:9: Amino acid sequence of uridine phosphorylase-dependent glycosyltransferase-720 (UGT720).

[0093] SEQ ID NO:10: Nucleic acid sequence of uridine phosphorylase-dependent glycosyltransferase-94 (UGT94).

[0094] SEQ ID NO:11: Amino acid sequence of uridine phosphorylase-dependent glycosyltransferase-94 (UGT94).

[0095] SEQ ID NO:12: Nucleic acid sequence of squalene epoxidase (SQE).

[0096] SEQ ID NO:13: Amino acid sequence of squalene epoxidase (SQE).

[0097] SEQ ID NO:14: Nucleic acid sequence of epoxide hydrolase (EPH).

[0098] SEQ ID NO:15: Amino acid sequence of epoxide hydrolase (EPH).

[0099] SEQ ID NO:16: Nucleic acid sequence of truncated 3-hydroxy-3-methylglutaryl-CoA reductase (tHMGR).

[0100] SEQ ID NO:17: Amino acid sequence of truncated 3-hydroxy-3-methylglutaryl-CoA reductase (tHMGR).

[0101] SEQ ID NO:18: Nucleic acid sequence of NADPH:cytochrome P450 reductase (SgCPR2).

[0102] SEQ ID NO:19: Amino acid sequence of NADPH:cytochrome P450 reductase (SgCPR2).

[0103] SEQ ID NO:20: Nucleic acid sequence of uridine phosphorylase-dependent glycosyltransferase 74 (UGT74_3).

[0104] SEQ ID NO:21: Amino acid sequence of uridine phosphorylase-dependent glycosyltransferase 74 (UGT74_3).

[0105] SEQ ID NO:22: Nucleic acid sequence of uridine phosphorylase-dependent glycosyltransferase 74 (UGT74_4).

[0106] SEQ ID NO:23: Amino acid sequence of uridine phosphorylase-dependent glycosyltransferase 74 (UGT74_4).

[0107] SEQ ID NO:24: Nucleic acid sequence of cytochrome P450-87 (ClCYP87D18_B m3) from watermelon (Citrullus lanatus).

[0108] SEQ ID NO:25: Amino acid sequence of cytochrome P450-87 (ClCYP87D18_Bm3) from watermelon.

[0109] SEQ ID NO:26: Nucleic acid sequence of mutant cytochrome P450-87 (CYP87D17 m2).

[0110] SEQ ID NO:27: Amino acid sequence of mutant cytochrome P450-87 (CYP87D17 m2).

[0111] SEQ ID NO:28: Nucleic acid sequence of mutant cytochrome P450-87 (CYP87D17 m3).

[0112] SEQ ID NO:29: Amino acid sequence of mutant cytochrome P450-87 (CYP87D17 m3).

[0113] SEQ ID NO:30: Nucleic acid sequence of mutant cytochrome P450-87 (CYP87D20 m2).

[0114] SEQ ID NO:31: Amino acid sequence of mutant cytochrome P450-87 (CYP87D20 m2).

[0115] SEQ ID NO:32: Nucleic acid sequence of mutant cytochrome P450-87 (CYP87D20 m3).

[0116] SEQ ID NO:33: Amino acid sequence of mutant cytochrome P450-87 (CYP87D20 m3).

[0117] SEQ ID NO:34: Cytochrome P450-72 (CYP72)-2A-cytochrome P450-72 (CYP72) dicistronic nucleic acid sequence.

[0118] SEQ ID NO:35: The first CYP72 amino acid sequence, which can be generated from the cleaved cytochrome P450-72 (CYP72)-2A-cytochrome P450-72 (CYP72) dicistronic nucleic acid sequence.

[0119] SEQ ID NO:36: The second CYP72 amino acid sequence, which can be generated from the cleaved cytochrome P450-72 (CYP72)-2A-cytochrome P450-72 (CYP72) dicistronic nucleic acid sequence.

[0120] SEQ ID NO:37: The full-length CYP72-2A-CYP72 amino acid sequence from the cytochrome P450-72 (CYP72)-2A-cytochrome P450-72 (CYP72) dicistronic nucleic acid sequence.

[0121] SEQ ID NO:38: Uridine phosphorylase-dependent glycosyltransferase-720 (UGT720)-2A-uridine phosphorylase-dependent glycosyltransferase-720 (UGT720) dicistronic nucleic acid sequence.

[0122] SEQ ID NO:39: The first UGT720 amino acid sequence, which can be generated from the cleaved uridine phosphorylase-dependent glycosyltransferase-720 (UGT720)-2A-uridine phosphorylase-dependent glycosyltransferase-720 (UGT720) dicistronic nucleic acid sequence.

[0123] SEQ ID NO:40: The second UGT720 amino acid sequence, which can be generated from the cleaved uridine phosphorylase-dependent glycosyltransferase-720 (UGT720)-2A-uridine phosphorylase-dependent glycosyltransferase-720 (UGT720) dicistronic nucleic acid sequence.

[0124] SEQ ID NO:41: The full-length UGT720 amino acid sequence from the uridine phosphorylase-dependent glycosyltransferase-720 (UGT720)-2A-uridine phosphorylase-dependent glycosyltransferase-720 (UGT720) dicistronic nucleic acid sequence.

[0125] SEQ ID NO:42: Uridine phosphorylase-dependent glycosyltransferase-94 (UGT94)-2A-uridine phosphorylase-dependent glycosyltransferase-94 (UGT94) dicistronic nucleic acid sequence.

[0126] SEQ ID NO:43: The first UGT94 amino acid sequence, which can be produced from the cleaved uridine phosphorylase-dependent glycosyltransferase-94 (UGT94)-2A-uridine phosphorylase-dependent glycosyltransferase-94 (UGT94) dicistronic nucleic acid sequence.

[0127] SEQ ID NO:44: The second UGT94 amino acid sequence, which can be produced from the cleaved uridine phosphorylase-dependent glycosyltransferase-94 (UGT94)-2A-uridine phosphorylase-dependent glycosyltransferase-94 (UGT94) dicistronic nucleic acid sequence.

[0128] SEQ ID NO:45: The full-length UGT94-2A-UGT94 amino acid sequence, which can be produced from the cleaved uridine phosphorylase-dependent glycosyltransferase-94 (UGT94)-2A-uridine phosphorylase-dependent glycosyltransferase-94 (UGT94) dicistronic nucleic acid sequence.

[0129] SEQ ID NO:46: The truncated 3-hydroxy-3-methylglutaryl-CoA reductase (tHMGR)-2A-truncated 3-hydroxy-3-methylglutaryl-CoA reductase (tHMGR) dicistronic nucleic acid sequence.

[0130] SEQ ID NO:47: The first tHMGR amino acid sequence, which can be produced from the cleaved truncated 3-hydroxy-3-methylglutaryl-CoA reductase (tHMGR)-2A-truncated 3-hydroxy-3-methylglutaryl-CoA reductase (tHMGR) dicistronic nucleic acid sequence.

[0131] SEQ ID NO:48: The second tHMGR amino acid sequence, which can be produced from the cleaved truncated 3-hydroxy-3-methylglutaryl-CoA reductase (tHMGR)-2A-truncated 3-hydroxy-3-methylglutaryl-CoA reductase (tHMGR) dicistronic nucleic acid sequence.

[0132] SEQ ID NO:49: The full-length tHMGR-2A-tHMGR amino acid sequence, which can be produced from the cleaved truncated 3-hydroxy-3-methylglutaryl-CoA reductase (tHMGR)-2A-truncated 3-hydroxy-3-methylglutaryl-CoA reductase (tHMGR) dicistronic nucleic acid sequence.

[0133] SEQ ID NO:50: The uridine phosphorylase-dependent glycosyltransferase-720 (UGT720)-2A-cytochrome P450-72 (CYP72) dicistronic nucleic acid sequence.

[0134] SEQ ID NO:51: The amino acid sequence of UGT720, which can be produced from the cleaved uridine phosphorylase-dependent glycosyltransferase-720 (UGT720)-2A-cytochrome P450-72 (CYP72) dicistronic nucleic acid sequence.

[0135] SEQ ID NO:52: The amino acid sequence of CYP72, which can be produced from the cleaved uridine phosphorylase-dependent glycosyltransferase-720 (UGT720)-2A-cytochrome P450-72 (CYP72) dicistronic nucleic acid sequence.

[0136] SEQ ID NO:53: The full-length UGT720-2A-CYP72 amino acid sequence from the uridine phosphorylase-dependent glycosyltransferase-720 (UGT720)-2A-cytochrome P450-72 (CYP72) dicistronic nucleic acid sequence.

[0137] SEQ ID NO:54: The uridine phosphorylase-dependent glycosyltransferase-94 (UGT94)-2A-truncated 3-hydroxy-3-methylglutaryl-CoA reductase (tHMGR) dicistronic nucleic acid sequence.

[0138] SEQ ID NO:55: The amino acid sequence of UGT94, which can be produced from the cleaved uridine phosphorylase-dependent glycosyltransferase-94 (UGT94)-2A-truncated 3-hydroxy-3-methylglutaryl-CoA reductase (tHMGR) dicistronic nucleic acid sequence.

[0139] SEQ ID NO:56: The amino acid sequence of tHMGR, which can be produced from the cleaved uridine phosphorylase-dependent glycosyltransferase-94 (UGT94)-2A-truncated 3-hydroxy-3-methylglutaryl-CoA reductase (tHMGR) dicistronic nucleic acid sequence.

[0140] SEQ ID NO:57: The UGT94-2A-tHMGR amino acid sequence from the uridine phosphorylase-dependent glycosyltransferase-94 (UGT94)-2A-truncated 3-hydroxy-3-methylglutaryl-CoA reductase (tHMGR) dicistronic nucleic acid sequence.

[0141] SEQ ID NO:58: The hygromycin resistance (HygR) gene nucleic acid sequence.

[0142] SEQ ID NO:59: The hygromycin resistance (HygR) gene amino acid sequence.

[0143] SEQ ID NO:60: The HLVH12 promoter nucleic acid sequence.

[0144] SEQ ID NO:61: Nucleic acid sequence of the DCMV promoter.

[0145] SEQ ID NO:62: Nucleic acid sequence of the FMVSgt:PCLSVFlt (also known as FSgt / PFLt) chimeric promoter.

[0146] SEQ ID NO:63: Nucleic acid sequence of the replicated MMV (dMMV) promoter.

[0147] SEQ ID NO:64: Nucleic acid sequence of the CmYLCV promoter.

[0148] SEQ ID NO:65: Nucleic acid sequence of the CaMV e35S (e35S) promoter.

[0149] SEQ ID NO:66: Nucleic acid sequence of the NOS promoter.

[0150] SEQ ID NO:67: Nucleic acid sequence of the ScBV promoter.

[0151] SEQ ID NO:68: Nucleic acid sequence of the CsVMV promoter.

[0152] SEQ ID NO:69: Nucleic acid sequence of the FMVSgt promoter.

[0153] SEQ ID NO:70: Nucleic acid sequence of the FS1_1 promoter.

[0154] SEQ ID NO:71: Nucleic acid sequence of the FE_3 promoter.

[0155] SEQ ID NO:72: Nucleic acid sequence of the Pea3A terminator.

[0156] SEQ ID NO:73: Nucleic acid sequence of the AtUBQ3 terminator.

[0157] SEQ ID NO:74: Nucleic acid sequence of the Gmax MYB2 terminator.

[0158] SEQ ID NO:75: Nucleic acid sequence of the AtRBCS2B terminator.

[0159] SEQ ID NO:76: Nucleic acid sequence of the pea E9 terminator.

[0160] SEQ ID NO:77: Nucleic acid sequence of the AtHSP18.2 terminator.

[0161] SEQ ID NO:78: Nucleic acid sequence of the potato Ubi3 terminator.

[0162] SEQ ID NO:79: Nucleic acid sequence of the terminator of At tubulin B9 (AtTub).

[0163] SEQ ID NO:80: Nucleic acid sequence of the 35S terminator.

[0164] SEQ ID NO:81: Nucleic acid sequence of SynJ 5UTR.

[0165] SEQ ID NO:82: Nucleic acid sequence of the TM6 MAR insulator.

[0166] SEQ ID NO:83: Nucleic acid sequence of the 2A self-cleaving peptide.

[0167] SEQ ID NO:84: Amino acid sequence of the 2A self-cleaving peptide.

[0168] SEQ ID NO:85: Nucleic acid sequence of cytochrome P450-72 (CYP72 V1).

[0169] SEQ ID NO:86: Amino acid sequence of cytochrome P450-72 (CYP72 V1).

[0170] SEQ ID NO:87: Nucleic acid sequence of the alternative squalene epoxidase (SQE).

[0171] SEQ ID NO:88: Nucleic acid sequence of the alternative cytochrome P450-87 (CYP87).

[0172] SEQ ID NO:89: Nucleic acid sequence of the alternative cucurbitadienol synthase (CDS).

[0173] SEQ ID NO:90: Nucleic acid sequence of the alternative epoxide hydrolase (EPH).

[0174] SEQ ID NO:91: Nucleic acid sequence of the alternative uridine phosphorylase-dependent glycosyltransferase-720 (UGT720).

[0175] SEQ ID NO:92: Nucleic acid sequence of the alternative uridine phosphorylase-dependent glycosyltransferase-94 (UGT94).

[0176] SEQ ID NO:93: Nucleic acid sequence of the upstream terpenoid biosynthetic enzyme (FPS; SP0231), watermelon cDNA.

[0177] SEQ ID NO:94: Amino acid sequence of the upstream terpenoid biosynthetic enzyme (FPS; SP0231), watermelon.

[0178] SEQ ID NO:95: Cytochrome P450 biosynthetic enzyme, high GC version (CYP87D20 m2(V2-I46L-A49L-C343Y)GC63; SP0577), Siraitia grosvenorii cDNA, nucleic acid sequence.

[0179] SEQ ID NO:96: Cytochrome P450 biosynthetic enzyme, high GC version (CYP87D20 m2(V2-I46L-A49L-C343Y)GC63; SP0577), Siraitia grosvenorii, nucleic acid sequence.

[0180] SEQ ID NO:97: Upstream squalene biosynthetic enzyme (SQS; SP0951), Citrullus lanatus cDNA, nucleic acid sequence.

[0181] SEQ ID NO:98: Upstream squalene biosynthetic enzyme (SQS; SP0951), Citrullus lanatus, amino acid sequence.

[0182] SEQ ID NO:99: Cytochrome P450 biosynthetic enzyme, mutant, high GC version (CYP87D17 m3 GC63; SP1333), Siraitia grosvenorii cDNA, nucleic acid sequence.

[0183] SEQ ID NO:100: Cytochrome P450 biosynthetic enzyme, mutant, high GC version (CYP87D17 m3GC63; SP1333), Siraitia grosvenorii, amino acid sequence.

[0184] SEQ ID NO:101: Cytochrome P450 biosynthetic enzyme, mutant, high GC version (CYP87D17 m2GC63; SP2503), Siraitia grosvenorii cDNA, nucleic acid sequence.

[0185] SEQ ID NO:102: Cytochrome P450 biosynthetic enzyme, mutant high GC version (CYP87D17 m2 GC63; SP2503), Siraitia grosvenorii, protein.

[0186] SEQ ID NO:103: Cytochrome P450 reductase biosynthetic enzyme, high GC version (SgCPR2 GC66; SP2571), Siraitia grosvenorii cDNA, nucleic acid sequence.

[0187] SEQ ID NO:104: Cytochrome P450 reductase biosynthetic enzyme, high GC version (SgCPR2 GC66; SP2571), Siraitia grosvenorii, amino acid sequence.

[0188] SEQ ID NO:105: Uridine phosphorylase-dependent glycosyltransferase 74, high GC version (SgUGT74_406_2GC64; SP2666), Siraitia grosvenorii cDNA, nucleic acid sequence.

[0189] SEQ ID NO:106: Uridine phosphorylase-dependent glycosyltransferase 74, high GC version (SgUGT74_406_2GC64; SP2666), Siraitia grosvenorii, amino acid sequence.

[0190] SEQ ID NO:107: Uridine phosphorylase-dependent glycosyltransferase 74, high GC version (SgUGT74_345_2GC65; SP3201), Siraitia grosvenorii cDNA, nucleic acid sequence.

[0191] SEQ ID NO:108: Uridine phosphorylase-dependent glycosyltransferase 74, high GC version (SgUGT74_345_2GC65; SP3201), Siraitia grosvenorii, amino acid sequence.

[0192] SEQ ID NO:109: Cytochrome P450 biosynthetic enzyme, mutant, high GC version (ClCYP87D18_B m3GC62; SP4900), Citrullus lanatus cDNA, nucleic acid sequence.

[0193] SEQ ID NO:110: Cytochrome P450 biosynthetic enzyme, mutant, high GC version (ClCYP87D18_B m3GC62; SP4900), Citrullus lanatus, amino acid sequence.

[0194] SEQ ID NO:111: Cytochrome P450 biosynthetic enzyme (CYP87D20 m3(V2-I46L-A49L-C343YL193KO)GC63; SP4910), Siraitia grosvenorii cDNA, nucleic acid sequence.

[0195] SEQ ID NO:112: Cytochrome P450 biosynthetic enzyme (CYP87D20m3(V2-I46L-A49L-C343YL193KO)GC63; SP4910), Siraitia grosvenorii, amino acid sequence.

[0196] SEQ ID NO:113: Uridine phosphorylase-dependent glycosyltransferase (Solyc01g107825.1UGT, SP5024), Solanum lycopersicum cDNA, nucleic acid sequence.

[0197] SEQ ID NO:114: Uridine phosphorylase-dependent glycosyltransferase (Solyc01g107825.1 UGT, SP5024), tomato, amino acid sequence.

[0198] SEQ ID NO:115: Uridine phosphorylase-dependent glycosyltransferase (Solyc02g070020.1 UGT; SP5025), tomato cDNA, nucleic acid sequence.

[0199] SEQ ID NO:116: Uridine phosphorylase-dependent glycosyltransferase (Solyc02g070020.1 UGT; SP5025), tomato, amino acid sequence.

[0200] SEQ ID NO:117: Uridine phosphorylase-dependent glycosyltransferase (Solyc09g092500.1 UGT; SP5026), tomato cDNA, nucleic acid sequence.

[0201] SEQ ID NO:118: Uridine phosphorylase-dependent glycosyltransferase (Solyc09g092500.1 UGT; SP5026), tomato, amino acid sequence.

[0202] SEQ ID NO:119: Uridine phosphorylase-dependent glycosyltransferase (Solyc10g085230.2 UGT; SP5027), tomato cDNA, nucleic acid sequence.

[0203] SEQ ID NO:120: Uridine phosphorylase-dependent glycosyltransferase (Solyc10g085230.2 UGT; SP5027), tomato, amino acid sequence.

[0204] SEQ ID NO:121: Uridine phosphorylase-dependent glycosyltransferase (Solyc10g085880.1 UGT; SP5028), tomato cDNA, nucleic acid sequence.

[0205] SEQ ID NO:122: Uridine phosphorylase-dependent glycosyltransferase (Solyc10g085880.1 UGT; SP5028), tomato, amino acid sequence.

[0206] SEQ ID NO:123: Uridine phosphorylase-dependent glycosyltransferase 94 (SgUGT94-289-3-MS1; SP5034), Siraitia grosvenorii cDNA, nucleic acid sequence.

[0207] SEQ ID NO:124: Uridine phosphorylase-dependent glycosyltransferase 94 (SGUGT94-289-3-MS1; SP5034), Siraitia grosvenorii, amino acid sequence.

[0208] SEQ ID NO:125: Upstream terpene biosynthetic enzyme (GPS1; SP5035), watermelon cDNA, nucleic acid sequence.

[0209] SEQ ID NO:126: Upstream terpene biosynthetic enzyme (GPS1; SP5035), watermelon, amino acid sequence.

[0210] SEQ ID NO:127: Uridine phosphorylase-dependent glycosyltransferase, high GC version (SgUGT75-281-2GC65; SP5036), Siraitia grosvenorii cDNA, nucleic acid sequence.

[0211] SEQ ID NO:128: Uridine phosphorylase-dependent glycosyltransferase, high GC version (SgUGT75-281-2GC65; SP5036), Siraitia grosvenorii, amino acid sequence.

[0212] SEQ ID NO:129: Uridine phosphorylase-dependent glycosyltransferase 74 (SgUGT74AC1 (without termination); SP5037), Siraitia grosvenorii cDNA, nucleic acid sequence.

[0213] SEQ ID NO:130: Uridine phosphorylase-dependent glycosyltransferase 74 (SgUGT74AC1 (without termination); SP5037), Siraitia grosvenorii, amino acid sequence.

[0214] SEQ ID NO:131: Uridine phosphorylase-dependent glycosyltransferase (Sg MG1 GC63; SP5038), Siraitia grosvenorii cDNA, nucleic acid sequence.

[0215] SEQ ID NO:132: Uridine phosphorylase-dependent glycosyltransferase (Sg MG1 GC63; SP5038), Siraitia grosvenorii, amino acid sequence.

[0216] SEQ ID NO:133: Uridine phosphorylase-dependent glycosyltransferase (t134248; SP5039), Stevia rebaudiana cDNA, nucleic acid sequence.

[0217] SEQ ID NO:134: Uridine phosphorylase-dependent glycosyltransferase (t134248; SP5039), Stevia rebaudiana, amino acid sequence.

[0218] SEQ ID NO:135: Uridine phosphorylase-dependent glycosyltransferase (t72140; SP5040), Stevia cDNA, nucleic acid sequence.

[0219] SEQ ID NO:136: Uridine phosphorylase-dependent glycosyltransferase (t72140; SP5040), Stevia, amino acid sequence.

[0220] SEQ ID NO:137: Uridine phosphorylase-dependent glycosyltransferase (t72143; SP5041), Stevia cDNA, nucleic acid sequence.

[0221] SEQ ID NO:138: Uridine phosphorylase-dependent glycosyltransferase (t72143; SP5041), Stevia, amino acid sequence.

[0222] SEQ ID NO:139: Uridine phosphorylase-dependent glycosyltransferase (t74692; SP5042), Stevia cDNA, nucleic acid sequence.

[0223] SEQ ID NO:140: Uridine phosphorylase-dependent glycosyltransferase (t74692; SP5042), Stevia, amino acid sequence.

[0224] SEQ ID NO:141: Uridine phosphorylase-dependent glycosyltransferase (t 85004; SP5043), Stevia cDNA, nucleic acid sequence.

[0225] SEQ ID NO:142: Uridine phosphorylase-dependent glycosyltransferase (t 85004; SP5043), Stevia, amino acid sequence.

[0226] SEQ ID NO:143: Upstream terpenoid biosynthetic enzyme (Sg 00000109.2_FPS1; SP5044), Momordica grosvenori cDNA, nucleic acid sequence.

[0227] SEQ ID NO:144: Upstream terpenoid biosynthetic enzyme (Sg 00000109.2_FPS1; SP5044), Momordica grosvenori, amino acid sequence.

[0228] SEQ ID NO:145: Upstream squalene biosynthetic enzyme (Sg00000892.729_SQS1; SP5045), Momordica grosvenori cDNA, nucleic acid sequence.

[0229] SEQ ID NO:146: Upstream squalene biosynthetic enzyme (Sg00000892.729_SQS1; SP5045), Momordica grosvenori, amino acid sequence.

[0230] SEQ ID NO:147: Upstream terpenoid biosynthetic enzyme (Bj HMGS mutant; SP5046), Brassica juncea cDNA, nucleic acid sequence.

[0231] SEQ ID NO:148: Upstream terpenoid biosynthetic enzyme (Bj HMGS mutant; SP5046), Brassica juncea, amino acid sequence.

[0232] SEQ ID NO:149: UDP-glucose pyrophosphorylase (Ta UDP-glucose pyrophosphorylase; SP5047), Thermocrispum agreste cDNA, nucleic acid sequence.

[0233] SEQ ID NO:150: UDP-glucose pyrophosphorylase (Ta UDP-glucose pyrophosphorylase; SP5047), Thermocrispum agreste, amino acid sequence.

[0234] SEQ ID NO:151: Upstream terpenoid biosynthetic enzyme (ClGPS_2; SP5048), Citrullus lanatus cDNA, nucleic acid sequence.

[0235] SEQ ID NO:152: Upstream terpenoid biosynthetic enzyme (ClGPS_2; SP5048), Citrullus lanatus, amino acid sequence.

[0236] SEQ ID NO:153: Upstream terpenoid biosynthetic enzyme (MpGPS-SSU; SP5049), Mentha x piperita cDNA, nucleic acid sequence.

[0237] SEQ ID NO:154: Upstream terpenoid biosynthetic enzyme (MpGPS-SSU; SP5049), Mentha x piperita, amino acid sequence.

[0238] SEQ ID NO:155: Upstream terpenoid biosynthetic enzyme (MpGPS-SSU; SP5050), Litsea cubeba cDNA, nucleic acid sequence.

[0239] SEQ ID NO:156: Upstream terpenoid biosynthetic enzyme (MpGPS-SSU; SP5050), Litsea cubeba, amino acid sequence.

[0240] SEQ ID NO:157: Hydrolase biosynthetic enzyme (DbExg1; SP5051), Dekkera bruxellensis cDNA, nucleic acid sequence.

[0241] SEQ ID NO:158: Hydrolytic enzyme biosynthetic enzyme (DbExg1; SP5051), Dekkera bruxellensis, amino acid sequence.

[0242] SEQ ID NO:159: Upstream terpenoid biosynthetic enzyme (ClCG09G011560.1_PMK; SP5072), watermelon cDNA, nucleic acid sequence.

[0243] SEQ ID NO:160: Upstream terpenoid biosynthetic enzyme (ClCG09G011560.1_PMK; SP5072), watermelon, amino acid sequence.

[0244] SEQ ID NO:161: Upstream terpenoid biosynthetic enzyme (ClCG10G001230.1_PMK; SP5073), watermelon cDNA, nucleic acid sequence.

[0245] SEQ ID NO:162: Upstream terpenoid biosynthetic enzyme (ClCG10G001230.1_PMK; SP5073), watermelon, amino acid sequence.

[0246] SEQ ID NO:163: Upstream terpenoid biosynthetic enzyme (ClCG09G022040.1_IPK; SP5074), watermelon cDNA, nucleic acid sequence.

[0247] SEQ ID NO:164: Upstream terpenoid biosynthetic enzyme (ClCG09G022040.1_IPK; SP5074), watermelon, amino acid sequence.

[0248] SEQ ID NO:165: Upstream terpenoid biosynthetic enzyme (ClCG05G021710.1_MVD; SP5075), watermelon cDNA, nucleic acid sequence.

[0249] SEQ ID NO:166: Upstream terpenoid biosynthetic enzyme (ClCG05G021710.1_MVD; SP5075), watermelon, amino acid sequence.

[0250] SEQ ID NO:167: Upstream terpenoid biosynthetic enzyme (Sg 00153574.101_FPS1; SP5076), Siraitia grosvenorii cDNA, nucleic acid sequence.

[0251] SEQ ID NO:168: Upstream terpenoid biosynthetic enzyme (Sg 00153574.101_FPS1; SP5076), Siraitia grosvenorii, amino acid sequence.

[0252] SEQ ID NO:169: Upstream terpene biosynthetic enzyme (Sg 00010190.2_SQS1; SP5077), Siraitia grosvenorii cDNA, nucleic acid sequence.

[0253] SEQ ID NO:170: Upstream terpene biosynthetic enzyme (Sg 00010190.2_SQS1; SP5077), Siraitia grosvenorii, amino acid sequence.

[0254] SEQ ID NO:171: Upstream terpene biosynthetic enzyme (Sg 00153449.125_PMK; SP5078), Siraitia grosvenorii cDNA, nucleic acid sequence.

[0255] SEQ ID NO:172: Upstream terpene biosynthetic enzyme (Sg 00153449.125_PMK; SP5078), Siraitia grosvenorii, amino acid sequence.

[0256] SEQ ID NO:173: Upstream terpene biosynthetic enzyme, high GC version (tHMGR GC69; SP5079), Avena strigose cDNA, nucleic acid sequence.

[0257] SEQ ID NO:174: Upstream terpene biosynthetic enzyme, high GC version (tHMGR GC69; SP5079), Avena strigose, amino acid sequence.

[0258] SEQ ID NO:175: Uridine phosphorylase-dependent glycosyltransferase, high GC version (SgUGT720-269-1Itkin GC65; SP5080), Siraitia grosvenorii cDNA, nucleic acid sequence.

[0259] SEQ ID NO:176: Uridine phosphorylase-dependent glycosyltransferase, high GC version (SgUGT720-269-1Itkin GC65; SP5080), Siraitia grosvenorii, amino acid sequence.

[0260] SEQ ID NO:177: Uridine phosphorylase-dependent glycosyltransferase, high GC version (SgUGT720-269-4Itkin GC65; SP5081), Siraitia grosvenorii cDNA, nucleic acid sequence.

[0261] SEQ ID NO:178: Uridine phosphorylase-dependent glycosyltransferase, high GC version (SgUGT720-269-4Itkin GC65; SP5081), Siraitia grosvenorii, amino acid sequence.

[0262] SEQ ID NO:179: Uridine phosphorylase-dependent glycosyltransferase (SgUGT94-289-2; SP5082), Momordica grosvenori cDNA, nucleic acid sequence.

[0263] SEQ ID NO:180: Uridine phosphorylase-dependent glycosyltransferase (SgUGT94-289-2; SP5082), Momordica grosvenori, amino acid sequence.

[0264] SEQ ID NO:181: Uridine phosphorylase-dependent glycosyltransferase (SgUGT94-289-2Itkin; SP5083), Momordica grosvenori cDNA, nucleic acid sequence.

[0265] SEQ ID NO:182: Uridine phosphorylase-dependent glycosyltransferase (SgUGT94-289-2Itkin; SP5083), Momordica grosvenori, amino acid sequence.

[0266] SEQ ID NO:183: Uridine phosphorylase-dependent glycosyltransferase (SgUGT94-289-3Itkin; SP5084), Momordica grosvenori cDNA, nucleic acid sequence.

[0267] SEQ ID NO:184: Uridine phosphorylase-dependent glycosyltransferase (SgUGT94-289-3Itkin; SP5084), Momordica grosvenori, amino acid sequence.

[0268] SEQ ID NO:185: Uridine phosphorylase-dependent glycosyltransferase (t134583; SP5085), Momordica grosvenori cDNA, nucleic acid sequence.

[0269] SEQ ID NO:186: Uridine phosphorylase-dependent glycosyltransferase (t134583; SP5085), Momordica grosvenori, amino acid sequence.

[0270] SEQ ID NO:187: Uridine phosphorylase-dependent glycosyltransferase (t74645; SP5086), Momordica grosvenori cDNA, nucleic acid sequence.

[0271] SEQ ID NO:188: Uridine phosphorylase-dependent glycosyltransferase (t74645; SP5086), Momordica grosvenori, amino acid sequence.

[0272] SEQ ID NO:189: Uridine phosphorylase-dependent glycosyltransferase (t74693; SP5087), Momordica grosvenori cDNA, nucleic acid sequence.

[0273] SEQ ID NO:190: Uridine phosphorylase-dependent glycosyltransferase (t74693; SP5087), Siraitia grosvenorii, amino acid sequence.

[0274] SEQ ID NO:191: Upstream terpenoid biosynthetic enzyme (Sg 00001291.26_PMK; SP5088), Siraitia grosvenorii cDNA, nucleic acid sequence.

[0275] SEQ ID NO:192: Upstream terpenoid biosynthetic enzyme (Sg 00001291.26_PMK; SP5088), Siraitia grosvenorii, amino acid sequence.

[0276] SEQ ID NO:193: Upstream terpenoid biosynthetic enzyme (Sg 00154122.1_IPK; SP5089), Siraitia grosvenorii cDNA, nucleic acid sequence.

[0277] SEQ ID NO:194: Upstream terpenoid biosynthetic enzyme (Sg 00154122.1_IPK; SP5089), Siraitia grosvenorii, amino acid sequence.

[0278] SEQ ID NO:195: Upstream terpenoid biosynthetic enzyme (Sg 27366_MVD; SP5090), Siraitia grosvenorii cDNA, nucleic acid sequence.

[0279] SEQ ID NO:196: Upstream terpenoid biosynthetic enzyme (Sg 27366_MVD; SP5090), Siraitia grosvenorii, amino acid sequence.

[0280] SEQ ID NO:197: Hydrolase biosynthetic enzyme (E142A-RRK67; SP5091), Aspergillus oryzae cDNA, nucleic acid sequence.

[0281] SEQ ID NO:198: Hydrolase biosynthetic enzyme (E142A-RRK67; SP5091), Aspergillus oryzae, amino acid sequence.

[0282] SEQ ID NO:199: Upstream terpenoid biosynthetic enzyme (McCDS; SP5092), Momordica charantia cDNA, nucleic acid sequence.

[0283] SEQ ID NO:200: Upstream terpenoid biosynthetic enzyme (McCDS; SP5092), Momordica charantia, amino acid sequence.

[0284] SEQ ID NO:201: Upstream terpene biosynthetic enzyme (CpCDS; SP5093), cDNA of Cucurbita pepo, nucleic acid sequence.

[0285] SEQ ID NO:202: Upstream terpene biosynthetic enzyme (CpCDS; SP5093), Cucurbita pepo, amino acid sequence.

[0286] SEQ ID NO:203: Upstream terpene biosynthetic enzyme (TcCDS; SP5094), cDNA of Trichosanthes cucumerina, nucleic acid sequence.

[0287] SEQ ID NO:204: Upstream terpene biosynthetic enzyme (TcCDS; SP5094), Trichosanthes cucumerina, amino acid sequence.

[0288] SEQ ID NO:205: Upstream terpene biosynthetic enzyme (CcCDS; SP5095), cDNA of Citrullus colocynthis, nucleic acid sequence.

[0289] SEQ ID NO:206: Upstream terpene biosynthetic enzyme (CcCDS; SP5095), Citrullus colocynthis, amino acid sequence.

[0290] SEQ ID NO:207: Upstream terpene biosynthetic enzyme (Ac SQS with intron 6; SP5096), cDNA of Amaranthus cruentus, nucleic acid sequence.

[0291] SEQ ID NO:208: Upstream terpene biosynthetic enzyme (Ac SQS with intron 6; SP5096), Amaranthus cruentus, amino acid sequence.

[0292] SEQ ID NO:209: Upstream terpene biosynthetic enzyme (Oe SQS; SP5097), cDNA of Olea europaea, nucleic acid sequence.

[0293] SEQ ID NO:210: Upstream terpene biosynthetic enzyme (Oe SQS; SP5097), Olea europaea, amino acid sequence.

[0294] SEQ ID NO:211: Upstream terpene biosynthetic enzyme (Cr SQS; SP5098), cDNA of Chlamydomonas reinhardtii, nucleic acid sequence.

[0295] SEQ ID NO:212: Upstream terpenoid biosynthetic enzyme (Cr SQS; SP5098), Chlamydomonas reinhardtii, amino acid sequence.

[0296] SEQ ID NO:213: Epoxidase biosynthetic enzyme (Cp SQE1; SP5099), Cucurbita pepo cDNA, nucleic acid sequence.

[0297] SEQ ID NO:214: Epoxidase biosynthetic enzyme (Cp SQE1; SP5099), Cucurbita pepo, amino acid sequence.

[0298] SEQ ID NO:215: Epoxidase biosynthetic enzyme (Mc SQE1; SP5100), Momordica charantia cDNA, nucleic acid sequence.

[0299] SEQ ID NO:216: Epoxidase biosynthetic enzyme (Mc SQE1; SP5100), Momordica charantia, amino acid sequence.

[0300] SEQ ID NO:217: Epoxidase biosynthetic enzyme (Pg SQE1; SP5101), Panax ginseng cDNA, nucleic acid sequence.

[0301] SEQ ID NO:218: Epoxidase biosynthetic enzyme (Pg SQE1; SP5101), Panax ginseng, amino acid sequence.

[0302] SEQ ID NO:219: Upstream terpenoid biosynthetic enzyme (Ab tHMGR; SP5102), Achyranthes bidentata cDNA, nucleic acid sequence.

[0303] SEQ ID NO:220: Upstream terpenoid biosynthetic enzyme (Ab tHMGR; SP5102), Achyranthes bidentata, amino acid sequence.

[0304] SEQ ID NO:221: Upstream terpenoid biosynthetic enzyme (Hb tHMGR; SP5103), Hevea brasiliensis cDNA, nucleic acid sequence.

[0305] SEQ ID NO:222: Upstream terpenoid biosynthetic enzyme (Hb tHMGR; SP5103), Hevea brasiliensis, amino acid sequence.

[0306] SEQ ID NO:223: Upstream terpenoid biosynthetic enzyme (Pg tHMGR; SP5104), Panax ginseng cDNA, nucleic acid sequence.

[0307] SEQ ID NO:224: Upstream terpenoid biosynthetic enzyme (Pg tHMGR; SP5104), Panax ginseng, amino acid sequence.

[0308] SEQ ID NO:225: Transcription factor (At MYC2D105N mutant; SP5105), Arabidopsis thaliana cDNA, nucleic acid sequence.

[0309] SEQ ID NO:226: Transcription factor (At MYC2D105N mutant; SP5105), Arabidopsis thaliana, amino acid sequence.

[0310] SEQ ID NO:227: Transcription factor (Cs bHLH; SP5106), Cucumis sativus cDNA, nucleic acid sequence.

[0311] SEQ ID NO:228: Transcription factor (Cs bHLH; SP5106), Cucumis sativus, amino acid sequence.

[0312] SEQ ID NO:229: Transcription factor (Bh bHLH; Sp5107), Benincasa hispida cDNA, nucleic acid sequence.

[0313] SEQ ID NO:230: Transcription factor (Bh bHLH; Sp5107), Benincasa hispida, amino acid sequence.

[0314] SEQ ID NO:231: Transcription factor 2 (Cs bHLH; SP5108), Cucumis sativus cDNA, nucleic acid sequence.

[0315] SEQ ID NO:232: Transcription factor 2 (Cs bHLH; SP5108), Cucumis sativus, amino acid sequence.

[0316] SEQ ID NO:233: Transcription factor 2 (Bh bHLH; SP5109), Benincasa hispida cDNA, nucleic acid sequence.

[0317] SEQ ID NO:234: Transcription factor 2 (Bh bHLH; SP5109), Benincasa hispida, amino acid sequence.

[0318] SEQ ID NO:235: Populus alba sucrose synthase (SP5110), Populus alba cDNA, nucleic acid sequence.

[0319] SEQ ID NO:236: Populus alba sucrose synthase (SP5110), Populus alba, amino acid sequence.

[0320] SEQ ID NO:237: UDP-glucose pyrophosphorylase (Lg UDP-glucose pyrophosphorylase; SP5111), cDNA of Larix gmelinii, nucleic acid sequence.

[0321] SEQ ID NO:238: UDP-glucose pyrophosphorylase (Lg UDP-glucose pyrophosphorylase; SP5111), Larix gmelinii, amino acid sequence.

[0322] SEQ ID NO:239: Transcription factor (At PAP2; SP5112), cDNA of Arabidopsis thaliana, nucleic acid sequence.

[0323] SEQ ID NO:240: Transcription factor (At PAP2; SP5112), Arabidopsis thaliana, amino acid sequence.

[0324] SEQ ID NO:241: Heat shock protein (HSP17.6; SP5113), cDNA of Medicago sativa, nucleic acid sequence.

[0325] SEQ ID NO:242: Heat shock protein (HSP17.6; SP5113), Medicago sativa, amino acid sequence.

[0326] SEQ ID NO:243: Transcription factor (At JUNGBRUNNEN1 Nac factor; SP5114), cDNA of Arabidopsis thaliana, nucleic acid sequence.

[0327] SEQ ID NO:244: Transcription factor (At JUNGBRUNNEN1 Nac factor; SP5114), Arabidopsis thaliana, amino acid sequence.

[0328] SEQ ID NO:245: Upstream terpenoid biosynthetic enzyme (AmGPS-SSU (no stop); SP5115), cDNA of Antirrhinum majus, nucleic acid sequence.

[0329] SEQ ID NO:246: Upstream terpenoid biosynthetic enzyme (AmGPS-SSU (no stop); SP5115), Antirrhinum majus, amino acid sequence.

[0330] SEQ ID NO:247: Upstream terpenoid biosynthetic enzyme (PgFPS; SP5116), cDNA of Panax ginseng, nucleic acid sequence.

[0331] SEQ ID NO:248: Upstream terpenoid biosynthetic enzyme (PgFPS; SP5116), Panax ginseng, amino acid sequence.

[0332] SEQ ID NO:249: Upstream terpenoid biosynthetic enzyme (PgSQS; SP5117), Panax ginseng cDNA, nucleic acid sequence.

[0333] SEQ ID NO:250: Upstream terpenoid biosynthetic enzyme (PgSQS; SP5117), Panax ginseng, amino acid sequence.

[0334] SEQ ID NO:251: Upstream terpenoid biosynthetic enzyme (gbHMGS1; SP5118), Ginkgo biloba cDNA, nucleic acid sequence.

[0335] SEQ ID NO:252: Upstream terpenoid biosynthetic enzyme (gbHMGS1; SP5118), Ginkgo biloba, amino acid sequence.

[0336] SEQ ID NO:253: Uridine phosphorylase-dependent glycosyltransferase (UGT73AM3; SP5263), Cucumis sativus cDNA, nucleic acid sequence.

[0337] SEQ ID NO:254: Uridine phosphorylase-dependent glycosyltransferase (UGT73AM3; SP5263), Cucumis sativus, amino acid sequence.

[0338] SEQ ID NO:255: Uridine phosphorylase-dependent glycosyltransferase, high GC version (Solyc10g085230.2UGT GC61; SP5265), Solanum lycopersicum cDNA, nucleic acid sequence.

[0339] SEQ ID NO:256: Uridine phosphorylase-dependent glycosyltransferase, high GC version (Solyc10g085230.2UGT GC61; SP5265), Solanum lycopersicum, amino acid sequence.

[0340] SEQ ID NO:257: Uridine phosphorylase-dependent glycosyltransferase, high GC version (UGT73AM3 GC64; SP5350), Cucumis sativus cDNA, nucleic acid sequence.

[0341] SEQ ID NO:258: Uridine phosphorylase-dependent glycosyltransferase, high GC version (UGT73AM3 GC64; SP5350), Cucumis sativus, amino acid sequence.

[0342] SEQ ID NO:259: AtUBQ10 promoter, Arabidopsis thaliana, nucleic acid sequence.

[0343] SEQ ID NO:260: PCLSV promoter, Peanut chlorotic mottle virus, nucleic acid sequence.

[0344] SEQ ID NO:261: FS4 promoter, watermelon, nucleic acid sequence.

[0345] SEQ ID NO:262: AtACT2 promoter, Arabidopsis thaliana, nucleic acid sequence.

[0346] SEQ ID NO:263: Enhanced AtEf-1A promoter, Cauliflower mosaic virus / Arabidopsis thaliana, nucleic acid sequence.

[0347] SEQ ID NO:264: FuasFScp promoter, Scrophulariaceae mosaic virus hybrid P, nucleic acid sequence.

[0348] SEQ ID NO:265: AtFAD2 terminator, Arabidopsis thaliana, nucleic acid sequence.

[0349] SEQ ID NO:266: AtNDUFA8 terminator, Arabidopsis thaliana, nucleic acid sequence.

[0350] SEQ ID NO:267: CsHSP17.3 terminator, cucumber, nucleic acid sequence.

[0351] SEQ ID NO:268: CsHSP22 terminator, cucumber, nucleic acid sequence.

[0352] SEQ ID NO:269: FE4 promoter, watermelon, nucleic acid sequence.

[0353] SEQ ID NO:270: Cucumin promoter, Cucumis melo L., nucleic acid sequence.

[0354] SEQ ID NO:271: SgCDS promoter, Momordica grosvenori, nucleic acid sequence.

[0355] SEQ ID NO:272: Upstream terpenoid biosynthetic enzyme, high GC version (CltHMGR GC; SP2040), watermelon cDNA, nucleic acid sequence.

[0356] SEQ ID NO:273: Upstream terpenoid biosynthetic enzyme, high GC version (CltHMGR GC 2A; SP2040), Citrullus vulgaris, amino acid sequence.

[0357] SEQ ID NO:274: Upstream terpenoid biosynthetic enzyme (CltHMGR GC 2A; SP3351), watermelon cDNA, nucleic acid sequence.

[0358] SEQ ID NO:275: Upstream terpenoid biosynthetic enzyme (CltHMGR GC 2A; SP3351), Citrullus vulgaris, amino acid sequence.

[0359] SEQ ID NO:276: Upstream terpenoid biosynthetic enzyme (CltHMGR Zm (for 2A); SP4989), watermelon cDNA, nucleic acid sequence.

[0360] SEQ ID NO:277: Upstream terpenoid biosynthetic enzyme (CltHMGR Zm (for 2A); SP4989), watermelon, amino acid sequence.

[0361] SEQ ID NO:278: Cytochrome P450 biosynthetic enzyme, optimized for maize (mf CYP72A459v1a Zm (GC48); SP2015), Momordica grosvenori cDNA, nucleic acid sequence.

[0362] SEQ ID NO:279: Cytochrome P450 biosynthetic enzyme, optimized for maize (mf CYP72A459v1a Zm (GC48); SP2015), Momordica grosvenori, amino acid sequence.

[0363] SEQ ID NO:280: Uridine phosphorylase-dependent glycosyltransferase (mfUGT720; SP4263), Momordica grosvenori cDNA, nucleic acid sequence.

[0364] SEQ ID NO:281: Uridine phosphorylase-dependent glycosyltransferase (mfUGT720; SP4263), Momordica grosvenori, amino acid sequence.

[0365] SEQ ID NO:282: Uridine phosphorylase-dependent glycosyltransferase, high GC version (Sg UGT94-289-1GC65; SP4332), Momordica grosvenori cDNA, nucleic acid sequence.

[0366] SEQ ID NO:283: Uridine phosphorylase-dependent glycosyltransferase, high GC version (Sg UGT94-289-1GC65; SP4332), Momordica grosvenori, amino acid sequence.

[0367] SEQ ID NO:284: Upstream terpene biosynthetic enzyme, optimized for maize (Sg CDS Zmays GC51; SP5029), Momordica grosvenori cDNA, nucleic acid sequence.

[0368] SEQ ID NO:285: Upstream terpene biosynthetic enzyme, optimized for maize (Sg CDS Zmays GC51; SP5029), Momordica grosvenori, amino acid sequence.

[0369] SEQ ID NO:286: Uridine phosphorylase-dependent glycosyltransferase, high GC version (mfUGT720 GC; SP5030), Siraitia grosvenorii cDNA, nucleic acid sequence.

[0370] SEQ ID NO:287: Uridine phosphorylase-dependent glycosyltransferase, high GC version (mfUGT720 GC; SP5030), Siraitia grosvenorii, amino acid sequence.

[0371] SEQ ID NO:288: Cytochrome P450 biosynthetic enzyme, high GC version (Sg CYP87D18 GC62; SP5031), Siraitia grosvenorii cDNA, nucleic acid sequence.

[0372] SEQ ID NO:289: Cytochrome P450 biosynthetic enzyme, high GC version (Sg CYP87D18 GC62; SP5031), Siraitia grosvenorii, amino acid sequence.

[0373] SEQ ID NO:290: Upstream squalene biosynthetic enzyme, high GC version (Sg SQE1 GC66; SP5032), Siraitia grosvenorii cDNA, nucleic acid sequence.

[0374] SEQ ID NO:291: Upstream squalene biosynthetic enzyme, high GC version (Sg SQE1 GC66; SP5032), Siraitia grosvenorii, amino acid sequence.

[0375] SEQ ID NO:292: Epoxide hydrolase biosynthetic enzyme, high GC version (Sg EPH3 GC64; SP5033), Siraitia grosvenorii cDNA, nucleic acid sequence.

[0376] SEQ ID NO:293: Epoxide hydrolase biosynthetic enzyme, high GC version (Sg EPH3 GC64; SP5033), Siraitia grosvenorii, amino acid sequence.

[0377] SEQ ID NO:294: Uridine phosphorylase-dependent glycosyltransferase, high GC version (Sg UGT720 GC 2A; SP3186), Siraitia grosvenorii cDNA, nucleic acid sequence.

[0378] SEQ ID NO:295: Uridine phosphorylase-dependent glycosyltransferase, high GC version (Sg UGT720 GC 2A; SP3186), Siraitia grosvenorii, amino acid sequence.

[0379] SEQ ID NO:296: Cytochrome P450 biosynthetic enzyme (Sg SgCYP72v1aZm (for 2A, GC47); SP3204), Momordica grosvenori cDNA, nucleic acid sequence.

[0380] SEQ ID NO:297: Cytochrome P450 biosynthetic enzyme (Sg SgCYP72v1aZm (for 2A, GC47); SP3204), Momordica grosvenori, amino acid sequence.

[0381] SEQ ID NO:298: Uridine phosphorylase-dependent glycosyltransferase (SgUGT94-1 GC 2A; SP3807), Momordica grosvenori cDNA, nucleic acid sequence.

[0382] SEQ ID NO:299: Uridine phosphorylase-dependent glycosyltransferase (SgUGT94-1 GC 2A; SP3807), Momordica grosvenori, amino acid sequence.

[0383] SEQ ID NO:300: Uridine phosphorylase-dependent glycosyltransferase (Sg UGT720 Zm (for 2A); SP3897), Momordica grosvenori cDNA, nucleic acid sequence.

[0384] SEQ ID NO:301: Uridine phosphorylase-dependent glycosyltransferase (Sg UGT720 Zm (for 2A); SP3897), Momordica grosvenori, amino acid sequence.

[0385] SEQ ID NO:302: Uridine phosphorylase-dependent glycosyltransferase (mfUGT720; SP4263), Momordica grosvenori cDNA, nucleic acid sequence.

[0386] SEQ ID NO:303: Uridine phosphorylase-dependent glycosyltransferase (mfUGT720; SP4263), Momordica grosvenori, amino acid sequence.

[0387] SEQ ID NO:304: Uridine phosphorylase-dependent glycosyltransferase, high GC version (Sg UGT94-289-1GC65; SP4332), Momordica grosvenori cDNA, nucleic acid sequence.

[0388] SEQ ID NO:305: Uridine phosphorylase-dependent glycosyltransferase, high GC version (Sg UGT94-289-1GC65; SP4332), Momordica grosvenori, amino acid sequence. Detailed implementation mode

[0389] The present disclosure generally describes transgenic plants and biosynthetic systems for producing mogrosol / mogroside pathway enzymes and mogrosides, and methods for producing such transgenic plants. The following sections provide more detailed descriptions of embodiments of the subject matter.

[0390] I. Mogroside Pathway Enzymes and Mogrosides

[0391] Mogrosides are highly stable molecules based on a triterpene backbone, formed by 1 - 6 different numbers of glucose units attached to the triterpene backbone. Mogrosides can also contain non - glucose moieties, such as momordicoside I. Figure 1 The mogroside biosynthetic pathway from Siraitia grosvenorii is shown (Itkin et al., Proc Nat Acad Sci USA 113:E7619 - E7628, 2016; Seki et al., Biosci. Biotechnol. Biochem. 82:927 - 934, 2018), and certain enzymes capable of catalyzing the reactions in the pathway. Importantly, the enzyme pathway for producing mogrosides according to the present disclosure is not limited by Figure 1 the mechanism shown. Other terpene structures, mogrosol precursors, enzyme - catalyzed reactions, or transformation mechanisms are also possible. Additionally, certain enzymes can catalyze more than one type of reaction, and one or more additional genes can be used to produce any of the mogrosol intermediates, mogrosol, or any of the mogroside compounds.

[0392] Exemplary nucleic acid and protein sequences are provided herein for certain enzymes of the mogroside pathway (also referred to as the mogroside biosynthetic pathway) that are used to convert a mogrosol precursor such as squalene into mogrosol and ultimately various mogroside compounds, including but not limited to mogroside V. Enzymes of the mogroside pathway include but are not limited to squalene epoxidase (SQE), cucurbitadienol synthase (CDS), epoxide or epoxide hydrolase (EPH), various cytochrome P450 enzymes (CYP) including but not limited to CYP72 and CYP87, uridine phosphorylase - dependent glycosyltransferases (UGT) including but not limited to UGT720, UGT94, and UGT74, and may additionally include 3 - hydroxy - 3 - methylglutaryl - CoA reductase (HMGR) or truncated forms thereof, and NADPH:cytochrome P450 reductase (CPR2).

[0393] SQE, CDS, CYP, and EPH are involved in successive steps of producing and converting mogroside precursors such as squalene into mogrol. Intermediate products of the enzymatic pathway include, but are not limited to, 2,3-oxidosqualene, 2,3;22,23-diepoxysqualene, 24,25-epoxycucurbitadienol, and 24,25-dihydroxycucurbitadienol. CDS is an oxidosqualene cyclase that uses 2,3;22,23-diepoxysqualene as its substrate to produce 24,25-epoxycucurbitadienol. Genome analysis shows that Momordica grosvenori has five genes that can encode SQE. Among them, two are strongly expressed at the initial stage of fruit development, such as the CDS, CYP enzymes, and EPH that catalyze subsequent steps. The Momordica grosvenori genome contains eight genes encoding EPH, which catalyze the conversion of 24,25-epoxycucurbitadienol into 24,25-dihydroxycucurbitadienol. Some enzymes can catalyze more than one type of reaction.

[0394] After the formation of mogrol, a series of glycosylations occur to add glucose molecules at the C-3 and C-24 positions, thereby producing mogrosides I-VI with different degrees of glycosylation. The Roman numerals I, II, III, IV, V, and VI represent the number of glucose units in the corresponding glycosylated mogrosides, isomogrosides, or oxymogrosides, respectively. Two UGTs contribute to these steps. One is UGT720, which is strongly expressed at the initial stage of fruit development and transfers one glucose molecule each to the hydroxyl groups at the C-24 and C-3 positions of mogrol. The second is UGT94, which is strongly expressed at the later stage of fruit development and adds sugars to other sugars already present on the receptor molecule.

[0395] Although the mogroside pathway was initially described in Momordica grosvenori (Siraitia grosvenorii), certain non-Momordica plants can prepare tetracyclic triterpenoids similar to mogrol because at least one intermediate such as triterpenoids is present in the cellular pathway. In addition, given that the related pathways for modifying tetracyclic triterpenoids require related enzymes such as reductases, these plants have expressed certain related network enzymes. Although these non-Momordica plants can express enzymes that produce mogrol precursors, these plants cannot naturally produce all the enzymes in a cooperative manner required to produce mogrosides. For example, plants such as cucumber, melon, and watermelon naturally express cucurbitadienol synthase that can produce cucurbitadienol. However, other enzymes such as CYP enzymes that can modify the cucurbitadienol skeleton redirect this intermediate to other terpene derivatives. Therefore, altering the genomes of these non-Momordica plants by recombinant, gene editing, or other modern plant breeding techniques can allow these non-Momordica plants to produce mogrol and mogrosides. Thus, in certain embodiments, one or more additional genes can be introduced into non-Momordica plants, or the enzymes native to such non-Momordica plants can be upregulated to allow the production of intermediate metabolites, thereby producing mogrol, mogrosides, and mogroside-based sweeteners.

[0396] The term "mogroside precursor" broadly encompasses all possible terpene derivatives and intermediates that give rise to mogrol and mogroside compounds, including but not limited to 2,3-oxidosqualene, 2,3;22,23-dioxidosqualene, 24,25-epoxycucurbitadienol and 24,25-dihydroxycucurbitadienol, cucurbitadienol, 11-hydroxy-cucurbitadienol and 11-oxo-cucurbitadienol. Mogrosides refer to any possible glycosylated products of mogrol, including but not limited to: mogroside I, Siratose (a stereoisomer of mogroside I), mogroside VI, mogroside V, isomogroside V, mogroside IV, mogroside III, mogroside IIIE, mogroside IIE, mogroside IIA, mogroside IE and mogroside IA. Other examples of mogrosides include but are not limited to mogroside IIB, 7-oxomogroside IIE, 11-oxomogroside A1, mogroside IIIA2, 11-deoxymogroside III, 11-oxomogroside IVA, 7-oxomogroside V and 11-oxo-mogroside V. Metabolites and derivatives of mogrosides refer to any close variants of mogrosides resulting from metabolic reactions, naturally occurring reactions or non-naturally occurring reactions. Compared with standard mogrosides, derivatives of mogrosides may contain deletions, alterations or additions of atoms or functional groups. However, the metabolites and derivatives of mogrosides substantially retain the same functions and characteristics as standard mogrosides. In some cases, mogroside compounds produced by the transgenic plants and organisms disclosed by the present invention may also undergo non-enzymatic (spontaneous) conversion to other mogroside compounds.

[0397] One or more of these mogroside compounds can be isolated, purified or partially purified from the disclosed transgenic plants or transgenic organisms grown by fermentation techniques. For example, one or more mogroside compounds can be harvested via wet extraction or from an aqueous layer containing mogroside compounds in a large-scale processing step that does not necessarily result in the isolation of a single mogroside compound. Alternatively, the entire transgenic plant (or a part thereof) or transgenic organism that produces mogroside compounds can be dried and ground into a powder or extracted, or the entire transgenic plant (or a part thereof) or transgenic organism that produces mogroside compounds can be minimally processed and used as a food ingredient.

[0398] In certain embodiments, the transgenic plants and organisms disclosed by the present invention will produce unique ratios of mogroside compounds. In some embodiments, the transgenic plants and organisms disclosed by the present invention can produce more of one or more mogroside compounds and less of other mogroside compounds, such as a greater amount of mogroside V compared with other mogroside compounds.

[0399] In a further embodiment, the complete mogroside biosynthetic pathway can be established in a selected plant or organism using a combinatorial approach, for example by endogenously activating one or more nucleic acid sequences of the mogroside biosynthetic pathway that are naturally present in the plant or organism, and providing any nucleic acid sequences of the mogroside biosynthetic pathway that are not naturally present in the plant or organism via one or more expression vectors comprising non-endogenous mogroside biosynthetic pathway nucleic acid sequences.

[0400] In a further embodiment, the transgenic plant or organism can also be engineered to express one or more nucleic acids involved in the biosynthesis of other sweeteners, such as genes involved in the production of serendipide I, α-serenin, steviol glycosides (stevia), rebaudioside M, or glycyrrhizin. Additionally, the transgenic plant or organism can be engineered to produce mogrosides and other sweeteners together in the same plant or organism, such as producing mogrosides and rebaudioside M.

[0401] II. Recombinant Host Cells and Species

[0402] Although transgenic plants are generally described in this disclosure, other host cells and organisms are also contemplated for certain embodiments of this disclosure.

[0403] As used herein, the term "recombinant host cell" is intended to refer to any host cell whose genome has been engineered to contain at least one nucleic acid sequence of the mogroside biosynthetic pathway disclosed herein, which nucleic acid sequences encode one or more polypeptides in certain embodiments. These sequences include, but are not limited to, nucleic acid or amino acid sequences that are not naturally present in the host cell or organism, DNA sequences that are not normally transcribed into RNA or translated into protein ("expressed"), and other sequences that have been altered from those normally present in the host cell, such as by increasing the copy number of a DNA sequence or altering the expression pattern or level of expression.

[0404] In addition to the plant species disclosed herein, many prokaryotes and additional eukaryotes are suitable for use as recombinant hosts in different aspects of this disclosure. In addition to any plant species, the recombinant host cell can be a bacterium, yeast, or fungus. Host cells or species selected for the production of mogroside compounds can be analyzed to determine whether any of the mogroside biosynthetic pathway genes are endogenous to the host cell or species and which mogroside biosynthetic pathway genes are absent. Genes that do not have an endogenous counterpart in the host cell or organism are typically assembled in one or more recombinant constructs and then transformed into the host cell or organism to provide the missing function.

[0405] Exemplary prokaryotic and eukaryotic species that can be used in certain aspects of the present disclosure include, but are not limited to, Agaricus, Aspergillus, Bacillus, Candida, Corynebacterium, Escherichia, Fusarium / Gibberella, Kluyveromyces, Laetiporus, Lentinus, Phaffia, Phanerochaete, Pichia, Physcomitrella, Rhodoturula, Saccharomyces, Sphaceloma, Schizosaccharomyces, Xanthophyllomyces, and Yarrowia. In some embodiments, the recombinant host can be a microorganism, such as Pichia pastoris, Schizosaccharomyces pombe, Aspergillus niger, or Saccharomyces cerevisiae. In some embodiments, the recombinant host can be a microorganism, such as Escherichia coli or Agrobacterium tumefaciens. It should be understood that certain microorganisms can be used for screening and testing target genes in a high-throughput manner, while other microorganisms with desired productivity or growth characteristics can be used for large-scale production of mogroside compounds. In certain embodiments, food-grade microorganisms can be used for large-scale production purposes.

[0406] III. Nucleic Acid and Polypeptide Sequences

[0407] Certain embodiments of the present disclosure relate to nucleic acid sequences (polynucleotides) of genes in the mogroside biosynthetic pathway and the corresponding amino acid sequences (proteins or polypeptides). Complementary sequences of any nucleic acid or protein sequence described herein are also provided.

[0408] As is well known in the art, "identity" is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences as determined by comparing the sequences. In the art, "identity" also refers to the degree of sequence relatedness between polypeptide or polynucleotide sequences as determined by the matches between these sequence strings. Methods for determining "identity" are designed to give the maximum match between the test sequences. In addition, methods for determining identity are incorporated into publicly available programs. "Identity" can be readily calculated by any of a number of methods known to those skilled in the art. Computer programs can be used to determine the "identity" between two sequences, and these programs include, but are not limited to, GCG; the 5 BLAST program suites, 3 designed for nucleotide sequence queries (BLASTN, BLASTX, and TBLASTX), and 2 designed for protein sequence queries (BLASTP and TBLASTN). The BLASTX program is publicly available from NCBI and other sources (BLAST Manual, NCBI NLM NIH, Bethesda, Md. 20894). The well-known Smith Waterman algorithm can also be used to determine identity.

[0409] According to the present disclosure, a polynucleotide or polypeptide sequence as described herein may exhibit at least about 34%, 40%, 50%, 60%, 62% or 70% to about 100% sequence identity with at least one sequence described herein. For example, in one embodiment, a mogroside biosynthetic pathway gene as described herein may have sequence identity of, for example, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 45%, 46%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% with a sequence selected from SEQ ID NO: 1, 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 38, 42, 46, 50, 54, 85 or 87 - 92, or its complement. In other embodiments, a mogroside biosynthetic pathway protein as described herein may have sequence identity of, for example, 34%, 35%, 36%, 37%, 38%, 99% or 100% with a sequence selected from SEQ ID NO: 2, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35 - 37, 39 - 41, 43 - 45, 47 - 49, 51 - 53, 55 - 57 or 86.

[0410] Parameters for polypeptide sequence comparison include the following: algorithm: Needleman and Wunsch (J. Mol. Biol. 48:443 - 453, 1970); comparison matrix: BLOSUM62 from Hentikoff and Hentikoff, (Proc. Natl. Acad. Sci. USA 89:10915 - 10919, 1992); gap penalty: 12; and gap length penalty: 4. Programs that can be used with these parameters are publicly available as the "GAP" program from Genetics Computer Group, Madison WI. The above parameters and no penalty for terminal gaps can be used as the default parameters for peptide comparison.

[0411] Parameters for nucleic acid sequence comparison include the following: algorithm: Needleman and Wunsch (ibid.); comparison matrix: match = +10; mismatch = 0; gap penalty: 50; and gap length penalty: 3. Programs that can be used with these parameters are publicly available as the "Gap" program from Genetics Computer Group, Madison Wis. The above parameters can be used as the default parameters for nucleic acid comparison.

[0412] As used herein, "hybridize", "hybridized", or "capable of hybridizing" shall be understood to mean the formation of a double-stranded or triple-stranded molecule or a molecule having a partially double-stranded or triple-stranded nature. Such hybridization can be carried out under relatively high stringency conditions, including low salt and / or high temperature conditions, for example by washing for 10 minutes at a temperature of about 50°C to about 70°C in about 0.02M to about 0.15M NaCl. In one embodiment of the present disclosure, the conditions are 0.15M NaCl and 70°C. Stringent conditions allow for little or no mismatch between the nucleic acid and the target strand. Such conditions are well known to those of ordinary skill in the art and are preferred for applications requiring high selectivity. Non-limiting applications include isolating nucleic acids, such as genes or nucleic acid fragments thereof, or detecting at least one specific mRNA transcript or nucleic acid fragment thereof, etc. Also included are proteins or polypeptides, or fragments thereof, such as any of those described herein.

[0413] With respect to the nucleic acid sequences disclosed herein, "fragment" refers to any portion of a polynucleotide molecule that retains useful functional properties. Useful fragments include oligonucleotides and polynucleotides that can be used as probes or primers in hybridization or amplification techniques or in the regulation of replication, transcription, or translation. A polynucleotide fragment refers to any subsequence of a polynucleotide, typically at least about 15 consecutive nucleotides, at least about 16 consecutive nucleotides, at least about 17 consecutive nucleotides, at least about 18 consecutive nucleotides, at least about 19 consecutive nucleotides, at least about 20 consecutive nucleotides, at least about 21 consecutive nucleotides, at least about 22 consecutive nucleotides, at least about 23 consecutive nucleotides, at least about 24 consecutive nucleotides, at least about 25 consecutive nucleotides, at least about 30 consecutive nucleotides, at least about 35 nucleotides, at least about 40 consecutive nucleotides, at least about 45 consecutive nucleotides, or at least about 50 or more nucleotides of any nucleic acid sequence provided herein.

[0414] As disclosed herein, a fragment can also include a subsequence of a polypeptide and protein molecule, or a subsequence of a polypeptide. The fragment can have antigenic potential, or can be a subsequence of a polypeptide that performs at least one biological function of the full-length polypeptide in substantially the same manner or to a similar extent as the full-length polypeptide. The size of the fragment can vary from as few as 5 amino acids to the full length of the full-length polypeptide, but is preferably at least about 10 amino acid lengths, at least about 15 amino acid lengths, at least about 20 amino acid lengths, at least about 25 amino acid lengths, at least about 30 amino acid lengths, at least about 35 amino acid lengths, at least about 40 amino acid lengths, at least about 45 amino acid lengths, at least about 50 amino acid lengths, at least about 55 amino acid lengths, or at least about 60 amino acid lengths or more of any of the amino acid sequences provided herein.

[0415] The nucleic acids of SEQ ID NO:1, 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 38, 42, 46, 50, 54, 58, 85 or 87 - 92 provided herein and the amino acids of SEQ ID NO:2, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35 - 37, 39 - 41, 43 - 45, 47 - 49, 51 - 53, 55 - 57, 59 or 86 provided herein can be from any source, such as identified as naturally occurring in plants, or synthetic, such as by mutagenizing SEQ ID NO:1, 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 38, 42, 46, 50, 54, 58, 85 or 87 - 92, for example to produce a coding sequence having a G / C content more like that of a naturally occurring gene from a particular plant. The naturally occurring sequences can be from any plant or algal species described herein.

[0416] IV. Transformation Constructs

[0417] Vectors for plant transformation or transformation of other host cells or organisms can include, for example, plasmids, cosmids, YACs (yeast artificial chromosomes), BACs (bacterial artificial chromosomes), or any other suitable cloning system, as well as DNA fragments thereof. Thus, when the terms "vector" or "expression vector" are used, they include all of the above types of vectors and nucleic acid sequences isolated therefrom. It is expected that using cloning systems with large insertion capacities will allow the introduction of large DNA sequences containing more than one selected gene. According to the present disclosure, this can be used to introduce genes corresponding to an entire biosynthetic pathway into plants. The introduction of these sequences can be facilitated by using bacterial or yeast artificial chromosomes (BACs or YACs, respectively), or even plant artificial chromosomes. For example, Hamilton et al. disclosed the use of BACs for Agrobacterium-mediated transformation (Proc. Natl. Acad. Sci. USA 93:9975-9979, 1996).

[0418] Expression cassettes isolated from these vectors are particularly useful for transformation. Of course, the DNA fragments used to transform plant cells will generally contain cDNAs, genes, or multiple genes that are desired to be introduced into and expressed in the host cell. As needed, these DNA fragments can further include structures such as the desired promoters, enhancers, polylinkers, terminators, or even regulatory genes. The DNA fragments or genes selected for introduction into the cell will generally encode proteins expressed in the resulting recombinant cell, producing a screenable or selectable trait and / or conferring an improved phenotype on the resulting transgenic plant. However, this may not always be the case, and the present disclosure also encompasses transgenic plants incorporating non-expressing transgenes. As previously mentioned, in addition to plant cells, the host cells in certain aspects of the present disclosure can be bacterial cells, such as Escherichia coli or Agrobacterium tumefaciens, yeast cells, fungi, algae, or cyanobacterial cells. Those skilled in the art know the genetic elements that must be present on the vector for successful transformation, selection, and propagation of the host cell containing the sequence of interest. The components that can be included in the vectors used in the present disclosure are as follows.

[0419] A. Promoters and other regulatory elements

[0420] In certain embodiments, the expression cassettes of the present disclosure further comprise one or more promoters, such as one or more nucleotide sequences shown in SEQ ID NO: 60-71, or nucleotide sequences having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to one or more nucleotide sequences shown in SEQ ID NO: 60-71.

[0421] In addition to the promoter sequences disclosed in the sequence listing, other exemplary promoters for expressing nucleic acid sequences include plant promoters such as the CaMV 35S promoter (Odell et al., Nature 313:810-812, 1985), or other promoters such as CaMV19S ((Lawton et al., Plant Mol. Biol. 9:315-324, 1987), nos (Ebert et al., Proc. Natl. Acad. Sci. USA 84:5745-5749, 1987), Adh (Walker et al., Proc. Natl. Acad. Sci. USA 84:6624-6628, 1987), sucrose synthase (Yang and Russell, Proc. Natl. Acad. Sci. USA 87:4144-4148, 1990), α-tubulin, actin (Wang et al., Mol. Cell Biol. 12:3399-3406, 1992), cab (Sullivan et al., Mol. Gen. Genet. 215:431-440, 1989), PEPCase (Hudspeth and Grula, Plant Mol. Biol. 12:579-589, 1989) or those associated with the R gene complex (Chandler et al., Plant Cell 1:1175-1183, 1989). Tissue-specific promoters such as root cell promoters (Conkling et al., Plant Physiol. 93:1203-1211, 1990) and tissue-specific enhancers are also expected to be useful, such as inducible promoters such as ABA- and swelling-inducible promoters. The PAL2 promoter is particularly useful for the present disclosure (U.S. Patent Application Publication No. 2004 / 0049802, the entire disclosure of which is specifically incorporated herein by reference). In one embodiment of the present disclosure, the native promoter of one or more mogroside pathway genes is used. In some embodiments, the promoter is a strong promoter or a weak promoter.

[0422] The DNA sequence between the transcription start site and the start of the coding sequence, i.e., the untranslated leader sequence, can also affect gene expression. Thus, one may wish to use a specific leader sequence with the transformation constructs of the present disclosure. Leader sequences are expected to include those that contain sequences predicted to direct optimal expression of the linked gene, i.e., consensus leader sequences that include those that can increase or maintain mRNA stability and prevent inappropriate translation initiation. The selection of these sequences is known to those skilled in the art according to the present disclosure. Sequences from genes highly expressed in plants may be desirable.

[0423] It is contemplated that vectors for use in accordance with the present disclosure can be constructed to include the ocs enhancer element. This element was first identified as a 16 bp palindromic enhancer of the octopine synthase (ocs) gene from Agrobacterium (Ellis et al., EMBO J. 6:3203-3208, 1987), and is present in at least 10 other promoters (Bouchez et al., EMBO J. 8:4197-4204, 1989). When enhancer elements (e.g., the ocs element, particularly multiple copies of this element) are applied to plant transformation, the transcriptional level of adjacent promoters can be increased.

[0424] It is contemplated that the mogroside biosynthetic pathway coding sequences can be introduced under the control of a novel promoter or enhancer, etc., or a homologous or tissue-specific promoter or control element. Vectors for tissue-specific targeting of genes in transgenic plants typically include tissue-specific promoters and may also include other tissue-specific control elements such as enhancer sequences. In view of the present disclosure, promoters that direct specific or enhanced expression in certain plant tissues are known to those skilled in the art. These promoters include, for example, the rbcS promoter specific to green tissues; the ocs, nos, and mas promoters that have higher activity in root or wounded leaf tissues.

[0425] B. Terminator

[0426] In certain embodiments, the expression cassette of the present disclosure further comprises one or more terminators, such as one or more nucleotide sequences shown in SEQ ID NO: 72-80, or nucleotide sequences having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with one or more nucleotide sequences shown in SEQ ID NO: 72-80.

[0427] Conversion constructs prepared according to the present disclosure generally include 3'-end DNA sequences which serve as signals for terminating transcription and allow polyadenylation of the mRNA produced from the coding sequence operably linked to the promoter. In one embodiment of the present disclosure, the native terminator of the mogroside biosynthetic pathway coding sequence is used. Alternatively, a heterologous 3'-end can enhance the expression of sense or antisense mogroside biosynthetic pathway coding sequences. In addition to the terminator sequences disclosed in the sequence listing, other examples of terminators considered useful herein include those from the nopaline synthase gene (nos 3'-end) of Agrobacterium tumefaciens (Bevan et al., Nucl. Acids Res. 11:369-385, 1983), the terminator of the T7 transcript of the octopine synthase gene of Agrobacterium tumefaciens, and the 3'-ends of the protease inhibitor I or II genes from potato or tomato. Regulatory elements such as the Adh intron (Callis et al., Genes Dev. 1:1183-1200, 1987), the sucrose synthase intron (Vasil et al., Plant Physiol. 91:1575-1579, 1989) or the TMV omega element (Gallie and Kado, Proc. Natl. Acad. Sci. USA 86:129-132, 1989) can be further included if desired.

[0428] C. Transit or signal peptides

[0429] In certain embodiments of the present disclosure, transit or signal sequences can be incorporated into the mogroside biosynthetic pathway coding sequences. Sequences linked to the coding sequence of an expressed gene are referred to as transit sequences (usually into vacuoles, vesicles, plastids and other intracellular organelles) and signal sequences (usually to the endoplasmic reticulum, Golgi apparatus and outside the cell membrane), which are removed from the initial translation product after translation and facilitate the transport of the protein into or through intracellular or extracellular membranes. By facilitating the transport of the protein into and out of intracellular and extracellular compartments, these sequences can increase the accumulation of gene products that protect them from proteolytic degradation. These sequences also allow additional mRNA sequences from highly expressed genes to attach to the coding sequence of the gene. Since mRNA translated by ribosomes is more stable than naked mRNA, the presence of translatable mRNA in front of the gene can increase the overall stability of the mRNA transcript from the gene, thereby increasing the synthesis of the gene product. Since transit and signal sequences are usually removed from the initial translation product after translation, the use of these sequences allows the addition of additional translation sequences that may not appear on the final polypeptide. It is also contemplated that for enhancing the stability of the protein, the targeting of certain proteins may be desirable (U.S. Patent No. 5,545,818, incorporated herein by reference in its entirety).

[0430] Alternatively, vectors can be constructed and used to intracellularly target specific gene products in transgenic plant cells or direct proteins to the extracellular environment. This is typically accomplished by ligating a DNA sequence encoding a transit or signal peptide sequence to the coding sequence of a specific gene. The resulting transit or signal peptide will transport the protein to a specific intracellular or extracellular destination, respectively, and will then be removed post-translationally.

[0431] D. Marker Genes

[0432] The ability to identify transformants can be provided or enhanced by using selectable or screenable marker proteins. A "marker gene" is a gene that confers a unique phenotype on cells expressing the marker protein, thus allowing such transformed cells to be distinguished from cells that do not have the marker. Such genes can encode selectable or screenable markers, depending on whether the marker confers a trait that can be "selected" by a person skilled in the art by chemical means, i.e., by using a selection agent (e.g., herbicide, antibiotic, etc.), or whether it is merely a trait that can be identified by a person skilled in the art by observation or testing, i.e., by "screening" (e.g., green fluorescent protein). In addition to the marker genes disclosed in the sequence listing, many additional examples of suitable marker proteins are known in the art and can be used in the practice of the present disclosure.

[0433] The terms "selectable" or "screenable" marker also include genes encoding "secretable markers", the secretion of which can be detected as a means of identifying or selecting transformed cells. Examples include markers for secretable antigens that can be identified by antibody interaction, or even secretable enzymes that can be detected by their catalytic activity. Secretable proteins fall into many categories, including small diffusible proteins that can be detected, for example, by ELISA; small active enzymes that can be detected in extracellular solutions (e.g., α - amylase, β - lactamase, phosphinothricin acetyltransferase); and proteins that are inserted or trapped in the cell wall (e.g., proteins including leader sequences found in expression units such as extensin or tobacco PR S).

[0434] Many selectable marker coding regions are known and available for use in the present disclosure, including but not limited to neo (Potrykus et al., Mol. Gen. Genet. 199:169-177, 1985), which confers kanamycin resistance and can be selected with kanamycin, G418, paromomycin, etc.; bar, which confers bialaphos or phosphinothricin resistance; a mutant EPSP synthase protein that confers glyphosate resistance; nitrilase such as bxn from Klebsiella ozaenae, which confers resistance to bromoxynil (Stalker et al., J. Biol. Chem. 263:6310-6314, 1988); mutant acetolactate synthase (ALS), which confers resistance to imidazolinones, sulfonylureas or other ALS-inhibiting chemicals (European Patent Application 154,204, 1985); methotrexate-resistant DHFR (Thillet et al., J. Biol. Chem. 263:12500-12508, 1988), bromoxynil dehalogenase that confers resistance to the herbicide bromoxynil; or a mutant anthranilate synthase that confers resistance to 5-methyltryptophan, or a sequence that confers resistance to dicamba.

[0435] Exemplary embodiments of selectable markers that can be used to select transformants in the system are those encoding phosphinothricin acetyltransferase, such as the bar gene from Streptomyces hygroscopicus or the pat gene from Streptomyces viridochromogenes. Phosphinothricin acetyltransferase (PAT) inactivates the active ingredient in the herbicides bialaphos and phosphinothricin (PPT). PPT inhibits glutamine synthetase, causing rapid accumulation of ammonia and cell death.

[0436] Useful selectable markers include the β-glucuronidase (GUS) or uidA gene, which encodes an enzyme known for various chromogenic substrates; the R-locus gene, which encodes a product that regulates the production of anthocyanin pigments (red) in plant tissues; the β-lactamase gene (Sutcliffe, Proc. Natl. Acad. Sci. USA 75:3737-3741, 1978), which encodes an enzyme known for various chromogenic substrates (e.g., PADAC, a chromogenic cephalosporin); the xylE gene (Zukowsky et al., Proc. Natl. Acad. Sci. USA 80:1101-1105, 1983), which encodes a catechol dioxygenase capable of converting the chromogenic catechol; the α-amylase gene (Ikuta et al., Biotechnology 8:241-242, 1990); the tyrosinase gene (Katz et al., J. Gen. Microbiol. 129:2703-2714, 1983), which encodes an enzyme capable of oxidizing tyrosine to DOPA and dopaquinone, which in turn condense to form the easily detectable compound melanin; the β-galactosidase gene, which encodes an enzyme in the presence of chromogenic substrates; the luciferase (lux) gene (Ow et al., Science 234:856-859, 1986), which allows bioluminescence detection; the aequorin gene (Prasher et al., Biochem. Biophys. Res. Commun. 126:1259-1268, 1985), which can be used for calcium-sensitive bioluminescence detection; or a gene encoding green fluorescent protein (GFP; Sheen et al., Plant J. 8:777-784, 1995; Haseloff et al., Proc. Natl. Acad. Sci. USA 94:2122-2127, 1997; Reichel et al., Proc. Natl. Acad. Sci. USA 93:5888-5893, 1996; WO 97 / 41228) is also considered a useful reporter gene. The expression of green fluorescent protein can be visualized as fluorescence after irradiation with light of a specific wavelength in cells or plants.

[0437] E. Additional agronomic traits

[0438] In certain embodiments, one or more additional agronomically beneficial traits are engineered into the transgenic plants or organisms of the present disclosure. A "trait" refers to a physiological, morphological, biochemical, or physical characteristic of a plant or organism or of a particular plant material or cell. In some cases, the trait is visible to the human eye, such as seed or plant size, or can be measured by biochemical techniques, such as detecting the protein, starch, or oil content of a seed or leaf, or by observing a metabolic or physiological process, such as measuring carbon dioxide uptake, or by observing the expression level of one or more genes, such as by using northern analysis, RT-PCR, microarray gene expression assays, or reporter gene expression systems, or by agricultural observations such as stress tolerance, yield, or pathogen tolerance. However, any technique can be used to measure the amount, relative level, or difference of any selected compound or macromolecule in a transgenic plant.

[0439] "Trait modification" refers to a detectable difference in the characteristics of a plant or organism that expresses or ectopically expresses a polynucleotide or polypeptide relative to a plant or organism that does not express or ectopically expresses the polynucleotide or polypeptide (e.g., a wild-type or other control plant or organism). In some cases, trait modification can be quantitatively evaluated. For example, compared to a wild-type or other control plant or organism, trait modification can result in an increase or decrease in the observed trait (difference) of at least about 2%, at least 5% difference, at least about 10% difference, at least about 20% difference, at least about 30%, at least about 50%, at least about 70%, or at least about 100%, or even greater differences. Natural variation is known to exist in modified traits. Thus, the observed trait modification results in a change in the normal distribution of the trait in the plant or organism compared to the distribution observed in a wild-type plant or organism.

[0440] Trait modifications of particular interest in the plants of the present disclosure include those to seeds (e.g., embryo or endosperm), fruits, roots, flowers, leaves, stems, branches, seedlings, etc., including: enhanced tolerance to environmental conditions (including freezing, cold, heat, drought, water saturation, radiation, and ozone); improved tolerance to microbial, fungal, or viral diseases; improved tolerance to pest infestations including insects, nematodes, mollusks, parasitic higher plants, etc.; reduced herbicide sensitivity or increased herbicide tolerance, e.g., increased glyphosate or dicamba tolerance; improved heavy metal tolerance or enhanced ability to absorb heavy metals; improved growth under poor light conditions (e.g., low light and / or short day), or altered expression levels of target genes. Other phenotypes that can be modified relate to the production of plant metabolites such as changes in the production of paclitaxel, tocopherols, tocotrienols, sterols, phytosterols, vitamins, wax monomers, antioxidants, amino acids, lignin, cellulose, tannins, prenyl lipids (such as chlorophyll and carotenoids), glucosinolates, and terpenoids, enhanced or constitutively altered protein or oil production (especially in seeds), or modified sugar (insoluble or soluble) and / or starch composition. Physical plant characteristics that can be modified include cell development (e.g., number of trichomes), fruit and seed size and number, yield of plant parts (e.g., stems, leaves, inflorescences, and roots), seed stability during storage, characteristics of seed pods (e.g., sensitivity to breaking), root hair length and number, internode distance, or seed coat quality. Plant growth characteristics that can be modified include growth rate, seed germination rate, vigor of plants and seedlings, leaf and flower senescence, male sterility, apomixis, flowering time, flower abscission, nitrogen uptake rate, osmotic sensitivity to soluble sugar concentration, biomass, or transpiration characteristics, as well as plant architectural characteristics such as apical dominance, branching pattern, number of organs, organ characteristics, organ shape, or size. Additionally, the amount of natural sugars can be reduced, and color can be reduced or removed.

[0441] V. Gene Editing

[0442] One method of generating the transgenic plants of the present disclosure is by genomic modification using site-specific integration or genome editing. Targeted modification of the plant genome by using genome editing methods can be used to generate improved plant lines by modifying the plant genomic DNA. As used herein, "site-directed integration" refers to genome editing methods capable of targeting the insertion of one or more nucleic acids of interest into the plant genome. Suitable methods for altering wild-type DNA sequences or pre-existing transgenic sequences or inserting DNA into the plant genome at a predetermined chromosomal locus include any methods known in the art. Exemplary methods include the use of sequence-specific nucleases such as zinc finger nucleases, engineered or native meganucleases, TALE-endonucleases, or RNA-guided endonucleases (e.g., clustered regularly interspaced short palindromic repeats (CRISPR) / Cas9 system, CRISPR / Cpf1 system, CRISPR / CasX system, CRISPR / CasY system, or CRISPR / Cascade system). Some embodiments relate to methods of genome editing by introducing precise base pair modifications into the plant genome by using single-stranded oligonucleotides. Genome editing methods for modifying, deleting, or inserting nucleic acid sequences into genomic DNA are known in the art.

[0443] In certain embodiments, the present disclosure provides for modifying or replacing existing coding sequences within the plant genome, such as existing transgenic insertions, with sequences encoding different proteins or expression cassettes comprising such proteins. Some embodiments relate to the use of known genome editing methods such as zinc finger nucleases, engineered or native meganucleases, TALE-endonucleases, or RNA-guided endonucleases (e.g., clustered regularly interspaced short palindromic repeats (CRISPR) / Cas9 system, CRISPR / Cpf1 system, CRISPR / CasX system, CRISPR / CasY system, or CRISPR / Cascade system).

[0444] Accordingly, some embodiments can relate to recombinant DNA constructs that contain an expression cassette encoding a site-specific nuclease and optionally any associated proteins for genome modification. These nuclease expression cassettes can be present as donor templates for template editing in the same molecule or vector. Several methods for site-directed integration are known in the art, including different sequence-specific nucleases (or proteins or guide RNAs or complexes of both) that cleave genomic DNA to generate double-strand breaks (DSBs) or nicks at desired genomic sites or loci. As understood in the art, during the repair of DSBs or nicks introduced by nucleases, donor template DNA, transgenes, or expression cassettes can be integrated into the genome at the site of the DSB or nick. The presence of homologous arms in the DNA to be integrated can facilitate the reception and targeting of the inserted sequence into the plant genome by homologous recombination during repair, although the insertion event can occur by non-homologous end joining (NHEJ). As used herein, the term "double-strand break inducer" refers to any reagent that can induce a double-strand break (DSB) in a DNA molecule. In some embodiments, the double-strand break inducer is a site-specific genome modification enzyme.

[0445] As used herein, the term "site-specific genome modification enzyme" refers to any enzyme capable of modifying a nucleotide sequence in a sequence-specific manner. In some embodiments, the site-specific genome modification enzyme modifies the genome by inducing a single-strand break. In some embodiments, the site-specific genome modification enzyme modifies the genome by inducing a double-strand break. In some embodiments, the site-specific genome modification enzyme comprises a cytidine deaminase. In some embodiments, the site-specific genome modification enzyme comprises an adenine deaminase. Site-specific genome modification enzymes include endonucleases, recombinases, transposases, deaminases, helicases, and any combination thereof. In some embodiments, the site-specific genome modification enzyme is a sequence-specific nuclease.

[0446] In one aspect, the endonuclease is selected from the group consisting of meganucleases, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), Argonaute (non-limiting examples of Argonaute proteins include Thermus thermophilus Argonaute (TtAgo), Pyrococcus furiosus Argonaute (PfAgo), and Natronobacterium gregoryi Argonaute (NgAgo)), RNA-guided nucleases such as CRISPR-associated nucleases (non-limiting examples of CRISPR-associated nucleases include, but are not limited to, Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, Cpf1, CasX, CasY, homologs thereof, or modified versions thereof).

[0447] In some embodiments, the site-specific genome modification enzyme is a recombinase. Non-limiting examples of recombinases include tyrosine recombinases associated with DNA recognition motifs, which are selected from the group consisting of Cre recombinase, Gin recombinase, Flp recombinase, and Tnp1 recombinase. In one aspect, Cre recombinase or Gin recombinase is tethered to a zinc finger DNA-binding domain, or a TALE DNA-binding domain, or a Cas9 nuclease. In another aspect, serine recombinases associated with DNA recognition motifs are selected from the group consisting of PhiC31 integrase, R4 integrase, and TP-901 integrase. In yet another aspect, DNA transposases associated with the DNA-binding domains provided herein are selected from the group consisting of TALE-piggyBa and TALE-mutants.

[0448] By introducing a DNA of interest and the disclosed site-specific genome modification enzyme, any DNA of interest provided herein can be integrated into a target site of a chromosomal sequence. Any method provided herein can utilize any site-specific genome modification enzyme disclosed herein.

[0449] VI. Antisense and RNAi Constructs

[0450] Antisense and RNAi treatments represent one way to alter the activity of genes in the mogroside biosynthetic pathway according to the present disclosure (e.g., by downregulating genes or transcription factors that inhibit the expression of genes in the mogroside biosynthetic pathway).

[0451] Techniques for RNAi are well known in the art and are described, for example, in Lehner et al. (Brief Funct. Genomic Proteomic 3:68 - 83, 2004) and Downward (BMJ 328:1245 - 1248, 2004). This technique is based on the fact that double-stranded RNA can direct the degradation of messenger RNA having a sequence complementary to one or the other strand (Fire et al., Nature 391:806 - 811, 1998). Thus, by expressing a specific coding sequence in both the sense and antisense directions, as a fragment or longer portion of the corresponding coding sequence, the expression of that coding sequence can be downregulated.

[0452] Antisense (and in some aspects RNAi) methods take advantage of the fact that nucleic acids tend to pair with "complementary" sequences. Complementary means that a polynucleotide is a polynucleotide capable of base pairing according to the standard Watson-Crick complementary rules. That is, the larger purine will pair with the smaller pyrimidine base to form combinations where guanine pairs with cytosine (G:C) and in the case of DNA thymine pairs with adenine (A:T) or in the case of RNA thymine pairs with uracil (A:U). The inclusion of less common bases such as inosine, 5-methylcytosine, 6-methyladenine, hypoxanthine, and other bases in the hybridizing sequences does not interfere with pairing.

[0453] Targeting double-stranded (ds) DNA with a polynucleotide results in triple helix formation; targeting RNA results in double helix formation. When introduced into target cells, antisense oligonucleotides specifically bind to their target polynucleotides and interfere with transcription, RNA processing, transport, translation, and / or stability. Antisense and RNAi constructs, or DNA encoding such RNA, can be used to inhibit gene transcription or translation or both in vitro or in vivo, such as within a host plant cell. In certain embodiments of the present disclosure, such oligonucleotides can comprise any unique portion of the nucleic acid sequences provided herein. In certain embodiments of the present disclosure, such sequences comprise at least 18, 20, 25, 30, 50, 75, or 100 or more contiguous nucleic acids of the nucleic acid sequence of interest and / or its complementary sequence, which can be in the sense and / or antisense direction. By including sequences in both the sense and antisense directions, increased inhibition of the corresponding coding sequence can be achieved.

[0454] Constructs can be designed that are complementary to all or part of the promoter and other control regions, exons, introns, or even exon-intron boundaries of a gene. The most effective constructs are expected to include regions complementary to intron / exon splice junctions. Thus, one embodiment is proposed to include constructs complementary to regions within 50-200 bases of an intron-exon splice junction. It has been observed that some exon sequences can be included in the construct without severely affecting its target selectivity. The amount of exon material included will vary depending on the specific exon and intron sequences used. Whether too much exon DNA is included can be easily tested simply by testing the construct in vitro to determine whether normal cell function is affected or whether the expression of the relevant gene with the complementary sequence is affected.

[0455] As described above, "complementary" or "antisense" refers to polynucleotide sequences that are substantially complementary over their entire length and have very few base mismatches. For example, when sequences of 15 bases in length have complementary nucleotides at 13 or 14 positions, they can be said to be complementary. Naturally, completely complementary sequences will be sequences that are completely complementary over their entire length and have no base mismatches. Other sequences with lower degrees of homology are also expected. For example, RNAi or antisense constructs can be designed that have limited regions of high homology but also contain non-homologous regions (e.g., ribozymes; see above). Methods for selecting and designing sequences that produce RNAi are well known in the art (e.g., Reynolds et al., Nat. Biotechnol. 22:326-330, 2004). These molecules, although having less than 50% homology, will bind to the target sequence under appropriate conditions.

[0456] It may be advantageous to combine portions of genomic DNA with cDNA or synthetic sequences to generate specific constructs. For example, genomic clones can be used when introns are required in the final construct. cDNA or synthetic polynucleotides can provide more convenient restriction sites for the remainder of the construct and will thus be used for the rest of the sequence. Constructs for generating RNAi can also contain tandem repeats of subsequences that exhibit gene regulatory activity.

[0457] VII. Transformation

[0458] In some embodiments, the transgenic plants of the present disclosure are produced by transforming selected natural plants with one or more expression cassettes disclosed herein. Natural plants before transformation do not naturally produce all of the mogrosanol / mogroside pathway enzymes, nor do they produce non-natural mogrosanol and mogroside compounds. Although natural plants can produce one or more enzymes capable of producing mogrosanol precursors or mogrosanol, these plants do not naturally produce non-natural mogrosides. In certain embodiments, the natural plants selected for transformation include wild-type, or untransformed, or non-transformed watermelons, which do not naturally produce detectable amounts of mogrosanol or mogroside compounds. Other plants that can be transformed with one or more expression cassettes disclosed herein include, but are not limited to, cantaloupe, honeydew melon, winter melon, casaba melon, Persian melon, citron melon, musk melon, honeydew melon, crenshaw melon, Christmas melon, fairy melon, caravelle melon, hami melon, rock melon, golden Langkawi melon, Korean melon, saticoy melon, Galia melon, gyokuro melon, gold medal melon, ten melon, new century melon, banana melon, yubari king melon, sugar melon, tiger melon, climbing melon, horned melon, cucamelon, casabanana melon, melon), ginseng fruit, pineapple melon, camouflage melon, golden loofah, bitter melon, charentaismelon, crane melon, arrow melon, honey melon, rambutan melon, autumn melon, quick melon, cucumber, tomato, lettuce, spinach, rice, oats, corn, sorghum, bitter apple (Citrullus colocynthis), pumpkin, beet, tobacco, miscanthus, bush cherry, peach, nectarine, apricot, radish, plum, sour cherry, apple, pear, sweet cherry, citrus, zucchini, pea, turnip or Nicotiana benthamiana plant. Mogroside compounds can be isolated from any part of the transformed plant, including but not limited to fruit (juice or skin), leaves, roots, seeds or flowers.

[0459] Suitable methods for the transformation of plants or other cells of the present disclosure are considered to practically include any method that can introduce DNA into cells, such as by direct delivery of DNA, for example, by PEG-mediated protoplast transformation (Omirulleh et al., Plant. Mol. Biol. 21:414-428, 1993), by desiccation / inhibition-mediated DNA uptake (Potrykus et al., Mol. Gen. Genet. 199:169-177, 1985), by electroporation (U.S. Patent No. 5,384,253, which is hereby incorporated by reference in its entirety), by agitation with silicon carbide fibers (U.S. Patent Nos. 5,302,523 and 5,464,765, which are hereby incorporated by reference in their entireties), by Agrobacterium-mediated transformation (U.S. Patent No. 5,591,616 and U.S. Patent No. 5,563,055; both of which are hereby incorporated by reference in their entireties), and by acceleration of DNA-coated particles (U.S. Patent No. 5,550,318; U.S. Patent No. 5,538,877; and U.S. Patent No. 5,538,880; each of which is hereby incorporated by reference in its entirety), etc. By applying these techniques, cells of almost any plant species can be transiently or stably transformed, and these cells can be developed into transgenic plants.

[0460] A. Agrobacterium-mediated transformation

[0461] Agrobacterium-mediated transfer is a widely applicable system for introducing genes into plant cells because DNA can be introduced into whole plant tissues, thus bypassing the need to regenerate whole plants from protoplasts. The introduction of DNA into plant cells using Agrobacterium-mediated plant integration vectors is well known in the art. See, for example, Frale et al. (Proc. Natl. Acad. Sci. USA 80:4803-4807, 1985), and U.S. Patent No. 5,563,055, which is hereby incorporated by reference in its entirety.

[0462] Agrobacterium-mediated transformation is most effective in dicotyledonous plants and is an efficient method for transforming dicotyledonous plants (including Arabidopsis, tobacco, tomato, alfalfa, and potato). In fact, although Agrobacterium-mediated transformation has been routinely used for dicotyledonous plants for many years, it has only recently become applicable to monocotyledonous plants. Advances in Agrobacterium-mediated transformation technology now allow the technique to be applied to almost all monocotyledonous plants. For example, Agrobacterium-mediated transformation has now been applied to rice (Hiei et al., Plant Mol. Biol. 35:205-218, 1997; U.S. Patent No. 5,591,616, which is hereby incorporated by reference in its entirety), wheat and barley (McCormac et al., Mol. Biotechnol. 9:155-159, 1998), alfalfa, and maize (Ishida et al., Nat. Biotechnol. 14:745-750, 1996). Similarly, Agrobacterium-mediated transformation has also been shown to be effective in Miscanthus.

[0463] Modern Agrobacterium transformation vectors are able to replicate in E. coli. E. coli as well as Agrobacterium allow for convenient manipulation. In addition, recent technological advances in vectors for Agrobacterium-mediated gene transfer have improved the arrangement of genes and restriction sites in the vector to facilitate the construction of vectors capable of expressing a variety of polypeptide-encoding genes. The vectors have convenient multiple cloning regions flanked by a promoter and a polyadenylation site for direct expression of the inserted polypeptide-encoding gene and are suitable for the purposes of the present invention. Additionally, Agrobacterium containing armed and disarmed Ti genes can be used for transformation. This is the method of choice in those plant lines in which Agrobacterium-mediated transformation is effective because of the ease and well-defined nature of its gene transfer.

[0464] B. Electroporation

[0465] For transformation by electroporation, friable tissues such as suspensions of cells or embryogenic callus can be used, or immature embryos or other organized tissues can be transformed directly. In this technique, the technician partially degrades the cell wall of the selected cells by exposing the selected cells to a pectin-degrading enzyme (pectin lyase) or by mechanically damaging them in a controlled manner. Examples of some species that have been transformed by electroporation of intact cells include maize (U.S. Patent No. 5,384,253, which is hereby incorporated by reference in its entirety; Rhodes et al., Methods Mol. Biol. 55:121-131, 1995; D’Halluin et al., Plant Cell 4:1495-1505, 1992), wheat (Zhou et al., Plant Cell Rep. 12:612-616, 1993), tomato (Tsukada et al., Plant Cell Physiol. 30:599-603, 1989), soybean (Christou et al., Proc. Natl. Acad. Sci. USA 84:3962-3966, 1987), and tobacco (Riggs and Bates, Proc. Natl. Acad. Sci. USA 83:5602-5606, 1986).

[0466] Electroporation-mediated transformation of plants can also be performed using protoplasts (Bates, Mol. Biotechnol. 2:135-145, 1994; Lazzeri, Methods Mol. Biol. 49:95-106, 1995). For example, the generation of transgenic soybean plants by electroporation of cotyledon-derived protoplasts is described in WO 9217598 (which is hereby incorporated by reference in particular). Other examples of species for which protoplast transformation has been described include barley (Lazzeri, ibid.), sorghum (Battraw et al., Theor. Appl. Genet. 82:161-168, 1991), maize (Rhodes et al., Science 240:204-207, 1988), wheat (He et al., Plant Cell Rep. 14:192-196, 1994), and tomato (Tsukada, ibid.).

[0467] C. Particle Bombardment

[0468] Another method of delivering a transforming DNA fragment to a plant cell according to the present disclosure is microprojectile bombardment (U.S. Patent No. 5,550,318; U.S. Patent No. 5,538,880; U.S. Patent No. 5,610,042; and PCT Application WO94 / 09699, each of which is incorporated herein by reference in its entirety). In this method, particles can be coated with nucleic acid and delivered into cells by a propulsive force. Exemplary particles include those composed of tungsten, platinum, and commonly gold. It is contemplated that in some cases, precipitation of DNA onto the metal particles is not necessary for delivering DNA to recipient cells using microprojectile bombardment. However, it is contemplated that the particles can contain DNA rather than being coated with DNA. Thus, it is proposed that DNA-coated particles can increase the level of DNA delivery by particle bombardment, but they are not essential per se.

[0469] For bombardment, the cells in suspension are concentrated on a filter or solid medium. Alternatively, immature embryos or other target cells can be arranged on solid medium. The cells to be bombarded are located at an appropriate distance below the stopping plate of the macroprojectile.

[0470] An exemplary embodiment of a method for accelerating the delivery of DNA into plant cells is the biolistic particle delivery system, which can be used to propel particles coated with DNA or cells through a screen (e.g., a stainless steel or Nytex screen) onto the surface of a filter covered with suspension-cultured monocotyledonous plant cells. The screen disperses the particles so that they are not delivered to the recipient cells as large aggregates. The microprojectile bombardment technique is widely applicable and can be used to transform almost any plant species. Examples of species that have been transformed by microprojectile bombardment include monocotyledonous species such as maize (PCT application WO95 / 06128), barley (Ritala et al., Plant Mol. Biol. 24:317-325, 1994; Hensgens et al., Plant Mol. Biol. 22:1101-1127, 1993), wheat (U.S. Patent No. 5,563,055, which is hereby incorporated by reference in its entirety), rice (Hensgens et al., ibid.), oats (Torbet et al., Crop Science 38:226-231, 1998), rye (Hensgens et al., ibid.), sugarcane (Bower et al., Plant J. 2:409-416, 1992), and sorghum (Casas et al., Proc. Natl. Acad. Sci. USA 90:11212-11216, 1993; Hagio et al., Plant Cell Rep. 10:260-264, 1991); and many dicotyledonous plants, including tobacco (Tomes et al., Plant Mol. Biol. 14:261-268, 1990), soybean (U.S. Patent No. 5,322,783, which is hereby incorporated by reference in its entirety), sunflower (Knittel et al., Plant Cell Rep. 14:81-86, 1994), peanut ((Singsit et al., Transgenic Res. 6:169-176, 1997), cotton (McCabe and Martinell, Nat. Biotechnol. 11:596-598, 1993), tomato (Van Eck et al., Plant Cell. Rep. 14:299-304, 1995), Miscanthus (Richards et al., Plant Cell Rep. 20:48-54, 2001), and common bean (U.S. Patent No. 5,563,055, which is hereby incorporated by reference in its entirety).

[0471] D. Other transformation methods

[0472] Transformation of protoplasts can be achieved using methods based on calcium phosphate precipitation, polyethylene glycol treatment, electroporation, and combinations of these treatments (see, e.g., Potrykus et al., supra; Omirulleh et al., supra). The application of these systems in different plant lines depends on the ability to regenerate that particular plant line from protoplasts. Exemplary methods for regenerating cereals from protoplasts have been described (Toriyama et al., Nat. Biotechnol. 6:1072-1074, 1988; Abdullah et al., Nat. Biotechnol. 4:1087-1090, 1986; Omirulleh et al., supra, and U.S. Patent No. 5,508,184; each incorporated herein by reference in its entirety). Examples of transformation using direct uptake by cereal protoplasts include transformation of rice (Ghosh-Biswas et al., J. Biotechnol. 32:1-10, 1994), sorghum (Battraw et al., supra), barley (Lazzeri, supra), oats, and maize (Omirulleh et al., supra).

[0473] To transform plant lines that cannot be successfully regenerated from protoplasts, other methods for introducing DNA into intact cells or tissues can be utilized. For example, regeneration of cereals from immature embryos or explants can be carried out as described (Vasil, supra). In addition, silicon carbide fiber-mediated transformation can be used with or without protoplasting (Kaeppler et al., Theor. Appl. Genet. 84:560-566, 1992; U.S. Patent No. 5,563,055, incorporated herein by reference in its entirety). Transformation with this technique is accomplished by agitating silicon carbide fibers and cells in a DNA solution. When the cells are punctured, the DNA enters passively. This technique has been successfully used, for example, for monocot cereal maize (PCT application WO95 / 06128, incorporated herein by reference in its entirety) and rice (Nagatani et al., Biotechnol. Tech. 11:471-473, 1997).

[0474] E. Tissue Culture

[0475] Tissue culture can be used in certain transformation techniques to prepare cells for transformation and to regenerate plants therefrom. Maintenance of tissue culture requires the use of culture media and a controlled environment. A "culture medium" refers to a variety of nutrient mixtures used to grow cells in vitro, i.e., outside of a whole, living organism. Culture media are typically suspensions of the various components (salts, amino acids, growth regulators, sugars, buffers) required for the growth of most cell types. However, each specific cell type requires a specific range of proportions of components for growth, and an even more specific range of formulations for optimal growth. The cell growth rate will also vary with cultures initiated with a series of media that permit the growth of that cell type.

[0476] Nutrient media are prepared as liquids, but this can be solidified by adding the liquid to materials that can provide a solid support. Agar is most commonly used for this purpose. AGAR, and are specific types of solid supports suitable for culturing plant cells in tissue culture.

[0477] Some cell types will grow and divide in liquid suspension or on solid media. As disclosed herein, plant cells will grow in suspension or on solid media, but regenerating plants from suspension cultures generally requires transfer from liquid media to solid media at certain points in development. The type and degree of cell differentiation in a culture are affected not only by the type of medium and environment (e.g., pH) used, but also by whether the medium is solid or liquid.

[0478] Tissues that can be grown in culture include meristematic cells, type I, II, and III callus, immature embryos, and gamete cells such as microspores, pollen, sperm, and egg cells. Type I, II, and III callus can be initiated from tissue sources including, but not limited to, immature embryos, shoot apical meristems of seedlings, roots, leaves, microspores, etc. Those cells that are capable of proliferating as callus are also recipient cells for genetic transformation.

[0479] Somatic cells come in various types. Embryogenic cells are an example of somatic cells that can be induced to regenerate plants by embryogenesis. Non-embryogenic cells are those that do not typically respond in this way. Certain techniques can be used to enrich for recipient cells in a cell population. For example, development of type II callus, followed by manual selection and culturing of friable embryogenic tissue, generally results in cell enrichment. Artificial selection techniques that can be used to select target cells can include, for example, assessing cell morphology and differentiation, or various physical or biological means can be used. Cryopreservation is also a possible method for selecting recipient cells.

[0480] Artificially selecting recipient cells, for example by selecting embryogenic cells from the surface of type II callus, is a method that can be used to attempt to enrich specific cells prior to culturing (whether on solid medium or in suspension).

[0481] When used, the cultured cells can be grown on a solid support or in the form of a liquid suspension. In either case, nutrients can be provided to the cells in the form of a culture medium, and the environmental conditions can be controlled. There are many types of tissue culture media composed of various amino acids, salts, sugars, growth regulators, and vitamins. Most of the media employed in the practice of the present disclosure will have some similar components, but depending on the specific application contemplated, their composition and proportions can vary. For example, various cell types typically grow in more than one type of medium, but will exhibit different growth rates and different morphologies depending on the growth medium. In some media, the cells survive but do not divide. Various types of media suitable for culturing plant cells have been described previously. Examples of such media include, but are not limited to, the N6 medium described by Chu et al., (Sci. Sin. [Peking] 18:659 - 668, 1975) and the MS medium (Murashige and Skoog, Physiol. Plant 15:473 - 479, 1962).

[0482] VIII. Production and Characterization of Stably Transformed Plants

[0483] After delivering the foreign DNA into the recipient cells, the next step generally involves identifying the transformed cells for further culturing and plant regeneration. To improve the ability to identify transformants, it may be desirable to use a selectable or screenable marker gene in conjunction with the transformation vector prepared according to the present disclosure. In this case, the population of potentially transformed cells will typically be analyzed by exposing the cells to one or more selection agents, or the desired marker gene trait of the cells will be screened.

[0484] A. Selection

[0485] It is believed that in any given study, DNA is only introduced into a small percentage of the target cells. To provide an effective system for identifying those cells that have received the DNA and integrated it into their genome, a method for selecting those cells that are stably transformed can be employed. An exemplary embodiment of such a method is to introduce a marker gene into the host cell that confers resistance to some common inhibitors such as antibiotics or herbicides. Examples of antibiotics that can be used include the aminoglycoside antibiotics neomycin, kanamycin, and paromomycin, or the antibiotic hygromycin. Aminoglycoside phosphotransferase enzymes such as neomycin phosphotransferase II (NPT II) or NPT I confer resistance to aminoglycoside antibiotics, while hygromycin phosphotransferase confers resistance to hygromycin.

[0486] The cells that may be transformed are then exposed to a selection agent. Among the surviving cell population, typically those are the cells that have integrated the resistance-conferring gene and express it at a sufficient level to allow cell survival. The cells can be further tested to confirm the stable integration of the foreign DNA.

[0487] One herbicide that constitutes the required selection agent is the broad-spectrum herbicide bialaphos. Bialaphos is a tripeptide antibiotic produced by Streptomyces hygroscopicus and consists of phosphinothricin (PPT), an analogue of L-glutamic acid, and two L-alanine residues. After removal of the L-alanine residues by intracellular peptidases, PPT is released and is a potent inhibitor of glutamine synthetase (GS), a key enzyme involved in ammonia assimilation and nitrogen metabolism (Ogawa et al., Sci. Rep. Meiji Seika 13:42-48, 1973). Synthetic PPT (the active ingredient in the herbicide Liberty TM ) is also effective as a selection agent. Inhibition of GS in plants by PPT leads to rapid accumulation of ammonia and death of plant cells.

[0488] The organisms that produce bialaphos and other Streptomyces species also synthesize the enzyme phosphinothricin acetyltransferase (PAT), which is encoded by the bar gene in Streptomyces hygroscopicus and the pat gene in Streptomyces viridochromogenes. The use of the herbicide resistance gene encoding phosphinothricin acetyltransferase (PAT) is described in DE3642829A, in which the gene was isolated from Streptomyces viridochromogenes. In organisms of bacterial origin, the enzyme acetylates the free amino group of PPT to prevent autotoxicity (Thompson et al., EMBO J. 6:2519-2523, 1987). The bar gene has been cloned (Thompson et al., ibid.) and expressed in transgenic tobacco, tomato, potato (De Block et al., Plant Physiol. 91:694-701, 1989), Brassica (De block et al., plantphysiol. 91:694-701, 1989) and maize (U.S. Patent No. 5,550,318, which is incorporated herein by reference in its entirety).

[0489] Another example of a herbicide that can be used to select transformed cell lines in the practice of the present disclosure is the broad-spectrum herbicide glyphosate. Glyphosate inhibits the action of the enzyme EPSPS, which is active in the aromatic amino acid biosynthesis pathway. Inhibition of this enzyme results in starvation for the amino acids phenylalanine, tyrosine, and tryptophan and their derived secondary metabolites. U.S. Patent No. 4,535,060 (incorporated herein by reference in its entirety) describes the isolation of EPSPS mutants that confer glyphosate resistance to the aroA gene of Salmonella typhimurium for EPSPS. The EPSPS gene was cloned from maize and mutations similar to those found in the glyphosate-resistant aroA gene were introduced in vitro. Mutant genes encoding glyphosate-resistant EPSPS enzymes are described, for example, in International Patent WO97 / 4103.

[0490] To use the bar-bialaphos or EPSPS-glyphosate selection system, the transformed tissue is cultured on non-selective medium for 0 - 28 days and then transferred to medium containing 1 - 3 mg / l bialaphos or 1 - 3 mM glyphosate, as appropriate. While the range of 1 - 3 mg / l bialaphos or 1 - 3 mM glyphosate may be beneficial, it is proposed that a range of 0.1 - 50 mg / l bialaphos or 0.1 - 50 mM glyphosate would be useful.

[0491] An example of a selectable marker trait is luciferase. In the presence of the substrate luciferin, cells expressing luciferase emit light that can be detected on photographic or X-ray film, in a photometer (or liquid scintillation counter), by a device that enhances night vision, or by a highly sensitive video camera such as a photon-counting camera. These assays are non-destructive and the transformed cells can be further cultured after identification. A photon-counting camera is particularly valuable because it allows the identification of specific cells or cell populations expressing luciferase and their manipulation in real time. Another selectable marker that can be used in a similar manner is the gene encoding green fluorescent protein.

[0492] B. Regeneration and Seed Production

[0493] Cells that survive exposure to a selection agent, or cells that score positive in a screening assay, can be cultured in a medium that supports plant regeneration. In one exemplary embodiment, MS and N6 media can be modified by including other substances such as growth regulators. One such growth regulator is dicamba or 2,4-D. However, other growth regulators can be used, including NAA, NAA + 2,4-D, or picloram. It has been found that medium modifications in these and similar ways promote cell growth at specific developmental stages. Tissue can be maintained on a basal medium containing a growth regulator until there is sufficient tissue available to initiate plant regeneration, or after repeated rounds of manual selection, until the morphology of the tissue is suitable for regeneration (at least 2 weeks), and then transferred to a medium that aids in embryoid maturation. The cultures are transferred to this medium every 2 weeks. Shoot development will indicate the time to transfer to a medium lacking growth regulators.

[0494] Then, the transformed cells identified by selection or screening and cultured in a suitable medium that supports regeneration are matured into plants. The developing seedlings are transferred to a soilless plant growth mixture and hardened, for example, in an environmentally controlled chamber (e.g., at approximately 85% relative humidity, 600 ppm CO 2 and 25 - 250 microeinsteins m 2 s -1 light). The plants can be matured in a growth chamber or greenhouse. Depending on the initial tissue, plants can be regenerated approximately 6 weeks to 10 months after identification of the transformants. During the regeneration process, the cells are grown on solid medium in tissue culture containers. Exemplary embodiments of such containers are petri dishes and Plant Cons. The regenerated plants can be grown at approximately 19 to 28 °C. After the regenerated plants reach the shoot and root development stages, they can be transferred to a greenhouse for further growth and testing.

[0495] Due to seed development arrest and premature senescence of the plants, seeds on transformed plants may occasionally require embryo rescue. To rescue the developing embryos, they are excised from surface-sterilized seeds 10 - 20 days after pollination and cultured. An embodiment of the medium used for culturing at this stage contains MS salts, 2% sucrose, and 5.5 g / l agarose. In embryo rescue, large embryos (defined as having a length greater than 3 mm) germinate directly on a suitable medium. Embryos smaller than this length can be cultured on a medium containing the above components and 10 -5 M abscisic acid for 1 week and then transferred to a medium lacking growth regulators for germination.

[0496] C. Characterization

[0497] To confirm the presence of foreign DNA or "transgenes" in regenerated plants, a variety of assays can be performed. Such assays include, for example, "molecular biology" assays such as Southern and Northern blotting and PCR TM ; "biochemical" assays, such as detecting the presence of protein products by immunological means (ELISA and Western blotting) or by enzyme function; plant part assays, such as leaf or root assays; and also by analyzing the phenotype of the entire regenerated plant.

[0498] D. DNA integration, RNA expression, and inheritance

[0499] Using techniques well known to those skilled in the art, genomic DNA can be isolated from cell lines or any plant part to assay for the presence of foreign genes. Note that complete sequences are not always present, presumably due to rearrangements or deletions of sequences in the cell. The presence of DNA elements introduced by the methods of the present disclosure can be assayed, for example, by polymerase chain reaction (PCR TM ). Using this technique, discrete fragments of DNA are amplified and detected by gel electrophoresis. This type of analysis allows determination of whether a gene is present in a stable transformant, but does not prove that the introduced gene has integrated into the host cell genome. However, typically the DNA has integrated into the genomes of all transformants, and the presence of the gene is confirmed by PCR TM analysis. In addition, it is generally not possible to use PCR TM techniques to determine whether a transformant has foreign genes introduced at different sites in the genome, i.e., whether the transformant has an independent origin. It is expected that using PCR TM techniques, it may be possible to clone fragments of host genomic DNA adjacent to the introduced gene.

[0500] Positive evidence of DNA integration into the host genome and the independent nature of the transformant can be determined using Southern hybridization techniques. Using this technique, specific DNA sequences introduced into the host genome and flanking host DNA sequences can be identified. Thus, the Southern hybridization pattern of a given transformant serves as an identifying characteristic of that transformant. In addition, the presence of the introduced gene in high molecular weight DNA can be confirmed by Southern hybridization, i.e., it is confirmed that the introduced gene has integrated into the host cell genome. Southern hybridization techniques provide the information obtained using PCR TM such as the presence of a gene, but also prove integration into the genome and characterize each individual transformant.

[0501] It is expected that using dot or slot blot hybridization techniques (which are modifications of Southern hybridization techniques), information of the same origin as that obtained by PCR TM such as the presence of a gene can be obtained.

[0502] PCR TM Both PCR and Southern hybridization techniques can be used to confirm the transmission of transgenes to offspring. In most cases, the characteristic Southern hybridization pattern of a given transformant segregates in the offspring as one or more Mendelian genes (Spencer1 et al., 1992), indicating stable inheritance of the transgene.

[0503] Although DNA analysis techniques can be performed using DNA isolated from any part of the plant, RNA is only expressed in specific cell or tissue types, and thus RNA for analysis needs to be prepared from these tissues. PCR TM techniques can also be used to detect and quantify the RNA produced by the introduced gene. In this application of PCR TM it is first necessary to reverse transcribe the RNA into DNA using an enzyme such as reverse transcriptase, and then amplify the DNA by using conventional PCR TM techniques. In most cases, although PCR TM techniques are useful, they cannot prove the integrity of the RNA product. Further information on the nature of the RNA product can be obtained by Northern blotting. This technique will demonstrate the presence of the RNA species and give information on the integrity of that RNA. Dot or slot blot Northern hybridization can also be used to determine the presence of the RNA species. These techniques are modifications of Northern blotting and will only demonstrate the presence or absence of the RNA species.

[0504] E. Gene expression

[0505] Although Southern blotting and PCR TM can be used to detect the gene in question, they do not provide information on whether the corresponding protein is expressed. Expression can be evaluated by specifically identifying the protein product of the introduced gene or by assessing the phenotypic changes caused by its expression.

[0506] Assays for the production and identification of specific proteins can utilize the physicochemical, structural, functional, or other properties of the protein. Unique physicochemical or structural properties allow the separation and identification of proteins by electrophoretic methods (such as native or denaturing gel electrophoresis or isoelectric focusing) or by chromatographic techniques (such as ion exchange or gel exclusion chromatography). The unique structure of individual proteins provides the opportunity to detect their presence using specific antibodies in the form of assays such as ELISA. Combinations of methods with even greater specificity can be used, such as western blotting, where antibodies are used to localize individual gene products that have been separated by electrophoretic techniques. Additional techniques can be used to absolutely confirm the identity of the target product, such as evaluation by amino acid sequencing after purification. Although these are the most commonly used, other procedures can alternatively be used.

[0507] The assay procedures can also be used to identify the expression of a protein by its functionality, especially the ability of an enzyme to catalyze a specific chemical reaction involving specific substrates and products. These reactions can then be followed by physical or chemical steps to provide and quantify the loss of substrate or the production of reaction products. The examples are as variable as the enzyme to be analyzed and can include, for example, determining PAT enzyme activity after production of radiolabeled acetylated phosphinothricin from phosphinothricin and 14 C-acetyl coenzyme A, or determining anthranilate synthase activity after loss of fluorescence of anthranilate.

[0508] The expression of a gene product is often determined by assessing the phenotypic consequences of its expression. These assays can also take many forms, including but not limited to analyzing changes in the chemical composition, morphology, or physiological properties of a plant. The chemical composition can be altered by the expression of an enzyme that modifies the amino acid composition or a storage protein and can be detected by amino acid analysis, or by an enzyme that alters the amount of starch, which can be analyzed by near-infrared reflectance spectroscopy. Morphological changes can include greater height or thicker stems. Most commonly, changes in the response of a plant or plant part to an applied treatment are evaluated under carefully controlled conditions called a bioassay.

[0509] IX. Sweeteners and consumables containing mogrosides

[0510] In some embodiments, the present disclosure generally relates to a sweetener or sweetening composition comprising mogrosides and / or their metabolites or derivatives, wherein the sweetener or sweetening composition is derived from a transgenic plant that produces and contains non-natural mogrol / mogrosides. As used herein, the term "sweetener" refers to a consumable product that produces a sweet taste when consumed alone. In certain embodiments, the sweetener or sweetening composition is derived from a mogrol / mogroside pathway transgenic plant prepared according to the present disclosure. In some embodiments, the sweetener is a high-intensity or low-intensity sweetener. The sweetener containing mogrosides can be derived from the mogrol / mogroside pathway transgenic plant of the present disclosure after appropriate processing. The resulting sweetener can be used to provide low- or no-calorie sweetness for many purposes. Examples of such uses for providing sweetness are in beverages (such as tea, coffee, fruit juices, and fruit drinks), foods (such as jams and jellies), peanut butter, pies, puddings, cereals, candies, ice cream, yogurt, baked products; health products (such as toothpaste, mouthwash, cough drops, cough syrups); chewing gum; and sugar substitutes.

[0511] In certain embodiments, the sweetener is in the juice from the fruit of a transgenic plant according to the present disclosure. Applications of juice containing one or more mogroside compounds (such as watermelon juice) include, but are not limited to, use as a beverage (including, for example, pre-mixed cocktails and dairy alternatives), as an ingredient (such as sprayed onto bars or cereals), or for sweetening ketchup or other common products. In such embodiments, the juice can be inactivated, with the protein removed or concentrated. Additionally, concentrated fruit or vegetable syrups, such as watermelon syrup produced from the watermelon disclosed in the present invention, can be used to replace high fructose corn syrup in a variety of foods and beverages. In some embodiments of the present disclosure, mogroside compounds are produced in transgenic tomatoes, which can then be used, for example, to produce low-calorie ketchup or other tomato-based sauces or soups.

[0512] In some embodiments, the present disclosure also relates to methods for preparing sweeteners derived from transgenic plants of the present disclosure that produce non-natural mogrol / mogroside. The methods generally encompass the following steps, which can include, but are not limited to: pre-treatment of cleaning and crushing the transgenic plant or its parts, extraction of the transgenic plant or its parts, sedimentation and / or centrifugation, adsorption and / or separation, concentration and recovery to produce a crude sweetener, further purification, optional concentration / drying and formulation. Extraction methods include water extraction at room temperature, or at a heated temperature, or at a frozen temperature; extraction by organic solvents such as ethanol, etc. Methods of separation and purification include centrifugation, maceration, gravitational sedimentation, filtration, microfiltration, nanofiltration, ultrafiltration, reverse osmosis, chromatography, absorption chromatography, purification by ion exchange resins, etc.

[0513] In further embodiments, the transgenic plants disclosed in the present invention can be processed to produce ingredients containing mogrosides, such as by whole plant extraction, tissue extraction, fruit processing, aqueous separation of small molecules with mogroside fractions, removal of residual proteins to produce an aqueous fraction free of any genetically engineered components. The resulting ingredients containing mogrosides can be in any form, including but not limited to powders, liquids, syrups, concentrates or extracts. Additionally, in some embodiments, fruits or vegetables containing intact mogrosides are consumable.

[0514] In certain embodiments, the sweetener is obtained from the leaves of a transgenic plant prepared according to the present disclosure. In other embodiments, the sweetener is obtained from the fruit of a transgenic plant prepared according to the present disclosure, fruit parts (such as the peel), or other parts of an organ or tissue.

[0515] In addition, mogroside compounds produced by the transgenic plants and organisms disclosed by the present invention can be blended with one or more other naturally occurring or artificial sweeteners, such as steviol glycosides, cymenoside I, α-cymenoside I, sucrose, glucose, fructose, lactose, maltose, sorbitol, galactose, thaumatin, sucrooctate, bernadame, sucrononic acid, carrelame, lugduname, high fructose corn syrup, RealSweet TM Sugarcane RebM, erythritol, xylitol, yacon syrup, allulose, saccharin, aspartame, acesulfame potassium, sucralose, neotame, advantame, cyclamate or glycyrrhizin. The ratio of mogroside compounds to other sweeteners in the final formulation can be, for example, 10 / 90, 20 / 80, 30 / 70, 40 / 60, 50 / 50, 60 / 40, 70 / 30, 80 / 20 or 90 / 10, or any other desired ratio. In one embodiment, the ratio is about 80% mogroside V, about 15% 11-oxo-mogroside V and about 5% mogroside IIIA1. In another embodiment, the ratio is about 40% cymenoside I, about 40% mogroside V and about 20% 11-oxo-mogroside V.

[0516] In certain embodiments, one or more additional sweeteners can be carbohydrate sweeteners. Non-limiting examples of suitable carbohydrate sweeteners include sucrose, fructose, glucose, erythritol, maltitol, lactitol, sorbitol, mannitol, rhamnose, cyclodextrins (such as α-cyclodextrin, β-cyclodextrin, and γ-cyclodextrin), ribulose, threose, arabinose, xylose, lyxose, allose, altrose, mannose, invert sugar, isotrehalose, neotrehalose, palatinose or isomaltulose, erythrose, deoxyribose, gulose, idose, talose, erythrulose, xylulose, allulose, turanose, cellobiose, glucosamine, mannosamine, fucose, fucoidan, glucuronic acid, gluconic acid, gluconolactone, abequose, galactosamine, xylo-oligosaccharides (such as xylotriose, xylobiose, etc.), gentiobiose-oligosaccharides (such as gentiobiose, gentiotriose, gentiotetrose, etc.), galacto-oligosaccharides, sorbose, ketotriose (dihydroxyacetone), aldose (glyceraldehyde), Aspergillus niger oligosaccharides, fructo-oligosaccharides (such as kestose, nystose, etc.), maltotetraose, maltotriitol, tetrasaccharides, mannan-oligosaccharides, malt-oligosaccharides (such as maltotriose, maltotetraose, maltopentaose, maltohexaose, maltoheptaose, etc.), dextrin, lactulose, melibiose, raffinose, rhamnose, ribose, isomerized liquid sugars such as high fructose corn / starch syrup (HFCS / HFSS) (such as HFCS55, HFCS42, or HFCS90), conjugated sugars, soy-oligosaccharides, glucose syrup, and combinations thereof. When applicable, D- or L-configurations can be used. In other embodiments, the additional sweetener is a carbohydrate sweetener selected from glucose, fructose, sucrose, and combinations thereof. In another embodiment, the additional sweetener is a carbohydrate sweetener selected from D-allose, D-allulose, L-ribose, D-talose, L-glucose, L-fucose, L-arabinose, turanose, and combinations thereof.

[0517] In other embodiments, one or more additional sweeteners are not directly derived from natural extracts. Such sweeteners characteristically have a greater sweetening potency than sucrose, fructose, or glucose, but have fewer calories. Non-limiting examples of such sweeteners suitable for the embodiments of the present disclosure include sucralose, acesulfame potassium, acesulfamic acid and its salts, aspartame, alitame, saccharin and its salts, neohesperidin dihydrochalcone, cyclamate, cyclohexanesulfamic acid and its salts, neotame, advantame, glucosylated steviol glycosides (GSGs), and combinations thereof. When present in a sweetening composition such as a food, other consumable, or beverage, at least one sweetener not directly derived from natural extracts is present in the sweetener composition in an amount effective to provide a concentration of from about 0.3 ppm to about 3,500 ppm. In one embodiment, when present in a sweetened beverage, at least one sweetener not directly derived from natural extracts is present in the sweetener composition in an amount effective to provide a concentration of from about 0.5 ppm to about 3,000 ppm, from about 1.0 ppm to about 2,500 ppm, from about 5.0 ppm to about 2,000 ppm, from about 10 ppm to about 1,500 ppm, from about 50 ppm to about 1,000 ppm, from about 100 ppm to about 800 ppm, or from about 400 ppm to about 600 ppm. In another embodiment, in at least one embodiment, when present in a sweetening composition such as a food, other consumable, or beverage, at least one sweetener not directly derived from natural extracts is present in the sweetener composition in an amount effective to provide a concentration greater than about 0.3 ppm, greater than about 0.5 ppm, greater than about 1.0 ppm, greater than about 5.0 ppm, greater than about 10 ppm, greater than about 20 ppm, greater than about 50 ppm, greater than about 100 ppm, greater than about 250 ppm, greater than about 500 ppm, or greater than about 1,000 ppm.

[0518] In other embodiments, the additional sweetener can be a natural high-intensity sweetener. Suitable natural high-intensity sweeteners include, but are not limited to, rebaudioside A, rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside E, rebaudioside F, rebaudioside I, rebaudioside H, rebaudioside L, rebaudioside K, rebaudioside J, rebaudioside M, rebaudioside N, rebaudioside O, dulcoside A, dulcoside B, rubusoside, stevia, stevioside, mogroside IV, mogroside V, mogroside, thaumatin, glycyrrhizic acid and its salts (monatin SS, RR, RS, SR), curculin, glycyrrhizin and its salts, thaumatin, monellin, mablin, brazzein, hernandulcin, phyllodulcin, litseae, phloridzin, trilobatin, leucocyanidin, osladin, multinoside A, pterocaryoside A, pterocaryoside B, mukurozioside, phlomisoside I, periandrin I, abrusoside A, steviolbioside, and cyclocarya paliurus glycoside I. The natural high-intensity sweetener can be provided as a pure compound or as part of an extract. For example, rebaudioside A can be provided as a single compound or as part of a stevia extract. When present in a sweetening composition, such as a food, other consumable, or beverage, the natural high-intensity sweetener is present in the sweetener composition in an amount effective to provide a concentration of from about 0.1 ppm to about 3,000 ppm. In one embodiment, when present in a sweetening composition, such as a food, other consumable, or beverage, the natural high-intensity sweetener is present in the sweetener composition in an amount effective to provide a concentration of from about 0.5 ppm to about 2500 ppm, from about 1.0 ppm to about 2000 ppm, from about 5 ppm to about 1500 ppm, from about 10 ppm to about 1000 ppm, or from about 25 ppm to about 500 ppm. In one embodiment, when present in a sweetening composition such as a food, other consumable, or beverage, the natural high-intensity sweetener is present in the sweetener composition in an amount effective to provide a concentration greater than about 0.1 ppm, about 0.5 ppm, about 1.0 ppm, about 2.5 ppm, about 5.0 ppm, about 10 ppm, about 20 ppm, about 25 ppm, about 50 ppm, about 75 ppm, about 100 ppm, about 200 ppm, about 500 ppm, about 1000 ppm, about 2000 ppm, or about 300 ppm.

[0519] In other embodiments, the additional sweetener can be a chemically or enzymatically modified natural high-intensity sweetener. Modified natural high-intensity sweeteners include glycosylated natural high-intensity sweeteners, such as glucosyl-, galactosyl- or fructosyl-derivatives containing 1-50 glycoside residues. Glycosylated natural high-intensity sweeteners can be prepared by enzymatic transglycosylation reactions catalyzed by various enzymes having transglycosylase activity.

[0520] When the sweetener composition contains more than one sweetener, these sweeteners can exhibit a synergistic effect when combined and have improved flavor and temporal characteristics compared to each individual sweetener alone. As used herein, the term "temporal characteristics" of a composition refers to the intensity of sweetness perceived by a person over time when tasting the composition. The term "flavor profile" or "taste profile" as commonly used herein refers to the intensity of the various flavor / taste attributes of a sweetener or sweetened composition. Exemplary flavor / taste attributes are sweetness intensity, bitterness intensity, saltiness intensity, licorice intensity, cooling intensity, and licorice intensity. Methods for determining the flavor profile of a given sweetener or sweetened composition are known in the art. The term "synergistic" or "synergistic effect" refers to an effect (e.g., flavor, temporal characteristics) achieved with a combination of two or more sweeteners that is greater than the sum of the effects produced by using the individual sweeteners alone or separately. Advantageously, this synergistic effect between two or more sweeteners allows for the use of a smaller dose of one or both sweeteners or provides a greater effect at the same amount. The amount or degree of the synergistic effect can vary.

[0521] The amount of sucrose in the reference solution can be described in degrees Brix (Bx). One degree Brix is 1 gram of sucrose in 100 grams of solution and represents the strength of the solution as a weight percentage (% w / w) (strictly speaking, by mass). In one embodiment, the sweetener composition contains one or more of the sweetener compounds disclosed herein, which, when present in the sweetened composition, are effective in providing a sweetness equivalent of at least about 5 degrees Brix, such as at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, or at least about 15 or more degrees Brix.

[0522] The sweetness of non-sucrose sweeteners can also be measured by determining the sucrose equivalent of the non-sucrose sweetener relative to a sucrose reference. Typically, a tasting panel is trained to detect the sweetness of a reference sucrose solution containing 1-15% sucrose (w / v). Other non-sucrose sweeteners are then tasted at a series of dilutions to determine the concentration of the non-sucrose sweetener that is as sweet as a given percentage of sucrose reference. For example, if a 1% solution of a sweetener is as sweet as a 10% solution of sucrose, the potency of the sweetener is said to be 10 times that of sucrose.

[0523] Sweetener compositions can be customized to provide a desired calorie content. For example, sweetener compositions can be "full calorie" such that they impart a desired sweetness when added to a sweetenable composition (e.g., a food, other consumable, or beverage) and have about 120 calories per 8 ounce serving. Alternatively, sweetener compositions can be "medium calorie" such that they impart a desired sweetness when added to a sweetenable composition and have less than about 60 calories per 8 ounce serving. Or, sweetener compositions can be "low calorie" such that they impart a desired sweetness when added to a sweetenable composition and have less than 40 calories per 8 ounce serving. In other embodiments, sweetener compositions can be "zero calorie" such that they impart a desired sweetness when added to a sweetenable composition and have less than 5 calories per 8 ounce serving.

[0524] The sweetener compositions disclosed herein can optionally include one or more additional additives. In some embodiments, the sweetener composition contains additives including, but not limited to, carbohydrates, polyols, amino acids and their corresponding salts, polyamino acids and their corresponding salts, sugar acids and their corresponding salts, nucleotides, organic acids, inorganic acids, organic salts (including organic acid salts and organic base salts), inorganic salts, bitter compounds, flavorants and flavor components, astringent compounds, proteins or protein hydrolysates, surfactants, emulsifiers, bulking agents, gums, antioxidants, colorants, flavonoids, alcohols, polymers, and combinations thereof. In some embodiments, the additives are used to improve the temporal and flavor characteristics of the sweetener to provide a sweetener composition having a taste similar to sucrose. The sweetenable composition can contain one or more functional ingredients as detailed above. Functional ingredients include, but are not limited to, vitamins, minerals, antioxidants, preservatives, glucosamine, polyphenols, and combinations thereof. Any suitable functional ingredient described herein can be used.

[0525] Compared to existing monk fruit products, the mogroside sweeteners disclosed in the present invention have beneficial environmental impacts. Compared to, for example, harvesting and processing only in China and transporting globally to food companies, the monk fruit sweeteners disclosed in the present invention can be produced locally, resulting in less transportation and fewer food miles. The disclosed monk fruit sweeteners of the present invention also require minimal processing as the monk fruit sweeteners are readily obtainable in the disclosed transgenic plants or organisms compared to, for example, factory processing of monk fruit sweeteners in China.

[0526] X. Sweetenable Compositions

[0527] The sweetener compositions disclosed herein can be incorporated into any known edible material (referred to herein as "sweetenable compositions"), such as pharmaceutical compositions, edible gel mixtures and compositions, dental compositions, foods (candies, condiments, chewing gums, cereal compositions, baked goods, dairy products, and tabletop sweetener compositions), beverages, and beverage products.

[0528] The sweetenable compositions disclosed herein include beverages, i.e., ready-to-drink liquid formulations, beverage concentrates, etc. In certain embodiments, a beverage concentrate is prepared with an initial volume of liquid (such as water), to which additional ingredients are added. A full-strength beverage composition can be formed from the beverage concentrate by adding a greater volume of liquid (such as water) to the concentrate.

[0529] In embodiments using a mogroside-containing filled juice concentrate (about 80% mogroside V, about 15% 11-oxo-mogroside V, and about 5% mogroside III A1) as the sweetenable composition, studies have shown it to be the sweetest and purest natural sweetener. The mogroside-containing filled juice concentrate disclosed herein can be produced from consumer-friendly fruits that can be sustainably grown locally, is the only sweetener that achieves high sugar reduction while maintaining a 100% juice label, and is an affordable drop-in solution. The filled juice can be used as a single strength or concentrated to deliver pure sweetness at various inclusion levels. When used in various formulation inclusions, the filled juice mogroside concentration can deliver an equivalent of ~10 sucrose equivalent values (SEV).

[0530] In addition to concentration, other juice parameters can also be readily altered, resulting in different sweetener products. For example, the natural sugars in a fruit (such as watermelon) can be partially or fully removed, the juice color and / or flavor can be minimized or removed, the pulp can be removed as is typically done, or fully or partially retained in a puree, and the acidity can be reduced, or a combination of one or more of these parameters can be altered.

[0531] Filled juice applications include, but are not limited to, juices, nectars, fruit / flavored distilled beverages, energy and sports drinks, carbonated soft drinks, flavored waters, nutritional beverages, vitamins and dietary supplements, or oral rehydration in liquid or chewable / gum form, snacks such as snack bars or fruit snacks, confections and gum confections in jelly and chewable form, dairy products such as spoonable yogurt, drinking yogurt, and flavored beverages, desserts, ice cream, frozen yogurt, water-based popsicles and sorbets, breakfast cereals and other cold cereals, tabletop sweeteners, sweet spreads such as syrups and fruit spreads, sauces and condiments such as table sauces and cooking sauces, and processed and packaged fruits and vegetables.

[0532] In embodiments using a sweetenable composition containing a dry powder of mogrosides (about 40% of mogroside I, about 40% of mogroside V, and about 20% of 11-oxo-mogroside V), studies have shown that the taste is pure, with a high level of sweetness in high-demand applications and no off-flavors. The dry powder of mogrosides disclosed in the present invention can be produced from consumer-friendly vegetables, which can be grown locally in a sustainable manner, resulting in a reduction in sugar and calories, a strong positive association with health benefits, and being a part equivalent to monk fruit / sucrose. The dry powder can be used in a wide range of food and beverage applications to deliver the purest and sweetest taste at low inclusion levels. When used at various purity levels, the dry powder concentration can deliver an equivalent of about 10 SEV.

[0533] Dry powder applications include, but are not limited to: health and functional beverages such as energy and sports drinks, carbonated soft drinks, flavored waters, juices, nectars, fruit / flavored distilled beverages, protein and meal replacement drinks, drink mixes, drink concentrates, ready-to-drink teas and coffees; dietary supplements and over-the-counter products such as vitamins and dietary supplements, oral hydration, cold relief, digestive treatments, sleep aids, capsules, tablets, liquids, powders, chewables / gums, lozenges, and other forms of pain relievers; snacks such as snack bars, fruit snacks, nuts, trail mixes, rice cakes, potato and wheat snacks; baked products such as cookies, cakes, and desserts, baking mixes and ingredients, and breads; dairy products and desserts such as spoonable and drinkable yogurts, flavored beverages, creamers, ice cream and frozen yogurt, water-based popsicles and sorbets, shelf-stable desserts and dessert toppings; hot and cold breakfast cereals; artificial and other natural sweeteners (tabletop sweeteners); sugar and chocolate confections such as jellies and chews, mints, gums, toffees and caramels, marshmallows, and various forms of chocolate; sweet spreads such as syrups, fruit, nut, and chocolate spreads; sauces and seasonings such as table, cooking, and pasta sauces, vinegars and seasonings, and pickling seasonings; meals and processed meats such as prepared meals, meal kits, sandwiches and wraps, and poultry and meat products; and processed and packaged fruits and vegetables.

[0534] A. Beverages and Beverage Products

[0535] In one embodiment, the sweetening composition is a beverage or a beverage product. As used herein, "beverage product" is a ready-to-drink beverage, a beverage concentrate, a beverage syrup, or a powdered beverage. Suitable ready-to-drink beverages include carbonated and non-carbonated beverages. Carbonated beverages include, but are not limited to, frozen carbonated beverages, enhanced foaming beverages, colas, fruit-flavored foaming beverages (such as lemon-lime, orange, grape, strawberry, and pineapple), ginger ales, soft drinks, and root beers. Non-carbonated beverages include, but are not limited to, fruit juices, fruit-flavored juices or waters, juice drinks, nectars, fruit / flavor-distilled beverages, energy and sports drinks, vegetable juices, vegetable-flavored juices, sports drinks, energy drinks, nutritional drinks, enhanced water beverages, enhanced water with vitamins, near-water beverages (such as water with natural or synthetic flavorants), coconut water, tea beverages (such as black tea, green tea, red tea, oolong tea), coffee, cocoa beverages, beverages containing milk components (such as milk drinks, coffee containing milk components, café au lait, milk tea, fruit milk drinks), beverages containing cereal extracts and mousses.

[0536] In certain embodiments, the beverage is a juice drink that has been modified to remove at least some sucrose. In certain embodiments, such juice can be modified to remove at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, or more of the sucrose in the unmodified juice. In certain embodiments, the modification is carried out by filtering the juice to remove sucrose. In certain embodiments, the sucrose in the juice is broken down into fructose and glucose before adding the sweetening composition described herein.

[0537] The beverage contains a matrix, i.e., a base component in which the components including the composition of the present disclosure are dissolved. In one embodiment, the beverage contains beverage-quality water as the matrix, such as deionized water, distilled water, reverse osmosis water, carbon-treated water, purified water, softened water, and combinations thereof. Other suitable matrices include, but are not limited to, phosphoric acid, phosphate buffer, citric acid, citrate buffer, and carbon-treated water. Beverage concentrates and beverage syrups are prepared with an initial volume of a liquid matrix (such as water) and the desired beverage ingredients. Then, full-strength beverages are prepared by adding more volume of water. Powdered beverages are prepared by dry-blending all beverage ingredients in the absence of a liquid matrix. Then, full-strength beverages are prepared by adding the full volume of water.

[0538] It is expected that the pH of the beverage will not substantially or adversely affect the taste of the sweetener. Non-limiting examples of the pH range of the beverage can be from about 1.8 to about 10. In one embodiment, the pH of the beverage is about 4. In another embodiment, the pH of the beverage is less than about 4. In a specific embodiment, the pH of the beverage is less than about 3.8, less than about 3.6, less than about 3.4, less than about 3.2, less than about 3.0, less than about 2.8, less than about 2.6, less than about 2.4 or less than about 2.2. In another embodiment, the pH of the beverage is about 3.8, about 3.6, about 3.4, about 3.2, about 3.0, about 2.8, about 2.6, about 2.4 or about 2.2 or lower.

[0539] B. Edible Gel Mixes and Edible Gel Compositions

[0540] In one embodiment, the sweetening composition is an edible gel or an edible gel mix. An edible gel is a gel that can be eaten. Non-limiting examples of edible gel compositions for a particular embodiment include gel desserts, puddings, jellies, pastes, trifles, spics, marshmallows, gum candies / chews, and the like. An edible gel mix is generally a powdery or granular solid to which a fluid can be added to form an edible gel composition. Non-limiting examples of fluids for a particular embodiment include water, dairy fluids, dairy analog fluids, fruit juices, alcohols, alcoholic beverages, and combinations thereof. Non-limiting examples of dairy liquids that can be used for a particular embodiment include milk, fermented milk, cream, liquid whey, and mixtures thereof. Non-limiting examples of dairy analog fluids that can be used for a particular embodiment include, for example, soy milk and non-dairy coffee whiteners.

[0541] C. Candies

[0542] In one embodiment, the sweetening composition is a confectionery. As referred to herein, "confectionery" can mean sugar, lollipops, confectionery products, or similar terms. Confectionery typically contains a base composition component and a sweetening component. According to certain embodiments of the present disclosure, the confectionery can be a dessert, such as yogurt, jelly, drinkable jelly, pudding, Bavarian cream, blancmange, cake, brownie, mousse, etc., a sweetened food item consumed at tea time or after a meal; a frozen food; a frozen dessert, such as ice cream types like ice cream, ice milk, sherbet, etc., and frozen treats like water ice, frozen confections, etc.; common confectionery, such as baked confectionery or steamed confectionery, like pancakes, cookies, buns with bean paste fillings, halvah, alfajor, etc.; rice cakes and snacks; table products; common confectionery, such as chewing gum, hard candy, gummy candy, mints, nougat, jelly beans, fudge, English toffee, American toffee, Swiss milk tablets, licorice, chocolate, gelatin candy, marshmallows, marzipan, almond paste, olive paste, cotton candy, etc.; sauces, including fruit - flavored sauces, chocolate sauce, etc.; edible gels; cheeses, including cream cheese, flour paste, whipped cream, etc.; jams, including strawberry jam, marmalade, etc.; and breads, including sweet breads, etc. or other starch products, and combinations thereof.

[0543] D. Flavoring Composition

[0544] In one embodiment, the sweetening composition is a flavoring composition. As used herein, a flavoring is a composition used to enhance or improve the flavor of a food or beverage. Non - limiting examples of flavorings include ketchup; mustard; barbecue sauce; butter; chili sauce; chutney; cocktail sauce; curry; dips; fish sauce; horseradish; hot sauce; jellies, jams, marmalades, or preserves; mayonnaise; peanut butter; sauce; flavored mayonnaise; salad dressing, salad; sauerkraut; soy sauce; steak sauce; syrup; tartar sauce; and Worcestershire sauce. A flavoring base typically contains a mixture of different ingredients, non - limiting examples of which include vehicles (such as water and vinegar); spices or seasonings (such as salt, pepper, garlic, mustard, onion, chili, turmeric, and combinations thereof); fruits, vegetables, or their products (such as tomato or tomato - based products (paste, puree), fruit juices, fruit peels, and combinations thereof); oils or oil emulsions, particularly vegetable oils; thickeners (such as xanthan gum, food starches, other hydrocolloids, and combinations thereof); and emulsifiers (such as egg yolk solids, proteins, gum arabic, carob gum, guar gum, gum tragacanth, gum acacia, carrageenan, pectin, propylene glycol alginate, sodium carboxymethyl cellulose, polysorbates, and combinations thereof). The formulation of the flavoring base and methods for preparing the flavoring base are well - known to those of ordinary skill in the art.

[0545] E. Chewing Gum Composition

[0546] In one embodiment, the sweetening composition is a chewing gum composition. Chewing gum compositions typically comprise a water-soluble portion and a water-insoluble chewable gum base portion. During a period of chewing, the water-soluble portion dissipates along with a portion of the flavoring agent, while the insoluble gum base portion remains in the mouth. The insoluble gum base generally determines whether the chewing gum is considered a chewing gum, a bubble gum, or a functional chewing gum.

[0547] Flavoring agents can be used in the insoluble gum base or the soluble portion of the chewing gum composition. Such flavoring agents can be natural or artificial flavoring agents. In a specific embodiment, the flavoring agent comprises essential oils, such as oils derived from plants or fruits, peppermint oil, spearmint oil, other mint oils, clove oil, cinnamon oil, wintergreen oil, bay oil, thyme oil, cedar leaf oil, nutmeg, allspice, sage, mace, and almond. In another specific embodiment, the flavoring agent includes plant extracts or fruit essences, such as apple, banana, watermelon, pear, peach, grape, strawberry, raspberry, cherry, plum, pineapple, apricot, and mixtures thereof. In another specific embodiment, the flavoring agent includes citrus flavorings, such as extracts, essences, or oils of lemon, lime, orange, tangerine, grapefruit, lemon, or kumquat.

[0548] F. Cereal Compositions

[0549] In one embodiment, the sweetening composition is a cereal composition. Cereal compositions are typically consumed as a staple food or a snack. Non-limiting examples of cereal compositions for a particular embodiment include ready-to-eat cereals and hot cereals. Ready-to-eat cereals are cereals that can be consumed without further processing (i.e., cooking) by the consumer. Examples of ready-to-eat cereals include breakfast cereals and snack bars. Breakfast cereals are typically processed into shredded, flaked, puffed, or extruded forms. Breakfast cereals are typically eaten cold and are often mixed with milk and / or fruit. Snack bars include, for example, energy bars, rice cakes, granola bars, and nutrition bars. Hot cereals are typically cooked before consumption, usually in milk or water. Non-limiting examples of hot cereals include grits, porridge, polenta, rice, and oatmeal.

[0550] Cereal compositions typically comprise at least one cereal ingredient. As used herein, the term "cereal ingredient" refers to materials such as whole or partial grains, whole or partial seeds, and whole or partial grasses. Non-limiting examples of cereal ingredients for a particular embodiment include corn, wheat, rice, barley, bran, wheat germ, bulgur, sorghum, millet, oats, rye, triticale, buckwheat, fonio, quinoa, beans, soybeans, amaranth, teff, spelt, and kaniwa.

[0551] G. Baked Goods

[0552] In one embodiment, the sweetening composition is a baked good. As used herein, "baked good" includes ready-to-eat and all ready-to-eat baked products, flours, and mixtures that require preparation before serving. Non-limiting examples of baked goods include cakes, cookies, biscuits, brownies, muffins, rolls, bagels, doughnuts, puff pastries, pastries, croissants, crackers, breads, bread products, and buns.

[0553] Baked goods according to certain embodiments of the present disclosure generally contain a combination of a sweetener, water, fat, and a leavening agent. Baked goods prepared according to many embodiments of the present disclosure also contain flour to form a dough or batter.

[0554] According to certain embodiments of the present disclosure, the leavening agent may include a chemical leavening agent or a yeast leavening agent. Non-limiting examples of chemical leavening agents suitable for specific embodiments of the present disclosure include baking soda (e.g., sodium bicarbonate, potassium bicarbonate, or aluminum bicarbonate), baking acid (e.g., sodium aluminum phosphate, monocalcium phosphate, or dicalcium phosphate), and combinations thereof.

[0555] H. Dairy products

[0556] In one embodiment, the sweetening composition is a dairy product. Dairy products and methods of preparing dairy products suitable for the present disclosure are well known to those of ordinary skill in the art. Dairy products as used herein include milk or foods produced from milk. Non-limiting examples of dairy products suitable for embodiments of the present disclosure include milk, cream, sour cream, fresh cream, buttermilk, fermented buttermilk, milk powder, condensed milk, evaporated milk, butter, cheese, low-fat cheese, cream cheese, yogurt, ice cream, frozen cheesecake, frozen yogurt, gelato, custard (vla), healthy yogurt (piima), filmjolk, kajmak, kefir, viili, kumiss, airag, ice milk, casein, ayran, lassi, khoa, or combinations thereof. Dairy products can be produced by conventional methods or can be filtered or further modified to adjust flavor properties. In certain embodiments, the dairy product can be a liquid dairy product in which one or more carbohydrate sugars (lactose or its breakdown products galactose or glucose) are reduced or substantially removed compared to the milk prior to such processing and supplemented with the sweetening composition described herein. The reduction in carbohydrates can be about 5% or about 10% or about 20% or about 50% or about 70% or more compared to untreated milk.

[0557] According to specific embodiments of the present disclosure, the dairy composition may further comprise other additives. Non-limiting examples of suitable additives include sweeteners and flavorings as disclosed herein, such as chocolate, strawberry, and banana. Particular embodiments of the dairy composition provided herein may also comprise additional nutritional supplements such as vitamins (e.g., vitamin D) and minerals (e.g., calcium) to improve the nutritional composition of the milk.

[0558] I. Tabletop Sweetener Composition

[0559] In one embodiment, the sweetening composition is a tabletop sweetener. The tabletop sweetener may further comprise at least one bulking agent, additive, anticaking agent, functional ingredient, or a combination thereof.

[0560] Suitable "bulking agents" include, but are not limited to, maltodextrin (10DE, 18DE, or 5DE), corn syrup solids (20 or 36DE), sucrose, fructose, glucose, invert sugar, sorbitol, xylose, ribose, mannose, xylitol, mannitol, galactitol, erythritol, maltitol, lactitol, isomaltitol, maltose, tagatose, lactose, inulin, glycerin, propylene glycol, polyols, polydextrose, fructooligosaccharides, cellulose and its derivatives, etc., and mixtures thereof. Additionally, according to other embodiments of the present disclosure, granulated sugar (sucrose) or other caloric sweeteners such as crystalline fructose, other carbohydrates, or sugar alcohols may be used as bulking agents since they provide good content uniformity without adding a large number of calories.

[0561] As used herein, the phrases "anticaking agent" and "flow agent" refer to any composition that aids in content uniformity and uniform dissolution. According to specific embodiments, non-limiting examples of anticaking agents include dental gypsum, calcium silicate, silica, microcrystalline cellulose (Avicel, FMC BioPolymer, Philadelphia, PA), and tricalcium phosphate. In one embodiment, the anticaking agent is present in the tabletop sweetener composition in an amount of from about 0.001% to about 3% by weight of the tabletop sweetener composition.

[0562] The tabletop sweetener composition can be packaged in any form known in the art. Non-limiting forms include, but are not limited to, powder form, granule form, sachets, tablets, pouches, pellets, cubes, solids, and liquids.

[0563] In one embodiment, the tabletop sweetener composition is a single-serve (portion-controlled) package containing a dry blend. Dry blend formulations typically may include powders or granules. Although the tabletop sweetener composition can be in packets of any size, illustrative non-limiting examples of conventional portion-controlled tabletop sweetener packets are about 2.5 x 1.5 inches and contain about 1 gram of sweetener composition having a sweetness equivalent to 2 teaspoons of granulated sugar (about 8 g). In a specific embodiment, the dry blend tabletop sweetener formulation may contain from about 1% (w / w) to about 10% (w / w) of sweetener.

[0564] The tabletop sweetener composition may also be embodied in liquid form, wherein the compositions of the present disclosure are combined with a liquid carrier. Suitable non-limiting examples of carrier agents for liquid tabletop sweeteners include water, alcohols, polyols, glycerol-based or citrate-based dissolved in water, and mixtures thereof. The sweetness equivalent of any form of tabletop sweetener composition described herein or known in the art can vary to obtain the desired sweetness profile. For example, the tabletop sweetener composition may contain a sweetness equivalent to an equal amount of standard sugar. In another embodiment, the sweetness of the tabletop sweetener composition can be up to 100 times the sweetness of an equal amount of sugar. In another embodiment, the sweetness of the tabletop sweetener composition can be at most 90 times, 80 times, 70 times, 60 times, 50 times, 40 times, 30 times, 20 times, 10 times, 9 times, 8 times, 7 times, 6 times, 5 times, 4 times, 3 times, and 2 times that of an equal amount of sugar.

[0565] J. Delivery Systems

[0566] The sweetener compositions disclosed herein may also be formulated into various delivery systems having improved ease of handling and dissolution rates. Non-limiting examples of suitable delivery systems include sweetener compositions co-crystallized with sugars or polyols, agglomerated sweetener compositions, compacted sweetener compositions, dried sweetener compositions, particulate sweetener compositions, spherical sweetener compositions, granular sweetener compositions, and liquid sweetener compositions.

[0567] XI. Breeding Plants

[0568] In addition to directly transforming a particular plant genotype with a construct prepared according to the present disclosure, transgenic plants can also be prepared by crossing a plant having the selected DNA of the present disclosure with a second plant lacking the construct. For example, the selected mogroside biosynthetic pathway coding sequences can be introduced into a particular plant variety by hybridization without directly transforming the plants of that given variety. Thus, the present disclosure encompasses not only plants directly transformed or regenerated from cells transformed according to the present disclosure, but also the progeny of such plants.

[0569] As used herein, the term "progeny" refers to any generation of progeny of a parental plant prepared according to the present disclosure, wherein the progeny contains the selected DNA construct. As disclosed herein, a "hybrid" plant to provide a plant line having one or more added transgenes relative to a starting plant line is defined as a technique in which the starting line is hybridized with a donor plant line containing the transgene of the present disclosure, resulting in the introduction of the transgene of the present disclosure into the plant line. To achieve this, for example, the following steps can be performed:

[0570] (a) Plant seeds of a first parental plant (starting line) and a second parental plant (donor plant line containing the transgene of the present disclosure);

[0571] (b) Growing the seeds of the first and second parental plants into plants with flowers;

[0572] (c) Pollinating the flowers from the first parental plant with pollen from the second parental plant; and

[0573] (d) Harvesting the seeds produced on the parental plant with fertilized flowers.

[0574] Backcrossing is defined herein as a process that includes the following steps:

[0575] (a) Crossing a plant of a first genotype containing a desired gene, DNA sequence, or element with a plant of a second genotype lacking the desired gene, DNA sequence, or element;

[0576] (b) Selecting one or more progeny plants containing the desired gene, DNA sequence, or element;

[0577] (c) Crossing the progeny plant with a plant of the second genotype; and

[0578] (d) Repeating steps (b) and (c) in order to transfer the desired DNA sequence from the plant of the first genotype to the plant of the second genotype.

[0579] The introgression of a DNA element into a plant genotype is defined as the result of a backcross conversion process. The plant genotype into which the DNA sequence is introgressed can be referred to as the backcross-converted genotype, line, inbred, or hybrid. Similarly, a plant genotype lacking the desired DNA sequence can be referred to as the non-converted genotype, line, inbred, or hybrid.

[0580] XII. Other Definitions

[0581] The definitions or explanations of the following technical terms will be used throughout the present disclosure. The technical terms used herein are generally given the meanings that are commonly applied to them in the relevant fields of plant biology, molecular biology, bioinformatics, and plant breeding. All of the following term definitions apply to the entire content of this application.

[0582] For ease of understanding the present disclosure, a number of terms are defined below. The terms defined herein have the meanings commonly understood by those of ordinary skill in the relevant fields of the present disclosure. When used in the claims and / or the specification in conjunction with the term "comprising", the use of the word "a" or "an" can mean "one", but it is also consistent with the meanings of "one or more", "at least one", and "one or more than one". The term "or" used in the claims is used to mean "and / or", unless explicitly stated to refer only to alternatives or the alternatives are mutually exclusive, although the present disclosure supports definitions that refer only to alternatives and "and / or". In the present application, the term "about" is used to mean that a value includes the inherent error variations of the device, the method for measuring the value, or the variations present in the subject under study.

[0583] As used in this specification and the claims, the words "comprising" (and any form of comprising, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "includes" and "include"), or "containing" (and any form of containing, such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. In any of the embodiments of the compositions and methods provided herein, "comprising" may be replaced with "consisting essentially of" or "consisting of". As used herein, the phrase "consisting essentially of" requires the specified integers or steps and those that do not substantially affect the characteristics or functions of the claimed invention. As used herein, the term "consisting of" is used only to refer to the presence of the recited integers (e.g., features, elements, characteristics, properties, method / process steps or limitations) or groups of integers (e.g., features, elements, characteristics, properties, method / process steps or limitations).

[0584] The term "or combinations thereof" as used herein refers to all permutations and combinations of the items listed prior to that term. For example, "A, B, C, or combinations thereof" is intended to include at least one of the following: A, B, C, AB, AC, BC, or ABC, and, if order is important in a particular case, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB. Continuing with this example, explicitly included are combinations that contain one or more repetitions of an item or term, such as BB, AAA, AB, BBC, AAABCCCC, CBBAAA, CABABB, etc. Those skilled in the art will understand that there is generally no limit to the number of items or terms in any combination, unless it is apparent from the context.

[0585] As used herein, approximate terms such as, but not limited to, "about," "substantially," or "essentially" refer to conditions that when so modified are understood to not necessarily be absolute or perfect, but will be considered close enough by one of ordinary skill in the art to ensure the presence of the specified condition. The degree to which the description may vary will depend on how much change can be made and still be recognized by one of ordinary skill in the art as having the required characteristics and capabilities of the unmodified feature. Generally, but subject to the foregoing discussion, a numerical value modified by an approximate term such as "about" in this disclosure may vary from the stated value by at least ±1, 2, 3, 4, 5, 6, 7, 10, 12, or 15%.

[0586] The terms "peptide," "oligopeptide," "polypeptide," "protein," or "enzyme" are used interchangeably herein and refer to a polymeric form of amino acids of any length joined together by peptide bonds, unless otherwise indicated herein. The terms "gene sequence," "polynucleotide," "nucleic acid sequence," "nucleoside sequence," "nucleic acid," "nucleic acid molecule" are used interchangeably herein and refer to a polymeric, unbranched form of nucleotides of any length, ribonucleotides or deoxyribonucleotides or a combination of both.

[0587] Endogenous. "Endogenous" or "native" nucleic acids and / or proteins refer to nucleic acids and / or proteins that exist in a plant or other organism in their native form (i.e., without any human intervention such as recombinant DNA engineering techniques).

[0588] Exogenous. The term "exogenous" (as opposed to "endogenous") refers to a nucleic acid or protein that has been introduced into a plant or other organism by recombinant DNA technology. An "exogenous" nucleic acid or protein may not exist in the plant in its native form, may be different from the nucleic acid or protein that exists in the plant in its native form, may exist at a higher or lower level than the nucleic acid or protein that exists naturally in the plant, or in the case of a nucleic acid may be the same as the nucleic acid that exists in the plant in its native form but integrated at a location different from its native genetic environment.

[0589] Expression: The combination of intracellular processes, including transcription and translation undergone by a coding DNA molecule such as a structural gene to produce a polypeptide.

[0590] Expression cassette. A target nucleic acid sequence operably linked to one or more control sequences described herein (linked at least to a promoter). The expression cassette may also include additional transcriptional and / or translational enhancers. The expression cassette may also include terminator, silencer, and enhancer sequences, intron sequences added to the 5' untranslated region (UTR) or the coding sequence of the nucleic acid sequence, and / or other control sequences such as protein and / or RNA stabilizing elements. The expression cassette may be integrated into the genome of the host cell and replicated with the genome of the host cell, or may transiently exist in the host cell.

[0591] Genetic transformation: A method of introducing a DNA sequence or construct (such as a vector or expression cassette) into a cell or protoplast, where the foreign DNA is incorporated into the chromosome or is capable of autonomous replication.

[0592] Heterologous: A sequence that is not normally present in a given host genome in the genetic context in which it is currently found. In this regard, the sequence can be native to the host genome but rearranged relative to other genetic sequences within the host sequence. For example, a regulatory sequence can be heterologous because it is linked to a different coding sequence relative to the native regulatory sequence.

[0593] Regulate. The term regulate means that the expression level is altered compared to that seen in a control plant. Regulate refers to an increased or decreased expression level.

[0594] Obtain: When used in conjunction with a transgenic plant cell or transgenic plant, obtain means transforming a non-transgenic plant cell or plant to produce a transgenic plant cell or plant, or growing transgenic plant seeds to produce a transgenic plant cell or plant. Such transgenic plant seeds can be from an R0 transgenic plant or can be from any generation of its offspring that inherit the given transgenic sequence from the starting transgenic parental plant.

[0595] Operably linked. The terms "operably linked" or "functionally linked" are used interchangeably and, as used herein, refer to, for example, a functional linkage between a promoter sequence and a target nucleic acid sequence such that the promoter sequence is capable of directing the transcription of the target nucleic acid sequence, or a functional linkage between a terminator sequence and a target nucleic acid sequence such that the terminator sequence is capable of terminating or halting the transcription of the target nucleic acid sequence.

[0596] Plant. The term "plant" as used herein includes whole plants, ancestors and descendants of plants, and plant parts, including fruits, seeds, buds, stems, leaves, roots (including tubers), flowers, and tissues and organs, where each of the foregoing contains the target gene / nucleic acid. The term "plant" also includes plant cells, suspension cultures, callus, embryos, meristematic regions, gametophytes, sporophytes, pollen, and microspores, where each of the foregoing contains the target gene / nucleic acid.

[0597] Ploidy. Ploidy or chromosome ploidy refers to the number of complete sets of chromosomes present in the cell nucleus. Somatic cells, tissues, and individual organisms can be described according to the number of chromosome sets present ("ploidy level"): haploid (1 set), diploid (2 sets), triploid (3 sets), tetraploid (4 sets), pentaploid (5 sets), hexaploid (6 sets), heptaploid (7 sets), etc. The general term polyploidy is used herein to describe cells having three or more chromosome sets.

[0598] Promoter: A recognition site on a DNA sequence or group of DNA sequences that provides expression control elements for a structural gene and to which RNA polymerase specifically binds and initiates RNA synthesis (transcription) of the gene.

[0599] R0 transgenic plant: A plant that has been genetically transformed or regenerated from one or more plant cells that have been genetically transformed.

[0600] Recombinant. A nucleic acid sequence, expression cassette, genetic construct or vector that contains a nucleic acid sequence disclosed herein, or an organism transformed with such a nucleic acid sequence, expression cassette or vector, which is produced by genetic engineering techniques, wherein (a) the nucleic acid sequence or a portion thereof, or (b) a genetic control sequence operably linked to the nucleic acid sequence, such as a promoter or terminator, or (c) a combination of (a) and (b) is not in its natural genetic environment or has been artificially modified and / or inserted by genetic engineering methods.

[0601] Regeneration: The process of growing a plant from plant cells (such as plant protoplasts, callus or explants).

[0602] Selected DNA: A DNA fragment that is desired to be introduced or has been introduced into the plant genome by genetic transformation.

[0603] Terminator. A DNA control sequence at the end of a transcription unit that signals 3'-processing and polyadenylation of the primary transcript and termination of transcription.

[0604] Transformation construct: A chimeric DNA molecule designed to be introduced into the host genome by genetic transformation. A transformation construct typically contains all the genetic elements necessary to direct the expression of one or more foreign genes. In certain embodiments of the present disclosure, it may be necessary to introduce the transformation construct into the host cell in the form of an expression cassette.

[0605] Transformed cell: A cell whose DNA complement has been altered by the introduction of an exogenous DNA molecule into the cell.

[0606] Transgene: A DNA fragment that has been integrated into the host genome or is capable of autonomous replication in a host cell and is capable of causing the expression of one or more coding sequences. Exemplary transgenes will provide a new phenotype to the host cell or a plant regenerated therefrom relative to the corresponding non-transformed cell or plant. A transgene can be introduced directly into a plant by genetic transformation, or it can be inherited from any previous plant transformed with the DNA fragment.

[0607] Transgenic plant: A plant or any subsequent generation of progeny plants derived therefrom, wherein the DNA of the plant or its progeny contains an introduced exogenous DNA fragment that does not naturally occur in non-transgenic plants of the same strain. A transgenic plant may additionally contain sequences that are native to the plant being transformed, but wherein the "exogenous" gene has been altered to change the level or pattern of gene expression, for example by using one or more heterologous regulatory or other elements.

[0608] Vector: A DNA molecule designed to be transformed into a host cell. Some vectors are capable of replicating in a host cell. Plasmids are exemplary vectors, as are expression cassettes isolated therefrom.

[0609] Examples

[0610] The following examples are included to illustrate illustrative embodiments of the present disclosure. Those skilled in the art should understand that the techniques disclosed in the following examples represent techniques that the inventors have found to function well in the practice of the present disclosure and that, accordingly, can be considered to constitute one embodiment of a mode for its practice. However, in light of the present disclosure, those skilled in the art should understand that many changes can be made to the specific embodiments disclosed without departing from the spirit and scope of the present disclosure and still obtain similar or analogous results.

[0611] Example 1

[0612] Construction of expression cassettes

[0613] Construct various expression cassettes having different combinations of nucleotide sequences encoding momordica grosvenori glycoside pathway enzymes and regulatory elements. The construction of these expression cassettes is carried out according to standard genetic engineering methods. The following expression cassettes are constructed.

[0614] SP1463: Use the TM6 MAR insulator sequence, followed by the HLVH12 promoter operably linked to the SQE nucleic acid sequence, which is operably linked to the Pea3A terminator, the DCMV promoter operably linked to the CYP87 nucleic acid sequence, which is operably linked to the AtUBQ3 terminator, the FSgt / PFLt promoter operably linked to the CDS nucleic acid sequence, which is operably linked to the GmaxMYB2 terminator, the e35S promoter operably linked to the CYP72:2A:CYP72 dicistronic nucleic acid sequence, which is operably linked to the AtRBCS2B terminator, the e35S promoter operably linked to the UGT720:2A:UGT720 dicistronic nucleic acid sequence, which is operably linked to the E9 terminator, the CmYLCV promoter operably linked to the UGT94:2A:UGT94 dicistronic nucleic acid sequence, which is operably linked to the ATHSP18.2 terminator, the NOS promoter operably linked to the EPH nucleic acid sequence, which is operably linked to the Ubi3 terminator, the ScBV promoter operably linked to the tHMGR:2A:tHMGR dicistronic nucleic acid sequence, which is operably linked to the AtTub89 terminator, the CsVMV promoter operably linked to the HygR nucleic acid sequence, which is operably linked to the 35S terminator, followed by the TM6 MAR insulator sequence, and assemble the expression cassette.

[0615] SP3139: Use the TM6 MAR insulator sequence, followed by the HLVH12 promoter operably linked to the SQE nucleic acid sequence, which is operably linked to the Pea3A terminator, the DCMV promoter operably linked to the CYP87 nucleic acid sequence, which is operably linked to the AtUBQ3 terminator, the FSgt / PFLt promoter operably linked to the CDS nucleic acid sequence, which is operably linked to the GmaxMYB2 terminator, the dMMV promoter operably linked to the CYP72 Zm nucleic acid sequence, which is operably linked to the AtRBCS2B terminator, the CmYLCV promoter operably linked to the UGT720 nucleic acid sequence, which is operably linked to the E9 terminator, the e35S promoter operably linked to the UGT94 nucleic acid sequence, which is operably linked to the ATHSP18.2 terminator, the NOS promoter operably linked to the EPH nucleic acid sequence, which is operably linked to the Ubi3 terminator, the ScBV promoter operably linked to the tHMGR nucleic acid sequence, which is operably linked to the AtTub89 terminator, the e35S promoter operably linked to the HygR nucleic acid sequence, which is operably linked to the 35S terminator, followed by the TM6 MAR insulator sequence, and assemble the expression cassette.

[0616] SP1908: Use the TM6 MAR insulator sequence, followed by the HLVH12 promoter operably linked to the SQE nucleic acid sequence, which is operably linked to the Pea3A terminator, the DCMV promoter operably linked to the CYP87 nucleic acid sequence, which is operably linked to the AtUBQ3 terminator, the FSgt / PFLt promoter operably linked to the CDS nucleic acid sequence, which is operably linked to the GmaxMYB2 terminator, the dMMV promoter operably linked to the CYP72 Zm nucleic acid sequence, which is operably linked to the AtRBCS2B terminator, the CmYLCV promoter operably linked to the UGT720 nucleic acid sequence, which is operably linked to the E9 terminator, the e35S promoter operably linked to the UGT94 nucleic acid sequence, which is operably linked to the ATHSP18.2 terminator, the NOS promoter operably linked to the EPH nucleic acid sequence, which is operably linked to the Ubi3 terminator, the e35S promoter operably linked to the HygR nucleic acid sequence, which is operably linked to the 35S terminator, followed by the TM6 MAR insulator sequence, and assemble the expression cassette.

[0617] SP3488: Use the TM6 MAR insulator sequence, followed by the HLVH12 promoter operably linked to the SQE nucleic acid sequence, which is operably linked to the Pea3A terminator, the DCMV promoter operably linked to the CYP87 nucleic acid sequence, which is operably linked to the AtUBQ3 terminator, the FSgt / PFLt promoter operably linked to the CDS nucleic acid sequence, which is operably linked to the GmaxMYB2 terminator, the e35S promoter operably linked to the CYP72:2A:CYP72 dicistronic nucleic acid sequence, which is operably linked to the AtRBCS2B terminator, the CmYLCV promoter operably linked to the UGT720:2A:UGT720 dicistronic nucleic acid sequence, which is operably linked to the E9 terminator, the dMMV promoter operably linked to the UGT94:2A:UGT94 dicistronic nucleic acid sequence, which is operably linked to the ATHSP18.2 terminator, the NOS promoter operably linked to the EPH nucleic acid sequence, which is operably linked to the Ubi3 terminator, the ScBV promoter operably linked to the tHMGR:2A:tHMG dicistronic nucleic acid sequence, which is operably linked to the AtTub89 terminator, the CsVMV promoter operably linked to the HygR nucleic acid sequence, which is operably linked to the 35S terminator, followed by the TM6 MAR insulator sequence, and assemble the expression cassette.

[0618] SP3015: Use the TM6 MAR insulator sequence, followed by the dMMV promoter operably linked to the CYP72 nucleic acid sequence, which is operably linked to the AtRBCS2B terminator, the DCMV promoter operably linked to the CYP87 nucleic acid sequence, which is operably linked to the AtUBQ3 terminator, the FSgt / PFLt promoter operably linked to the CDS nucleic acid sequence, which is operably linked to the GmaxMYB2 terminator, the HLVH12 promoter operably linked to the SQE nucleic acid sequence, which is operably linked to the Pea3A terminator, the e35S promoter operably linked to the UGT720 nucleic acid sequence, which is operably linked to the E9 terminator, the CmYLCV promoter operably linked to the UGT94 nucleic acid sequence, which is operably linked to the ATHSP18.2 terminator, the NOS promoter operably linked to the EPH nucleic acid sequence, which is operably linked to the Ubi3 terminator, the ScBV promoter operably linked to the tHMGR nucleic acid sequence, which is operably linked to the AtTub89 terminator, the CsVMV promoter operably linked to the HygR nucleic acid sequence, which is operably linked to the 35S terminator, followed by the TM6 MAR insulator sequence, and assemble the expression cassette.

[0619] SP3432: Use the TM6 MAR insulator sequence, followed by the CmYLCV promoter operably linked to the SQE nucleic acid sequence, which is operably linked to the Pea3A terminator, the DCMV promoter operably linked to the CYP87 nucleic acid sequence, which is operably linked to the AtUBQ3 terminator, the FSgt / PFLt promoter operably linked to the CDS nucleic acid sequence, which is operably linked to the GmaxMYB2 terminator, the e35S promoter operably linked to the CYP72:2A:CYP72 dicistronic nucleic acid sequence, which is operably linked to the AtRBCS2B terminator, the e35S promoter operably linked to the UGT720:2A:UGT720 dicistronic nucleic acid sequence, which is operably linked to the E9 terminator, the dMMV promoter operably linked to the UGT94:2A:UGT94 dicistronic nucleic acid sequence, which is operably linked to the ATHSP18.2 terminator, the NOS promoter operably linked to the EPH nucleic acid sequence, which is operably linked to the Ubi3 terminator, the e35S promoter operably linked to the tHMGR:2A:tHMGR dicistronic nucleic acid sequence, which is operably linked to the AtTub89 terminator, the CsVMV promoter operably linked to the HygR nucleic acid sequence, which is operably linked to the 35S terminator, followed by the TM6 MAR insulator sequence, and assemble the expression cassette.

[0620] SP1160: Use the TM6 MAR insulator sequence, followed by the e35S promoter operably linked to the CYP72:2A:CYP72 dicistronic nucleic acid sequence, which is operably linked to the AtRBCS2B terminator, the CmYLCV promoter operably linked to the UGT720:2A:UGT720 dicistronic nucleic acid sequence, which is operably linked to the E9 terminator, the FSgt / PFLt promoter operably linked to the CDS nucleic acid sequence, which is operably linked to the GmaxMYB2 terminator, the HLVH12 promoter operably linked to the SQE nucleic acid sequence, which is operably linked to the Pea3A terminator, the DCMV promoter operably linked to the CYP87 nucleic acid sequence, which is operably linked to the AtUBQ3 terminator, the dMMV promoter operably linked to the UGT94:2A:UGT94 dicistronic nucleic acid sequence, which is operably linked to the ATHSP18.2 terminator, the NOS promoter operably linked to the EPH nucleic acid sequence, which is operably linked to the Ubi3 terminator, the ScBV promoter operably linked to the tHMGR:2A:tHMGR dicistronic nucleic acid sequence, which is operably linked to the AtTub89 terminator, the CsVMV promoter operably linked to the HygR nucleic acid sequence, which is operably linked to the 35S terminator, followed by the TM6 MAR insulator sequence, and assemble the expression cassette.

[0621] SP2916: Use the TM6 MAR insulator sequence, followed by the e35S promoter operably linked to the CYP72:2A:CYP72 dicistronic nucleic acid sequence, which is operably linked to the AtRBCS2B terminator (all in reverse), the CmYLCV promoter operably linked to the UGT720:2A:UGT720 dicistronic nucleic acid sequence, which is operably linked to the E9 terminator, the FSgt / PFLt promoter operably linked to the CDS nucleic acid sequence, which is operably linked to the GmaxMYB2 terminator, the HLVH12 promoter operably linked to the SQE nucleic acid sequence, which is operably linked to the Pea3A terminator, the DCMV promoter operably linked to the CYP87 nucleic acid sequence, which is operably linked to the AtUBQ3 terminator, the dMMV promoter operably linked to the UGT94:2A:UGT94 dicistronic nucleic acid sequence, which is operably linked to the ATHSP18.2 terminator, the NOS promoter operably linked to the EPH nucleic acid sequence, which is operably linked to the Ubi3 terminator, the ScBV promoter operably linked to the tHMGR:2A:tHMGR dicistronic nucleic acid sequence, which is operably linked to the AtTub89 terminator, the CsVMV promoter operably linked to the HygR nucleic acid sequence, which is operably linked to the 35S terminator, followed by the TM6 MAR insulator sequence, and assemble the expression cassette.

[0622] SP4643: Using the TM6 MAR insulator sequence, followed by the e35S promoter operably linked to the CYP72:2A:CYP72 dicistronic nucleic acid sequence, which is operably linked to the AtRBCS2B terminator, the DCMV promoter operably linked to the CYP87 nucleic acid sequence, which is operably linked to the AtUBQ3 terminator, the FSgt / PFLt promoter operably linked to the CDS nucleic acid sequence, which is operably linked to the GmaxMYB2 terminator, the HLVH12 promoter operably linked to the SQE nucleic acid sequence, which is operably linked to the Pea3A terminator, the CmYLCV promoter operably linked to the UGT720:2A:UGT720 dicistronic nucleic acid sequence, which is operably linked to the E9 terminator, the dMMV promoter operably linked to the UGT94:2A:UGT94 dicistronic nucleic acid sequence, which is operably linked to the ATHSP18.2 terminator, the NOS promoter operably linked to the EPH nucleic acid sequence, which is operably linked to the Ubi3 terminator, the ScBV promoter operably linked to the tHMGR:2A:tHMGR dicistronic nucleic acid sequence, which is operably linked to the AtTub89 terminator, the CsVMV promoter operably linked to the HygR nucleic acid sequence, which is operably linked to the 35S terminator, followed by the TM6 MAR insulator sequence, and assembling the expression cassette.

[0623] SP4870: Use the TM6 MAR insulator sequence, followed by the e35S promoter operably linked to the CYP72:2A:CYP72 dicistronic nucleic acid sequence, which is operably linked to the AtRBCS2B terminator, the CmYLCV promoter operably linked to the UGT720:2A:UGT720 dicistronic nucleic acid sequence, which is operably linked to the E9 terminator, the FMVSgt promoter operably linked to the CDS nucleic acid sequence, which is operably linked to the GmaxMYB2 terminator, the HLVH12 promoter operably linked to the SQE nucleic acid sequence, which is operably linked to the Pea3A terminator, the DCMV promoter operably linked to the CYP87 nucleic acid sequence, which is operably linked to the AtUBQ3 terminator, the dMMV promoter operably linked to the UGT94:2A:UGT94 dicistronic nucleic acid sequence, which is operably linked to the ATHSP18.2 terminator, the NOS promoter operably linked to the EPH nucleic acid sequence, which is operably linked to the Ubi3 terminator, the ScBV promoter operably linked to the tHMGR:2A:tHMGR dicistronic nucleic acid sequence, which is operably linked to the AtTub89 terminator, the CsVMV promoter operably linked to the HygR nucleic acid sequence, which is operably linked to the 35S terminator, followed by the TM6 MAR insulator sequence, and assemble the expression cassette.

[0624] SP1603: Use the TM6 MAR insulator sequence, followed by the e35S promoter operably linked to the CYP72:2A:CYP72 dicistronic nucleic acid sequence, which is operably linked to the AtRBCS2B terminator (all in reverse), the CmYLCV promoter operably linked to the UGT720:2A:UGT720 dicistronic nucleic acid sequence, which is operably linked to the E9 terminator, the FMVSgt promoter operably linked to the CDS nucleic acid sequence, which is operably linked to the GmaxMYB2 terminator, the HLVH12 promoter operably linked to the SQE nucleic acid sequence, which is operably linked to the Pea3A terminator, the DCMV promoter operably linked to the CYP87 nucleic acid sequence, which is operably linked to the AtUBQ3 terminator, the dMMV promoter operably linked to the UGT94:2A:UGT94 dicistronic nucleic acid sequence, which is operably linked to the ATHSP18.2 terminator, the NOS promoter operably linked to the EPH nucleic acid sequence, which is operably linked to the Ubi3 terminator, the ScBV promoter operably linked to the tHMGR:2A:tHMGR dicistronic nucleic acid sequence, which is operably linked to the AtTub89 terminator, the CsVMV promoter operably linked to the HygR nucleic acid sequence, which is operably linked to the 35S terminator, followed by the TM6 MAR insulator sequence, and assemble the expression cassette.

[0625] SP3095: Use the TM6 MAR insulator sequence, followed by the e35S promoter operably linked to the CYP72:2A:CYP72 dicistronic nucleic acid sequence, which is operably linked to the AtRBCS2B terminator (all in reverse), the CmYLCV promoter operably linked to the UGT720:2A:UGT720 dicistronic nucleic acid sequence, which is operably linked to the E9 terminator, the FMVSgt promoter operably linked to the CDS nucleic acid sequence, which is operably linked to the GmaxMYB2 terminator, the HLVH12 promoter operably linked to the SQE nucleic acid sequence, which is operably linked to the Pea3A terminator, the DCMV promoter operably linked to the CYP87 nucleic acid sequence, which is operably linked to the AtUBQ3 terminator, the dMMV promoter operably linked to the UGT94:2A:UGT94 dicistronic nucleic acid sequence, which is operably linked to the ATHSP18.2 terminator, the NOS promoter operably linked to the EPH nucleic acid sequence, which is operably linked to the Ubi3 terminator, the FE3 promoter operably linked to the tHMGR:2A:tHMGR dicistronic nucleic acid sequence, which is operably linked to the AtTub89 terminator, the CsVMV promoter operably linked to the HygR nucleic acid sequence, which is operably linked to the 35S terminator, followed by the TM6 MAR insulator sequence, and assemble the expression cassette.

[0626] SP0265: Use the TM6 MAR insulator sequence, followed by the CmYLCV promoter operably linked to the SQE nucleic acid sequence, which is operably linked to the Pea3A terminator, the DCMV promoter operably linked to the CYP87 nucleic acid sequence, which is operably linked to the AtUBQ3 terminator, the FSgt / PFLt promoter operably linked to the CDS nucleic acid sequence, which is operably linked to the GmaxMYB2 terminator, the e35S promoter operably linked to the UGT720:2A:CYP72 dicistronic nucleic acid sequence, which is operably linked to the E9 terminator, the dMMV promoter operably linked to the UGT94:2A:tHMGR dicistronic nucleic acid sequence, which is operably linked to the ATHSP18.2 terminator, the NOS promoter operably linked to the EPH nucleic acid sequence, which is operably linked to the Ubi3 terminator, the CsVMV promoter operably linked to the HygR nucleic acid sequence, which is operably linked to the 35S terminator, followed by the TM6 MAR insulator sequence, and assemble the expression cassette.

[0627] SP4406: Use the TM6 MAR insulator sequence, followed by the HLVH12 promoter operably linked to the SQE nucleic acid sequence, which is operably linked to the Pea3A terminator, the DCMV promoter operably linked to the CYP87 nucleic acid sequence, which is operably linked to the AtUBQ3 terminator, the FSgt / PFLt promoter operably linked to the CDS nucleic acid sequence, which is operably linked to the GmaxMYB2 terminator, the e35S promoter operably linked to the CYP72:2A:CYP72 dicistronic nucleic acid sequence, which is operably linked to the AtRBCS2B terminator, the CmYLCV promoter operably linked to the UGT720:2A:UGT720 dicistronic nucleic acid sequence, which is operably linked to the E9 terminator, the dMMV promoter operably linked to the UGT94:2A:UGT94 dicistronic nucleic acid sequence, which is operably linked to the ATHSP18.2 terminator, the NOS promoter operably linked to the EPH nucleic acid sequence, which is operably linked to the Ubi3 terminator, the ScBV promoter operably linked to the tHMGR:2A:tHMGR dicistronic nucleic acid sequence, which is operably linked to the AtTub89 terminator, the CsVMV promoter operably linked to the HygR nucleic acid sequence, which is operably linked to the 35S terminator, followed by the TM6 MAR insulator sequence, and assemble the expression cassette.

[0628] SP2152: Use the TM6 MAR insulator sequence, followed by the HLVH12 promoter operably linked to the SQE nucleic acid sequence, which is operably linked to the Pea3A terminator, the DCMV promoter operably linked to the CYP87 nucleic acid sequence, which is operably linked to the AtUBQ3 terminator, the FSgt / PFLt promoter operably linked to the CDS nucleic acid sequence, which is operably linked to the GmaxMYB2 terminator, the e35S promoter operably linked to the CYP72:2A:CYP72 dicistronic nucleic acid sequence, which is operably linked to the AtRBCS2B terminator, the CmYLCV promoter operably linked to the UGT720:2A:UGT720 dicistronic nucleic acid sequence, which is operably linked to the E9 terminator, the dMMV promoter operably linked to the UGT94:2A:UGT94 dicistronic nucleic acid sequence, which is operably linked to the ATHSP18.2 terminator, the NOS promoter operably linked to the EPH nucleic acid sequence, which is operably linked to the Ubi3 terminator, the FE3 promoter operably linked to the tHMGR:2A:tHMGR dicistronic nucleic acid sequence, which is operably linked to the AtTub89 terminator, the CsVMV promoter operably linked to the HygR nucleic acid sequence, which is operably linked to the 35S terminator, followed by the TM6 MAR insulator sequence, and assemble the expression cassette.

[0629] SP4311: Use the TM6 MAR insulator sequence, followed by the e35S promoter operably linked to the CYP72:2A:CYP72 dicistronic nucleic acid sequence, which is operably linked to the AtRBCS2B terminator, the CmYLCV promoter operably linked to the UGT720:2A:UGT720 dicistronic nucleic acid sequence, which is operably linked to the E9 terminator, the FSgt / PFLt promoter operably linked to the CDS nucleic acid sequence, which is operably linked to the GmaxMYB2 terminator, the HLVH12 promoter operably linked to the SQE nucleic acid sequence, which is operably linked to the Pea3A terminator, the DCMV promoter operably linked to the CYP87 nucleic acid sequence, which is operably linked to the AtUBQ3 terminator, the dMMV promoter operably linked to the UGT94:2A:UGT94 dicistronic nucleic acid sequence, which is operably linked to the ATHSP18.2 terminator, the NOS promoter operably linked to the EPH nucleic acid sequence, which is operably linked to the Ubi3 terminator, the ScBV promoter operably linked to the tHMGR nucleic acid sequence, which is operably linked to the AtTub89 terminator, the CsVMV promoter operably linked to the HygR nucleic acid sequence, which is operably linked to the 35S terminator, followed by the TM6 MAR insulator sequence, and assemble the expression cassette.

[0630] SP4378: Use the TM6 MAR insulator sequence, followed by the e35S promoter operably linked to the CYP72:2A:CYP72 dicistronic nucleic acid sequence, which is operably linked to the AtRBCS2B terminator (all in reverse), the CmYLCV promoter operably linked to the UGT720:2A:UGT720 dicistronic nucleic acid sequence, which is operably linked to the E9 terminator, the FSgt / PFLt promoter operably linked to the CDS nucleic acid sequence, which is operably linked to the GmaxMYB2 terminator, the HLVH12 promoter operably linked to the SQE nucleic acid sequence, which is operably linked to the Pea3A terminator, the DCMV promoter operably linked to the CYP87 nucleic acid sequence, which is operably linked to the AtUBQ3 terminator, the dMMV promoter operably linked to the UGT94:2A:UGT94 dicistronic nucleic acid sequence, which is operably linked to the ATHSP18.2 terminator, the NOS promoter operably linked to the EPH nucleic acid sequence, which is operably linked to the Ubi3 terminator, the ScBV promoter operably linked to the tHMGR nucleic acid sequence, which is operably linked to the AtTub89 terminator, the CsVMV promoter operably linked to the HygR nucleic acid sequence, which is operably linked to the 35S terminator, followed by the TM6 MAR insulator sequence, and assemble the expression cassette.

[0631] SP3132: Use the TM6 MAR insulator sequence, followed by the e35S promoter operably linked to the CYP72:2A:CYP72 dicistronic nucleic acid sequence, which is operably linked to the AtRBCS2B terminator, the DCMV promoter operably linked to the CYP87 nucleic acid sequence, which is operably linked to the AtUBQ3 terminator, the FSgt / PFLt promoter operably linked to the CDS nucleic acid sequence, which is operably linked to the GmaxMYB2 terminator, the HLVH12 promoter operably linked to the SQE nucleic acid sequence, which is operably linked to the Pea3A terminator, the CmYLCV promoter operably linked to the UGT720:2A:UGT720 dicistronic nucleic acid sequence, which is operably linked to the E9 terminator, the dMMV promoter operably linked to the UGT94:2A:UGT94 dicistronic nucleic acid sequence, which is operably linked to the ATHSP18.2 terminator, the NOS promoter operably linked to the EPH nucleic acid sequence, which is operably linked to the Ubi3 terminator, the ScBV promoter operably linked to the tHMGR nucleic acid sequence, which is operably linked to the AtTub89 terminator, the CsVMV promoter operably linked to the HygR nucleic acid sequence, which is operably linked to the 35S terminator, followed by the TM6 MAR insulator sequence, and assemble the expression cassette.

[0632] SP2355: Use the TM6 MAR insulator sequence, followed by the e35S promoter operably linked to the CYP72:2A:CYP72 dicistronic nucleic acid sequence, which is operably linked to the AtRBCS2B terminator, the CmYLCV promoter operably linked to the UGT720:2A:UGT720 dicistronic nucleic acid sequence, which is operably linked to the E9 terminator, the FMVSgt promoter operably linked to the CDS nucleic acid sequence, which is operably linked to the GmaxMYB2 terminator, the HLVH12 promoter operably linked to the SQE nucleic acid sequence, which is operably linked to the Pea3A terminator, the DCMV promoter operably linked to the CYP87 nucleic acid sequence, which is operably linked to the AtUBQ3 terminator, the dMMV promoter operably linked to the UGT94:2A:UGT94 dicistronic nucleic acid sequence, which is operably linked to the ATHSP18.2 terminator, the NOS promoter operably linked to the EPH nucleic acid sequence, which is operably linked to the Ubi3 terminator, the ScBV promoter operably linked to the tHMGR nucleic acid sequence, which is operably linked to the AtTub89 terminator, the CsVMV promoter operably linked to the HygR nucleic acid sequence, which is operably linked to the 35S terminator, followed by the TM6 MAR insulator sequence, and assemble the expression cassette.

[0633] SP4762: Using the TM6 MAR insulator sequence, followed by the e35S promoter operably linked to the CYP72:2A:CYP72 dicistronic nucleic acid sequence, which is operably linked to the AtRBCS2B terminator, the CmYLCV promoter operably linked to the UGT720:2A:UGT720 dicistronic nucleic acid sequence, which is operably linked to the E9 terminator, the FMVSgt promoter operably linked to the CDS nucleic acid sequence, which is operably linked to the GmaxMYB2 terminator, the HLVH12 promoter operably linked to the SQE nucleic acid sequence, which is operably linked to the Pea3A terminator, the DCMV promoter operably linked to the CYP87 nucleic acid sequence, which is operably linked to the AtUBQ3 terminator, the dMMV promoter operably linked to the UGT94:2A:UGT94 dicistronic nucleic acid sequence, which is operably linked to the ATHSP18.2 terminator, the NOS promoter operably linked to the EPH nucleic acid sequence, which is operably linked to the Ubi3 terminator, the ScBV promoter operably linked to the tHMGR nucleic acid sequence, which is operably linked to the AtTub89 terminator, the CsVMV promoter operably linked to the HygR nucleic acid sequence, which is operably linked to the 35S terminator, followed by the TM6 MAR insulator sequence, assemble the expression cassette.

[0634] SP0892: Using the TM6 MAR insulator sequence, followed by the HLVH12 promoter operably linked to the SQE nucleic acid sequence, which is operably linked to the Pea3A terminator, the DCMV promoter operably linked to the CYP87 nucleic acid sequence, which is operably linked to the AtUBQ3 terminator, the FSgt / PFLt promoter operably linked to the CDS nucleic acid sequence, which is operably linked to the GmaxMYB2 terminator, the dMMV promoter operably linked to the CYP72 nucleic acid sequence, which is operably linked to the AtRBCS2B terminator, the CmYLCV promoter operably linked to the UGT720 nucleic acid sequence, which is operably linked to the E9 terminator, the e35S promoter operably linked to the UGT94 nucleic acid sequence, which is operably linked to the ATHSP18.2 terminator, the NOS promoter operably linked to the EPH nucleic acid sequence, which is operably linked to the Ubi3 terminator, the ScBV promoter operably linked to the tHMGR nucleic acid sequence, which is operably linked to the AtTub89 terminator, the CsVMV promoter operably linked to the HygR nucleic acid sequence, which is operably linked to the 35S terminator, followed by the TM6 MAR insulator sequence, assemble the expression cassette.

[0635] SP2249: Use the TM6 MAR insulator sequence, followed by the HLVH12 promoter operably linked to the SQE nucleic acid sequence, which is operably linked to the Pea3A terminator, the DCMV promoter operably linked to the CYP87 nucleic acid sequence, which is operably linked to the AtUBQ3 terminator, the FSgt / PFLt promoter operably linked to the CDS nucleic acid sequence, which is operably linked to the GmaxMYB2 terminator, the dMMV promoter operably linked to the CYP72 nucleic acid sequence, which is operably linked to the AtRBCS2B terminator, the e35S promoter operably linked to the UGT720 nucleic acid sequence, which is operably linked to the E9 terminator, the CmYLCV promoter operably linked to the UGT94 nucleic acid sequence, which is operably linked to the ATHSP18.2 terminator, the NOS promoter operably linked to the EPH nucleic acid sequence, which is operably linked to the Ubi3 terminator, the ScBV promoter operably linked to the tHMGR nucleic acid sequence, which is operably linked to the AtTub89 terminator, the CsVMV promoter operably linked to the HygR nucleic acid sequence, which is operably linked to the 35S terminator, followed by the TM6 MAR insulator sequence, and assemble the expression cassette.

[0636] SP0796: Use the TM6 MAR insulator sequence, followed by the dMMV promoter operably linked to the CYP72 nucleic acid sequence, which is operably linked to the AtRBCS2B terminator, the DCMV promoter operably linked to the CYP87 nucleic acid sequence, which is operably linked to the AtUBQ3 terminator, the FSgt / PFLt promoter operably linked to the CDS nucleic acid sequence, which is operably linked to the GmaxMYB2 terminator, the HLVH12 promoter operably linked to the SQE nucleic acid sequence, which is operably linked to the Pea3A terminator, the CmYLCV promoter operably linked to the UGT720 nucleic acid sequence, which is operably linked to the E9 terminator, the e35S promoter operably linked to the UGT94 nucleic acid sequence, which is operably linked to the ATHSP18.2 terminator, the NOS promoter operably linked to the EPH nucleic acid sequence, which is operably linked to the Ubi3 terminator, the ScBV promoter operably linked to the tHMGR nucleic acid sequence, which is operably linked to the AtTub89 terminator, the CsVMV promoter operably linked to the HygR nucleic acid sequence, which is operably linked to the 35S terminator, followed by the TM6 MAR insulator sequence, and assemble the expression cassette.

[0637] SP2057: Use the TM6 MAR insulator sequence, followed by the HLVH12 promoter operably linked to the SQE nucleic acid sequence, which is operably linked to the Pea3A terminator, the DCMV promoter operably linked to the CYP87 nucleic acid sequence, which is operably linked to the AtUBQ3 terminator, the FSgt / PFLt promoter operably linked to the CDS nucleic acid sequence, which is operably linked to the GmaxMYB2 terminator, the dMMV promoter operably linked to the CYP72 nucleic acid sequence, which is operably linked to the AtRBCS2B terminator, the NOS promoter operably linked to the EPH nucleic acid sequence, which is operably linked to the Ubi3 terminator, the e35S promoter operably linked to the UGT94 nucleic acid sequence, which is operably linked to the ATHSP18.2 terminator, the CmYLCV promoter operably linked to the UGT720 nucleic acid sequence, which is operably linked to the E9 terminator, the ScBV promoter operably linked to the tHMGR nucleic acid sequence, which is operably linked to the AtTub89 terminator, the CsVMV promoter operably linked to the HygR nucleic acid sequence, which is operably linked to the 35S terminator, followed by the TM6 MAR insulator sequence, and assemble the expression cassette.

[0638] SP3308: Use the TM6 MAR insulator sequence, followed by the HLVH12 promoter operably linked to the SQE nucleic acid sequence, which is operably linked to the Pea3A terminator, the DCMV promoter operably linked to the CYP87 nucleic acid sequence, which is operably linked to the AtUBQ3 terminator, the FSgt / PFLt promoter operably linked to the CDS nucleic acid sequence, which is operably linked to the GmaxMYB2 terminator, the e35S promoter operably linked to the CYP72 nucleic acid sequence, which is operably linked to the AtRBCS2B terminator, the CmYLCV promoter operably linked to the UGT720 nucleic acid sequence, which is operably linked to the E9 terminator, the dMMV promoter operably linked to the UGT94 nucleic acid sequence, which is operably linked to the ATHSP18.2 terminator, the NOS promoter operably linked to the EPH nucleic acid sequence, which is operably linked to the Ubi3 terminator, the ScBV promoter operably linked to the tHMGR nucleic acid sequence, which is operably linked to the AtTub89 terminator, the CsVMV promoter operably linked to the HygR nucleic acid sequence, which is operably linked to the 35S terminator, followed by the TM6 MAR insulator sequence, and assemble the expression cassette.

[0639] SP1379: Use the TM6 MAR insulator sequence, followed by the dMMV promoter operably linked to the CYP72 nucleic acid sequence, which is operably linked to the AtRBCS2B terminator, the CmYLCV promoter operably linked to the UGT720 nucleic acid sequence, which is operably linked to the E9 terminator, the FSgt / PFLt promoter operably linked to the CDS nucleic acid sequence, which is operably linked to the GmaxMYB2 terminator, the HLVH12 promoter operably linked to the SQE nucleic acid sequence, which is operably linked to the Pea3A terminator, the DCMV promoter operably linked to the CYP87 nucleic acid sequence, which is operably linked to the AtUBQ3 terminator, the e35S promoter operably linked to the UGT94 nucleic acid sequence, which is operably linked to the ATHSP18.2 terminator, the NOS promoter operably linked to the EPH nucleic acid sequence, which is operably linked to the Ubi3 terminator, the ScBV promoter operably linked to the tHMGR nucleic acid sequence, which is operably linked to the AtTub89 terminator, the CsVMV promoter operably linked to the HygR nucleic acid sequence, which is operably linked to the 35S terminator, followed by the TM6 MAR insulator sequence, and assemble the expression cassette.

[0640] SP3494: Use the TM6 MAR insulator sequence, followed by the dMMV promoter operably linked to the CYP72 nucleic acid sequence, which is operably linked to the AtRBCS2B terminator (all in reverse), the CmYLCV promoter operably linked to the UGT720 nucleic acid sequence, which is operably linked to the E9 terminator, the FSgt / PFLt promoter operably linked to the CDS nucleic acid sequence, which is operably linked to the GmaxMYB2 terminator, the HLVH12 promoter operably linked to the SQE nucleic acid sequence, which is operably linked to the Pea3A terminator, the DCMV promoter operably linked to the CYP87 nucleic acid sequence, which is operably linked to the AtUBQ3 terminator, the e35S promoter operably linked to the UGT94 nucleic acid sequence, which is operably linked to the ATHSP18.2 terminator, the NOS promoter operably linked to the EPH nucleic acid sequence, which is operably linked to the Ubi3 terminator, the ScBV promoter operably linked to the tHMGR nucleic acid sequence, which is operably linked to the AtTub89 terminator, the CsVMV promoter operably linked to the HygR nucleic acid sequence, which is operably linked to the 35S terminator, followed by the TM6 MAR insulator sequence, and assemble the expression cassette.

[0641] SP2585: Use the TM6 MAR insulator sequence, followed by the CmYLCV promoter operably linked to the SQE nucleic acid sequence, which is operably linked to the Pea3A terminator, the DCMV promoter operably linked to the CYP87 nucleic acid sequence, which is operably linked to the AtUBQ3 terminator, the FSgt / PFLt promoter operably linked to the CDS nucleic acid sequence, which is operably linked to the GmaxMYB2 terminator, the dMMV promoter operably linked to the CYP72 nucleic acid sequence, which is operably linked to the AtRBCS2B terminator, the e35S promoter operably linked to the UGT720 nucleic acid sequence, which is operably linked to the E9 terminator, the e35S promoter operably linked to the UGT94 nucleic acid sequence, which is operably linked to the ATHSP18.2 terminator, the NOS promoter operably linked to the EPH nucleic acid sequence, which is operably linked to the Ubi3 terminator, the e35S promoter operably linked to the tHMGR nucleic acid sequence, which is operably linked to the AtTub89 terminator, the CsVMV promoter operably linked to the HygR nucleic acid sequence, which is operably linked to the 35S terminator, followed by the TM6 MAR insulator sequence, and assemble the expression cassette.

[0642] SP3635: Use the TM6 MAR insulator sequence, followed by the CmYLCV promoter operably linked to the SQE nucleic acid sequence, which is operably linked to the Pea3A terminator, the DCMV promoter operably linked to the CYP87 nucleic acid sequence, which is operably linked to the AtUBQ3 terminator, the FSgt / PFLt promoter operably linked to the CDS nucleic acid sequence, which is operably linked to the GmaxMYB2 terminator, the e35S promoter operably linked to the UGT720:2A:CYP72 dicistronic nucleic acid sequence, which is operably linked to the E9 terminator, the e35S promoter operably linked to the UGT94:2A:tHMGR dicistronic nucleic acid sequence, which is operably linked to the ATHSP18.2 terminator, the NOS promoter operably linked to the EPH nucleic acid sequence, which is operably linked to the Ubi3 terminator, the CsVMV promoter operably linked to the HygR nucleic acid sequence, which is operably linked to the 35S terminator, followed by the TM6 MAR insulator sequence, and assemble the expression cassette.

[0643] SP3800: Use the TM6 MAR insulator sequence, followed by the CmYLCV promoter operably linked to the SQE nucleic acid sequence, which is operably linked to the Pea3A terminator, the DCMV promoter operably linked to the CYP87 nucleic acid sequence, which is operably linked to the AtUBQ3 terminator, the FSgt / PFLt promoter operably linked to the CDS nucleic acid sequence, which is operably linked to the GmaxMYB2 terminator, the e35S promoter operably linked to the CYP72:2A:CYP72 dicistronic nucleic acid sequence, which is operably linked to the AtRBCS2B terminator, the e35S promoter operably linked to the UGT720:2A:UGT720 dicistronic nucleic acid sequence, which is operably linked to the E9 terminator, the e35S promoter operably linked to the UGT94:2A:UGT94 dicistronic nucleic acid sequence, which is operably linked to the ATHSP18.2 terminator, the NOS promoter operably linked to the EPH nucleic acid sequence, which is operably linked to the Ubi3 terminator, the e35S promoter operably linked to the tHMGR:2A:tHMGR dicistronic nucleic acid sequence, which is operably linked to the AtTub89 terminator, the CsVMV promoter operably linked to the HygR nucleic acid sequence, which is operably linked to the 35S terminator, followed by the TM6 MAR insulator sequence, and assemble the expression cassette.

[0644] SP0981: Use the TM6 MAR insulator sequence, followed by the HLVH12 promoter operably linked to the SQE nucleic acid sequence, which is operably linked to the Pea3A terminator, the DCMV promoter operably linked to the CYP87 nucleic acid sequence, which is operably linked to the AtUBQ3 terminator, the FSgt / PFLt promoter operably linked to the CDS nucleic acid sequence, which is operably linked to the GmaxMYB2 terminator, the dMMV promoter operably linked to the CYP72:2A:CYP72 dicistronic nucleic acid sequence, which is operably linked to the AtRBCS2B terminator, the CmYLCV promoter operably linked to the UGT720:2A:UGT720 dicistronic nucleic acid sequence, which is operably linked to the E9 terminator, the e35S promoter operably linked to the UGT94:2A:UGT94 dicistronic nucleic acid sequence, which is operably linked to the ATHSP18.2 terminator, the NOS promoter operably linked to the EPH nucleic acid sequence, which is operably linked to the Ubi3 terminator, the ScBV promoter operably linked to the tHMGR:2A:tHMGR dicistronic nucleic acid sequence, which is operably linked to the AtTub89 terminator, the CsVMV promoter operably linked to the HygR nucleic acid sequence, which is operably linked to the 35S terminator, followed by the TM6 MAR insulator sequence, and assemble the expression cassette.

[0645] SP0137: Use the TM6 MAR insulator sequence, followed by the HLVH12 promoter operably linked to the SQE nucleic acid sequence, which is operably linked to the Pea3A terminator, the DCMV promoter operably linked to the CYP87 nucleic acid sequence, which is operably linked to the AtUBQ3 terminator, the FSgt / PFLt promoter operably linked to the CDS nucleic acid sequence, which is operably linked to the GmaxMYB2 terminator, the dMMV promoter operably linked to the CYP72:2A:CYP72 dicistronic nucleic acid sequence, which is operably linked to the AtRBCS2B terminator, the CmYLCV promoter operably linked to the UGT720:2A:UGT720 dicistronic nucleic acid sequence, which is operably linked to the E9 terminator, the e35S promoter operably linked to the UGT94:2A:UGT94 dicistronic nucleic acid sequence, which is operably linked to the ATHSP18.2 terminator, the NOS promoter operably linked to the EPH nucleic acid sequence, which is operably linked to the Ubi3 terminator, the FE3 promoter operably linked to the tHMGR:2A:tHMGR dicistronic nucleic acid sequence, which is operably linked to the AtTub89 terminator, the CsVMV promoter operably linked to the HygR nucleic acid sequence, which is operably linked to the 35S terminator, followed by the TM6 MAR insulator sequence, and assemble the expression cassette.

[0646] SP2154: Use the TM6 MAR insulator sequence, followed by the dMMV promoter operably linked to the CYP72:2A:CYP72 dicistronic nucleic acid sequence, which is operably linked to the AtRBCS2B terminator, the CmYLCV promoter operably linked to the UGT720:2A:UGT720 dicistronic nucleic acid sequence, which is operably linked to the E9 terminator, the FSgt / PFLt promoter operably linked to the CDS nucleic acid sequence, which is operably linked to the GmaxMYB2 terminator, the HLVH12 promoter operably linked to the SQE nucleic acid sequence, which is operably linked to the Pea3A terminator, the DCMV promoter operably linked to the CYP87 nucleic acid sequence, which is operably linked to the AtUBQ3 terminator, the e35S promoter operably linked to the UGT94:2A:UGT94 dicistronic nucleic acid sequence, which is operably linked to the ATHSP18.2 terminator, the NOS promoter operably linked to the EPH nucleic acid sequence, which is operably linked to the Ubi3 terminator, the ScBV promoter operably linked to the tHMGR:2A:tHMGR dicistronic nucleic acid sequence, which is operably linked to the AtTub89 terminator, the CsVMV promoter operably linked to the HygR nucleic acid sequence, which is operably linked to the 35S terminator, followed by the TM6 MAR insulator sequence, and assemble the expression cassette.

[0647] SP1727: Use the TM6 MAR insulator sequence, followed by the dMMV promoter operably linked to the CYP72:2A:CYP72 dicistronic nucleic acid sequence, which is operably linked to the AtRBCS2B terminator (all in reverse), the CmYLCV promoter operably linked to the UGT720:2A:UGT720 dicistronic nucleic acid sequence, which is operably linked to the E9 terminator, the FSgt / PFLt promoter operably linked to the CDS nucleic acid sequence, which is operably linked to the GmaxMYB2 terminator, the HLVH12 promoter operably linked to the SQE nucleic acid sequence, which is operably linked to the Pea3A terminator, the DCMV promoter operably linked to the CYP87 nucleic acid sequence, which is operably linked to the AtUBQ3 terminator, the e35S promoter operably linked to the UGT94:2A:UGT94 dicistronic nucleic acid sequence, which is operably linked to the ATHSP18.2 terminator, the NOS promoter operably linked to the EPH nucleic acid sequence, which is operably linked to the Ubi3 terminator, the ScBV promoter operably linked to the tHMGR:2A:tHMGR dicistronic nucleic acid sequence, which is operably linked to the AtTub89 terminator, the CsVMV promoter operably linked to the HygR nucleic acid sequence, which is operably linked to the 35S terminator, followed by the TM6 M AR insulator sequence, and assemble the expression cassette.

[0648] SP0075: Use the TM6 MAR insulator sequence, followed by the HLVH12 promoter operably linked to the SQE nucleic acid sequence, which is operably linked to the Pea3A terminator, the DCMV promoter operably linked to the CYP87 nucleic acid sequence, which is operably linked to the AtUBQ3 terminator, the FSgt / PFLt promoter operably linked to the CDS nucleic acid sequence, which is operably linked to the GmaxMYB2 terminator, the dMMV promoter operably linked to the CYP72:2A:CYP72 dicistronic nucleic acid sequence, which is operably linked to the AtRBCS2B terminator, the e35S promoter operably linked to the UGT720:2A:UGT720 dicistronic nucleic acid sequence, which is operably linked to the E9 terminator, the CmYLCV promoter operably linked to the UGT94:2A:UGT94 dicistronic nucleic acid sequence, which is operably linked to the ATHSP18.2 terminator, the NOS promoter operably linked to the EPH nucleic acid sequence, which is operably linked to the Ubi3 terminator, the ScBV promoter operably linked to the tHMGR:2A:tHMGR dicistronic nucleic acid sequence, which is operably linked to the AtTub89 terminator, the CsVMV promoter operably linked to the HygR nucleic acid sequence, which is operably linked to the 35S terminator, followed by the TM6 MAR insulator sequence, and assemble the expression cassette.

[0649] SP4305: Use the TM6 MAR insulator sequence, followed by the dMMV promoter operably linked to the CYP72:2A:CYP72 dicistronic nucleic acid sequence, which is operably linked to the AtRBCS2B terminator, the DCMV promoter operably linked to the CYP87 nucleic acid sequence, which is operably linked to the AtUBQ3 terminator, the FSgt / PFLt promoter operably linked to the CDS nucleic acid sequence, which is operably linked to the GmaxMYB2 terminator, the HLVH12 promoter operably linked to the SQE nucleic acid sequence, which is operably linked to the Pea3A terminator, the CmYLCV promoter operably linked to the UGT720:2A:UGT720 dicistronic nucleic acid sequence, which is operably linked to the E9 terminator, the e35S promoter operably linked to the UGT94:2A:UGT94 dicistronic nucleic acid sequence, which is operably linked to the ATHSP18.2 terminator, the NOS promoter operably linked to the EPH nucleic acid sequence, which is operably linked to the Ubi3 terminator, the ScBV promoter operably linked to the tHMGR:2A:tHMGR dicistronic nucleic acid sequence, which is operably linked to the AtTub89 terminator, the CsVMV promoter operably linked to the HygR nucleic acid sequence, which is operably linked to the 35S terminator, followed by the TM6 MAR insulator sequence, and assemble the expression cassette.

[0650] SP4221: Use the TM6 MAR insulator sequence, followed by the dMMV promoter operably linked to the CYP72:2A:CYP72 dicistronic nucleic acid sequence, which is operably linked to the AtRBCS2B terminator, the DCMV promoter operably linked to the CYP87 nucleic acid sequence, which is operably linked to the AtUBQ3 terminator, the FSgt / PFLt promoter operably linked to the CDS nucleic acid sequence, which is operably linked to the GmaxMYB2 terminator, the HLVH12 promoter operably linked to the SQE nucleic acid sequence, which is operably linked to the Pea3A terminator, the e35S promoter operably linked to the UGT720:2A:UGT720 dicistronic nucleic acid sequence, which is operably linked to the E9 terminator, the CmYLCV promoter operably linked to the UGT94:2A:UGT94 dicistronic nucleic acid sequence, which is operably linked to the ATHSP18.2 terminator, the NOS promoter operably linked to the EPH nucleic acid sequence, which is operably linked to the Ubi3 terminator, the ScBV promoter operably linked to the tHMGR:2A:tHMGR dicistronic nucleic acid sequence, which is operably linked to the AtTub89 terminator, the CsVMV promoter operably linked to the HygR nucleic acid sequence, which is operably linked to the 35S terminator, followed by the TM6 MAR insulator sequence, and assemble the expression cassette.

[0651] SP3488: Use the TM6 MAR insulator sequence, followed by the HLVH12 promoter operably linked to the SQE nucleic acid sequence, which is operably linked to the Pea3A terminator, the DCMV promoter operably linked to the CYP87 nucleic acid sequence, which is operably linked to the AtUBQ3 terminator, the FSgt / PFLt promoter operably linked to the CDS nucleic acid sequence, which is operably linked to the GmaxMYB2 terminator, the e35S promoter operably linked to the CYP72:2A:CYP72 dicistronic nucleic acid sequence, which is operably linked to the AtRBCS2B terminator, the CmYLCV promoter operably linked to the UGT720:2A:UGT720 dicistronic nucleic acid sequence, which is operably linked to the E9 terminator, the dMMV promoter operably linked to the UGT94:2A:UGT94 dicistronic nucleic acid sequence, which is operably linked to the ATHSP18.2 terminator, the NOS promoter operably linked to the EPH nucleic acid sequence, which is operably linked to the Ubi3 terminator, the ScBV promoter operably linked to the tHMGR:2A:tHMGR dicistronic nucleic acid sequence, which is operably linked to the AtTub89 terminator, the CsVMV promoter operably linked to the HygR nucleic acid sequence, which is operably linked to the 35S terminator, followed by the TM6 MAR insulator sequence, and assemble the expression cassette.

[0652] SP4094: Use the TM6 MAR insulator sequence, followed by the dMMV promoter operably linked to the CYP72:2A:CYP72 dicistronic nucleic acid sequence, which is operably linked to the AtRBCS2B terminator, the CmYLCV promoter operably linked to the UGT720:2A:UGT720 dicistronic nucleic acid sequence, which is operably linked to the E9 terminator, the FMVSgt promoter operably linked to the CDS nucleic acid sequence, which is operably linked to the GmaxMYB2 terminator, the HLVH12 promoter operably linked to the SQE nucleic acid sequence, which is operably linked to the Pea3A terminator, the DCMV promoter operably linked to the CYP87 nucleic acid sequence, which is operably linked to the AtUBQ3 terminator, the e35S promoter operably linked to the UGT94:2A:UGT94 dicistronic nucleic acid sequence, which is operably linked to the ATHSP18.2 terminator, the NOS promoter operably linked to the EPH nucleic acid sequence, which is operably linked to the Ubi3 terminator, the ScBV promoter operably linked to the tHMGR:2A:tHMGR dicistronic nucleic acid sequence, which is operably linked to the AtTub89 terminator, the CsVMV promoter operably linked to the HygR nucleic acid sequence, which is operably linked to the 35S terminator, followed by the TM6 MAR insulator sequence, and assemble the expression cassette.

[0653] SP2971: Use the TM6 MAR insulator sequence, followed by the dMMV promoter operably linked to the CYP72:2A:CYP72 dicistronic nucleic acid sequence, which is operably linked to the AtRBCS2B terminator (all in reverse), the CmYLCV promoter operably linked to the UGT720:2A:UGT720 dicistronic nucleic acid sequence, which is operably linked to the E9 terminator, the FMVSgt promoter operably linked to the CDS nucleic acid sequence, which is operably linked to the GmaxMYB2 terminator, the HLVH12 promoter operably linked to the SQE nucleic acid sequence, which is operably linked to the Pea3A terminator, the DCMV promoter operably linked to the CYP87 nucleic acid sequence, which is operably linked to the AtUBQ3 terminator, the e35S promoter operably linked to the UGT94:2A:UGT94 dicistronic nucleic acid sequence, which is operably linked to the ATHSP18.2 terminator, the NOS promoter operably linked to the EPH nucleic acid sequence, which is operably linked to the Ubi3 terminator, the ScBV promoter operably linked to the tHMGR:2A:tHMGR dicistronic nucleic acid sequence, which is operably linked to the AtTub89 terminator, the CsVMV promoter operably linked to the HygR nucleic acid sequence, which is operably linked to the 35S terminator, followed by the TM6 MAR insulator sequence, and assemble the expression cassette.

[0654] SP2049: Use the TM6 MAR insulator sequence, followed by the HLVH12 promoter operably linked to the SQE nucleic acid sequence, which is operably linked to the Pea3A terminator, the DCMV promoter operably linked to the CYP87 nucleic acid sequence, which is operably linked to the AtUBQ3 terminator, the FMVSgt promoter operably linked to the CDS nucleic acid sequence, which is operably linked to the GmaxMYB2 terminator, the dMMV promoter operably linked to the CYP72:2A:CYP72 dicistronic nucleic acid sequence, which is operably linked to the AtRBCS2B terminator, the e35S promoter operably linked to the UGT720:2A:UGT720 dicistronic nucleic acid sequence, which is operably linked to the E9 terminator, the CmYLCV promoter operably linked to the UGT94:2A:UGT94 dicistronic nucleic acid sequence, which is operably linked to the ATHSP18.2 terminator, the NOS promoter operably linked to the EPH nucleic acid sequence, which is operably linked to the Ubi3 terminator, the ScBV promoter operably linked to the tHMGR:2A:tHMGR dicistronic nucleic acid sequence, which is operably linked to the AtTub89 terminator, the CsVMV promoter operably linked to the HygR nucleic acid sequence, which is operably linked to the 35S terminator, followed by the TM6 MAR insulator sequence, and assemble the expression cassette.

[0655] SP4063: Use the TM6 MAR insulator sequence, followed by the dMMV promoter operably linked to the CYP72:2A:CYP72 dicistronic nucleic acid sequence, which is operably linked to the AtRBCS2B terminator (all in reverse), the CmYLCV promoter operably linked to the UGT720:2A:UGT720 dicistronic nucleic acid sequence, which is operably linked to the E9 terminator, the FMVSgt promoter operably linked to the CDS nucleic acid sequence, which is operably linked to the GmaxMYB2 terminator, the HLVH12 promoter operably linked to the SQE nucleic acid sequence, which is operably linked to the Pea3A terminator, the DCMV promoter operably linked to the CYP87 nucleic acid sequence, which is operably linked to the AtUBQ3 terminator, the e35S promoter operably linked to the UGT94:2A:UGT94 dicistronic nucleic acid sequence, which is operably linked to the ATHSP18.2 terminator, the NOS promoter operably linked to the EPH nucleic acid sequence, which is operably linked to the Ubi3 terminator, the FE3 promoter operably linked to the tHMGR:2A:tHMGR dicistronic nucleic acid sequence, which is operably linked to the AtTub89 terminator, the CsVMV promoter operably linked to the HygR nucleic acid sequence, which is operably linked to the 35S terminator, followed by the TM6 MAR insulator sequence, and assemble the expression cassette.

[0656] SP0121: Use the TM6 MAR insulator sequence, followed by the HLVH12 promoter operably linked to the SQE nucleic acid sequence, which is operably linked to the Pea3A terminator, the DCMV promoter operably linked to the CYP87 nucleic acid sequence, which is operably linked to the AtUBQ3 terminator, the FMVSgt promoter operably linked to the CDS nucleic acid sequence, which is operably linked to the GmaxMYB2 terminator, the FMVSgt promoter operably linked to the CYP72:2A:CYP72 dicistronic nucleic acid sequence, which is operably linked to the AtRBCS2B terminator, the e35S promoter operably linked to the UGT720:2A:UGT720 dicistronic nucleic acid sequence, which is operably linked to the E9 terminator, the CmYLCV promoter operably linked to the UGT94:2A:UGT94 dicistronic nucleic acid sequence, which is operably linked to the ATHSP18.2 terminator, the NOS promoter operably linked to the EPH nucleic acid sequence, which is operably linked to the Ubi3 terminator, the FE3 promoter operably linked to the tHMGR:2A:tHMGR dicistronic nucleic acid sequence, which is operably linked to the AtTub89 terminator, the CsVMV promoter operably linked to the HygR nucleic acid sequence, which is operably linked to the 35S terminator, followed by the TM6 MAR insulator sequence, and assemble the expression cassette.

[0657] SP3358: Use the TM6 MAR insulator sequence, followed by the CmYLCV promoter operably linked to the SQE nucleic acid sequence, which is operably linked to the Pea3A terminator, the DCMV promoter operably linked to the CYP87 nucleic acid sequence, which is operably linked to the AtUBQ3 terminator, the FSgt / PFLt promoter operably linked to the CDS nucleic acid sequence, which is operably linked to the GmaxMYB2 terminator, the e35S promoter operably linked to the CYP72:2A:CYP72 dicistronic nucleic acid sequence, which is operably linked to the AtRBCS2B terminator, the e35S promoter operably linked to the UGT720:2A:UGT720 dicistronic nucleic acid sequence, which is operably linked to the E9 terminator, the e35S promoter operably linked to the UGT94:2A:UGT94 dicistronic nucleic acid sequence, which is operably linked to the ATHSP18.2 terminator, the NOS promoter operably linked to the EPH nucleic acid sequence, which is operably linked to the Ubi3 terminator, the e35S promoter operably linked to the tHMGR:2A:tHMGR dicistronic nucleic acid sequence, which is operably linked to the AtTub89 terminator, the CsVMV promoter operably linked to the HygR nucleic acid sequence, which is operably linked to the 35S terminator, followed by the TM6 MAR insulator sequence, and assemble the expression cassette.

[0658] SP4513: Use the TM6 MAR insulator sequence, followed by the HLVH12 promoter operably linked to the SQE nucleic acid sequence, which is operably linked to the Pea3A terminator, the DCMV promoter operably linked to the CYP87 nucleic acid sequence, which is operably linked to the AtUBQ3 terminator, the FSgt / PFLt promoter operably linked to the CDS nucleic acid sequence, which is operably linked to the GmaxMYB2 terminator, the dMMV promoter operably linked to the CYP72:2A:CYP72 dicistronic nucleic acid sequence, which is operably linked to the AtRBCS2B terminator, the CmYLCV promoter operably linked to the UGT720:2A:UGT720 dicistronic nucleic acid sequence, which is operably linked to the E9 terminator, the e35S promoter operably linked to the UGT94:2A:UGT94 dicistronic nucleic acid sequence, which is operably linked to the ATHSP18.2 terminator, the NOS promoter operably linked to the EPH nucleic acid sequence, which is operably linked to the Ubi3 terminator, the ScBV promoter operably linked to the tHMGR:2A:tHMGR dicistronic nucleic acid sequence, which is operably linked to the AtTub89 terminator, the CsVMV promoter operably linked to the HygR nucleic acid sequence, which is operably linked to the 35S terminator, followed by the TM6 MAR insulator sequence, and assemble the expression cassette.

[0659] SP2221: Use the TM6 MAR insulator sequence, followed by the HLVH12 promoter operably linked to the SQE nucleic acid sequence, which is operably linked to the Pea3A terminator, the DCMV promoter operably linked to the CYP87 nucleic acid sequence, which is operably linked to the AtUBQ3 terminator, the FSgt / PFLt promoter operably linked to the CDS nucleic acid sequence, which is operably linked to the GmaxMYB2 terminator, the dMMV promoter operably linked to the CYP72:2A:CYP72 dicistronic nucleic acid sequence, which is operably linked to the AtRBCS2B terminator, the CmYLCV promoter operably linked to the UGT720:2A:UGT720 dicistronic nucleic acid sequence, which is operably linked to the E9 terminator, the e35S promoter operably linked to the UGT94:2A:UGT94 dicistronic nucleic acid sequence, which is operably linked to the ATHSP18.2 terminator, the NOS promoter operably linked to the EPH nucleic acid sequence, which is operably linked to the Ubi3 terminator, the FE3 promoter operably linked to the tHMGR:2A:tHMGR dicistronic nucleic acid sequence, which is operably linked to the AtTub89 terminator, the CsVMV promoter operably linked to the HygR nucleic acid sequence, which is operably linked to the 35S terminator, followed by the TM6 MAR insulator sequence, and assemble the expression cassette.

[0660] SP3925: Using the TM6 MAR insulator sequence, followed by the dMMV promoter operably linked to the CYP72:2A:CYP72 dicistronic nucleic acid sequence, which is operably linked to the AtRBCS2B terminator, the CmYLCV promoter operably linked to the UGT720:2A:UGT720 dicistronic nucleic acid sequence, which is operably linked to the E9 terminator, the FSgt / PFLt promoter operably linked to the CDS nucleic acid sequence, which is operably linked to the GmaxMYB2 terminator, the HLVH12 promoter operably linked to the SQE nucleic acid sequence, which is operably linked to the Pea3A terminator, the DCMV promoter operably linked to the CYP87 nucleic acid sequence, which is operably linked to the AtUBQ3 terminator, the e35S promoter operably linked to the UGT94:2A:UGT94 dicistronic nucleic acid sequence, which is operably linked to the ATHSP18.2 terminator, the NOS promoter operably linked to the EPH nucleic acid sequence, which is operably linked to the Ubi3 terminator, the ScBV promoter operably linked to the tHMGR:2A:tHMGR dicistronic nucleic acid sequence, which is operably linked to the AtTub89 terminator, the CsVMV promoter operably linked to the HygR nucleic acid sequence, which is operably linked to the 35S terminator, followed by the TM6 MAR insulator sequence, assemble the expression cassette.

[0661] SP3748: Use the TM6 MAR insulator sequence, followed by the dMMV promoter operably linked to the CYP72:2A:CYP72 dicistronic nucleic acid sequence, which is operably linked to the AtRBCS2B terminator (all in reverse), the CmYLCV promoter operably linked to the UGT720:2A:UGT720 dicistronic nucleic acid sequence, which is operably linked to the E9 terminator, the FSgt / PFLt promoter operably linked to the CDS nucleic acid sequence, which is operably linked to the GmaxMYB2 terminator, the HLVH12 promoter operably linked to the SQE nucleic acid sequence, which is operably linked to the Pea3A terminator, the DCMV promoter operably linked to the CYP87 nucleic acid sequence, which is operably linked to the AtUBQ3 terminator, the e35S promoter operably linked to the UGT94:2A:UGT94 dicistronic nucleic acid sequence, which is operably linked to the ATHSP18.2 terminator, the NOS promoter operably linked to the EPH nucleic acid sequence, which is operably linked to the Ubi3 terminator, the ScBV promoter operably linked to the tHMGR:2A:tHMGR dicistronic nucleic acid sequence, which is operably linked to the AtTub89 terminator, the CsVMV promoter operably linked to the HygR nucleic acid sequence, which is operably linked to the 35S terminator, followed by the TM6 MAR insulator sequence, and assemble the expression cassette.

[0662] SP4511: Use the TM6 MAR insulator sequence, followed by the dMMV promoter operably linked to the CYP72:2A:CYP72 dicistronic nucleic acid sequence, which is operably linked to the AtRBCS2B terminator, the DCMV promoter operably linked to the CYP87 nucleic acid sequence, which is operably linked to the AtUBQ3 terminator, the FSgt / PFLt promoter operably linked to the CDS nucleic acid sequence, which is operably linked to the GmaxMYB2 terminator, the HLVH12 promoter operably linked to the SQE nucleic acid sequence, which is operably linked to the Pea3A terminator, the CmYLCV promoter operably linked to the UGT720:2A:UGT720 dicistronic nucleic acid sequence, which is operably linked to the E9 terminator, the e35S promoter operably linked to the UGT94:2A:UGT94 dicistronic nucleic acid sequence, which is operably linked to the ATHSP18.2 terminator, the NOS promoter operably linked to the EPH nucleic acid sequence, which is operably linked to the Ubi3 terminator, the ScBV promoter operably linked to the tHMGR:2A:tHMGR dicistronic nucleic acid sequence, which is operably linked to the AtTub89 terminator, the CsVMV promoter operably linked to the HygR nucleic acid sequence, which is operably linked to the 35S terminator, followed by the TM6 MAR insulator sequence, and assemble the expression cassette.

[0663] SP3547: Use the TM6 MAR insulator sequence, followed by the dMMV promoter operably linked to the CYP72:2A:CYP72 dicistronic nucleic acid sequence, which is operably linked to the AtRBCS2B terminator, the CmYLCV promoter operably linked to the UGT720:2A:UGT720 dicistronic nucleic acid sequence, which is operably linked to the E9 terminator, the FMVSgt promoter operably linked to the CDS nucleic acid sequence, which is operably linked to the GmaxMYB2 terminator, the HLVH12 promoter operably linked to the SQE nucleic acid sequence, which is operably linked to the Pea3A terminator, the DCMV promoter operably linked to the CYP87 nucleic acid sequence, which is operably linked to the AtUBQ3 terminator, the e35S promoter operably linked to the UGT94:2A:UGT94 dicistronic nucleic acid sequence, which is operably linked to the ATHSP18.2 terminator, the NOS promoter operably linked to the EPH nucleic acid sequence, which is operably linked to the Ubi3 terminator, the ScBV promoter operably linked to the tHMGR:2A:tHMGR dicistronic nucleic acid sequence, which is operably linked to the AtTub89 terminator, the CsVMV promoter operably linked to the HygR nucleic acid sequence, which is operably linked to the 35S terminator, followed by the TM6 MAR insulator sequence, and assemble the expression cassette.

[0664] SP3481: Using the TM6 MAR insulator sequence, followed by the dMMV promoter operably linked to the CYP72:2A:CYP72 dicistronic nucleic acid sequence, which is operably linked to the AtRBCS2B terminator (all in reverse), the CmYLCV promoter operably linked to the UGT720:2A:UGT720 dicistronic nucleic acid sequence, which is operably linked to the E9 terminator, the FMVSgt promoter operably linked to the CDS nucleic acid sequence, which is operably linked to the GmaxMYB2 terminator, the HLVH12 promoter operably linked to the SQE nucleic acid sequence, which is operably linked to the Pea3A terminator, the DCMV promoter operably linked to the CYP87 nucleic acid sequence, which is operably linked to the AtUBQ3 terminator, the e35S promoter operably linked to the UGT94:2A:UGT94 dicistronic nucleic acid sequence, which is operably linked to the ATHSP18.2 terminator, the NOS promoter operably linked to the EPH nucleic acid sequence, which is operably linked to the Ubi3 terminator, the ScBV promoter operably linked to the tHMGR:2A:tHMGR dicistronic nucleic acid sequence, which is operably linked to the AtTub89 terminator, the CsVMV promoter operably linked to the HygR nucleic acid sequence, which is operably linked to the 35S terminator, followed by the TM6 MAR insulator sequence, assemble the expression cassette.

[0665] SP2185: Using the TM6 MAR insulator sequence, followed by the dMMV promoter operably linked to the CYP72:2A:CYP72 dicistronic nucleic acid sequence, which is operably linked to the AtRBCS2B terminator (all in reverse), the CmYLCV promoter operably linked to the UGT720:2A:UGT720 dicistronic nucleic acid sequence, which is operably linked to the E9 terminator, the FMVSgt promoter operably linked to the CDS nucleic acid sequence, which is operably linked to the GmaxMYB2 terminator, the HLVH12 promoter operably linked to the SQE nucleic acid sequence, which is operably linked to the Pea3A terminator, the DCMV promoter operably linked to the CYP87 nucleic acid sequence, which is operably linked to the AtUBQ3 terminator, the e35S promoter operably linked to the UGT94:2A:UGT94 dicistronic nucleic acid sequence, which is operably linked to the ATHSP18.2 terminator, the NOS promoter operably linked to the EPH nucleic acid sequence, which is operably linked to the Ubi3 terminator, the FE3 promoter operably linked to the tHMGR:2A:tHMGR dicistronic nucleic acid sequence, which is operably linked to the AtTub89 terminator, the CsVMV promoter operably linked to the HygR nucleic acid sequence, which is operably linked to the 35S terminator, followed by the TM6 MAR insulator sequence, assemble the expression cassette.

[0666] SP2792: Use the TM6 MAR insulator sequence, followed by the CmYLCV promoter operably linked to the SQE nucleic acid sequence, which is operably linked to the Pea3A terminator, the DCMV promoter operably linked to the CYP87 nucleic acid sequence, which is operably linked to the AtUBQ3 terminator, the FSgt / PFLt promoter operably linked to the CDS nucleic acid sequence, which is operably linked to the GmaxMYB2 terminator, the e35S promoter operably linked to the UGT720:2A:CYP72 dicistronic nucleic acid sequence, which is operably linked to the E9 terminator, the e35S promoter operably linked to the UGT94:2A:tHMGR dicistronic nucleic acid sequence, which is operably linked to the ATHSP18.2 terminator, the NOS promoter operably linked to the EPH nucleic acid sequence, which is operably linked to the Ubi3 terminator, the CsVMV promoter operably linked to the HygR nucleic acid sequence, which is operably linked to the 35S terminator, followed by the TM6 MAR insulator sequence, and assemble the expression cassette.

[0667] SP1000: Use the TM6 MAR insulator sequence, followed by the HLVH12 promoter operably linked to the SQE nucleic acid sequence, which is operably linked to the Pea3A terminator, the DCMV promoter operably linked to the CYP87 nucleic acid sequence, which is operably linked to the AtUBQ3 terminator, the FSgt / PFLt promoter operably linked to the CDS nucleic acid sequence, which is operably linked to the GmaxMYB2 terminator, the e35S promoter operably linked to the CYP72:2A:CYP72 dicistronic nucleic acid sequence, which is operably linked to the AtRBCS2B terminator, the CmYLCV promoter operably linked to the UGT720:2A:UGT720 dicistronic nucleic acid sequence, which is operably linked to the E9 terminator, the e35S promoter operably linked to the UGT94:2A:UGT94 dicistronic nucleic acid sequence, which is operably linked to the ATHSP18.2 terminator, the NOS promoter operably linked to the EPH nucleic acid sequence, which is operably linked to the Ubi3 terminator, the ScBV promoter operably linked to the tHMGR:2A:tHMGR dicistronic nucleic acid sequence, which is operably linked to the AtTub89 terminator, the CsVMV promoter operably linked to the HygR nucleic acid sequence, which is operably linked to the 35S terminator, followed by the TM6 MAR insulator sequence, and assemble the expression cassette.

[0668] SP3766: Use the TM6 MAR insulator sequence, followed by the HLVH12 promoter operably linked to the SQE nucleic acid sequence, which is operably linked to the Pea3A terminator, the DCMV promoter operably linked to the CYP87 nucleic acid sequence, which is operably linked to the AtUBQ3 terminator, the FSgt / PFLt promoter operably linked to the CDS nucleic acid sequence, which is operably linked to the GmaxMYB2 terminator, the e35S promoter operably linked to the CYP72:2A:CYP72 dicistronic nucleic acid sequence, which is operably linked to the AtRBCS2B terminator, the CmYLCV promoter operably linked to the UGT720:2A:UGT720 dicistronic nucleic acid sequence, which is operably linked to the E9 terminator, the e35S promoter operably linked to the UGT94:2A:UGT94 dicistronic nucleic acid sequence, which is operably linked to the ATHSP18.2 terminator, the NOS promoter operably linked to the EPH nucleic acid sequence, which is operably linked to the Ubi3 terminator, the FE3 promoter operably linked to the tHMGR:2A:tHMGR dicistronic nucleic acid sequence, which is operably linked to the AtTub89 terminator, the CsVMV promoter operably linked to the HygR nucleic acid sequence, which is operably linked to the 35S terminator, followed by the TM6 MAR insulator sequence, and assemble the expression cassette.

[0669] SP4353: Use the TM6 MAR insulator sequence, followed by the dMMV promoter operably linked to the CYP72:2A:CYP72 dicistronic nucleic acid sequence, which is operably linked to the AtRBCS2B terminator, the CmYLCV promoter operably linked to the UGT720:2A:UGT720 dicistronic nucleic acid sequence, which is operably linked to the E9 terminator, the FSgt / PFLt promoter operably linked to the CDS nucleic acid sequence, which is operably linked to the GmaxMYB2 terminator, the HLVH12 promoter operably linked to the SQE nucleic acid sequence, which is operably linked to the Pea3A terminator, the DCMV promoter operably linked to the CYP87 nucleic acid sequence, which is operably linked to the AtUBQ3 terminator, the e35S promoter operably linked to the UGT94:2A:UGT94 dicistronic nucleic acid sequence, which is operably linked to the ATHSP18.2 terminator, the NOS promoter operably linked to the EPH nucleic acid sequence, which is operably linked to the Ubi3 terminator, the ScBV promoter operably linked to the tHMGR nucleic acid sequence, which is operably linked to the AtTub89 terminator, the CsVMV promoter operably linked to the HygR nucleic acid sequence, which is operably linked to the 35S terminator, followed by the TM6 MAR insulator sequence, and assemble the expression cassette.

[0670] SP0255: Use the TM6 MAR insulator sequence, followed by the dMMV promoter operably linked to the CYP72:2A:CYP72 dicistronic nucleic acid sequence, which is operably linked to the AtRBCS2B terminator (all in reverse), the CmYLCV promoter operably linked to the UGT720:2A:UGT720 dicistronic nucleic acid sequence, which is operably linked to the E9 terminator, the FSgt / PFLt promoter operably linked to the CDS nucleic acid sequence, which is operably linked to the GmaxMYB2 terminator, the HLVH12 promoter operably linked to the SQE nucleic acid sequence, which is operably linked to the Pea3A terminator, the DCMV promoter operably linked to the CYP87 nucleic acid sequence, which is operably linked to the AtUBQ3 terminator, the e35S promoter operably linked to the UGT94:2A:UGT94 dicistronic nucleic acid sequence, which is operably linked to the ATHSP18.2 terminator, the NOS promoter operably linked to the EPH nucleic acid sequence, which is operably linked to the Ubi3 terminator, the ScBV promoter operably linked to the tHMGR nucleic acid sequence, which is operably linked to the AtTub89 terminator, the CsVMV promoter operably linked to the HygR nucleic acid sequence, which is operably linked to the 35S terminator, followed by the TM6 MAR insulator sequence, and assemble the expression cassette.

[0671] SP4815: Use the TM6 MAR insulator sequence, followed by the dMMV promoter operably linked to the CYP72:2A:CYP72 dicistronic nucleic acid sequence, which is operably linked to the AtRBCS2B terminator, the DCMV promoter operably linked to the CYP87 nucleic acid sequence, which is operably linked to the AtUBQ3 terminator, the FSgt / PFLt promoter operably linked to the CDS nucleic acid sequence, which is operably linked to the GmaxMYB2 terminator, the HLVH12 promoter operably linked to the SQE nucleic acid sequence, which is operably linked to the Pea3A terminator, the CmYLCV promoter operably linked to the UGT720:2A:UGT720 dicistronic nucleic acid sequence, which is operably linked to the E9 terminator, the e35S promoter operably linked to the UGT94:2A:UGT94 dicistronic nucleic acid sequence, which is operably linked to the ATHSP18.2 terminator, the NOS promoter operably linked to the EPH nucleic acid sequence, which is operably linked to the Ubi3 terminator, the ScBV promoter operably linked to the tHMGR nucleic acid sequence, which is operably linked to the AtTub89 terminator, the CsVMV promoter operably linked to the HygR nucleic acid sequence, which is operably linked to the 35S terminator, followed by the TM6 MAR insulator sequence, and assemble the expression cassette.

[0672] SP1073: Use the TM6 MAR insulator sequence, followed by the dMMV promoter operably linked to the CYP72:2A:CYP72 dicistronic nucleic acid sequence, which is operably linked to the AtRBCS2B terminator, the CmYLCV promoter operably linked to the UGT720:2A:UGT720 dicistronic nucleic acid sequence, which is operably linked to the E9 terminator, the FMVSgt promoter operably linked to the CDS nucleic acid sequence, which is operably linked to the GmaxMYB2 terminator, the HLVH12 promoter operably linked to the SQE nucleic acid sequence, which is operably linked to the Pea3A terminator, the DCMV promoter operably linked to the CYP87 nucleic acid sequence, which is operably linked to the AtUBQ3 terminator, the e35S promoter operably linked to the UGT94:2A:UGT94 dicistronic nucleic acid sequence, which is operably linked to the ATHSP18.2 terminator, the NOS promoter operably linked to the EPH nucleic acid sequence, which is operably linked to the Ubi3 terminator, the ScBV promoter operably linked to the tHMGR nucleic acid sequence, which is operably linked to the AtTub89 terminator, the CsVMV promoter operably linked to the HygR nucleic acid sequence, which is operably linked to the 35S terminator, followed by the TM6 MAR insulator sequence, and assemble the expression cassette.

[0673] SP4402: Use the TM6 MAR insulator sequence, followed by the dMMV promoter operably linked to the CYP72:2A:CYP72 dicistronic nucleic acid sequence, which is operably linked to the AtRBCS2B terminator (all in reverse), the CmYLCV promoter operably linked to the UGT720:2A:UGT720 dicistronic nucleic acid sequence, which is operably linked to the E9 terminator, the FMVSgt promoter operably linked to the CDS nucleic acid sequence, which is operably linked to the GmaxMYB2 terminator, the HLVH12 promoter operably linked to the SQE nucleic acid sequence, which is operably linked to the Pea3A terminator, the DCMV promoter operably linked to the CYP87 nucleic acid sequence, which is operably linked to the AtUBQ3 terminator, the e35S promoter operably linked to the UGT94:2A:UGT94 dicistronic nucleic acid sequence, which is operably linked to the ATHSP18.2 terminator, the NOS promoter operably linked to the EPH nucleic acid sequence, which is operably linked to the Ubi3 terminator, the ScBV promoter operably linked to the tHMGR nucleic acid sequence, which is operably linked to the AtTub89 terminator, the CsVMV promoter operably linked to the HygR nucleic acid sequence, which is operably linked to the 35S terminator, followed by the TM6 MAR insulator sequence, and assemble the expression cassette.

[0674] SP1415: Use the TM6 MAR insulator sequence, followed by the HLVH12 promoter operably linked to the SQE nucleic acid sequence, which is operably linked to the Pea3A terminator, the DCMV promoter operably linked to the CYP87 nucleic acid sequence, which is operably linked to the AtUBQ3 terminator, the FSgt / PFLt promoter operably linked to the CDS nucleic acid sequence, which is operably linked to the GmaxMYB2 terminator, the e35S promoter operably linked to the CYP72:2A:CYP72 dicistronic nucleic acid sequence, which is operably linked to the AtRBCS2B terminator, the CmYLCV promoter operably linked to the UGT720:2A:UGT720 dicistronic nucleic acid sequence, which is operably linked to the E9 terminator, the e35S promoter operably linked to the UGT94:2A:UGT94 dicistronic nucleic acid sequence, which is operably linked to the ATHSP18.2 terminator, the NOS promoter operably linked to the EPH nucleic acid sequence, which is operably linked to the Ubi3 terminator, the ScBV promoter operably linked to the tHMGR:2A:tHMGR dicistronic nucleic acid sequence, which is operably linked to the AtTub89 terminator, the CsVMV promoter operably linked to the HygR nucleic acid sequence, which is operably linked to the 35S terminator, followed by the TM6 MAR insulator sequence, and assemble the expression cassette.

[0675] SP2353: Use the TM6 MAR insulator sequence, followed by the HLVH12 promoter operably linked to the SQE nucleic acid sequence, which is operably linked to the Pea3A terminator, the DCMV promoter operably linked to the CYP87 nucleic acid sequence, which is operably linked to the AtUBQ3 terminator, the FSgt / PFLt promoter operably linked to the CDS nucleic acid sequence, which is operably linked to the GmaxMYB2 terminator, the e35S promoter operably linked to the CYP72:2A:CYP72 dicistronic nucleic acid sequence, which is operably linked to the AtRBCS2B terminator, the CmYLCV promoter operably linked to the UGT720:2A:UGT720 dicistronic nucleic acid sequence, which is operably linked to the E9 terminator, the e35S promoter operably linked to the UGT94:2A:UGT94 dicistronic nucleic acid sequence, which is operably linked to the ATHSP18.2 terminator, the NOS promoter operably linked to the EPH nucleic acid sequence, which is operably linked to the Ubi3 terminator, the FE3 promoter operably linked to the tHMGR:2A:tHMGR dicistronic nucleic acid sequence, which is operably linked to the AtTub89 terminator, the CsVMV promoter operably linked to the HygR nucleic acid sequence, which is operably linked to the 35S terminator, followed by the TM6 MAR insulator sequence, and assemble the expression cassette.

[0676] SP0565: Use the TM6 MAR insulator sequence, followed by the HLVH12 promoter operably linked to the SQE nucleic acid sequence, which is operably linked to the Pea3A terminator, the DCMV promoter operably linked to the CYP87 nucleic acid sequence, which is operably linked to the AtUBQ3 terminator, the FMVSgt promoter operably linked to the CDS nucleic acid sequence, which is operably linked to the GmaxMYB2 terminator, the e35S promoter operably linked to the CYP72:2A:CYP72 dicistronic nucleic acid sequence, which is operably linked to the AtRBCS2B terminator, the e35S promoter operably linked to the UGT720:2A:UGT720 dicistronic nucleic acid sequence, which is operably linked to the E9 terminator, the CmYLCV promoter operably linked to the UGT94:2A:UGT94 dicistronic nucleic acid sequence, which is operably linked to the ATHSP18.2 terminator, the NOS promoter operably linked to the EPH nucleic acid sequence, which is operably linked to the Ubi3 terminator, the ScBV promoter operably linked to the tHMGR:2A:tHMGR dicistronic nucleic acid sequence, which is operably linked to the AtTub89 terminator, the CsVMV promoter operably linked to the HygR nucleic acid sequence, which is operably linked to the 35S terminator, followed by the TM6 MAR insulator sequence, and assemble the expression cassette.

[0677] SP1202: Use the TM6 MAR insulator sequence, followed by the HLVH12 promoter operably linked to the SQE nucleic acid sequence, which is operably linked to the Pea3A terminator, the DCMV promoter operably linked to the CYP87 nucleic acid sequence, which is operably linked to the AtUBQ3 terminator, the FMVSgt promoter operably linked to the CDS nucleic acid sequence, which is operably linked to the GmaxMYB2 terminator, the e35S promoter operably linked to the CYP72:2A:CYP72 dicistronic nucleic acid sequence, which is operably linked to the AtRBCS2B terminator, the e35S promoter operably linked to the UGT720:2A:UGT720 dicistronic nucleic acid sequence, which is operably linked to the E9 terminator, the CmYLCV promoter operably linked to the UGT94:2A:UGT94 dicistronic nucleic acid sequence, which is operably linked to the ATHSP18.2 terminator, the NOS promoter operably linked to the EPH nucleic acid sequence, which is operably linked to the Ubi3 terminator, the FE3 promoter operably linked to the tHMGR:2A:tHMGR dicistronic nucleic acid sequence, which is operably linked to the AtTub89 terminator, the CsVMV promoter operably linked to the HygR nucleic acid sequence, which is operably linked to the 35S terminator, followed by the TM6 MAR insulator sequence, and assemble the expression cassette.

[0678] SP2808: Using the TM6 MAR insulator sequence, followed by the HLVH12 promoter operably linked to the SQE nucleic acid sequence, which is operably linked to the Pea3A terminator, the DCMV promoter operably linked to the CYP87 nucleic acid sequence, which is operably linked to the AtUBQ3 terminator, the FSgt / PFLt promoter operably linked to the CDS nucleic acid sequence, which is operably linked to the GmaxMYB2 terminator, the dMMV promoter operably linked to the CYP72 Zm nucleic acid sequence, which is operably linked to the AtRBCS2B terminator, the e35S promoter operably linked to the UGT720 nucleic acid sequence, which is operably linked to the ATHSP18.2 terminator, the e35S promoter operably linked to the UGT94 nucleic acid sequence, which is operably linked to the ATHSP18.2 terminator, the N...

Claims

1. A transgenic plant, plant part or seed, comprising: a) A first polynucleotide sequence encoding a cytochrome P450 polypeptide having at least 91% sequence identity with SEQ ID NO:2 or at least 95% sequence identity with SEQ ID NO:86; b) A second polynucleotide sequence encoding a cucurbitadienol synthase polypeptide having at least 90% sequence identity with SEQ ID NO:5; c) A third polynucleotide sequence encoding a cytochrome P450 polypeptide having at least 90% sequence identity with SEQ ID NO:7, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31 or SEQ ID NO:33; d) A fourth polynucleotide sequence encoding a uridine phosphorylase-dependent glycosyltransferase polypeptide having at least 90% sequence identity with SEQ ID NO:9; e) A fifth polynucleotide sequence encoding a uridine phosphorylase-dependent glycosyltransferase polypeptide having at least 90% sequence identity with SEQ ID NO:11; f) A sixth polynucleotide sequence encoding a squalene epoxidase polypeptide having at least 90% sequence identity with SEQ ID NO:13; g) A seventh polynucleotide sequence encoding an epoxide hydrolase polypeptide having at least 90% sequence identity with SEQ ID NO:15; or h) An eighth polynucleotide sequence encoding a truncated 3-hydroxy-3-methylglutaryl-CoA reductase polypeptide having at least 90% sequence identity with SEQ ID NO:17, SEQ ID NO:274, SEQ ID NO:276 or SEQ ID NO:278, wherein the first, second, third, fourth, fifth, sixth, seventh or eighth polynucleotide sequence is operably linked to a heterologous promoter, and wherein the transgenic plant, plant part or seed produces at least a first mogroside compound.

2. The transgenic plant, plant part or seed according to claim 1, wherein the first, second, third, fourth, fifth, sixth, seventh or eighth polynucleotide sequence is operably linked to a different heterologous promoter.

3. The transgenic plant, plant part or seed according to claim 1, wherein at least two of the first, second, third, fourth, fifth, sixth, seventh or eighth polynucleotide sequences are operably linked to a single heterologous promoter.

4. The transgenic plant, plant part or seed according to claim 1, wherein one or more of the first, second, third, fourth, fifth, sixth, seventh or eighth polynucleotide sequences are present in multiple copies.

5. The transgenic plant, plant part or seed according to claim 1, wherein the plant is a plant of the Cucurbitaceae, Solanaceae or Asteraceae family.

6. The transgenic plant, plant part or seed according to claim 5, wherein the plant is a plant of the genus Cucurbita, Citrullus, Cucumis, Momordica, Solanum or Lactuca.

7. The transgenic plant, plant part or seed of claim 6, wherein the plant is watermelon, cantaloupe, honeydew melon, winter melon, casaba melon, Persian melon, citron melon, musk melon, honeydew melon, crenshaw melon, Christmas melon, fairy melon, caravelle melon, hamimelon, rock melon, golden Langkawi melon, Korean melon, saticoy melon, Galia melon, gyokuro melon, gold medal melon, ten melon, new century melon, banana melon, yubari king melon, sugar melon, tiger melon, climbing melon, horned melon, cucamelon, casabanana melon, ginseng fruit, pineapple melon, camouflage melon, sponge melon, bitter melon, charentais melon melon, crane melon, arrow melon, honey melon, rambutan melon, autumn melon, quick melon, lettuce, spinach, rice, oats, corn, sorghum, bitter apple (Citrullus colocynthis), pumpkin, beet, tobacco, miscanthus, tomato, cucumber, potato, amaranth or Nicotiana benthamiana plants.

8. The transgenic plant, plant part or seed of claim 7, wherein the plant is a watermelon, tomato, lettuce or cucumber plant.

9. The transgenic plant, plant part or seed of claim 1, wherein the heterologous promoter is an inducible, plant, bacterial, viral, synthetic, constitutive, tissue-specific, developmentally regulated, cell cycle regulated, temporally regulated, spatially regulated and / or spatiotemporally regulated promoter.

10. The transgenic plant, plant part or seed according to claim 9, wherein the heterologous promoter is FSgt / PFLt (SEQ ID NO: 62), FMVSgt (SEQ ID NO: 69), CsVMV (SEQ ID NO: 68), dMMV (SEQ ID NO: 63), HLVH12 (SEQ ID NO: 60), NOS (SEQ ID NO: 66), ScBV (SEQ ID NO: 67), DCMV (SEQ ID NO: 61), CmYLCV (SEQ ID NO: 64), FS1_1 (SEQ ID NO: 70), FE_3 (SEQ ID NO: 71), e35S (SEQ ID NO: 65), AtUBQ10 (SEQ ID NO: 259), PCLSV (SEQ ID NO: 260), FS4 (SEQ ID NO: 261), AtACT2 (SEQ ID NO: 262), enhanced AtEf-1A (SEQ ID NO: 263), FuasFScp (SEQ ID NO: 264), FE4 (SEQ ID NO: 269), cucumisin (SEQ ID NO: 270) or SgCDS (SEQ ID NO: 271) promoter.

11. The transgenic plant, plant part or seed according to claim 1, wherein the first, second, third, fourth, fifth, sixth, seventh or eighth polynucleotide sequence is operably linked to a heterologous terminator.

12. The transgenic plant, plant part or seed according to claim 11, wherein the first, second, third, fourth, fifth, sixth, seventh or eighth heterologous terminator sequence is GmaxMYB2 (SEQ ID NO: 74), 35S_T (SEQ ID NO:), ATHSP18.2 (SEQ ID NO: 77), AtRBCS2b (SEQ ID NO: 75), AtUBQ3 (SEQ ID NO: 73), pea E9 (SEQ ID NO: 76), pea 3A (SEQ ID NO: 72), potato Ubi3 (SEQ ID NO: 78), AtTubB9 (SEQ ID NO: 79), AtFAD2 (SEQ ID NO: 265), AtNDUFA8 (SEQ ID NO: 266), CsHSP17.3 (SEQ ID NO: 267) or CsHSP22 (SEQ ID NO: 268) terminator.

13. The transgenic plant, plant part or seed according to claim 1, which further comprises a selectable marker sequence.

14. The transgenic plant, plant part or seed according to claim 13, wherein the selectable marker sequence is a β-glucuronidase, green fluorescent protein or antibiotic resistance sequence.

15. The transgenic plant, plant part or seed according to claim 14, wherein the selectable marker sequence is a hygromycin B phosphotransferase (HygR) or neomycin phosphotransferase II (nptII) selectable marker sequence.

16. The transgenic plant, plant part or seed according to claim 1, further comprising: i) a ninth polynucleotide sequence encoding a NADPH: cytochrome P450 reductase polypeptide having at least 90% sequence identity with SEQ ID NO: 19; j) a tenth polynucleotide sequence encoding a uridine phosphorylase-dependent glycosyltransferase polypeptide having at least 90% sequence identity with SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 120 or SEQ ID NO: 126; k) an eleventh polynucleotide sequence encoding a 3-hydroxy-3-methylglutaryl-CoA synthase polypeptide having at least 90% sequence identity with SEQ ID NO: 227; or l) a twelfth polynucleotide sequence encoding a geranyl diphosphate synthase polypeptide having at least 90% sequence identity with SEQ ID NO: 256: wherein the ninth, tenth, eleventh or twelfth polynucleotide sequence is operably linked to a heterologous promoter.

17. The transgenic plant, plant part or seed according to claim 1, further comprising a 2A linker, insulator, selectable marker or filler sequence.

18. The transgenic plant, plant part or seed according to claim 1, wherein the plant is a monocotyledon or dicotyledon.

19. The transgenic plant, plant part or seed according to claim 1, wherein the at least first mogroside compound is a non-natural mogrol precursor, mogrol, mogroside or a metabolite or derivative thereof.

20. The transgenic plant, plant part or seed according to claim 19, wherein the mogroside is mogroside IIA, mogroside IIA1, mogroside IIA2, mogroside IIE, 11-oxo-mogroside II, mogroside III, mogroside IIIA1, mogroside IIIA2, mogroside IIIE, 11-oxo-mogroside III, mogroside IV, mogroside IV A, 11-oxo-mogroside IV, semenicoside I, mogroside V, 11-oxo-mogroside V or mogroside VI, or an isomer thereof.

21. The transgenic plant, plant part or seed according to claim 1, wherein the plant produces at least 10 ng / g to 30 mg / g dry weight of the at least first mogroside compound.

22. The transgenic plant, plant part or seed according to claim 21, wherein the amount of the at least first mogroside compound is higher than the level in non-transgenic plants, plant parts or seeds of the same species.

23. The transgenic plant part according to claim 1, wherein the plant part is a fruit, leaf, root, flower, branch, cell, cell culture, cell suspension culture, endosperm, ovule or pollen.

24. A processed low-calorie food or beverage product produced from the transgenic plant, plant part or seed according to claim 1.

25. The processed low-calorie food or beverage product according to claim 24, wherein the product is produced from the juice or extract of the transgenic plant, plant part or seed.

26. A juice or extract produced from the transgenic plant, plant part or seed according to claim 1.

27. A recombinant DNA molecule comprising: a) a first polynucleotide sequence encoding a cytochrome P450 polypeptide having at least 91% sequence identity with SEQ ID NO:2 or at least 95% sequence identity with SEQ ID NO:86; b) a second polynucleotide sequence encoding a cucurbitadienol synthase polypeptide having at least 90% sequence identity with SEQ ID NO:5; c) a third polynucleotide sequence encoding a cytochrome P450 polypeptide having at least 90% sequence identity with SEQ ID NO:7, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31 or SEQ ID NO:33; d) a fourth polynucleotide sequence encoding a uridine phosphorylase-dependent glycosyltransferase polypeptide having at least 90% sequence identity with SEQ ID NO:9; e) a fifth polynucleotide sequence encoding a uridine phosphorylase-dependent glycosyltransferase polypeptide having at least 90% sequence identity with SEQ ID NO:11; f) a sixth polynucleotide sequence encoding a squalene epoxidase polypeptide having at least 90% sequence identity with SEQ ID NO:13; g) a seventh polynucleotide sequence encoding an epoxide hydrolase polypeptide having at least 90% sequence identity with SEQ ID NO:15; or h) an eighth polynucleotide sequence encoding a truncated 3-hydroxy-3-methylglutaryl-CoA reductase polypeptide having at least 90% sequence identity with SEQ ID NO:17, SEQ ID NO:274, SEQ ID NO:276 or SEQ ID NO:278, wherein the first, second, third, fourth, fifth, sixth, seventh or eighth polynucleotide sequence is operably linked to a heterologous promoter.

28. The recombinant DNA molecule according to claim 27, wherein the first, second, third, fourth, fifth, sixth, seventh or eighth polynucleotide sequence is operably linked to a different heterologous promoter.

29. The recombinant DNA molecule according to claim 27, wherein at least two of the first, second, third, fourth, fifth, sixth, seventh or eighth polynucleotide sequences are operably linked to a single heterologous promoter.

30. The recombinant DNA molecule according to claim 27, wherein one or more of the first, second, third, fourth, fifth, sixth, seventh or eighth polynucleotide sequences are present in multiple copies.

31. The recombinant DNA molecule according to claim 27, wherein the heterologous promoter is the FSgt / PFLt (SEQ ID NO: 62), FMVSgt (SEQ ID NO: 69), CsVMV (SEQ ID NO: 68), dMMV (SEQ ID NO: 63), HLVH12 (SEQ ID NO: 60), NOS (SEQ ID NO: 66), ScBV (SEQ ID NO: 67), DCMV (SEQ ID NO: 61), CmYLCV (SEQ ID NO: 64), FS1_1 (SEQ ID NO: 70), FE_3 (SEQ ID NO: 71), e35S (SEQ ID NO: 65), AtUBQ10 (SEQ ID NO: 259), PCLSV (SEQ ID NO: 260), FS4 (SEQ ID NO: 261), AtACT2 (SEQ ID NO: 262), enhanced AtEf-1A (SEQ ID NO: 263), FuasFScp (SEQ ID NO: 264), FE4 (SEQ ID NO: 269), cucumisin (SEQ ID NO: 270) or SgCDS (SEQ ID NO: 271) promoter.

32. The recombinant DNA molecule according to claim 27, wherein the first, second, third, fourth, fifth, sixth, seventh or eighth polynucleotide sequence is operably linked to a heterologous terminator.

33. The recombinant DNA molecule according to claim 32, wherein the heterologous terminator sequence is GmaxMYB2 (SEQ ID NO: 74), 35S_T (SEQ ID NO:), ATHSP18.2 (SEQ ID NO: 77), AtRBCS2b (SEQ ID NO: 75), AtUBQ3 (SEQ ID NO: 73), pea E9 (SEQ ID NO: 76), pea 3A (SEQ ID NO: 72), potato Ubi3 (SEQ ID NO: 78), AtTubB9 (SEQ ID NO: 79), AtFAD2 (SEQ ID NO: 265), AtNDUFA8 (SEQ ID NO: 266), CsHSP17.3 (SEQ ID NO: 267) or CsHSP22 (SEQ ID NO: 268) terminator.

34. The recombinant DNA molecule according to claim 27, which further comprises a 2A linker, an insulator, a selectable marker or a filler sequence.

35. A DNA molecule comprising a polynucleotide sequence encoding a cytochrome P450 polypeptide having at least 91% sequence identity with SEQ ID NO:2 or at least 95% sequence identity with SEQ ID NO:

86.

36. The DNA molecule according to claim 35, which is operably linked to a heterologous promoter.

37. A DNA molecule exhibiting gene regulatory functional activity, comprising a polynucleotide sequence selected from: a) a sequence having at least 90% sequence identity with SEQ ID NO:70 or 71 and exhibiting promoter activity; b) a sequence comprising SEQ ID NO:70 or 71; and c) a fragment of SEQ ID NO:70 or 71, wherein the fragment exhibits promoter activity; wherein the DNA molecule is operably linked to a heterologous transcribable polynucleotide molecule.

38. A method for producing at least a first mogroside compound, comprising cultivating a transgenic plant according to claim 1, wherein the transgenic plant produces the at least first mogroside compound.

39. The method according to claim 38, wherein the transgenic plant produces the at least first mogroside compound in a plant part or seed of the plant.

40. The method according to claim 38, wherein the transgenic plant produces the at least first mogroside compound in a fruit or leaf of the plant.

41. The method according to claim 38, further comprising the step of isolating the at least first mogroside compound from the transgenic plant.

42. The method according to claim 41, wherein the at least first mogroside compound is isolated from a fruit or leaf of the transgenic plant.

43. The method according to claim 41, wherein the at least first mogroside compound is isolated from a plant part or seed of the transgenic plant.

44. A recombinant host cell comprising: a) a first polynucleotide sequence encoding a cytochrome P450 polypeptide having at least 91% sequence identity with SEQ ID NO:2 or at least 95% sequence identity with SEQ ID NO:86; b) a second polynucleotide sequence encoding a cucurbitadienol synthase polypeptide having at least 90% sequence identity with SEQ ID NO:5; c) a third polynucleotide sequence encoding a cytochrome P450 polypeptide having at least 90% sequence identity with SEQ ID NO:7, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31 or SEQ ID NO:33; d) a fourth polynucleotide sequence encoding a uridine phosphorylase-dependent glycosyltransferase polypeptide having at least 90% sequence identity with SEQ ID NO:9; e) a fifth polynucleotide sequence encoding a uridine phosphorylase-dependent glycosyltransferase polypeptide having at least 90% sequence identity with SEQ ID NO:11; f) a sixth polynucleotide sequence encoding a squalene epoxidase polypeptide having at least 90% sequence identity with SEQ ID NO:13; g) a seventh polynucleotide sequence encoding an epoxide hydrolase polypeptide having at least 90% sequence identity with SEQ ID NO:15; or h) an eighth polynucleotide sequence encoding a truncated 3-hydroxy-3-methylglutaryl-CoA reductase polypeptide having at least 90% sequence identity with SEQ ID NO:17, SEQ ID NO:274, SEQ ID NO:276 or SEQ ID NO:278, wherein the first, second, third, fourth, fifth, sixth, seventh or eighth polynucleotide sequence is operably linked to a heterologous promoter.

45. The recombinant host cell according to claim 44, wherein the recombinant host cell produces at least a first mogroside compound.

46. A composition comprising: a) about 80% mogroside V, about 15% 11-oxo-mogroside V and about 5% mogroside III-A1; or b) about 40% esgoside I, about 40% mogroside V and about 20% 11-oxo-mogroside V.

47. The composition according to claim 46, wherein the composition is a liquid.

48. The composition according to claim 46, wherein the composition is a dry powder.

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