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Title:
HIGH-PURITY STEVIOL GLYCOSIDES
Document Type and Number:
WIPO Patent Application WO/2020/112957
Kind Code:
A1
Abstract:
Methods of preparing highly purified steviol glycosides, particularly steviolmonoside, steviolmonoside A, steviolbioside, steviolbioside D, rubusoside, steviolbioside A, steviolbioside B, rebaudioside B, stevioside, rebaudioside G, stevioside A, stevioside B, stevioside C, rebaudioside A, rebaudioside E, rebaudioside E2, rebaudioside E4, rebaudioside E6, rebaudioside E3, rebaudioside D, rebaudioside 1, rebaudioside AM, rebaudioside D7, rebaudioside M, 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 2a and/or SvG7 are described. The methods include utilizing enzyme preparations and recombinant microorganisms for converting various staring compositions to target steviol glycosides. The highly purified steviol glycosides are useful as non-caloric sweetener, flavor enhancer, sweetness enhancer, and foaming suppressor in edible and chewable compositions such as any beverages, confectioneries, bakery products, cookies, and chewing gums.

Inventors:
MARKOSYAN AVETIK (AM)
CHOW SIEW YIN (MY)
NIZAM BIN NAWI KHAIRUL (MY)
CHKHAN KRISTINA (RU)
AFZAAL BIN HASIM MOHAMAD (MY)
RAMANDACH SARAVANAN (MY)
Application Number:
PCT/US2019/063543
Publication Date:
June 04, 2020
Filing Date:
November 27, 2019
Export Citation:
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Assignee:
PURECIRCLE USA INC (US)
International Classes:
C07H1/00; C07H15/256; C12P19/56
Domestic Patent References:
WO2018213290A12018-11-22
WO2019178471A12019-09-19
Foreign References:
CN105838759A2016-08-10
US20170303566A12017-10-26
Attorney, Agent or Firm:
BABCOCK, Audrey J. et al. (US)
Download PDF:
Claims:
CLAIMS

We claim:

1. Steviol glycosides I-XVI with the following formulae:

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

No. R1 R2

No. R1 R2

No, R1 R2

2. A method for producing at least one steviol glycoside of claim 1 , comprising the steps of: a. providing a starting composition comprising an organic compound with at least one carbon atom;

b. providing an enzyme preparation or microorganism containing at least one enzyme selected from steviol biosynthesis enzymes, UDP-giucosyltransferases and optionally UDP-glucose recycling enzymes; c. contacting the enzyme preparation or microorganism with a medium containing the starting composition to produce a medium comprising at least one steviol glycoside of claim 1.

3. A method for producing at least one steviol glycoside of claim 1, comprising the steps of: a. providing a starting composition comprising an organic compound with at least one carbon atom;

b. providing a biocatalyst comprising at least one enzyme selected from steviol biosynthesis enzymes, UDP-glucosyltransferases and optionally UDP-glucose recycling enzymes;

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 step of: d. separating at least one steviol glycoside of claim 1 from the medium to provide a highly purified composition of at least one steviol glycoside of claim 1.

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

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

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

8. The method of claim 2, wherein the enzyme is selected from the group consisting of a mevalonate (MV A) pathway enzyme, a 2-C-methyl-D-erythritol-4-phosphate pathway (MEP/DOXP) enzyme, geranylgeranyl diphosphate synthase, copalyl diphosphate synthase, kaurene synthase, kaurene oxidase, kaurenoic acid 13-hydroxylase (KAH), steviol synthetase, deoxyxylulose 5 -phosphate synthase (DXS), D-l-deoxyxylulose 5- phosphate reductoisomerase (DXR), 4-diphosphocytidyl-2-C-methyl-D-erythritol synthase (CMS), 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase (CMK), 4-diphosphocytidyl- 2-C-methyl-D-erythritOl 2,4- cyclodiphosphate 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, EUGT1 1 , UGTS12, UGT76G 1, UGlyT91Cl or mutant variant 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, >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 content of at least one steviol glycoside in the highly purified composition of at least one steviol glycoside of claim 1 is greater than about 95% by weight on a dry basis.

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

1 1. The consumable product of claim 10, wherein the product is selected from the group consisting of beverages; natural juices; refreshing drinks; carbonated soft drinks; diet drinks; zero calorie drinks; reduced calorie drinks and foods; yogurt drinks; instant juices; instant coffee; powdered types of instant beverages; canned products; syrups; fermented soybean paste; soy sauce; vinegar; dressings; mayonnaise; ketchups; curry; soup; instant bouillon; powdered soy sauce; powdered vinegar; types of biscuits; rice biscuit; crackers; bread; chocolates; caramel; candy; chewing gum; jelly; pudding; preserved fruits and vegetables; fresh cream; jam; marmalade; flower paste; powdered milk; ice cream; sorbet; vegetables and fruits packed in bottles; canned and boiled beans; meat and foods boiled in sweetened sauce; agricultural vegetable food products; seafood; ham; sausage; fish ham; fish sausage; fish paste; deep fried fish products; dried seafood products; frozen food products; preserved seaweed; preserved meat; tobacco and medicinal products.

12. The consumable product of claim 10, further comprising at least one additive selected from the group consisting of carbohydrates, polyols, amino acids and then- corresponding salts, poly-amino 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, flavorants and flavoring ingredients, astringent compounds, proteins 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, hydration agents, probiotics, prebiotics, weight management agents, osteoporosis management agents, phytoestrogens, long chain primary aliphatic saturated alcohols, phytosterols and combinations thereof.

14. The consumable product of claim 10, further comprising a compound selected from the group consisting of steviolmonoside, steviolmonoside A, steviolbioside, steviolbioside A, steviolbioside B , steviolbioside C, steviolbioside D, steviolbioside E, rubusoside, dulcoside A, dulcoside B, dulcoside C, dulcoside D, stevioside, stevioside A, stevioside B, stevioside C, stevioside D, stevioside E, stevioside E2, stevioside F, stevioside G, stevioside H, rebaudioside A, rebaudioside A2, rebaudioside A3, rebaudioside A4, rebaudioside B, rebaudioside B2 , rebaudioside C, rebaudioside C2, rebaudioside C3, rebaudioside C4, rebaudioside C5, rebaudioside C6, rebaudioside D, rebaudioside D2, rebaudioside D3, rebaudioside D4, rebaudioside D5, rebaudioside D6, rebaudioside D7, rebaudioside D8, rebaudioside E, rebaudioside E2, rebaudioside E3, rebaudioside E4, rebaudioside E5, rebaudioside E6, rebaudioside E7, rebaudioside F, rebaudioside F2, rebaudioside F3, rebaudioside G, rebaudioside H, rebaudioside H2 , rebaudioside H3, rebaudioside H4 , rebaudioside H5, rebaudioside H6, rebaudioside /, rebaudioside 12, rebaudioside 13, rebaudioside J, rebaudioside K, rebaudioside K2, rebaudioside KA, rebaudioside L, rebaudioside M, rebaudioside M2, rebaudioside M3, rebaudioside N, rebaudioside N2, rebaudioside N3, rebaudioside N4, rebaudioside N5, rebaudioside O, rebaudioside 02, rebaudioside 03, rebaudioside 04, rebaudioside Q, rebaudioside Q2, rebaudioside Q3, rebaudioside R, rebaudioside S, rebaudioside T, rebaudioside Tl, rebaudioside U, rebaudioside U2, rebaudioside V, rebaudioside V2, rebaudioside V3, rebaudioside W, rebaudioside W2, rebaudioside W3, rebaudioside Y, rebaudioside Zl, rebaudioside Z2, rebaudioside AM, SvG7, NSF-02, Mogroside V, siratose, Luo Han Guo, allulose, D-allose, D-tagatose, erythritol, brazzein, neohesperidin dihydrochalcone, glycyrrhizic acid and its salts, thaumatin, perillartine, pernanduicin, mukuroziosides, baiyunoside, phlomisoside-/, dimethyl-hexahydrofluorene-dicarboxylic acid, abrusosides, periandrin, carnosiflosides, cyclocarioside, pterocaryosides, poiypodoside A, brazilin, hernandulcin, phillodulcin, glycyphyllin, phlorizin, trilobatin, dihydroflavonol, dihydroquercetin-3-acetate, neoastilibin, /rarw-cinnamaldehyde, monatin and its salts, selligueain A, hematoxylin, monellin, osladin, pterocaryoside A, pterocaryoside B, mabinlin, pentadin, lniraculin, curculin, neoculin, chlorogenic acid, cynarin, siamenoside, sucralose, potassium acesulfame, aspartame, alitame, saccharin, cyclamate, neotame, dulcin, suosan advantame, gymnemic acid, hodulcin, ziziphin, lactisole, glutamate, aspartic acid, glycine, alanine, threonine, proline, serine, lysine, tryptophan, 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 hydrolyzates, polyglycitols, sugar alcohols, L-sugars, L-sorbose, L-arabinose, trehalose, galactose, rhamnose, various cyclodextrins, cyclic oligosaccharides, various types of maltodextrins, dextran, sucrose, glucose, ribulose, fructose, threose, xylose, lyxose, altrose, mannose, idose, lactose, maltose, invert sugar, isotrehalose, neotrehalose, isomaltulose, erythrose, deoxyribose, gulose, talose, erythrulose, xylulose, cellobiose, amylopectin, glucosamine, mannosamine, glucuronic acid, gluconic acid, glucono-lactone, abequose, galactosamine, beet oligosaccharides, isomalto-oligosaccharides (isomaltose, isomaltotriose, panose and the like), xylo-oligosaccharides (xylotriose, xylobiose and the like), xylo-terminated oligosaccharides, gentio-oligosaccharides (gentiobiose, gentiotriose, gentiotetraose and the like), nigero-oligosaccharides, palatinose oligosaccharides, fructooligosaccharides (kestose, nystose and the like), maltotetraol, maltotriol, malto-oligosaccharides (maltotriose, maltotetraose, maltopentaose, maltohexaose, maltoheptaose and the like), starch, inulin, inulo-oligosaccharides, lactulose, melibiose, raffinose, isomerized liquid sugars such as high fructose corn syrups, coupling sugars, soybean oligosaccharides, D- psicose, D-ribose, L-glucose, L-fucose, D-turanose, D-leucrose.

15. A method for enhancing the sweetness of a beverage or food product, 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 at least one steviol glycoside of claim 1 is present in a concentration at or below the sweetness recognition threshold.

16. A method for modifying the flavor of a beverage or food product, comprising

a. providing a beverage or food product, and

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

17. A method for suppressing foaming of a beverage or food product, comprising

a. providing a beverage or a food product, and

b. adding a foam suppressor comprising at least one steviol glycoside of claim 1.

Description:
HIGH-PURITY STEVIOL GLYCOSIDES

SEQUENCE LISTING

The text file entitled“39227_80PROV_Sequence_Listing_ST25.txt,” created on November 27, 2018, having 15 kilobytes of data, and filed concurrently herewith, is hereby incorporated by reference in its entirety in this application.

TECHNICAL FIELD

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

BACKGROUND OF THE INVENTION High intensity sweeteners possess a sweetness level that is many times greater than the sweetness level of sucrose. They are essentially non-caloric and are commonly used in diet and reduced-calorie products, including foods and beverages. High intensity sweeteners do not elicit a glycemic response, making them suitable for use in products targeted to diabetics and others interested in controlling for their intake of carbohydrates. Steviol glycosides are a class of compounds found in the leaves of Stevia rebaudiana Bertoni, a perennial shrub of the Asteraceae ( Compositae ) family native to certain regions of South America. They are characterized structurally by a single base, steviol, differing by the presence of carbohydrate residues at positions C13 and Cl 9, They accumulate in Stevia leaves, composing approximately 10% - 20% of the total dry weight. On a dry weight basis, the four major glycosides found in the leaves of Stevia typically include stevioside (9.1 %), rebaudioside A (3.8%), rebaudioside C (0.6- 1 .0%) and dulcoside H (0.3%). Other known steviol glycosides include rebaudioside B, C, D, E, F and M, steviolbioside and rubusoside.

Although methods are known for preparing steviol glycosides from Stevia rebaudiana , many of these methods are unsuitable for use commercially.

Accordingly, there remains a need for simple, efficient, and economical methods for preparing compositions comprising steviol glycosides, including highly purified steviol glycoside compositions. SUMMARY OF THE INVENTION

The following applications are hereby incorporated by reference in their entireties in this application: International Application No. PCT/US2018/026920, filed April 10, 2018; U.S. Provisional Application No. 62/644,065, filed March 16, 2018; and U.S. Provisional Application No. 62/644,407, filed March 17, 2018.

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

As used herein,“biocatalysis” or“biocatalytic” refers to the use of natural or genetically engineered biocatalysts, such as enzymes, or cells comprising one or more enzyme, capable of single or multiple step chemical transformations on organic compounds. Biocatalysis processes include fermentation, biosynthesis, bioconversion and biotransformation processes. Both isolated enzyme, and whole-cell biocatalysis methods are known in the art. Biocatalyst protein enzymes can be naturally occurring or recombinant proteins.

As used herein, the term“steviol glycoside(s)” refers to a glycoside of steviol, including, but not limited to, naturally occurring steviol glycosides, e.g. steviolmonoside, steviolmonoside A, steviolbioside, steviolbioside D, rubusoside, steviolbioside A, steviolbioside B, rebaudioside B, stevioside, rebaudioside G, stevioside A, stevioside B, stevioside C, rebaudioside A, rebaudioside E, rebaudioside E2, rebaudioside FA, rebaudioside E6, rebaudioside E3, rebaudioside D, rebaudioside I, rebaudioside AM, rebaudioside D7, rebaudioside M, rebaudioside M4, rebaudioside la, rebaudioside lb, rebaudioside 7c, rebaudioside Id, rebaudioside le, rebaudioside If, rebaudioside lg, rebaudioside lh, rebaudioside li, rebaudioside lj, rebaudioside lk, rebaudioside 11, rebaudioside hn, rebaudioside In, rebaudioside 2a, synthetic steviol glycosides, e.g. enzymatically glucosylated steviol glycosides and combinations thereof.

As used herein, the term “SvG7” refers to any naturally occurring steviol glycosides or any synthetic steviol glycosides, including enzymatically glucosylated steviol glycosides and combinations thereof, specifically a molecule comprising steviol having seven glusose residues attached covalently including, but not limited to reb la, reb lb, reb 7c, reb Id, reb le, reb If reb lg, reb lh, reb li, reb lj, reb lk, reb 11, reb 1m, reb In, and/ or reb 2a. SvG7 can refer to a single steviol glycoside having seven glucose residues attached covalently or a mixture of steviol glycosides having seven glucose residues attached covalently.

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 enzyme preparation, thereby producing a composition comprising a target steviol glycoside.

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

The target steviol glycoside can be any steviol glycoside. In one embodiment, the target steviol glycoside is steviolmonoside, steviolmonoside A, steviolbioside, steviolbioside D, rubusoside, steviolbioside A, steviolbioside B, rebaudioside B, stevioside, rebaudioside G, stevioside A, stevioside B, stevioside C, rebaudioside A, rebaudioside E, rebaudioside E2, rebaudioside E4, rebaudioside E6, rebaudioside E3, rebaudioside D, rebaudioside /, rebaudioside AM, rebaudioside D7, rebaudioside M, rebaudioside M4, rebaudioside la, rebaudioside lb, rebaudioside lc, rebaudioside Id, rebaudioside le, rebaudioside If, rebaudioside Ig, rebaudioside lh, rebaudioside li, rebaudioside Ij, rebaudioside lk, rebaudioside //, rebaudioside lm, rebaudioside In, rebaudioside 2a,

SvG7 or a synthetic steviol glycoside.

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

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

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

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

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

In one embodiment, the target steviol glycoside is rebaudioside If

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

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

In one embodiment, the target steviol glycoside is rebaudioside li. In one embodiment, the target steviol glycoside is rebaudioside lj.

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

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

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

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

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

In one embodiment, the target steviol glycoside is SvG7.

In some preferred embodiments enzyme preparation comprising one or more enzymes, or a microbial cell comprising one or more enzymes, capable of converting the starting composition to target steviol glycosides are used. The enzyme can be located on the surface and/or inside the cell. The enzyme preparation can be provided in the form of a whole cell suspension, a crude lysate or as purified enzyme(s). The enzyme preparation can be in free form or immobilized to a solid support made from inorganic or organic materials.

In some embodiments, a microbial cell comprises the necessary enzymes and genes encoding thereof for converting the starting composition to target steviol glycosides. Accordingly, the present invention also 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 comprising at least one enzyme capable of converting the starting composition to target steviol glycosides, thereby producing a medium comprising at least one target steviol glycoside.

The enzymes necessary for converting the starting composition to target steviol glycosides include the steviol biosynthesis enzymes, NDP-glucosyltransferases (NGTs), ADP-glucosyltransferases (AGTs), CDP-glucosyltransferases (CGTs), GDP- glucosyltransferases (GGTs), TDP-glucosyltransferases (TDPs), UDP- glucosyltransferases (UGTs) 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 biosynthesis enzymes include mevalonate (MVA) pathway enzymes.

In another embodiment, the steviol biosynthesis enzymes include non-mevalonate 2-C-methyl-D-erythritol-4-phosphate pathway (MEP/DOXP) enzymes.

In one embodiment the steviol biosynthesis enzymes are selected from the group including geranylgeranyl diphosphate synthase, copalyl diphosphate synthase, kaurene synthase, kaurene oxidase, kaurenoic acid 13-hydroxylase (KAH), steviol synthetase, deoxyxylulose 5 -phosphate synthase (DXS), D-l -deoxyxylulose 5-phosphate reductoisomerase (DXR), 4-diphosphocytidyl-2-C-methyl-D-erythritol synthase (CMS), 4- diphosphocytidyl-2-C-methyl-D-erythritol kinase (CMK), 4-diphosphocytidyl-2-C- methyl-D-erythritol 2,4- cyclodiphosphate 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 etc.

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

As used hereinafter, the term“SuSy_AT”, unless specified otherwise, refers to sucrose synthase having amino-acid sequence“SEQ ID 1” as described in Example 1 , or a polypetide having substantial (>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 that code for those polypetides.

As used hereinafter, the term“UGTS12”, unless specified otherwise, refers to UDP-glucosyltransferase having amino-acid sequence “SEQ ID 2” as described in Example 1 or a polypetide having substantial (>85%, >86%, >87%, >88%, >89%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99%) amino-acid sequence identity to the SEQ ID 2 polypeptide as well as isolated nucleic acid molecules that code for those polypetides.

As used hereinafter, the term“UGT76G1”, unless specified otherwise, refers to UDP-glucosyltransferase having amino-acid sequence “SEQ ID 3” as described in Example 1 or a polypetide having substantial (>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 that code for those polypetides,

In one embodiment, steviol biosynthesis enzymes and UDP-glucosyltransferases are produced in a microbial cell. The microbial cell may be, for example, E. coli, Saccharomyces sp., Aspergillus sp., Pichia sp., Bacillus sp., Yarrowia sp. etc. In another embodiment, the UDP-glucosyltransferases are synthesized.

In one embodiment, the UDP-glucosyltransferase is selected from group including UGT74G1, UGT85C2, UGT76G1, UGT91D2, UGTS12, EUGT1 1 and UGTs having substantial (>85%, >86%, >87%, >88%, >89%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99%) amino-acid sequence identity to these polypeptides as well as isolated nucleic acid molecules that code for these UGTs.

In one embodiment, steviol biosynthesis enzymes, UGTs, and UDP-glucose recycling system are present in one microorganism (microbial cell). The microorganism may be for example, E, coli, Saccharomyces sp., Aspergillus sp., Pichia sp., Bacillus sp., Yarrowia sp.

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 an -OH functional group at Cl 3 to give a target steviol glycoside having an -O- glucose beta glucopyranoside glycosidic linkage at C l 3. In a particular embodiment, the UDP-glucosyltransferase is UGT85C2, or a UGT having >85% amino-acid sequence identity with UGT85C2.

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 a target steviol glycoside having a -COO-glucose beta-glucopyranoside glycosidic linkage at C l 9. In a particular embodiment, the UDP-glucosyltransferase is UGT74G 1 , or a 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 to give a target steviol glycoside with at least one additional glucose bearing at least one beta l ->2 glucopyranoside glycosidic linkage(s) at the newly formed glycosidic bond(s). In a particular embodiment, the UDP- glucosyltransferase is UGTS12, or a UGT having >85% amino-acid sequence identity with UGTS12. In another particular embodiment, the UDP-glucosyltransferase is EUGT1 1, or a UGT having >85% amino-acid sequence identity with EUGTl l . In yet another particular embodiment, the UDP-glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91D2.

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 to give a target steviol glycoside with at least one additional glucose bearing at least one beta 1~>3 glucopyranoside glycosidic linkage(s) at the newly formed bond glycosidic bond(s). In a particular embodiment, the UDP-glucosyltransferase is UGT76G1, or a 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 C19 side of any starting steviol glycoside to give a target steviol glycoside with at least one additional glucose bearing at least one beta 1— >4 glucopyranoside glycosidic linkage(s) at the newly formed glycosidic bond(s). In a particular embodiment, the UDP- glucosyltransferase is UGTS12, or a UGT having >85% amino-acid sequence identity with UGTS12. In another particular embodiment, the UDP-glucosyltransferase is EUGTl l, or a UGT having >85% amino-acid sequence identity with EUGTl l . In yet another particular embodiment, the UDP-glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91D2. In another particular embodiment, the UDP-glucosyltransferase is UGT76G1, or a UGT having >85% amino-acid sequence identity with UGT76G1. In another embodiment, the UDP-glucosyltransferase is any UDP- glucosyltransferase capable of adding at least one glucose unit to any existing glucose on the Cl 9 side of any starting steviol glycoside to give a target steviol glycoside with at least one additional glucose bearing at least one beta 1— >6 glucopyranoside glycosidic linkage(s) at the newly formed glycosidic bond(s). In a particular embodiment, the UDP- glucosyltransferase is UGTS12, or a UGT having >85% amino-acid sequence identity with UGTS12. In another particular embodiment, the UDP-glucosyltransferase is EUGT1 1, or a UGT having >85% amino-acid sequence identity with EUGT1 1. In yet another particular embodiment, the UDP-glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91D2.

In another embodiment, the UDP-glucosyltransferase is any UDP- glucosyltransferase capable of adding at least one glucose unit to any existing glucose on the Cl 3 side of any starting steviol glycoside to give a target steviol glycoside with at least one additional glucose bearing at least one beta l -»2 glucopyranoside glycosidic linkage(s) at the newly formed glycosidic bond(s). In a particular embodiment, the UDP- glucosyltransferase is UGTS12, or a UGT having >85% amino-acid sequence identity with UGTS12. In another particular embodiment, the UDP-glucosyltransferase is EUGT1 1 , or a UGT having >85% amino-acid sequence identity with EUGTl l . In yet another particular embodiment, the UDP-glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91D2.

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 a target steviol glycoside with at least one additional glucose bearing at least one beta l-»3 glucopyranoside glycosidic linkage(s) at the newly formed bond glycosidic bond(s). In a particular embodiment, the UDP-glucosyltransferase is UGT76G1, or a 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 Cl 3 side of any starting steviol glycoside to give a target steviol glycoside with at least one additional glucose bearing at least one beta 1 ®4 glucopyranoside glycosidic linkage(s) at the newly formed glycosidic bond(s). In a particular embodiment, the UDP- glucosyltransferase is UGTS12, or a UGT having >85% amino-acid sequence identity with UGTS12. In another particular embodiment, the UDP-glucosyltransferase is EUGT1 1, or a UGT having >85% amino-acid sequence identity with EUGTl l . In yet another particular embodiment, the UDP-glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91D2. In another particular embodiment, the UDP-glucosyltransferase is UGT76G 1 , or a 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 C 13 side of any starting steviol glycoside to give a target steviol glycoside with at least one additional glucose bearing at least one beta 1— >6 glucopyranoside glycosidic linkage(s) at the newly formed glycosidic bond(s). In a particular embodiment, the UDP- glucosyltransferase is UGTS12, or a UGT having >85% amino-acid sequence identity with UGTS12. In another particular embodiment, the UDP-glucosyltransferase is EUGTl l , or a UGT having >85% amino-acid sequence identity with EUGTl l . In yet another particular embodiment, the UDP-glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91D2.

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 particular embodiment, the UDP-glucosyltransferase is UGT85C2 or a UGT having >85% amino-acid sequence identity with UGT85C2 or a UGT having >85% amino-acid sequence identity with UGT85C2.

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

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 particular embodiment, the UDP-glucosyltransferase is UGTSI2 or a UGT having >85% amino-acid sequence identity with UGTS12. In another particular embodiment, the UDP-glucosyltransferase is EUGTl l , or a UGT having >85% amino- acid sequence identity with EUGT1 1. In yet another particular embodiment, the UDP- glucosyltransferase is UGT91 D2, or a UGT having >85% amino-acid sequence identity with UGT91 D2.

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 particular embodiment, the UDP-glucosyltransferase is UGT76G1 or a UGT having >85% amino-acid sequence identity with UGT76G1.

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 particular embodiment, the UDP-glucosyltransferase is UGT74G 1 or a 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 particular embodiment, the UDP-glucosyltransferase is UGT85C2 or a UGT having >85% amino-acid sequence identity with UGT85C2 or a UGT having >85% amino-acid sequence identity with UGT85C2.

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 particular embodiment, the UDP-glucosyltransferase is UGTS12 or a UGT having >85% amino-acid sequence identity with UGTS12. In another particular embodiment, the UDP-glucosyltransferase is EUGTl 1 , or a UGT having >85% amino-acid sequence identity with EUGTl 1. In yet another particular embodiment, the UDP-glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91D2.

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 particular embodiment, the UDP-glucosyltransferase is UGT76G1 or a 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 particular embodiment, the UDP-glucosyltransferase is UGT76G 1 or a UGT having >85% amino-acid sequence identity with UGT76G1.

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 particular embodiment, the UDP-glucosyltransferase is UGT74G1 or a 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 particular embodiment, the UDP-glucosyltransferase is UGTS12 or a UGT having >85% amino-acid sequence identity with UGTS12. In another particular embodiment, the UDP-glucosyltransferase is EUGT1 1 , or a UGT having >85% amino- acid sequence identity with EUGT1 1. In yet another particular embodiment, the UDP- glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91 D2.

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 particular embodiment, the UDP-glucosyltransferase is UGT74G1 or a UGT having >85% amino-acid sequence identity with UGT74G1.

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 particular embodiment, the UDP-glucosyltransferase is UGTS12 or a UGT having >85% amino-acid sequence identity with UGTS12. In another particular embodiment, the UDP-glucosyltransferase is EUGT1 1 , or a UGT having >85% amino- acid sequence identity with EUGT1 1. In yet another particular embodiment, the UDP- glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91 D2.

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 particular embodiment, the UDP-glucosyltransferase is UGT76G1 or a UGT having >85% amino-acid sequence identity with UGT76G1. 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 particular embodiment, the UDP-glucosyltransferase is UGTS12 or a UGT having >85% amino-acid sequence identity with UGTS12. In another particular embodiment, the UDP-glucosyltransferase is EUGTl l, or a UGT having >85% amino- acid sequence identity with EUGTl l . In yet another particular embodiment, the UDP- glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91D2.

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 particular embodiment, the UDP-glucosyltransferase is UGT76G1 or a 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 particular embodiment, the UDP-glucosyltransferase is UGT85C2 or a UGT having >85% amino-acid sequence identity with UGT85C2 or a UGT having >85% amino-acid sequence identity with UGT85C2.

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 particular embodiment, the UDP-glucosyltransferase is UGT76G1 or a UGT having >85% amino-acid sequence identity with UGT76G1.

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 particular embodiment, the UDP-glucosyltransferase is UGT85C2 or a UGT having >85% amino-acid sequence identity with UGT85C2 or a UGT having >85% amino-acid sequence identity with UGT85C2.

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 particular embodiment, the UDP-glucosyltransferase is UGTS12 or a UGT having >85% amino-acid sequence identity with UGTS12. In another particular embodiment, the UDP-glucosyltransferase is EUGTl l, or a UGT having >85% amino- acid sequence identity with EUGT1 1. In yet another particular embodiment, the UDP- glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91D2.

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 particular embodiment, the UDP-glucosyltransferase is UGT74G1 or a UGT having >85% amino-acid sequence identity with UGT74G1.

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 particular embodiment, the UDP-glucosyltransferase is UGT76G1 or a UGT having >85% amino-acid sequence identity with UGT76G1.

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 particular embodiment, the UDP-glucosyltransferase is UGTS12 or a UGT having >85% amino-acid sequence identity with UGTS12. In another particular embodiment, the UDP-glucosyltransferase is EUGT1 1 , or a UGT having >85% amino- acid sequence identity with EUGT1 1. In yet another particular embodiment, the UDP- glucosyltransferase is UGT91 D2, or a UGT having >85% amino-acid sequence identity with UGT91D2.

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 particular embodiment, the UDP-glucosyltransferase is UGT76G 1 or a 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 particular embodiment, the UDP-glucosyltransferase is UGTS12 or a UGT having >85% amino-acid sequence identity with UGTS12. In another particular embodiment, the UDP-glucosyltransferase is EUGT1 1, or a UGT having >85% amino- acid sequence identity with EUGT1 1. In yet another particular embodiment, the UDP- glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91 D2. 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 particular embodiment, the UDP-glucosyltransferase is UGTS12 or a UGT having >85% amino-acid sequence identity with UGTS12. In another particular embodiment, the UDP-glucosyltransferase is EUGT1 1 , or a UGT having >85% amino- acid sequence identity with EUGT1 1. In yet another particular embodiment, the UDP- glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91D2.

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 particular embodiment, the UDP-glucosyltransferase is UGT76G 1 or a UGT having >85% amino-acid sequence identity with UGT76G1.

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 particular embodiment, the UDP-glucosyltransferase is UGTS12 or a UGT having >85% amino-acid sequence identity with UGTS12. In another particular embodiment, the UDP-glucosyltransferase is EUGT1 1, or a UGT having >85% amino- acid sequence identity with EUGT1 1. In yet another particular embodiment, the UDP- glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91 D2.

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 particular embodiment, the UDP-glucosyltransferase is UGT76G1 or a UGT having >85% amino-acid sequence identity with UGT76G1.

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 particular embodiment, the UDP-glucosyltransferase is UGT76G1 or a UGT having >85% amino-acid sequence identity with UGT76G1.

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 particular embodiment, the UDP-glucosyltransferase is UGTS12 or a UGT having >85% amino-acid sequence identity with UGTS12. In another particular embodiment, the UDP-glucosyltransferase is EUGT1 1, or a UGT having >85% amino- acid sequence identity with EUGT1 1. In yet another particular embodiment, the UDP- glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91D2.

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 particular embodiment, the UDP-glucosyltransferase is UGT76G1 or a UGT having >85% amino-acid sequence identity with UGT76G1.

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 particular embodiment, the UDP-glucosyltransferase is UGTS12 or a UGT having >85% amino-acid sequence identity with UGTS12. In another particular embodiment, the UDP-glucosyltransferase is EUGT1 1, or a UGT having >85% amino- acid sequence identity with EUGT11. In yet another particular embodiment, the UDP- glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91D2.

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 particular embodiment, the UDP-glucosyltransferase is UGT85C2 or a UGT having >85% amino-acid sequence identity with UGT85C2 or a UGT having >85% amino-acid sequence identity with UGT85C2.

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 particular embodiment, the UDP-glucosyltransferase is UGTS12 or a UGT having >85% amino-acid sequence identity with UGTS12. In another particular embodiment, the UDP-glucosyltransferase is EUGT1 1, or a UGT having >85% amino- acid sequence identity with EUGT1 1. In yet another particular embodiment, the UDP- glucosyltransferase is UGT91 D2, or a UGT having >85% amino-acid sequence identity with UGT91 D2. 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 particular embodiment, the UDP-glucosyltransferase is UGT76G1 or a UGT having >85% amino-acid sequence identity with UGT76G1.

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 particular embodiment, the UDP-glucosyltransferase is UGT76G1 or a UGT having >85% amino-acid sequence identity with UGT76G1.

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 particular embodiment, the UDP-glucosyltransferase is UGT76G1 or a UGT having >85% amino-acid sequence identity with UGT76G1.

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 particular embodiment, the UDP-glucosyltransferase is UGT76G1 or a UGT having >85% amino-acid sequence identity with UGT76G1.

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 particular embodiment, the UDP-glucosyltransferase is UGTS12 or a UGT having >85% amino-acid sequence identity with UGTS12. In another particular embodiment, the UDP-glucosyltransferase is EUGT1 1 , or a UGT having >85% amino- acid sequence identity with EUGT1 1. In yet another particular embodiment, the UDP- glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91 D2.

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 particular embodiment, the UDP-glucosyltransferase is UGTS12 or a UGT having >85% amino-acid sequence identity with UGTS12. In another particular embodiment, the UDP-glucosyltransferase is EUGT1 1, or a UGT having >85% amino- acid sequence identity with EUGT1 1. In yet another particular embodiment, the UDP- glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91 D2.

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 particular embodiment, the UDP-glucosyltransferase is UGT76G1 or a UGT having >85% amino-acid sequence identity with UGT76G1.

In another embodiment, the UDP-glucosyltransferase is any UDP- glucosyltransferase capable of adding at least one glucose unit to rebaudioside E6 to form rebaudioside 1. In a particular embodiment, the UDP-glucosyltransferase is UGTS12 or a UGT having >85% amino-acid sequence identity with UGTS12. In another particular embodiment, the UDP-glucosyltransferase is EUGT1 1 , or a UGT having >85% amino- acid sequence identity with EUGT1 1. In yet another particular embodiment, the UDP- glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91 D2.

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 particular embodiment, the UDP-glucosyltransferase is UGTS12 or a UGT having >85% amino-acid sequence identity with UGTS12. In another particular embodiment, the UDP-glucosyltransferase is EUGT1 1 , or a UGT having >85% amino- acid sequence identity with EUGT1 1. In yet another particular embodiment, the UDP- glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91 D2.

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 particular embodiment, the UDP-glucosyltransferase is UGTS12 or a UGT having >85% amino-acid sequence identity with UGTS12. In another particular embodiment, the UDP-glucosyltransferase is EIJGT1 1, or a UGT having >85% amino- acid sequence identity with EUGT1 1 . In yet another particular embodiment, the UDP- glucosyltransferase is UGT91 D2, or a UGT having >85% amino-acid sequence identity with UGT91D2. 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 particular embodiment, the UDP-glucosyltransferase is UGT76G1 or a UGT having >85% amino-acid sequence identity with UGT76G1.

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 particular embodiment, the UDP-glucosyltransferase is UGT76G1 or a UGT having >85% amino-acid sequence identity with UGT76G1.

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 particular embodiment, the UDP-glucosyltransferase is UGTS12 or a UGT having >85% amino-acid sequence identity with UGTS12. In another particular embodiment, the UDP-glucosyltransferase is EUGT1 1 , or a UGT having >85% amino- acid sequence identity with EUGT1 1. In yet another particular embodiment, the UDP- glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91D2.

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 particular embodiment, the UDP-glucosyltransferase is UGT76G1 or a UGT having >85% amino-acid sequence identity with UGT76G1.

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 particular embodiment, the UDP-glucosyltransferase is UGTS12 or a UGT having >85% amino-acid sequence identity with UGTS12. In another particular embodiment, the UDP-glucosyltransferase is EIJGT1 1, or a UGT having >85% amino- acid sequence identity with EUGT1 1. In yet another particular embodiment, the UDP- glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91D2. In a particular embodiment, the UDP-glucosyltransferase is UGT76G1 or a UGT having >85% amino-acid sequence identity with UGT76G1.

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 particular embodiment, the UDP-glucosyltransferase is UGTS12 or a UGT having >85% amino-acid sequence identity with UGTS12. In another particular embodiment, the UDP-glucosyltransferase is EUGT1 1, or a UGT having >85% amino- acid sequence identity with EUGT1 1. In yet another particular embodiment, the UDP- glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91D2.

In another embodiment, the UDP-glucosyltransferase is any UDP- glucosyltransferase capable of adding at least one glucose unit to rebaudioside M to form rebaudioside la. In a particular embodiment, the UDP-glucosyltransferase is UGTS12 or a UGT having >85% amino-acid sequence identity with UGTS12. In another particular embodiment, the UDP-glucosyltransferase is EUGT1 1, or a UGT having >85% amino- acid sequence identity with EUGT1 1. In yet another particular embodiment, the UDP- glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91D2.

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

In another embodiment, the UDP-glucosyltransferase is any UDP- glucosyltransferase capable of adding at least one glucose unit to rebaudioside M to form rebaudioside lc. In a particular embodiment, the UDP-glucosyltransferase is UGTS12 or a UGT having >85% amino-acid sequence identity with UGTS12. In another particular embodiment, the UDP-glucosyltransferase is EUGT1 1 , or a UGT having >85% amino- acid sequence identity with EUGT1 1. In yet another particular embodiment, the UDP- glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91D2.

In another embodiment, the UDP-glucosyltransferase is any UDP- glucosyltransferase capable of adding at least one glucose unit to rebaudioside to form rebaudioside Id. In a particular embodiment, the UDP-glucosyltransferase is UGTS12 or a UGT having >85% amino-acid sequence identity with UGTS12. In another particular embodiment, the UDP-glucosyltransferase is EUGT1 1, or a UGT having >85% amino- acid sequence identity with EUGTl l . In yet another particular embodiment, the UDP- glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91D2.

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

In another embodiment, the UDP-glucosyltransferase is any UDP- glucosyltransferase capable of adding at least one glucose unit to rebaudioside to form rebaudioside If. In a particular embodiment, the UDP-glucosyltransferase is UGTS12 or a UGT having >85% amino-acid sequence identity with UGTS12. In another particular embodiment, the UDP-glucosyltransferase is EUGTl 1, or a UGT having >85% amino- acid sequence identity with EUGTl 1. In yet another particular embodiment, the UDP- glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91D2.

In another embodiment, the UDP-glucosyltransferase is any UDP- glucosyltransferase capable of adding at least one glucose unit to rebaudioside M to form rebaudioside lg. In a particular embodiment, the UDP-glucosyltransferase is UGTS12 or a UGT having >85% amino-acid sequence identity with UGTS12. In another particular embodiment, the UDP-glucosyltransferase is EUGTl l , or a UGT having >85% amino- acid sequence identity with EUGTl l . In yet another particular embodiment, the UDP- glucosyltransferase is UGT91 D2, or a UGT having >85% amino-acid sequence identity with UGT91 D2.

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

In another embodiment, the UDP-glucosyltransferase is any UDP- glucosyltransferase capable of adding at least one glucose unit to rebaudioside to form rebaudioside //. In a particular embodiment, the UDP-glucosyltransferase is UGTS12 or a UGT having >85% amino-acid sequence identity with UGTS12. In another particular embodiment, the UDP-glucosyltransferase is EUGT1 1 , or a UGT having >85% amino- acid sequence identity with EUGT1 1. In yet another particular embodiment, the UDP- glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91D2.

In another embodiment, the UDP-glucosyltransferase is any UDP- glucosyltransferase capable of adding at least one glucose unit to rebaudioside M to form rebaudioside lj. In a particular embodiment, the UDP-glucosyltransferase is UGTS12 or a UGT having >85% amino-acid sequence identity with UGTS12. In another particular embodiment, the UDP-glucosyltransferase is EUGT1 1, or a UGT having >85% amino- acid sequence identity with EUGT1 1. In yet another particular embodiment, the UDP- glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91D2.

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

In another embodiment, the UDP-glucosyltransferase is any UDP- glucosyltransferase capable of adding at least one glucose unit to rebaudioside to form rebaudioside 11. In a particular embodiment, the UDP-glucosyltransferase is UGTS12 or a UGT having >85% amino-acid sequence identity with UGTS12. In another particular embodiment, the UDP-glucosyltransferase is EUGT1 1, or a UGT having >85% amino- acid sequence identity with EUGT1 1. In yet another particular embodiment, the UDP- glucosyltransferase is UGT91 D2, or a UGT having >85% amino-acid sequence identity with UGT91 D2.

In another embodiment, the UDP-glucosyltransferase is any UDP- glucosyltransferase capable of adding at least one glucose unit to rebaudioside to form rebaudioside lin. In a particular embodiment, the UDP-glucosyltransferase is UGTS12 or a UGT having >85% amino-acid sequence identity with UGTS12. In another particular embodiment, the UDP-glucosyltransferase is EUGT1 1, or a UGT having >85% amino- acid sequence identity with EUGT1 1. In yet another particular embodiment, the UDP- glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91D2.

In another embodiment, the UDP-glucosyltransferase is any UDP- glucosyltransferase capable of adding at least one glucose unit to rebaudioside M to form rebaudioside In. In a particular embodiment, the UDP-glucosyltransferase is UGTS12 or a UGT having >85% amino-acid sequence identity with UGTS12. In another particular embodiment, the UDP-glucosyltransferase is EUGT1 1, or a UGT having >85% amino- acid sequence identity with EUGT1 1. In yet another particular embodiment, the UDP- glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91D2.

In another embodiment, the UDP-glucosyltransferase is any UDP- glucosyltransferase capable of adding at least one glucose unit to rebaudioside M4 to form rebaudioside 2a. In a particular embodiment, the UDP-glucosyltransferase is UGTS12 or a UGT having >85% amino-acid sequence identity with UGTS12. In another particular embodiment, the UDP-glucosyltransferase is EUGT1 1 , or a UGT having >85% amino- acid sequence identity with EUGT1 1. In yet another particular embodiment, the UDP- glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91D2. In a particular embodiment, the UDP-glucosyltransferase is UGT76G1 or a UGT having >85% amino-acid sequence identity with UGT76G1.

Optionally, the method of the present invention further comprises recycling UDP to provide UDP-glucose. In one embodiment, the method comprises recycling UDP by providing a recycling catalyst and a recycling substrate, such that the biotransformation of steviol and/or the steviol glycoside substrate to the target steviol glycoside is carried out using catalytic amounts of UDP-glucosyltransferase and UDP-glucose

In one embodiment, the recycling catalyst is sucrose synthase SuSy_At or a sucrose synthase having >85% amino-acid sequence identity with SuSy_At.

In one embodiment, the recycling substrate for UDP-glucose recycling catalyst is sucrose.

Optionally, the method of the present invention further comprises the use of transglycosidases that use oligo- or poly-saccharides as the sugar donor to modify recipient target steviol glycoside molecules. Non-limiting examples include cyclodextrin glycosyltransferase (CGTase), fructofuranosidase, amylase, saccharase, glucosucrase, beta-h-fructosidase, beta-fructosidase, sucrase, fructosyl invertase, alkaline invertase, acid invertase, fructofuranosidase. Gh some embodiments, glucose and sugar(s) other than glucose, including but not limited to fructose, xylose, rhamnose, arabinose, deoxyglucose, galactose are transferred to the recipient target steviol glycosides. In one embodiment, the recipient steviol glycoside is rebaudioside la, rebaudioside lb, rebaudioside lc, rebaudioside hi rebaudioside le, rebaudioside If, rebaudioside Ig, rebaudioside lh, rebaudioside li, rebaudioside lj, rebaudioside Ik, rebaudioside 11, rebaudioside lrn, and/or rebaudioside In. In another embodiment, the recipient steviol glycoside is rebaudioside 2a. In another embodiment, the recipient steviol glycoside is rebaudioside M4. In another embodiment, the recipient steviol glycoside is SvG7.

Optionally, the method of the present invention further comprises separating the target steviol glycoside from the medium to provide a highly purified target steviol glycoside composition. The target steviol glycoside can be separated by at least one suitable method, such as, for example, crystallization, separation by membranes, centrifugation, extraction, chromatographic separation or a combination of such methods.

In one embodiment, the target steviol glycoside can be produced within the microorganism. In another embodiment, the target steviol glycoside can be secreted out in the medium. In one another embodiment, the released steviol glycoside can be continuously removed from the medium. In yet another embodiment, the target steviol glycoside is separated after the completion of the conversion reaction.

In one embodiment, separation produces a composition comprising greater than about 80% by weight of the target steviol glycoside on an anhydrous basis, i.e., a highly purified steviol glycoside composition, In another embodiment, separation produces a composition comprising greater than about 90% by weight of the target steviol glycoside. In particular embodiments, the composition comprises greater than about 95% by weight of the target steviol glycoside. In other 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 hydrates, solvates, anhydrous or combinations thereof. Purified target steviol glycosides can be used in consumable products as a sweetener, flavor modifier, flavor with modifying properties and/or foaming suppressor. Suitable consumer products include, but are not limited to, food, beverages, pharmaceutical compositions, tobacco products, nutraceutical compositions, oral hygiene compositions, and cosmetic compositions.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. la thru FIG. lo show the chemical structure of some SvG7 steviol glycosides rebaudioside la, rebaudioside lb, rebaudioside lc, rebaudioside Id, rebaudioside le, rebaudioside If, rebaudioside Ig, rebaudioside lh, rebaudioside //, rebaudioside lj, rebaudioside Ik, rebaudioside 11, rebaudioside lm, rebaudioside In and rebaudioside 2a respectively.

FIG. I p shows the chemical structure of rebaudioside M4.

FIG. 2a thru FIG. 2k show the pathways of producing rebaudioside la, rebaudioside lb, rebaudioside lc, rebaudioside Id, rebaudioside le, rebaudioside If rebaudioside lg, rebaudioside lh, rebaudioside li, rebaudioside lj, rebaudioside Ik, rebaudioside 11, rebaudioside lm, rebaudioside In, rebaudioside 2a, rebaudioside M4 and various steviol glycosides from steviol and the various intermediary steviol glycosides.

FIG. 3a thru FIG. 3n show the biocatalytic production of rebaudioside la, rebaudioside lb, rebaudioside lc, rebaudioside Id, rebaudioside le, rebaudioside If rebaudioside lg, rebaudioside lh, rebaudioside li, rebaudioside lj, rebaudioside Ik, rebaudioside 11, rebaudioside lm and rebaudioside In, respectively, from rebaudioside A using the enzymes UGTS12 and UGT76G1 and concomitant recycling of UDP to UDP-glucose via sucrose synthase SuSy_At.

FIG. 3o and FIG. 3p show the biocatalytic production of rebaudioside 2a and rebaudioside M4, respectively, from stevioside using the enzymes UGTS12 and UGT76G1 and concomitant recycling of UDP to UDP-glucose via sucrose synthase SuSy_At.

FIG. 3q and FIG. 3r show the biocatalytic production of rebaudioside 2a and rebaudioside M4, respectively, from rebaudioside AM using the enzymes UGTS12 and UGT76G1 and concomitant recycling of UDP to UDP-glucose via sucrose synthase SuSy__At. FIG. 3s shows the biocatalytic production of rebaudioside 2a from rebaudioside M4 using the enzymes UGTS12 and UGT76G1 and concomitant recycling of UDP to UDP-glucose via sucrose synthase SuSy_At.

FIG. 4 shows the HPLC chromatogram of stevioside. The peak with retention time of 20.958 minutes corresponds to stevioside. The peak with retention time 20 725 minutes corresponds to rebaudioside A. The peak at 32.925 minutes corresponds to rebaudioside B. The peak at 33.930 minutes corresponds to steviolbioside.

FIG. 5 shows the HPLC chromatogram of the product of the biocatalytic production of SvG7 molecules from stevioside. The peak at 6.459 minutes corresponds to rebaudioside 2a. The peak at 9.825 minutes corresponds to rebaudioside AM. The peak at 13.845 minutes corresponds to rebaudioside M. The peak at 32.974 minutes corresponds to rebaudioside B. The peak at 33.979 minutes corresponds to steviolbioside.

FIG. 6 shows the HPLC chromatogram of rebaudioside 2a after purification by HPLC. The peak with retention time of 6.261 minutes correspond to rebaudioside 2a. FIG. 7 shows the 1H NMR spectrum of rebaudioside 2a (500 MHz, pyridine-r/5).

FIG. 8 shows the HSQC spectrum of rebaudioside 2a (500 MHz, pyridine-c/5).

FIG. 9 shows the H,H COSY spectrum of rebaudioside 2a (500 MHz, pyridine-r/5).

FIG. 10 shows the HMBC spectrum of rebaudioside 2a (500 MHz, pyridine-t/5).

FIG. 1 la shows the HSQC-TOCSY spectrum of rebaudioside 2a (500 MHz, pyridine-c/5). FIG. l i b shows the l D-NOESY spectrum of rebaudioside 2a (500 MHz, pyridine-r/5).

FIG. 12a and FIG. 12b show the LC chromatogram and mass spectrum of rebaudioside 2a respectively.

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 enzyme preparation, thereby producing a composition comprising a target steviol glycoside. One object of the invention is to provide an efficient biocatalytic method for preparing target steviol glycosides, particularly steviolmonoside, steviolmonoside A, steviolbioside, steviolbioside D, rubusoside, steviolbioside A, steviolbioside B, rebaudioside B, stevioside, rebaudioside G, stevioside A, stevioside B, stevioside C, rebaudioside A, rebaudioside E, rebaudioside E2, rebaudioside E4, rebaudioside E6, rebaudioside E3, rebaudioside D, rebaudioside /, rebaudioside AM, rebaudioside D7, rebaudioside M, rebaudioside M4, rebaudioside la, rebaudioside lb, rebaudioside lc, rebaudioside Id, rebaudioside le, rebaudioside If, rebaudioside Ig, rebaudioside Ih, rebaudioside li, rebaudioside lj, rebaudioside lk, rebaudioside 11, rebaudioside lm, rebaudioside In, rebaudioside 2a, and/or SvG7 or a synthetic steviol glycoside from various starting compositions.

Starting Composition

As used herein,“starting composition” refers to any composition (generally an aqueous solution) containing one or more organic compound comprising at least one carbon atom.

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

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

In one embodiment, the starting composition is steviol.

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

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

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

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

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

In another embodiment, the starting composition steviol glycoside is rebaudioside £. 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 that contains more than one hydroxyl group. A polyol may be a diol, triol, or a tetraol which contain 2, 3, and 4 hydroxyl groups, respectively. A polyol also may contain more than four hydroxyl groups, such as a pentaol, hexaol, heptaol, or the like, which contain 5, 6, or 7 hydroxyl groups, respectively. Additionally, a polyol also may be a sugar alcohol, polyhydric alcohol, or polyalcohol which is a reduced form of carbohydrate, wherein the carbonyl group (aldehyde or ketone, reducing sugar) has been reduced to a primary or secondary hydroxyl group. 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 hydrolyzates, polyglycitols and sugar alcohols or any other carbohydrates capable of being reduced.

The term“carbohydrate” refers to aldehyde or ketone compounds substituted with multiple hydroxyl groups, of the general formula (CH 2 0) n , wherein n is 3-30, as well as their oligomers and polymers. The carbohydrates of the present invention can, in addition, be substituted or deoxygenated at one or more positions. Carbohydrates, as used herein, encompass unmodified carbohydrates, carbohydrate derivatives, substituted carbohydrates, and modified carbohydrates. As used herein, the phrases “carbohydrate derivatives”, “substituted carbohydrate”, and “modified carbohydrates” are synonymous. Modified carbohydrate means any carbohydrate wherein at least one atom has been added, removed, or substituted, or combinations thereof. Thus, carbohydrate derivatives or substituted carbohydrates include substituted and unsubstituted monosaccharides, disaccharides, oligosaccharides, and polysaccharides. The carbohydrate derivatives or substituted carbohydrates optionally can be deoxygenated at any corresponding C-position, and/or substituted with one or more moieties such as hydrogen, halogen, haloalkyl, carboxyl, acyl, acyloxy, amino, amido, carboxyl derivatives, alkylamino, dialkylamino, arylamino, alkoxy, aryloxy, nitro, cyano, sulfo, mercapto, imino, sulfonyl, sulfenyl, sulfinyl, sulfamoyl, carboalkoxy, carboxamido, phosphonyl, phosphinyl, phosphoryl, phosphino, thioester, thioether, oximino, hydrazino, carbamyl, phospho, phosphonato, or any other viable functional group provided the carbohydrate derivative or substituted carbohydrate functions to improve the sweet taste of the sweetener composition.

Examples of carbohydrates which may be used in accordance with this invention include, but are not limited to, tagatose, trehalose, galactose, rhamnose, various cyclodextrins, cyclic oligosaccharides, various types of maltodextrins, dextran, sucrose, glucose, ribulose, fructose, threose, arabinose, xylose, lyxose, allose, altrose, mannose, idose, lactose, maltose, invert sugar, isotrehalose, neotrehalose, isomaltulose, erythrose, deoxyribose, gulose, idose, talose, erythrulose, xylulose, psicose, turanose, cellobiose, amylopectin, glucosamine, mannosamine, fucose, glucuronic acid, gluconic acid, glucono- lactone, abequose, galactosamine, beet oligosaccharides, isomalto-oligosaccharides (isomaltose, isomaltotriose, panose and the like), xylo-oligosaccharides (xylotriose, xylobiose and the like), xylo-terminated oligosaccharides, gentio-oligosaccharides (gentiobiose, gentiotriose, gentiotetraose and the like), sorbose, nigero-oligosaccharides, palatinose oligosaccharides, fructooligosaccharides (kestose, nystose and the like), maltotetraol, maltotriol, malto-oligosaccharides (maltotriose, maltotetraose, maltopentaose, maltohexaose, maltoheptaose and the like), starch, inulin, inulo- oligosaccharides, lactulose, melibiose, raffinose, ribose, isomerized liquid sugars such as high fructose com syrups, coupling sugars, and soybean oligosaccharides. Additionally, the carbohydrates as used herein may be in either the D- or L-configuration.

The starting composition may be synthetic or purified (partially or entirely), 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 still another embodiment, the starting composition is sucrose.

In yet another embodiment, the starting composition is starch.

In another embodiment, the starting composition is maltodextrin.

In yet another embodiment, the starting composition is cellulose.

In still another embodiment, the starting composition is amylose.

The organic compound(s) of starting composition serve as a substrate(s) for the production of the target steviol glycoside(s), as described herein.

Target Steviol Glycoside

The target steviol glycoside of the present method can be any steviol glycoside that 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, steviolbioside D, rubusoside, steviolbioside A, steviolbioside B, rebaudioside B, stevioside, rebaudioside G, stevioside A, stevioside B, stevioside C, rebaudioside A, rebaudioside E, rebaudioside E2, rebaudioside E4, rebaudioside E6, rebaudioside E3, rebaudioside D, rebaudioside I, rebaudioside AM, rebaudioside D7, rebaudioside M, rebaudioside M4, rebaudioside la, rebaudioside lb, rebaudioside 7c, rebaudioside Id, rebaudioside le, rebaudioside If, rebaudioside Ig, rebaudioside lh , rebaudioside li, rebaudioside lj, rebaudioside lk, rebaudioside 11, rebaudioside lm, rebaudioside In, rebaudioside 2a, SvG7 or other glycoside of steviol occurring in Stevia rebaudiana plant, synthetic steviol glycosides, e.g. enzymatically glucosylated 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 steviolbioside D.

In another embodiment, the target steviol glycoside is rubusoside.

In another embodiment, the target steviol glycoside is steviolbioside A.

In another embodiment, the target steviol glycoside is steviolbioside 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 1

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 la. In another embodiment, the target steviol glycoside is rebaudioside lb. In another embodiment, the target steviol glycoside is rebaudioside 7c. In another embodiment, the target steviol glycoside is rebaudioside Id. In another embodiment, the target steviol glycoside is rebaudioside le.

In another embodiment, the target steviol glycoside is rebaudioside If. In another embodiment, the target steviol glycoside is rebaudioside lg. In another embodiment, the target steviol glycoside is rebaudioside Ih. In another embodiment, the target steviol glycoside is rebaudioside li. In another embodiment, the target steviol glycoside is rebaudioside lj.

In another embodiment, the target steviol glycoside is rebaudioside lk. In another embodiment, the target steviol glycoside is rebaudioside 11. In another embodiment, the target steviol glycoside is rebaudioside lm. In another embodiment, the target steviol glycoside is rebaudioside In. In another embodiment, the target steviol glycoside is rebaudioside 2a.

In another embodiment, the target steviol glycoside is SvG7. The target steviol glycoside can be in any polymorphic or amorphous form, including hydrates, solvates, anhydrous or combinations 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 steviolbioside D.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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 the production of rebaudioside /.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

In one embodiment, the present invention is a biocatalytic process for the production of rebaudioside 11. In one embodiment, the present invention is a biocatalytic process for the production of rebaudioside Im.

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

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

In one embodiment, the present invention is a biocatalytic process for the production of SvG7.

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

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

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

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

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

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

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

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

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

In a particular embodiment, the present invention provides for the biocatalytic process for the production of rebaudioside 11 from a starting composition comprising rebaudioside A and UDP-glucose.

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

In a particular embodiment, the present invention provides for the biocatalytic process for the production of rebaudioside In from a starting composition comprising rebaudioside ! and UDP-glucose. In a particular embodiment, the present invention provides for the 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 for the 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 for the biocatalytic process for the production of rebaudioside 2a from a starting composition comprising rebausioside ^ 4 and UDP-glucose.

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

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

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

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

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

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

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

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

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

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

In a particular embodiment, the present invention provides for the biocatalytic process for the production of rebaudioside 11 from a starting composition comprising rebaudioside Mand UDP-glucose.

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

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

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

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

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

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

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

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

Optionally, the method of the present invention further comprises separating the target steviol glycoside from the medium to provide a highly purified target steviol glycoside composition. The target steviol glycoside can be separated by any suitable method, such as, for example, crystallization, separation by membranes, centrifugation, extraction, chromatographic separation or a combination of such methods.

In particular embodiments, the process described herein results in a highly purified target steviol glycoside composition. The term“highly purified”, as used herein, refers to a composition having greater than about 80% by weight of the target steviol glycoside on an anhydrous (dried) basis. In one embodiment, the highly purified target steviol glycoside composition contains greater than about 90% by weight of the target steviol glycoside on an anhydrous (dried) basis, such as, for example, 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% target steviol glycoside content on a dried basis.

In one embodiment, when the target steviol glycoside is rebaudioside M4, the process described herein provides a composition having greater than about 90% rebaudioside M4 content by weight on a dried basis. In another particular embodiment, when the target steviol glycoside is rebaudioside M4, the process described herein provides a composition comprising greater than about 95% content by weight on a dried basis.

In one embodiment, when the target steviol glycoside is rebaudioside 2a, the process described herein provides a composition having greater than about 90% rebaudioside 2a content by weight on a dried basis. In another particular embodiment, when the target steviol glycoside is rebaudioside 2a, the process described herein provides a composition comprising greater than about 95% content by weight on a dried basis.

In one embodiment, when the target steviol glycoside is SvG7, the process described herein provides a composition having greater than about 90% SvG7 content by weight on a dried basis. In another particular embodiment, when the target steviol glycoside is SvG7, the process described herein provides a composition comprising greater than about 95% SvG7 content by weight on a dried basis.

Microorganisms and enzyme preparations

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

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

In another embodiment, the biocatalyst is one or more microbial cells comprising enzyme(s) capable of converting the starting composition to the target steviol glycoside. The enzyme can be located on the surface of the cell, inside the cell or located both on the surface of the cell and inside the cell.

Suitable enzymes for converting the starting composition to target steviol glycosides include, but are not limited to, the steviol biosynthesis enzymes, NDP- glucosyltransferases (NGTs), ADP-glucosyltransferases (AGTs), CDP- glucosyltransferases (CGTs), GDP-glucosyltransferases (GGTs), TDP- glucosyltransferases (TDPs), UDP-glucosyltransferases (UGTs). Optionally it may include NDP-recycling enzyme(s), ADP-recycling enzyme(s), CDP-recycling enzyme(s), GDP-recycling enzyme(s), TDP-recycling enzyme(s), and/or UDP-recycling enzyme(s).

In one embodiment, the steviol biosynthesis enzymes include mevalonate (MVA) pathway enzymes.

In another embodiment, the steviol biosynthesis enzymes include non-mevalonate 2-C-methyl-D-erythritol-4-phosphate pathway (MEP/DOXP) enzymes.

In one embodiment the steviol biosynthesis enzymes are selected from the group including geranylgeranyl diphosphate synthase, copalyl diphosphate synthase, kaurene synthase, kaurene oxidase, kaurenoic acid 13-hydroxylase (KAH), steviol synthetase, deoxyxylulose 5 -phosphate synthase (DXS), D-l-deoxyxylulose 5-phosphate reductoisomerase (DXR), 4-diphosphocytidyl-2-C-methyl-D-erythritol synthase (CMS), 4- diphosphocytidyl-2-C-methyl-D-erythritol kinase (CMK), 4-diphosphocytidyl-2-C- methyl-D-erythritol 2,4- cyclodiphosphate synthase (MCS), l-hydroxy-2-methyl-2(E)- butenyl 4-diphosphate synthase (PIDS), 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 etc. The UDP-glucosyltransferase can be any UDP-glucosyltransferase capable of adding at least one glucose unit to steviol and/or a steviol glycoside substrate to provide the target steviol glycoside.

In one embodiment, steviol biosynthesis enzymes and UDP-glucosyltransferases are produced in a microbial cell. The microbial cell may be, for example, E. coli, Saccharomyces sp., Aspergillus sp., Pichia sp., Bacillus sp., Yarrowia sp. etc. In another embodiment, the UDP-glucosyltransferases are synthesized.

In one embodiment, the UDP-glucosyltransferase is selected from group including UGT74G1 , UGT85C2, UGT76G1, UGT91D2, UGTS12, EUGT1 1 and UGTs having substantial (>85%, >86%, >87%, >88%, >89%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99%) amino-acid sequence identity to these polypeptides as well as isolated nucleic acid molecules that code for these UGTs

In one embodiment, steviol biosynthesis enzymes, UGTs and UDP-glucose recycling system are present in one microorganism (microbial cell). The microorganism may be for example, E. coli, Saccharomyces sp., Aspergillus sp., Pichia sp., Bacillus sp., Yarrowia sp.

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 an -OH functional group at Cl 3 to give a target steviol glycoside having an -O- glucose beta glucopyranoside glycosidic linkage at Cl 3. In a particular embodiment, the UDP-glucosyltransferase is UGT85C2, or a UGT having >85% amino-acid sequence identity with UGT85C2.

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 C 19 to give a target steviol glycoside having a -COO-glucose beta-glucopyranoside glycosidic linkage at Cl 9. In a particular embodiment, the UDP-glucosyltransferase is UGT74G1, or a 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 C 19 side of any starting steviol glycoside to give a target steviol glycoside with at least one additional glucose bearing at least one beta 1— >2 glucopyranoside glycosidic linkage(s) at the newly formed glycosidic bond(s). In a particular embodiment, the UDP- glucosyltransferase is UGTS12, or a UGT having >85% amino-acid sequence identity with UGTS12. In another particular embodiment, the UDP-glucosyltransferase is EUGTl 1 , or a UGT having >85% amino-acid sequence identity with EUGTl 1. In yet another particular embodiment, the UDP-glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91D2.

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 to give a target steviol glycoside with at least one additional glucose bearing at least one beta 1— >3 glucopyranoside glycosidic linkage(s) at the newly formed bond glycosidic bond(s). In a particular embodiment, the UDP-glucosyltransferase is UGT76G1 , or a 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 Cl 9 side of any starting steviol glycoside to give a target steviol glycoside with at least one additional glucose bearing at least one beta 1— >4 glucopyranoside glycosidic linkage(s) at the newly formed glycosidic bond(s). In a particular embodiment, the UDP- glucosyltransferase is UGTS12, or a UGT having >85% amino-acid sequence identity with UGTS12. In another particular embodiment, the UDP-glucosyltransferase is EUGTl 1, or a UGT having >85% amino-acid sequence identity with EUGTl 1. In yet another particular embodiment, the UDP-glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91D2. In another particular embodiment, the UDP-glucosyltransferase is UGT76G1, or a UGT having >85% amino-acid sequence identity with UGT76G1 ,

In another embodiment, the UDP-glucosyltransferase is any UDP- glucosyltransferase capable of adding at least one glucose unit to any existing glucose on the C 19 side of any starting steviol glycoside to give a target steviol glycoside with at least one additional glucose bearing at least one beta 1— >6 glucopyranoside glycosidic linkage(s) at the newly formed glycosidic bond(s). In a particular embodiment, the UDP- glucosyltransferase is UGTS12, or a UGT having >85% amino-acid sequence identity with UGTSI2. In another particular embodiment, the UDP-glucosy!transferase is EUGT1 1, or a UGT having >85% amino-acid sequence identity with EUGT1 1. In yet another particular embodiment, the UDP-glucosyltransferase is UGT91 D2, or a UGT having >85% amino-acid sequence identity with UGT91 D2.

In another embodiment, the UDP-glucosyltransferase is any UDP- glucosyltransferase capable of adding at least one glucose unit to any existing glucose on the C 13 side of any starting steviol glycoside to give a target steviol glycoside with at least one additional glucose bearing at least one beta 1— >2 glucopyranoside glycosidic linkage(s) at the newly formed glycosidic bond(s). In a particular embodiment, the UDP- glucosyltransferase is UGTS12, or a UGT having >85% amino-acid sequence identity with UGTS12. In another particular embodiment, the UDP-glucosyltransferase is EUGTl 1, or a UGT having >85% amino-acid sequence identity with EUGTl 1. In yet another particular embodiment, the UDP-glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91D2.

In another embodiment, the UDP-glucosyltransferase is any UDP- glucosyltransferase capable of adding at least one glucose unit to any existing glucose on the Cl 3 side of any starting steviol glycoside to give a target steviol glycoside with at least one additional glucose bearing at least one beta l -»3 glucopyranoside glycosidic linkage(s) at the newly formed bond glycosidic bond(s). In a particular embodiment, the UDP-glucosyltransferase is UGT76G1, or a UGT having >85% amino-acid sequence identity with UGT76G1.

In another embodiment, the UDP-glucosyltransferase is any UDP- glucosyltransferase capable of adding at least one glucose unit to any existing glucose on the Cl 3 side of any starting steviol glycoside to give a target steviol glycoside with at least one additional glucose bearing at least one beta 1®4 glucopyranoside glycosidic linkage(s) at the newly formed glycosidic bond(s). In a particular embodiment, the UDP- glucosyltransferase is UGTS12, or a UGT having >85% amino-acid sequence identity with UGTS12. In another particular embodiment, the UDP-glucosyltransferase is EUGTl 1, or a UGT having >85% amino-acid sequence identity with EUGTl 1. In yet another particular embodiment, the UDP-glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91D2. In another particular embodiment, the UDP-glucosyltransferase is UGT76G1, or a UGT having >85% amino-acid sequence identity with UGT76G1.

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 a target steviol glycoside with at least one additional glucose bearing at least one beta l-»6 glucopyranoside glycosidic linkage(s) at the newly formed glycosidic bond(s). In a particular embodiment, the UDP- glucosyltransferase is UGTS12, or a UGT having >85% amino-acid sequence identity with UGTS12. In another particular embodiment, the UDP-glucosyltransferase is EUGT1 1, or a UGT having >85% amino-acid sequence identity with EUGT1 1 . In yet another particular embodiment, the UDP-glucosyltransferase is UGT91 D2, or a UGT having >85% amino-acid sequence identity with UGT91D2.

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 particular embodiment, the UDP-glucosyltransferase is UGT85C2 or a UGT having >85% amino-acid sequence identity with UGT85C2 or a UGT having >85% amino-acid sequence identity with UGT85C2.

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 particular embodiment, the UDP-glucosyltransferase is UGT74G 1 or a UGT having >85% amino-acid sequence identity with UGT74G1.

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 particular embodiment, the UDP-glucosyltransferase is UGTS12 or a UGT having >85% amino-acid sequence identity with UGTS12. In another particular embodiment, the UDP-glucosyltransferase is EUGT1 1, or a UGT having >85% amino- acid sequence identity with EUGT1 1. In yet another particular embodiment, the UDP- glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91D2.

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 particular embodiment, the UDP-glucosyltransferase is UGT76G1 or a UGT having >85% amino-acid sequence identity with UGT76G1.

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 particular embodiment, the UDP-glucosyltransferase is UGT74G1 or a UGT having >85% amino-acid sequence identity with UGT74G1.

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 particular embodiment, the UDP-glucosyltransferase is UGT85C2 or a UGT having >85% amino-acid sequence identity with UGT85C2 or a UGT having >85% amino-acid sequence identity with UGT85C2.

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 particular embodiment, the UDP-glucosyltransferase is UGTS12 or a UGT having >85% amino-acid sequence identity with UGTS12. In another particular embodiment, the UDP-glucosyltransferase is EUGT1 1 , or a UGT having >85% amino-acid sequence identity with EUGT1 1. In yet another particular embodiment, the UDP-glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91D2.

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 particular embodiment, the UDP-glucosyltransferase is UGT76G 1 or a UGT having >85% amino-acid sequence identity with UGT76G1.

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 particular embodiment, the UDP-glucosyltransferase is UGT76G 1 or a 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 particular embodiment, the UDP-glucosyltransferase is UGT74G 1 or a 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 particular embodiment, the UDP-glucosyltransferase is UGTS12 or a UGT having >85% amino-acid sequence identity with UGTS12. In another particular embodiment, the UDP-glucosyltransferase is EUGT1 1, or a UGT having >85% amino- acid sequence identity with EUGT1 1. In yet another particular embodiment, the UDP- glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91D2.

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 particular embodiment, the UDP-glucosyltransferase is UGT74G1 or a 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 particular embodiment, the UDP-glucosyltransferase is UGTS12 or a UGT having >85% amino-acid sequence identity with UGTS12. In another particular embodiment, the UDP-glucosyltransferase is EUGT1 1 , or a UGT having >85% amino- acid sequence identity with EUGT1 1. In yet another particular embodiment, the UDP- glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91D2.

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 particular embodiment, the UDP-glucosyltransferase is UGT76G1 or a UGT having >85% amino-acid sequence identity with UGT76G1.

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 particular embodiment, the UDP-glucosyltransferase is UGTS12 or a UGT having >85% amino-acid sequence identity with UGTS12. In another particular embodiment, the UDP-glucosyltransferase is EUGT1 1 , or a UGT having >85% amino- acid sequence identity with EUGT1 1. In yet another particular embodiment, the UDP- glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91D2.

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 particular embodiment, the UDP-glucosyltransferase is UGT76G1 or a UGT having >85% amino-acid sequence identity with UGT76G1.

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 particular embodiment, the UDP-glucosyltransferase is UGT85C2 or a UGT having >85% amino-acid sequence identity with UGT85C2 or a UGT having >85% amino-acid sequence identity with UGT85C2.

In another embodiment, the UDP-glucosyltransferase is any UDP- glucosyltransferase capable of adding at least one glucose unit to steviolbioside to form stevioside C. In a particular embodiment, the UDP-glucosyltransferase is UGT76G1 or a 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 particular embodiment, the UDP-glucosyltransferase is UGT85C2 or a UGT having >85% amino-acid sequence identity with UGT85C2 or a UGT having >85% amino-acid sequence identity with UGT85C2.

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 particular embodiment, the UDP-glucosyltransferase is UGTS12 or a UGT having >85% amino-acid sequence identity with UGTS12. In another particular embodiment, the UDP-glucosyltransferase is EUGT1 1, or a UGT having >85% amino- acid sequence identity with EUGT1 1. In yet another particular embodiment, the UDP- glucosyltransferase is UGT91 D2, or a UGT having >85% amino-acid sequence identity with UGT91 D2. 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 particular embodiment, the UDP-glucosyltransferase is UGT74G1 or a 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 particular embodiment, the UDP-glucosyltransferase is UGT76G1 or a UGT having >85% amino-acid sequence identity with UGT76G1.

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 particular embodiment, the UDP-glucosyltransferase is UGTS12 or a UGT having >85% amino-acid sequence identity with UGTS12. In another particular embodiment, the UDP-glucosyltransferase is EUGT1 1, or a UGT having >85% amino- acid sequence identity with EUGT1 1. In yet another particular embodiment, the UDP- glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91D2.

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 particular embodiment, the UDP-glucosyltransferase is UGT76G 1 or a UGT having >85% amino-acid sequence identity with UGT76G1.

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 particular embodiment, the UDP-glucosyltransferase is UGTS12 or a UGT having >85% amino-acid sequence identity with UGTS12, In another particular embodiment, the UDP-glucosyltransferase is EUGT1 1 , or a UGT having >85% amino- acid sequence identity with EUGT1 1. In yet another particular embodiment, the UDP- glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91D2.

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 particular embodiment, the UDP-glucosyltransferase is UGTS12 or a UGT having >85% amino-acid sequence identity with UGTS12. In another particular embodiment, the UDP-glucosyltransferase is EUGT1 1 , or a UGT having >85% amino- acid sequence identity with EUGT1 1. In yet another particular embodiment, the UDP- glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91D2.

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 particular embodiment, the UDP-glucosyltransferase is UGT76G1 or a UGT having >85% amino-acid sequence identity with UGT76G1.

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 particular embodiment, the UDP-glucosyltransferase is UGTS12 or a UGT having >85% amino-acid sequence identity with UGTS12. In another particular embodiment, the UDP-glucosyltransferase is EUGT1 1, or a UGT having >85% amino- acid sequence identity with EUGT1 1. In yet another particular embodiment, the UDP- glucosyltransferase is UGT91 D2, or a UGT having >85% amino-acid sequence identity with UGT91D2.

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 particular embodiment, the UDP-glucosyltransferase is UGT76G1 or a UGT having >85% amino-acid sequence identity with UGT76G1.

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 particular embodiment, the UDP-glucosyltransferase is UGT76G1 or a UGT having >85% amino-acid sequence identity with UGT76G1.

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 particular embodiment, the UDP-glucosyltransferase is UGTS12 or a UGT having >85% amino-acid sequence identity with UGTS12. In another particular embodiment, the UDP-glucosyltransferase is EUGT1 1, or a UGT having >85% amino- acid sequence identity with EUGT1 1. In yet another particular embodiment, the UDP- glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91D2.

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 particular embodiment, the UDP-glucosyltransferase is UGT76G1 or a UGT having >85% amino-acid sequence identity with UGT76G1.

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 particular embodiment, the UDP-glucosyltransferase is UGTS12 or a UGT having >85% amino-acid sequence identity with UGTS12. In another particular embodiment, the UDP-glucosyltransferase is EUGT1 1 , or a UGT having >85% amino- acid sequence identity with EUGT1 1. In yet another particular embodiment, the UDP- glucosyltransferase is UGT91 D2, or a UGT having >85% amino-acid sequence identity with UGT91 D2,

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 particular embodiment, the UDP-glucosyltransferase is UGT85C2 or a UGT having >85% amino-acid sequence identity with UGT85C2 or a UGT having >85% amino-acid sequence identity with UGT85C2.

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 particular embodiment, the UDP-glucosyltransferase is UGTS12 or a UGT having >85% amino-acid sequence identity with UGTS12. In another particular embodiment, the UDP-glucosyltransferase is EUGT1 1 , or a UGT having >85% amino- acid sequence identity with EUGT1 1. In yet another particular embodiment, the UDP- glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91D2.

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 particular embodiment, the UDP-glucosyltransferase is UGT76G1 or a UGT having >85% amino-acid sequence identity with UGT76G1. 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 particular embodiment, the UDP-glucosyltransferase is UGT76G1 or a 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 particular embodiment, the UDP-glucosyltransferase is UGT76G1 or a UGT having >85% amino-acid sequence identity with UGT76G1.

In another embodiment, the UDP-glucosyltransferase is any UDP- glucosyltransferase capable of adding at least one glucose unit to rebaudioside E2 to form rebaudioside /. In a particular embodiment, the UDP-glucosyltransferase is UGT76G1 or a 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 particular embodiment, the UDP-glucosyltransferase is UGTS12 or a UGT having >85% amino-acid sequence identity with UGTS12. In another particular embodiment, the UDP-glucosyltransferase is EUGT1 1 , or a UGT having >85% amino- acid sequence identity with EUGT1 1. In yet another particular embodiment, the UDP- glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91 D2.

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 particular embodiment, the UDP-glucosyltransferase is UGTS12 or a UGT having >85% amino-acid sequence identity with UGTS12. In another particular embodiment, the UDP-glucosyltransferase is EUGT1 1 , or a UGT having >85% amino- acid sequence identity with EUGT1 1. In yet another particular embodiment, the UDP- glucosyltransferase is UGT91 D2, or a UGT having >85% amino-acid sequence identity with UGT91 D2.

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 particular embodiment, the UDP-glucosyltransferase is UGT76G 1 or a UGT having >85% amino-acid sequence identity with UGT76G1.

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 particular embodiment, the UDP-glucosyltransferase is UGTS12 or a UGT having >85% amino-acid sequence identity with UGTS12, In another particular embodiment, the UDP-glucosyltransferase is EUGTl l, or a UGT having >85% amino- acid sequence identity with EUGT1 1. In yet another particular embodiment, the UDP- glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91D2.

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 particular embodiment, the UDP-glucosyltransferase is UGTS12 or a UGT having >85% amino-acid sequence identity with UGTS12. In another particular embodiment, the UDP-glucosyltransferase is EUGT1 1, or a UGT having >85% amino- acid sequence identity with EUGT1 1. In yet another particular embodiment, the UDP- glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91 D2,

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 particular embodiment, the UDP-glucosyltransferase is UGTS12 or a UGT having >85% amino-acid sequence identity with UGTS12. In another particular embodiment, the UDP-glucosyltransferase is EUGT11, or a UGT having >85% amino- acid sequence identity with EUGT1 1. In yet another particular embodiment, the UDP- glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91D2.

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 particular embodiment, the UDP-glucosyltransferase is UGT76G1 or a UGT having >85% amino-acid sequence identity with UGT76G1. 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 particular embodiment, the UDP-glucosyltransferase is UGT76G1 or a UGT having >85% amino-acid sequence identity with UGT76G1.

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 particular embodiment, the UDP-glucosyltransferase is UGTS12 or a UGT having >85% amino-acid sequence identity with UGTS12 In another particular embodiment, the UDP-glucosyltransferase is EUGT1 1, or a UGT having >85% amino- acid sequence identity with EUGT11. In yet another particular embodiment, the UDP- glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91D2,

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 particular embodiment, the UDP-glucosyltransferase is UGT76G1 or a UGT having >85% amino-acid sequence identity with UGT76G1.

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 particular embodiment, the UDP-glucosyltransferase is UGTS12 or a UGT having >85% amino-acid sequence identity with UGTS12. In another particular embodiment, the UDP-glucosyltransferase is EUGT1 1 , or a UGT having >85% amino- acid sequence identity with EUGT1 1. In yet another particular embodiment, the UDP- glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91D2. In a particular embodiment, the UDP-glucosyltransferase is UGT76G1 or a UGT having >85% amino-acid sequence identity with UGT76G1.

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 particular embodiment, the UDP-glucosyltransferase is UGTS12 or a UGT having >85% amino-acid sequence identity with UGTS12. In another particular embodiment, the UDP-glucosyltransferase is EUGT1 1 , or a UGT having >85% amino- acid sequence identity with EUGT1 1. In yet another particular embodiment, the UDP- glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91D2.

In another embodiment, the UDP-glucosyltransferase is any UDP- glucosyltransferase capable of adding at least one glucose unit to rebaudioside M to form rebaudioside la. In a particular embodiment, the UDP-glucosyltransferase is UGTS12 or a UGT having >85% amino-acid sequence identity with UGTS12. In another particular embodiment, the UDP-glucosyltransferase is EUGT1 1 , or a UGT having >85% amino- acid sequence identity with EUGT1 1. In yet another particular embodiment, the UDP- glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91 D2.

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

In another embodiment, the UDP-glucosyltransferase is any UDP- glucosyltransferase capable of adding at least one glucose unit to rebaudioside M to form rebaudioside lc. In a particular embodiment, the UDP-glucosyltransferase is UGTS12 or a UGT having >85% amino-acid sequence identity with UGTS12. In another particular embodiment, the UDP-glucosyltransferase is EUGT1 1 , or a UGT having >85% amino- acid sequence identity with EUGT1 1. In yet another particular embodiment, the UDP- glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91D2.

In another embodiment, the UDP-glucosyltransferase is any UDP- glucosyltransferase capable of adding at least one glucose unit to rebaudioside to form rebaudioside Id. In a particular embodiment, the UDP-glucosyltransferase is UGTS12 or a UGT having >85% amino-acid sequence identity with UGTS12. In another particular embodiment, the UDP-glucosyltransferase is EUGT1 1 , or a UGT having >85% amino- acid sequence identity with EUGT1 1. In yet another particular embodiment, the UDP- glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91D2. In another embodiment, the UDP-glucosyltransferase is any UDP- glucosyltransferase capable of adding at least one glucose unit to rebaudioside M to form rebaudioside le. In a particular embodiment, the UDP-glucosyltransferase is UGT76G 1 or a UGT having >85% amino-acid sequence identity with UGT76G1.

In another embodiment, the UDP-glucosyltransferase is any UDP- glucosyltransferase capable of adding at least one glucose unit to rebaudioside to form rebaudioside If. In a particular embodiment, the UDP-glucosyltransferase is UGTS12 or a UGT having >85% amino-acid sequence identity with UGTS12. In another particular embodiment, the UDP-glucosyltransferase is EUGT1 1, or a UGT having >85% amino- acid sequence identity with EUGT1 1. In yet another particular embodiment, the UDP- glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91D2.

In another embodiment, the UDP-glucosyltransferase is any UDP- glucosyltransferase capable of adding at least one glucose unit to rebaudioside M to form rebaudioside Ig. In a particular embodiment, the UDP-glucosyltransferase is UGTS12 or a UGT having >85% amino-acid sequence identity with UGTS12. In another particular embodiment, the UDP-glucosyltransferase is EUGT1 1, or a UGT having >85% amino- acid sequence identity with EUGT1 1. In yet another particular embodiment, the UDP- glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91 D2.

In another embodiment, the UDP-glucosyltransferase is any UDP- glucosyltransferase capable of adding at least one glucose unit to rebaudioside M to form rebaudioside lh In a particular embodiment, the UDP-glucosyltransferase is UGT76G1 or a 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 li. In a particular embodiment, the UDP-glucosyltransferase is UGTS12 or a UGT having >85% amino-acid sequence identity with UGTS12. In another particular embodiment, the UDP-glucosyltransferase is EUGT1 1, or a UGT having >85% amino- acid sequence identity with EUGT1 1. In yet another particular embodiment, the UDP- glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91 D2.

In another embodiment, the UDP-glucosyltransferase is any UDP- glucosyltransferase capable of adding at least one glucose unit to rebaudioside M to form rebaudioside Ij. In a particular embodiment, the UDP-glucosyltransferase is UGTS12 or a UGT having >85% amino-acid sequence identity with UGTS12. In another particular embodiment, the UDP-glucosyltransferase is EUGT1 1, or a UGT having >85% amino- acid sequence identity with EUGT1 1. In yet another particular embodiment, the UDP- glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91 D2.

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

In another embodiment, the UDP-glucosyltransferase is any UDP- glucosyltransferase capable of adding at least one glucose unit to rebaudioside M to form rebaudioside 11. In a particular embodiment, the UDP-glucosyltransferase is UGTS12 or a UGT having >85% amino-acid sequence identity with UGTS12. In another particular embodiment, the UDP-glucosyltransferase is EUGT1 1, or a UGT having >85% amino- acid sequence identity with EUGT1 1. In yet another particular embodiment, the UDP- glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91D2.

In another embodiment, the UDP-glucosyltransferase is any UDP- glucosyltransferase capable of adding at least one glucose unit to rebaudioside M to form rebaudioside lm. In a particular embodiment, the UDP-glucosyltransferase is UGTS12 or a UGT having >85% amino-acid sequence identity with UGTS12. In another particular embodiment, the UDP-glucosyltransferase is EUGT1 1 , or a UGT having >85% amino- acid sequence identity with EIJGT1 1. In yet another particular embodiment, the UDP- glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91 D2. In another embodiment, the UDP-glucosyltransferase is any UDP- glucosyltransferase capable of adding at least one glucose unit to rebaudioside M to form rebaudioside In. In a particular embodiment, the UDP-glucosyltransferase is UGTS12 or a UGT having >85% amino-acid sequence identity with UGTS12. In another particular embodiment, the UDP-glucosyltransferase is EUGT1 1, or a UGT having >85% amino- acid sequence identity with EUGT1 1. In yet another particular embodiment, the UDP- glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91D2.

In another embodiment, the UDP-glucosyltransferase is any UDP- glucosyltransferase capable of adding at least one glucose unit to rebaudioside M4 to form rebaudioside 2a. In a particular embodiment, the UDP-glucosyltransferase is UGTS12 or a UGT having >85% amino-acid sequence identity with UGTS12. In another particular embodiment, the UDP-glucosyltransferase is EIJGT1 1 , or a UGT having >85% amino- acid sequence identity with EUGT1 1. In yet another particular embodiment, the UDP- glucosyltransferase is UGT91D2, or a UGT having >85% amino-acid sequence identity with UGT91D2. In a particular embodiment, the UDP-glucosyltransferase is UGT76G 1 or a UGT having >85% amino-acid sequence identity with UGT76G1.

Optionally, the method of the present invention further comprises recycling UDP to provide UDP-glucose. In one embodiment, the method comprises recycling UDP by providing a recycling catalyst and a recycling substrate, such that the biotransformation of steviol and/or the steviol glycoside substrate to the target steviol glycoside is carried out using catalytic amounts of UDP-glucosyltransferase and UDP-glucose.

In one embodiment, the recycling catalyst is sucrose synthase SuSy_At or a sucrose synthase having >85% amino-acid sequence identity with SuSy_At.

In one embodiment, the recycling substrate for UDP-glucose recycling catalyst is sucrose.

Optionally, the method of the present invention further comprises the use of transglycosidases that use oligo- or poly-saccharides as the sugar donor to modify recipient target steviol glycoside molecules. Non-limiting examples include cyclodextrin glycosyltransferase (CGTase), fructofuranosidase, amylase, saccharase, glucosucrase, beta-h-fructosidase, beta-fructosidase, sucrase, fructosyl invertase, alkaline invertase, acid invertase, fructofuranosidase. In some embodiments, glucose and sugar(s) other than glucose, including but not limited to fructose, xylose, rhamnose, arabinose, deoxyglucose, galactose are transferred to the recipient target steviol glycosides. In one embodiment, the recipient steviol glycoside is rebaudioside la, rebaudioside lb, rebaudioside lc, rebaudioside Id, rebaudioside le, rebaudioside If, rebaudioside Ig, rebaudioside Ih, rebaudioside li, rebaudioside //, rebaudioside Ik, rebaudioside 11, rebaudioside 1m, and/or rebaudioside In. In another embodiment, the recipient steviol glycoside is rebaudioside 2a. In another embodiment, the recipient steviol glycoside is rebaudioside M4. In another embodiment, the recipient steviol glycoside is SvG7. In another embodiment, the UDP-glucosyltransferase capable of adding at least one glucose unit to starting composition steviol glycoside has >85% amino-acid sequence identity with UGTs selected from the following listing of Genlnfo identifier numbers, preferably from the group presented in Table 1, and Table 2.

397567 30680413 1 15480946 147798902 218193594 225443294

454245 32816174 1 16310259 14781 1764 218193942 225444853

1359905 32816178 1 16310985 147827151 219885307 225449296

1685003 34393978 1 16788066 147836230 222615927 225449700

1685005 37993665 1 16788606 147839909 222619587 225454338

2191 136 37993671 1 16789315 147846163 222623 142 225454340

2501497 37993675 119394507 147855977 222625633 225454342

291 1049 39104603 1 19640480 148905778 222625635 225454473

4218003 41469414 122209731 148905999 222636620 225454475

4314356 41469452 125526997 148906835 222636621 225458362

13492674 42566366 125534279 148907340 222636628 225461551

13492676 42570280 125534461 148908935 222636629 225461556

15217773 42572855 125540090 148909182 224053242 225461558

15217796 44890129 125541516 148909920 224053386 225469538

15223396 46806235 125545408 148910082 224055535 225469540

15223589 50284482 125547340 148910154 224056138 226316457

15227766 51090402 125547520 148910612 224056160 226492603

15230017 51090594 125554547 148910769 224067918 226494221

15231757 52839682 125557592 156138791 224072747 226495389

15234056 56550539 125557593 156138797 224080189 226495945

15234195 62734263 125557608 156138799 224091845 226502400

15234196 62857204 125559566 156138803 224094703 226507980

15238503 62857206 125563266 165972256 224100653 226531 147

15239523 62857210 125571055 168016721 224100657 226532094

15239525 62857212 125579728 171674071 224101569 238477377

15239543 75265643 125588307 171906258 224103105 240254512

15239937 75285934 125589492 183013901 224103633 242032615

15240305 75288884 125599469 183013903 224103637 242032621

15240534 77550661 125601477 186478321 224109218 242038423 15982889 77556148 126635837 187373030 2241 14583 242043290

18086351 82791223 126635845 187373042 2241 16284 242044836

18418378 83778990 126635847 190692175 224120552 242051252

18418380 89953335 126635863 194701936 224121288 242056217

18418382 1 10741436 126635867 195620060 224121296 242056219

19743740 1 10743955 126635883 209954691 224121300 242056663

1991 1201 1 15438196 126635887 209954719 224130358 242059339

20149064 1 15438785 133874210 209954725 224140703 242059341

20260654 115441237 133874212 209954733 224143404 242060922

21435782 1 15454819 145358033 210063105 224143406 24206741 1

21553613 115456047 147772508 210063107 224144306 242067413

21593514 1 15457492 147776893 212275846 224285244 242076258

22759895 1 15459312 147776894 216296854 225431707 242076396

23955910 1 15464719 147776895 217074506 225435532 242084750

26452040 1 15471069 147786916 218185693 225436321 242091005

28393204 1 15471071 147798900 218187075 225440041 242095206

30679796 1 15474009 147798901 218189427 225441 1 16 242345159

242345161 297724601 326492035 356523945 357140904 359486938

255536859 297725463 326493430 356523957 357165849 359487055

255538228 297728331 326500410 356523959 357165852 359488135

255541676 297738632 326506816 356523961 357168415 359488708

255547075 297745347 326507826 356523963 357437837 359493630

255552620 297745348 326508394 356524387 357442755 359493632

255552622 297795735 326509445 356524403 357442757 359493634

255555343 297796253 32651 1261 356527181 357445729 359493636

255555361 297796257 32651 1866 356533209 357445731 359493815

255555363 297796261 326512412 356533852 357445733 359495856

255555365 297797587 326517673 356534718 357446799 359495858

255555369 297798502 326518800 356535480 357446805 359495869

255555373 297799226 326521 124 356542996 357452779 359495871

255555377 297805988 326525567 356543136 357452781 359497638

255556812 297807499 326525957 356543932 357452783 359807261

255556818 297809125 326526607 356549841 357452787 374256637

255563008 297809127 326527141 356549843 357452789 377655465

255564074 29781 1403 326530093 356554358 357452791 378405177

255564531 297820040 326534036 356554360 357452797 378829085

255572878 297821483 326534312 356558606 357452799 387135070

255577901 297825217 332071 132 356560333 357470367 387135072

255583249 297832276 339715876 356560599 357472193 387135078

255583253 297832280 342306012 356560749 357472195 387135092

255583255 297832518 342306016 356566018 357474295 387135094

255585664 297832520 343457675 356566169 357474493 387135098

255585666 297840825 343457677 356566173 357474497 387135100

255634688 297840827 350534960 356567761 357474499 387135134

255644801 297847402 356498085 356574704 357490035 387135136

255645821 297849372 356499771 356576401 357493567 387135174

255647456 300078590 356499777 356577660 357497139 387135176

255648275 300669727 356499779 3571 14993 357497581 387135184

260279126 302142947 356501328 3571 15447 357497671 387135186

260279128 302142948 356502523 3571 15451 357500579 387135188 261343326 302142950 356503180 3571 15453 357504663 387135190

283132367 302142951 356503184 3571 16080 357504691 387135192

2833621 12 302765302 356503295 3571 16928 357504699 387135194

289188052 302796334 356504436 3571 17461 357504707 387135282

295841350 30281 1470 356504523 3571 17463 357505859 387135284

296088529 302821107 356504765 3571 17829 357510851 387135294

296090415 302821679 35651 1 1 13 3571 17839 357516975 387135298

296090524 319759260 356515120 357125059 359477003 387135300

296090526 319759266 356517088 357126015 359477998 387135302

297599503 320148814 356520732 357134488 359478043 387135304

297601531 326489963 356522586 357135657 359478286 387135312

29761 1791 326490273 356522588 357138503 359484299 387135314

297722841 326491 131 356522590 357139683 359486936 387135316

387135318 449440433 460376293 460413408 462423864 475546199

387135320 449445896 460378310 460416351 470101924 475556485

387135322 449446454 460380744 462394387 470102280 475559699

387135324 449447657 460381726 462394433 470102858 475578293

387135326 449449002 460382093 462394557 47010421 1 475591753

387135328 449449004 460382095 462395646 470104264 475593742

388493506 449449006 460382754 462395678 470104266 475612072

388495496 449451379 460384935 462396388 470106317 475622476

388498446 449451589 460384937 462396389 470106357 475622507

388499220 449451591 460385076 462396419 4701 15448 475623787

388502176 449451593 460385872 462396542 470130404 482550481

388517521 449453712 460386018 462397507 470131550 482550499

388519407 449453714 460389217 462399998 470136482 482550740

388521413 449453716 460394872 462400798 470136484 482550999

388827901 449453732 460396139 462401217 470136488 482552352

388827903 449457075 460397862 4624021 18 470136492 482554970

388827907 449467555 460397864 462402237 470137933 482555336

388827909 449468742 460398541 462402284 470137937 482555478

388827913 449495638 460403139 462402416 470140422 482556454

393887637 449495736 460403141 462404228 470140426 482557289

393887646 449499880 460403143 462406358 470140908 482558462

393887649 449502786 460403145 462408262 470141232 482558508

393990627 449503471 460405998 462409325 470142008 482558547

397746860 449503473 460407578 462409359 470142010 482561055

397789318 449515857 460407590 462409777 470142012 482561555

413924864 449518643 460409128 46241 1467 470143607 482562795

414590349 449519559 460409134 46241431 1 470143939 482562850

414590661 449522783 460409136 462414416 470145404 482565074

414591 157 449524530 460409459 462414476 473923244 482566269

414879558 449524591 460409461 462415526 4741 14354 482566296

414879559 449528823 460409463 462415603 474143634 482566307

414879560 449528825 460409465 462415731 474202268 482568689

414888074 449534021 460409467 462416307 474299266 482570049

431812559 460365546 460410124 462416920 4743631 19 482570572

449432064 460366882 460410126 462416922 474366157 482575121

449432066 460369823 460410128 462416923 474429346

449433069 460369829 460410130 462416924 475432777 449436944 460369831 460410132 462417401 475473002

449438665 460369833 460410134 462419769 475489790

449438667 460370755 460410213 462420317 47551 1330

449440431 460374714 46041 1200 462423366 475516200

Table 1

Table 2

One embodiment of the present invention is a microbial cell comprising an enzyme, i.e. an enzyme capable of converting the starting composition to the target steviol glycoside. Accordingly, some embodiments of the present method include contacting a microorganism with a medium containing the starting composition to provide a medium comprising at least one target steviol glycoside.

The microorganism can be any microorganism possessing the necessary enzyme(s) for converting the starting composition to target steviol glycoside(s). These enzymes are encoded within the microorganism’s genome.

Suitable microoganisms include, but are not limited to, E.coli, Saccharomyces sp., Aspergillus sp., Pichia sp., Bacillus sp., Yarrowia sp etc.

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 microorganism may be immobilized to a solid support made from inorganic or organic materials. Non-limiting examples of solid supports suitable to immobilize the microorganism include derivatized cellulose or glass, ceramics, metal oxides or membranes. The microorganism may be immobilized to the solid support, for example, by covalent attachment, adsorption, cross-linking, entrapment or encapsulation.

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

The target steviol glycoside is optionally purified, Purification of the target steviol glycoside from the reaction medium can be achieved by at least one suitable method to provide a highly purified target steviol glycoside composition. Suitable methods include crystallization, separation by membranes, centrifugation, extraction (liquid or solid phase), chromatographic separation, HPLC (preparative or analytical) or a combination of such methods.

Uses

Highly purified target glycoside(s), particularly steviolmonoside, steviolmonoside A, steviolbioside, steviolbioside D, rubusoside, steviolbioside A, steviolbioside B, rebaudioside B , stevioside, rebaudioside G, stevioside A, stevioside B, stevioside C, rebaudioside A, rebaudioside E, rebaudioside E2, rebaudioside E4, rebaudioside E6, rebaudioside E3, rebaudioside D, rebaudioside /, rebaudioside AM, rebaudioside D7, rebaudioside M, rebaudioside M4, rebaudioside la, rebaudioside lb, rebaudioside lc, rebaudioside Id, rebaudioside le, rebaudioside If, rebaudioside lg, rebaudioside Ih, rebaudioside li, rebaudioside lj, rebaudioside Ik, rebaudioside II, rebaudioside lm, rebaudioside In, rebaudioside 2a and/or SvG7 obtained according to this invention can be used “as-is” or in combination with other sweeteners, flavors, food ingredients and combinations thereof.

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

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

Highly purified target glycoside(s), particularly steviolmonoside, steviolmonoside A, steviolbioside, steviolbioside D, rubusoside, steviolbioside A, steviolbioside B, rebaudioside B, stevioside, rebaudioside G, stevioside A, stevioside B, stevioside C, rebaudioside A, rebaudioside E, rebaudioside E2, rebaudioside E4, rebaudioside E6, rebaudioside E3, rebaudioside D, rebaudioside I, rebaudioside AM, rebaudioside D7, rebaudioside M, rebaudioside M4, rebaudioside la, rebaudioside lb, rebaudioside lc, rebaudioside Id, rebaudioside le, rebaudioside If, rebaudioside lg, rebaudioside lh, rebaudioside li, rebaudioside lj, rebaudioside Ik, rebaudioside 11, rebaudioside lm, rebaudioside In, rebaudioside 2a and/or SvG7 obtained according to this invention can be prepared in various polymorphic forms, including but not limited to hydrates, solvates, anhydrous, amorphous forms and combinations thereof.

Highly purified target glycoside(s), particularly steviolmonoside, steviolmonoside A, steviolbioside, steviolbioside D, rubusoside, steviolbioside A, steviolbioside B, rebaudioside B, stevioside, rebaudioside G, stevioside A, stevioside B, stevioside C, rebaudioside A, rebaudioside E, rebaudioside E2, rebaudioside E4, rebaudioside E6, rebaudioside E3, rebaudioside D, rebaudioside /, rebaudioside AM, rebaudioside D7, rebaudioside M, rebaudioside M4, rebaudioside la, rebaudioside lb, rebaudioside lc, rebaudioside Id, rebaudioside le, rebaudioside If, rebaudioside lg, rebaudioside lh, rebaudioside li, rebaudioside Ij, rebaudioside Ik, rebaudioside 11, rebaudioside lm, rebaudioside In, rebaudioside 2a and/or SvG7 obtained according to this invention may be incorporated as a high intensity natural sweetener in foodstuffs, beverages, pharmaceutical compositions, cosmetics, chewing gums, table top products, cereals, dairy products, toothpastes and other oral cavity compositions, etc.

In some embodiments, the highly purified target glycoside(s) of present invention are present in foodstuffs, beverages, pharmaceutical compositions, cosmetics, chewing gums, table top products, cereals, dairy products, toothpastes and other oral cavity compositions, etc in an amount from about 0.0001 % to about 12% by weight, such as, for example, 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 1 1.0% by weight, about 1 1.5% by weight or about 12.0% by weight.

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

Highly purified target glycoside(s), particularly steviolmonoside, steviolmonoside A, steviolbioside, steviolbioside D, rubusoside, steviolbioside A, steviolbioside B, rebaudioside B, stevioside, rebaudioside G, stevioside A, stevioside B, stevioside C, rebaudioside A, rebaudioside E, rebaudioside E2, rebaudioside E4, rebaudioside E6, rebaudioside E3, rebaudioside D, rebaudioside I, rebaudioside AM, rebaudioside D7, rebaudioside M, rebaudioside M4, rebaudioside la, rebaudioside lb, rebaudioside lc, rebaudioside Id, rebaudioside le, rebaudioside If, rebaudioside Ig, rebaudioside lh, rebaudioside li, rebaudioside lj, rebaudioside Ik, rebaudioside 11, rebaudioside lm, rebaudioside In, rebaudioside 2a, SvG7 and/or combinations thereof, obtained according to this invention, may be employed as a sweetening compound, or it may be used together with at least one naturally occurring high intensity sweeteners such as dulcoside A, dulcoside B, dulcoside C, dulcoside D , rebaudioside A2, rebaudioside A3, rebaudioside A4, rebaudioside B2, rebaudioside C, rebaudioside C2, rebaudioside C3, rebaudioside C4, rebaudioside C5, rebaudioside C6, rebaudioside D2, rebaudioside D3, rebaudioside D4, rebaudioside D5, rebaudioside D6, rebaudioside D8, rebaudioside E5, rebaudioside E7, rebaudioside F, rebaudioside F2, rebaudioside F3, rebaudioside H, rebaudioside 112, rebaudioside H3, rebaudioside H4, rebaudioside H5, rebaudioside H6, rebaudioside 12, rebaudioside 13, rebaudioside J, rebaudioside K, rebaudioside K2, rebaudioside KA, rebaudioside L, rebaudioside M2, rebaudioside M3, rebaudioside N, rebaudioside N2, rebaudioside N3, rebaudioside N4, rebaudioside N5, rebaudioside O, rebaudioside 02, rebaudioside 03, rebaudioside 04, rebaudioside Q, rebaudioside Q2, rebaudioside Q3, rebaudioside R, rebaudioside S, rebaudioside T, rebaudioside Tl, rebaudioside U, rebaudioside U2, rebaudioside V, rebaudioside V2, rebaudioside V3, rebaudioside W, rebaudioside W2, rebaudioside W3, rebaudioside Y, rebaudioside Zl, rebaudioside Z2, steviolbioside C, steviolbioside E, stevioside D, stevioside E, stevioside E2, stevioside F, stevioside G, stevioside H, mogrosides, brazzein, neohesperidin dihydrochalcone, glycyrrhizic acid and its salts, thaumatin, perillartine, pernandulcin, mukuroziosides, baiyunoside, phlomisoside-7, dimethyl-hexahydrofluorene-dicarboxylic acid, abrusosides, periandrin, carnosiflosides, cyclocarioside, pterocaryosides, polypodoside A, brazilin, hernandulcin, phillodulcin, glycyphyllin, phlorizin, trilobatin, dihydroflavonol, dihydroquercetin-3-acetate, neoastilibin, /rani-cinnamaldehyde, monatin, monatin salts, other indole derivative sweeteners, selligueain A, hematoxylin, monellin, osladin, pterocaryoside A, pterocaryoside B, mabinlin, pentadin, miraculin, curculin, neoculin, chlorogenic acid, cynarin, Luo Han Guo sweetener, mogroside V, sia enoside, siratose and combinations thereof.

In a particular embodiment, steviolmonoside, steviolmonoside A, steviolbioside, steviolbioside D, rubusoside, steviolbioside A, steviolbioside B, rebaudioside B, stevioside, rebaudioside G, stevioside A, stevioside B , stevioside C, rebaudioside A, rebaudioside E, rebaudioside E2, rebaudioside E4, rebaudioside E6, rebaudioside E3, rebaudioside D, rebaudioside I, rebaudioside AM, rebaudioside D7, rebaudioside M, rebaudioside M4, rebaudioside la, rebaudioside lb, rebaudioside lc, rebaudioside Id, rebaudioside le, rebaudioside If, rebaudioside Ig, rebaudioside Ih, rebaudioside li, rebaudioside lj, rebaudioside lk, rebaudioside 11, rebaudioside Jin, rebaudioside In, rebaudioside 2a and/or SvG7 can be used in a sweetener composition comprising a compound selected from the group consisting of dulcoside A, dulcoside B, dulcoside C, dulcoside D, rebaudioside A2, rebaudioside A3, rebaudioside A4, rebaudioside B2, rebaudioside C, rebaudioside C2, rebaudioside C3, rebaudioside C4, rebaudioside C5, rebaudioside C6, rebaudioside D2, rebaudioside D3, rebaudioside D4, rebaudioside D5, rebaudioside D6, rebaudioside D8, rebaudioside E5, rebaudioside E7, rebaudioside F, rebaudioside F2, rebaudioside F3, rebaudioside H, rebaudioside H2, rebaudioside H3, rebaudioside 114, rebaudioside H5, rebaudioside H6, rebaudioside 12, rebaudioside 13, rebaudioside J, rebaudioside K, rebaudioside K2, rebaudioside KA, rebaudioside L, rebaudioside M2, rebaudioside M3, rebaudioside N, rebaudioside N2, rebaudioside N3, rebaudioside N4, rebaudioside N5, rebaudioside O, rebaudioside 02, rebaudioside 03, rebaudioside 04, rebaudioside Q, rebaudioside Q2, rebaudioside Q3, rebaudioside R, rebaudioside S, rebaudioside T, rebaudioside 77, rebaudioside U, rebaudioside U2, rebaudioside V, rebaudioside V2, rebaudioside V3, rebaudioside W, rebaudioside W2, rebaudioside W3, rebaudioside Y, rebaudioside Zl, rebaudioside Z2, steviolbioside C, steviolbioside E, stevioside D, stevioside E, stevioside E2, stevioside F, stevioside G, stevioside H, NSF- 02, Mogroside V, siratose, Luo Han Guo, allulose, allose, D-tagatose, erythritol and combinations thereof.

Highly purified target glycoside(s), particularly steviolmonoside, steviolmonoside A, steviolbioside, steviolbioside D, rubusoside, steviolbioside A, steviolbioside B, rebaudioside B, stevioside, rebaudioside G, stevioside A, stevioside B, stevioside C, rebaudioside A, rebaudioside E, rebaudioside E2, rebaudioside E4, rebaudioside E6, rebaudioside E3, rebaudioside D, rebaudioside I, rebaudioside AM, rebaudioside D7, rebaudioside M, rebaudioside M4, rebaudioside la, rebaudioside lb, rebaudioside lc, rebaudioside Id, rebaudioside le, rebaudioside If, rebaudioside lg, rebaudioside lh, rebaudioside li, rebaudioside lj, rebaudioside lk, rebaudioside 11, rebaudioside lm, rebaudioside In, rebaudioside 2a and/or SvG7 may also be used in combination with synthetic high intensity sweeteners such as sucralose, potassium acesulfame, aspartame, alitame, saccharin, neohesperidin dihydrochalcone, cyclamate, neotame, dulcin, suosan advantame, salts thereof, and combinations thereof.

Moreover, highly purified target steviol glycoside(s) particularly steviolmonoside, steviolmonoside A, steviolbioside, steviolbioside D, rubusoside, steviolbioside A, steviolbioside B, rebaudioside B , stevioside, rebaudioside G, stevioside A, stevioside B, stevioside C, rebaudioside A, rebaudioside E, rebaudioside E2, rebaudioside E4, rebaudioside E6, rebaudioside E3, rebaudioside D, rebaudioside /, rebaudioside AM, rebaudioside D7, rebaudioside M, rebaudioside M4, rebaudioside la, rebaudioside lb, rebaudioside lc, rebaudioside Id, rebaudioside le, rebaudioside If rebaudioside lg, rebaudioside lh, rebaudioside li, rebaudioside lj, rebaudioside lk, rebaudioside 11, rebaudioside lm, rebaudioside In, rebaudioside 2a and/or SvG7 can be used in combination with natural sweetener suppressors such as gymnemic acid, hodulcin, ziziphin, lactisole, and others. Steviolmonoside, steviolmonoside A, steviolbioside, steviolbioside D, rubusoside, steviolbioside A, steviolbioside B, rebaudioside B, stevioside, rebaudioside G, stevioside A, stevioside B, stevioside C, rebaudioside A, rebaudioside E, rebaudioside E2, rebaudioside E4, rebaudioside E6, rebaudioside E3, rebaudioside D, rebaudioside I, rebaudioside AM, rebaudioside D7, rebaudioside M, rebaudioside M4, rebaudioside la, rebaudioside lb, rebaudioside lc, rebaudioside Id, rebaudioside le, rebaudioside If rebaudioside lg, rebaudioside lh, rebaudioside li, rebaudioside lj, rebaudioside lk, rebaudioside 11, rebaudioside lm, rebaudioside In, rebaudioside 2a and/or SvG7 may also be combined with various umami taste enhancers. Steviolmonoside, steviolmonoside A, steviolbioside, steviolbioside D, rubusoside, steviolbioside A, steviolbioside B, rebaudioside B, stevioside, rebaudioside G, stevioside A, stevioside B, stevioside C, rebaudioside A, rebaudioside E, rebaudioside E2, rebaudioside E4, rebaudioside E6, rebaudioside E3, rebaudioside D, rebaudioside /, rebaudioside AM , rebaudioside D7, rebaudioside M, rebaudioside M4, rebaudioside la, rebaudioside lb, rebaudioside lc, rebaudioside Id, rebaudioside le, rebaudioside If, rebaudioside Ig, rebaudioside Ih, rebaudioside li, rebaudioside lj, rebaudioside lk, rebaudioside 11, rebaudioside lm, rebaudioside In, rebaudioside 2a and/or SvG7 can be mixed with umami tasting and sweet amino acids such as glutamate, aspartic acid, glycine, alanine, threonine, proline, serine, glutamate, lysine, tryptophan and combinations thereof.

Highly purified target steviol glycoside(s) particularly, steviolmonoside, steviolmonoside A, steviolbioside, steviolbioside D, rubusoside, steviolbioside A, steviolbioside B, rebaudioside B, stevioside, rebaudioside G, stevioside A, stevioside B, stevioside C, rebaudioside A, rebaudioside E, rebaudioside E2, rebaudioside E4, rebaudioside E6, rebaudioside E3, rebaudioside D, rebaudioside /, rebaudioside AM, rebaudioside D7, rebaudioside M, rebaudioside M4, rebaudioside la, rebaudioside lb, rebaudioside lc, rebaudioside Id, rebaudioside le, rebaudioside If rebaudioside lg, rebaudioside lh, rebaudioside li, rebaudioside lj, rebaudioside lk, rebaudioside 11, rebaudioside lm, rebaudioside In, rebaudioside 2a and/or SvG7 can be used in combination with one or more additive selected from the group consisting of carbohydrates, polyols, amino acids and their corresponding salts, poly-amino acids and their corresponding salts, sugar acids and their corresponding salts, nucleotides, organic acids, inorganic acids, organic salts including organic acid salts and organic base salts, inorganic salts, bitter compounds, flavorants and flavoring ingredients, astringent compounds, proteins or protein hydrolysates, surfactants, emulsifiers, flavonoids, alcohols, polymers and combinations thereof.

Highly purified target steviol glycoside(s) particularly, steviolmonoside, steviolmonoside A, steviolbioside, steviolbioside D, rubusoside, steviolbioside A, steviolbioside B, rebaudioside B, stevioside, rebaudioside G, stevioside A, stevioside B, stevioside C, rebaudioside A, rebaudioside E, rebaudioside E2, rebaudioside E4, rebaudioside E6, rebaudioside E3, rebaudioside D, rebaudioside /, rebaudioside AM, rebaudioside D7, rebaudioside M, rebaudioside M4, rebaudioside la , rebaudioside lb, rebaudioside lc, rebaudioside Id, rebaudioside le, rebaudioside If, rebaudioside lg, rebaudioside lh, rebaudioside li, rebaudioside lj, rebaudioside lk, rebaudioside 11, rebaudioside lm, rebaudioside In, rebaudioside 2a and/or SvG7 may be combined with polyols or sugar alcohols. The term“polyol” refers to a molecule that contains more than one hydroxyl group. A polyol may be a diol, triol, or a tetraol which contain 2, 3, and 4 hydroxyl groups, respectively. A polyol also may contain more than four hydroxyl groups, such as a pentaol, hexaol, heptaol, or the like, which contain 5, 6, or 7 hydroxyl groups, respectively. Additionally, a polyol also may be a sugar alcohol, polyhydric alcohol, or polyalcohol which is a reduced form of carbohydrate, wherein the carbonyl group (aldehyde or ketone, reducing sugar) has been reduced to a primary or secondary hydroxyl group. 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 hydrolyzates, polyglycitols and sugar alcohols or any other carbohydrates capable of being reduced which do not adversely affect the taste of the sweetener composition.

Highly purified target steviol glycoside(s), particularly steviolmonoside, steviolmonoside A, steviolbioside, steviolbioside D, rubusoside, steviolbioside A, steviolbioside B, rebaudioside B, stevioside, rebaudioside G, stevioside A, stevioside B, stevioside C, rebaudioside A, rebaudioside E, rebaudioside E2, rebaudioside E4, rebaudioside E6, rebaudioside E3, rebaudioside D, rebaudioside I, rebaudioside AM, rebaudioside D7, rebaudioside M, rebaudioside M4, rebaudioside la, rebaudioside lb, rebaudioside lc, rebaudioside Id, rebaudioside le, rebaudioside If rebaudioside lg, rebaudioside lh, rebaudioside li, rebaudioside lj, rebaudioside lk, rebaudioside 11, rebaudioside lm, rebaudioside In, rebaudioside 2a and/or SvG7 may be combined with reduced calorie sweeteners such as, for example, D-tagatose, L-sugars, L-sorbose, L- arabinose and combinations thereof.

Highly purified target steviol glycoside(s), particularly steviolmonoside, steviolmonoside A, steviolbioside, steviolbioside D, rubusoside, steviolbioside A, steviolbioside B, rebaudioside B, stevioside, rebaudioside G, stevioside A, stevioside B, stevioside C, rebaudioside A, rebaudioside E, rebaudioside E2, rebaudioside E4 , rebaudioside E6, rebaudioside E3, rebaudioside D, rebaudioside I, rebaudioside AM, rebaudioside D7, rebaudioside M, rebaudioside M4, rebaudioside la, rebaudioside lb, rebaudioside lc, rebaudioside Id, rebaudioside le, rebaudioside If, rebaudioside Ig, rebaudioside lh, rebaudioside li, rebaudioside lj, rebaudioside lk, rebaudioside 11, rebaudioside 1m, rebaudioside In, rebaudioside 2a and/or SvG7 may also be combined with various carbohydrates. The term“carbohydrate” generally refers to aldehyde or ketone compounds substituted with multiple hydroxyl groups, of the general formula (CH 0) n , wherein n is 3-30, as well as their oligomers and polymers. The carbohydrates of the present invention can, in addition, be substituted or deoxygenated at one or more positions. Carbohydrates, as used herein, encompass unmodified carbohydrates, carbohydrate derivatives, substituted carbohydrates, and modified carbohydrates. As used herein, the phrases“carbohydrate derivatives”,“substituted carbohydrate”, and“modified carbohydrates” are synonymous. Modified carbohydrate means any carbohydrate wherein at least one atom has been added, removed, or substituted, or combinations thereof. Thus, carbohydrate derivatives or substituted carbohydrates include substituted and unsubstituted monosaccharides, disaccharides, oligosaccharides, and polysaccharides. The carbohydrate derivatives or substituted carbohydrates optionally can be deoxygenated at any corresponding C-position, and/or substituted with one or more moieties such as hydrogen, halogen, haloalkyl, carboxyl, acyl, acyloxy, amino, amido, carboxyl derivatives, alkylamino, dialkylamino, arylamino, alkoxy, aryloxy, nitro, cyano, sulfo, mercapto, imino, sulfonyl, sulfenyl, sulf yl, sulfamoyl, carboalkoxy, carboxamido, phosphonyl, phosphinyl, phosphoryl, phosphino, thioester, thioether, oximino, hydrazino, carbamyl, phospho, phosphonato, or any other viable functional group provided the carbohydrate derivative or substituted carbohydrate functions to improve the sweet taste of the sweetener composition.

Examples of carbohydrates which may be used in accordance with this invention include, but are not limited to, psicose, turanose, allose, tagatose, trehalose, galactose, rhamnose, various cyclodextrins, cyclic oligosaccharides, various types of maltodextrins, dextran, sucrose, glucose, ribulose, fructose, threose, arabinose, xylose, lyxose, altrose, mannose, idose, lactose, maltose, invert sugar, isotrehalose, neotrehalose, isomaltulose, erythrose, deoxyribose, gulose, idose, talose, erythrulose, xylulose, psicose, turanose, cellobiose, amylopectin, glucosamine, mannosamine, fucose, glucuronic acid, gluconic acid, glucono-lactone, abequose, galactosamine, beet oligosaccharides, isomalto- oligosaccharides (isomaltose, isomaltotriose, panose and the like), xylo-oligosaccharides (xylotriose, xylobiose and the like), xylo-terminated oligosaccharides, gentio- oligosaccharides (gentiobiose, gentiotriose, gentiotetraose and the like), sorbose, nigero- oligosaccharides, palatinose oligosaccharides, fructooligosaccharides (kestose, nystose and the like), maltotetraol, maltotriol, malto-oligosaccharides (maltotriose, maltotetraose, maltopentaose, maltohexaose, maltoheptaose and the like), starch, inulin, inulo- oligosaccharides, lactulose, melibiose, raffinose, ribose, isomerized liquid sugars such as high fructose corn syrups, coupling sugars, and soybean oligosaccharides. Additionally, the carbohydrates as used herein may be in either the D- or L-configuration.

Highly purified target steviol glycoside(s), particularly steviolmonoside, steviolmonoside A, steviolbioside, steviolbioside D, rubusoside, steviolbioside A, steviolbioside B, rebaudioside B, stevioside, rebaudioside G, stevioside A, stevioside B, stevioside C, rebaudioside A, rebaudioside E , rebaudioside E2, rebaudioside E4, rebaudioside E6, rebaudioside E3, rebaudioside D, rebaudioside I, rebaudioside AM, rebaudioside D7, rebaudioside M, rebaudioside M4, rebaudioside la, rebaudioside lb, rebaudioside lc, rebaudioside Id, rebaudioside le, rebaudioside If, rebaudioside Ig, rebaudioside lh, rebaudioside li, rebaudioside lj, rebaudioside lk, rebaudioside 11, rebaudioside lm, rebaudioside In, rebaudioside 2a and/or SvG7 obtained according to this invention can be used in combination with various physiologically active substances or functional ingredients. Functional ingredients generally are classified into categories such as carotenoids, dietary fiber, fatty acids, saponins, antioxidants, nutraceuticals, flavonoids, isothiocyanates, phenols, plant sterols and stands (phytosterols and phytostanols); polyols; prebiotics, probiotics; phytoestrogens; soy protein; sulfides/thiols; amino acids; proteins; vitamins; and minerals. Functional ingredients also may be classified based on their health benefits, such as cardiovascular, cholesterol-reducing, and anti-inflammatory. Exemplary functional ingredients are provided in WO2013/096420, the contents of which is hereby incorporated by reference.

Highly purified target steviol glycoside(s), particularly steviolmonoside, steviolmonoside A, steviolbioside, steviolbioside D, rubusoside, steviolbioside A, steviolbioside B, rebaudioside B, stevioside, rebaudioside G, stevioside A, stevioside B, stevioside C, rebaudioside A, rebaudioside E, rebaudioside E2, rebaudioside E4, rebaudioside E6 , rebaudioside E3, rebaudioside D, rebaudioside 1, rebaudioside AM, rebaudioside D7, rebaudioside M, rebaudioside M4, rebaudioside la, rebaudioside lb, rebaudioside lc, rebaudioside Id, rebaudioside le, rebaudioside If, rebaudioside lg, rebaudioside lh, rebaudioside li, rebaudioside lj, rebaudioside lk, rebaudioside 11, rebaudioside 1m, rebaudioside In, rebaudioside 2a and/or SvG7 obtained according to this invention may be applied as a high intensity sweetener to produce zero calorie, reduced calorie or diabetic beverages and food products with improved taste characteristics. It may also be used in drinks, foodstuffs, pharmaceuticals, and other products in which sugar cannot be used. In addition, highly purified target steviol glycoside(s), particularly steviolmonoslde, steviolmonoside A, steviolbioside, steviolbioside D, rubusoside, steviolbioside A, steviolbioside B, rebaudioside B, stevioside, rebaudioside G, stevioside A, stevioside B, stevioside C, rebaudioside A, rebaudioside E, rebaudioside E2, rebaudioside E4, rebaudioside E6, rebaudioside E3, rebaudioside D, rebaudioside 1, rebaudioside AM, rebaudioside D7, rebaudioside M, rebaudioside M4, rebaudioside la, rebaudioside lb, rebaudioside lc, rebaudioside Id, rebaudioside le, rebaudioside If rebaudioside lg, rebaudioside lh, rebaudioside li, rebaudioside lj, rebaudioside lk, rebaudioside 11, rebaudioside lm, rebaudioside In, rebaudioside 2a and/or SvG7 can be used as a sweetener not only for drinks, foodstuffs, and other products dedicated for human consumption, but also in animal feed and fodder with improved characteristics.

Highly purified target steviol glycoside(s), particularly steviolmonoside, steviolmonoside A, steviolbioside, steviolbioside D, rubusoside, steviolbioside A, steviolbioside B, rebaudioside B, stevioside, rebaudioside G, stevioside A, stevioside B, stevioside C, rebaudioside A, rebaudioside E, rebaudioside E2, rebaudioside E4, rebaudioside E6, rebaudioside E3, rebaudioside D, rebaudioside I, rebaudioside AM, rebaudioside D7, rebaudioside M, rebaudioside M4, rebaudioside la, rebaudioside lb, rebaudioside lc, rebaudioside Id, rebaudioside le, rebaudioside If rebaudioside lg, rebaudioside lh, rebaudioside li, rebaudioside lj, rebaudioside lk, rebaudioside 11, rebaudioside lm, rebaudioside In, rebaudioside 2a and/or SvG7 obtained according to this invention may be applied as a foaming suppressor to produce zero calorie, reduced calorie or diabetic beverages and food products.

Examples of consumable products in which highly purified target steviol glycoside(s), particularly steviolmonoside, steviolmonoside A, steviolbioside, steviolbioside D, rubusoside, steviolbioside A, steviolbioside B, rebaudioside B, stevioside, rebaudioside G, stevioside A, stevioside B, stevioside C, rebaudioside A, rebaudioside E, rebaudioside E2, rebaudioside E4, rebaudioside E6, rebaudioside E3, rebaudioside D, rebaudioside /, rebaudioside AM, rebaudioside D7, rebaudioside M, rebaudioside M4, rebaudioside la, rebaudioside lb, rebaudioside lc, rebaudioside Id, rebaudioside le, rebaudioside If, rebaudioside lg, rebaudioside lh, rebaudioside li, rebaudioside lj, rebaudioside lk, rebaudioside 11, rebaudioside lm, rebaudioside In, rebaudioside 2a and/or SvG7 may be used as a sweetening compound include, but are not limited to, alcoholic beverages such as vodka, wine, beer, liquor, and sake, etc.; natural juices; refreshing drinks; carbonated soft drinks; diet drinks; zero calorie drinks; reduced calorie drinks and foods; yogurt drinks; instant juices; instant coffee; powdered types of instant beverages; canned products; syrups; fermented soybean paste; soy sauce; vinegar; dressings; mayonnaise; ketchups; curry; soup; instant bouillon; powdered soy sauce; powdered vinegar; types of biscuits; rice biscuit; crackers; bread; chocolates; caramel; candy; chewing gum; jelly; pudding; preserved fruits and vegetables; fresh cream; jam; marmalade; flower paste; powdered milk; ice cream; sorbet; vegetables and fruits packed in bottles; canned and boiled beans; meat and foods boiled in sweetened sauce; agricultural vegetable food products; seafood; ham; sausage; fish ham; fish sausage; fish paste; deep fried fish products; dried seafood products; frozen food products; preserved seaweed; preserved meat; tobacco; medicinal products; and many others. In principle it can have unlimited applications.

Examples of consumable products in which highly purified target steviol glycoside(s), particularly steviolmonoside, steviolmonoside A, steviolbioside, steviolbioside D, rubusoside, steviolbioside A, steviolbioside B, rebaudioside B, stevioside, rebaudioside G, stevioside A, stevioside B, stevioside C, rebaudioside A, rebaudioside E, rebaudioside E2, rebaudioside E4, rebaudioside E6, rebaudioside E3, rebaudioside D, rebaudioside I, rebaudioside AM, rebaudioside D7, rebaudioside M, rebaudioside M4, rebaudioside la, rebaudioside lb, rebaudioside lc, rebaudioside Id, rebaudioside le, rebaudioside If, rebaudioside lg, rebaudioside lh, rebaudioside li, rebaudioside lj, rebaudioside lk, rebaudioside 11, rebaudioside lm, rebaudioside In, rebaudioside 2a and/or SvG7 may be used as a flavor modifier or flavor with modifying properties include, but are not limited to, alcoholic beverages such as vodka, wine, beer, liquor, and sake, etc.; natural juices; refreshing drinks; carbonated soft drinks; diet drinks; zero calorie drinks; reduced calorie drinks and foods; yogurt drinks; instant juices; instant coffee; powdered types of instant beverages; canned products; syrups; fermented soybean paste; soy sauce; vinegar; dressings; mayonnaise; ketchups; curry; soup; instant bouillon; powdered soy sauce; powdered vinegar; types of biscuits; rice biscuit; crackers; bread; chocolates; caramel; candy; chewing gum; jelly; pudding; preserved fruits and vegetables; fresh cream; jam; marmalade; flower paste; powdered milk; ice cream; sorbet; vegetables and fruits packed in bottles; canned and boiled beans; meat and foods boiled in sweetened sauce; agricultural vegetable food products; seafood; ham; sausage; fish ham; fish sausage; fish paste; deep fried fish products; dried seafood products; frozen food products; preserved seaweed; preserved meat; tobacco; medicinal products; and many others. In principle it can have unlimited applications.

During the manufacturing of products such as foodstuffs, drinks, pharmaceuticals, cosmetics, table top products, and chewing gum, the conventional methods such as mixing, kneading, dissolution, pickling, permeation, percolation, sprinkling, atomizing, infusing and other methods may be used.

Moreover, the highly purified target steviol glycoside(s) steviolmonoside, steviolmonoside A, steviolbioside, steviolbioside D, rubusoside, steviolbioside A, steviolbioside B, rebaudioside B, stevioside, rebaudioside G, stevioside A, stevioside B , stevioside C, rebaudioside A, rebaudioside E, rebaudioside E2, rebaudioside E4 , rebaudioside E6, rebaudioside E3 , rebaudioside D, rebaudioside /, rebaudioside AM, rebaudioside D7, rebaudioside M, rebaudioside M4, rebaudioside la, rebaudioside lb, rebaudioside lc, rebaudioside Id, rebaudioside le, rebaudioside If, rebaudioside lg, rebaudioside lh, rebaudioside li, rebaudioside lj, rebaudioside Ik, rebaudioside 11, rebaudioside lm, rebaudioside In, rebaudioside 2a and/or SvG7 obtained in this invention may be used in dry or liquid forms.

The highly purified target steviol glycoside can be added before or after heat treatment of food products. The amount of the highly purified target steviol glycoside(s), particularly steviolmonoside, steviolmonoside A, steviolbioside, steviolbioside D, rubusoside, steviolbioside A, steviolbioside B, rebaudioside B, stevioside, rebaudioside G, stevioside A, stevioside B, stevioside C, rebaudioside A, rebaudioside E, rebaudioside E2, rebaudioside E4, rebaudioside E6, rebaudioside E3, rebaudioside D, rebaudioside I, rebaudioside AM, rebaudioside D7, rebaudioside M, rebaudioside M4, rebaudioside la, rebaudioside lb, rebaudioside lc, rebaudioside Id, rebaudioside le, rebaudioside If, rebaudioside lg, rebaudioside lh, rebaudioside li, rebaudioside lj, rebaudioside lk, rebaudioside 11, rebaudioside lm, rebaudioside In, rebaudioside 2a and/or SvG7 depends on the purpose of usage. As discussed above, it can be added alone or in combination with other compounds.

The present invention is also directed to sweetness enhancement in beverages using steviolmonoside, steviolmonoside A, steviolbioside, steviolbioside D, rubusoside, steviolbioside A, steviolbioside B, rebaudioside B, stevioside, rebaudioside G, stevioside A, stevioside B, stevioside C, rebaudioside A, rebaudioside E, rebaudioside E2, rebaudioside E4, rebaudioside E6, rebaudioside E3, rebaudioside D, rebaudioside 1, rebaudioside AM, rebaudioside D7, rebaudioside M, rebaudioside M4, rebaudioside la, rebaudioside lb, rebaudioside lc, rebaudioside Id, rebaudioside le, rebaudioside If rebaudioside lg, rebaudioside lh, rebaudioside li, rebaudioside lj, rebaudioside lk, rebaudioside 11, rebaudioside lm, rebaudioside In, rebaudioside 2a and/or SvG7 as a sweetness enhancer, wherein steviolmonoside, steviolmonoside A, steviolbioside, steviolbioside D, rubusoside, steviolbioside A, steviolbioside B, rebaudioside B, stevioside, rebaudioside G, stevioside A, stevioside B, stevioside C , rebaudioside A, rebaudioside E, rebaudioside E2, rebaudioside E4, rebaudioside E6, rebaudioside E3, rebaudioside D, rebaudioside 1, rebaudioside AM, rebaudioside D7, rebaudioside M, rebaudioside M4, rebaudioside la, rebaudioside lb, rebaudioside lc, rebaudioside Id, rebaudioside le, rebaudioside If rebaudioside lg, rebaudioside lh, rebaudioside li, rebaudioside lj, rebaudioside lk, rebaudioside 11, rebaudioside lm, rebaudioside In, rebaudioside 2a and/or SvG7 is present in a concentration at or below their respective sweetness recognition thresholds.

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

The term “sweetness recognition threshold concentration,” as generally used herein, is the lowest known concentration of a sweet compound that is perceivable by the human sense of taste, typically around 1.0% sucrose equivalence (1.0% SE). Generally, the sweetness enhancers may enhance or potentiate the sweet taste of sweeteners without providing any noticeable sweet taste by themselves when present at or below the sweetness recognition threshold concentration of a given sweetness enhancer; however, the sweetness enhancers may themselves provide sweet taste at concentrations above their sweetness recognition threshold concentration. The sweetness recognition threshold concentration is specific for a particular enhancer and can vary based on the beverage matrix. The sweetness recognition threshold concentration can be easily determined by taste testing increasing concentrations of a given enhancer until greater than 1.0% sucrose equivalence in a given beverage matrix is detected. The concentration that provides about 1.0% sucrose equivalence is considered the sweetness recognition threshold.

In some embodiments, sweetener is present in the beverage in an amount from about 0.0001% to about 12% by weight, such as, for example, 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 1 1.0% by weight, about 1 1.5% by weight or about 12.0% by weight.

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

In another embodiment, the sweetener is erythritol.

In still 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 a sweetener can be used alone, or in combination with other sweeteners.

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

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

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

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

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

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

In yet another embodiment, the rare sugar is D-turanose. In a more particular embodiment, D-turanose is present in the beverage in an amount of about 0.5% to about 10% by weight, such as, for example, from about 2% to about 8%,

In yet another embodiment, the rare sugar is D-leucrose. In a more particular embodiment, D-leucrose is present in the beverage in an amount of about 0.5% to about 10% by weight, such as, for example, from about 2% to about 8%.

The addition of the sweetness enhancer at a concentration at or below its sweetness recognition threshold increases the detected sucrose equivalence of the beverage comprising the sweetener and the sweetness enhancer compared to a corresponding beverage in the absence of the sweetness enhancer. Moreover, sweetness can be increased by an amount more than the detectable sweetness of a solution containing the same concentration of the at least one sweetness enhancer in the absence of any sweetener.

Accordingly, the present invention also provides a method for enhancing the sweetness of a beverage comprising a sweetener comprising providing a beverage comprising a sweetener and adding a sweetness enhancer selected from steviolmonoside, steviolmonoside A, steviolbioside, steviolbioside D, rubusoside, steviolbioside A, steviolbioside B, rebaudioside B, stevioside, rebaudioside G, stevioside A, stevioside B, stevioside C, rebaudioside A, rebaudioside E, rebaudioside E2, rebaudioside E4, rebaudioside E6, rebaudioside E3, rebaudioside D, rebaudioside /, rebaudioside AM, rebaudioside D7, rebaudioside M, rebaudioside M4 , rebaudioside la, rebaudioside lb, rebaudioside lc, rebaudioside Id, rebaudioside le, rebaudioside If, rebaudioside Ig, rebaudioside lh, rebaudioside li, rebaudioside lj, rebaudioside lk, rebaudioside 11, rebaudioside lm, rebaudioside In, rebaudioside 2a and/or SvG7 or a combination thereof, wherein steviolmonoside, steviolmonoside A, steviolbioside, steviolbioside D, rubusoside, steviolbioside A, steviolbioside B, rebaudioside B, stevioside, rebaudioside G, stevioside A, stevioside B, stevioside C, rebaudioside A, rebaudioside E, rebaudioside E2, rebaudioside E4, rebaudioside E6, rebaudioside E3, rebaudioside D, rebaudioside 1, rebaudioside AM, rebaudioside D7, rebaudioside M, rebaudioside M4, rebaudioside la, rebaudioside lb, rebaudioside lc, rebaudioside Id, rebaudioside le, rebaudioside If, rebaudioside lg, rebaudioside Ih, rebaudioside li, rebaudioside lj, rebaudioside Ik, rebaudioside 11, rebaudioside 1m, rebaudioside In, rebaudioside 2a and/or SvG7 are present in a concentration at or below their sweetness recognition thresholds.

Addition of steviolmonoside, steviolmonoside A, steviolbioside, steviolbioside D, rubusoside, steviolbioside A, steviolbioside B, rebaudioside B, stevioside, rebaudioside G, stevioside A, stevioside B, stevioside C, rebaudioside A, rebaudioside E, rebaudioside E2, rebaudioside E4, rebaudioside E6, rebaudioside E3, rebaudioside D, rebaudioside 1, rebaudioside AM, rebaudioside D7, rebaudioside M, rebaudioside M4, rebaudioside la, rebaudioside lb, rebaudioside lc, rebaudioside Id, rebaudioside le, rebaudioside If rebaudioside lg, rebaudioside lh, rebaudioside 11, rebaudioside lj, rebaudioside lk, rebaudioside 11, rebaudioside hn, rebaudioside In, rebaudioside 2a and/or SvG7 in a concentration at or below the sweetness recognition threshold to a beverage containing a sweetener may increase the detected sucrose equivalence from about 1.0% to about 5.0%, such as, for example, about 1.0%, about 1.5%, about 2.0%, about 2.5%, about 3.0%, about 3.5%, about 4.0%, about 4.5% or about 5.0%.

The following examples illustrate preferred embodiments of the invention for the preparation of highly purified target steviol glycoside(s), particularly steviolmonoside, steviolmonoside A, steviolbioside, steviolbioside D, rubusoside, steviolbioside A, steviolbioside B, rebaudioside B, stevioside, rebaudioside G, stevioside A, stevioside B, stevioside C, rebaudioside A, rebaudioside E, rebaudioside E2, rebaudioside E4, rebaudioside E6, rebaudioside E3, rebaudioside D, rebaudioside /, rebaudioside AM, rebaudioside D7, rebaudioside M, rebaudioside M4, rebaudioside la, rebaudioside lb, rebaudioside lc, rebaudioside Id, rebaudioside le, rebaudioside If rebaudioside lg, rebaudioside lh, rebaudioside li, rebaudioside lj, rebaudioside lk, rebaudioside 11, rebaudioside lm, rebaudioside In, rebaudioside 2a and/or SvG7. It will be understood that the invention is not limited to the materials, proportions, conditions and procedures set forth in the examples, which are only illustrative.

EXAMPLES EXAMPLE 1

Protein sequences of engineered enzymes used in the biocatalytic process

SEQ ID 1 :

>SuSy_At, variant PM1 -54-2-E05 (engineered sucrose synthase; source of WT gene: Arabidopsis thaliana)

MANAERMITRVHSQRERLNETLVSERNEVLALLSRVEAKGKGILQQNQII AEFEALPEQTRKKLEGGPFFDLLKSTQEAIVLPPWVALAVRPRPGVWEYL RVNLHALVVEELQPAEFLHFKEELVDGVKNGNFTLELDFEPFNASI PRPT LHKYIGNGVDFLNRHLSAKLFHDKESLLPLLDFLRLHSHQGKNLMLSEKI QNLNTLQHTLRKAEEYLAELKSETLYEEFEAKFEEIGLERGWGDNAERVL DMIRLLLDLLEAPDPSTLETFLGRVPMVFNVVILSPHGYFAQDNVLGYPD TGGQVVYILDQVRALEIEMLQRIKQQGLNIKPRILILTRLLPDAVGTTCG ERLERVYDSEYCDILRVPFRTEKGIVRKWISRFEVWPYLETYTEDAAVEL SKELNGKPDLI IGNYS DGNLVASLLAHKLGVTQCTIAHALEKTKYPDSDI YWKKLDDKYHFSCQFTADIFAMNHTDFI ITSTFQEIAGSKETVGQYESHT

AFTLPGLYRVVHGIDVFDPKFNIVSPGADMS IYFPYTEEKRRLTKFHSEI EELLYSDVENDEHLCVLKDKKKPILFTMARLDRVKNLSGLVEWYGKNTRL RELVNLVVVGGDRRKESKDNEEKAEMKKMYDLIEEYKLNGQFRWISSQMD RVRNGELYRYICDTKGAFVQPALYEAFGLTVVEAMTCGLPTFATCKGGPA EIIVHGKSGFHI DPYHGDQAADLLADFFTKCKEDPSHWDEISKGGLQRIE EKYTWQIYSQRLLTLTGVYGFWKHVSNLDRLEHRRYLEMFYALKYRPLAQ AVPLAQDD

SEQ ID 2:

>UGTS12 variant 0234 (engineered glucosyltransferase; source of WT gene: Solatium lycopersicum )

MATNLRVLMFPWLAYGHISPFLNIAKQLADRGFLIYLCSTRINLESI IKK IPEKYADSIHLIELQLPELPELPPHYHTTNGLPPHLNPTLHKALKMSKPN FSRILQNLKPDLLIYDVLQPWAEHVANEQGIPAGKLLVSCAAVFSYFFSF RKNPGVEFPFPAIHLPEVEKVKIREILAKEPEEGGRLDEGNKQMMLMCTS RTIEAKYIDYCTELCNWKVVPVGPPFQDLITNDADNKELIDWLGTKPENS TVFVSFGSEYFLSKEDMEEIAFALEASNVNFIWVVRFPKGEERNLEDALP EGFLERIGERGRVLDKFAPQPRILNHPSTGGFI SHCG NSVMESIDFGVP IIAMPIHNDQPINAKLMVELGVAVEIVRDDDGKIHRGEIAEALKSVVTGE TGEILRAKVREISKNLKS IRDEEMDAVAEELIQLCRNSNKSK

SEQ ID 3:

>UGT76G1 variant 0042 (engineered glucosyltransferase; source of WT gene: Stevia rebaudiand)

MENKTETTVRRRRRI ILFPVPFQGHINPILQLANVLYSKGFAITILHTNFNKPKTSNYPH FTFRFILDNDPQDERISNLPTHGPLAGMRI PI INEHGADELRRELELLMLASEEDEEVSC LITDALWYFAQDVADSLNLRRLVLMTSSLFNFHAHVSLPQFDELGYLDPDDKTRLEEQAS GFPMLKVKDIKSAYSNWQIGKEILGKMIKQTKASSGVIWNSFKELEESELETVIREI PAP SFLIPLPKHLTASSSSLLDHDRTVFEWLDQQAPSSVLYVSFGSTSEVDEKDFLEIARGLV DSGQSFLWVVRPGFVKGSTWVEPLPDGFLGERGKIVKWVPQQEVLAHPAIGAFWTHSGWN STLESVCEGVPMI FSSFGGDQPLNARYMSDVLRVGVYLENGWERGEVVNAIRRVMVDEEG EYIRQNARVLKQKADVSLMKGGSSYESLESLVSYISSL EXAMPLE 2

Expression and formulation of SuSy At variant of SEQ ID 1

The gene coding for the SuSy_At variant of SEQ ID 1 (EXAMPLE 1) was cloned into the expression vector pLElA17 (derivative of pRSF-l b, Novagen). The resulting plasmid was used for transformation of E.coli BL21(DE3) cells.

Cells were cultivated in ZYM505 medium (F. William Studier, Protein Expression and Purification 41 (2005) 207-234) supplemented with kanamycin (50 mg/1) at 37°C. Expression of the genes was induced at logarithmic phase by IPTG (0.2 mM) and carried out at 30°C and 200 rpm for 16-18 hours.

Cells were harvested by centrifugation (3220 x g, 20 min, 4°C) and re-suspended to an optical density of 200 (measured at 600nm (OD 6 oo)) with cell lysis buffer (100 M Tris-HCl pH 7.0; 2 mM MgCl 2 , DNA nuclease 20 U/mL, lysozyme 0.5 mg/mL). Cells were then disrupted by sonication and crude extracts were separated from cell debris by centrifugation (18000 x g 40 min, 4°C). The supernatant was sterilized by filtration through a 0.2 pm filter and diluted 50:50 with distilled water, resulting in an enzymatic active preparation.

For enzymatic active preparations of SuSy_At, activity in Units is defined as follows: 1 mU of SuSy_At turns over 1 nmol of sucrose into fructose in 1 minute. Reaction conditions for the assay are 30°C, 50 mM potassium phosphate buffer pH 7.0, 400 M sucrose at t 0 , 3 mM MgCfe, and 15 mM uridine diphosphate (UDP).

EXAMPLE 3

Expression and formulation of UGTS12 variant of SEQ ID 2

The gene coding for the UGTS12 variant of SEQ ID 2 (EXAMPLE 1 ) was cloned into the expression vector pLElA17 (derivative of pRSF- l b, Novagen). The resulting plasmid was used for transformation of E.coli BL21(DE3) cells.

Cells were cultivated in ZYM505 medium (F, William Studier, Protein Expression and Purification 41 (2005) 207-234) supplemented with kanamycin (50 mg/1) at 37°C. Expression of the genes was induced at logarithmic phase by IPTG (0.1 mM) and carried out at 30°C and 200 rpm for 16-18 hours. Cells were harvested by centrifugation (3220 x g, 20 min, 4°C) and re-suspended to an optical density of 200 (measured at 600nm (OD 6 oo)) with cell lysis buffer (100 mM Tris-HCl pH 7.0; 2 mM MgCl 2 , DNA nuclease 20 U/mL, lysozyme 0.5 mg/mL). Cells were then disrupted by sonication and crude extracts were separated from cell debris by centrifugation (18000 x g 40 min, 4°C). The supernatant was sterilized by filtration through a 0.2 pm filter and diluted 50:50 with 1 M sucrose solution, resulting in an enzymatic active preparation.

For enzymatic active preparations of UGTS12, activity in Units is defined as follows: 1 mU of UGTS12 turns over 1 nmol of rebaudioside A (Reb A) into rebaudioside D (Reb D) in 1 minute. Reaction conditions for the assay are 30°C, 50 mM potassium phosphate buffer pH 7.0, 10 mM Reb A at to, 500 mM sucrose, 3 mM MgCl 2 , 0.25 mM uridine diphosphate (UDP) and 3 U/mL of SuSy_At.

EXAMPLE 4

Expression and formulation of UGT76G1 variant of SEQ ID 3

The gene coding for the UGT76G1 variant of SEQ ID 3 (EXAMPLE 1) was cloned into the expression vector pLElA17 (derivative of pRSF-lb, Novagen). The resulting plasmid was used for transformation of E.coli BL21(DE3) cells.

Cells were cultivated in ZYM505 medium (F. William Studier, Protein Expression and Purification 41 (2005) 207-234) supplemented with kanamycin (50 mg/1) at 37°C. Expression of the genes was induced at logarithmic phase by IPTG (0.1 mM) and carried out at 30°C and 200 rpm for 16-18 hours.

Cells were harvested by centrifugation (3220 x g, 20 in in, 4°C) and re-suspended to an optical density of 200 (measured at 600nm (OD 60 o)) with cell lysis buffer (100 mM Tris-HCl pH 7.0; 2 mM MgCl 2 , DNA nuclease 20 U/mL, lysozyme 0.5 mg/mL). Cells were then disrupted by sonication and crude extracts were separated from cell debris by centrifugation (18000 x g 40 min, 4°C). The supernatant was sterilized by filtration through a 0.2 pm filter and diluted 50:50 with 1 M sucrose solution, resulting in an enzymatic active preparation.

For enzymatic active preparations of UGT76G1, activity in Units is defined as follows: 1 mU of UGT76G1 turns over 1 nmol of rebaudioside D (Reb D ) into rebaudioside M (Reb M) in 1 minute. Reaction conditions for the assay are 30°C, 50 mM potassium phosphate buffer pH 7.0, 10 mM Reb D at to, 500 mM sucrose, 3 mM MgCl 2 , 0.25 mM uridine diphosphate (UDP) and 3 U/mL of SuSy_At.

EXAMPLE 5

Synthesis of SvG7 in a one-pot reaction, adding UGTSI2, SuSy At and UGT76G1 at the same time.

Various SvG7 molecules were synthesized directly from stevioside (see Fig. 4) in a one-pot reaction, utilizing the three enzymes (see EXAMPLES 1, 2, 3 and 4): UGTS12 (variant of SEQ ID 2), SuSy_At (variant of SEQ ID 1) and UGT76G1 (variant of SEQ ID 3).

The final reaction solution contained 348 U/L UGTS12, 1341 U/L SuSy_At, 10 U/L UGT76G1, 47 mM stevioside, 0.32 mM uridine diphosphate (UDP), 0.99 M sucrose, 3.9 mM MgCl 2 and potassium phosphate buffer (pH 6.6). First, 206 mL of distilled water were mixed with 0.24 g MgCl 2 .6H 2 0, 102 g sucrose, 9.8 mL of 1.5 M potassium phosphate buffer (pH 6.6) and 15 g stevioside. The final volume of the reaction mixture was adjusted to 300 mL.

After dissolving the components, the temperature was adjusted to 45 °C and UGTS12, SuSy_At, UGT76G1 and 39 mg UDP were added. The reaction mixture was incubated at 45°C shaker for 24 hrs. Additional 39 mg UDP was added at 12 hours, 24 hours, and 36 hours. The content of reb 2a and various SvG7 at the end of the reaction (48 hours) was analyzed by HPLC.

EXAMPLE 6

HPLC Analysis

For analysis, biotransformation samples were inactivated by adjusting the reaction mixture to pH5.5 using 17% H 3 PO 4 and then boiled for 10 minutes. Resulting samples were filtered, the filtrates were diluted 10 times and used as samples for HPLC analysis. HPLC assay was carried out on Agilent HP 1200 HPLC system, comprised of a pump, a column thermostat, an auto sampler, a UV detector capable of background correction and a data acquisition system. Analytes were separated using Agilent Poroshell 120 SB- C18, 4.6 m x 150 m , 2.7 pm at 40°C. The mobile phase consisted of two premixes:

- premix 1 containing 75% 10 mM phosphate buffer (pH2.6) and 25% acetonitrile, and premix 2 containing 68% 10 mM phosphate buffer (pH2.6) and 32% acetonitrile.

Elution gradient started with premix 1, changed to premix 2 to 50% at 12.5 minute, changed to premix 2 to 100% at 13 minutes. Total run time was 45 minutes. The column temperature was maintained at 40 °C. The injection volume was 5 pL. Rebaudioside species were detected by UV at 210 nm.

Table 3 shows for each time point the conversion of stevioside into identified rebaudioside species (area percentage). The chromatograms of the starting material stevioside and the reaction mixture at 48 hours are shown in Fig. 4 and Fig. 5 respectively. Those with skill in the art will appreciate that retention times can occasionally vary with changes in solvent and/or equipment.

Table 3

Biotransformation of stevioside to reb 2a (rt 6.459) and various SvG7

EXAMPLE 7

Purification of rebaudioside 2a and various SvG7

300 mL of the reaction mixture of EXAMPLE 5, (after 48 hrs), was inactivated by adjusting the pH to pH 5.5 with H 3 PO 4 and then boiled for 10 minutes and filtered. The filtrate was loaded into a column containing 500 mL YWD03 (Cangzhou Yuanwei, China) resin pre-equilibrated with water. The resin was washed with 2.5 L water and the water effluent from this step was discarded.

The steviol glycosides were eluted from the YWD03 resin column by elution with 2.5 L 70 % v/v ethanol/water. The effluent from this step was collected and dried under vacuum at 60°C to yield 20g of dried solid product. This sample was dissolved in water and subjected to further fractionation and separation by HPLC, using the conditions listed in Table 4 below.

HPLC fractions that corresponded to individual compounds from multiple runs were combined according to retention time. The fractions were freeze-dried.

Table 4

Conditions for HPLC

The purity of obtained fractions was evaluated by analytical HPLC method described in EXAMPLE 6. The chromatogram of purified rebaudioside 2a is shown in Fig 6. EXAMPLE 8

Structure elucidation of rebaudioside 2a

NMR experiments were performed on a Brulcer 500 MHz spectrometer, with the sample dissolved in pyridine-r/5. Along with signals from the sample, signals from pyridine-e/J at 5c 123.5, 135.5, 149.9 ppm and 8 H 7.19, 7.55, 8.71 ppm were observed. ‘H-NMR spectrum of rebaudioside 2a recorded in pyridine-r/ confirmed the excellent quality of the sample (see Fig. 7). HSQC (see Fig. 8) shows the presence of an exomethylene group in the sugar region with a long-range coupling to C-15, observable in the H,H-COSY (Fig. 9). Other deep-fielded signals of the quaternary carbons (C-13, C-16 and C-19) are detected by the HMBC (Fig. 10). Correlation of the signals in the HSQC, HMBC and H,H-COSY reveal the presence of steviol glycoside with the following aglycone structure:

Correlation of HSQC and HMBC shows the presence seven anomeric signals, marked with l i, l ii, l iii, l iv, lv, lvi and lvii. The coupling constant of the anomeric protons of about 8 Hz, the broad signals of their sugar linkage and the NOE-correlations of the anomeric protons allow the identification of these seven sugars as b-D- glucopyranosides.

Combined data from HSQC and HMBC reveal the sugar-sugar linkages and sugar- aglycone linkages. The assignment of the sugar sequence was confirmed by using the combination of HSQC-TOCSY (Fig 1 la) and NOESY (Figl l b).

Altogether, results from NMR experiments above were used to assign the chemical shifts of the protons and carbons of the structure of rebaudioside 2a (see Table 5). Table 5

Chemical shifts of rebaudioside 2a

Table 5 (continued) Chemical shifts of rebaudioside 2a Table 5 (continued)

Chemical shifts of rebaudioside 2a

Correlation of all NMR results indicates rebaudioside 2a with seven b-D-glucoses attached to steviol aglycone, as depicted with the following chemical structure:

LCMS (Fig 12a and Fig 12b) analysis of rebaudioside 2a showed a [M-H] ion at m/z 1451.6, in good agreement with the expected molecular formula of C62H100O38 (calculated for [C62H99O38] monoisotopic ioir 1451.6 -). The MS data confirms that rebaudioside 2a has a molecular formula of Ce 2H100O38. LCMS analysis was performed in the following conditions listed in Table 6.

Table 6

Conditions for LCMS analysis