阅读说明：本技术 高纯度甜菊糖苷 (High purity steviol glycosides ) 是由 A·马科斯雅恩 S·Y·周 K·尼扎姆宾纳维 K·克汗 M·阿弗扎尔宾哈希姆 S·A/L· 于 2019-03-15 设计创作，主要内容包括：描述了制备高度纯化的甜菊糖苷的方法,所述甜菊糖苷特别是甜菊单糖苷,甜菊单糖苷A,甜菊双糖苷,甜菊双糖苷D,甜茶苷,甜菊双糖苷A,甜菊双糖苷B,瑞鲍迪苷B,甜菊苷,瑞鲍迪苷G,甜菊苷A,甜菊苷B,甜菊苷C,瑞鲍迪苷A,瑞鲍迪苷E,瑞鲍迪苷E2,瑞鲍迪苷E4,瑞鲍迪苷E6,瑞鲍迪苷E3,瑞鲍迪苷D,瑞鲍迪苷I,瑞鲍迪苷AM,瑞鲍迪苷D7,瑞鲍迪苷M,瑞鲍迪苷M4,瑞鲍迪苷1a,瑞鲍迪苷1b,瑞鲍迪苷1c,瑞鲍迪苷1d,瑞鲍迪苷1e,瑞鲍迪苷1f,瑞鲍迪苷1g,瑞鲍迪苷1h,瑞鲍迪苷1i,瑞鲍迪苷1j,瑞鲍迪苷1k,瑞鲍迪苷1l,瑞鲍迪苷1m,瑞鲍迪苷1n,瑞鲍迪苷1o,瑞鲍迪苷1p,瑞鲍迪苷1q,瑞鲍迪苷1r,瑞鲍迪苷1s,瑞鲍迪苷1t,瑞鲍迪苷2a,瑞鲍迪苷2b,瑞鲍迪苷2c,瑞鲍迪苷2d,瑞鲍迪苷2e,瑞鲍迪苷2f,瑞鲍迪苷2g,瑞鲍迪苷2h,瑞鲍迪苷2i,瑞鲍迪苷2j,瑞鲍迪苷2k,瑞鲍迪苷2l,瑞鲍迪苷2m,瑞鲍迪苷2n,瑞鲍迪苷2o,瑞鲍迪苷2p,瑞鲍迪苷2q,瑞鲍迪苷2r,瑞鲍迪苷2s和/或SvG7。该方法包括使用酶制剂和重组微生物将不同的起始组合物转化为目标甜菊糖苷。高度纯化的甜菊糖苷用作可食用和可咀嚼组合物例如任意饮料,糖食,焙烤食品,饼干和口香糖中的无热量甜味剂,风味增强剂,甜味增强剂和泡沫抑制剂。(Described is a process for the preparation of highly purified steviol glycosides, in particular steviolmonoside, steviolmonoside a, steviolbioside D, rubusoside, steviolbioside a, steviolbioside B, rebaudioside B, stevioside, rebaudioside G, stevioside a, stevioside B, stevioside C, rebaudioside a, rebaudioside E2, rebaudioside E4, rebaudioside E6, rebaudioside E3, rebaudioside D, rebaudioside I, rebaudioside AM, rebaudioside D7, rebaudioside M4, rebaudioside 1a, rebaudioside 1B, rebaudioside 1C, rebaudioside 1D, rebaudioside 1E, rebaudioside 1f, rebaudioside 1G, rebaudioside 1h, rebaudioside 1I, rebaudioside 1j, rebaudioside 1k, rebaudioside 1l, rebaudioside 1m, rebaudioside 1n, rebaudioside 1o, rebaudioside 1p, rebaudioside 1q, rebaudioside 1r, rebaudioside 1s, rebaudioside 1t, rebaudioside 2a, rebaudioside 2b, rebaudioside 2c, rebaudioside 2d, rebaudioside 2e, rebaudioside 2f, rebaudioside 2g, rebaudioside 2h, rebaudioside 2i, rebaudioside 2j, rebaudioside 2k, rebaudioside 2l, rebaudioside 2m, rebaudioside 2n, rebaudioside 2o, rebaudioside 2p, rebaudioside 2q, rebaudioside 2r, rebaudioside 2s, and/or SvG 7. The method comprises converting different starting compositions into the target steviol glycoside using an enzyme preparation and a recombinant microorganism. The highly purified steviol glycosides are useful as non-caloric sweeteners, flavor enhancers, sweetness enhancers and foam inhibitors in edible and chewable compositions such as any beverages, confections, baked goods, cookies and chewing gum.)

1. Steviol glycoside I-XL having the formula:

wherein the R1 and R2 sugar chains are as defined in the table below;

2. a process for producing at least one steviol glycoside according to claim 1, comprising the steps of:

a. providing a starting composition comprising an organic compound having at least one carbon atom;

b. providing an enzyme preparation or microorganism containing at least one enzyme selected from the group consisting of a steviol biosynthetic enzyme, an NDP-glucosyltransferase and optionally an NDP-glucose recycling enzyme;

c. Contacting the enzyme preparation or microorganism with a medium containing the starting composition to produce a medium comprising at least one steviol glycoside according to claim 1.

3. A process for producing at least one steviol glycoside according to claim 1, comprising the steps of:

a. providing a starting composition comprising an organic compound having at least one carbon atom;

b. providing a biocatalyst comprising at least one enzyme selected from the group consisting of a steviol biosynthetic enzyme, an NDP-glucosyltransferase, and optionally an NDP-glucose recycling enzyme;

c. contacting the biocatalyst with a medium containing the starting composition to produce a medium comprising at least one steviol glycoside of claim 1.

4. The method of claim 2 or 3, further comprising the steps of:

d. separating at least one steviol glycoside according to claim 1 from the medium, providing a highly purified composition of at least one steviol glycoside according to claim 1.

5. The process of claim 2, 3 or 4, wherein the starting composition is selected from the group consisting of steviol, steviolmonoside A, steviolbioside D, rubusoside, steviolbioside A, steviolbioside B, rebaudioside B, stevioside, rebaudioside G, stevioside A, stevioside B, stevioside C, rebaudioside A, rebaudioside E2, rebaudioside E4, rebaudioside E6, rebaudioside E3, rebaudioside D, rebaudioside I, rebaudioside AM, rebaudioside D7, rebaudioside M4, other steviolbiosides, polyols, sugars and combinations thereof.

6. The method of claim 2, wherein the microorganism is selected from the group consisting of Escherichia coli (E.coli), Saccharomyces (Saccharomyces sp.), Aspergillus (Aspergillus sp.), Pichia (Pichia sp.), Bacillus (Bacillus sp.) and Yarrowia (Yarrowia sp.).

7. The process of claim 3, wherein the biocatalyst is an enzyme or a cell comprising one or more enzymes capable of converting a starting composition into at least one steviol glycoside according to claim 1.

8. The method of claim 2, wherein the enzyme is selected from the group consisting of Mevalonate (MVA) pathway enzymes, 2-C-methyl-D-erythritol-4-phosphate pathway (MEP/DOXP) enzymes, geranylgeranyl diphosphate synthase, copalyl diphosphate synthase, kaurene oxidase, kaurene 13-hydroxylase (KAH), steviol synthase, deoxyxylulose 5-phosphate synthase (DXS), D-1-deoxyxylulose 5-phosphate reductoisomerase (DXR), cytosinyl-2-C-methyl-D-erythritol synthase (CMS), cytosinyl-2-C-methyl-D-erythritol 4-diphosphate kinase (CMK), cytosinyl-2-C-methyl-D-erythritol 4-diphosphate (D-erythritol), and the like Sugar alcohol 2, 4-cyclic diphosphate synthase (MCS), l-hydroxy-2-methyl-2 (E) -butenyl 4-diphosphate synthase (HDS), l-hydroxy-2-methyl-2 (E) -butenyl 4-diphosphate reductase (HDR), acetoacetyl-CoA thiolase, truncated HMG-CoA reductase, mevalonate kinase, phosphomevalonate kinase, mevalonate pyrophosphate decarboxylase, cytochrome P450 reductase, UGT74G1, UGT85C2, UGT91D2, EUGT11, UGTSl2, UGT76G1, UGlyT91C1 or salts thereof having > 85% amino acid sequence identity, > 86% amino acid sequence identity, > 87% amino acid sequence identity, > 88% amino acid sequence identity, > 89% amino acid sequence identity, > 90% amino acid sequence identity, > 91% amino acid sequence identity, a mutant variant of > 92% amino acid sequence identity, > 93% amino acid sequence identity, > 94% amino acid sequence identity, > 95% amino acid sequence identity, > 96% amino acid sequence identity, > 97% amino acid sequence identity, > 98% amino acid sequence identity, > 99% amino acid sequence identity.

9. The method of claim 4, wherein the at least one steviol glycoside of claim 1 is present in the highly purified composition in an amount of greater than about 95% by weight on a dry weight basis.

10. A consumable product comprising at least one steviol glycoside according to claim 1, wherein the product is selected from the group consisting of a food product, a beverage, a pharmaceutical composition, a tobacco product, a nutraceutical composition, an oral hygiene composition and a cosmetic composition.

11. The consumable product of claim 10, wherein said product is selected from the group consisting of a beverage; natural juice; refreshing beverage; a carbonated soft drink; a sugar-free beverage; a zero-calorie beverage; low-calorie beverages and foods; a yogurt drink; instant juice; instant coffee; instant beverages of the powdered type; can products; syrup; fermenting the soybean paste; soy sauce; vinegar; a seasoning; mayonnaise; tomato sauce; curry food; soup; instant soup; powdery soy sauce; powdery vinegar; various types of biscuits; rice biscuits; a thin crisp biscuit; bread; chocolate; caramel; a candy; a chewing gum; jelly; pudding; preserved fruits and pickled vegetables; fresh cream; jam; citrus pulp; flower sauce; milk powder; ice cream; a snowflake; bottling vegetables and fruits; canning and boiling beans; cooking meat and food with sweet juice; agricultural vegetable food; seafood; ham; sausages; fish ham; fish sausages; fish paste; a fried fish product; drying marine products; freezing the food; pickling the seaweed; pickling meat; tobacco and pharmaceutical products.

12. The consumer product of claim 10, further comprising at least one additive selected from the group consisting of sugars, 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, caffeine, flavor and flavor ingredients, astringent compounds, protein or protein hydrolysates, surfactants, emulsifiers, flavonoids, alcohols, polymers, and combinations thereof.

13. The consumable product of claim 10, further comprising at least one functional ingredient selected from the group consisting of saponins, antioxidants, dietary fiber sources, fatty acids, vitamins, glucosamine, minerals, preservatives, hydrating agents, probiotics, prebiotics, body weight management agents, osteoporosis management agents, phytoestrogens, long chain primary aliphatic saturated alcohols, phytosterols, and combinations thereof.

14. The consumer product of claim 10, further comprising a compound selected from the group consisting of: steviolmonoside, steviolmonoside A, steviolbioside A, steviolbioside B, steviolbioside C, steviolbioside D, steviolbioside E, rubusoside, dulcoside A, dulcoside B, dulcoside C, dulcoside D, stevioside A, stevioside B, stevioside C, stevioside D, stevioside E2, stevioside F, stevioside G, stevioside H, rebaudioside A2, rebaudioside A3, rebaudioside A4, rebaudioside B2, rebaudioside C2, rebaudioside C3, rebaudioside C4, rebaudioside C5, rebaudioside C6, rebaudioside D2, rebaudioside D3, rebaudioside D4, rebaudioside D5, rebaudioside D6, rebaudioside D5848, rebaudioside D7, rebaudioside E, rebaudioside D7, rebaudioside D, rebaudioside E8, rebaudioside D7, rebaudioside E, rebaudioside E3, rebaudioside E4, rebaudioside E5, rebaudioside E6, rebaudioside E7, rebaudioside F2, rebaudioside F3, rebaudioside G, rebaudioside H2, rebaudioside H3, rebaudioside H4, rebaudioside H5, rebaudioside H6, rebaudioside I2, rebaudioside I3, rebaudioside J, rebaudioside K2, rebaudioside KA, rebaudioside L, rebaudioside M2, rebaudioside M3, rebaudioside N2, rebaudioside N3, rebaudioside O3, rebaudioside T3, rebaudioside T, rebaudioside U2, rebaudioside V2, rebaudioside V3, rebaudioside W2, rebaudioside W3, rebaudioside Y, rebaudioside Z1, rebaudioside Z2, rebaudioside AM, SvG7, NSF-02, mogroside V, siratose, Lo Han Guo, psicose, D-allose, D-tagatose, erythritol, brazzein, neohesperidin dihydrochalcone, glycyrrhizic acid and salts thereof, thaumatin, perillaseed, pinosylvin, sessile sesquiterpene, sinomenine, phlomisoside I, dimethyl-hexahydrofluorene-dicarboxylic acid, abrin, brassinosteroid, biochanin, cyclocarioside, polybopogoside A, rubusoside, lanoside, dulcoside, sarsasaponin, cyclocarioside, sarsasaponin, secodarabine-type triterpene, polypodoside A, rubusoside, brazilin, glucoronide, glucoroniside, sarsasaponin, sarsasannside, sarsasanqua, sarin, sarsasanqua, dihydroflavonol, dihydroquercetin-3-acetate, neoastin, trans-cinnamaldehyde, monatin and salts thereof, trueranin A, hematoxylin, monellin, caritin, pterocarpin A, pterocarpin B, mabinlin, pethidine, flavoured glycoprotein, curculin, neocurculin, chlorogenic acid, cynarin, siamenoside, sucralose, acesulfame potassium, aspartame, alitame, saccharin, cyclamate, neotame, dulcin, suosan advantame, gymnemic acid, dulcin, ziziphin, nordulcin, aspartic acid, glycine, alanine, threonine, proline, serine, lysine, tryptophan, maltitol, mannitol, sorbitol, lactitol, xylitol, inositol, isomalt, propylene glycol, glycerol, galactitol, dulcitol, hydrogenated isomaltulose, reduced isomalto-oligosaccharides, reduced xylo-oligosaccharides, reduced gentio-oligosaccharides, reduced maltose syrup, reduced glucose syrup, hydrogenated starch hydrolysate, polyglucitol, sugar alcohol, L-sugar, L-sorbose, L-arabinose, trehalose, galactose, rhamnose, various cyclodextrins, cyclic oligosaccharides, various types of maltodextrin, dextran, sucrose, glucose, ribulose, fructose, threose, xylose, lyxose, altrose, mannose, idose, lactose, maltose, invert sugar, isohalose, neotrehalose, isomaltulose, erythrose, deoxyribose, gulose, talose, erythrulose, xylulose, cellobiose, amylopectin, glucosamine, mannosamine, glucuronic acid, gluconic acid, gluconolactone, abicose, galactosamine, beet oligosaccharides, isomalto-oligosaccharides (isomaltose, isomaltotriose, panose, etc.), xylo-oligosaccharides (xylotriose, xylobiose, etc.), xylose-terminated oligosaccharides, gentiobiose oligosaccharides (gentiobiose, gentiotriose, gentiotetraose, etc.), aspergillus niger oligosaccharides, palatinose oligosaccharides, fructo-oligosaccharides (kestose, kestotetraose, etc.), maltotetraol, maltotriose, malto-oligosaccharides (maltotriose, maltotetraose, maltopentaose, maltohexaose, maltoheptaose, etc.), starch, inulin oligosaccharide, lactulose, melibiose, raffinose, isomerized liquid sugars such as high fructose corn syrup, coupling sugars, soy-oligosaccharides, D-psicose, D-ribose, L-glucose, L-fucose, D-melilotose, D-leuconostoc disaccharide.

15. A method of enhancing the sweetness of a beverage or foodstuff comprising a sweetener, comprising:

a. providing a beverage or food product comprising a sweetener; and

b. adding a sweetness enhancer comprising at least one steviol glycoside of claim 1, wherein the at least one steviol glycoside of claim 1 is present at a concentration at or below the sweetness recognition threshold.

16. A method of modifying the flavor of a beverage or foodstuff comprising:

a. providing a beverage or food, and

b. adding a composition comprising at least one steviol glycoside according to claim 1.

17. A method for inhibiting foaming of a beverage or food product comprising:

a. providing a beverage or food, and

b. adding a foam inhibitor comprising at least one steviol glycoside according to claim 1.

Technical Field

The present invention relates to methods for preparing compositions comprising steviol glycosides, including highly purified steviol glycoside compositions.

Background

High intensity sweeteners have sweetness levels many times greater than that of sucrose. They are largely non-caloric and are commonly used in dietary and low-calorie products, including foods and beverages. High intensity sweeteners do not elicit an ascending insulin response, making them suitable for use in products targeted to diabetes and other products where it is beneficial to control carbohydrate intake.

Steviol glycosides are a class of compounds found in the leaves of the perennial shrub Stevia (Stevia rebaudiana Bertoni) of the family Asteraceae (Compositae) of the earth-grown plants in certain areas of south america. They are structurally characterized by the single base steviol, which differs by the presence of saccharide residues at the C13 and C19 positions. They accumulate in Stevia (Stevia) leaves, accounting for approximately 10% -20% of the total dry weight. The 4 major glycosides found in stevia leaves typically include stevioside (9.1%), rebaudioside A (3.8%), rebaudioside C (0.6-1.0%), and dulcoside A (0.3%) on a dry weight basis. Other known steviol glycosides include rebaudiosides B, C, D, E, F and M, steviolbioside and rubusoside.

Although methods of preparing steviol glycosides from stevia are known, many of these methods are not commercially applicable.

Thus, there remains a need for simple, efficient and economical processes for preparing steviol glycoside-containing compositions, including highly purified steviol glycoside compositions.

Summary of The Invention

As used herein, the abbreviated term "reb" refers to "rebaudioside". Both terms have the same meaning and may be used interchangeably.

As used herein, "biocatalysis" or "biocatalysis" refers to the use of natural or genetically engineered biocatalysts, such as enzymes or cells comprising one or more enzymes, that are capable of performing a single or multiple step chemical transformation of organic compounds. Biocatalytic processes include fermentation, biosynthesis, bioconversion, and bioconversion processes. Isolated enzymes and whole cell biocatalysis are well known in the art. The biocatalyst protease may be a naturally occurring or recombinant protein.

As used herein, the term "steviol glycoside" refers to a glycoside of steviol, including, but not limited to: naturally occurring steviol glycosides, e.g., steviolmonoside A, steviolbioside D, rubusoside, steviolbioside A, steviolbioside B, rebaudioside B, stevioside, rebaudioside G, stevioside A, stevioside B, stevioside C, rebaudioside A, rebaudioside E2, rebaudioside E4, rebaudioside E6, rebaudioside E3, rebaudioside D, rebaudioside I, rebaudioside AM, rebaudioside D7, rebaudioside M4, rebaudioside 1a, rebaudioside 1B, rebaudioside 1C, rebaudioside 1D, rebaudioside 1E, rebaudioside 1f, rebaudioside 1G, rebaudioside 1h, rebaudioside 1I, rebaudioside 1j, rebaudioside 1k, rebaudioside 1l, rebaudioside 1M, rebaudioside 1n, rebaudioside 1o, rebaudioside 1p, rebaudioside 1q, rebaudioside 1r, rebaudioside 1s, rebaudioside 1t, rebaudioside 2a, rebaudioside 2b, rebaudioside 2c, rebaudioside 2d, rebaudioside 2e, rebaudioside 2f, rebaudioside 2g, rebaudioside 2h, rebaudioside 2i, rebaudioside 2j, rebaudioside 2k, rebaudioside 2l, rebaudioside 2m, rebaudioside 2n, rebaudioside 2o, rebaudioside 2p, rebaudioside 2q, rebaudioside 2r, rebaudioside 2s, synthetic steviol glycosides, e.g., enzymatically glycosylated steviol glycosides and combinations thereof.

As used herein, the term "SvG 7" refers to any naturally occurring steviol glycoside or any synthetic steviol glycoside, including enzymatically glycosylated steviol glycosides and combinations thereof, particularly molecules comprising steviol having 7 glucose residues covalently linked, including, but not limited to, reb 1a, reb 1b, reb 1c, reb 1d, reb 1e, reb 1f, reb 1g, reb 1h, reb 1i, reb 1j, reb 1k, reb 1l, reb 1m, reb 1n, reb 1o, reb 1p, reb 1q, reb 1r, reb 1s, reb 1t, reb 2a, reb 2b, reb 2c, reb 2d, reb 2e, reb 2f, reb 2g, reb 2h, reb 2i, reb 2j, reb 2k, reb 2l, reb 2d, reb 2o, reb 2p, reb 2q, reb 2r, and/or reb 2 s. SvG7 may refer to a single steviol glycoside having 7 glucose residues covalently linked or a mixture of steviol glycosides having 7 glucose residues covalently linked.

The present invention provides a process for preparing a composition comprising a target steviol glycoside by contacting a starting composition comprising an organic substrate with a microbial cell and/or an enzyme preparation, thereby producing a composition comprising the target steviol glycoside.

The starting composition may be any organic compound comprising at least one carbon atom. In one embodiment, the starting composition is selected from steviol glycosides, polyols or sugar alcohols, various sugars.

The target steviol glycoside can be any steviol glycoside. In one embodiment, the target steviol glycoside is steviolmonoside, steviolmonoside A, steviolbioside D, rubusoside, steviolbioside A, steviolbioside B, rebaudioside B, stevioside, rebaudioside G, stevioside A, stevioside B, stevioside C, rebaudioside A, rebaudioside E2, rebaudioside E4, rebaudioside E6, rebaudioside E3, rebaudioside D, rebaudioside I, rebaudioside AM, rebaudioside D7, rebaudioside M4, rebaudioside 1a, rebaudioside 1B, rebaudioside 1C, rebaudioside 1D, rebaudioside 1E, rebaudioside 1f, rebaudioside 1G, rebaudioside 1h, rebaudioside 1I, rebaudioside 1j, rebaudioside 1k, rebaudioside 1M, rebaudioside 1o, rebaudioside 1p, rebaudioside 1q, rebaudioside 1r, rebaudioside 1s, rebaudioside 1t, rebaudioside 2a, rebaudioside 2b, rebaudioside 2c, rebaudioside 2d, rebaudioside 2e, rebaudioside 2f, rebaudioside 2g, rebaudioside 2h, rebaudioside 2i, rebaudioside 2j, rebaudioside 2k, rebaudioside 2l, rebaudioside 2m, rebaudioside 2n, rebaudioside 2o, rebaudioside 2p, rebaudioside 2q, rebaudioside 2r, rebaudioside 2s, SvG7, or a synthetic steviol glycoside.

In one embodiment, the target steviol glycoside is rebaudioside 1 a.

In one embodiment, the target steviol glycoside is rebaudioside 1 b.

In one embodiment, the target steviol glycoside is rebaudioside 1 c.

In one embodiment, the target steviol glycoside is rebaudioside 1 d.

In one embodiment, the target steviol glycoside is rebaudioside 1 e.

In one embodiment, the target steviol glycoside is rebaudioside 1 f.

In one embodiment, the target steviol glycoside is rebaudioside 1 g.

In one embodiment, the target steviol glycoside is rebaudioside 1 h.

In one embodiment, the target steviol glycoside is rebaudioside 1 i.

In one embodiment, the target steviol glycoside is rebaudioside 1 j.

In one embodiment, the target steviol glycoside is rebaudioside 1 k.

In one embodiment, the target steviol glycoside is rebaudioside 1 l.

In one embodiment, the target steviol glycoside is rebaudioside 1 m.

In one embodiment, the target steviol glycoside is rebaudioside 1 n.

In one embodiment, the target steviol glycoside is rebaudioside 1 o.

In one embodiment, the target steviol glycoside is rebaudioside 1 p.

In one embodiment, the target steviol glycoside is rebaudioside 1 q.

In one embodiment, the target steviol glycoside is rebaudioside 1 r.

In one embodiment, the target steviol glycoside is rebaudioside 1 s.

In one embodiment, the target steviol glycoside is rebaudioside 1 t.

In one embodiment, the target steviol glycoside is rebaudioside 2 a.

In one embodiment, the target steviol glycoside is rebaudioside 2 b.

In one embodiment, the target steviol glycoside is rebaudioside 2 c.

In one embodiment, the target steviol glycoside is rebaudioside 2 d.

In one embodiment, the target steviol glycoside is rebaudioside 2 e.

In one embodiment, the target steviol glycoside is rebaudioside 2 f.

In one embodiment, the target steviol glycoside is rebaudioside 2 g.

In one embodiment, the target steviol glycoside is rebaudioside 2 h.

In one embodiment, the target steviol glycoside is rebaudioside 2 i.

In one embodiment, the target steviol glycoside is rebaudioside 2 j.

In one embodiment, the target steviol glycoside is rebaudioside 2 k.

In one embodiment, the target steviol glycoside is rebaudioside 2 l.

In one embodiment, the target steviol glycoside is rebaudioside 2 m.

In one embodiment, the target steviol glycoside is rebaudioside 2 n.

In one embodiment, the target steviol glycoside is rebaudioside 2 o.

In one embodiment, the target steviol glycoside is rebaudioside 2 p.

In one embodiment, the target steviol glycoside is rebaudioside 2 q.

In one embodiment, the target steviol glycoside is rebaudioside 2 r.

In one embodiment, the target steviol glycoside is rebaudioside 2 s.

In one embodiment, the target steviol glycoside is rebaudioside M4.

In one embodiment, the target steviol glycoside is SvG 7.

In some preferred embodiments, an enzyme preparation comprising one or more enzymes or a microbial cell comprising one or more enzymes is used, which is capable of converting the starting composition into the target steviol glycoside. The enzyme may be located on the surface of the cell and/or within it. The enzyme preparation may be provided as a whole cell suspension, crude lysate or as purified enzyme. The enzyme preparation may be in free form or immobilized on a solid support made of inorganic or organic material.

In some embodiments, the microbial cells comprise the requisite enzymes and genes encoding them to convert the starting composition to the target steviol glycoside. Accordingly, the present invention also provides a process for preparing a composition comprising a target steviol glycoside by contacting a microbial cell, comprising an organic substrate, with a starting composition comprising at least one enzyme capable of converting the starting composition into the target steviol glycoside, thereby producing a medium comprising the at least one target steviol glycoside.

Enzymes essential for converting the starting composition into the target steviol glycoside include steviol biosynthetic enzymes, NDP-glucosyltransferase (NGT), ADP-glucosyltransferase (AGT), CDP-glucosyltransferase (CGT), GDP-glucosyltransferase (GGT), TDP-glucosyltransferase (TDP), UDP-glucosyltransferase (UGT) and/or NDP-recycling enzyme, ADP-recycling enzyme, CDP-recycling enzyme, GDP-recycling enzyme, TDP-recycling enzyme and/or UDP-recycling enzyme.

In one embodiment, the steviol biosynthetic enzyme comprises a Mevalonate (MVA) pathway enzyme.

In another embodiment, the steviol biosynthetic enzyme comprises a non-mevalonate 2-C-methyl-D-erythritol 4-phosphate pathway (MEP/DOXP) enzyme.

In one embodiment, the steviol biosynthetic enzyme is selected from: geranylgeranyl diphosphate synthase, copalyl diphosphate synthase (copalyl diphosphate synthase), kaurene synthase, kaurene oxidase, kaurene 13-hydroxylase (KAH), steviol synthase, deoxyxylulose 5-phosphate synthase (DXS), D-1-deoxyxylulose 5-phosphate reductoisomerase (DXR), 4-cytosinyl diphosphate (diphosphocytidyl) -2-C-methyl-D-erythritol synthase (CMS), 4-cytosinyl diphosphate-2-C-methyl-D-erythritol kinase (CMK), 4-cytosinyl diphosphate synthase (MCS), l-hydroxy-2-methyl-2 (E) -butenyl 4-diphosphate synthase (HDS), l-hydroxy-2-methyl-2 (E) -butenyl 4-diphosphate reductase (HDR), acetoacetyl-CoA thiolase, truncated HMG-CoA reductase, mevalonate kinase, phosphomevalonate kinase, mevalonate pyrophosphate decarboxylase, cytochrome P450 reductase, and the like.

The UDP-glucosyltransferase can be any UDP-glucosyltransferase capable of adding at least one glucose unit to a steviol and/or steviol glycoside substrate to provide a target steviol glycoside.

As used below, unless otherwise specified, the term "SuSy _ AT" refers to a sucrose synthase having the amino acid sequence "SEQ ID 1" as described in example 1 or a polypeptide having significant (> 85%, > 86%, > 87%, > 88%, > 89%, > 90%, > 91%, > 92%, > 93%, > 94%, > 95%, > 96%, > 97%, > 98%, > 99%) amino acid sequence identity to the SEQ ID 1 polypeptide, as well as isolated nucleic acid molecules encoding those polypeptides.

As used below, unless otherwise specified, the term "UGTSl 2" refers to a UDP-glucosyltransferase enzyme having the amino acid sequence "SEQ ID 2" as described in example 1 or a polypeptide having significant (> 85%, > 86%, > 87%, > 88%, > 89%, > 90%, > 91%, > 92%, > 93%, > 94%, > 95%, > 96%, > 97%, > 98%, > 99%) amino acid sequence identity to a SEQ ID 2 polypeptide, as well as isolated nucleic acid molecules encoding those polypeptides.

As used herein below, the term "UGT 76G 1" refers to, unless otherwise specified, a UDP-glucosyltransferase having the amino acid sequence "SEQ ID 3" as described in example 1 or a polypeptide having significant (> 85%, > 86%, > 87%, > 88%, > 89%, > 90%, > 91%, > 92%, > 93%, > 94%, > 95%, > 96%, > 97%, > 98%, > 99%) amino acid sequence identity to the SEQ ID 3 polypeptide, as well as isolated nucleic acid molecules encoding those polypeptides.

In one embodiment, the steviol biosynthetic enzyme and UDP-glucosyltransferase are produced in a microbial cell. The microbial cell may be, for example, Escherichia coli (E.coli), Saccharomyces (Saccharomyces sp.), Aspergillus (Aspergillus sp.), Pichia (Pichia sp.), Bacillus (Bacillus sp.), Yarrowia (Yarrowia sp.), or the like. In another embodiment, a UDP-glucosyltransferase enzyme is synthesized.

In one embodiment, the UDP-glucosyltransferase is selected from: UGT74G1, UGT85C2, UGT76G1, UGT91D2, UGTSl2, EUGT11 and UGTs having significant (> 85%, > 86%, > 87%, > 88%, > 89%, > 90%, > 91%, > 92%, > 93%, > 94%, > 95%, > 96%, > 97%, > 98%, > 99%) amino acid sequence identity to these polypeptides and isolated nucleic acid molecules encoding these UGTs.

In one embodiment, the steviol biosynthetic enzymes, UGT and UDP-glucose recycling system are present in one microorganism (microbial cell). The microorganism may be, for example, Escherichia coli, Saccharomyces, Aspergillus, Pichia, Bacillus, yarrowia.

In one embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to steviol or to any starting steviol glycoside having a-OH functionality at C13, resulting in a target steviol glycoside having a glycosidic bond of-O-glucose beta glucopyranoside at C13. In a specific embodiment, the UDP-glucosyltransferase is UGT85C2 or UGT having > 85% amino acid sequence identity with UGT85C 2.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to steviol or to any starting steviol glycoside having a-COOH functional group at C19 to give the target steviol glycoside having a glycosidic bond of-COO-glucose β -glucopyranoside at C19. In a specific embodiment, the UDP-glucosyltransferase is UGT74G1 or UGT having > 85% amino acid sequence identity with UGT74G 1.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to any existing glucose on the C19 side of any starting steviol glycoside, resulting in a target steviol glycoside with at least one additional glucose having at least one glycosidic bond of β 1 → 2 glucopyranoside at the newly formed glycosidic bond. In a specific embodiment, the UDP-glucosyltransferase is UGTSl2 or UGT with > 85% amino acid sequence identity to UGTSl 2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11 or UGT having > 85% amino acid sequence identity with EUGT 11. In another specific embodiment, the UDP-glucosyltransferase is UGT91D2 or UGT having > 85% amino acid sequence identity with UGT91D 2.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to the C19 position of any starting steviol glycoside to give the target steviol glycoside with at least one additional glucose having at least one glycosidic bond of β 1 → 3 glucopyranoside on the newly formed glycosidic bond. In a specific embodiment, the UDP-glucosyltransferase is UGT76G1 or UGT having > 85% amino acid sequence identity with UGT76G 1.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to the C19 position of any starting steviol glycoside, resulting in a target steviol glycoside with at least one additional glucose having at least one glycosidic bond of β 1 → 4 glucopyranoside at the newly formed glycosidic bond. In a specific embodiment, the UDP-glucosyltransferase is UGTSl2 or UGT with > 85% amino acid sequence identity to UGTSl 2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11 or UGT having > 85% amino acid sequence identity with EUGT 11. In another specific embodiment, the UDP-glucosyltransferase is UGT91D2 or UGT having > 85% amino acid sequence identity with UGT91D 2. In another specific embodiment, the UDP-glucosyltransferase is UGT76G1 or UGT having > 85% amino acid sequence identity with UGT76G 1.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to the C19 position of any starting steviol glycoside, resulting in a target steviol glycoside with at least one additional glucose having at least one glycosidic bond of β 1 → 6 glucopyranoside at the newly formed glycosidic bond. In a specific embodiment, the UDP-glucosyltransferase is UGTSl2 or UGT with > 85% amino acid sequence identity to UGTSl 2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11 or UGT having > 85% amino acid sequence identity with EUGT 11. In another specific embodiment, the UDP-glucosyltransferase is UGT91D2 or UGT having > 85% amino acid sequence identity with UGT91D 2.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to any existing glucose on the C13 side of any starting steviol glycoside, resulting in the target steviol glycoside bearing at least one additional glucose having at least one glycosidic bond of β 1 → 2 glucopyranoside at the newly formed glycosidic bond. In a specific embodiment, the UDP-glucosyltransferase is UGTSl2 or UGT with > 85% amino acid sequence identity to UGTSl 2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11 or UGT having > 85% amino acid sequence identity with EUGT 11. In another specific embodiment, the UDP-glucosyltransferase is UGT91D2 or UGT having > 85% amino acid sequence identity with UGT91D 2.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to any existing glucose on the C13 side of any starting steviol glycoside, resulting in a target steviol glycoside with at least one additional glucose having at least one glycosidic bond of β 1 → 3 glucopyranoside on the newly formed glycosidic bond. In a specific embodiment, the UDP-glucosyltransferase is UGT76G1 or UGT having > 85% amino acid sequence identity with UGT76G 1.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to any existing glucose on the C13 side of any starting steviol glycoside, resulting in the target steviol glycoside bearing at least one additional glucose having at least one glycosidic bond of β 1 → 4 glucopyranoside at the newly formed glycosidic bond. In a specific embodiment, the UDP-glucosyltransferase is UGTSl2 or UGT with > 85% amino acid sequence identity to UGTSl 2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11 or UGT having > 85% amino acid sequence identity with EUGT 11. In another specific embodiment, the UDP-glucosyltransferase is UGT91D2 or UGT having > 85% amino acid sequence identity with UGT91D 2. In another specific embodiment, the UDP-glucosyltransferase is UGT76G1 or UGT having > 85% amino acid sequence identity with UGT76G 1.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to any existing glucose on the C13 side of any starting steviol glycoside, resulting in the target steviol glycoside bearing at least one additional glucose having at least one glycosidic bond of β 1 → 6 glucopyranoside at the newly formed glycosidic bond. In a specific embodiment, the UDP-glucosyltransferase is UGTSl2 or UGT with > 85% amino acid sequence identity to UGTSl 2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11 or UGT having > 85% amino acid sequence identity with EUGT 11. In another specific embodiment, the UDP-glucosyltransferase is UGT91D2 or UGT having > 85% amino acid sequence identity with UGT91D 2.

In one embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to steviol to form steviolmonoside. In a specific embodiment, the UDP-glucosyltransferase is UGT85C2 or a UGT with > 85% amino acid sequence identity to UGT85C2 or a UGT with > 85% amino acid sequence identity to UGT85C 2.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to steviol to form steviolmonoside a. In a specific embodiment, the UDP-glucosyltransferase is UGT74G1 or UGT having > 85% amino acid sequence identity with UGT74G 1.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to steviolmonoside to form steviolbioside. In a specific embodiment, the UDP-glucosyltransferase is UGTSl2 or UGT with > 85% amino acid sequence identity to UGTSl 2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11 or UGT having > 85% amino acid sequence identity with EUGT 11. In another specific embodiment, the UDP-glucosyltransferase is UGT91D2 or UGT having > 85% amino acid sequence identity with UGT91D 2.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to steviolmonoside to form steviolbioside D. In a specific embodiment, the UDP-glucosyltransferase is UGT76G1 or UGT having > 85% amino acid sequence identity with UGT76G 1.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to steviolmonoside to form rubusoside. In a specific embodiment, the UDP-glucosyltransferase is UGT74G1 or UGT having > 85% amino acid sequence identity with UGT74G 1.

In one embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to steviolmonoside a to form rubusoside. In a specific embodiment, the UDP-glucosyltransferase is UGT85C2 or a UGT with > 85% amino acid sequence identity to UGT85C2 or a UGT with > 85% amino acid sequence identity to UGT85C 2.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to steviolmonoside a to form steviolbioside a. In a specific embodiment, the UDP-glucosyltransferase is UGTSl2 or UGT with > 85% amino acid sequence identity to UGTSl 2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11 or UGT having > 85% amino acid sequence identity with EUGT 11. In another specific embodiment, the UDP-glucosyltransferase is UGT91D2 or UGT having > 85% amino acid sequence identity with UGT91D 2.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to steviolmonoside a to form steviolbioside B. In a specific embodiment, the UDP-glucosyltransferase is UGT76G1 or UGT having > 85% amino acid sequence identity with UGT76G 1.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to steviolbioside to form rebaudioside B. In a specific embodiment, the UDP-glucosyltransferase is UGT76G1 or UGT having > 85% amino acid sequence identity with UGT76G 1.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to steviolbioside to form stevioside. In a specific embodiment, the UDP-glucosyltransferase is UGT74G1 or UGT having > 85% amino acid sequence identity with UGT74G 1.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to steviolbioside D to form rebaudioside B. In a specific embodiment, the UDP-glucosyltransferase is UGTSl2 or UGT with > 85% amino acid sequence identity to UGTSl 2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11 or UGT having > 85% amino acid sequence identity with EUGT 11. In another specific embodiment, the UDP-glucosyltransferase is UGT91D2 or UGT having > 85% amino acid sequence identity with UGT91D 2.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to steviolbioside D to form rebaudioside G. In a specific embodiment, the UDP-glucosyltransferase is UGT74G1 or UGT having > 85% amino acid sequence identity with UGT74G 1.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rubusoside to form stevioside. In a specific embodiment, the UDP-glucosyltransferase is UGTSl2 or UGT with > 85% amino acid sequence identity to UGTSl 2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11 or UGT having > 85% amino acid sequence identity with EUGT 11. In another specific embodiment, the UDP-glucosyltransferase is UGT91D2 or UGT having > 85% amino acid sequence identity with UGT91D 2.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rubusoside to form rebaudioside G. In a specific embodiment, the UDP-glucosyltransferase is UGT76G1 or UGT having > 85% amino acid sequence identity with UGT76G 1.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rubusoside to form stevioside a. In a specific embodiment, the UDP-glucosyltransferase is UGTSl2 or UGT with > 85% amino acid sequence identity to UGTSl 2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11 or UGT having > 85% amino acid sequence identity with EUGT 11. In another specific embodiment, the UDP-glucosyltransferase is UGT91D2 or UGT having > 85% amino acid sequence identity with UGT91D 2.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rubusoside to form stevioside B. In a specific embodiment, the UDP-glucosyltransferase is UGT76G1 or UGT having > 85% amino acid sequence identity with UGT76G 1.

In one embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to steviolbioside a to form stevioside a. In a specific embodiment, the UDP-glucosyltransferase is UGT85C2 or a UGT with > 85% amino acid sequence identity to UGT85C2 or a UGT with > 85% amino acid sequence identity to UGT85C 2.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to steviolbioside a to form stevioside C. In a specific embodiment, the UDP-glucosyltransferase is UGT76G1 or UGT having > 85% amino acid sequence identity with UGT76G 1.

In one embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to steviolbioside B to form stevioside B. In a specific embodiment, the UDP-glucosyltransferase is UGT85C2 or a UGT with > 85% amino acid sequence identity to UGT85C2 or a UGT with > 85% amino acid sequence identity to UGT85C 2.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to steviolbioside B to form stevioside C. In a specific embodiment, the UDP-glucosyltransferase is UGTSl2 or UGT with > 85% amino acid sequence identity to UGTSl 2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11 or UGT having > 85% amino acid sequence identity with EUGT 11. In another specific embodiment, the UDP-glucosyltransferase is UGT91D2 or UGT having > 85% amino acid sequence identity with UGT91D 2.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside B to form rebaudioside a. In a specific embodiment, the UDP-glucosyltransferase is UGT74G1 or UGT having > 85% amino acid sequence identity with UGT74G 1.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to stevioside to form rebaudioside A. In a specific embodiment, the UDP-glucosyltransferase is UGT76G1 or UGT having > 85% amino acid sequence identity with UGT76G 1.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to stevioside to form rebaudioside E. In a specific embodiment, the UDP-glucosyltransferase is UGTSl2 or UGT with > 85% amino acid sequence identity to UGTSl 2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11 or UGT having > 85% amino acid sequence identity with EUGT 11. In another specific embodiment, the UDP-glucosyltransferase is UGT91D2 or UGT having > 85% amino acid sequence identity with UGT91D 2.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to stevioside to form rebaudioside E2. In a specific embodiment, the UDP-glucosyltransferase is UGT76G1 or UGT having > 85% amino acid sequence identity with UGT76G 1.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside G to form rebaudioside a. In a specific embodiment, the UDP-glucosyltransferase is UGTSl2 or UGT with > 85% amino acid sequence identity to UGTSl 2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11 or UGT having > 85% amino acid sequence identity with EUGT 11. In another specific embodiment, the UDP-glucosyltransferase is UGT91D2 or UGT having > 85% amino acid sequence identity with UGT91D 2.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside G to form rebaudioside E4. In a specific embodiment, the UDP-glucosyltransferase is UGTSl2 or UGT with > 85% amino acid sequence identity to UGTSl 2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11 or UGT having > 85% amino acid sequence identity with EUGT 11. In another specific embodiment, the UDP-glucosyltransferase is UGT91D2 or UGT having > 85% amino acid sequence identity with UGT91D 2.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside G to form rebaudioside E6. In a specific embodiment, the UDP-glucosyltransferase is UGT76G1 or UGT having > 85% amino acid sequence identity with UGT76G 1.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to stevioside A to form rebaudioside E. In a specific embodiment, the UDP-glucosyltransferase is UGTSl2 or UGT with > 85% amino acid sequence identity to UGTSl 2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11 or UGT having > 85% amino acid sequence identity with EUGT 11. In another specific embodiment, the UDP-glucosyltransferase is UGT91D2 or UGT having > 85% amino acid sequence identity with UGT91D 2.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to stevioside A to form rebaudioside E4. In a specific embodiment, the UDP-glucosyltransferase is UGT76G1 or UGT having > 85% amino acid sequence identity with UGT76G 1.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to stevioside A to form rebaudioside E3. In a specific embodiment, the UDP-glucosyltransferase is UGT76G1 or UGT having > 85% amino acid sequence identity with UGT76G 1.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to stevioside B to form rebaudioside E2. In a specific embodiment, the UDP-glucosyltransferase is UGTSl2 or UGT with > 85% amino acid sequence identity to UGTSl 2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11 or UGT having > 85% amino acid sequence identity with EUGT 11. In another specific embodiment, the UDP-glucosyltransferase is UGT91D2 or UGT having > 85% amino acid sequence identity with UGT91D 2.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to stevioside B to form rebaudioside E6. In a specific embodiment, the UDP-glucosyltransferase is UGT76G1 or UGT having > 85% amino acid sequence identity with UGT76G 1.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to stevioside B to form rebaudioside E3. In a specific embodiment, the UDP-glucosyltransferase is UGTSl2 or UGT with > 85% amino acid sequence identity to UGTSl 2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11 or UGT having > 85% amino acid sequence identity with EUGT 11. In another specific embodiment, the UDP-glucosyltransferase is UGT91D2 or UGT having > 85% amino acid sequence identity with UGT91D 2.

In one embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to steviolbioside C to form rebaudioside E3. In a specific embodiment, the UDP-glucosyltransferase is UGT85C2 or a UGT with > 85% amino acid sequence identity to UGT85C2 or a UGT with > 85% amino acid sequence identity to UGT85C 2.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside a to form rebaudioside D. In a specific embodiment, the UDP-glucosyltransferase is UGTSl2 or UGT with > 85% amino acid sequence identity to UGTSl 2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11 or UGT having > 85% amino acid sequence identity with EUGT 11. In another specific embodiment, the UDP-glucosyltransferase is UGT91D2 or UGT having > 85% amino acid sequence identity with UGT91D 2.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside a to form rebaudioside I. In a specific embodiment, the UDP-glucosyltransferase is UGT76G1 or UGT having > 85% amino acid sequence identity with UGT76G 1.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside E to form rebaudioside D. In a specific embodiment, the UDP-glucosyltransferase is UGT76G1 or UGT having > 85% amino acid sequence identity with UGT76G 1.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside E to form rebaudioside AM. In a specific embodiment, the UDP-glucosyltransferase is UGT76G1 or UGT having > 85% amino acid sequence identity with UGT76G 1.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside E2 to form rebaudioside I. In a specific embodiment, the UDP-glucosyltransferase is UGT76G1 or UGT having > 85% amino acid sequence identity with UGT76G 1.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside E2 to form rebaudioside AM. In a specific embodiment, the UDP-glucosyltransferase is UGTSl2 or UGT with > 85% amino acid sequence identity to UGTSl 2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11 or UGT having > 85% amino acid sequence identity with EUGT 11. In another specific embodiment, the UDP-glucosyltransferase is UGT91D2 or UGT having > 85% amino acid sequence identity with UGT91D 2.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside E4 to form rebaudioside D. In a specific embodiment, the UDP-glucosyltransferase is UGTSl2 or UGT with > 85% amino acid sequence identity to UGTSl 2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11 or UGT having > 85% amino acid sequence identity with EUGT 11. In another specific embodiment, the UDP-glucosyltransferase is UGT91D2 or UGT having > 85% amino acid sequence identity with UGT91D 2.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside E4 to form rebaudioside D7. In a specific embodiment, the UDP-glucosyltransferase is UGT76G1 or UGT having > 85% amino acid sequence identity with UGT76G 1.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside E6 to form rebaudioside I. In a specific embodiment, the UDP-glucosyltransferase is UGTSl2 or UGT with > 85% amino acid sequence identity to UGTSl 2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11 or UGT having > 85% amino acid sequence identity with EUGT 11. In another specific embodiment, the UDP-glucosyltransferase is UGT91D2 or UGT having > 85% amino acid sequence identity with UGT91D 2.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside E6 to form rebaudioside D7. In a specific embodiment, the UDP-glucosyltransferase is UGTSl2 or UGT with > 85% amino acid sequence identity to UGTSl 2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11 or UGT having > 85% amino acid sequence identity with EUGT 11. In another specific embodiment, the UDP-glucosyltransferase is UGT91D2 or UGT having > 85% amino acid sequence identity with UGT91D 2.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside E3 to form rebaudioside AM. In a specific embodiment, the UDP-glucosyltransferase is UGTSl2 or UGT with > 85% amino acid sequence identity to UGTSl 2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11 or UGT having > 85% amino acid sequence identity with EUGT 11. In another specific embodiment, the UDP-glucosyltransferase is UGT91D2 or UGT having > 85% amino acid sequence identity with UGT91D 2.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside E3 to form rebaudioside D7. In a specific embodiment, the UDP-glucosyltransferase is UGT76G1 or UGT having > 85% amino acid sequence identity with UGT76G 1.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside D to form rebaudioside M. In a specific embodiment, the UDP-glucosyltransferase is UGT76G1 or UGT having > 85% amino acid sequence identity with UGT76G 1.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside I to form rebaudioside M. In a specific embodiment, the UDP-glucosyltransferase is UGTSl2 or UGT with > 85% amino acid sequence identity to UGTSl 2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11 or UGT having > 85% amino acid sequence identity with EUGT 11. In another specific embodiment, the UDP-glucosyltransferase is UGT91D2 or UGT having > 85% amino acid sequence identity with UGT91D 2.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside AM to form rebaudioside M. In a specific embodiment, the UDP-glucosyltransferase is UGT76G1 or UGT having > 85% amino acid sequence identity with UGT76G 1.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside AM to form rebaudioside M4. In a specific embodiment, the UDP-glucosyltransferase is UGTSl2 or UGT with > 85% amino acid sequence identity to UGTSl 2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11 or UGT having > 85% amino acid sequence identity with EUGT 11. In another specific embodiment, the UDP-glucosyltransferase is UGT91D2 or UGT having > 85% amino acid sequence identity with UGT91D 2. In a specific embodiment, the UDP-glucosyltransferase is UGT76G1 or UGT having > 85% amino acid sequence identity with UGT76G 1.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside D7 to form rebaudioside M. In a specific embodiment, the UDP-glucosyltransferase is UGTSl2 or UGT with > 85% amino acid sequence identity to UGTSl 2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11 or UGT having > 85% amino acid sequence identity with EUGT 11. In another specific embodiment, the UDP-glucosyltransferase is UGT91D2 or UGT having > 85% amino acid sequence identity with UGT91D 2.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside M to form rebaudioside 1 a. In a specific embodiment, the UDP-glucosyltransferase is UGTSl2 or UGT with > 85% amino acid sequence identity to UGTSl 2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11 or UGT having > 85% amino acid sequence identity with EUGT 11. In another specific embodiment, the UDP-glucosyltransferase is UGT91D2 or UGT having > 85% amino acid sequence identity with UGT91D 2.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside M to form rebaudioside 1 b. In a specific embodiment, the UDP-glucosyltransferase is UGT76G1 or UGT having > 85% amino acid sequence identity with UGT76G 1.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside M to form rebaudioside 1 c. In a specific embodiment, the UDP-glucosyltransferase is UGTSl2 or UGT with > 85% amino acid sequence identity to UGTSl 2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11 or UGT having > 85% amino acid sequence identity with EUGT 11. In another specific embodiment, the UDP-glucosyltransferase is UGT91D2 or UGT having > 85% amino acid sequence identity with UGT91D 2.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside M to form rebaudioside 1 d. In a specific embodiment, the UDP-glucosyltransferase is UGTSl2 or UGT with > 85% amino acid sequence identity to UGTSl 2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11 or UGT having > 85% amino acid sequence identity with EUGT 11. In another specific embodiment, the UDP-glucosyltransferase is UGT91D2 or UGT having > 85% amino acid sequence identity with UGT91D 2.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside M to form rebaudioside 1 e. In a specific embodiment, the UDP-glucosyltransferase is UGT76G1 or UGT having > 85% amino acid sequence identity with UGT76G 1.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside M to form rebaudioside 1 f. In a specific embodiment, the UDP-glucosyltransferase is UGTSl2 or UGT with > 85% amino acid sequence identity to UGTSl 2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11 or UGT having > 85% amino acid sequence identity with EUGT 11. In another specific embodiment, the UDP-glucosyltransferase is UGT91D2 or UGT having > 85% amino acid sequence identity with UGT91D 2.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside M to form 1g of rebaudioside M. In a specific embodiment, the UDP-glucosyltransferase is UGTSl2 or UGT with > 85% amino acid sequence identity to UGTSl 2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11 or UGT having > 85% amino acid sequence identity with EUGT 11. In another specific embodiment, the UDP-glucosyltransferase is UGT91D2 or UGT having > 85% amino acid sequence identity with UGT91D 2.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside M to form rebaudioside 1 h. In a specific embodiment, the UDP-glucosyltransferase is UGT76G1 or UGT having > 85% amino acid sequence identity with UGT76G 1.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside M to form rebaudioside 1 i. In a specific embodiment, the UDP-glucosyltransferase is UGTSl2 or UGT with > 85% amino acid sequence identity to UGTSl 2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11 or UGT having > 85% amino acid sequence identity with EUGT 11. In another specific embodiment, the UDP-glucosyltransferase is UGT91D2 or UGT having > 85% amino acid sequence identity with UGT91D 2.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside M to form rebaudioside 1 j. In a specific embodiment, the UDP-glucosyltransferase is UGTSl2 or UGT with > 85% amino acid sequence identity to UGTSl 2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11 or UGT having > 85% amino acid sequence identity with EUGT 11. In another specific embodiment, the UDP-glucosyltransferase is UGT91D2 or UGT having > 85% amino acid sequence identity with UGT91D 2.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside M to form rebaudioside 1 k. In a specific embodiment, the UDP-glucosyltransferase is UGT76G1 or UGT having > 85% amino acid sequence identity with UGT76G 1.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside M to form 1 i of rebaudioside M. In a specific embodiment, the UDP-glucosyltransferase is UGTSl2 or UGT with > 85% amino acid sequence identity to UGTSl 2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11 or UGT having > 85% amino acid sequence identity with EUGT 11. In another specific embodiment, the UDP-glucosyltransferase is UGT91D2 or UGT having > 85% amino acid sequence identity with UGT91D 2.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside M to form rebaudioside 1M. In a specific embodiment, the UDP-glucosyltransferase is UGTSl2 or UGT with > 85% amino acid sequence identity to UGTSl 2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11 or UGT having > 85% amino acid sequence identity with EUGT 11. In another specific embodiment, the UDP-glucosyltransferase is UGT91D2 or UGT having > 85% amino acid sequence identity with UGT91D 2.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside M to form rebaudioside 1 n. In a specific embodiment, the UDP-glucosyltransferase is UGTSl2 or UGT with > 85% amino acid sequence identity to UGTSl 2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11 or UGT having > 85% amino acid sequence identity with EUGT 11. In another specific embodiment, the UDP-glucosyltransferase is UGT91D2 or UGT having > 85% amino acid sequence identity with UGT91D 2.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside M to form rebaudioside 1 o. In a specific embodiment, the UDP-glucosyltransferase is UGTSl2 or UGT with > 85% amino acid sequence identity to UGTSl 2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11 or UGT having > 85% amino acid sequence identity with EUGT 11. In another specific embodiment, the UDP-glucosyltransferase is UGT91D2 or UGT having > 85% amino acid sequence identity with UGT91D 2. In a specific embodiment, the UDP-glucosyltransferase is UGT76G1 or UGT having > 85% amino acid sequence identity with UGT76G 1.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside M to form rebaudioside 1 p. In a specific embodiment, the UDP-glucosyltransferase is UGTSl2 or UGT with > 85% amino acid sequence identity to UGTSl 2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11 or UGT having > 85% amino acid sequence identity with EUGT 11. In another specific embodiment, the UDP-glucosyltransferase is UGT91D2 or UGT having > 85% amino acid sequence identity with UGT91D 2. In a specific embodiment, the UDP-glucosyltransferase is UGT76G1 or UGT having > 85% amino acid sequence identity with UGT76G 1.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside M to form rebaudioside 1 q. In a specific embodiment, the UDP-glucosyltransferase is UGTSl2 or UGT with > 85% amino acid sequence identity to UGTSl 2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11 or UGT having > 85% amino acid sequence identity with EUGT 11. In another specific embodiment, the UDP-glucosyltransferase is UGT91D2 or UGT having > 85% amino acid sequence identity with UGT91D 2. In a specific embodiment, the UDP-glucosyltransferase is UGT76G1 or UGT having > 85% amino acid sequence identity with UGT76G 1.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside M to form rebaudioside 1 r. In a specific embodiment, the UDP-glucosyltransferase is UGTSl2 or UGT with > 85% amino acid sequence identity to UGTSl 2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11 or UGT having > 85% amino acid sequence identity with EUGT 11. In another specific embodiment, the UDP-glucosyltransferase is UGT91D2 or UGT having > 85% amino acid sequence identity with UGT91D 2. In a specific embodiment, the UDP-glucosyltransferase is UGT76G1 or UGT having > 85% amino acid sequence identity with UGT76G 1.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside M to form rebaudioside 1 s. In a specific embodiment, the UDP-glucosyltransferase is UGTSl2 or UGT with > 85% amino acid sequence identity to UGTSl 2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11 or UGT having > 85% amino acid sequence identity with EUGT 11. In another specific embodiment, the UDP-glucosyltransferase is UGT91D2 or UGT having > 85% amino acid sequence identity with UGT91D 2. In a specific embodiment, the UDP-glucosyltransferase is UGT76G1 or UGT having > 85% amino acid sequence identity with UGT76G 1.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside M to form rebaudioside 1 t. In a specific embodiment, the UDP-glucosyltransferase is UGTSl2 or UGT with > 85% amino acid sequence identity to UGTSl 2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11 or UGT having > 85% amino acid sequence identity with EUGT 11. In another specific embodiment, the UDP-glucosyltransferase is UGT91D2 or UGT having > 85% amino acid sequence identity with UGT91D 2. In a specific embodiment, the UDP-glucosyltransferase is UGT76G1 or UGT having > 85% amino acid sequence identity with UGT76G 1.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside M4 to form rebaudioside 2 a. In a specific embodiment, the UDP-glucosyltransferase is UGTSl2 or UGT with > 85% amino acid sequence identity to UGTSl 2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11 or UGT having > 85% amino acid sequence identity with EUGT 11. In another specific embodiment, the UDP-glucosyltransferase is UGT91D2 or UGT having > 85% amino acid sequence identity with UGT91D 2.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside M4 to form rebaudioside 2 b. In a specific embodiment, the UDP-glucosyltransferase is UGT76G1 or UGT having > 85% amino acid sequence identity with UGT76G 1.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside M4 to form rebaudioside 2 c. In a specific embodiment, the UDP-glucosyltransferase is UGTSl2 or UGT with > 85% amino acid sequence identity to UGTSl 2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11 or UGT having > 85% amino acid sequence identity with EUGT 11. In another specific embodiment, the UDP-glucosyltransferase is UGT91D2 or UGT having > 85% amino acid sequence identity with UGT91D 2.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside M4 to form rebaudioside 2 d. In a specific embodiment, the UDP-glucosyltransferase is UGTSl2 or UGT with > 85% amino acid sequence identity to UGTSl 2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11 or UGT having > 85% amino acid sequence identity with EUGT 11. In another specific embodiment, the UDP-glucosyltransferase is UGT91D2 or UGT having > 85% amino acid sequence identity with UGT91D 2.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside M4 to form rebaudioside 2 e. In a specific embodiment, the UDP-glucosyltransferase is UGT76G1 or UGT having > 85% amino acid sequence identity with UGT76G 1.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside M4 to form rebaudioside 2 f. In a specific embodiment, the UDP-glucosyltransferase is UGTSl2 or UGT with > 85% amino acid sequence identity to UGTSl 2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11 or UGT having > 85% amino acid sequence identity with EUGT 11. In another specific embodiment, the UDP-glucosyltransferase is UGT91D2 or UGT having > 85% amino acid sequence identity with UGT91D 2.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside M4 to form 2g of rebaudioside. In a specific embodiment, the UDP-glucosyltransferase is UGTSl2 or UGT with > 85% amino acid sequence identity to UGTSl 2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11 or UGT having > 85% amino acid sequence identity with EUGT 11. In another specific embodiment, the UDP-glucosyltransferase is UGT91D2 or UGT having > 85% amino acid sequence identity with UGT91D 2.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside M4 to form rebaudioside 2 h. In a specific embodiment, the UDP-glucosyltransferase is UGT76G1 or UGT having > 85% amino acid sequence identity with UGT76G 1.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside M4 to form rebaudioside 2 i. In a specific embodiment, the UDP-glucosyltransferase is UGTSl2 or UGT with > 85% amino acid sequence identity to UGTSl 2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11 or UGT having > 85% amino acid sequence identity with EUGT 11. In another specific embodiment, the UDP-glucosyltransferase is UGT91D2 or UGT having > 85% amino acid sequence identity with UGT91D 2.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside M4 to form rebaudioside 2 j. In a specific embodiment, the UDP-glucosyltransferase is UGTSl2 or UGT with > 85% amino acid sequence identity to UGTSl 2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11 or UGT having > 85% amino acid sequence identity with EUGT 11. In another specific embodiment, the UDP-glucosyltransferase is UGT91D2 or UGT having > 85% amino acid sequence identity with UGT91D 2.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside M4 to form rebaudioside 2 k. In a specific embodiment, the UDP-glucosyltransferase is UGT76G1 or UGT having > 85% amino acid sequence identity with UGT76G 1.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside M4 to form 2 i of rebaudioside. In a specific embodiment, the UDP-glucosyltransferase is UGTSl2 or UGT with > 85% amino acid sequence identity to UGTSl 2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11 or UGT having > 85% amino acid sequence identity with EUGT 11. In another specific embodiment, the UDP-glucosyltransferase is UGT91D2 or UGT having > 85% amino acid sequence identity with UGT91D 2.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside M4 to form rebaudioside 2M. In a specific embodiment, the UDP-glucosyltransferase is UGTSl2 or UGT with > 85% amino acid sequence identity to UGTSl 2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11 or UGT having > 85% amino acid sequence identity with EUGT 11. In another specific embodiment, the UDP-glucosyltransferase is UGT91D2 or UGT having > 85% amino acid sequence identity with UGT91D 2.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside M4 to form rebaudioside 2 n. In a specific embodiment, the UDP-glucosyltransferase is UGTSl2 or UGT with > 85% amino acid sequence identity to UGTSl 2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11 or UGT having > 85% amino acid sequence identity with EUGT 11. In another specific embodiment, the UDP-glucosyltransferase is UGT91D2 or UGT having > 85% amino acid sequence identity with UGT91D 2.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside M4 to form rebaudioside 2 o. In a specific embodiment, the UDP-glucosyltransferase is UGTSl2 or UGT with > 85% amino acid sequence identity to UGTSl 2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11 or UGT having > 85% amino acid sequence identity with EUGT 11. In another specific embodiment, the UDP-glucosyltransferase is UGT91D2 or UGT having > 85% amino acid sequence identity with UGT91D 2. In a specific embodiment, the UDP-glucosyltransferase is UGT76G1 or UGT having > 85% amino acid sequence identity with UGT76G 1.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside M4 to form rebaudioside 2 p. In a specific embodiment, the UDP-glucosyltransferase is UGTSl2 or UGT with > 85% amino acid sequence identity to UGTSl 2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11 or UGT having > 85% amino acid sequence identity with EUGT 11. In another specific embodiment, the UDP-glucosyltransferase is UGT91D2 or UGT having > 85% amino acid sequence identity with UGT91D 2. In a specific embodiment, the UDP-glucosyltransferase is UGT76G1 or UGT having > 85% amino acid sequence identity with UGT76G 1.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside M4 to form rebaudioside 2 q. In a specific embodiment, the UDP-glucosyltransferase is UGTSl2 or UGT with > 85% amino acid sequence identity to UGTSl 2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11 or UGT having > 85% amino acid sequence identity with EUGT 11. In another specific embodiment, the UDP-glucosyltransferase is UGT91D2 or UGT having > 85% amino acid sequence identity with UGT91D 2. In a specific embodiment, the UDP-glucosyltransferase is UGT76G1 or UGT having > 85% amino acid sequence identity with UGT76G 1.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside M4 to form rebaudioside 2 r. In a specific embodiment, the UDP-glucosyltransferase is UGTSl2 or UGTSl2 and UGT having > 85% amino acid sequence identity. In another specific embodiment, the UDP-glucosyltransferase is EUGT11 or UGT having > 85% amino acid sequence identity with EUGT 11. In another specific embodiment, the UDP-glucosyltransferase is UGT91D2 or UGT having > 85% amino acid sequence identity with UGT91D 2. In a specific embodiment, the UDP-glucosyltransferase is UGT76G1 or UGT having > 85% amino acid sequence identity with UGT76G 1.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside M4 to form rebaudioside 2 s. In a specific embodiment, the UDP-glucosyltransferase is UGTSl2 or UGT with > 85% amino acid sequence identity to UGTSl 2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11 or UGT having > 85% amino acid sequence identity with EUGT 11. In another specific embodiment, the UDP-glucosyltransferase is UGT91D2 or UGT having > 85% amino acid sequence identity with UGT91D 2. In a specific embodiment, the UDP-glucosyltransferase is UGT76G1 or UGT having > 85% amino acid sequence identity with UGT76G 1.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside M4 to form rebaudioside 1 q. In a specific embodiment, the UDP-glucosyltransferase is UGTSl2 or UGT with > 85% amino acid sequence identity to UGTSl 2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11 or UGT having > 85% amino acid sequence identity with EUGT 11. In another specific embodiment, the UDP-glucosyltransferase is UGT91D2 or UGT having > 85% amino acid sequence identity with UGT91D 2. In a specific embodiment, the UDP-glucosyltransferase is UGT76G1 or UGT having > 85% amino acid sequence identity with UGT76G 1. Optionally, the process of the invention further comprises recycling UDP to obtain UDP-glucose. In one embodiment, the method comprises recycling UDP by providing a recycle catalyst and recycling the substrate such that catalytic amounts of UDP-glucosyltransferase and UDP-glucose are used to convert steviol and/or steviol glycoside substrate to the target steviol glycoside.

In one embodiment, the recycling catalyst is a sucrose synthase SuSy _ At or a sucrose synthase with > 85% amino acid sequence identity to SuSy _ At.

In one embodiment, the recycle substrate of the UDP-glucose recycle catalyst is sucrose.

Optionally, the method of the invention further comprises the use of a transglycosidase enzyme using an oligosaccharide or polysaccharide as a sugar donor to modify the acceptor target steviol glycoside molecule. Non-limiting examples include cyclodextrin glycosyltransferase (CGTase), fructofuranosidase, amylase, sucrase, glucosucrase, beta-h-fructosidase, beta-fructosidase, sucrase, fructosyl converting enzyme (fructosylinvertase), alkaline converting enzyme, acid converting enzyme, fructofuranosidase. In some embodiments, glucose and non-glucose sugars include, but are not limited to, fructose, xylose, rhamnose, arabinose, deoxyglucose, galactose, and conversion to the acceptor target steviol glycoside. In one embodiment, the acceptor steviol glycoside is rebaudioside 1a, rebaudioside 1b, rebaudioside 1c, rebaudioside 1d, rebaudioside 1e, rebaudioside 1f, rebaudioside 1g, rebaudioside 1h, rebaudioside 1i, rebaudioside 1j, rebaudioside 1k, rebaudioside 1l, rebaudioside 1m, rebaudioside 1n, rebaudioside 1o, rebaudioside 1p, rebaudioside 1q, rebaudioside 1r, rebaudioside 1s, rebaudioside 1t, rebaudioside 2a, rebaudioside 2b, rebaudioside 2c, rebaudioside 2d, rebaudioside 2e, rebaudioside 2f, rebaudioside 2g, rebaudioside 2h, rebaudioside 2i, rebaudioside 2j, rebaudioside 2k, rebaudioside 2l, rebaudioside 2m, rebaudioside 2n, rebaudioside 2o, rebaudioside 2p, rebaudioside 2q, rebaudioside 2r and/or rebaudioside 2 s. In another embodiment, the acceptor steviol glycoside is rebaudioside M4. In another embodiment, the steviol glycoside is SvG 7.

Optionally, the process of the present invention further comprises separating the target steviol glycoside from the medium to obtain a highly purified target steviol glycoside composition. The target steviol glycoside can be isolated by at least one suitable method, such as crystallization, by membrane separation, centrifugation, extraction, chromatography or a combination of such methods.

In one embodiment, the target steviol glycoside can be produced in a microorganism. In another embodiment, the steviol glycoside of interest may be secreted in a medium. In another embodiment, the released steviol glycosides may be continuously removed from the medium. In another embodiment, the target steviol glycoside is isolated after the conversion reaction is complete.

In one embodiment, the separation results in a composition comprising greater than about 80% by weight of the steviol glycoside of interest on an anhydrous basis, i.e., a highly purified steviol glycoside composition. In another embodiment, the separating produces a composition comprising greater than about 90% by weight of the target steviol glycoside. In a specific embodiment, the composition comprises greater than about 95% by weight of the target steviol glycoside. In further embodiments, the composition comprises greater than about 99% by weight of the target steviol glycoside.

The target steviol glycoside can be in any polymorphic or amorphous form, including a hydrate, a solvate, an anhydrate or a combination thereof.

The purified target steviol glycosides can be used in consumer products as sweeteners, flavor modifiers, flavors with modified properties and/or foam inhibitors. Suitable consumer products include, but are not limited to, food products, beverages, pharmaceutical compositions, tobacco products, nutraceutical compositions, oral hygiene compositions, and cosmetic compositions.

Brief Description of Drawings

Fig. 1 a-fig. 1t show the chemical structures of some SvG7 steviol glycosides, rebaudioside 1a, rebaudioside 1b, rebaudioside 1c, rebaudioside 1d, rebaudioside 1e, rebaudioside 1f, rebaudioside 1g, rebaudioside 1h, rebaudioside 1i, rebaudioside 1j, rebaudioside 1k, rebaudioside 1l, rebaudioside 1m, rebaudioside 1n, rebaudioside 1o, rebaudioside 1p, rebaudioside 1q, rebaudioside 1r, rebaudioside 1s, and rebaudioside 1t, respectively.

Fig. 2 a-fig. 2s show the chemical structures of some SvG7 steviol glycosides, rebaudioside 2a, rebaudioside 2b, rebaudioside 2c, rebaudioside 2d, rebaudioside 2e, rebaudioside 2f, rebaudioside 2g, rebaudioside 2h, rebaudioside 2i, rebaudioside 2j, rebaudioside 2k, rebaudioside 2l, rebaudioside 2m, rebaudioside 2n, rebaudioside 2o, rebaudioside 2p, rebaudioside 2q, rebaudioside 2r, and rebaudioside 2s, respectively.

Figure 3 shows the chemical structure of rebaudioside M4.

FIGS. 4 a-4 s show the production of rebaudioside 1a, rebaudioside 1b, rebaudioside 1c, rebaudioside 1d, rebaudioside 1e, rebaudioside 1f, rebaudioside 1g, rebaudioside 1h, rebaudioside 1i, rebaudioside 1j, rebaudioside 1k, rebaudioside 1l, rebaudioside 1m, rebaudioside 1n, rebaudioside 1o, rebaudioside 1p, rebaudioside 1q, rebaudioside 1r, rebaudioside 1s, and rebaudioside 1t, rebaudioside 2a, rebaudioside 2b, rebaudioside 2c, rebaudioside 2d, rebaudioside 2e, rebaudioside 2f, rebaudioside 2g, rebaudioside 2h, rebaudioside 2i, rebaudioside 2j, rebaudioside 2k, rebaudioside 2l, rebaudioside 2m, rebaudioside 2n, rebaudioside 2o, rebaudioside 2p, rebaudioside 2q, rebaudioside 2r and rebaudioside 2s, rebaudioside M4 and a different steviol glycoside pathway.

Fig. 5 a-5 t show the biocatalytic production of rebaudioside 1a, rebaudioside 1b, rebaudioside 1c, rebaudioside 1d, rebaudioside 1e, rebaudioside 1f, rebaudioside 1G, rebaudioside 1h, rebaudioside 1i, rebaudioside 1j, rebaudioside 1k, rebaudioside 1l, rebaudioside 1m and rebaudioside 1n, rebaudioside 1o, rebaudioside 1p, rebaudioside 1q, rebaudioside 1r, rebaudioside 1s and rebaudioside 1t by the enzymes UGTSl2 and UGT76G1 and concomitant recycling of UDP to UDP-glucose by the sucrose synthase SuSy _ At.

Fig. 6 a-6 t show biocatalytic production of rebaudioside 2a, rebaudioside 2b, rebaudioside 2c, rebaudioside 2d, rebaudioside 2e, rebaudioside 2f, rebaudioside 2G, rebaudioside 2h, rebaudioside 2i, rebaudioside 2j, rebaudioside 2k, rebaudioside 2l, rebaudioside 2m, rebaudioside 2n, rebaudioside 2o, rebaudioside 2p, rebaudioside 2q, rebaudioside 2r, rebaudioside 2s, and rebaudioside 1q by using UGTSl2 and UGT76G1 and concomitant recycling of UDP to UDP-glucose by the sucrose synthase SuSy _ At, respectively.

Figure 7 shows the biocatalytic production of rebaudioside M4 from stevioside using UGTSl2 and UGT76G1 with concomitant recycling of UDP to UDP-glucose by the sucrose synthase SuSy _ At.

Figures 8 a-8 t show the biocatalytic production of rebaudioside 2a, rebaudioside 2b, rebaudioside 2c, rebaudioside 2d, rebaudioside 2e, rebaudioside 2f, rebaudioside 2G, rebaudioside 2h, rebaudioside 2i, rebaudioside 2j, rebaudioside 2k, rebaudioside 2l, rebaudioside 2m, rebaudioside 2n, rebaudioside 2o, rebaudioside 2p, rebaudioside 2q, rebaudioside 2r, rebaudioside 2s, and rebaudioside 1q from rebaudioside AM using UGTSl2 and UGT76G1 and concomitant recycling of UDP to UDP-glucose by the sucrose synthase SuSy _ At.

Figure 9 shows the biocatalytic production of rebaudioside M4 from rebaudioside AM using UGTSl2 and UGT76G1 with concomitant recycling of UDP to UDP-glucose by the sucrose synthase SuSy _ At.

Fig. 10 a-fig. 10t show the biocatalytic production of rebaudioside 2a rebaudioside 2b, rebaudioside 2c, rebaudioside 2d, rebaudioside 2e, rebaudioside 2f, rebaudioside 2G, rebaudioside 2h, rebaudioside 2i, rebaudioside 2j, rebaudioside 2k, rebaudioside 2l, rebaudioside 2M, rebaudioside 2n, rebaudioside 2o, rebaudioside 2p, rebaudioside 2q, rebaudioside 2r, rebaudioside 2s, and rebaudioside 1q using UGTSl2 and UGT76G1 and concomitant UDP-to-UDP-glucose recycling by the sucrose synthase SuSy _ At.

FIG. 11a shows an HPLC chromatogram of stevioside. The peak with a retention time of 20.958 minutes corresponded to stevioside. The peak with a retention time of 20.725 minutes corresponded to rebaudioside a. The peak at 32.925 min corresponds to rebaudioside B. The peak at 33.930 minutes corresponds to steviolbioside.

FIG. 11b shows an HPLC chromatogram of biocatalytic production of SvG7 from stevioside. The peak at 5.089 min corresponds to rebaudioside 2 m. The peak at 6.459 min corresponds to rebaudioside 2 a. The peak at 9.825 min corresponds to rebaudioside AM. The peak at 13.845 min corresponds to rebaudioside M. The peak at 32.974 min corresponds to rebaudioside B. The peak at 33.979 minutes corresponds to steviolbioside.

Figure 11c shows an HPLC chromatogram of rebaudioside 2a after purification by HPLC. The peak with a retention time of 6.261 minutes corresponded to rebaudioside 2 a.

Figure 11d shows an HPLC chromatogram of rebaudioside 2m after purification by HPLC. The peak with retention time of 5.197 min corresponds to rebaudioside 2 m.

FIG. 12a shows the 1H NMR spectrum (500MHz, pyridine-d 5) of rebaudioside 2 a.

FIG. 12b shows the HSQC spectrum (500MHz, pyridine-d 5) of rebaudioside 2 a.

FIG. 12c shows the H, H COSY spectrum (500MHz, pyridine-d 5) of rebaudioside 2 a.

Figure 12d shows the HMBC spectrum (500MHz, pyridine-d 5) of rebaudioside 2 a.

FIG. 12e shows the HSQC-TOCSY spectrum (500MHz, pyridine-d 5) of rebaudioside 2 a.

FIG. 12f shows the 1D-NOESY spectrum (500MHz, pyridine-D5) of rebaudioside 2 a.

Figure 12g shows a LC chromatogram of rebaudioside 2 a.

Figure 12h shows the mass spectrum of rebaudioside 2 a.

FIG. 13a shows the 1H NMR spectrum (500MHz, pyridine-d 5) of rebaudioside 2 m.

FIG. 13b shows the HSQC spectrum (500MHz, pyridine-d 5) for rebaudioside 2 m.

FIG. 13c shows the H, H COSY spectrum (500MHz, pyridine-d 5) of rebaudioside 2 m.

FIG. 13d shows the HMBC spectrum (500MHz, pyridine-d 5) of rebaudioside 2 m.

FIG. 13e shows the HSQC-TOCSY spectrum (500MHz, pyridine-d 5) of rebaudioside 2 m.

FIG. 13f shows the 1D-NOESY spectrum (500MHz, pyridine-D5) of rebaudioside 2 m.

Figure 13g shows an LC chromatogram of rebaudioside 2 m.

Figure 13h shows the mass spectrum of rebaudioside 2 m.

Detailed Description

The present invention provides a process for preparing a composition comprising a target steviol glycoside by contacting a starting composition comprising an organic substrate with a microbial cell and/or an enzyme preparation, thereby producing a composition comprising the target steviol glycoside.

One object of the present invention resides in a biocatalytic process for the preparation of a target steviol glycoside, in particular steviolmonoside, steviolmonoside a, steviolbioside D, rubusoside, steviolbioside a, steviolbioside B, rebaudioside B, stevioside, rebaudioside G, stevioside a, stevioside B, stevioside C, rebaudioside a, rebaudioside E2, rebaudioside E4, rebaudioside E6, rebaudioside E3, rebaudioside D, rebaudioside I, rebaudioside AM, rebaudioside D7, rebaudioside M4, rebaudioside 1a, rebaudioside 1B, rebaudioside 1C, rebaudioside 1D, rebaudioside 1E, rebaudioside 1f, rebaudioside 1G, rebaudioside 1h, rebaudioside 1I, rebaudioside 1j, rebaudioside 1k, rebaudioside 1l, rebaudioside 1m, rebaudioside 1n, rebaudioside 1o, rebaudioside 1p, rebaudioside 1q, rebaudioside 1r, rebaudioside 1s, rebaudioside 1t, rebaudioside 2a, rebaudioside 2b, rebaudioside 2c, rebaudioside 2d, rebaudioside 2e, rebaudioside 2f, rebaudioside 2g, rebaudioside 2h, rebaudioside 2i, rebaudioside 2j, rebaudioside 2k, rebaudioside 2l, rebaudioside 2m, rebaudioside 2n, rebaudioside 2o, rebaudioside 2p, rebaudioside 2q, rebaudioside 2r, rebaudioside 2s, and/or SvG7 or synthetic steviol glycosides.

Starting composition

As used herein, a "starting composition" refers to any composition (typically an aqueous solution) comprising one or more organic compounds containing at least one carbon atom.

In one embodiment, the starting composition is selected from the group consisting of steviol, steviol glycosides, polyols and various sugars.

The starting composition steviol glycoside is selected from the group consisting of steviol, steviolmonoside a, steviolbioside D, rubusoside, steviolbioside a, steviolbioside B, rebaudioside B, stevioside, rebaudioside G, stevioside a, stevioside B, stevioside C, rebaudioside a, rebaudioside E2, rebaudioside E4, rebaudioside E6, rebaudioside E3, rebaudioside D, rebaudioside I, rebaudioside AM, rebaudioside D7, rebaudioside M, and/or rebaudioside M4 or other glycosides of steviol present in the stevia plant, synthetic steviol glycosides, e.g., enzymatically glycosylated steviol glycosides and combinations thereof.

In one embodiment, the starting composition is steviol.

In another embodiment, the starting composition steviol glycoside is steviolmonoside.

In another embodiment, the starting composition steviol glycoside is steviolmonoside a.

In another embodiment, the starting composition steviol glycoside is steviolbioside.

In another embodiment, the starting composition steviol glycoside is rebaudioside D.

In another embodiment, the starting composition steviol glycoside is rubusoside.

In another embodiment, the starting composition steviol glycoside is rubusoside.

In another embodiment, the starting composition steviol glycoside is rebaudioside a.

In another embodiment, the starting composition steviol glycoside is rebaudioside B.

In another embodiment, the starting composition steviol glycoside is rebaudioside B.

In another embodiment, the starting composition steviol glycoside is stevioside.

In another embodiment, the starting composition steviol glycoside is rebaudioside G.

In another embodiment, the starting composition steviol glycoside is stevioside a.

In another embodiment, the starting composition steviol glycoside is stevioside B.

In another embodiment, the starting composition steviol glycoside is stevioside C.

In another embodiment, the starting composition steviol glycoside is rebaudioside a.

In another embodiment, the starting composition steviol glycoside is rebaudioside E.

In another embodiment, the starting composition steviol glycoside is rebaudioside E2.

In another embodiment, the starting composition steviol glycoside is rebaudioside E4.

In another embodiment, the starting composition steviol glycoside is rebaudioside E6.

In another embodiment, the starting composition steviol glycoside is rebaudioside E3.

In another embodiment, the starting composition steviol glycoside is rebaudioside D.

In another embodiment, the starting composition steviol glycoside is rebaudioside I.

In another embodiment, the starting composition steviol glycoside is rebaudioside AM.

In another embodiment, the starting composition steviol glycoside is rebaudioside D7.

In another embodiment, the starting composition steviol glycoside is rebaudioside M.

In another embodiment, the starting composition steviol glycoside is rebaudioside M4.

The term "polyol" refers to a molecule comprising more than one hydroxyl group. The polyol may be a diol, triol or tetraol containing 2, 3 and 4 hydroxyl groups respectively. The polyol may also contain more than 4 hydroxyl groups, such as pentahydric, hexahydric, heptahydric, and the like, which contain 5, 6, or 7 hydroxyl groups, respectively. In addition, the polyol may also be a sugar alcohol, a polyol or a polyol which is a reduced form of a saccharide in which carbonyl groups (aldehydes or ketones, reducing sugars) have been reduced to primary or secondary hydroxyl groups. Examples of polyols include, but are not limited to erythritol, maltitol, mannitol, sorbitol, lactitol, xylitol, inositol, isomalt, propylene glycol, glycerol, threitol, galactitol, hydrogenated isomaltulose, reduced isomalto-oligosaccharides, reduced xylo-oligosaccharides, reduced gentio-oligosaccharides, reduced maltose syrup, reduced glucose syrup, hydrogenated starch hydrolysates, polyalditol (polyglycitol) and sugar alcohols or any other saccharide capable of being reduced.

The term "saccharide" refers to a compound of formula (CH)₂O)_n(wherein n is 3 to 30) aldehyde or ketone compounds substituted with a plurality of hydroxyl groups, and oligomers and polymers thereof. In addition, the saccharides of the present invention are substituted or deoxygenated at one or more positions. As used herein, saccharides include unmodified saccharides, saccharide derivatives, substituted saccharides and modified saccharides. As used herein, the phrases "saccharide derivative," "substituted saccharide," and "modified saccharide" are synonymous. Modified saccharide refers to any saccharide, or a combination thereof, in which at least one atom is added, removed, or substituted. Thus, derivatives of saccharides or substituted saccharides include substituted and unsubstituted monosaccharides, disaccharides, oligosaccharides and polysaccharides. Said saccharide derivatives or substituted saccharides optionally in any phaseThe corresponding C-position is deoxy and/or substituted with one or more moieties such as hydrogen, halo, haloalkyl, carboxy, acyl, acyloxy, amino, amido, carboxy derivatives, alkylamino, dialkylamino, arylamino, alkoxy, aryloxy, nitro, cyano, sulfo, mercapto, imino, sulfonyl, sulfinyl, sulfenyl, sulfinyl, sulfamoyl, carboalkoxy, carboxamido, phosphonyl, phosphinyl, phosphoryl, phosphino, thioester, thioether, oximo, hydrazino, carbamate, phospho, phosphonate, or any other viable functional group that can provide a carbohydrate derivative or substituted carbohydrate function to improve the sweet taste of the sweetener composition.

Examples of saccharides that can be used according to the present invention include, but are not limited to, tagatose, trehalose, galactose, rhamnose, various cyclodextrins, cyclic oligosaccharides, various types of maltodextrin, dextran, sucrose, glucose, ribulose, fructose, threose, arabinose, xylose, lyxose, allose, altrose, mannose, idose, lactose, maltose, invert sugar, isohexose, neotrehalose, isomaltulose, erythrose, deoxyribose, gulose, idose, talose, erythrulose, xylulose, psicose, turanose, cellobiose, pullulan, glucosamine, mannosamine, fucose, glucuronic acid, gluconic acid, gluconolactone, abicose, galactosamine, beet oligosaccharides, isomaltooligosaccharides (isomaltose, isomaltotriose, panose, etc.), xylooligosaccharides (xylotriose, xylobiose, etc.), xylose-terminated oligosaccharides, gentiooligosaccharides (gentiobiose, gentiotriose, gentiotetraose and the like), sorbose, aspergillus niger oligosaccharides, palatinose oligosaccharides, fructooligosaccharides (kestose, kestotetraose and the like), maltotetraol, maltotriol, maltooligosaccharides (maltotriose, maltotetraose, maltopentaose, maltohexaose, maltoheptaose and the like), starch, inulin-oligosaccharides, lactulose, melibiose, raffinose, ribose, isomerized liquid sugars such as high fructose corn syrup, coupling sugars and soybean oligosaccharides. In addition, the saccharides used herein may be in the D-configuration or the L-configuration.

The starting composition may be synthetic or purified (partial or complete), commercially available or prepared.

In one embodiment, the starting composition is glycerol.

In another embodiment, the starting composition is glucose.

In another embodiment, the starting composition is rhamnose.

In another embodiment, the starting composition is sucrose.

In another embodiment, the starting composition is starch.

In another embodiment, the starting composition is maltodextrin.

In another embodiment, the starting composition is cellulose.

In another embodiment, the starting composition is amylose.

The organic compounds of the starting composition are used as substrates for the production of the target steviol glycosides as described herein.

Target steviol glycosides

The steviol glycoside of the invention which is the object of the invention can be any steviol glycoside which can be prepared by the process disclosed herein. In one embodiment, the target steviol glycoside is selected from the group consisting of steviolmonoside, steviolmonoside A, steviolbioside D, rubusoside, steviolbioside A, steviolbioside B, rebaudioside B, stevioside, rebaudioside G, stevioside A, stevioside B, stevioside C, rebaudioside A, rebaudioside E2, rebaudioside E4, rebaudioside E6, rebaudioside E3, rebaudioside D, rebaudioside I, rebaudioside AM, rebaudioside D7, rebaudioside M4, rebaudioside 1a, rebaudioside 1B, rebaudioside 1C, rebaudioside 1D, rebaudioside 1E, rebaudioside 1f, rebaudioside 1G, rebaudioside 1h, rebaudioside 1I, rebaudioside 1j, rebaudioside 1k, rebaudioside 1M, rebaudioside 1o, rebaudioside 1p, rebaudioside 1q, rebaudioside 1r, rebaudioside 1s, rebaudioside 1t, rebaudioside 2a, rebaudioside 2b, rebaudioside 2c, rebaudioside 2d, rebaudioside 2e, rebaudioside 2f, rebaudioside 2g, rebaudioside 2h, rebaudioside 2i, rebaudioside 2j, rebaudioside 2k, rebaudioside 2l, rebaudioside 2m, rebaudioside 2n, rebaudioside 2o, rebaudioside 2p, rebaudioside 2q, rebaudioside 2r, rebaudioside 2s, SvG7, or other glycosides of steviol found in stevia rebaudiana plants, synthetic steviol glycosides, e.g., enzymatically glycosylated steviol glycosides and combinations thereof.

In one embodiment, the target steviol glycoside is steviolmonoside.

In another embodiment, the target steviol glycoside is steviolmonoside a.

In another embodiment, the target steviol glycoside is steviolbioside.

In another embodiment, the target steviol glycoside is rebaudioside D.

In another embodiment, the target steviol glycoside is rubusoside.

In another embodiment, the target steviol glycoside is rebaudioside a.

In another embodiment, the target steviol glycoside is rebaudioside B.

In another embodiment, the target steviol glycoside is rebaudioside B.

In another embodiment, the target steviol glycoside is stevioside.

In another embodiment, the target steviol glycoside is rebaudioside G.

In another embodiment, the target steviol glycoside is stevioside a.

In another embodiment, the target steviol glycoside is stevioside B.

In another embodiment, the target steviol glycoside is stevioside C.

In another embodiment, the target steviol glycoside is rebaudioside a.

In another embodiment, the target steviol glycoside is rebaudioside E.

In another embodiment, the target steviol glycoside is rebaudioside E2.

In another embodiment, the target steviol glycoside is rebaudioside E4.

In another embodiment, the target steviol glycoside is rebaudioside E6.

In another embodiment, the target steviol glycoside is rebaudioside E3.

In another embodiment, the target steviol glycoside is rebaudioside D.

In another embodiment, the target steviol glycoside is rebaudioside I.

In another embodiment, the target steviol glycoside is rebaudioside AM.

In another embodiment, the target steviol glycoside is rebaudioside D7.

In another embodiment, the target steviol glycoside is rebaudioside M.

In another embodiment, the target steviol glycoside is rebaudioside M4.

In another embodiment, the target steviol glycoside is rebaudioside 1 a.

In another embodiment, the target steviol glycoside is rebaudioside 1 b.

In another embodiment, the target steviol glycoside is rebaudioside 1 c.

In another embodiment, the target steviol glycoside is rebaudioside 1 d.

In another embodiment, the target steviol glycoside is rebaudioside 1 e.

In another embodiment, the target steviol glycoside is rebaudioside 1 f.

In another embodiment, the target steviol glycoside is rebaudioside 1 g.

In another embodiment, the target steviol glycoside is rebaudioside 1 h.

In another embodiment, the target steviol glycoside is rebaudioside 1 i.

In another embodiment, the target steviol glycoside is rebaudioside 1 j.

In another embodiment, the target steviol glycoside is rebaudioside 1 k.

In another embodiment, the target steviol glycoside is rebaudioside 1 l.

In another embodiment, the target steviol glycoside is rebaudioside 1 m.

In another embodiment, the target steviol glycoside is rebaudioside 1 n.

In another embodiment, the target steviol glycoside is rebaudioside 1 o.

In another embodiment, the target steviol glycoside is rebaudioside 1 p.

In another embodiment, the target steviol glycoside is rebaudioside 1 q.

In another embodiment, the target steviol glycoside is rebaudioside 1 r.

In another embodiment, the target steviol glycoside is rebaudioside 1 s.

In another embodiment, the target steviol glycoside is rebaudioside 1 t.

In another embodiment, the target steviol glycoside is rebaudioside 2 a.

In another embodiment, the target steviol glycoside is rebaudioside 2 b.

In another embodiment, the target steviol glycoside is rebaudioside 2 c.

In another embodiment, the target steviol glycoside is rebaudioside 2 d.

In another embodiment, the target steviol glycoside is rebaudioside 2 e.

In another embodiment, the target steviol glycoside is rebaudioside 2 f.

In another embodiment, the target steviol glycoside is rebaudioside 2 g.

In another embodiment, the target steviol glycoside is rebaudioside 2 h.

In another embodiment, the target steviol glycoside is rebaudioside 2 i.

In another embodiment, the target steviol glycoside is rebaudioside 2 j.

In another embodiment, the target steviol glycoside is rebaudioside 2 k.

In another embodiment, the target steviol glycoside is rebaudioside 2 l.

In another embodiment, the target steviol glycoside is rebaudioside 2 m.

In another embodiment, the target steviol glycoside is rebaudioside 2 n.

In another embodiment, the target steviol glycoside is rebaudioside 2 o.

In another embodiment, the target steviol glycoside is rebaudioside 2 p.

In another embodiment, the target steviol glycoside is rebaudioside 2 q.

In another embodiment, the target steviol glycoside is rebaudioside 2 r.

In another embodiment, the target steviol glycoside is rebaudioside 2 s.

In another embodiment, the target steviol glycoside is SvG 7.

The target steviol glycoside can be in any polymorphic or amorphous form, including a hydrate, a solvate, an anhydrate or a combination thereof.

In one embodiment, the present invention is a biocatalytic process for the production of steviolmonoside.

In one embodiment, the present invention is a biocatalytic process for the production of steviolmonoside a.

In one embodiment, the present invention is a biocatalytic process for the production of steviolbioside.

In one embodiment, the present invention is a biocatalytic process for the production of rebaudioside D.

In one embodiment, the present invention is a biocatalytic process for production of rubusoside.

In one embodiment, the invention is a biocatalytic process rebaudioside a for production.

In one embodiment, the present invention is a biocatalytic process for the production of rebaudioside B.

In one embodiment, the invention is a biocatalytic process for the production of rebaudioside B.

In one embodiment, the invention is a biocatalytic method for producing stevioside.

In one embodiment, the invention is a biocatalytic process for the production of rebaudioside G.

In one embodiment, the invention is a biocatalytic method for the production of stevioside A.

In one embodiment, the invention is a biocatalytic method for the production of stevioside B.

In one embodiment, the invention is a biocatalytic method for the production of stevioside C.

In one embodiment, the invention is a biocatalytic process for the production of rebaudioside a.

In one embodiment, the invention is a biocatalytic process for the production of rebaudioside E.

In one embodiment, the invention is a biocatalytic process for the production of rebaudioside E2.

In one embodiment, the invention is a biocatalytic process for the production of rebaudioside E4.

In one embodiment, the invention is a biocatalytic process for the production of rebaudioside E6.

In one embodiment, the invention is a biocatalytic process for the production of rebaudioside E3.

In one embodiment, the invention is a biocatalytic process for the production of rebaudioside D.

In one embodiment, the invention is a biocatalytic process for the production of rebaudioside I.

In one embodiment, the invention is a biocatalytic process for the production of rebaudioside AM.

In one embodiment, the invention is a biocatalytic process for the production of rebaudioside D7.

In one embodiment, the invention is a biocatalytic process for the production of rebaudioside E3.

In one embodiment, the invention is a biocatalytic process for the production of rebaudioside M.

In one embodiment, the invention is a biocatalytic process for the production of rebaudioside M4.

In one embodiment, the invention is a biocatalytic process for the production of rebaudioside 1 a.

In one embodiment, the invention is a biocatalytic process for the production of rebaudioside 1 b.

In one embodiment, the invention is a biocatalytic process for the production of rebaudioside 1 c.

In one embodiment, the invention is a biocatalytic process for the production of rebaudioside 1 d.

In one embodiment, the invention is a biocatalytic process for the production of rebaudioside 1 e.

In one embodiment, the invention is a biocatalytic process for the production of rebaudioside 1 f.

In one embodiment, the invention is a biocatalytic process for the production of 1g of rebaudioside.

In one embodiment, the invention is a biocatalytic process for the production of rebaudioside 1 h.

In one embodiment, the invention is a biocatalytic process for the production of rebaudioside 1 i.

In one embodiment, the invention is a biocatalytic process for the production of rebaudioside 1 j.

In one embodiment, the invention is a biocatalytic process for the production of rebaudioside 1 k.

In one embodiment, the invention is a biocatalytic process for the production of rebaudioside 1 i.

In one embodiment, the invention is a biocatalytic process for the production of rebaudioside 1 m.

In one embodiment, the invention is a biocatalytic process for the production of rebaudioside 1 n.

In one embodiment, the invention is a biocatalytic process for the production of rebaudioside 1 o.

In one embodiment, the invention is a biocatalytic process for the production of rebaudioside 1 p.

In one embodiment, the invention is a biocatalytic process for the production of rebaudioside 1 q.

In one embodiment, the invention is a biocatalytic process for the production of rebaudioside 1 r.

In one embodiment, the invention is a biocatalytic process for the production of rebaudioside 1 s.

In one embodiment, the invention is a biocatalytic process for the production of rebaudioside 1 t.

In one embodiment, the invention is a biocatalytic process for the production of rebaudioside 2 a.

In one embodiment, the invention is a biocatalytic process for the production of rebaudioside 2 b.

In one embodiment, the invention is a biocatalytic process for the production of rebaudioside 2 c.

In one embodiment, the invention is a biocatalytic process for the production of rebaudioside 2 d.

In one embodiment, the invention is a biocatalytic process for the production of rebaudioside 2 e.

In one embodiment, the invention is a biocatalytic process for the production of rebaudioside 2 f.

In one embodiment, the invention is a biocatalytic process for the production of 2g of rebaudioside.

In one embodiment, the invention is a biocatalytic process for the production of rebaudioside 2 h.

In one embodiment, the invention is a biocatalytic process for the production of rebaudioside 2 i.

In one embodiment, the invention is a biocatalytic process for the production of rebaudioside 2 j.

In one embodiment, the invention is a biocatalytic process for the production of rebaudioside 2 k.

In one embodiment, the invention is a biocatalytic process for the production of rebaudioside 2 i.

In one embodiment, the invention is a biocatalytic process for the production of rebaudioside 2 m.

In one embodiment, the invention is a biocatalytic process for the production of rebaudioside 2 n.

In one embodiment, the invention is a biocatalytic process for the production of rebaudioside 2 o.

In one embodiment, the invention is a biocatalytic process for the production of rebaudioside 2 p.

In one embodiment, the invention is a biocatalytic process for the production of rebaudioside 2 q.

In one embodiment, the invention is a biocatalytic process for the production of rebaudioside 2 r.

In one embodiment, the invention is a biocatalytic process for the production of rebaudioside 2 s.

In one embodiment, the invention is a biocatalytic process for producing SvG 7.

In a particular embodiment, the present invention provides a biocatalytic process for the production of rebaudioside 1a from a starting composition comprising rebaudioside a and UDP-glucose.

In a particular embodiment, the present invention provides a biocatalytic process for the production of rebaudioside 1b from a starting composition comprising rebaudioside a and UDP-glucose.

In a particular embodiment, the present invention provides a biocatalytic process for the production of rebaudioside 1c from a starting composition comprising rebaudioside a and UDP-glucose.

In a specific embodiment, the present invention provides a biocatalytic process for the production of rebaudioside 1d from a starting composition comprising rebaudioside a and UDP-glucose.

In a particular embodiment, the present invention provides a biocatalytic process for the production of rebaudioside 1e from a starting composition comprising rebaudioside a and UDP-glucose.

In a particular embodiment, the present invention provides a biocatalytic process for the production of rebaudioside 1f from a starting composition comprising rebaudioside a and UDP-glucose.

In a particular embodiment, the invention provides a biocatalytic process for the production of rebaudioside 1g from a starting composition comprising rebaudioside a and UDP-glucose.

In a particular embodiment, the invention provides a biocatalytic process for the production of rebaudioside 1h from a starting composition comprising rebaudioside a and UDP-glucose.

In a specific embodiment, the present invention provides a biocatalytic process for the production of rebaudioside 1i from a starting composition comprising rebaudioside a and UDP-glucose.

In a specific embodiment, the present invention provides a biocatalytic process for the production of rebaudioside 1j from a starting composition comprising rebaudioside a and UDP-glucose.

In a specific embodiment, the present invention provides a biocatalytic process for the production of rebaudioside 1k from a starting composition comprising rebaudioside a and UDP-glucose.

In a particular embodiment, the present invention provides a biocatalytic process for the production of rebaudioside 1i from a starting composition comprising rebaudioside a and UDP-glucose.

In a particular embodiment, the invention provides a biocatalytic process for the production of rebaudioside 1m from a starting composition comprising rebaudioside a and UDP-glucose.

In a particular embodiment, the present invention provides a biocatalytic process for the production of rebaudioside 1n from a starting composition comprising rebaudioside a and UDP-glucose.

In a specific embodiment, the present invention provides a biocatalytic process for the production of rebaudioside 1o from a starting composition comprising rebaudioside a and UDP-glucose.

In a specific embodiment, the present invention provides a biocatalytic process for the production of rebaudioside 1p from a starting composition comprising rebaudioside a and UDP-glucose.

In a particular embodiment, the present invention provides a biocatalytic process for the production of rebaudioside 1q from a starting composition comprising rebaudioside a and UDP-glucose.

In a particular embodiment, the invention provides a biocatalytic process for the production of rebaudioside 1r from a starting composition comprising rebaudioside a and UDP-glucose.

In a specific embodiment, the present invention provides a biocatalytic process for the production of rebaudioside 1s from a starting composition comprising rebaudioside a and UDP-glucose.

In a particular embodiment, the present invention provides a biocatalytic process for the production of rebaudioside 1t from a starting composition comprising rebaudioside a and UDP-glucose.

In a particular embodiment, the present invention provides a biocatalytic process for the production of rebaudioside 2a from a starting composition comprising stevioside and UDP-glucose.

In a particular embodiment, the present invention provides a biocatalytic process for the production of rebaudioside 2b from a starting composition comprising stevioside and UDP-glucose.

In a particular embodiment, the present invention provides a biocatalytic process for the production of rebaudioside 2c from a starting composition comprising stevioside and UDP-glucose.

In a particular embodiment, the present invention provides a biocatalytic process for the production of rebaudioside 2d from a starting composition comprising stevioside and UDP-glucose.

In a particular embodiment, the present invention provides a biocatalytic process for the production of rebaudioside 2e from a starting composition comprising stevioside and UDP-glucose.

In a particular embodiment, the present invention provides a biocatalytic process for the production of rebaudioside 2f from a starting composition comprising stevioside and UDP-glucose.

In a particular embodiment, the present invention provides a biocatalytic process for the production of rebaudioside 2g from a starting composition comprising stevioside and UDP-glucose.

In a particular embodiment, the present invention provides a biocatalytic process for the production of rebaudioside 2h from a starting composition comprising stevioside and UDP-glucose.

In a particular embodiment, the present invention provides a biocatalytic process for the production of rebaudioside 2i from a starting composition comprising stevioside and UDP-glucose.

In a particular embodiment, the present invention provides a biocatalytic process for the production of rebaudioside 2j from a starting composition comprising stevioside and UDP-glucose.

In a particular embodiment, the present invention provides a biocatalytic process for the production of rebaudioside 2k from a starting composition comprising stevioside and UDP-glucose.

In a particular embodiment, the present invention provides a biocatalytic process for the production of rebaudioside 2 i from a starting composition comprising stevioside and UDP-glucose.

In a particular embodiment, the present invention provides a biocatalytic process for the production of rebaudioside 2m from a starting composition comprising stevioside and UDP-glucose.

In a particular embodiment, the present invention provides a biocatalytic process for the production of rebaudioside 2n from a starting composition comprising stevioside and UDP-glucose.

In a particular embodiment, the present invention provides a biocatalytic process for the production of rebaudioside 2o from a starting composition comprising stevioside and UDP-glucose.

In a particular embodiment, the present invention provides a biocatalytic method for the production of rebaudioside 2p from a starting composition comprising stevioside and UDP-glucose.

In a particular embodiment, the present invention provides a biocatalytic process for the production of rebaudioside 2q from a starting composition comprising stevioside and UDP-glucose.

In a particular embodiment, the present invention provides a biocatalytic process for the production of rebaudioside 2r from a starting composition comprising stevioside and UDP-glucose.

In a particular embodiment, the present invention provides a biocatalytic method for the production of rebaudioside 2s from a starting composition comprising stevioside and UDP-glucose.

In a particular embodiment, the present invention provides a biocatalytic process for the production of rebaudioside 1q from a starting composition comprising stevioside and UDP-glucose.

In a particular embodiment, the present invention provides a biocatalytic process for the production of rebaudioside M4 from a starting composition comprising stevioside and UDP-glucose.

In a particular embodiment, the present invention provides a biocatalytic process for the production of rebaudioside 2a from a starting composition comprising rebaudioside AM and UDP-glucose.

In a particular embodiment, the invention provides a biocatalytic process for the production of rebaudioside 2b from a starting composition comprising rebaudioside AM and UDP-glucose.

In a specific embodiment, the present invention provides a biocatalytic process for the production of rebaudioside 2c from a starting composition comprising rebaudioside AM and UDP-glucose.

In a specific embodiment, the invention provides a biocatalytic process for the production of rebaudioside 2d from a starting composition comprising rebaudioside AM and UDP-glucose.

In a particular embodiment, the present invention provides a biocatalytic process for the production of rebaudioside 2e from a starting composition comprising rebaudioside AM and UDP-glucose.

In a specific embodiment, the present invention provides a biocatalytic process for the production of rebaudioside 2f from a starting composition comprising rebaudioside AM and UDP-glucose.

In a specific embodiment, the invention provides a biocatalytic process for the production of rebaudioside 2g from a starting composition comprising rebaudioside AM and UDP-glucose.

In a particular embodiment, the invention provides a biocatalytic process for the production of rebaudioside 2h from a starting composition comprising rebaudioside AM and UDP-glucose.

In a specific embodiment, the present invention provides a biocatalytic process for the production of rebaudioside 2i from a starting composition comprising rebaudioside AM and UDP-glucose.

In a specific embodiment, the present invention provides a biocatalytic process for the production of rebaudioside 2j from a starting composition comprising rebaudioside AM and UDP-glucose.

In a specific embodiment, the present invention provides a biocatalytic process for the production of rebaudioside 2k from a starting composition comprising rebaudioside AM and UDP-glucose.

In a particular embodiment, the present invention provides a biocatalytic process for the production of rebaudioside 2 i from a starting composition comprising rebaudioside AM and UDP-glucose.

In a specific embodiment, the invention provides a biocatalytic process for the production of rebaudioside 2m from a starting composition comprising rebaudioside AM and UDP-glucose.

In a specific embodiment, the present invention provides a biocatalytic process for the production of rebaudioside 2n from a starting composition comprising rebaudioside AM and UDP-glucose.

In a specific embodiment, the present invention provides a biocatalytic process for the production of rebaudioside 2o from a starting composition comprising rebaudioside AM and UDP-glucose.

In a specific embodiment, the present invention provides a biocatalytic process for the production of rebaudioside 2p from a starting composition comprising rebaudioside AM and UDP-glucose.

In a specific embodiment, the present invention provides a biocatalytic process for the production of rebaudioside 2q from a starting composition comprising rebaudioside AM and UDP-glucose.

In a specific embodiment, the invention provides a biocatalytic process for the production of rebaudioside 2r from a starting composition comprising rebaudioside AM and UDP-glucose.

In a specific embodiment, the present invention provides a biocatalytic process for the production of rebaudioside 2s from a starting composition comprising rebaudioside AM and UDP-glucose.

In a particular embodiment, the present invention provides a biocatalytic process for the production of rebaudioside 1q from a starting composition comprising rebaudioside AM and UDP-glucose.

In a particular embodiment, the present invention provides a biocatalytic process for the production of rebaudioside M4 from a starting composition comprising rebaudioside AM and UDP-glucose.

In a particular embodiment, the invention provides a biocatalytic process for the production of rebaudioside 2a from a starting composition comprising rebaudioside M4 and UDP-glucose.

In a particular embodiment, the invention provides a biocatalytic process for the production of rebaudioside 2b from a starting composition comprising rebaudioside M4 and UDP-glucose.

In a particular embodiment, the invention provides a biocatalytic process for the production of rebaudioside 2c from a starting composition comprising rebaudioside M4 and UDP-glucose.

In a particular embodiment, the invention provides a biocatalytic process for the production of rebaudioside 2d from a starting composition comprising rebaudioside M4 and UDP-glucose.

In a particular embodiment, the invention provides a biocatalytic process for the production of rebaudioside 2e from a starting composition comprising rebaudioside M4 and UDP-glucose.

In a particular embodiment, the invention provides a biocatalytic process for the production of rebaudioside 2f from a starting composition comprising rebaudioside M4 and UDP-glucose.

In a particular embodiment, the invention provides a biocatalytic process for the production of rebaudioside 2g from a starting composition comprising rebaudioside M4 and UDP-glucose.

In a particular embodiment, the invention provides a biocatalytic process for the production of rebaudioside 2h from a starting composition comprising rebaudioside M4 and UDP-glucose.

In a particular embodiment, the invention provides a biocatalytic process for the production of rebaudioside 2i from a starting composition comprising rebaudioside M4 and UDP-glucose.

In a particular embodiment, the invention provides a biocatalytic process for the production of rebaudioside 2j from a starting composition comprising rebaudioside M4 and UDP-glucose.

In a particular embodiment, the invention provides a biocatalytic process for the production of rebaudioside 2k from a starting composition comprising rebaudioside M4 and UDP-glucose.

In a particular embodiment, the invention provides a biocatalytic process for the production of rebaudioside 2 i from a starting composition comprising rebaudioside M4 and UDP-glucose.

In a particular embodiment, the invention provides a biocatalytic process for the production of rebaudioside 2M from a starting composition comprising rebaudioside M4 and UDP-glucose.

In a particular embodiment, the invention provides a biocatalytic process for the production of rebaudioside 2n from a starting composition comprising rebaudioside M4 and UDP-glucose.

In a particular embodiment, the invention provides a biocatalytic process for the production of rebaudioside 2o from a starting composition comprising rebaudioside M4 and UDP-glucose.

In a particular embodiment, the invention provides a biocatalytic process for the production of rebaudioside 2p from a starting composition comprising rebaudioside M4 and UDP-glucose.

In a particular embodiment, the invention provides a biocatalytic process for the production of rebaudioside 2q from a starting composition comprising rebaudioside M4 and UDP-glucose.

In a particular embodiment, the invention provides a biocatalytic process for the production of rebaudioside 2r from a starting composition comprising rebaudioside M4 and UDP-glucose.

In a particular embodiment, the invention provides a biocatalytic process for the production of rebaudioside 2s from a starting composition comprising rebaudioside M4 and UDP-glucose.

In a particular embodiment, the invention provides a biocatalytic process for the production of rebaudioside 1q from a starting composition comprising rebaudioside M4 and UDP-glucose.

In a particular embodiment, the present invention provides a biocatalytic process for the production of rebaudioside 1a from a starting composition comprising rebaudioside M and UDP-glucose.

In a particular embodiment, the invention provides a biocatalytic method of production of rebaudioside 1b from a starting composition comprising rebaudioside M and UDP-glucose.

In a particular embodiment, the present invention provides a biocatalytic process for the production of rebaudioside 1c from a starting composition comprising rebaudioside M and UDP-glucose.

In a particular embodiment, the invention provides a biocatalytic process for the production of rebaudioside 1d from a starting composition comprising rebaudioside M and UDP-glucose.

In a particular embodiment, the present invention provides a biocatalytic process for the production of rebaudioside 1e from a starting composition comprising rebaudioside M and UDP-glucose.

In a particular embodiment, the invention provides a biocatalytic method of production of rebaudioside 1f from a starting composition comprising rebaudioside M and UDP-glucose.

In a particular embodiment, the invention provides a biocatalytic process for the production of rebaudioside 1g from a starting composition comprising rebaudioside M and UDP-glucose.

In a particular embodiment, the invention provides a biocatalytic process for the production of rebaudioside 1h from a starting composition comprising rebaudioside M and UDP-glucose.

In a particular embodiment, the invention provides a biocatalytic method of production of rebaudioside 1i from a starting composition comprising rebaudioside M and UDP-glucose.

In a particular embodiment, the present invention provides a biocatalytic process for the production of rebaudioside 1j from a starting composition comprising rebaudioside M and UDP-glucose.

In a particular embodiment, the present invention provides a biocatalytic process for the production of rebaudioside 1k from a starting composition comprising rebaudioside M and UDP-glucose.

In a particular embodiment, the present invention provides a biocatalytic process for the production of rebaudioside 1 i from a starting composition comprising rebaudioside M and UDP-glucose.

In a particular embodiment, the invention provides a biocatalytic process for the production of rebaudioside 1M from a starting composition comprising rebaudioside M and UDP-glucose.

In a particular embodiment, the present invention provides a biocatalytic process for the production of rebaudioside 1n from a starting composition comprising rebaudioside M and UDP-glucose.

In a particular embodiment, the present invention provides a biocatalytic process for the production of rebaudioside 1o from a starting composition comprising rebaudioside M and UDP-glucose.

In a particular embodiment, the present invention provides a biocatalytic process for the production of rebaudioside 1p from a starting composition comprising rebaudioside M and UDP-glucose.

In a particular embodiment, the present invention provides a biocatalytic process for the production of rebaudioside 1q from a starting composition comprising rebaudioside M and UDP-glucose.

In a particular embodiment, the invention provides a biocatalytic process for the production of rebaudioside 1r from a starting composition comprising rebaudioside M and UDP-glucose.

In a particular embodiment, the present invention provides a biocatalytic process for the production of rebaudioside 1s from a starting composition comprising rebaudioside M and UDP-glucose.

In a particular embodiment, the invention provides a biocatalytic process for the production of rebaudioside 1t from a starting composition comprising rebaudioside M and UDP-glucose.

In a particular embodiment, the present invention provides a biocatalytic method for producing SvG7 from a starting composition comprising stevioside and UDP-glucose.

In a specific embodiment, the invention provides a biocatalytic process for producing SvG7 from a starting composition comprising rebaudioside a and UDP-glucose.

In a particular embodiment, the invention provides a biocatalytic process for producing SvG7 from a starting composition comprising stevioside, rebaudioside a and UDP-glucose.

In a specific embodiment, the invention provides a biocatalytic process for the production SvG7 from a starting composition comprising rebaudioside AM and UDP-glucose.

In a specific embodiment, the invention provides a biocatalytic process for producing SvG7 from a starting composition comprising rebaudioside M and UDP-glucose.

In a specific embodiment, the invention provides a biocatalytic process for the production of SvG7 from a starting composition comprising rebaudioside M4 and UDP-glucose.

Optionally, the process of the present invention further comprises separating the target steviol glycoside from the medium to obtain a highly purified target steviol glycoside composition. The target steviol glycoside can be isolated by any suitable method, e.g., crystallization, separation by membrane separation, centrifugation, extraction, chromatography or a combination of such methods.

In particular embodiments, the methods described herein produce highly purified target steviol glycoside compositions. As used herein, the term "highly purified" refers to a composition having greater than about 80% by weight of a steviol glycoside of interest, on an anhydrous (dry) basis. In one embodiment, the highly purified target steviol glycoside composition comprises greater than about 90% by weight of the target steviol glycoside on an anhydrous (dry) basis, e.g., a target steviol glycoside content of greater than about 91%, greater than about 92%, greater than about 93%, greater than about 94%, greater than about 95%, greater than about 96%, greater than about 97%, greater than about 98% or greater than about 99% on a dry weight basis.

In one embodiment, when the target steviol glycoside is rebaudioside M4, the methods described herein provide a composition having a rebaudioside M4 content of greater than about 90% on a dry weight basis. In another specific embodiment, when the target steviol glycoside is rebaudioside M4, the methods described herein provide a composition comprising an amount greater than about 95% on a dry weight basis.

In one embodiment, when the target steviol glycoside is rebaudioside 1a, the methods described herein provide a composition having a rebaudioside 1a content of greater than about 90% on a dry weight basis. In another specific embodiment, when the target steviol glycoside is rebaudioside 1a, the methods described herein provide a composition comprising an amount of greater than about 95% on a dry weight basis.

In one embodiment, when the target steviol glycoside is rebaudioside 1b, the methods described herein provide a composition having a content of greater than about 90% rebaudioside 1b on a dry weight basis. In another specific embodiment, when the target steviol glycoside is rebaudioside 1b, the methods described herein provide a composition comprising an amount of greater than about 95% on a dry weight basis.

In one embodiment, when the target steviol glycoside is rebaudioside 1c, the methods described herein provide a composition having a rebaudioside 1c content of greater than about 90% on a dry weight basis. In another specific embodiment, when the target steviol glycoside is rebaudioside 1c, the methods described herein provide a composition comprising an amount of greater than about 95% on a dry weight basis.

In one embodiment, when the target steviol glycoside is rebaudioside 1d, the methods described herein provide a composition having a rebaudioside 1d content of greater than about 90% on a dry weight basis. In another specific embodiment, when the target steviol glycoside is rebaudioside 1d, the methods described herein provide a composition comprising an amount of greater than about 95% on a dry weight basis.

In one embodiment, when the target steviol glycoside is rebaudioside 1e, the methods described herein provide a composition having a rebaudioside 1e content of greater than about 90% on a dry weight basis. In another specific embodiment, when the target steviol glycoside is rebaudioside 1e, the methods described herein provide a composition comprising an amount of greater than about 95% on a dry weight basis.

In one embodiment, when the target steviol glycoside is rebaudioside 1f, the methods described herein provide a composition having a rebaudioside 1f content of greater than about 90% on a dry weight basis. In another specific embodiment, when the target steviol glycoside is rebaudioside 1f, the methods described herein provide a composition comprising an amount of greater than about 95% on a dry weight basis.

In one embodiment, when the target steviol glycoside is rebaudioside 1g, the methods described herein provide a composition having a content of greater than about 90% rebaudioside 1g on a dry weight basis. In another specific embodiment, when the target steviol glycoside is rebaudioside 1g, the methods described herein provide a composition comprising an amount of greater than about 95% on a dry weight basis.

In one embodiment, when the target steviol glycoside is rebaudioside 1h, the methods described herein provide a composition having a rebaudioside 1h content of greater than about 90% on a dry weight basis. In another specific embodiment, when the target steviol glycoside is rebaudioside 1h, the methods described herein provide a composition comprising an amount of greater than about 95% on a dry weight basis.

In one embodiment, when the target steviol glycoside is rebaudioside 1i, the methods described herein provide a composition having a rebaudioside 1i content of greater than about 90% on a dry weight basis. In another specific embodiment, when the target steviol glycoside is rebaudioside 1i, the methods described herein provide a composition comprising an amount of greater than about 95% on a dry weight basis.

In one embodiment, when the target steviol glycoside is rebaudioside 1j, the methods described herein provide a composition having a content of greater than about 90% rebaudioside 1j on a dry weight basis. In another specific embodiment, when the target steviol glycoside is rebaudioside 1j, the methods described herein provide a composition comprising an amount greater than about 95% on a dry weight basis.

In one embodiment, when the target steviol glycoside is rebaudioside 1k, the methods described herein provide a composition having a rebaudioside 1k content of greater than about 90% on a dry weight basis. In another specific embodiment, when the target steviol glycoside is rebaudioside 1k, the methods described herein provide a composition comprising an amount of greater than about 95% on a dry weight basis.

In one embodiment, when the target steviol glycoside is rebaudioside 1 i, the methods described herein provide a composition having a content of greater than about 90% rebaudioside 1 i on a dry weight basis. In another specific embodiment, when the target steviol glycoside is rebaudioside 1 i, the methods described herein provide a composition comprising an amount greater than about 95% on a dry weight basis.

In one embodiment, when the target steviol glycoside is rebaudioside 1m, the methods described herein provide a composition having a content of greater than about 90% rebaudioside 1m on a dry weight basis. In another specific embodiment, when the target steviol glycoside is rebaudioside 1m, the methods described herein provide a composition comprising an amount of greater than about 95% on a dry weight basis.

In one embodiment, when the target steviol glycoside is rebaudioside 1n, the methods described herein provide a composition having a rebaudioside 1n content of greater than about 90% on a dry weight basis. In another specific embodiment, when the target steviol glycoside is rebaudioside 1n, the methods described herein provide a composition comprising an amount greater than about 95% on a dry weight basis.

In one embodiment, when the target steviol glycoside is rebaudioside 1o, the methods described herein provide a composition having a rebaudioside 1o content of greater than about 90% on a dry weight basis. In another specific embodiment, when the target steviol glycoside is rebaudioside 1o, the methods described herein provide a composition comprising an amount of greater than about 95% on a dry weight basis.

In one embodiment, when the target steviol glycoside is rebaudioside 1p, the methods described herein provide a composition having a content of greater than about 90% rebaudioside 1p on a dry weight basis. In another specific embodiment, when the target steviol glycoside is rebaudioside 1p, the methods described herein provide a composition comprising an amount greater than about 95% on a dry weight basis.

In one embodiment, when the target steviol glycoside is rebaudioside 1q, the methods described herein provide a composition having a content of greater than about 90% rebaudioside 1q on a dry weight basis. In another specific embodiment, when the target steviol glycoside is rebaudioside 1q, the methods described herein provide a composition comprising an amount of greater than about 95% on a dry weight basis.

In one embodiment, when the target steviol glycoside is rebaudioside 1r, the methods described herein provide a composition having a content of greater than about 90% rebaudioside 1r on a dry weight basis. In another specific embodiment, when the target steviol glycoside is rebaudioside 1r, the methods described herein provide a composition comprising an amount of greater than about 95% on a dry weight basis.

In one embodiment, when the target steviol glycoside is rebaudioside 1s, the methods described herein provide a composition having a content of greater than about 90% rebaudioside 1s on a dry weight basis. In another specific embodiment, when the target steviol glycoside is rebaudioside 1s, the methods described herein provide a composition comprising an amount greater than about 95% on a dry weight basis.

In one embodiment, when the target steviol glycoside is rebaudioside 1t, the methods described herein provide a composition having a rebaudioside 1t content of greater than about 90% on a dry weight basis. In another specific embodiment, when the target steviol glycoside is rebaudioside 1t, the methods described herein provide a composition comprising an amount greater than about 95% on a dry weight basis.

In one embodiment, when the target steviol glycoside is rebaudioside 2a, the methods described herein provide a composition having a rebaudioside 2a content of greater than about 90% on a dry weight basis. In another specific embodiment, when the target steviol glycoside is rebaudioside 2a, the methods described herein provide a composition comprising an amount of greater than about 95% on a dry weight basis.

In one embodiment, when the target steviol glycoside is rebaudioside 2b, the methods described herein provide a composition having a rebaudioside 2b content of greater than about 90% on a dry weight basis. In another specific embodiment, when the target steviol glycoside is rebaudioside 2b, the methods described herein provide a composition comprising an amount of greater than about 95% on a dry weight basis.

In one embodiment, when the target steviol glycoside is rebaudioside 2c, the methods described herein provide a composition having a rebaudioside 2c content of greater than about 90% on a dry weight basis. In another specific embodiment, when the target steviol glycoside is rebaudioside 2c, the methods described herein provide a composition comprising an amount of greater than about 95% on a dry weight basis.

In one embodiment, when the target steviol glycoside is rebaudioside 2d, the methods described herein provide a composition having a rebaudioside 2d content of greater than about 90% on a dry weight basis. In another specific embodiment, when the target steviol glycoside is rebaudioside 2d, the methods described herein provide a composition comprising an amount of greater than about 95% on a dry weight basis.

In one embodiment, when the target steviol glycoside is rebaudioside 2e, the methods described herein provide a composition having a rebaudioside 2e content of greater than about 90% on a dry weight basis. In another specific embodiment, when the target steviol glycoside is rebaudioside 2e, the methods described herein provide a composition comprising an amount of greater than about 95% on a dry weight basis.

In one embodiment, when the target steviol glycoside is rebaudioside 2f, the methods described herein provide a composition having a rebaudioside 2f content of greater than about 90% on a dry weight basis. In another specific embodiment, when the target steviol glycoside is rebaudioside 2f, the methods described herein provide a composition comprising an amount of greater than about 95% on a dry weight basis.

In one embodiment, when the target steviol glycoside is rebaudioside 2g, the methods described herein provide a composition having a content of greater than about 90% rebaudioside 2g on a dry weight basis. In another specific embodiment, when the target steviol glycoside is rebaudioside 2g, the methods described herein provide a composition comprising an amount greater than about 95% on a dry weight basis.

In one embodiment, when the target steviol glycoside is rebaudioside 2h, the methods described herein provide a composition having a rebaudioside 2h content of greater than about 90% on a dry weight basis. In another specific embodiment, when the target steviol glycoside is rebaudioside 2h, the methods described herein provide a composition comprising an amount of greater than about 95% on a dry weight basis.

In one embodiment, when the target steviol glycoside is rebaudioside 2i, the methods described herein provide a composition having a rebaudioside 2i content of greater than about 90% on a dry weight basis. In another specific embodiment, when the target steviol glycoside is rebaudioside 2i, the methods described herein provide a composition comprising an amount of greater than about 95% on a dry weight basis.

In one embodiment, when the target steviol glycoside is rebaudioside 2j, the methods described herein provide a composition having a rebaudioside 2j content of greater than about 90% on a dry weight basis. In another specific embodiment, when the target steviol glycoside is rebaudioside 2j, the methods described herein provide a composition comprising an amount greater than about 95% on a dry weight basis.

In one embodiment, when the target steviol glycoside is rebaudioside 2k, the methods described herein provide a composition having a rebaudioside 2k content of greater than about 90% on a dry weight basis. In another specific embodiment, when the target steviol glycoside is rebaudioside 2k, the methods described herein provide a composition comprising an amount greater than about 95% on a dry weight basis.

In one embodiment, when the target steviol glycoside is rebaudioside 2 i, the methods described herein provide a composition having a content of greater than about 90% rebaudioside 2 i on a dry weight basis. In another specific embodiment, when the target steviol glycoside is rebaudioside 2 i, the methods described herein provide a composition comprising an amount greater than about 95% on a dry weight basis.

In one embodiment, when the target steviol glycoside is rebaudioside 2m, the methods described herein provide a rebaudioside 2m content of greater than about 90% on a dry weight basis. Composition in another specific embodiment, when the target steviol glycoside is rebaudioside 2m, the methods described herein provide a composition comprising a content of greater than about 95% on a dry weight basis.

In one embodiment, when the target steviol glycoside is rebaudioside 2n, the methods described herein provide a composition having a rebaudioside 2n content of greater than about 90% on a dry weight basis. In another specific embodiment, when the target steviol glycoside is rebaudioside 2n, the methods described herein provide a composition comprising an amount greater than about 95% on a dry weight basis.

In one embodiment, when the target steviol glycoside is rebaudioside 2o, the methods described herein provide a composition having a rebaudioside 2o content of greater than about 90% on a dry weight basis. In another specific embodiment, when the target steviol glycoside is rebaudioside 2o, the methods described herein provide a composition comprising an amount greater than about 95% on a dry weight basis.

In one embodiment, when the target steviol glycoside is rebaudioside 2p, the methods described herein provide a composition having a content of greater than about 90% rebaudioside 2p on a dry weight basis. In another specific embodiment, when the target steviol glycoside is rebaudioside 2p, the methods described herein provide a composition comprising an amount greater than about 95% on a dry weight basis.

In one embodiment, when the target steviol glycoside is rebaudioside 2q, the methods described herein provide a composition having a rebaudioside 2q content of greater than about 90% on a dry weight basis. In another specific embodiment, when the target steviol glycoside is rebaudioside 2q, the methods described herein provide a composition comprising an amount greater than about 95% on a dry weight basis.

In one embodiment, when the target steviol glycoside is rebaudioside 2r, the methods described herein provide a composition having a rebaudioside 2r content of greater than about 90% on a dry weight basis. In another specific embodiment, when the target steviol glycoside is rebaudioside 2r, the methods described herein provide a composition comprising an amount of greater than about 95% on a dry weight basis.

In one embodiment, when the target steviol glycoside is rebaudioside 2s, the methods described herein provide a composition having a rebaudioside 2s content of greater than about 90% on a dry weight basis. In another specific embodiment, when the target steviol glycoside is rebaudioside 2s, the methods described herein provide a composition comprising an amount greater than about 95% on a dry weight basis.

In one embodiment, when the target steviol glycoside is SvG7, the methods described herein provide a composition having a content of greater than about 90% SvG7 on a dry weight basis. In another specific embodiment, when the target steviol glycoside is SvG7, the methods described herein provide a composition comprising a content of greater than about 95% SvG7 on a dry weight basis.

Microbial and enzyme preparations

In one embodiment of the present invention, a microorganism (microbial cell) and/or an enzyme preparation is contacted with a medium comprising a starting composition to produce a target steviol glycoside.

The enzyme is provided as a whole cell suspension, crude lysate, purified enzyme, or a combination thereof. In one embodiment, the biocatalyst is a purified enzyme capable of converting a starting composition into a target steviol glycoside. In another embodiment, the biocatalyst is a crude lysate comprising at least one enzyme capable of converting a starting composition into a target steviol glycoside. In another embodiment, the biocatalyst is a whole cell suspension comprising at least one enzyme capable of converting a starting composition into a target steviol glycoside.

In another embodiment, the biocatalyst is one or more microbial cells comprising an enzyme capable of converting a starting composition into a target steviol glycoside. The enzyme is located on the cell surface, within the cell or both on the cell surface and within the cell.

Suitable enzymes for converting the starting composition to the target steviol glycoside include, but are not limited to, steviol biosynthetic enzymes, NDP-glucosyltransferase (NGT), ADP-glucosyltransferase (AGT), CDP-glucosyltransferase (CGT), GDP-glucosyltransferase (GGT), TDP-glucosyltransferase (TDP), UDP-glucosyltransferase (UGT). Optionally, NDP-recycling enzyme, ADP-recycling enzyme, CDP-recycling enzyme, GDP-recycling enzyme, TDP-recycling enzyme and/or UDP-recycling enzyme may be included.

In one embodiment, the steviol biosynthetic enzyme comprises a Mevalonate (MVA) pathway enzyme.

In another embodiment, the steviol biosynthetic enzyme comprises a non-mevalonate 2-C-methyl-D-erythritol 4-phosphate pathway (MEP/DOXP) enzyme.

In one embodiment, the steviol biosynthetic enzyme is selected from: geranylgeranyl diphosphate synthase, copalyl diphosphate synthase, kaurene oxidase, kaurene 13-hydroxylase (KAH), steviol synthase, deoxyxylulose 5-phosphate synthase (DXS), D-1-deoxyxylulose 5-phosphate reductoisomerase (DXR), cytosinyl-2-C-methyl-D-erythritol synthase (CMS), cytosinyl-2-C-methyl-D-erythritol kinase 4-diphosphate (CMK), cytosinyl-2-C-methyl-D-erythritol kinase 4-diphosphate (MCS), l-hydroxy-2-methyl-2 (E) -butenyl 4-diphosphate synthase (HDS), l-hydroxy-2-methyl-2 (E) -butenyl 4-diphosphate reductase (HDR), acetoacetyl-CoA thiolase, truncated HMG-CoA reductase, mevalonate kinase, phosphomevalonate kinase, mevalonate pyrophosphate decarboxylase, cytochrome P450 reductase, and the like.

The UDP-glucosyltransferase can be any UDP-glucosyltransferase capable of adding at least one glucose unit to a steviol and/or steviol glycoside substrate to provide a target steviol glycoside.

In one embodiment, the steviol biosynthetic enzyme and UDP-glucosyltransferase are produced in a microbial cell. The microbial cell may be, for example, Escherichia coli, Saccharomyces, Aspergillus, Pichia, Bacillus, yarrowia, or the like. In another embodiment, the UDP-glucosyltransferase is synthetic.

In one embodiment, the UDP-glucosyltransferase is selected from: UGT74G1, UGT85C2, UGT76G1, UGT91D2, UGTSl2, EUGT11 and UGTs having significant (> 85%, > 86%, > 87%, > 88%, > 89%, > 90%, > 91%, > 92%, > 93%, > 94%, > 95%, > 96%, > 97%, > 98%, > 99%) amino acid sequence identity to these polypeptides and isolated nucleic acid molecules encoding these UGTs.

In one embodiment, the steviol biosynthetic enzymes, UGT and UDP-glucose recycling system are present in one microorganism (microbial cell). The microorganism may be, for example, Escherichia coli, Saccharomyces, Aspergillus, Pichia, Bacillus and yarrowia.

In one embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to steviol or any starting steviol glycoside bearing a functional group of-OH at C13 to give the target steviol glycoside having the glycosidic bond of-O-glucose beta glucopyranoside at C13. In a specific embodiment, the UDP-glucosyltransferase is UGT85C2 or UGT having > 85% amino acid sequence identity with UGT85C 2.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to steviol or any starting steviol glycoside bearing a-COOH functional group at C19 to give the target steviol glycoside having the glycosidic bond of-COO-glucose β -glucopyranoside at C19. In a specific embodiment, the UDP-glucosyltransferase is UGT74G1 or UGT having > 85% amino acid sequence identity with UGT74G 1.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to the C19 position of any starting steviol glycoside, resulting in a target steviol glycoside with at least one additional glucose having at least one glycosidic bond of β 1 → 2 glucopyranoside at the newly formed glycosidic bond. In a specific embodiment, the UDP-glucosyltransferase is UGTSl2 or UGT with > 85% amino acid sequence identity to UGTSl 2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11 or UGT having > 85% amino acid sequence identity with EUGT 11. In another specific embodiment, the UDP-glucosyltransferase is UGT91D2 or UGT having > 85% amino acid sequence identity with UGT91D 2.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to the C19 position of any starting steviol glycoside to give the target steviol glycoside with at least one additional glucose having at least one glycosidic bond of β 1 → 3 glucopyranoside on the newly formed glycosidic bond. In a specific embodiment, the UDP-glucosyltransferase is UGT76G1 or UGT having > 85% amino acid sequence identity with UGT76G 1.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to the C19 position of any starting steviol glycoside, resulting in a target steviol glycoside with at least one additional glucose having at least one glycosidic bond of β 1 → 4 glucopyranoside at the newly formed glycosidic bond. In a specific embodiment, the UDP-glucosyltransferase is UGTSl2 or UGT with > 85% amino acid sequence identity to UGTSl 2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11 or UGT having > 85% amino acid sequence identity with EUGT 11. In another specific embodiment, the UDP-glucosyltransferase is UGT91D2 or UGT having > 85% amino acid sequence identity with UGT91D 2. In another specific embodiment, the UDP-glucosyltransferase is UGT76G1 or UGT having > 85% amino acid sequence identity with UGT76G 1.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to the C19 position of any starting steviol glycoside, resulting in a target steviol glycoside with at least one additional glucose having at least one glycosidic bond of β 1 → 6 glucopyranoside at the newly formed glycosidic bond. In a specific embodiment, the UDP-glucosyltransferase is UGTSl2 or UGT with > 85% amino acid sequence identity to UGTSl 2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11 or UGT having > 85% amino acid sequence identity with EUGT 11. In another specific embodiment, the UDP-glucosyltransferase is UGT91D2 or UGT having > 85% amino acid sequence identity with UGT91D 2.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to any existing glucose on the C13 side of any starting steviol glycoside, resulting in the target steviol glycoside bearing at least one additional glucose having at least one glycosidic bond of β 1 → 2 glucopyranoside at the newly formed glycosidic bond. In a specific embodiment, the UDP-glucosyltransferase is UGTSl2 or UGT with > 85% amino acid sequence identity to UGTSl 2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11 or UGT having > 85% amino acid sequence identity with EUGT 11. In another specific embodiment, the UDP-glucosyltransferase is UGT91D2 or UGT having > 85% amino acid sequence identity with UGT91D 2.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to any existing glucose on the C13 side of any starting steviol glycoside to give the target steviol glycoside with at least one additional glucose having at least one glycosidic bond of β 1 → 3 glucopyranoside on the newly formed glycosidic bond. In a specific embodiment, the UDP-glucosyltransferase is UGT76G1 or UGT having > 85% amino acid sequence identity with UGT76G 1.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to any existing glucose on the C13 side of any starting steviol glycoside, resulting in the target steviol glycoside bearing at least one additional glucose having at least one glycosidic bond of β 1 → 4 glucopyranoside at the newly formed glycosidic bond. In a specific embodiment, the UDP-glucosyltransferase is UGTSl2 or UGT with > 85% amino acid sequence identity to UGTSl 2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11 or EUGT11 and UGT having > 85% amino acid sequence identity. In another specific embodiment, the UDP-glucosyltransferase is UGT91D2 or UGT having > 85% amino acid sequence identity with UGT91D 2. In another specific embodiment, the UDP-glucosyltransferase is UGT76G1 or UGT having > 85% amino acid sequence identity with UGT76G 1.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to any existing glucose on the C13 side of any starting steviol glycoside, resulting in the target steviol glycoside bearing at least one additional glucose having at least one glycosidic bond of β 1 → 6 glucopyranoside at the newly formed glycosidic bond. In a specific embodiment, the UDP-glucosyltransferase is UGTSl2 or UGT with > 85% amino acid sequence identity to UGTSl 2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11 or UGT having > 85% amino acid sequence identity with EUGT 11. In another specific embodiment, the UDP-glucosyltransferase is UGT91D2 or UGT having > 85% amino acid sequence identity with UGT91D 2.

In one embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to steviol to form steviolmonoside. In a specific embodiment, the UDP-glucosyltransferase is UGT85C2 or a UGT with > 85% amino acid sequence identity to UGT85C2 or a UGT with > 85% amino acid sequence identity to UGT85C 2.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to steviol to form steviolmonoside a. In a specific embodiment, the UDP-glucosyltransferase is UGT74G1 or UGT having > 85% amino acid sequence identity with UGT74G 1.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to steviolmonoside to form steviolbioside. In a specific embodiment, the UDP-glucosyltransferase is UGTSl2 or UGT with > 85% amino acid sequence identity to UGTSl 2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11 or UGT having > 85% amino acid sequence identity with EUGT 11. In another specific embodiment, the UDP-glucosyltransferase is UGT91D2 or UGT having > 85% amino acid sequence identity with UGT91D 2.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to steviolmonoside to form steviolbioside D. In a specific embodiment, the UDP-glucosyltransferase is UGT76G1 or UGT having > 85% amino acid sequence identity with UGT76G 1.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to steviolmonoside to form rubusoside. In a specific embodiment, the UDP-glucosyltransferase is UGT74G1 or UGT having > 85% amino acid sequence identity with UGT74G 1.

In one embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to steviolmonoside a to form rubusoside. In a specific embodiment, the UDP-glucosyltransferase is UGT85C2 or a UGT with > 85% amino acid sequence identity to UGT85C2 or a UGT with > 85% amino acid sequence identity to UGT85C 2.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to steviolmonoside a to form steviolbioside a. In a specific embodiment, the UDP-glucosyltransferase is UGTSl2 or UGT with > 85% amino acid sequence identity to UGTSl 2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11 or UGT having > 85% amino acid sequence identity with EUGT 11. In another specific embodiment, the UDP-glucosyltransferase is UGT91D2 or UGT having > 85% amino acid sequence identity with UGT91D 2.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to steviolmonoside a to form steviolbioside B. In a specific embodiment, the UDP-glucosyltransferase is UGT76G1 or UGT having > 85% amino acid sequence identity with UGT76G 1.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to steviolbioside to form rebaudioside B. In a specific embodiment, the UDP-glucosyltransferase is UGT76G1 or UGT having > 85% amino acid sequence identity with UGT76G 1.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to steviolbioside to form stevioside. In a specific embodiment, the UDP-glucosyltransferase is UGT74G1 or UGT having > 85% amino acid sequence identity with UGT74G 1.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to steviolbioside D to form rebaudioside B. In a specific embodiment, the UDP-glucosyltransferase is UGTSl2 or UGT with > 85% amino acid sequence identity to UGTSl 2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11 or UGT having > 85% amino acid sequence identity with EUGT 11. In another specific embodiment, the UDP-glucosyltransferase is UGT91D2 or UGT having > 85% amino acid sequence identity with UGT91D 2.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to steviolbioside D to form rebaudioside G. In a specific embodiment, the UDP-glucosyltransferase is UGT74G1 or UGT having > 85% amino acid sequence identity with UGT74G 1.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rubusoside to form stevioside. In a specific embodiment, the UDP-glucosyltransferase is UGTSl2 or UGT with > 85% amino acid sequence identity to UGTSl 2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11 or UGT having > 85% amino acid sequence identity with EUGT 11. In another specific embodiment, the UDP-glucosyltransferase is UGT91D2 or UGT having > 85% amino acid sequence identity with UGT91D 2.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rubusoside to form rebaudioside G. In a specific embodiment, the UDP-glucosyltransferase is UGT76G1 or UGT having > 85% amino acid sequence identity with UGT76G 1.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rubusoside to form stevioside a. In a specific embodiment, the UDP-glucosyltransferase is UGTSl2 or UGT with > 85% amino acid sequence identity to UGTSl 2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11 or UGT having > 85% amino acid sequence identity with EUGT 11. In another specific embodiment, the UDP-glucosyltransferase is UGT91D2 or UGT having > 85% amino acid sequence identity with UGT91D 2.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rubusoside to form stevioside B. In a specific embodiment, the UDP-glucosyltransferase is UGT76G1 or UGT having > 85% amino acid sequence identity with UGT76G 1.

In one embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to steviolbioside a to form stevioside a. In a specific embodiment, the UDP-glucosyltransferase is UGT85C2 or a UGT with > 85% amino acid sequence identity to UGT85C2 or a UGT with > 85% amino acid sequence identity to UGT85C 2.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to steviolbioside a to form stevioside C. In a specific embodiment, the UDP-glucosyltransferase is UGT76G1 or UGT having > 85% amino acid sequence identity with UGT76G 1.

In one embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to steviolbioside B to form stevioside B. In a specific embodiment, the UDP-glucosyltransferase is UGT85C2 or a UGT with > 85% amino acid sequence identity to UGT85C2 or a UGT with > 85% amino acid sequence identity to UGT85C 2.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to steviolbioside B to form stevioside C. In a specific embodiment, the UDP-glucosyltransferase is UGTSl2 or UGT with > 85% amino acid sequence identity to UGTSl 2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11 or UGT having > 85% amino acid sequence identity with EUGT 11. In another specific embodiment, the UDP-glucosyltransferase is UGT91D2 or UGT having > 85% amino acid sequence identity with UGT91D 2.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside B to form rebaudioside a. In a specific embodiment, the UDP-glucosyltransferase is UGT74G1 or UGT having > 85% amino acid sequence identity with UGT74G 1.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to stevioside to form rebaudioside A. In a specific embodiment, the UDP-glucosyltransferase is UGT76G1 or UGT having > 85% amino acid sequence identity with UGT76G 1.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to stevioside to form rebaudioside E. In a specific embodiment, the UDP-glucosyltransferase is UGTSl2 or UGT with > 85% amino acid sequence identity to UGTSl 2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11 or UGT having > 85% amino acid sequence identity with EUGT 11. In another specific embodiment, the UDP-glucosyltransferase is UGT91D2 or UGT having > 85% amino acid sequence identity with UGT91D 2.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to stevioside to form rebaudioside E2. In a specific embodiment, the UDP-glucosyltransferase is UGT76G1 or UGT having > 85% amino acid sequence identity with UGT76G 1.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside G to form rebaudioside a. In a specific embodiment, the UDP-glucosyltransferase is UGTSl2 or UGT with > 85% amino acid sequence identity to UGTSl 2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11 or UGT having > 85% amino acid sequence identity with EUGT 11. In another specific embodiment, the UDP-glucosyltransferase is UGT91D2 or UGT having > 85% amino acid sequence identity with UGT91D 2.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside G to form rebaudioside E4. In a specific embodiment, the UDP-glucosyltransferase is UGTSl2 or UGT with > 85% amino acid sequence identity to UGTSl 2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11 or UGT having > 85% amino acid sequence identity with EUGT 11. In another specific embodiment, the UDP-glucosyltransferase is UGT91D2 or UGT having > 85% amino acid sequence identity with UGT91D 2.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside G to form rebaudioside E6. In a specific embodiment, the UDP-glucosyltransferase is UGT76G1 or UGT having > 85% amino acid sequence identity with UGT76G 1.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to stevioside A to form rebaudioside E. In a specific embodiment, the UDP-glucosyltransferase is UGTSl2 or UGT with > 85% amino acid sequence identity to UGTSl 2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11 or UGT having > 85% amino acid sequence identity with EUGT 11. In another specific embodiment, the UDP-glucosyltransferase is UGT91D2 or UGT having > 85% amino acid sequence identity with UGT91D 2.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to stevioside A to form rebaudioside E4. In a specific embodiment, the UDP-glucosyltransferase is UGT76G1 or UGT having > 85% amino acid sequence identity with UGT76G 1.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to stevioside A to form rebaudioside E3. In a specific embodiment, the UDP-glucosyltransferase is UGT76G1 or UGT having > 85% amino acid sequence identity with UGT76G 1.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to stevioside B to form rebaudioside E2. In a specific embodiment, the UDP-glucosyltransferase is UGTSl2 or UGT with > 85% amino acid sequence identity to UGTSl 2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11 or UGT having > 85% amino acid sequence identity with EUGT 11. In another specific embodiment, the UDP-glucosyltransferase is UGT91D2 or UGT having > 85% amino acid sequence identity with UGT91D 2.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to stevioside B to form rebaudioside E6. In a specific embodiment, the UDP-glucosyltransferase is UGT76G1 or UGT having > 85% amino acid sequence identity with UGT76G 1.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to stevioside B to form rebaudioside E3. In a specific embodiment, the UDP-glucosyltransferase is UGTSl2 or UGT with > 85% amino acid sequence identity to UGTSl 2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11 or UGT having > 85% amino acid sequence identity with EUGT 11. In another specific embodiment, the UDP-glucosyltransferase is UGT91D2 or UGT having > 85% amino acid sequence identity with UGT91D 2.

In one embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to steviolbioside C to form rebaudioside E3. In a specific embodiment, the UDP-glucosyltransferase is UGT85C2 or a UGT with > 85% amino acid sequence identity to UGT85C2 or a UGT with > 85% amino acid sequence identity to UGT85C 2.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside a to form rebaudioside D. In a specific embodiment, the UDP-glucosyltransferase is UGTSl2 or UGT with > 85% amino acid sequence identity to UGTSl 2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11 or UGT having > 85% amino acid sequence identity with EUGT 11. In another specific embodiment, the UDP-glucosyltransferase is UGT91D2 or UGT having > 85% amino acid sequence identity with UGT91D 2.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside a to form rebaudioside I. In a specific embodiment, the UDP-glucosyltransferase is UGT76G1 or UGT having > 85% amino acid sequence identity with UGT76G 1.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside E to form rebaudioside D. In a specific embodiment, the UDP-glucosyltransferase is UGT76G1 or UGT having > 85% amino acid sequence identity with UGT76G 1.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside E to form rebaudioside AM. In a specific embodiment, the UDP-glucosyltransferase is UGT76G1 or UGT having > 85% amino acid sequence identity with UGT76G 1.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside E2 to form rebaudioside I. In a specific embodiment, the UDP-glucosyltransferase is UGT76G1 or UGT having > 85% amino acid sequence identity with UGT76G 1.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside E2 to form rebaudioside AM. In a specific embodiment, the UDP-glucosyltransferase is UGTSl2 or UGT with > 85% amino acid sequence identity to UGTSl 2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11 or UGT having > 85% amino acid sequence identity with EUGT 11. In another specific embodiment, the UDP-glucosyltransferase is UGT91D2 or UGT having > 85% amino acid sequence identity with UGT91D 2.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside E4 to form rebaudioside D. In a specific embodiment, the UDP-glucosyltransferase is UGTSl2 or UGT with > 85% amino acid sequence identity to UGTSl 2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11 or UGT having > 85% amino acid sequence identity with EUGT 11. In another specific embodiment, the UDP-glucosyltransferase is UGT91D2 or UGT having > 85% amino acid sequence identity with UGT91D 2.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside E4 to form rebaudioside D7. In a specific embodiment, the UDP-glucosyltransferase is UGT76G1 or UGT having > 85% amino acid sequence identity with UGT76G 1.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside E6 to form rebaudioside I. In a specific embodiment, the UDP-glucosyltransferase is UGTSl2 or UGT with > 85% amino acid sequence identity to UGTSl 2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11 or UGT having > 85% amino acid sequence identity with EUGT 11. In another specific embodiment, the UDP-glucosyltransferase is UGT91D2 or UGT having > 85% amino acid sequence identity with UGT91D 2.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside E6 to form rebaudioside D7. In a specific embodiment, the UDP-glucosyltransferase is UGTSl2 or UGT with > 85% amino acid sequence identity to UGTSl 2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11 or UGT having > 85% amino acid sequence identity with EUGT 11. In another specific embodiment, the UDP-glucosyltransferase is UGT91D2 or UGT having > 85% amino acid sequence identity with UGT91D 2.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside E3 to form rebaudioside AM. In a specific embodiment, the UDP-glucosyltransferase is UGTSl2 or UGT with > 85% amino acid sequence identity to UGTSl 2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11 or UGT having > 85% amino acid sequence identity with EUGT 11. In another specific embodiment, the UDP-glucosyltransferase is UGT91D2 or UGT having > 85% amino acid sequence identity with UGT91D 2.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside E3 to form rebaudioside D7. In a specific embodiment, the UDP-glucosyltransferase is UGT76G1 or UGT having > 85% amino acid sequence identity with UGT76G 1.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside D to form rebaudioside M. In a specific embodiment, the UDP-glucosyltransferase is UGT76G1 or UGT having > 85% amino acid sequence identity with UGT76G 1.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside I to form rebaudioside M. In a specific embodiment, the UDP-glucosyltransferase is UGTSl2 or UGT with > 85% amino acid sequence identity to UGTSl 2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11 or UGT with > 85% amino acid sequence identity to EUGT 11. In another specific embodiment, the UDP-glucosyltransferase is UGT91D2 or UGT having > 85% amino acid sequence identity with UGT91D 2.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside AM to form rebaudioside M. In a specific embodiment, the UDP-glucosyltransferase is UGT76G1 or UGT having > 85% amino acid sequence identity with UGT76G 1.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside AM to form rebaudioside M4. In a specific embodiment, the UDP-glucosyltransferase is UGTSl2 or UGT with > 85% amino acid sequence identity to UGTSl 2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11 or UGT having > 85% amino acid sequence identity with EUGT 11. In another specific embodiment, the UDP-glucosyltransferase is UGT91D2 or UGT having > 85% amino acid sequence identity with UGT91D 2. In a specific embodiment, the UDP-glucosyltransferase is UGT76G1 or UGT having > 85% amino acid sequence identity with UGT76G 1.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside D7 to form rebaudioside M. In a specific embodiment, the UDP-glucosyltransferase is UGTSl2 or UGT with > 85% amino acid sequence identity to UGTSl 2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11 or UGT having > 85% amino acid sequence identity with EUGT 11. In another specific embodiment, the UDP-glucosyltransferase is UGT91D2 or UGT having > 85% amino acid sequence identity with UGT91D 2.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside M to form rebaudioside 1 a. In a specific embodiment, the UDP-glucosyltransferase is UGTSl2 or UGT with > 85% amino acid sequence identity to UGTSl 2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11 or UGT having > 85% amino acid sequence identity with EUGT 11. In another specific embodiment, the UDP-glucosyltransferase is UGT91D2 or UGT having > 85% amino acid sequence identity with UGT91D 2.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside M to form rebaudioside 1 b. In a specific embodiment, the UDP-glucosyltransferase is UGT76G1 or UGT having > 85% amino acid sequence identity with UGT76G 1.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside M to form rebaudioside 1 c. In a specific embodiment, the UDP-glucosyltransferase is UGTSl2 or UGT with > 85% amino acid sequence identity to UGTSl 2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11 or UGT having > 85% amino acid sequence identity with EUGT 11. In another specific embodiment, the UDP-glucosyltransferase is UGT91D2 or UGT having > 85% amino acid sequence identity with UGT91D 2.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside M to form rebaudioside 1 d. In a specific embodiment, the UDP-glucosyltransferase is UGTSl2 or UGT with > 85% amino acid sequence identity to UGTSl 2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11 or UGT having > 85% amino acid sequence identity with EUGT 11. In another specific embodiment, the UDP-glucosyltransferase is UGT91D2 or UGT having > 85% amino acid sequence identity with UGT91D 2.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside M to form rebaudioside 1 e. In a specific embodiment, the UDP-glucosyltransferase is UGT76G1 or UGT having > 85% amino acid sequence identity with UGT76G 1.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside M to form rebaudioside 1 f. In a specific embodiment, the UDP-glucosyltransferase is UGTSl2 or UGT with > 85% amino acid sequence identity to UGTSl 2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11 or UGT having > 85% amino acid sequence identity with EUGT 11. In another specific embodiment, the UDP-glucosyltransferase is UGT91D2 or UGT having > 85% amino acid sequence identity with UGT91D 2.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside M to form 1g of rebaudioside M. In a specific embodiment, the UDP-glucosyltransferase is UGTSl2 or UGT with > 85% amino acid sequence identity to UGTSl 2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11 or UGT having > 85% amino acid sequence identity with EUGT 11. In another specific embodiment, the UDP-glucosyltransferase is UGT91D2 or UGT having > 85% amino acid sequence identity with UGT91D 2.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside M to form rebaudioside 1 h. In a specific embodiment, the UDP-glucosyltransferase is UGT76G1 or UGT having > 85% amino acid sequence identity with UGT76G 1.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside M to form rebaudioside 1 i. In a specific embodiment, the UDP-glucosyltransferase is UGTSl2 or UGT with > 85% amino acid sequence identity to UGTSl 2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11 or UGT having > 85% amino acid sequence identity with EUGT 11. In another specific embodiment, the UDP-glucosyltransferase is UGT91D2 or UGT having > 85% amino acid sequence identity with UGT91D 2.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside M to form rebaudioside 1 j. In a specific embodiment, the UDP-glucosyltransferase is UGTSl2 or UGT with > 85% amino acid sequence identity to UGTSl 2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11 or UGT having > 85% amino acid sequence identity with EUGT 11. In another specific embodiment, the UDP-glucosyltransferase is UGT91D2 or UGT having > 85% amino acid sequence identity with UGT91D 2.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside M to form rebaudioside 1 k. In a specific embodiment, the UDP-glucosyltransferase is UGT76G1 or UGT having > 85% amino acid sequence identity with UGT76G 1.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside M to form 1 i of rebaudioside M. In a specific embodiment, the UDP-glucosyltransferase is UGTSl2 or UGT with > 85% amino acid sequence identity to UGTSl 2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11 or UGT having > 85% amino acid sequence identity with EUGT 11. In another specific embodiment, the UDP-glucosyltransferase is UGT91D2 or UGT having > 85% amino acid sequence identity with UGT91D 2.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside M to form rebaudioside 1M. In a specific embodiment, the UDP-glucosyltransferase is UGTSl2 or UGT with > 85% amino acid sequence identity to UGTSl 2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11 or UGT having > 85% amino acid sequence identity with EUGT 11. In another specific embodiment, the UDP-glucosyltransferase is UGT91D2 or UGT having > 85% amino acid sequence identity with UGT91D 2.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside M to form rebaudioside 1 n. In a specific embodiment, the UDP-glucosyltransferase is UGTSl2 or UGT with > 85% amino acid sequence identity to UGTSl 2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11 or UGT having > 85% amino acid sequence identity with EUGT 11. In another specific embodiment, the UDP-glucosyltransferase is UGT91D2 or UGT having > 85% amino acid sequence identity with UGT91D 2.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside M to form rebaudioside 1 o. In a specific embodiment, the UDP-glucosyltransferase is UGTSl2 or UGT with > 85% amino acid sequence identity to UGTSl 2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11 or UGT having > 85% amino acid sequence identity with EUGT 11. In another specific embodiment, the UDP-glucosyltransferase is UGT91D2 or UGT having > 85% amino acid sequence identity with UGT91D 2. In a specific embodiment, the UDP-glucosyltransferase is UGT76G1 or UGT having > 85% amino acid sequence identity with UGT76G 1.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside M to form rebaudioside 1 p. In a specific embodiment, the UDP-glucosyltransferase is UGTSl2 or UGT with > 85% amino acid sequence identity to UGTSl 2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11 or UGT having > 85% amino acid sequence identity with EUGT 11. In another specific embodiment, the UDP-glucosyltransferase is UGT91D2 or UGT having > 85% amino acid sequence identity with UGT91D 2. In a specific embodiment, the UDP-glucosyltransferase is UGT76G1 or UGT having > 85% amino acid sequence identity with UGT76G 1.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside M to form rebaudioside 1 q. In a specific embodiment, the UDP-glucosyltransferase is UGTSl2 or UGT with > 85% amino acid sequence identity to UGTSl 2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11 or UGT having > 85% amino acid sequence identity with EUGT 11. In another specific embodiment, the UDP-glucosyltransferase is UGT91D2 or UGT having > 85% amino acid sequence identity with UGT91D 2. In a specific embodiment, the UDP-glucosyltransferase is UGT76G1 or UGT having > 85% amino acid sequence identity with UGT76G 1.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside M to form rebaudioside 1 r. In a specific embodiment, the UDP-glucosyltransferase is UGTSl2 or UGT with > 85% amino acid sequence identity to UGTSl 2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11 or UGT having > 85% amino acid sequence identity with EUGT 11. In another specific embodiment, the UDP-glucosyltransferase is UGT91D2 or UGT having > 85% amino acid sequence identity with UGT91D 2. In a specific embodiment, the UDP-glucosyltransferase is UGT76G1 or UGT having > 85% amino acid sequence identity with UGT76G 1.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside M to form rebaudioside 1 s. In a specific embodiment, the UDP-glucosyltransferase is UGTSl2 or UGT with > 85% amino acid sequence identity to UGTSl 2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11 or UGT having > 85% amino acid sequence identity with EUGT 11. In another specific embodiment, the UDP-glucosyltransferase is UGT91D2 or UGT having > 85% amino acid sequence identity with UGT91D 2. In a specific embodiment, the UDP-glucosyltransferase is UGT76G1 or UGT having > 85% amino acid sequence identity with UGT76G 1.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside M to form rebaudioside 1 t. In a specific embodiment, the UDP-glucosyltransferase is UGTSl2 or UGT with > 85% amino acid sequence identity to UGTSl 2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11 or UGT having > 85% amino acid sequence identity with EUGT 11. In another specific embodiment, the UDP-glucosyltransferase is UGT91D2 or UGT having > 85% amino acid sequence identity with UGT91D 2. In a specific embodiment, the UDP-glucosyltransferase is UGT76G1 or UGT having > 85% amino acid sequence identity with UGT76G 1.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside M4 to form rebaudioside 2 a. In a specific embodiment, the UDP-glucosyltransferase is UGTSl2 or UGT with > 85% amino acid sequence identity to UGTSl 2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11 or UGT having > 85% amino acid sequence identity with EUGT 11. In another specific embodiment, the UDP-glucosyltransferase is UGT91D2 or UGT having > 85% amino acid sequence identity with UGT91D 2.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside M4 to form rebaudioside 2 b. In a specific embodiment, the UDP-glucosyltransferase is UGT76G1 or UGT having > 85% amino acid sequence identity with UGT76G 1.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside M4 to form rebaudioside 2 c. In a specific embodiment, the UDP-glucosyltransferase is UGTSl2 or UGT with > 85% amino acid sequence identity to UGTSl 2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11 or UGT having > 85% amino acid sequence identity with EUGT 11. In another specific embodiment, the UDP-glucosyltransferase is UGT91D2 or UGT having > 85% amino acid sequence identity with UGT91D 2.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside M4 to form rebaudioside 2 d. In a specific embodiment, the UDP-glucosyltransferase is UGTSl2 or UGT with > 85% amino acid sequence identity to UGTSl 2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11 or UGT having > 85% amino acid sequence identity with EUGT 11. In another specific embodiment, the UDP-glucosyltransferase is UGT91D2 or UGT having > 85% amino acid sequence identity with UGT91D 2.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside M4 to form rebaudioside 2 e. In a specific embodiment, the UDP-glucosyltransferase is UGT76G1 or UGT having > 85% amino acid sequence identity with UGT76G 1.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside M4 to form rebaudioside 2 f. In a specific embodiment, the UDP-glucosyltransferase is UGTSl2 or UGT with > 85% amino acid sequence identity to UGTSl 2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11 or UGT having > 85% amino acid sequence identity with EUGT 11. In another specific embodiment, the UDP-glucosyltransferase is UGT91D2 or UGT having > 85% amino acid sequence identity with UGT91D 2.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside M4 to form 2g of rebaudioside. In a specific embodiment, the UDP-glucosyltransferase is UGTSl2 or UGT with > 85% amino acid sequence identity to UGTSl 2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11 or UGT having > 85% amino acid sequence identity with EUGT 11. In another specific embodiment, the UDP-glucosyltransferase is UGT91D2 or UGT having > 85% amino acid sequence identity with UGT91D 2.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside M4 to form rebaudioside 2 h. In a specific embodiment, the UDP-glucosyltransferase is UGT76G1 or UGT having > 85% amino acid sequence identity with UGT76G 1.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside M4 to form rebaudioside 2 i. In a specific embodiment, the UDP-glucosyltransferase is UGTSl2 or UGT with > 85% amino acid sequence identity to UGTSl 2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11 or UGT having > 85% amino acid sequence identity with EUGT 11. In another specific embodiment, the UDP-glucosyltransferase is UGT91D2 or UGT having > 85% amino acid sequence identity with UGT91D 2.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside M4 to form rebaudioside 2 j. In a specific embodiment, the UDP-glucosyltransferase is UGTSl2 or UGT with > 85% amino acid sequence identity to UGTSl 2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11 or UGT having > 85% amino acid sequence identity with EUGT 11. In another specific embodiment, the UDP-glucosyltransferase is UGT91D2 or UGT having > 85% amino acid sequence identity with UGT91D 2.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside M4 to form rebaudioside 2 k. In a specific embodiment, the UDP-glucosyltransferase is UGT76G1 or UGT having > 85% amino acid sequence identity with UGT76G 1.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside M4 to form 2 i of rebaudioside. In a specific embodiment, the UDP-glucosyltransferase is UGTSl2 or UGT with > 85% amino acid sequence identity to UGTSl 2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11 or UGT having > 85% amino acid sequence identity with EUGT 11. In another specific embodiment, the UDP-glucosyltransferase is UGT91D2 or UGT having > 85% amino acid sequence identity with UGT91D 2.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside M4 to form rebaudioside 2M. In a specific embodiment, the UDP-glucosyltransferase is UGTSl2 or UGT with > 85% amino acid sequence identity to UGTSl 2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11 or UGT having > 85% amino acid sequence identity with EUGT 11. In another specific embodiment, the UDP-glucosyltransferase is UGT91D2 or UGT having > 85% amino acid sequence identity with UGT91D 2.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside M4 to form rebaudioside 2 n. In a specific embodiment, the UDP-glucosyltransferase is UGTSl2 or UGT with > 85% amino acid sequence identity to UGTSl 2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11 or UGT having > 85% amino acid sequence identity with EUGT 11. In another specific embodiment, the UDP-glucosyltransferase is UGT91D2 or UGT having > 85% amino acid sequence identity with UGT91D 2.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside M4 to form rebaudioside 2 o. In a specific embodiment, the UDP-glucosyltransferase is UGTSl2 or UGT with > 85% amino acid sequence identity to UGTSl 2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11 or UGT having > 85% amino acid sequence identity with EUGT 11. In another specific embodiment, the UDP-glucosyltransferase is UGT91D2 or UGT having > 85% amino acid sequence identity with UGT91D 2. In a specific embodiment, the UDP-glucosyltransferase is UGT76G1 or UGT having > 85% amino acid sequence identity with UGT76G 1.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside M4 to form rebaudioside 2 p. In a specific embodiment, the UDP-glucosyltransferase is UGTSl2 or UGT with > 85% amino acid sequence identity to UGTSl 2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11 or UGT having > 85% amino acid sequence identity with EUGT 11. In another specific embodiment, the UDP-glucosyltransferase is UGT91D2 or UGT having > 85% amino acid sequence identity with UGT91D 2. In a specific embodiment, the UDP-glucosyltransferase is UGT76G1 or UGT having > 85% amino acid sequence identity with UGT76G 1.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside M4 to form rebaudioside 2 q. In a specific embodiment, the UDP-glucosyltransferase is UGTSl2 or UGT with > 85% amino acid sequence identity to UGTSl 2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11 or UGT having > 85% amino acid sequence identity with EUGT 11. In another specific embodiment, the UDP-glucosyltransferase is UGT91D2 or UGT having > 85% amino acid sequence identity with UGT91D 2. In a specific embodiment, the UDP-glucosyltransferase is UGT76G1 or UGT having > 85% amino acid sequence identity with UGT76G 1.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside M4 to form rebaudioside 2 r. In a specific embodiment, the UDP-glucosyltransferase is UGTSl2 or UGT with > 85% amino acid sequence identity to UGTSl 2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11 or UGT having > 85% amino acid sequence identity with EUGT 11. In another specific embodiment, the UDP-glucosyltransferase is UGT91D2 or UGT having > 85% amino acid sequence identity with UGT91D 2. In a specific embodiment, the UDP-glucosyltransferase is UGT76G1 or UGT having > 85% amino acid sequence identity with UGT76G 1.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside M4 to form rebaudioside 2 s. In a specific embodiment, the UDP-glucosyltransferase is UGTSl2 or UGT with > 85% amino acid sequence identity to UGTSl 2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11 or UGT having > 85% amino acid sequence identity with EUGT 11. In another specific embodiment, the UDP-glucosyltransferase is UGT91D2 or UGT having > 85% amino acid sequence identity with UGT91D 2. In a specific embodiment, the UDP-glucosyltransferase is UGT76G1 or UGT having > 85% amino acid sequence identity with UGT76G 1.

In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside M4 to form rebaudioside 1 q. In a specific embodiment, the UDP-glucosyltransferase is UGTSl2 or UGT with > 85% amino acid sequence identity to UGTSl 2. In another specific embodiment, the UDP-glucosyltransferase is EUGT11 or UGT having > 85% amino acid sequence identity with EUGT 11. In another specific embodiment, the UDP-glucosyltransferase is UGT91D2 or UGT having > 85% amino acid sequence identity with UGT91D 2. In a specific embodiment, the UDP-glucosyltransferase is UGT76G1 or UGT having > 85% amino acid sequence identity with UGT76G 1.

Optionally, the process of the invention further comprises recycling UDP to obtain UDP-glucose. In one embodiment, the method comprises recycling UDP by providing a recycle catalyst and a recycle substrate to biologically convert steviol and/or a steviol glycoside substrate to a target steviol glycoside using a catalytic amount of UDP-glucosyltransferase and UDP-glucose.

In one embodiment, the recycling catalyst is a sucrose synthase SuSy _ At or a sucrose synthase with > 85% amino acid sequence identity to SuSy _ At.

In one embodiment, the recycle substrate of the UDP-glucose recycle catalyst is sucrose.

Optionally, the method of the invention further comprises the application of a transglycosidase enzyme using an oligosaccharide or polysaccharide as a sugar donor in order to modify the acceptor target steviol glycoside molecule. Non-limiting examples include cyclodextrin glycosyltransferase (CGTase), fructofuranosidase, amylase, sucrase, glucosucrase, beta-h-fructosidase, beta-fructosidase, sucrase, fructosyl converting enzyme, alkaline converting enzyme, acid converting enzyme, fructofuranosidase. In some embodiments, glucose and non-glucose sugars include, but are not limited to, fructose, xylose, rhamnose, arabinose, deoxyglucose, galactose, and conversion to the acceptor target steviol glycoside. In one embodiment, the acceptor steviol glycoside is rebaudioside 1a, rebaudioside 1b, rebaudioside 1c, rebaudioside 1d, rebaudioside 1e, rebaudioside 1f, rebaudioside 1g, rebaudioside 1h, rebaudioside 1i, rebaudioside 1j, rebaudioside 1k, rebaudioside 1l, rebaudioside 1m, rebaudioside 1n, rebaudioside 1o, rebaudioside 1p, rebaudioside 1q, rebaudioside 1r, rebaudioside 1s, rebaudioside 1t, rebaudioside 2a, rebaudioside 2b, rebaudioside 2c, rebaudioside 2d, rebaudioside 2e, rebaudioside 2f, rebaudioside 2g, rebaudioside 2h, rebaudioside 2i, rebaudioside 2j, rebaudioside 2k, rebaudioside 2l, rebaudioside 2m, rebaudioside 2n, rebaudioside 2o, rebaudioside 2p, rebaudioside 2q, rebaudioside 2r and/or rebaudioside 2 s. In another embodiment, the acceptor steviol glycoside is rebaudioside M4. In another embodiment, the acceptor steviol glycoside is SvG 7.

In another embodiment, the UDP-glucosyltransferase enzyme capable of adding at least one glucose unit to a steviol glycoside of the starting composition has > 85% amino acid sequence identity with a UGT selected from the list GenInfo identifier below, preferably from tables 1 and 2.

TABLE 1

TABLE 2

One embodiment of the invention is a microbial cell comprising an enzyme, i.e. an enzyme capable of converting a starting composition into a target steviol glycoside. Thus, some embodiments of the present methods comprise contacting the microorganism with a medium comprising the starting composition to obtain a medium comprising at least one steviol glycoside of interest.

The microorganism may be any microorganism having the requisite enzymes to convert the starting composition to the target steviol glycoside. These enzymes are encoded within the microbial genome.

Suitable microorganisms include, but are not limited to, Escherichia coli, Saccharomyces, Aspergillus, Pichia, Bacillus, and yarrowia, among others.

In one embodiment, the microorganism is free when contacted with the starting composition.

In another embodiment, the microorganism is immobilized when contacted with the starting composition. For example, the microorganisms may be immobilized on a solid support made of inorganic or organic materials. Non-limiting examples of solid supports include derivatized cellulose or glass, metal oxides or membranes. For example, the microorganisms may be immobilized on the solid support by covalent binding, adsorption, cross-linking, capture or encapsulation.

In another embodiment, the enzyme capable of converting the starting composition to the target steviol glycoside is secreted from the microorganism and into the reaction medium.

Optionally purifying the target steviol glycoside. Purification of the target steviol glycoside from the reaction medium can be carried out by at least one suitable method to obtain a highly purified target steviol glycoside composition. Suitable methods include crystallization, separation by membrane, centrifugation, extraction (liquid or solid phase), chromatographic separation, HPLC (preparative or analytical) or a combination of such methods.

Use of

The highly purified target glycosides obtained according to the present invention, in particular steviolmonoside, steviolmonoside A, steviolbioside D, rubusoside, steviolbioside A, steviolbioside B, rebaudioside B, stevioside, rebaudioside G, steviolbioside A, stevioside B, stevioside C, rebaudioside A, rebaudioside E2, rebaudioside E4, rebaudioside E6, rebaudioside E3, rebaudioside D, rebaudioside I, rebaudioside AM, rebaudioside D7, rebaudioside M4, rebaudioside 1a, rebaudioside 1B, rebaudioside 1C, rebaudioside 1D, rebaudioside 1E, rebaudioside 1f, rebaudioside 1G, rebaudioside 1h, rebaudioside 1I, rebaudioside 1j, rebaudioside 1k, rebaudioside 1M, rebaudioside 1n, rebaudioside 1o, rebaudioside 1p, rebaudioside 1q, rebaudioside 1r, rebaudioside 1s, rebaudioside 1t, rebaudioside 2a, rebaudioside 2b, rebaudioside 2c, rebaudioside 2d, rebaudioside 2e, rebaudioside 2f, rebaudioside 2g, rebaudioside 2h, rebaudioside 2i, rebaudioside 2j, rebaudioside 2k, rebaudioside 2l, rebaudioside 2m, rebaudioside 2n, rebaudioside 2o, rebaudioside 2p, rebaudioside 2q, rebaudioside 2r, rebaudioside 2s, and/or SvG7 may be used "as such," or in combination with additional sweeteners, flavorants, comestible ingredients, and combinations thereof.

Non-limiting examples of flavorants include, but are not limited to, lime, lemon, orange, fruit, banana, grape, pear, pineapple, mango, berry, almond, cola, cinnamon, sugar, raffinose, vanilla, and combinations thereof.

Non-limiting examples of additional food ingredients include, but are not limited to, acidulants, organic and amino acids, colorants, fillers, modified starches, gums, texturizers, preservatives, caffeine, antioxidants, emulsifiers, stabilizers, thickeners, gelling agents, and combinations thereof.

Highly purified target glycosides obtained according to the invention, in particular steviolmonoside, steviolmonoside a, steviolbioside D, rubusoside, steviolbioside a, steviolbioside B, rebaudioside B, stevioside, rebaudioside G, stevioside a, stevioside B, stevioside C, rebaudioside a, rebaudioside E2, rebaudioside E4, rebaudioside E6, rebaudioside E3, rebaudioside D, rebaudioside I, rebaudioside AM, rebaudioside D7, rebaudioside M4, rebaudioside 1a, rebaudioside 1B, rebaudioside 1C, rebaudioside 1D, rebaudioside 1E, rebaudioside 1f, rebaudioside 1G, rebaudioside 1h, rebaudioside 1I, rebaudioside 1j, rebaudioside 1k, rebaudioside 1M, rebaudioside 1n, rebaudioside 1o, rebaudioside 1p, rebaudioside 1q, rebaudioside 1r, rebaudioside 1s, rebaudioside 1t, rebaudioside 2a, rebaudioside 2b, rebaudioside 2c, rebaudioside 2d, rebaudioside 2e, rebaudioside 2f, rebaudioside 2g, rebaudioside 2h, rebaudioside 2i, rebaudioside 2j, rebaudioside 2k, rebaudioside 2l, rebaudioside 2m, rebaudioside 2n, rebaudioside 2o, rebaudioside 2p, rebaudioside 2q, rebaudioside 2r, rebaudioside 2s, and/or SvG7 are prepared in various polymorphic forms, including, but not limited to, hydrates, solvates, anhydrous forms, amorphous forms, and combinations thereof.

The highly purified target glycosides obtained according to the present invention, in particular steviolmonoside, steviolmonoside A, steviolbioside D, rubusoside, steviolbioside A, steviolbioside B, rebaudioside B, stevioside, rebaudioside G, steviolbioside A, stevioside B, stevioside C, rebaudioside A, rebaudioside E2, rebaudioside E4, rebaudioside E6, rebaudioside E3, rebaudioside D, rebaudioside I, rebaudioside AM, rebaudioside D7, rebaudioside M4, rebaudioside 1a, rebaudioside 1B, rebaudioside 1C, rebaudioside 1D, rebaudioside 1E, rebaudioside 1f, rebaudioside 1G, rebaudioside 1h, rebaudioside 1I, rebaudioside 1j, rebaudioside 1k, rebaudioside 1M, rebaudioside 1n, rebaudioside 1o, rebaudioside 1p, rebaudioside 1q, rebaudioside 1r, rebaudioside 1s, rebaudioside 1t, rebaudioside 2a, rebaudioside 2b, rebaudioside 2c, rebaudioside 2d, rebaudioside 2e, rebaudioside 2f, rebaudioside 2g, rebaudioside 2h, rebaudioside 2i, rebaudioside 2j, rebaudioside 2k, rebaudioside 2l, rebaudioside 2m, rebaudioside 2n, rebaudioside 2o, rebaudioside 2p, rebaudioside 2q, rebaudioside 2r, rebaudioside 2s, and/or SvG7 may be incorporated as high-intensity natural sweeteners into foods, beverages, pharmaceutical compositions, cosmetics, chewing gum, tabletop products (tablop products), cereals, dairy products, toothpaste, and other oral compositions, and the like.

In some embodiments, the highly purified target glycosides of the present invention may be present in foods, beverages, pharmaceutical compositions, cosmetics, cereals, dairy products, toothpastes and other oral compositions in amounts of from about 0.0001% to about 12% by weight, for example, about 0.0001%, about 0.0005%, about 0.001%, about 0.005%, about 0.01%, about 0.05%, about 0.1%, about 0.5%, about 1.0%, about 1.5%, about 2.0%, about 2.5%, about 3.0%, about 3.5%, about 4.0%, about 4.5%, about 5.0%, about 5.5%, about 6.0%, about 6.5%, about 7.0%, about 7.5%, about 8.0%, about 8.5%, about 9.0%, about 9.5%, about 10.0%, about 10.5%, about 11.0%, about 11.5%, or about 12.0% by weight.

In a particular embodiment, the sweetener is present in the beverage in an amount from about 0.0001% to about 8% by weight, e.g., from about 0.0001% to about 0.0005% by weight, from about 0.0005% to about 0.001% by weight, from about 0.001% to about 0.005% by weight, from about 0.005% to about 0.01% by weight, from about 0.01% to about 0.05% by weight, from about 0.05% to about 0.1% by weight, from about 0.1% to about 0.5% by weight, from about 0.5% to about 1% by weight, from about 1% to about 2% by weight, from about 2% to about 3% by weight, from about 3% to about 4% by weight, from about 4% to about 5% by weight, from about 5% to about 6% by weight, from about 6% to about 7% by weight, and from about 7% to about 8% by weight.

The highly purified target glycosides obtained according to the present invention, in particular steviolmonoside, steviolmonoside A, steviolbioside D, rubusoside, steviolbioside A, steviolbioside B, rebaudioside B, stevioside, rebaudioside G, steviolbioside A, stevioside B, stevioside C, rebaudioside A, rebaudioside E2, rebaudioside E4, rebaudioside E6, rebaudioside E3, rebaudioside D, rebaudioside I, rebaudioside AM, rebaudioside D7, rebaudioside M4, rebaudioside 1a, rebaudioside 1B, rebaudioside 1C, rebaudioside 1D, rebaudioside 1E, rebaudioside 1f, rebaudioside 1G, rebaudioside 1h, rebaudioside 1I, rebaudioside 1j, rebaudioside 1k, rebaudioside 1M, rebaudioside 1n, rebaudioside 1o, rebaudioside 1p, rebaudioside 1q, rebaudioside 1r, rebaudioside 1s, rebaudioside 1t, rebaudioside 2a, rebaudioside 2B, rebaudioside 2C, rebaudioside 2D, rebaudioside 2e, rebaudioside 2f, rebaudioside 2g, rebaudioside 2h, rebaudioside 2i, rebaudioside 2j, rebaudioside 2k, rebaudioside 2l, rebaudioside 2m, rebaudioside 2n, rebaudioside 2o, rebaudioside 2p, rebaudioside 2q, rebaudioside 2r, rebaudioside 2s, SvG7, and/or combinations thereof or can be used as sweetening compounds or in combination with at least one naturally occurring high intensity sweetener, such as rebaudioside A, rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside A2, rebaudioside A3, rebaudioside A, rebaudioside B, rebaudioside D, rebaudioside E, rebaudioside F, rebaudioside H, rebaudioside I, rebaudioside J, rebaudioside K, rebaudioside KA, rebaudioside L, rebaudioside M, rebaudioside N, rebaudioside O, rebaudioside Q, rebaudioside Q2, rebaudioside Q3, rebaudioside R, rebaudioside S, rebaudioside T1, rebaudioside U2, rebaudioside V2, rebaudioside V3, rebaudioside W2, rebaudioside W3, rebaudioside Y, rebaudioside Z1, rebaudioside Z2, steviolbioside C, steviolbioside E, stevioside D, stevioside E2, stevioside F, stevioside G, stevioside H, mogroside, brazzein, neohesperidin dihydrochalcone, glycyrrhizic acid and salts thereof, thaumatin, perillartine, pileusine (pernandulcin), sapindoside (mukurosides), sinomenide, rebaudioside I, rebaudioside-L, rebaudioside-D, rebaudioside-C, rebaudioside D, glycyrrhizic acid, rebaudioside D, rebaudioside E, rebaudioside F, rebaudioside G, stevioside H, rebaudioside D, rebaudioside E, rebaudioside D, rebaudioside A, rebaudioside I, rebaudioside D-rebaudioside A, rebaudioside D, rebaudioside A, cyclocarioside, secodammarane-type triterpene glycosides (pterocaryosides), polyboloside A, brazilin, hernandulcin (hernandulcin), philodicin (philosocin), sarsasaponin, phloridzin, trilobatin, dihydroflavonol, dihydroquercetin-3-acetate, neoastin (neoastibin), trans-cinnamaldehyde, monatin salt, other indole derivative sweeteners, seliguanin A (seliguanin A), monellin, caritin, pterocaryosin A (pterocaryoside A), pterocaryosin B, mabinlin, peimin (pendadin), modified glycoproteins, curculin, neocurculin (neocurculin), chlorogenic acid, sinalin, momordica, sweeteners, mogroside V, siamenoside, sinoside and combinations thereof.

In a particular embodiment, steviolmonoside A, steviolbioside D, rubusoside, steviolbioside A, steviolbioside B, rebaudioside B, stevioside, rebaudioside G, stevioside A, stevioside B, stevioside C, rebaudioside A, rebaudioside E2, rebaudioside E4, rebaudioside E6, rebaudioside E3, rebaudioside D, rebaudioside I, rebaudioside AM, rebaudioside D7, rebaudioside M4, rebaudioside 1a, rebaudioside 1B, rebaudioside 1C, rebaudioside 1D, rebaudioside 1E, rebaudioside 1f, rebaudioside 1G, rebaudioside 1h, rebaudioside 1I, rebaudioside 1j, rebaudioside 1k, rebaudioside 1M, rebaudioside 1n, rebaudioside 1o, rebaudioside 1p, rebaudioside 1q, rebaudioside 1r, rebaudioside 1s, rebaudioside 1t, rebaudioside 2a, rebaudioside 2b, rebaudioside 2c, rebaudioside 2d, rebaudioside 2e, rebaudioside 2f, rebaudioside 2g, rebaudioside 2h, rebaudioside 2i, rebaudioside 2j, rebaudioside 2k, rebaudioside 2l, rebaudioside 2m, rebaudioside 2n, rebaudioside 2o, rebaudioside 2p, rebaudioside 2q, rebaudioside 2r, rebaudioside 2s, and/or SvG7 may be used in a sweetener composition comprising a compound selected from the group consisting of: rebaudioside A, rebaudioside B, rebaudioside C2, rebaudioside D2, rebaudioside E2, rebaudioside F2, rebaudioside H2, rebaudioside I2, rebaudioside J, rebaudioside K, rebaudioside N2, rebaudioside K2, rebaudioside K, rebaudioside E2, rebaudioside K, rebaudioside E2, rebaudioside K, rebaudioside, rebaudioside N5, rebaudioside O2, rebaudioside O3, rebaudioside O4, rebaudioside Q2, rebaudioside Q3, rebaudioside R, rebaudioside S, rebaudioside T1, rebaudioside U2, rebaudioside V2, rebaudioside V3, rebaudioside W2, rebaudioside W3, rebaudioside Y, rebaudioside Z1, rebaudioside Z2, steviolbioside C, steviolbioside E, stevioside D, stevioside E2, stevioside F, stevioside G, stevioside H, NSF-02, mogroside V, siratose, lo han guo, psicose, D-D, erythritol, and combinations thereof.

Highly purified target glycosides, in particular steviolmonoside, steviolmonoside A, steviolbioside D, rubusoside, steviolbioside A, steviolbioside B, rebaudioside B, stevioside, rebaudioside G, stevioside A, stevioside B, stevioside C, rebaudioside A, rebaudioside E2, rebaudioside E4, rebaudioside E6, rebaudioside E3, rebaudioside D, rebaudioside I, rebaudioside AM, rebaudioside D7, rebaudioside M4, rebaudioside 1a, rebaudioside 1B, rebaudioside 1C, rebaudioside 1D, rebaudioside 1E, rebaudioside 1f, rebaudioside 1G, rebaudioside 1h, rebaudioside 1I, rebaudioside 1j, rebaudioside 1k, rebaudioside 1l, rebaudioside 1M, rebaudioside 1n, rebaudioside 1o, rebaudioside 1p, rebaudioside 1q, rebaudioside 1r, rebaudioside 1s, rebaudioside 1t, rebaudioside 2a, rebaudioside 2b, rebaudioside 2c, rebaudioside 2d, rebaudioside 2e, rebaudioside 2f, rebaudioside 2g, rebaudioside 2h, rebaudioside 2i, rebaudioside 2j, rebaudioside 2k, rebaudioside 2l, rebaudioside 2m, rebaudioside 2n, rebaudioside 2o, rebaudioside 2p, rebaudioside 2q, rebaudioside 2r, rebaudioside 2s, and/or SvG7 may also be used in combination with synthetic high intensity sweeteners, such as sucralose, acesulfame potassium, aspartame, alitame, saccharin, neohesperidin dihydrochalcone, cyclamate, neotame, dulcin, sulosavadamine, and salts and combinations thereof.

Further, highly purified target steviol glycosides, in particular steviolmonoside, steviolmonoside A, steviolbioside D, rubusoside, steviolbioside A, steviolbioside B, rebaudioside B, stevioside, rebaudioside G, stevioside A, stevioside B, stevioside C, rebaudioside A, rebaudioside E2, rebaudioside E4, rebaudioside E6, rebaudioside E3, rebaudioside D, rebaudioside I, rebaudioside AM, rebaudioside D7, rebaudioside M4, rebaudioside 1a, rebaudioside 1B, rebaudioside 1C, rebaudioside 1D, rebaudioside 1E, rebaudioside 1f, rebaudioside 1G, rebaudioside 1h, rebaudioside 1I, rebaudioside 1j, rebaudioside 1k, rebaudioside 1M, rebaudioside 1o, rebaudioside 1p, rebaudioside 1q, rebaudioside 1r, rebaudioside 1s, rebaudioside 1t, rebaudioside 2a, rebaudioside 2b, rebaudioside 2c, rebaudioside 2d, rebaudioside 2e, rebaudioside 2f, rebaudioside 2g, rebaudioside 2h, rebaudioside 2i, rebaudioside 2j, rebaudioside 2k, rebaudioside 2l, rebaudioside 2m, rebaudioside 2n, rebaudioside 2o, rebaudioside 2p, rebaudioside 2q, rebaudioside 2r, rebaudioside 2s, and/or SvG7 may be used in combination with natural sweetener inhibitors, such as, for example, dulcoside, ziphin, nordulcitol (lactisola), and the like. Steviolmonoside, steviolmonoside A, steviolbioside D, rubusoside, steviolbioside A, steviolbioside B, rebaudioside B, stevioside, rebaudioside G, stevioside A, stevioside B, stevioside C, rebaudioside A, rebaudioside E2, rebaudioside E4, rebaudioside E6, rebaudioside E3, rebaudioside D, rebaudioside I, rebaudioside AM, rebaudioside D7, rebaudioside M4, rebaudioside 1a, rebaudioside 1B, rebaudioside 1C, rebaudioside 1D, rebaudioside 1E, rebaudioside 1f, rebaudioside 1G, rebaudioside 1h, rebaudioside 1I, rebaudioside 1j, rebaudioside 1k, rebaudioside 1l, rebaudioside 1M, rebaudioside 1n, rebaudioside 1p, rebaudioside 1q, rebaudioside 1r, rebaudioside 1s, rebaudioside 1t, rebaudioside 2a, rebaudioside 2b, rebaudioside 2c, rebaudioside 2d, rebaudioside 2e, rebaudioside 2f, rebaudioside 2g, rebaudioside 2h, rebaudioside 2i, rebaudioside 2j, rebaudioside 2k, rebaudioside 2l, rebaudioside 2m, rebaudioside 2n, rebaudioside 2o, rebaudioside 2p, rebaudioside 2q, rebaudioside 2r, rebaudioside 2s, and/or SvG7 may also be used in combination with various umami taste enhancers. Steviolmonoside, steviolmonoside A, steviolbioside D, rubusoside, steviolbioside A, steviolbioside B, rebaudioside B, stevioside, rebaudioside G, stevioside A, stevioside B, stevioside C, rebaudioside A, rebaudioside E2, rebaudioside E4, rebaudioside E6, rebaudioside E3, rebaudioside D, rebaudioside I, rebaudioside AM, rebaudioside D7, rebaudioside M4, rebaudioside 1a, rebaudioside 1B, rebaudioside 1C, rebaudioside 1D, rebaudioside 1E, rebaudioside 1f, rebaudioside 1G, rebaudioside 1h, rebaudioside 1I, rebaudioside 1j, rebaudioside 1k, rebaudioside 1l, rebaudioside 1M, rebaudioside 1n, rebaudioside 1p, rebaudioside 1q, rebaudioside 1r, rebaudioside 1s, rebaudioside 1t, rebaudioside 2a, rebaudioside 2b, rebaudioside 2c, rebaudioside 2d, rebaudioside 2e, rebaudioside 2f, rebaudioside 2g, rebaudioside 2h, rebaudioside 2i, rebaudioside 2j, rebaudioside 2k, rebaudioside 2l, rebaudioside 2m, rebaudioside 2n, rebaudioside 2o, rebaudioside 2p, rebaudioside 2q, rebaudioside 2r, rebaudioside 2s, and/or SvG7 may be mixed with umami and sweet amino acids, such as glutamic acid, aspartic acid, glycine, alanine, threonine, proline, serine, glutamic acid, lysine, tryptophan, and combinations thereof.

Highly purified target steviol glycosides, in particular steviolmonoside, steviolmonoside A, steviolbioside D, rubusoside, steviolbioside A, steviolbioside B, rebaudioside B, stevioside, rebaudioside G, stevioside A, stevioside B, stevioside C, rebaudioside A, rebaudioside E2, rebaudioside E4, rebaudioside E6, rebaudioside E3, rebaudioside D, rebaudioside I, rebaudioside AM, rebaudioside D7, rebaudioside M4, rebaudioside 1a, rebaudioside 1B, rebaudioside 1C, rebaudioside 1D, rebaudioside 1E, rebaudioside 1f, rebaudioside 1G, rebaudioside 1h, rebaudioside 1I, rebaudioside 1j, rebaudioside 1k, rebaudioside 1l, rebaudioside 1M, rebaudioside 1n, rebaudioside 1o, rebaudioside 1p, rebaudioside 1q, rebaudioside 1r, rebaudioside 1s, rebaudioside 1t, rebaudioside 2a, rebaudioside 2b, rebaudioside 2c, rebaudioside 2d, rebaudioside 2e, rebaudioside 2f, rebaudioside 2g, rebaudioside 2h, rebaudioside 2i, rebaudioside 2j, rebaudioside 2k, rebaudioside 2l, rebaudioside 2m, rebaudioside 2n, rebaudioside 2o, rebaudioside 2p, rebaudioside 2q, rebaudioside 2r, rebaudioside 2s, and/or SvG7 may be used in combination with one or more additives selected from the group consisting of sugars, polyols, amino acids and corresponding salts thereof, polyamino acids and corresponding salts thereof, sugar acids and corresponding salts thereof, nucleotides, organic acids, inorganic acids, organic salts, including organic acid salts and organic base salts, inorganic salts, bitter compounds, flavor and flavor ingredients, astringent compounds, protein or protein hydrolysates, surfactants, emulsifiers, flavonoids, alcohols, polymers and combinations thereof.

Highly purified target steviol glycosides, in particular steviolmonoside, steviolmonoside A, steviolbioside D, rubusoside, steviolbioside A, steviolbioside B, rebaudioside B, stevioside, rebaudioside G, stevioside A, stevioside B, stevioside C, rebaudioside A, rebaudioside E2, rebaudioside E4, rebaudioside E6, rebaudioside E3, rebaudioside D, rebaudioside I, rebaudioside AM, rebaudioside D7, rebaudioside M4, rebaudioside 1a, rebaudioside 1B, rebaudioside 1C, rebaudioside 1D, rebaudioside 1E, rebaudioside 1f, rebaudioside 1G, rebaudioside 1h, rebaudioside 1I, rebaudioside 1j, rebaudioside 1k, rebaudioside 1l, rebaudioside 1M, rebaudioside 1n, rebaudioside 1o, rebaudioside 1p, rebaudioside 1q, rebaudioside 1r, rebaudioside 1s, rebaudioside 1t, rebaudioside 2a, rebaudioside 2b, rebaudioside 2c, rebaudioside 2d, rebaudioside 2e, rebaudioside 2f, rebaudioside 2g, rebaudioside 2h, rebaudioside 2i, rebaudioside 2j, rebaudioside 2k, rebaudioside 2l, rebaudioside 2m, rebaudioside 2n, rebaudioside 2o, rebaudioside 2p, rebaudioside 2q, rebaudioside 2r, rebaudioside 2s, and/or SvG7 may be used in combination with a polyol or a sugar alcohol. The term "polyol" refers to a molecule comprising more than one hydroxyl group. The polyol may be a diol, triol or tetraol containing 2, 3 and 4 hydroxyl groups respectively. The polyols may also contain more than four hydroxyl groups, such as pentahydric, hexahydric, heptahydric, and the like, which contain 5, 6, or 7 hydroxyl groups, respectively. In addition, the polyol may also be a sugar alcohol, a polyol or a polyol which is a reduced form of a sugar in which carbonyl groups (aldehydes or ketones, reducing sugars) have been reduced to primary or secondary hydroxyl groups. Examples of polyols include, but are not limited to erythritol, maltitol, mannitol, sorbitol, lactitol, xylitol, inositol, isomaltulose, propylene glycol, glycerol, threitol, galactitol, hydrogenated isomaltulose, reduced isomalto-oligosaccharides, reduced xylo-oligosaccharides, reduced gentio-oligosaccharides, reduced maltose syrup, reduced glucose syrup, hydrogenated starch hydrolysates, polyglycols and sugar alcohols or any other sugar capable of reduction, which do not adversely affect the taste of the sweetener composition.

Highly purified target steviol glycosides, in particular steviolmonoside, steviolmonoside A, steviolbioside D, rubusoside, steviolbioside A, steviolbioside B, rebaudioside B, stevioside, rebaudioside G, stevioside A, stevioside B, stevioside C, rebaudioside A, rebaudioside E2, rebaudioside E4, rebaudioside E6, rebaudioside E3, rebaudioside D, rebaudioside I, rebaudioside AM, rebaudioside D7, rebaudioside M4, rebaudioside 1a, rebaudioside 1B, rebaudioside 1C, rebaudioside 1D, rebaudioside 1E, rebaudioside 1f, rebaudioside 1G, rebaudioside 1h, rebaudioside 1I, rebaudioside 1j, rebaudioside 1k, rebaudioside 1l, rebaudioside 1M, rebaudioside 1n, rebaudioside 1o, rebaudioside 1p, rebaudioside 1q, rebaudioside 1r, rebaudioside 1s, rebaudioside 1t, rebaudioside 2a, rebaudioside 2b, rebaudioside 2c, rebaudioside 2D, rebaudioside 2e, rebaudioside 2f, rebaudioside 2g, rebaudioside 2h, rebaudioside 2i, rebaudioside 2j, rebaudioside 2k, rebaudioside 2L, rebaudioside 2m, rebaudioside 2n, rebaudioside 2o, rebaudioside 2p, rebaudioside 2q, rebaudioside 2r, rebaudioside 2s, and/or SvG7 may be used in combination with a low calorie sweetener, such as D-tagatose, L-sugar, L-sorbose, L-arabinose, and combinations thereof.

Highly purified target steviol glycosides, in particular steviolmonoside, steviolmonoside A, steviolbioside D, rubusoside, steviolbioside A, steviolbioside B, rebaudioside B, stevioside, rebaudioside G, stevioside A, stevioside B, stevioside C, rebaudioside A, rebaudioside E2, rebaudioside E4, rebaudioside E6, rebaudioside E3, rebaudioside D, rebaudioside I, rebaudioside AM, rebaudioside D7, rebaudioside M4, rebaudioside 1a, rebaudioside 1B, rebaudioside 1C, rebaudioside 1D, rebaudioside 1E, rebaudioside 1f, rebaudioside 1G, rebaudioside 1h, rebaudioside 1I, rebaudioside 1j, rebaudioside 1k, rebaudioside 1l, rebaudioside 1M, rebaudioside 1n, rebaudioside 1o, rebaudioside 1p, rebaudioside 1q, rebaudioside 1r, rebaudioside 1s, rebaudioside 1t, rebaudioside 2a, rebaudioside 2b, rebaudioside 2c, rebaudioside 2d,rebaudioside 2e, rebaudioside 2f, rebaudioside 2g, rebaudioside 2h, rebaudioside 2i, rebaudioside 2j, rebaudioside 2k, rebaudioside 2l, rebaudioside 2m, rebaudioside 2n, rebaudioside 2o, rebaudioside 2p, rebaudioside 2q, rebaudioside 2r, rebaudioside 2s, and/or SvG7 may also be used in combination with various sugars. The term "saccharide" is generally meant to have the formula (CH) ₂O)_n(wherein n is 3 to 30) aldehyde or ketone compounds substituted with a plurality of hydroxyl groups, and oligomers and polymers thereof. In addition, the saccharides of the present invention may be substituted or deoxygenated at one or more positions. As used herein, saccharides include unmodified saccharides, saccharide derivatives, substituted saccharides and modified saccharides. As used herein, the phrases "saccharide derivative," "substituted saccharide," and "modified saccharide" are synonymous. Modified saccharide means any saccharide in which at least one atom or a combination thereof has been added, removed or substituted. Thus, saccharide derivatives or substituted saccharides include substituted and unsubstituted monosaccharides, disaccharides, oligosaccharides and polysaccharides. The carbohydrate derivative or substituted carbohydrate may be optionally deoxygenated at any corresponding C-position and/or substituted with one or more moieties (e.g., hydrogen, halogen, haloalkyl, carboxyl, acyl, acyloxy, amino, amido, carboxyl derivative, alkylamino, dialkylamino, arylamino, alkoxy, aryloxy, nitro, cyano, sulfo, thiol, imino, sulfonyl, sulfinyl, sulfoxy, sulfamoyl, alkoxycarbonyl, carboxamide, phosphono, phosphinyl, phosphoryl, phosphino, thioester, thioether, oximino, hydrazino, carbamoyl, phosphorous, phosphonate, or any other viable functional group) so long as the carbohydrate derivative or substituted carbohydrate acts to improve the sweetness of the sweetener composition.

Examples of saccharides for use according to the invention include, but are not limited to: psicose, melibiose, allose, tagatose, trehalose, galactose, rhamnose, various cyclodextrins, cyclic oligosaccharides, various types of maltodextrin, dextran, sucrose, glucose, ribulose, fructose, threose, arabinose, xylose, lyxose, allose, altrose, mannose, idose, lactose, maltose, invert sugar, isohydralose, neotrehalose, isomaltulose, erythrose, deoxyribose, gulose, idose, talose, erythrulose, xylulose, psicose, turanose, cellobiose, amylopectin, glucosamine, mannosamine, fucose, glucuronic acid, gluconic acid, gluconolactone, abicose, galactosamine, oligosaccharose, isomaltooligosaccharides (isomaltose, isomaltotriose, panose, etc.), xylooligosaccharides (xylotriose, xylobiose, etc.), xylose-terminated oligosaccharides, gentiooligosaccharides (gentiobiose, gentiotriose, gentiotetraose and the like), sorbose, aspergillus niger oligosaccharides, palatinose oligosaccharides, fructooligosaccharides (kestose, kestotetraose and the like), maltotetraol, maltotriol, maltooligosaccharides (maltotriose, maltotetraose, maltopentaose, maltohexaose, maltoheptaose and the like), starch, inulin oligosaccharides, lactulose, melibiose, raffinose, ribose, isomerized liquid sugars such as high fructose corn syrup, coupling sugars and soybean oligosaccharides. In addition, the saccharides used herein may be in the D-configuration or the L-configuration.

The highly purified target steviol glycosides, in particular steviolmonoside, steviolmonoside A, steviolbioside D, rubusoside, steviolbioside A, steviolbioside B, rebaudioside B, stevioside, rebaudioside G, stevioside A, stevioside B, stevioside C, rebaudioside A, rebaudioside E2, rebaudioside E4, rebaudioside E6, rebaudioside E3, rebaudioside D, rebaudioside I, rebaudioside AM, rebaudioside D7, rebaudioside M4, rebaudioside 1a, rebaudioside 1B, rebaudioside 1C, rebaudioside 1D, rebaudioside 1E, rebaudioside 1f, rebaudioside 1G, rebaudioside 1h, rebaudioside 1I, rebaudioside 1j, rebaudioside 1k, rebaudioside 1M, rebaudioside 1n, rebaudioside 1o, rebaudioside 1p, rebaudioside 1q, rebaudioside 1r, rebaudioside 1s, rebaudioside 1t, rebaudioside 2a, rebaudioside 2b, rebaudioside 2c, rebaudioside 2d, rebaudioside 2e, rebaudioside 2f, rebaudioside 2g, rebaudioside 2h, rebaudioside 2i, rebaudioside 2j, rebaudioside 2k, rebaudioside 2l, rebaudioside 2m, rebaudioside 2n, rebaudioside 2o, rebaudioside 2p, rebaudioside 2q, rebaudioside 2r, rebaudioside 2s, and/or SvG7 may be used in combination with various physiologically active substances or functional components. Functional ingredients are generally classified into, for example, the following categories: carotenoids, dietary fibers, fatty acids, saponins, antioxidants, nutraceuticals, flavonoids, isothiocyanates, phenols, phytosterols and stanols (phytosterols and phytostanols); a polyol; prebiotics, probiotics; phytoestrogens; soy protein; sulfides/mercaptans; an amino acid; a protein; vitamins and minerals. Functional ingredients may also be classified based on their health benefits (e.g., cardiovascular, cholesterol lowering and anti-inflammatory). Exemplary functional ingredients are provided in WO2013/096420 (the contents of which are hereby incorporated by reference).

The highly purified target steviol glycosides, in particular steviolmonoside, steviolmonoside A, steviolbioside D, rubusoside, steviolbioside A, steviolbioside B, rebaudioside B, stevioside, rebaudioside G, stevioside A, stevioside B, stevioside C, rebaudioside A, rebaudioside E2, rebaudioside E4, rebaudioside E6, rebaudioside E3, rebaudioside D, rebaudioside I, rebaudioside AM, rebaudioside D7, rebaudioside M4, rebaudioside 1a, rebaudioside 1B, rebaudioside 1C, rebaudioside 1D, rebaudioside 1E, rebaudioside 1f, rebaudioside 1G, rebaudioside 1h, rebaudioside 1I, rebaudioside 1j, rebaudioside 1k, rebaudioside 1M, rebaudioside 1n, rebaudioside 1o, rebaudioside 1p, rebaudioside 1q, rebaudioside 1r, rebaudioside 1s, rebaudioside 1t, rebaudioside 2a, rebaudioside 2b, rebaudioside 2c, rebaudioside 2d, rebaudioside 2e, rebaudioside 2f, rebaudioside 2g, rebaudioside 2h, rebaudioside 2i, rebaudioside 2j, rebaudioside 2k, rebaudioside 2l, rebaudioside 2m, rebaudioside 2n, rebaudioside 2o, rebaudioside 2p, rebaudioside 2q, rebaudioside 2r, rebaudioside 2s, and/or SvG7 may be used as high intensity sweeteners to produce zero calorie, low calorie, or diabetic beverages and food products with improved taste profiles. It can also be used in beverages, foods, pharmaceuticals and other products where sugar is not used. Further, highly purified target steviol glycosides, in particular steviolmonoside, steviolmonoside A, steviolbioside D, rubusoside, steviolbioside A, steviolbioside B, rebaudioside B, stevioside, rebaudioside G, stevioside A, stevioside B, stevioside C, rebaudioside A, rebaudioside E2, rebaudioside E4, rebaudioside E6, rebaudioside E3, rebaudioside D, rebaudioside I, rebaudioside AM, rebaudioside D7, rebaudioside M4, rebaudioside 1a, rebaudioside 1B, rebaudioside 1C, rebaudioside 1D, rebaudioside 1E, rebaudioside 1f, rebaudioside 1G, rebaudioside 1h, rebaudioside 1I, rebaudioside 1j, rebaudioside 1k, rebaudioside 1M, rebaudioside 1o, rebaudioside 1p, rebaudioside 1q, rebaudioside 1r, rebaudioside 1s, rebaudioside 1t, rebaudioside 2a, rebaudioside 2b, rebaudioside 2c, rebaudioside 2d, rebaudioside 2e, rebaudioside 2f, rebaudioside 2g, rebaudioside 2h, rebaudioside 2i, rebaudioside 2j, rebaudioside 2k, rebaudioside 2l, rebaudioside 2m, rebaudioside 2n, rebaudioside 2o, rebaudioside 2p, rebaudioside 2q, rebaudioside 2r, rebaudioside 2s, and/or SvG7 may be used as sweeteners not only in beverages, food products, and other products intended for human consumption, but also in animal feeds and forage materials having improved characteristics.

The highly purified target steviol glycosides, in particular steviolmonoside, steviolmonoside A, steviolbioside D, rubusoside, steviolbioside A, steviolbioside B, rebaudioside B, stevioside, rebaudioside G, stevioside A, stevioside B, stevioside C, rebaudioside A, rebaudioside E2, rebaudioside E4, rebaudioside E6, rebaudioside E3, rebaudioside D, rebaudioside I, rebaudioside AM, rebaudioside D7, rebaudioside M4, rebaudioside 1a, rebaudioside 1B, rebaudioside 1C, rebaudioside 1D, rebaudioside 1E, rebaudioside 1f, rebaudioside 1G, rebaudioside 1h, rebaudioside 1I, rebaudioside 1j, rebaudioside 1k, rebaudioside 1M, rebaudioside 1n, rebaudioside 1o, rebaudioside 1p, rebaudioside 1q, rebaudioside 1r, rebaudioside 1s, rebaudioside 1t, rebaudioside 2a, rebaudioside 2b, rebaudioside 2c, rebaudioside 2d, rebaudioside 2e, rebaudioside 2f, rebaudioside 2g, rebaudioside 2h, rebaudioside 2i, rebaudioside 2j, rebaudioside 2k, rebaudioside 2l, rebaudioside 2m, rebaudioside 2n, rebaudioside 2o, rebaudioside 2p, rebaudioside 2q, rebaudioside 2r, rebaudioside 2s, and/or SvG7 may be used as a foaming inhibitor to produce zero calorie, low calorie, or diabetic beverages and food products.

Examples of consumer products (wherein highly purified target steviol glycosides, in particular steviolmonoside, steviolmonoside A, steviolbioside D, rubusoside, steviolbioside A, steviolbioside B, rebaudioside B, stevioside, rebaudioside G, stevioside A, stevioside B, stevioside C, rebaudioside A, rebaudioside E2, rebaudioside E4, rebaudioside E6, rebaudioside E3, rebaudioside D, rebaudioside I, rebaudioside AM, rebaudioside D7, rebaudioside M4, rebaudioside 1a, rebaudioside 1B, rebaudioside 1C, rebaudioside 1D, rebaudioside 1E, rebaudioside 1f, rebaudioside 1G, rebaudioside 1h, rebaudioside 1I, rebaudioside 1j, rebaudioside 1k, rebaudioside 1M, rebaudioside 1n, rebaudioside 1o, rebaudioside 1p, rebaudioside 1q, rebaudioside 1r, rebaudioside 1s, rebaudioside 1t, rebaudioside 2a, rebaudioside 2b, rebaudioside 2c, rebaudioside 2d, rebaudioside 2e, rebaudioside 2f, rebaudioside 2g, rebaudioside 2h, rebaudioside 2i, rebaudioside 2j, rebaudioside 2k, rebaudioside 2l, rebaudioside 2m, rebaudioside 2n, rebaudioside 2o, rebaudioside 2p, rebaudioside 2q, rebaudioside 2r, rebaudioside 2s, and/or SvG7 may be used as a sweet compound) include, but are not limited to: alcoholic beverages such as vodka, wine, beer, white spirit and sake and the like; natural juice; refreshing beverage; a carbonated soft drink; a sugar-free beverage; a zero-calorie beverage; reduced-calorie beverages and foods; a yogurt drink; instant juice; instant coffee; a powdered instant beverage; can products; syrup; fermenting the soybean paste; soy sauce; vinegar; a seasoning; mayonnaise; tomato sauce; curry; soup; an instant bouillon; soy sauce powder; vinegar powder; various types of biscuits; rice biscuits; a thin crisp biscuit; bread; chocolate; caramel; a candy; a chewing gum; jelly; pudding; preserved fruit and vegetable; fresh cream; jam; citrus pulp; flower sauce; milk powder; ice cream; a snowflake; bottling vegetables and fruits; canning and boiling beans; cooking meat and food with sweet juice; agricultural vegetable food; seafood; ham; sausages; fish ham; fish sausages; fish paste; a fried fish product; drying marine products; freezing the food; pickling the seaweed; pickling meat; tobacco; a pharmaceutical product; and many other food products. Which in principle can be used in an unlimited number of applications.

Examples of consumer products (wherein highly purified target steviol glycosides, in particular steviolmonoside, steviolmonoside A, steviolbioside D, rubusoside, steviolbioside A, steviolbioside B, rebaudioside B, stevioside, rebaudioside G, stevioside A, stevioside B, stevioside C, rebaudioside A, rebaudioside E2, rebaudioside E4, rebaudioside E6, rebaudioside E3, rebaudioside D, rebaudioside I, rebaudioside AM, rebaudioside D7, rebaudioside M4, rebaudioside 1a, rebaudioside 1B, rebaudioside 1C, rebaudioside 1D, rebaudioside 1E, rebaudioside 1f, rebaudioside 1G, rebaudioside 1h, rebaudioside 1I, rebaudioside 1j, rebaudioside 1k, rebaudioside 1M, rebaudioside 1n, rebaudioside 1o, rebaudioside 1p, rebaudioside 1q, rebaudioside 1r, rebaudioside 1s, rebaudioside 1t, rebaudioside 2a, rebaudioside 2b, rebaudioside 2c, rebaudioside 2d, rebaudioside 2e, rebaudioside 2f, rebaudioside 2g, rebaudioside 2h, rebaudioside 2i, rebaudioside 2j, rebaudioside 2k, rebaudioside 2l, rebaudioside 2m, rebaudioside 2n, rebaudioside 2o, rebaudioside 2p, rebaudioside 2q, rebaudioside 2r, rebaudioside 2s, and/or SvG7 may be used as a flavor enhancer or a flavorant having improved properties) include, but are not limited to: alcoholic beverages such as vodka, wine, beer, white spirit and sake and the like; natural juice; refreshing beverage; a carbonated soft drink; a sugar-free beverage; a zero-calorie beverage; reduced-calorie beverages and foods; a yogurt drink; instant juice; instant coffee; a powdered instant beverage; can products; syrup; fermenting the soybean paste; soy sauce; vinegar; a seasoning; mayonnaise; tomato sauce; curry; soup; an instant bouillon; soy sauce powder; vinegar powder; various types of biscuits; rice biscuits; a thin crisp biscuit; bread; chocolate; caramel; a candy; a chewing gum; jelly; pudding; preserved fruit and vegetable; fresh cream; jam; citrus pulp; flower sauce; milk powder; ice cream; a snowflake; bottling vegetables and fruits; canning and boiling beans; cooking meat and food with sweet juice; agricultural vegetable food; seafood; ham; sausages; fish ham; fish sausages; fish paste; a fried fish product; drying marine products; freezing the food; pickling the seaweed; pickling meat; tobacco; a pharmaceutical product; and many other food products. Which in principle can be used in an unlimited number of applications.

In the manufacture of products such as food, beverages, pharmaceuticals, cosmetics, table top products and chewing gum, conventional methods such as mixing, kneading, dissolution, acid leaching, osmosis, percolation, spraying, atomising, infusing and other methods may be used.

Further, the highly purified target steviol glycosides, steviolmonoside A, steviolbioside D, rubusoside, steviolbioside A, steviolbioside B, rebaudioside B, stevioside, rebaudioside G, stevioside A, stevioside B, stevioside C, rebaudioside A, rebaudioside E2, rebaudioside E4, rebaudioside E6, rebaudioside E3, rebaudioside D, rebaudioside I, rebaudioside AM, rebaudioside D7, rebaudioside M4, rebaudioside 1a, rebaudioside 1B, rebaudioside 1C, rebaudioside 1D, rebaudioside 1E, rebaudioside 1f, rebaudioside 1G, rebaudioside 1h, rebaudioside 1I, rebaudioside 1j, rebaudioside 1k, rebaudioside 1M, rebaudioside 1n, rebaudioside 1o, rebaudioside 1p, rebaudioside 1q, rebaudioside 1r, rebaudioside 1s, rebaudioside 1t, rebaudioside 2a, rebaudioside 2b, rebaudioside 2c, rebaudioside 2d, rebaudioside 2e, rebaudioside 2f, rebaudioside 2g, rebaudioside 2h, rebaudioside 2i, rebaudioside 2j, rebaudioside 2k, rebaudioside 2l, rebaudioside 2m, rebaudioside 2n, rebaudioside 2o, rebaudioside 2p, rebaudioside 2q, rebaudioside 2r, rebaudioside 2s, and/or SvG7 may be used in dry or liquid form.

Highly purified target steviol glycosides can be added before or after heat treatment of the food product. Highly purified target steviol glycosides, in particular steviolmonoside, steviolmonoside A, steviolbioside D, rubusoside, steviolbioside A, steviolbioside B, rebaudioside B, stevioside, rebaudioside G, stevioside A, stevioside B, stevioside C, rebaudioside A, rebaudioside E2, rebaudioside E4, rebaudioside E6, rebaudioside E3, rebaudioside D, rebaudioside I, rebaudioside AM, rebaudioside D7, rebaudioside M4, rebaudioside 1a, rebaudioside 1B, rebaudioside 1C, rebaudioside 1D, rebaudioside 1E, rebaudioside 1f, rebaudioside 1G, rebaudioside 1h, rebaudioside 1I, rebaudioside 1j, rebaudioside 1k, rebaudioside 1l, rebaudioside 1M, rebaudioside 1n, the amounts of rebaudioside 1o, rebaudioside 1p, rebaudioside 1q, rebaudioside 1r, rebaudioside 1s, rebaudioside 1t, rebaudioside 2a, rebaudioside 2b, rebaudioside 2c, rebaudioside 2d, rebaudioside 2e, rebaudioside 2f, rebaudioside 2g, rebaudioside 2h, rebaudioside 2i, rebaudioside 2j, rebaudioside 2k, rebaudioside 2l, rebaudioside 2m, rebaudioside 2n, rebaudioside 2o, rebaudioside 2p, rebaudioside 2q, rebaudioside 2r, rebaudioside 2s and/or SvG7 depend on the purpose of use. As mentioned above, it can be used alone or in combination with other compounds.

The present invention also relates to a method of sweetness enhancement in a beverage using as sweetness enhancer the following compounds: steviolmonoside, steviolmonoside A, steviolbioside D, rubusoside, steviolbioside A, steviolbioside B, rebaudioside B, stevioside, rebaudioside G, stevioside A, stevioside B, stevioside C, rebaudioside A, rebaudioside E2, rebaudioside E4, rebaudioside E6, rebaudioside E3, rebaudioside D, rebaudioside I, rebaudioside AM, rebaudioside D7, rebaudioside M4, rebaudioside 1a, rebaudioside 1B, rebaudioside 1C, rebaudioside 1D, rebaudioside 1E, rebaudioside 1f, rebaudioside 1G, rebaudioside 1h, rebaudioside 1I, rebaudioside 1j, rebaudioside 1k, rebaudioside 1l, rebaudioside 1M, rebaudioside 1n, rebaudioside 1p, rebaudioside 1q, rebaudioside 1r, rebaudioside 1s, rebaudioside 1t, rebaudioside 2a, rebaudioside 2B, rebaudioside 2C, rebaudioside 2D, rebaudioside 2E, rebaudioside 2f, rebaudioside 2G, rebaudioside 2h, rebaudioside 2i, rebaudioside 2j, rebaudioside 2k, rebaudioside 2l, rebaudioside 2m, rebaudioside 2n, rebaudioside 2o, rebaudioside 2p, rebaudioside 2q, rebaudioside 2r, rebaudioside 2s, and/or SvG7, wherein steviolmonoside, steviolmonoside A, steviolbioside D, rubusoside, steviolbioside A, steviolbioside B, rebaudioside B, stevioside, rebaudioside G, rebaudioside A, stevioside B, steviolbioside C, rebaudioside A, rebaudioside E2, rebaudioside E, rebaudioside E4, rebaudioside E6, rebaudioside E3, rebaudioside D, rebaudioside I, rebaudioside AM, rebaudioside D7, rebaudioside M4, rebaudioside 1a, rebaudioside 1b, rebaudioside 1c, rebaudioside 1D, rebaudioside 1E, rebaudioside 1f, rebaudioside 1g, rebaudioside 1h, rebaudioside 1I, rebaudioside 1j, rebaudioside 1k, rebaudioside 1l, rebaudioside 1M, rebaudioside 1n, rebaudioside 1o, rebaudioside 1p, rebaudioside 1q, rebaudioside 1r, rebaudioside 1s, rebaudioside 1t, rebaudioside 2a, rebaudioside 2b, rebaudioside 2c, rebaudioside 2D, rebaudioside 2E, rebaudioside 2f, rebaudioside 2g, rebaudioside 2h, rebaudioside 2j, rebaudioside 2k, rebaudioside 2l, rebaudioside 2m, rebaudioside 2n, rebaudioside 2o, rebaudioside 2p, rebaudioside 2q, rebaudioside 2r, rebaudioside 2s, and/or SvG7 are present at a concentration at or below their respective sweetness recognition threshold.

As used herein, the term "sweetness enhancer" refers to a compound that is capable of enhancing or potentiating the perception of sweetness in a composition, such as a beverage. The term "sweet taste enhancer (sweet taste enhancer)" is synonymous with the terms "sweet taste enhancer (sweet taste promoter)", "sweet taste potentiator (sweet taste potentiator)", "sweet taste potentiator (sweet taste amplifier)" and "sweet taste potentiator (sweet taste intensifier)".

As generally used herein, the term "sweet taste recognition threshold concentration" is the lowest known concentration of sweet compounds that can be perceived by human taste, typically about 1.0% sucrose equivalent (1.0% SE). Generally, a sweetness enhancer, when present at or below the sweetness recognition threshold concentration for a given sweetness enhancer, can enhance or potentiate the sweetness of the sweetener without providing any noticeable sweetness by itself; however, the sweetness enhancer itself may provide sweetness at a concentration above its sweetness recognition threshold concentration. The sweetness recognition threshold concentration is specific to a particular enhancer and may vary depending on the beverage matrix. The sweetness recognition threshold concentration can be readily determined by taste testing by increasing the concentration of a given enhancer until a sucrose equivalent greater than 1.0% is detected in a given beverage base. A concentration that provides about 1.0% sucrose equivalent is considered a sweet taste recognition threshold.

In some embodiments, the sweetener is present in the beverage in an amount from about 0.0001% to about 12% by weight, e.g., about 0.0001% by weight, about 0.0005% by weight, about 0.001% by weight, about 0.005% by weight, about 0.01% by weight, about 0.05% by weight, about 0.1% by weight, about 0.5% by weight, about 1.0% by weight, about 1.5% by weight, about 2.0% by weight, about 2.5% by weight, about 3.0% by weight, about 3.5% by weight, about 4.0% by weight, about 4.5% by weight, about 5.0% by weight, about 5.5% by weight, about 6.0% by weight, about 6.5% by weight, about 7.0% by weight, about 7.5% by weight, about 8.0% by weight, about 8.5% by weight, about 9.0% by weight, about 9.5% by weight, about 10.0% by weight, about 10.5% by weight, about 11.0% by weight, or about 11.0% by weight.

In a particular embodiment, the sweetener is present in the beverage in an amount from about 0.0001% to about 10% by weight, e.g., from about 0.0001% to about 0.0005%, from about 0.0005% to about 0.001%, from about 0.001% to about 0.005%, from about 0.005% to about 0.01%, from about 0.01% to about 0.05%, from about 0.05% to about 0.1%, from about 0.1% to about 0.5%, from about 0.5% to about 1%, from about 1% to about 2%, from about 2% to about 3%, from about 3% to about 4%, from about 4% to about 5%, from about 5% to about 6%, from about 6% to about 7%, from about 7% to about 8%, from about 8% to about 9%, or from about 10%. In a particular embodiment, the sweetener is present in the beverage in an amount from about 0.5% to about 10% by weight. In another specific embodiment, the sweetener is present in the beverage in an amount from about 2% to about 8% by weight.

In one embodiment, the sweetener is a traditional caloric sweetener. Suitable sweeteners include, but are not limited to, sucrose, fructose, glucose, high fructose corn syrup and high fructose corn syrup.

In another embodiment, the sweetener is erythritol.

In another embodiment, the sweetener is a rare sugar. Suitable rare sugars include, but are not limited to, D-allose, D-psicose, D-ribose, D-tagatose, L-glucose, L-fucose, L-arabinose, D-turanose, D-leucrose, and combinations thereof.

It is contemplated that the sweetener may be used alone or in combination with other sweeteners.

In one embodiment, the rare sugar is D-allose. In a more specific embodiment, D-allose is present in the beverage in an amount of from about 0.5% to about 10% by weight, for example from about 2% to about 8%.

In another embodiment, the rare sugar is D-psicose. In a more specific embodiment, the D-psicose is present in the beverage in an amount of about 0.5% to about 10% by weight, for example about 2% to about 8%.

In another embodiment, the rare sugar is D-ribose. In a more specific embodiment, the D-ribose is present in the beverage in an amount of about 0.5% to about 10% by weight, for example about 2% to about 8%.

In another embodiment, the rare sugar is D-tagatose. In a more specific embodiment, D-tagatose is present in the beverage in an amount of about 0.5% to about 10% by weight, for example about 2% to about 8%.

In another embodiment, the rare sugar is L-glucose. In a more specific embodiment, the L-glucose is present in the beverage in an amount of about 0.5% to about 10% by weight, for example about 2% to about 8%.

In one embodiment, the rare sugar is L-fucose. In a more specific embodiment, L-fucose is present in the beverage in an amount from about 0.5% to about 10% by weight, for example from about 2% to about 8%.

In another embodiment, the rare sugar is L-arabinose. In a more specific embodiment, the L-arabinose is present in the beverage in an amount of from about 0.5% to about 10% by weight, for example from about 2% to about 8%.

In another embodiment, the rare sugar is D-turanose. In a more specific embodiment, D-turanose is present in the beverage in an amount from about 0.5% to about 10% by weight, e.g., from about 2% to about 8%.

In another embodiment, the rare sugar is D-leucrose. In a more specific embodiment, D-leuconostoc disaccharide is present in the beverage in an amount of about 0.5% to about 10% by weight, for example about 2% to about 8%.

Adding the sweetness enhancer at a concentration at or below its sweetness recognition threshold increases the detected sucrose equivalent for a beverage comprising the sweetener and sweetness enhancer as compared to a corresponding beverage in the absence of the sweetness enhancer. Furthermore, the sweetness increase may be greater than the detectable sweetness of a solution comprising the same concentration of the at least one sweetness enhancer without any sweetener.

Accordingly, the invention also provides a method for enhancing the sweetness of a beverage comprising a sweetener, the method comprising providing a beverage comprising a sweetener, and adding a sweetness enhancer selected from the group consisting of steviolmonoside, steviolmonoside a, steviolbioside D, rubusoside, steviolbioside a, steviolbioside B, rebaudioside B, stevioside, rebaudioside G, stevioside a, stevioside B, stevioside C, rebaudioside a, rebaudioside E2, rebaudioside E4, rebaudioside E6, rebaudioside E3, rebaudioside D, rebaudioside I, rebaudioside AM, rebaudioside D7, rebaudioside M4, rebaudioside 1a, rebaudioside 1B, rebaudioside 1C, rebaudioside 1D, rebaudioside 1E, rebaudioside 1f, rebaudioside h, rebaudioside 1G, rebaudioside 1i, rebaudioside 1j, rebaudioside 1k, rebaudioside 1l, rebaudioside 1m, rebaudioside 1n, rebaudioside 1o, rebaudioside 1p, rebaudioside 1q, rebaudioside 1r, rebaudioside 1s, rebaudioside 1t, rebaudioside 2a, rebaudioside 2b, rebaudioside 2c, rebaudioside 2D, rebaudioside 2e, rebaudioside 2f, rebaudioside 2g, rebaudioside 2h, rebaudioside 2i, rebaudioside 2j, rebaudioside 2k, rebaudioside 2l, rebaudioside 2m, rebaudioside 2n, rebaudioside 2o, rebaudioside 2p, rebaudioside 2q, rebaudioside 2r, rebaudioside 2s, and/or SvG7 or combinations thereof, wherein steviolmonoside, steviolmonoside A, steviolbioside D, steviolbioside, rebaudioside A, rebaudioside B, rebaudioside E2, rebaudioside E4, rebaudioside E6, rebaudioside E3, rebaudioside D, rebaudioside I, rebaudioside AM, rebaudioside D7, rebaudioside M4, rebaudioside 1a, rebaudioside 1B, rebaudioside 1C, rebaudioside 1D, rebaudioside 1E, rebaudioside 1f, rebaudioside 1G, rebaudioside 1h, rebaudioside 1I, rebaudioside 1j, rebaudioside 1k, rebaudioside 1l, rebaudioside 1M, rebaudioside 1n, rebaudioside 1o, rebaudioside 1p, rebaudioside 1q, rebaudioside 1r, rebaudioside 1t 1s, rebaudioside 1s, rebaudioside 2a, rebaudioside 2b, rebaudioside 2c, rebaudioside 2d, rebaudioside 2e, rebaudioside 2f, rebaudioside 2g, rebaudioside 2h, rebaudioside 2i, rebaudioside 2j, rebaudioside 2k, rebaudioside 2l, rebaudioside 2m, rebaudioside 2n, rebaudioside 2o, rebaudioside 2p, rebaudioside 2q, rebaudioside 2r, rebaudioside 2s, and/or SvG7, present at or below their sweetness recognition threshold.

Adding to a beverage comprising a sweetener, steviolmonoside A, steviolbioside D, rubusoside, steviolbioside A, steviolbioside B, rebaudioside B, stevioside, rebaudioside G, stevioside A, stevioside B, stevioside C, rebaudioside A, rebaudioside E2, rebaudioside E4, rebaudioside E6, rebaudioside E3, rebaudioside D, rebaudioside I, rebaudioside AM, rebaudioside D7, rebaudioside M4, rebaudioside 1a, rebaudioside 1B, rebaudioside 1C, rebaudioside 1D, rebaudioside 1E, rebaudioside 1f, rebaudioside 1G, rebaudioside 1h, rebaudioside 1I, rebaudioside 1j, rebaudioside 1k, rebaudioside 1m, rebaudioside 1n, rebaudioside 1o, rebaudioside 1p, rebaudioside 1q, rebaudioside 1r, rebaudioside 1s, rebaudioside 1t, rebaudioside 2a, rebaudioside 2b, rebaudioside 2c, rebaudioside 2d, rebaudioside 2e, rebaudioside 2f, rebaudioside 2g, rebaudioside 2h, rebaudioside 2i, rebaudioside 2j, rebaudioside 2k, rebaudioside 2l, rebaudioside 2m, rebaudioside 2n, rebaudioside 2o, rebaudioside 2p, rebaudioside 2q, rebaudioside 2r, rebaudioside 2s, and/or SvG7 may increase the sucrose equivalent detected by about 1.0% to about 5.0%, e.g., about 1.0%, about 1.5%, about 2.0%, about 2.5%, about 3.0%, about 3.5%, about 4.0%, or about 4.0%.

The following embodiments illustrate preferred embodiments of the invention for preparing highly purified target steviol glycosides, in particular steviolmonoside, steviolmonoside A, steviolbioside D, rubusoside, steviolbioside A, steviolbioside B, rebaudioside B, stevioside, rebaudioside G, stevioside A, stevioside B, stevioside C, rebaudioside A, rebaudioside E2, rebaudioside E4, rebaudioside E6, rebaudioside E3, rebaudioside D, rebaudioside I, rebaudioside AM, rebaudioside D7, rebaudioside M4, rebaudioside 1a, rebaudioside 1B, rebaudioside 1C, rebaudioside 1D, rebaudioside 1E, rebaudioside 1f, rebaudioside 1G, rebaudioside 1h, rebaudioside 1I, rebaudioside 1j, rebaudioside 1k, rebaudioside 1i, rebaudioside 1m, rebaudioside 1n, rebaudioside 1o, rebaudioside 1p, rebaudioside 1q, rebaudioside 1r, rebaudioside 1s, rebaudioside 1t, rebaudioside 2a, rebaudioside 2b, rebaudioside 2c, rebaudioside 2d, rebaudioside 2e, rebaudioside 2f, rebaudioside 2g, rebaudioside 2h, rebaudioside 2i, rebaudioside 2j, rebaudioside 2k, rebaudioside 2l, rebaudioside 2m, rebaudioside 2n, rebaudioside 2o, rebaudioside 2p, rebaudioside 2q, rebaudioside 2r, rebaudioside 2s, and/or SvG 7. It is to be understood that this invention is not limited to the materials, proportions, conditions and methods recited in the examples, which are exemplary only.

Examples

Example 1

Protein sequences of engineered enzymes for use in biocatalytic processes

SEQ ID 1：

(> SuSy _ At, variant PM1-54-2-E05 (engineered sucrose synthase; WT gene source: Arabidopsis)

MANAERMITRVHSQRERLNETLVSERNEVLALLSRVEAKGKGILQQNQIIAEFEALPEQTRKKLEGGPFFDLLKSTQEAIVLPPWVALAVRPRPGVWEYLRVNLHALVVEELQPAEFLHFKEELVDGVKNGNFTLELDFEPFNASIPRPTLHKYIGNGVDFLNRHLSAKLFHDKESLLPLLDFLRLHSHQGKNLMLSEKIQNLNTLQHTLRKAEEYLAELKSETLYEEFEAKFEEIGLERGWGDNAERVLDMIRLLLDLLEAPDPSTLETFLGRVPMVFNVVILSPHGYFAQDNVLGYPDTGGQVVYILDQVRALEIEMLQRIKQQGLNIKPRILILTRLLPDAVGTTCGERLERVYDSEYCDILRVPFRTEKGIVRKWISRFEVWPYLETYTEDAAVELSKELNGKPDLIIGNYSDGNLVASLLAHKLGVTQCTIAHALEKTKYPDSDIYWKKLDDKYHFSCQFTADIFAMNHTDFIITSTFQEIAGSKETVGQYESHTAFTLPGLYRVVHGIDVFDPKFNIVSPGADMSIYFPYTEEKRRLTKFHSEIEELLYSDVENDEHLCVLKDKKKPILFTMARLDRVKNLSGLVEWYGKNTRLRELVNLVVVGGDRRKESKDNEEKAEMKKMYDLIEEYKLNGQFRWISSQMDRVRNGELYRYICDTKGAFVQPALYEAFGLTVVEAMTCGLPTFATCKGGPAEIIVHGKSGFHIDPYHGDQAADLLADFFTKCKEDPSHWDEISKGGLQRIEEKYTWQIYSQRLLTLTGVYGFWKHVSNLDRLEHRRYLEMFYALKYRPLAQAVPLAQDD

SEQ ID 2:

' UGTSl2 variant 0234 (engineered glucosyltransferase; WT gene source: tomato)

MATNLRVLMFPWLAYGHISPFLNIAKQLADRGFLIYLCSTRINLESIIKKIPEKYADSIHLIELQLPELPELPPHYHTTNGLPPHLNPTLHKALKMSKPNFSRILQNLKPDLLIYDVLQPWAEHVANEQGIPAGKLLVSCAAVFSYFFSFRKNPGVEFPFPAIHLPEVEKVKIREILAKEPEEGGRLDEGNKQMMLMCTSRTIEAKYIDYCTELCNWKVVPVGPPFQDLITNDADNKELIDWLGTKPENSTVFVSFGSEYFLSKEDMEEIAFALEASNVNFIWVVRFPKGEERNLEDALPEGFLERIGERGRVLDKFAPQPRILNHPSTGGFISHCGWNSVMESIDFGVPIIAMPIHNDQPINAKLMVELGVAVEIVRDDDGKIHRGEIAEALKSVVTGETGEILRAKVREISKNLKSIRDEEMDAVAEELIQLCRNSNKSK

SEQ ID 3：

UGT76G1 variant 0042 (engineered glucosyltransferase; WT gene source: stevia rebaudiana Bertoni)

MENKTETTVRRRRRIILFPVPFQGHINPILQLANVLYSKGFAITILHTNFNKPKTSNYPHFTFRFILDNDPQDERISNLPTHGPLAGMRIPIINEHGADELRRELELLMLASEEDEEVSCLITDALWYFAQDVADSLNLRRLVLMTSSLFNFHAHVSLPQFDELGYLDPDDKTRLEEQASGFPMLKVKDIKSAYSNWQIGKEILGKMIKQTKASSGVIWNSFKELEESELETVIREIPAPSFLIPLPKHLTASSSSLLDHDRTVFEWLDQQAPSSVLYVSFGSTSEVDEKDFLEIARGLVDSGQSFLWVVRPGFVKGSTWVEPLPDGFLGERGKIVKWVPQQEVLAHPAIGAFWTHSGWNSTLESVCEGVPMIFSSFGGDQPLNARYMSDVLRVGVYLENGWERGEVVNAIRRVMVDEEGEYIRQNARVLKQKADVSLMKGGSSYESLESLVSYISSL

Example 2

Expression and formulation of the SuSy _ At variant of SEQ ID 1

The gene encoding the SuSy _ At variant of SEQ ID 1 (example 1) was cloned into the expression vector pLE1A17 (derivative of pRSF-1b, Novagen). The resulting plasmid was used to transform E.coli BL21(DE3) cells.

Cells were cultured at 37 ℃ in ZYM505 medium (F. William student, Protein Expression and Purification 41 (2005)) supplemented with kanamycin (50mg/l) 207-234. IPTG (0.2mM) induced gene expression in log phase and was carried out at 30 ℃ and 200rpm for 16-18 hours.

Cells were harvested by centrifugation (3220Xg, 20min, 4 ℃) and used in cell lysis buffer (100mM Tris-HCl pH7.0; 2mM MgCl)₂20U/mL DNA nuclease, lysisEnzyme 0.5mg/mL) was resuspended at an optical density of 200 (at 600nm (OD)₆₀₀) Measured at (b). The cells were then disrupted by sonication and the crude extract was separated from the cell debris by centrifugation (18000Xg 40min, 4 ℃). The supernatant was sterilized by filtration through a 0.2 μm filter and diluted with distilled water at 50:50 to obtain an enzyme activity preparation.

For the enzymatically active preparation of SuSy _ At, the unit activity is defined as follows: 1mU of SuSy _ At converted 1nmol of sucrose to fructose in 1 minute. The reaction conditions for this assay were 30 ℃, 50mM potassium phosphate buffer pH7.0, t₀400mM sucrose, 3mM MgCl₂And 15mM Uridine Diphosphate (UDP).

Example 3

Expression and formulation of UGTSl2 variant of SEQ ID 2

The gene encoding the UGTSl2 variant of SEQ ID 2 (example 1) was cloned into the expression vector pLE1A17 (derivative of pRSF-1b, Novagen). The resulting plasmid was used to transform E.coli BL21(DE3) cells.

Cells were cultured at 37 ℃ in ZYM505 medium (F. William student, Protein Expression and Purification 41 (2005)) supplemented with kanamycin (50mg/l) 207-234. Gene expression was induced in log phase with IPTG (0.1mM) and at 30 ℃ and 200rpm for 16-18 hours.

Cells were harvested by centrifugation (3220Xg, 20min, 4 ℃) and used in cell lysis buffer (100mM Tris-HCl pH 7.0; 2mM MgCl)₂DNA nuclease 20U/mL, lysozyme 0.5mg/mL) was resuspended to an optical density of 200 (measured at 600nm (OD)₆₀₀)). The cells were then disrupted by sonication and the crude extract was separated from the cell debris by centrifugation (18000Xg 40min, 4 ℃). The supernatant was sterilized by filtration through a 0.2 μ M filter and diluted 50:50 with 1M sucrose solution to give an enzyme active preparation.

For the enzyme activity preparation of UGTSl2, the unit activity is defined as follows: 1mU UGTSl2 converted 1nmol of rebaudioside A (Reb A) to rebaudioside D (Reb D) in 1 minute. The reaction conditions for this assay were 30 ℃, 50mM potassium phosphate buffer pH7.0, t₀10mM of RebA, 500mM of sucrose, 3mM of MgCl₂And 0.25mM Uridine Diphosphate (UDP) and 3U/mLSuSy_At。

Example 4

Expression and formulation of UGT76G1 variant of SEQ ID 3

The gene encoding the UGT76G1 variant of SEQ ID 3 (example 1) was cloned into the expression vector pLE1A17 (derivative of pRSF-1b, Novagen). The resulting plasmid was used to transform E.coli BL21(DE3) cells.

Cells were cultured at 37 ℃ in ZYM505 medium (F. William student, Protein Expression and Purification 41 (2005)) supplemented with kanamycin (50mg/l) 207-234. Gene expression was induced in log phase with IPTG (0.1mM) and at 30 ℃ and 200rpm for 16-18 hours.

Cells were harvested by centrifugation (3220Xg, 20min, 4 ℃) and used in cell lysis buffer (100mM Tris-HCl pH 7.0; 2mM MgCl)₂DNA nuclease 20U/mL, lysozyme 0.5mg/mL) was resuspended to an optical density of 200 (measured at 600nm (OD)₆₀₀)). The cells were then disrupted by sonication and the crude extract was separated from the cell debris by centrifugation (18000Xg 40min, 4 ℃). The supernatant was sterilized by filtration through a 0.2 μ M filter and diluted 50:50 with 1M sucrose solution to give an enzyme active preparation.

For the enzyme activity preparation of UGT76G1, the unit activity is defined as follows: 1mU UGT76G1 converted 1nmol of rebaudioside D (Reb D) to rebaudioside M (Reb M) in 1 minute. The reaction conditions for this assay were 30 ℃, 50mM potassium phosphate buffer pH 7.0, t₀10mM Reb D, 500mM sucrose, 3mM MgCl₂And 0.25mM Uridine Diphosphate (UDP) and 3U/mL of SuSy _ At.

Example 5

Synthesis of SvG7 in a one-pot reaction with addition of UGTSl2, SuSy _ At and UGT76G1

Different SvG7 molecules were synthesized directly from stevioside in a one-pot reaction using 3 enzymes (see examples 1, 2, 3 and 4) (see FIG. 11 a): UGTSl2 (variant of SEQ ID 2), SuSy _ At (variant of SEQ ID 1) and UGT76G1 (variant of SEQ ID 3).

The final reaction solution contained 348U/L UGTSl2, 1341U/L SuSy _ At, 10U/L UGT76G1, 47mM stevioside, 0.32mM Uridine Diphosphate (UDP), 0.99M sucrose, 3.9mM MgCl₂And potassium phosphate buffer (pH 6.6). First, 206mL of distilled water was mixed with 0.24g of MgCl_2·6H₂O, 102g sucrose, 9.8mL 1.5M potassium phosphate buffer (pH 6.6) and 15g stevioside. The final volume of the reaction mixture was adjusted to 300 mL.

After dissolving the components, the temperature was adjusted to 45 ℃ and UGTSl2, SuSy _ At, UGT76G1 and 39mg UDP were added. The reaction mixture was incubated in a 45 ℃ shake flask for 24 hr. An additional 39mg of UDP was added at 12 hours, 24 hours and 36 hours. At the end of the reaction (48 hours) the content of reb 2a, reb 2m and the difference SvG7 was analyzed by HPLC.

Example 6

HPLC analysis

For analysis, 17% H was used₃PO₄The biotransformation sample was inactivated by adjusting the reaction mixture to ph5.5 and then boiled for 10 minutes. The resulting sample was filtered, and the filtrate was diluted 10-fold for HPLC analysis. HPLC analysis was performed on an Agilent HP 1200HPLC system consisting of a pump, column thermostat, autosampler, UV detector capable of background correction and data acquisition system. The analytes were isolated using an Agilent Poroshell 120SB-C18, 4.6mmx150mm, 2.7 μm at 40 ℃. The mobile phase consisted of two premixes:

premix 1 comprising 75% 10mM phosphate buffer (ph2.6) and 25% acetonitrile; and

premix 2 containing 68% 10mM phosphate buffer (ph2.6) and 32% acetonitrile.

The elution gradient started from premix 1, becoming premix 2 to 50% at 12.5 minutes and premix 2 to 100% at 13 minutes. The total run time was 45 minutes. The column temperature was maintained at 40 ℃. The amount of sample was 5. mu.L. The rebaudioside species were detected by UV at 210 nm.

Table 3 shows the process of converting stevioside to the identified rebaudioside species (area percentage) at each time point. FIGS. 11a and 11b show chromatograms of starting material stevioside and the reaction mixture at 48 hours, respectively. One skilled in the art will appreciate that retention times can sometimes vary with solvent and/or equipment.

TABLE 3

The stevioside is biologically converted into reb 2a (rt 6.459), reb 2m (rt 5.089) and a different SvG7

Example 7

Purification of rebaudiosides 2a, 2m and each SvG7

300mL of the reaction mixture of example 5 (after 48 hr) was washed with H₃PO₄The pH was inactivated by adjusting to pH 5.5, then boiled for 10 minutes and filtered. The filtrate was loaded onto a column containing 500mL of YWD03(Cangzhou Yuanwei, China) resin pre-equilibrated with water. The resin was washed with 2.5L of water and the effluent eluent from this step was discarded.

The steviol glycosides were eluted from the YWD03 resin column by eluting with 2.5L of 70% v/v ethanol/water. The eluate from this step was collected and dried under vacuum at 60 ℃ to give 20g of a dried solid product. The sample was dissolved in water and further fractionated by HPLC using the conditions listed in table 4 below.

HPLC fractions corresponding to and from each compound from multiple runs were pooled according to retention time. These fractions were freeze-dried.

TABLE 4

HPLC conditions

Column	Agilent Prodigy 3u ODS (3)100A, 4.6mmx250mm, 3 micron
		Temperature of	40℃
Mobile phase	Isocratic-water 77% acetonitrile 23%
		Flow rate of flow	0.5mL/min
Injection of drugs	10μL
		Residence time	45min
Auto sampler temperature	Environment(s)
		Detection of	UV at 210nm

The purity of the fractions obtained was evaluated by the analytical HPLC method described in example 6. The chromatogram of purified rebaudioside 2a is shown in figure 11 c. The chromatogram of purified rebaudioside 2m is shown in figure 11 d.

Example 8

Structural elucidation of rebaudioside 2a

NMR experiments were performed using a Bruker 500MHz spectrometer, where the samples were dissolved in pyridine-d 5. Observed from delta_C123.5, 135.5, 149.9ppm and delta_HSignals from 7.19, 7.55, 8.71ppm pyridine-d 5 and from the sample.

In pyridine-d₅Of rebaudioside 2a recorded in¹The H-NMR spectrum confirmed the excellent quality of the sample (see FIG. 12 a). HSQC (see FIG. 12b) shows the presence of an exo-methylene group in the sugar domain with long-range coupling to C-15, which can be observed in H, H-COSY (FIG. 12C).The deep field signals of the other quaternary carbons (C-13, C-16 and C-19) were detected by HMBC (FIG. 12 d). The correlation of the signals in HSQC, HMBC and H, H-COSY reveals the presence of steviol glycosides with the following aglycone structure:

the correlation between HSQC and HMBC showed the presence of 7 anomeric signals, labeled 1i, 1ii, 1iii, 1iv, 1v, 1vi and 1vii, respectively. The coupling constant of the anomeric proton at about 8Hz, the broad signal of its sugar bond and the NOE-correlation of the anomeric proton allowed the identification of these 7 sugars as beta-D-glucopyranosides.

Pooled data from HSQC and HMBC revealed sugar-sugar and sugar-aglycone linkages. Assignment of the sugar sequences was confirmed by using a combination of HSQC-TOCSY (FIG. 12e) and NOESY (FIG. 12 f).

In summary, the results from NMR experiments were used to determine the chemical shifts of the protons and carbon atoms of the structure of rebaudioside 2a (see table 5).

TABLE 5

Chemical shift of rebaudioside 2a

TABLE 5 (continuation)

Chemical shift of rebaudioside 2a

TABLE 5 (continuation)

Chemical shift of rebaudioside 2a

Correlation of all NMR results showed rebaudioside 2a with seven β -D-glucose linked to the stevioside aglycone as shown in the following chemical structure:

LCMS (FIGS. 12g and 12H) analysis of rebaudioside 2a showed [ M-H at M/z 1451.6]^-Ion, with the expected C₆₂H₁₀₀O₃₈Molecular formula ([ C)₆₂H₉₉O₃₈]^-Calculated monoisotopic ion: 1451.6) the consistency was good. MS data confirm that rebaudioside 2a has C₆₂H₁₀₀O₃₈The molecular formula (II) is shown in the specification. LCMS analysis was performed under the following conditions listed in table 6.

TABLE 6

LCMS analysis conditions

Example 9

Structural elucidation of rebaudioside 2m

NMR experiments were performed using a Bruker 500MHz spectrometer, where the samples were dissolved in pyridine-d 5. Observed from delta_C123.5, 135.5, 149.9ppm and delta_HSignals from 7.19, 7.55, 8.71ppm pyridine-d 5 and from the sample.

In pyridine-d₅Of rebaudioside 2m¹The H-NMR spectrum confirmed the excellent quality of the sample (see FIG. 13 a). HSQC (see FIG. 13b) shows the presence of an exo-methylene group in the sugar domain with long-range coupling to C-15, which can be observed in H, H-COSY (FIG. 13C). The deep field signals of the other quaternary carbons (C-13, C-16 and C-19) were detected by HMBC (FIG. 13 d). The correlation of the signals in HSQC, HMBC and H, H-COSY reveals the presence of steviol glycosides with the following aglycone structure:

The correlation between HSQC and HMBC showed the presence of 7 anomeric signals, labeled 1i, 1ii, 1iii, 1iv, 1v, 1vi and 1vii, respectively. The coupling constant of the anomeric proton at about 8Hz, the broad signal of its sugar bond and the NOE-correlation of the anomeric proton allowed the identification of these 7 sugars as beta-D-glucopyranosides.

Pooled data from HSQC and HMBC revealed sugar-sugar and sugar-aglycone linkages. Assignment of the sugar sequences was confirmed by using a combination of HSQC-TOCSY (FIG. 13e) and NOESY (FIG. 13 f).

In summary, the results from NMR experiments were used to determine the chemical shifts of the protons and carbon atoms of the structure of rebaudioside 2m (see table 7).

TABLE 7

Chemical shift of rebaudioside 2m

Table 7 (continuation)

Chemical shift of rebaudioside 2m

Table 7 (continuation)

Chemical shift of rebaudioside 2m

Correlation of all NMR results showed that the stevia compound 2m with seven β -D-glucose linked to the stevioside aglycone is shown by the following chemical structure:

LCMS (FIGS. 13g and 13H) analysis of rebaudioside 2M showed [ M-H at M/z 1451.6]^-Ion, with the expected C₆₂H₁₀₀O₃₈Molecular formula ([ C)₆₂H₉₉O₃₈]^-Calculated monoisotopic ion: 1451.6) the consistency was good. MS data confirmed that stevia rebaudiana acid ester 2m has C₆₂H₁₀₀O₃₈The molecular formula (II) is shown in the specification. LCMS analysis was performed under the conditions listed in table 6.

Although the present invention and its advantages have been described in detail, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims. Furthermore, it is not intended that the scope of the application be limited to the specific embodiments of the invention described in this specification. As one of ordinary skill in the art will readily appreciate from the disclosure of the present invention, compositions, processes, methods, and steps, presently existing or later to be developed that perform substantially the same function or achieve substantially the same result as the corresponding embodiments described herein may be utilized according to the present invention.

Sequence listing

<110> PureCircle USA Inc.

<120> high purity steviol glycosides

<130> 39227-77WO

<160> 3

<170> PatentIn version 3.5

<210> 1

<211> 808

<212> PRT

<213> Arabidopsis thaliana

<400> 1

Met Ala Asn Ala Glu Arg Met Ile Thr Arg Val His Ser Gln Arg Glu

1 5 10 15

Arg Leu Asn Glu Thr Leu Val Ser Glu Arg Asn Glu Val Leu Ala Leu

20 25 30

Leu Ser Arg Val Glu Ala Lys Gly Lys Gly Ile Leu Gln Gln Asn Gln

35 40 45

Ile Ile Ala Glu Phe Glu Ala Leu Pro Glu Gln Thr Arg Lys Lys Leu

50 55 60

Glu Gly Gly Pro Phe Phe Asp Leu Leu Lys Ser Thr Gln Glu Ala Ile

65 70 75 80

Val Leu Pro Pro Trp Val Ala Leu Ala Val Arg Pro Arg Pro Gly Val

85 90 95

Trp Glu Tyr Leu Arg Val Asn Leu His Ala Leu Val Val Glu Glu Leu

100 105 110

Gln Pro Ala Glu Phe Leu His Phe Lys Glu Glu Leu Val Asp Gly Val

115 120 125

Lys Asn Gly Asn Phe Thr Leu Glu Leu Asp Phe Glu Pro Phe Asn Ala

130 135 140

Ser Ile Pro Arg Pro Thr Leu His Lys Tyr Ile Gly Asn Gly Val Asp

145 150 155 160

Phe Leu Asn Arg His Leu Ser Ala Lys Leu Phe His Asp Lys Glu Ser

165 170 175

Leu Leu Pro Leu Leu Asp Phe Leu Arg Leu His Ser His Gln Gly Lys

180 185 190

Asn Leu Met Leu Ser Glu Lys Ile Gln Asn Leu Asn Thr Leu Gln His

195 200 205

Thr Leu Arg Lys Ala Glu Glu Tyr Leu Ala Glu Leu Lys Ser Glu Thr

210 215 220

Leu Tyr Glu Glu Phe Glu Ala Lys Phe Glu Glu Ile Gly Leu Glu Arg

225 230 235 240

Gly Trp Gly Asp Asn Ala Glu Arg Val Leu Asp Met Ile Arg Leu Leu

245 250 255

Leu Asp Leu Leu Glu Ala Pro Asp Pro Ser Thr Leu Glu Thr Phe Leu

260 265 270

Gly Arg Val Pro Met Val Phe Asn Val Val Ile Leu Ser Pro His Gly

275 280 285

Tyr Phe Ala Gln Asp Asn Val Leu Gly Tyr Pro Asp Thr Gly Gly Gln

290 295 300

Val Val Tyr Ile Leu Asp Gln Val Arg Ala Leu Glu Ile Glu Met Leu

305 310 315 320

Gln Arg Ile Lys Gln Gln Gly Leu Asn Ile Lys Pro Arg Ile Leu Ile

325 330 335

Leu Thr Arg Leu Leu Pro Asp Ala Val Gly Thr Thr Cys Gly Glu Arg

340 345 350

Leu Glu Arg Val Tyr Asp Ser Glu Tyr Cys Asp Ile Leu Arg Val Pro

355 360 365

Phe Arg Thr Glu Lys Gly Ile Val Arg Lys Trp Ile Ser Arg Phe Glu

370 375 380

Val Trp Pro Tyr Leu Glu Thr Tyr Thr Glu Asp Ala Ala Val Glu Leu

385 390 395 400

Ser Lys Glu Leu Asn Gly Lys Pro Asp Leu Ile Ile Gly Asn Tyr Ser

405 410 415

Asp Gly Asn Leu Val Ala Ser Leu Leu Ala His Lys Leu Gly Val Thr

420 425 430

Gln Cys Thr Ile Ala His Ala Leu Glu Lys Thr Lys Tyr Pro Asp Ser

435 440 445

Asp Ile Tyr Trp Lys Lys Leu Asp Asp Lys Tyr His Phe Ser Cys Gln

450 455 460

Phe Thr Ala Asp Ile Phe Ala Met Asn His Thr Asp Phe Ile Ile Thr

465 470 475 480

Ser Thr Phe Gln Glu Ile Ala Gly Ser Lys Glu Thr Val Gly Gln Tyr

485 490 495

Glu Ser His Thr Ala Phe Thr Leu Pro Gly Leu Tyr Arg Val Val His

500 505 510

Gly Ile Asp Val Phe Asp Pro Lys Phe Asn Ile Val Ser Pro Gly Ala

515 520 525

Asp Met Ser Ile Tyr Phe Pro Tyr Thr Glu Glu Lys Arg Arg Leu Thr

530 535 540

Lys Phe His Ser Glu Ile Glu Glu Leu Leu Tyr Ser Asp Val Glu Asn

545 550 555 560

Asp Glu His Leu Cys Val Leu Lys Asp Lys Lys Lys Pro Ile Leu Phe

565 570 575

Thr Met Ala Arg Leu Asp Arg Val Lys Asn Leu Ser Gly Leu Val Glu

580 585 590

Trp Tyr Gly Lys Asn Thr Arg Leu Arg Glu Leu Val Asn Leu Val Val

595 600 605

Val Gly Gly Asp Arg Arg Lys Glu Ser Lys Asp Asn Glu Glu Lys Ala

610 615 620

Glu Met Lys Lys Met Tyr Asp Leu Ile Glu Glu Tyr Lys Leu Asn Gly

625 630 635 640

Gln Phe Arg Trp Ile Ser Ser Gln Met Asp Arg Val Arg Asn Gly Glu

645 650 655

Leu Tyr Arg Tyr Ile Cys Asp Thr Lys Gly Ala Phe Val Gln Pro Ala

660 665 670

Leu Tyr Glu Ala Phe Gly Leu Thr Val Val Glu Ala Met Thr Cys Gly

675 680 685

Leu Pro Thr Phe Ala Thr Cys Lys Gly Gly Pro Ala Glu Ile Ile Val

690 695 700

His Gly Lys Ser Gly Phe His Ile Asp Pro Tyr His Gly Asp Gln Ala

705 710 715 720

Ala Asp Leu Leu Ala Asp Phe Phe Thr Lys Cys Lys Glu Asp Pro Ser

725 730 735

His Trp Asp Glu Ile Ser Lys Gly Gly Leu Gln Arg Ile Glu Glu Lys

740 745 750

Tyr Thr Trp Gln Ile Tyr Ser Gln Arg Leu Leu Thr Leu Thr Gly Val

755 760 765

Tyr Gly Phe Trp Lys His Val Ser Asn Leu Asp Arg Leu Glu His Arg

770 775 780

Arg Tyr Leu Glu Met Phe Tyr Ala Leu Lys Tyr Arg Pro Leu Ala Gln

785 790 795 800

Ala Val Pro Leu Ala Gln Asp Asp

805

<210> 2

<211> 442

<212> PRT

<213> tomato

<400> 2

Met Ala Thr Asn Leu Arg Val Leu Met Phe Pro Trp Leu Ala Tyr Gly

1 5 10 15

His Ile Ser Pro Phe Leu Asn Ile Ala Lys Gln Leu Ala Asp Arg Gly

20 25 30

Phe Leu Ile Tyr Leu Cys Ser Thr Arg Ile Asn Leu Glu Ser Ile Ile

35 40 45

Lys Lys Ile Pro Glu Lys Tyr Ala Asp Ser Ile His Leu Ile Glu Leu

50 55 60

Gln Leu Pro Glu Leu Pro Glu Leu Pro Pro His Tyr His Thr Thr Asn

65 70 75 80

Gly Leu Pro Pro His Leu Asn Pro Thr Leu His Lys Ala Leu Lys Met

85 90 95

Ser Lys Pro Asn Phe Ser Arg Ile Leu Gln Asn Leu Lys Pro Asp Leu

100 105 110

Leu Ile Tyr Asp Val Leu Gln Pro Trp Ala Glu His Val Ala Asn Glu

115 120 125

Gln Gly Ile Pro Ala Gly Lys Leu Leu Val Ser Cys Ala Ala Val Phe

130 135 140

Ser Tyr Phe Phe Ser Phe Arg Lys Asn Pro Gly Val Glu Phe Pro Phe

145 150 155 160

Pro Ala Ile His Leu Pro Glu Val Glu Lys Val Lys Ile Arg Glu Ile

165 170 175

Leu Ala Lys Glu Pro Glu Glu Gly Gly Arg Leu Asp Glu Gly Asn Lys

180 185 190

Gln Met Met Leu Met Cys Thr Ser Arg Thr Ile Glu Ala Lys Tyr Ile

195 200 205

Asp Tyr Cys Thr Glu Leu Cys Asn Trp Lys Val Val Pro Val Gly Pro

210 215 220

Pro Phe Gln Asp Leu Ile Thr Asn Asp Ala Asp Asn Lys Glu Leu Ile

225 230 235 240

Asp Trp Leu Gly Thr Lys Pro Glu Asn Ser Thr Val Phe Val Ser Phe

245 250 255

Gly Ser Glu Tyr Phe Leu Ser Lys Glu Asp Met Glu Glu Ile Ala Phe

260 265 270

Ala Leu Glu Ala Ser Asn Val Asn Phe Ile Trp Val Val Arg Phe Pro

275 280 285

Lys Gly Glu Glu Arg Asn Leu Glu Asp Ala Leu Pro Glu Gly Phe Leu

290 295 300

Glu Arg Ile Gly Glu Arg Gly Arg Val Leu Asp Lys Phe Ala Pro Gln

305 310 315 320

Pro Arg Ile Leu Asn His Pro Ser Thr Gly Gly Phe Ile Ser His Cys

325 330 335

Gly Trp Asn Ser Val Met Glu Ser Ile Asp Phe Gly Val Pro Ile Ile

340 345 350

Ala Met Pro Ile His Asn Asp Gln Pro Ile Asn Ala Lys Leu Met Val

355 360 365

Glu Leu Gly Val Ala Val Glu Ile Val Arg Asp Asp Asp Gly Lys Ile

370 375 380

His Arg Gly Glu Ile Ala Glu Ala Leu Lys Ser Val Val Thr Gly Glu

385 390 395 400

Thr Gly Glu Ile Leu Arg Ala Lys Val Arg Glu Ile Ser Lys Asn Leu

405 410 415

Lys Ser Ile Arg Asp Glu Glu Met Asp Ala Val Ala Glu Glu Leu Ile

420 425 430

Gln Leu Cys Arg Asn Ser Asn Lys Ser Lys

435 440

<210> 3

<211> 458

<212> PRT

<213> stevia rebaudiana

<400> 3

Met Glu Asn Lys Thr Glu Thr Thr Val Arg Arg Arg Arg Arg Ile Ile

1 5 10 15

Leu Phe Pro Val Pro Phe Gln Gly His Ile Asn Pro Ile Leu Gln Leu

20 25 30

Ala Asn Val Leu Tyr Ser Lys Gly Phe Ala Ile Thr Ile Leu His Thr

35 40 45

Asn Phe Asn Lys Pro Lys Thr Ser Asn Tyr Pro His Phe Thr Phe Arg

50 55 60

Phe Ile Leu Asp Asn Asp Pro Gln Asp Glu Arg Ile Ser Asn Leu Pro

65 70 75 80

Thr His Gly Pro Leu Ala Gly Met Arg Ile Pro Ile Ile Asn Glu His

85 90 95

Gly Ala Asp Glu Leu Arg Arg Glu Leu Glu Leu Leu Met Leu Ala Ser

100 105 110

Glu Glu Asp Glu Glu Val Ser Cys Leu Ile Thr Asp Ala Leu Trp Tyr

115 120 125

Phe Ala Gln Asp Val Ala Asp Ser Leu Asn Leu Arg Arg Leu Val Leu

130 135 140

Met Thr Ser Ser Leu Phe Asn Phe His Ala His Val Ser Leu Pro Gln

145 150 155 160

Phe Asp Glu Leu Gly Tyr Leu Asp Pro Asp Asp Lys Thr Arg Leu Glu

165 170 175

Glu Gln Ala Ser Gly Phe Pro Met Leu Lys Val Lys Asp Ile Lys Ser

180 185 190

Ala Tyr Ser Asn Trp Gln Ile Gly Lys Glu Ile Leu Gly Lys Met Ile

195 200 205

Lys Gln Thr Lys Ala Ser Ser Gly Val Ile Trp Asn Ser Phe Lys Glu

210 215 220

Leu Glu Glu Ser Glu Leu Glu Thr Val Ile Arg Glu Ile Pro Ala Pro

225 230 235 240

Ser Phe Leu Ile Pro Leu Pro Lys His Leu Thr Ala Ser Ser Ser Ser

245 250 255

Leu Leu Asp His Asp Arg Thr Val Phe Glu Trp Leu Asp Gln Gln Ala

260 265 270

Pro Ser Ser Val Leu Tyr Val Ser Phe Gly Ser Thr Ser Glu Val Asp

275 280 285

Glu Lys Asp Phe Leu Glu Ile Ala Arg Gly Leu Val Asp Ser Gly Gln

290 295 300

Ser Phe Leu Trp Val Val Arg Pro Gly Phe Val Lys Gly Ser Thr Trp

305 310 315 320

Val Glu Pro Leu Pro Asp Gly Phe Leu Gly Glu Arg Gly Lys Ile Val

325 330 335

Lys Trp Val Pro Gln Gln Glu Val Leu Ala His Pro Ala Ile Gly Ala

340 345 350

Phe Trp Thr His Ser Gly Trp Asn Ser Thr Leu Glu Ser Val Cys Glu

355 360 365

Gly Val Pro Met Ile Phe Ser Ser Phe Gly Gly Asp Gln Pro Leu Asn

370 375 380

Ala Arg Tyr Met Ser Asp Val Leu Arg Val Gly Val Tyr Leu Glu Asn

385 390 395 400

Gly Trp Glu Arg Gly Glu Val Val Asn Ala Ile Arg Arg Val Met Val

405 410 415

Asp Glu Glu Gly Glu Tyr Ile Arg Gln Asn Ala Arg Val Leu Lys Gln

420 425 430

Lys Ala Asp Val Ser Leu Met Lys Gly Gly Ser Ser Tyr Glu Ser Leu

435 440 445

Glu Ser Leu Val Ser Tyr Ile Ser Ser Leu

450 455