High Purity Steviol Glycosides

Abstract

Highly purified steviol glycosides, especially 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 [0013] Methods for preparing rebaudioside I, 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 to convert various steviol glycosides into target steviol glycosides. High-purity steviol glycosides are useful as non-caloric sweeteners, flavor enhancers, sweetness enhancers, and foam suppressors in edible and chewable compositions such as any beverage, confectionery, bakery products, cookies, and chewing gum.

Description

[Technical field]

[0001] (Sequence Listing) A text file entitled "39227_80PROV_Sequence_Listing_ST25.txt", created on November 27, 2018, having 15 kilobytes of data, and submitted contemporaneously herewith, is hereby incorporated by reference in its entirety into this application.

[0002] FIELD OF THEINVENTION The present invention relates to processes for preparing compositions that include steviol glycosides, such as high purity steviol glycoside compositions. [Background technology]

[0003] High-intensity sweeteners have a sweetness level several times higher than that of sucrose.They are essentially non-caloric and are commonly used in diet and low-calorie foods and beverages.High-intensity sweeteners do not induce glycemic response, and therefore are suitable for use in products targeted at diabetics and others who are concerned about controlling their carbohydrate intake.

[0004] Steviol glycosides are a class of compounds found in the leaves of Stevia rebaudiana Bertoni, a perennial shrub of the Asteraceae family (Compositae family) native to certain regions of South America. They are structurally characterized by a single base, steviol, which differs by the presence of carbohydrate residues at the C13 and C19 positions. They accumulate in Stevia leaves and constitute approximately 10%-20% of the total dry weight. On a dry weight basis, the four major glycosides found in Stevia leaves typically include stevioside (9.1%), rebaudioside A (3.8%), rebaudioside C (0.6-1.0%), and dulcoside A (0.3%). Other known steviol glycosides include rebaudiosides B, C, D, E, F and M, steviolbioside, and rubusoside.

[0005] Although methods are known for preparing steviol glycosides from Stevia rebaudiana, many of these methods are not suitable for commercial use.

[0006] Thus, there remains a need for simple, efficient, and economical methods for preparing compositions that include steviol glycosides, such as high purity steviol glycoside compositions. Summary of the Invention

[0007] The following applications are hereby incorporated by reference in their entireties into 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.

[0008] As used herein, the abbreviation "reb" refers to "rebaudioside." Both terms have the same meaning and may be used interchangeably.

[0009] As used herein, "biocatalysis" or "biocatalytic" refers to the use of natural or genetically engineered biocatalysts, such as enzymes, or cells containing one or more enzymes, capable of single or multi-step chemical transformation of organic compounds. Biocatalytic processes include fermentation, biosynthesis, biological conversion, and biotransformation processes. Both isolated enzyme and whole cell biocatalytic methods are known in the art. Biocatalytic protein enzymes can be naturally occurring or recombinant proteins.

[0010] As used herein, the term "steviol glycoside" refers to naturally occurring steviol glycosides, such as 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 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, synthetic steviol glycosides, e.g., enzymatically glycosylated steviol glycosides, and combinations thereof.

[0011] As used herein, the term "SvG7" refers to any naturally occurring steviol glycoside or any synthetic steviol glycoside, including enzymatically glycosylated steviol glycosides and combinations thereof, specifically, molecules comprising steviol having seven covalently attached glucose residues, including, but not limited to, reb 1a, reb 1b, reb 1c, reb 1d, reb 1e, reb 1f, reb 1g, reb 1h, reb 1i, reb 1j, reb 1k, reb 1l, reb 1m, reb 1n, and / or reb 2a. SvG7 may refer to a single steviol glycoside having seven covalently attached glucose residues or a mixture of steviol glycosides having seven covalently attached glucose residues.

[0012] 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 microbial cells and / or an enzyme preparation, thereby producing a composition comprising a target steviol glycoside.

[0013] The starting composition can be any organic compound containing at least one carbon atom. In one embodiment, the starting composition is selected from the group consisting of steviol glycosides, polyols or sugar alcohols, and various carbohydrates.

[0014] The target steviol glycoside can be any steviol glycoside. In one embodiment, the target steviol glycoside is steviol monoside, steviol monoside A, steviol bioside, steviol bioside D, rubusoside, steviol bioside A, steviol bioside 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 E5, rebaudioside E6, rebaudioside E7, rebaudioside E8, rebaudioside E9, rebaudioside E10, rebaudioside E11, rebaudioside E12, rebaudioside E13, rebaudioside E14, rebaudioside E15, rebaudioside E16, rebaudioside E17, rebaudioside E18, rebaudioside E19, rebaudioside E20, rebaudioside E21, rebaudioside E22, rebaudioside E23, rebaudioside E24, rebaudioside E25, rebaudioside E26, rebaudioside E27, rebaudioside E28, rebaudioside E29, rebaudioside E30, rebaudioside E31, rebaudioside E32, rebaudioside E33, rebaudioside E34, rebaudioside E35, rebaudioside E36, rebaudioside E37, rebaudioside E38, rebaudioside E39, rebaudioside E40, rebaudioside E41, rebaudioside E4 Rebaudioside I, 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, SvG7, or synthetic steviol glycosides.

[0015] In one embodiment, the target steviol glycoside is rebaudioside 1a.

[0016] In one embodiment, the target steviol glycoside is rebaudioside 1b.

[0017] In one embodiment, the target steviol glycoside is rebaudioside 1c.

[0018] In one embodiment, the target steviol glycoside is rebaudioside 1d.

[0019] In one embodiment, the target steviol glycoside is rebaudioside 1e.

[0020] In one embodiment, the target steviol glycoside is rebaudioside 1f.

[0021] In one embodiment, the target steviol glycoside is rebaudioside 1g.

[0022] In one embodiment, the target steviol glycoside is rebaudioside 1h.

[0023] In one embodiment, the target steviol glycoside is rebaudioside 1i.

[0024] In one embodiment, the target steviol glycoside is rebaudioside 1j.

[0025] In one embodiment, the target steviol glycoside is rebaudioside 1k.

[0026] In one embodiment, the target steviol glycoside is rebaudioside 1l.

[0027] In one embodiment, the target steviol glycoside is rebaudioside 1m.

[0028] In one embodiment, the target steviol glycoside is rebaudioside 1n.

[0029] In one embodiment, the target steviol glycoside is rebaudioside 2a.

[0030] In one embodiment, the target steviol glycoside is rebaudioside M4.

[0031] In one embodiment, the target steviol glycoside is SvG7.

[0032] In some preferred embodiments, an enzyme preparation containing one or more enzymes or microbial cells containing one or more enzymes capable of converting the starting composition to the target steviol glycosides is used. The enzymes can be located on the surface and / or inside the cells. The enzyme preparation can be provided in the form of a whole cell suspension, a crude lysate, or as purified enzymes. The enzyme preparation can be in free form or immobilized on a solid support made of inorganic or organic materials.

[0033] In some embodiments, the microbial cells contain the necessary enzymes and genes encoding them to convert a starting composition to a target steviol glycoside. Thus, 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 microbial cells comprising at least one enzyme capable of converting the starting composition to a target steviol glycoside, thereby producing a medium comprising at least one target steviol glycoside.

[0034] Enzymes necessary to convert the starting composition to the target steviol glycosides include steviol biosynthetic enzymes, NDP-glucosyltransferase (NGT), ADP-glucosyltransferase (AGT), CDP-glucosyltransferase (CGT), GDP-glucosyltransferase (GGT), TDP-glucosyltransferase (TDP), UDP-glucosyltransferase (UGT), and / or NDP-recycling enzymes, ADP-recycling enzymes, CDP-recycling enzymes, GDP-recycling enzymes, TDP-recycling enzymes, and / or UDP-recycling enzymes.

[0035] In one embodiment, steviol biosynthetic enzymes include mevalonate (MVA) pathway enzymes.

[0036] In another embodiment, steviol biosynthetic enzymes include non-mevalonate 2-C-methyl-D-erythritol-4-phosphate pathway (MEP / DOXP) enzymes.

[0037] In one embodiment, the steviol biosynthetic enzymes are geranylgeranyl diphosphate synthase, copalyl diphosphate synthase, kaurene synthase, kaurene oxidase, kaurenoic acid 13-hydroxylase (KAH), steviol synthetase, deoxyxylulose 5-phosphate synthase (DXS), D-1-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, and the like.

[0038] The UDP-glucosyltransferase can be any UDP-glucosyltransferase capable of adding at least one glucose unit to steviol and / or steviol glycoside substrates to provide the target steviol glycoside.

[0039] As used hereinafter, the term "SuSy_AT", unless otherwise specified, refers to a sucrose synthase having the amino acid sequence "SEQ ID NO:1" as described in Example 1, or a polypeptide 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 NO:1 polypeptide, as well as isolated nucleic acid molecules encoding these polypeptides.

[0040] As used hereinafter, the term "UGTSl2", unless otherwise specified, refers to a UDP-glucosyltransferase having the amino acid sequence "SEQ ID NO:2" as described in Example 1, or a polypeptide 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 NO:2 polypeptide, as well as isolated nucleic acid molecules encoding these polypeptides.

[0041] As used hereinafter, the term "UGT76G1," unless otherwise specified, refers to a UDP-glucosyltransferase having the amino acid sequence "SEQ ID NO:3" as described in Example 1, or a polypeptide 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 NO:3 polypeptide, as well as isolated nucleic acid molecules encoding these polypeptides.

[0042] In one embodiment, the steviol biosynthetic enzymes and UDP-glucosyltransferase are produced in a microbial cell, which may be, for example, E. coli, Saccharomyces sp., Aspergillus sp., Pichia sp., Bacillus sp., Yarrowia sp., etc. In another embodiment, the UDP-glucosyltransferase is synthetic.

[0043] In one embodiment, the UDP-glucosyltransferase is selected from the group including UGT74G1, UGT85C2, UGT76G1, UGT91D2, UGTSI2, EUGT11 and UGTs having significant (>85%, >86%, >87%, >88%, >89%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99%) amino acid sequence identity to these polypeptides, as well as isolated nucleic acid molecules encoding these UGTs.

[0044] In one embodiment, the steviol biosynthetic enzymes, UGTs, and UDP-glucose salvage system are present in one microorganism (microbial cell), which may be, for example, E. coli, Saccharomyces sp., Aspergillus sp., Pichia sp., Bacillus sp., Yarrowia sp.

[0045] 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 having an -OH functional group at C 13, to give a target steviol glycoside having an -O-glucose β-glucopyranoside glycosidic linkage at C 13. In a particular embodiment, the UDP-glucosyltransferase is UGT85C2 or a UGT having >85% amino acid sequence identity with UGT85C2.

[0046] 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 having a -COOH functional group at C19 to give a target steviol glycoside having a -COO-glucose β-glucopyranoside glycosidic linkage at C19. In a particular embodiment, the UDP-glucosyltransferase is UGT74G1 or a UGT having >85% amino acid sequence identity with UGT74G1.

[0047] In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to any existing glucose at the C19 side of any starting steviol glycoside to give a target steviol glycoside with at least one additional glucose having at least one β1→2 glucopyranoside glycosidic bond in the newly formed glycosidic bond. In a particular embodiment, the UDP-glucosyltransferase is UGTSl2 or a UGT having >85% amino acid sequence identity with UGTSl2. In another particular embodiment, the UDP-glucosyltransferase is EUGT11 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.

[0048] 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 having at least one β1→3 glucopyranoside glycosidic bond in the newly formed glycosidic bond. In a particular embodiment, the UDP-glucosyltransferase is UGT76G1 or a UGT having >85% amino acid sequence identity with UGT76G1.

[0049] In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to any existing glucose at the C19 side of any starting steviol glycoside to give a target steviol glycoside with at least one additional glucose having at least one β1→4 glucopyranoside glycosidic bond in the newly formed glycosidic bond. In a particular embodiment, the UDP-glucosyltransferase is UGTSl2 or a UGT having >85% amino acid sequence identity with UGTSl2. In another particular embodiment, the UDP-glucosyltransferase is EUGT11 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 specific embodiment, the UDP-glucosyltransferase is UGT76G1 or a UGT having >85% amino acid sequence identity with UGT76G1.

[0050] In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to any existing glucose at the C19 side of any starting steviol glycoside to give a target steviol glycoside with at least one additional glucose having at least one β1→6 glucopyranoside glycosidic bond in the newly formed glycosidic bond. In a particular embodiment, the UDP-glucosyltransferase is UGTSl2 or a UGT having >85% amino acid sequence identity with UGTSl2. In another particular embodiment, the UDP-glucosyltransferase is EUGT11 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.

[0051] In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to any existing glucose at the C13 side of any starting steviol glycoside to give a target steviol glycoside with at least one additional glucose having at least one β1→2 glucopyranoside glycosidic bond in the newly formed glycosidic bond. In a particular embodiment, the UDP-glucosyltransferase is UGTSl2 or a UGT having >85% amino acid sequence identity with UGTSl2. In another particular embodiment, the UDP-glucosyltransferase is EUGT11 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.

[0052] 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 having at least one β1→3 glucopyranoside glycosidic bond in the newly formed glycosidic bond. In a particular embodiment, the UDP-glucosyltransferase is UGT76G1 or a UGT having >85% amino acid sequence identity with UGT76G1.

[0053] In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to any existing glucose at the C13 side of any starting steviol glycoside to give a target steviol glycoside with at least one additional glucose having at least one β1→4 glucopyranoside glycosidic bond in the newly formed glycosidic bond. In a particular embodiment, the UDP-glucosyltransferase is UGTSl2 or a UGT having >85% amino acid sequence identity with UGTSl2. In another particular embodiment, the UDP-glucosyltransferase is EUGT11 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 specific embodiment, the UDP-glucosyltransferase is UGT76G1 or a UGT having >85% amino acid sequence identity with UGT76G1.

[0054] In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to any existing glucose at the C13 side of any starting steviol glycoside to give a target steviol glycoside with at least one additional glucose having at least one β1→6 glucopyranoside glycosidic bond in the newly formed glycosidic bond. In a particular embodiment, the UDP-glucosyltransferase is UGTSl2 or a UGT having >85% amino acid sequence identity with UGTSl2. In another particular embodiment, the UDP-glucosyltransferase is EUGT11 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.

[0055] In one embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to steviol to form a steviol monoside, hi certain embodiments, the UDP-glucosyltransferase is UGT85C2, or a UGT with >85% amino acid sequence identity with UGT85C2, or a UGT with >85% amino acid sequence identity with UGT85C2.

[0056] 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 UGT74G1 or a UGT having >85% amino acid sequence identity with UGT74G1.

[0057] 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 UGTSl2 or a UGT with >85% amino acid sequence identity to UGTSl2. In another particular embodiment, the UDP-glucosyltransferase is EUGT11 or a UGT with >85% amino acid sequence identity to EUGT11. In yet another particular embodiment, the UDP-glucosyltransferase is UGT91D2 or a UGT with >85% amino acid sequence identity to UGT91D2.

[0058] 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.

[0059] In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to steviolmonoside to form rubusoside, hi a particular embodiment, the UDP-glucosyltransferase is UGT74G1 or a UGT having >85% amino acid sequence identity with UGT74G1.

[0060] 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 with >85% amino acid sequence identity to UGT85C2, or a UGT with >85% amino acid sequence identity to UGT85C2.

[0061] 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 UGTSl2 or a UGT with >85% amino acid sequence identity to UGTSl2. In another particular embodiment, the UDP-glucosyltransferase is EUGT11 or a UGT with >85% amino acid sequence identity to EUGT11. In yet another particular embodiment, the UDP-glucosyltransferase is UGT91D2 or a UGT with >85% amino acid sequence identity to UGT91D2.

[0062] 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 UGT76G1.

[0063] 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 UGT76G1 or a UGT having >85% amino acid sequence identity with UGT76G1.

[0064] In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to steviolbioside to form stevioside, hi a particular embodiment, the UDP-glucosyltransferase is UGT74G1 or a UGT having >85% amino acid sequence identity with UGT74G1.

[0065] 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 UGTSl2 or a UGT with >85% amino acid sequence identity to UGTSl2. In another particular embodiment, the UDP-glucosyltransferase is EUGT11 or a UGT with >85% amino acid sequence identity to EUGT11. In yet another particular embodiment, the UDP-glucosyltransferase is UGT91D2 or a UGT with >85% amino acid sequence identity to UGT91D2.

[0066] 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.

[0067] 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 UGTSl2 or a UGT with >85% amino acid sequence identity to UGTSl2. In another particular embodiment, the UDP-glucosyltransferase is EUGT11 or a UGT with >85% amino acid sequence identity to EUGT11. In yet another particular embodiment, the UDP-glucosyltransferase is UGT91D2 or a UGT with >85% amino acid sequence identity to UGT91D2.

[0068] 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.

[0069] 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 UGTSl2 or a UGT with >85% amino acid sequence identity to UGTSl2. In another particular embodiment, the UDP-glucosyltransferase is EUGT11 or a UGT with >85% amino acid sequence identity to EUGT11. In yet another particular embodiment, the UDP-glucosyltransferase is UGT91D2 or a UGT with >85% amino acid sequence identity to UGT91D2.

[0070] 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.

[0071] 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 particular embodiments, the UDP-glucosyltransferase is UGT85C2, or a UGT with >85% amino acid sequence identity with UGT85C2, or a UGT with >85% amino acid sequence identity with UGT85C2.

[0072] 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.

[0073] 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 with >85% amino acid sequence identity to UGT85C2, or a UGT with >85% amino acid sequence identity to UGT85C2.

[0074] 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 UGTSl2 or a UGT with >85% amino acid sequence identity to UGTSl2. In another particular embodiment, the UDP-glucosyltransferase is EUGT11 or a UGT with >85% amino acid sequence identity to EUGT11. In yet another particular embodiment, the UDP-glucosyltransferase is UGT91D2 or a UGT with >85% amino acid sequence identity to UGT91D2.

[0075] 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.

[0076] 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.

[0077] 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 UGTSl2 or a UGT with >85% amino acid sequence identity to UGTSl2. In another particular embodiment, the UDP-glucosyltransferase is EUGT11 or a UGT with >85% amino acid sequence identity to EUGT11. In yet another particular embodiment, the UDP-glucosyltransferase is UGT91D2 or a UGT with >85% amino acid sequence identity to UGT91D2.

[0078] 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 UGT76G1 or a UGT having >85% amino acid sequence identity with UGT76G1.

[0079] 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 UGTSl2 or a UGT with >85% amino acid sequence identity to UGTSl2. In another particular embodiment, the UDP-glucosyltransferase is EUGT11 or a UGT with >85% amino acid sequence identity to EUGT11. In yet another particular embodiment, the UDP-glucosyltransferase is UGT91D2 or a UGT with >85% amino acid sequence identity to UGT91D2.

[0080] 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 UGTSl2 or a UGT with >85% amino acid sequence identity to UGTSl2. In another particular embodiment, the UDP-glucosyltransferase is EUGT11 or a UGT with >85% amino acid sequence identity to EUGT11. In yet another particular embodiment, the UDP-glucosyltransferase is UGT91D2 or a UGT with >85% amino acid sequence identity to UGT91D2.

[0081] 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.

[0082] 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 UGTSl2 or a UGT with >85% amino acid sequence identity to UGTSl2. In another particular embodiment, the UDP-glucosyltransferase is EUGT11 or a UGT with >85% amino acid sequence identity to EUGT11. In yet another particular embodiment, the UDP-glucosyltransferase is UGT91D2 or a UGT with >85% amino acid sequence identity to UGT91D2.

[0083] 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.

[0084] 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.

[0085] 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 UGTSl2 or a UGT with >85% amino acid sequence identity to UGTSl2. In another particular embodiment, the UDP-glucosyltransferase is EUGT11 or a UGT with >85% amino acid sequence identity to EUGT11. In yet another particular embodiment, the UDP-glucosyltransferase is UGT91D2 or a UGT with >85% amino acid sequence identity to UGT91D2.

[0086] 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.

[0087] 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 UGTSl2 or a UGT with >85% amino acid sequence identity to UGTSl2. In another particular embodiment, the UDP-glucosyltransferase is EUGT11 or a UGT with >85% amino acid sequence identity to EUGT11. In yet another particular embodiment, the UDP-glucosyltransferase is UGT91D2 or a UGT with >85% amino acid sequence identity to UGT91D2.

[0088] 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 with >85% amino acid sequence identity to UGT85C2, or a UGT with >85% amino acid sequence identity to UGT85C2.

[0089] 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 UGTSl2 or a UGT with >85% amino acid sequence identity to UGTSl2. In another particular embodiment, the UDP-glucosyltransferase is EUGT11 or a UGT with >85% amino acid sequence identity to EUGT11. In yet another particular embodiment, the UDP-glucosyltransferase is UGT91D2 or a UGT with >85% amino acid sequence identity to UGT91D2.

[0090] 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.

[0091] In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase that can add 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 to UGT76G1.

[0092] 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.

[0093] 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 to UGT76G1.

[0094] 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 UGTSl2 or a UGT with >85% amino acid sequence identity to UGTSl2. In another particular embodiment, the UDP-glucosyltransferase is EUGT11 or a UGT with >85% amino acid sequence identity to EUGT11. In yet another particular embodiment, the UDP-glucosyltransferase is UGT91D2 or a UGT with >85% amino acid sequence identity to UGT91D2.

[0095] 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 UGTSl2 or a UGT with >85% amino acid sequence identity to UGTSl2. In another particular embodiment, the UDP-glucosyltransferase is EUGT11 or a UGT with >85% amino acid sequence identity to EUGT11. In yet another particular embodiment, the UDP-glucosyltransferase is UGT91D2 or a UGT with >85% amino acid sequence identity to UGT91D2.

[0096] In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase that can add 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.

[0097] 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 UGTSl2 or a UGT with >85% amino acid sequence identity to UGTSl2. In another particular embodiment, the UDP-glucosyltransferase is EUGT11 or a UGT with >85% amino acid sequence identity to EUGT11. In yet another particular embodiment, the UDP-glucosyltransferase is UGT91D2 or a UGT with >85% amino acid sequence identity to UGT91D2.

[0098] 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 UGTSl2 or a UGT with >85% amino acid sequence identity to UGTSl2. In another particular embodiment, the UDP-glucosyltransferase is EUGT11 or a UGT with >85% amino acid sequence identity to EUGT11. In yet another particular embodiment, the UDP-glucosyltransferase is UGT91D2 or a UGT with >85% amino acid sequence identity to UGT91D2.

[0099] 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 UGTSl2 or a UGT with >85% amino acid sequence identity to UGTSl2. In another particular embodiment, the UDP-glucosyltransferase is EUGT11 or a UGT with >85% amino acid sequence identity to EUGT11. In yet another particular embodiment, the UDP-glucosyltransferase is UGT91D2 or a UGT with >85% amino acid sequence identity to UGT91D2.

[0100] In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase that can add 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.

[0101] In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase that can add 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 to UGT76G1.

[0102] 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 UGTSl2 or a UGT with >85% amino acid sequence identity to UGTSl2. In another particular embodiment, the UDP-glucosyltransferase is EUGT11 or a UGT with >85% amino acid sequence identity to EUGT11. In yet another particular embodiment, the UDP-glucosyltransferase is UGT91D2 or a UGT with >85% amino acid sequence identity to UGT91D2.

[0103] In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase that can add 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.

[0104] 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 UGTSl2 or a UGT with >85% amino acid sequence identity to UGTSl2. In another particular embodiment, the UDP-glucosyltransferase is EUGT11 or a UGT with >85% amino acid sequence identity to EUGT11. In yet another particular embodiment, the UDP-glucosyltransferase is UGT91D2 or a UGT with >85% amino acid sequence identity to UGT91D2. In a particular embodiment, the UDP-glucosyltransferase is UGT76G1 or a UGT with >85% amino acid sequence identity to UGT76G1.

[0105] 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 UGTSl2 or a UGT with >85% amino acid sequence identity to UGTSl2. In another particular embodiment, the UDP-glucosyltransferase is EUGT11 or a UGT with >85% amino acid sequence identity to EUGT11. In yet another particular embodiment, the UDP-glucosyltransferase is UGT91D2 or a UGT with >85% amino acid sequence identity to UGT91D2.

[0106] In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside M to form rebaudioside 1a. In a particular embodiment, the UDP-glucosyltransferase is UGTSl2 or a UGT with >85% amino acid sequence identity to UGTSl2. In another particular embodiment, the UDP-glucosyltransferase is EUGT11 or a UGT with >85% amino acid sequence identity to EUGT11. In yet another particular embodiment, the UDP-glucosyltransferase is UGT91D2 or a UGT with >85% amino acid sequence identity to UGT91D2.

[0107] In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside M to form rebaudioside 1b. In a particular embodiment, the UDP-glucosyltransferase is UGT76G1 or a UGT having >85% amino acid sequence identity to UGT76G1.

[0108] In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside M to form rebaudioside 1c. In a particular embodiment, the UDP-glucosyltransferase is UGTSl2 or a UGT with >85% amino acid sequence identity to UGTSl2. In another particular embodiment, the UDP-glucosyltransferase is EUGT11 or a UGT with >85% amino acid sequence identity to EUGT11. In yet another particular embodiment, the UDP-glucosyltransferase is UGT91D2 or a UGT with >85% amino acid sequence identity to UGT91D2.

[0109] In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside M to form rebaudioside 1d. In a particular embodiment, the UDP-glucosyltransferase is UGTSl2 or a UGT with >85% amino acid sequence identity to UGTSl2. In another particular embodiment, the UDP-glucosyltransferase is EUGT11 or a UGT with >85% amino acid sequence identity to EUGT11. In yet another particular embodiment, the UDP-glucosyltransferase is UGT91D2 or a UGT with >85% amino acid sequence identity to UGT91D2.

[0110] In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside M to form rebaudioside 1e. In a particular embodiment, the UDP-glucosyltransferase is UGT76G1 or a UGT having >85% amino acid sequence identity with UGT76G1.

[0111] In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside M to form rebaudioside 1f. In a particular embodiment, the UDP-glucosyltransferase is UGTSl2 or a UGT with >85% amino acid sequence identity to UGTSl2. In another particular embodiment, the UDP-glucosyltransferase is EUGT11 or a UGT with >85% amino acid sequence identity to EUGT11. In yet another particular embodiment, the UDP-glucosyltransferase is UGT91D2 or a UGT with >85% amino acid sequence identity to UGT91D2.

[0112] In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside M to form rebaudioside 1g. In a particular embodiment, the UDP-glucosyltransferase is UGTSl2 or a UGT with >85% amino acid sequence identity to UGTSl2. In another particular embodiment, the UDP-glucosyltransferase is EUGT11 or a UGT with >85% amino acid sequence identity to EUGT11. In yet another particular embodiment, the UDP-glucosyltransferase is UGT91D2 or a UGT with >85% amino acid sequence identity to UGT91D2.

[0113] In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase that can add at least one glucose unit to rebaudioside M to form rebaudioside 1h. In a particular embodiment, the UDP-glucosyltransferase is UGT76G1 or a UGT having >85% amino acid sequence identity to UGT76G1.

[0114] In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside M to form rebaudioside 1i. In a particular embodiment, the UDP-glucosyltransferase is UGTSl2 or a UGT with >85% amino acid sequence identity to UGTSl2. In another particular embodiment, the UDP-glucosyltransferase is EUGT11 or a UGT with >85% amino acid sequence identity to EUGT11. In yet another particular embodiment, the UDP-glucosyltransferase is UGT91D2 or a UGT with >85% amino acid sequence identity to UGT91D2.

[0115] In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside M to form rebaudioside 1j. In a particular embodiment, the UDP-glucosyltransferase is UGTSl2 or a UGT with >85% amino acid sequence identity to UGTSl2. In another particular embodiment, the UDP-glucosyltransferase is EUGT11 or a UGT with >85% amino acid sequence identity to EUGT11. In yet another particular embodiment, the UDP-glucosyltransferase is UGT91D2 or a UGT with >85% amino acid sequence identity to UGT91D2.

[0116] In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside M to form rebaudioside 1k. In a particular embodiment, the UDP-glucosyltransferase is UGT76G1 or a UGT having >85% amino acid sequence identity to UGT76G1.

[0117] In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside M to form rebaudioside 1l. In a particular embodiment, the UDP-glucosyltransferase is UGTSl2 or a UGT with >85% amino acid sequence identity to UGTSl2. In another particular embodiment, the UDP-glucosyltransferase is EUGT11 or a UGT with >85% amino acid sequence identity to EUGT11. In yet another particular embodiment, the UDP-glucosyltransferase is UGT91D2 or a UGT with >85% amino acid sequence identity to UGT91D2.

[0118] In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside M to form rebaudioside 1m. In a particular embodiment, the UDP-glucosyltransferase is UGTSl2 or a UGT with >85% amino acid sequence identity to UGTSl2. In another particular embodiment, the UDP-glucosyltransferase is EUGT11 or a UGT with >85% amino acid sequence identity to EUGT11. In yet another particular embodiment, the UDP-glucosyltransferase is UGT91D2 or a UGT with >85% amino acid sequence identity to UGT91D2.

[0119] In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside M to form rebaudioside 1n. In a particular embodiment, the UDP-glucosyltransferase is UGTSl2 or a UGT with >85% amino acid sequence identity to UGTSl2. In another particular embodiment, the UDP-glucosyltransferase is EUGT11 or a UGT with >85% amino acid sequence identity to EUGT11. In yet another particular embodiment, the UDP-glucosyltransferase is UGT91D2 or a UGT with >85% amino acid sequence identity to UGT91D2.

[0120] 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 UGTSl2 or a UGT with >85% amino acid sequence identity to UGTSl2. In another particular embodiment, the UDP-glucosyltransferase is EUGT11 or a UGT with >85% amino acid sequence identity to EUGT11. In yet another particular embodiment, the UDP-glucosyltransferase is UGT91D2 or a UGT with >85% amino acid sequence identity to UGT91D2. In a particular embodiment, the UDP-glucosyltransferase is UGT76G1 or a UGT with >85% amino acid sequence identity to UGT76G1.

[0121] Optionally, the method further comprises recycling UDP to provide UDP-glucose. In one embodiment, the method comprises recycling UDP by providing a recycled catalyst and a recycled substrate such that bioconversion of steviol and / or steviol glycoside substrates to target steviol glycosides occurs using catalytic amounts of UDP-glucosyltransferase and UDP-glucose.

[0122] In one embodiment, the recycled catalyst is sucrose synthase SuSy_At, or a sucrose synthase having >85% amino acid sequence identity with SuSy_At.

[0123] In one embodiment, the recycling substrate for the UDP-glucose recycling catalyst is sucrose.

[0124] Optionally, the method of the present invention further comprises the use of a transglycosidase that uses an oligosaccharide or polysaccharide as a sugar donor to modify the target acceptor steviol glycoside molecule. Non-limiting examples include cyclodextrin glycosyltransferase (CGTase), fructofuranosidase, amylase, saccharase, glucosucrase, β-h-fructosidase, β-fructosidase, sucrase, fructosyl invertase, alkaline invertase, acid invertase, and fructofuranosidase. In some embodiments, glucose and sugars other than glucose, including but not limited to fructose, xylose, rhamnose, arabinose, deoxyglucose, and galactose, are transferred to the target acceptor steviol glycoside. In one embodiment, the acceptor steviol glycoside is rebaudioside 1a, rebaudioside 1b, rebaudioside 1c, rebaudioside 1d, rebaudioside 1e, rebaudioside 1f, rebaudioside 1g, rebaudioside 1h, rebaudioside 1i, rebaudioside 1j, rebaudioside 1k, rebaudioside 1l, rebaudioside 1m, and / or rebaudioside 1n. In another embodiment, the acceptor steviol glycoside is rebaudioside 2a. In another embodiment, the acceptor steviol glycoside is rebaudioside M4. In another embodiment, the acceptor steviol glycoside is SvG7.

[0125] Optionally, the method of the present invention further comprises separating the target steviol glycoside from the culture medium to provide a high purity target steviol glycoside composition. The target steviol glycoside can be separated by at least one suitable method, such as, for example, crystallization, membrane separation, centrifugation, extraction, chromatographic separation, or a combination of such methods.

[0126] In one embodiment, the target steviol glycosides may be produced within a microorganism. In another embodiment, the target steviol glycosides may be secreted into the culture medium. In another embodiment, the released steviol glycosides may be continuously removed from the culture medium. In yet another embodiment, the target steviol glycosides are separated after completion of the conversion reaction.

[0127] In one embodiment, the separation produces a composition comprising greater than about 80% by weight of the target steviol glycoside on an anhydrous basis, i.e., a high purity steviol glycoside composition. In another embodiment, the separation produces a composition comprising greater than about 90% by weight of the target steviol glycoside. In certain 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.

[0128] The target steviol glycosides can be in any polymorphic or amorphous form, including hydrates, solvates, anhydrates, or combinations thereof.

[0129] The purified target steviol glycosides can be used in consumer products as sweeteners, flavor modifiers, flavor modifiers, and / or foam suppressants. Suitable consumer products include, but are not limited to, foods, beverages, pharmaceutical compositions, tobacco products, nutritional supplement compositions, oral hygiene compositions, and cosmetic compositions. [Brief description of the drawings]

[0130] [Figure 1a] FIG. 1 shows the chemical structure of SvG7 steviol glycoside, rebaudioside 1a. [Figure 1b] FIG. 1 shows the chemical structure of SvG7 steviol glycoside, rebaudioside 1b. [Figure 1c] FIG. 1 shows the chemical structure of SvG7 steviol glycoside rebaudioside 1c. [Figure 1d] FIG. 1 shows the chemical structure of SvG7 steviol glycoside rebaudioside 1d. [Figure 1e] FIG. 1 shows the chemical structure of SvG7 steviol glycoside, rebaudioside 1e. [Figure 1f] FIG. 1 shows the chemical structure of SvG7 steviol glycoside rebaudioside 1f. [Figure 1g] FIG. 1 shows the chemical structure of SvG7 steviol glycoside rebaudioside 1g. [Figure 1h] FIG. 1 shows the chemical structure of SvG7 steviol glycoside, rebaudioside 1h. [Figure 1i] FIG. 1 shows the chemical structure of rebaudioside 1i, an SvG7 steviol glycoside. [Figure 1j] FIG. 1 shows the chemical structure of SvG7 steviol glycoside rebaudioside 1j. [Figure 1k] FIG. 1 shows the chemical structure of SvG7 steviol glycoside, rebaudioside 1k. [Figure 1l] FIG. 1 shows the chemical structure of SvG7 steviol glycoside, rebaudioside 1l. [Figure 1m] FIG. 1 shows the chemical structure of SvG7 steviol glycoside rebaudioside 1m. [Figure 1n] FIG. 1 shows the chemical structure of SvG7 steviol glycoside, rebaudioside 1n. [Figure 1o] FIG. 1 shows the chemical structure of SvG7 steviol glycoside, rebaudioside 2a. [Figure 1p] FIG. 1 shows the chemical structure of Rebaudioside M4. [Figure 2a]FIG. 1 shows pathways to produce 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, rebaudioside M4 and various steviol glycosides from steviol and various intermediate steviol glycosides. [Figure 2b] FIG. 1 shows pathways to produce 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, rebaudioside M4 and various steviol glycosides from steviol and various intermediate steviol glycosides. [Figure 2c] FIG. 1 shows pathways to produce 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, rebaudioside M4 and various steviol glycosides from steviol and various intermediate steviol glycosides. [Figure 2d] FIG. 1 shows pathways to produce 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, rebaudioside M4 and various steviol glycosides from steviol and various intermediate steviol glycosides. [Figure 2e]FIG. 1 shows pathways to produce 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, rebaudioside M4 and various steviol glycosides from steviol and various intermediate steviol glycosides. [Figure 2f] FIG. 1 shows pathways to produce 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, rebaudioside M4 and various steviol glycosides from steviol and various intermediate steviol glycosides. [Figure 2g] FIG. 1 shows pathways to produce 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, rebaudioside M4 and various steviol glycosides from steviol and various intermediate steviol glycosides. [Figure 2h] FIG. 1 shows pathways to produce 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, rebaudioside M4 and various steviol glycosides from steviol and various intermediate steviol glycosides. [Figure 2i]FIG. 1 shows pathways to produce 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, rebaudioside M4 and various steviol glycosides from steviol and various intermediate steviol glycosides. [Figure 2j] FIG. 1 shows pathways to produce 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, rebaudioside M4 and various steviol glycosides from steviol and various intermediate steviol glycosides. [Figure 2k] FIG. 1 shows pathways to produce 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, rebaudioside M4 and various steviol glycosides from steviol and various intermediate steviol glycosides. [Figure 3a] FIG. 1 shows the biocatalytic production of rebaudioside 1a from rebaudioside A using enzymes UGTSl2 and UGT76G1 and the concomitant recycling of UDP to UDP-glucose via sucrose synthase SuSy_At. [Figure 3b] FIG. 1 shows the biocatalytic production of rebaudioside 1b from rebaudioside A using enzymes UGTSl2 and UGT76G1 and the concomitant recycling of UDP to UDP-glucose via sucrose synthase SuSy_At. [Figure 3c]FIG. 1 shows the biocatalytic production of rebaudioside 1c from rebaudioside A using enzymes UGTSl2 and UGT76G1 and the concomitant recycling of UDP to UDP-glucose via sucrose synthase SuSy_At. [Figure 3d] FIG. 1 shows the biocatalytic production of rebaudioside 1d from rebaudioside A using enzymes UGTSl2 and UGT76G1 and the concomitant recycling of UDP to UDP-glucose via sucrose synthase SuSy_At. [Figure 3e] FIG. 1 shows the biocatalytic production of rebaudioside 1e from rebaudioside A using enzymes UGTSl2 and UGT76G1 and the concomitant recycling of UDP to UDP-glucose via sucrose synthase SuSy_At. [Figure 3f] FIG. 1 shows the biocatalytic production of rebaudioside 1f from rebaudioside A using enzymes UGTSl2 and UGT76G1 and the concomitant recycling of UDP to UDP-glucose via sucrose synthase SuSy_At. [Figure 3g] FIG. 1 illustrates the biocatalytic production of rebaudioside 1g from rebaudioside A using enzymes UGTSl2 and UGT76G1 and the concomitant recycling of UDP to UDP-glucose via sucrose synthase SuSy_At. [Figure 3h] FIG. 1 shows the biocatalytic production of rebaudioside 1h from rebaudioside A using enzymes UGTSl2 and UGT76G1 and the concomitant recycling of UDP to UDP-glucose via sucrose synthase SuSy_At. [Figure 3i] FIG. 1 shows the biocatalytic production of rebaudioside 1i from rebaudioside A using enzymes UGTSl2 and UGT76G1 and the concomitant recycling of UDP to UDP-glucose via sucrose synthase SuSy_At. [Figure 3j]FIG. 1 shows the biocatalytic production of rebaudioside 1j from rebaudioside A using enzymes UGTSl2 and UGT76G1 and the concomitant recycling of UDP to UDP-glucose via sucrose synthase SuSy_At. [Figure 3k] FIG. 1 shows the biocatalytic production of rebaudioside 1k from rebaudioside A using enzymes UGTSl2 and UGT76G1 and the concomitant recycling of UDP to UDP-glucose via sucrose synthase SuSy_At. [Figure 3l] FIG. 1 shows the biocatalytic production of rebaudioside 1l from rebaudioside A using enzymes UGTSl2 and UGT76G1 and the concomitant recycling of UDP to UDP-glucose via sucrose synthase SuSy_At. [Figure 3m] FIG. 1 shows the biocatalytic production of rebaudioside 1m from rebaudioside A using enzymes UGTSl2 and UGT76G1 and the concomitant recycling of UDP to UDP-glucose via sucrose synthase SuSy_At. [Figure 3n] FIG. 1 shows the biocatalytic production of rebaudioside 1n from rebaudioside A using enzymes UGTSl2 and UGT76G1 and the concomitant recycling of UDP to UDP-glucose via sucrose synthase SuSy_At. [Figure 3o] FIG. 1 shows the biocatalytic production of rebaudioside 2a from stevioside using the enzymes UGTSl2 and UGT76G1 and the concomitant recycling of UDP to UDP-glucose via sucrose synthase SuSy_At. [Figure 3p] FIG. 1 shows the biocatalytic production of rebaudioside M4 from stevioside using enzymes UGTSl2 and UGT76G1 and the concomitant recycling of UDP to UDP-glucose via sucrose synthase SuSy_At. [Figure 3q]FIG. 1 shows the biocatalytic production of rebaudioside 2a from rebaudioside AM using enzymes UGTSl2 and UGT76G1 and the concomitant recycling of UDP to UDP-glucose via sucrose synthase SuSy_At. [Figure 3r] FIG. 1 shows the biocatalytic production of rebaudioside M4 from rebaudioside AM using enzymes UGTSl2 and UGT76G1 and the concomitant recycling of UDP to UDP-glucose via sucrose synthase SuSy_At. [Figure 3s] FIG. 1 shows the biocatalytic production of rebaudioside 2a from rebaudioside M4 using enzymes UGTSl2 and UGT76G1 and the concomitant recycling of UDP to UDP-glucose via sucrose synthase SuSy_At. [Figure 4] 1 shows an HPLC chromatogram of stevioside. The peak at retention time 20.958 min corresponds to stevioside. The peak at retention time 20.725 min corresponds to rebaudioside A. The peak at 32.925 min corresponds to rebaudioside B. The peak at 33.930 min corresponds to steviolbioside. [Diagram 5] 1 shows HPLC chromatograms of products from biocatalytic production of SvG7 molecule from stevioside. The peak at 6.459 min corresponds to rebaudioside 2a. The peak at 9.825 min corresponds to rebaudioside AM. The peak at 13.845 min corresponds to rebaudioside M. The peak at 32.974 min corresponds to rebaudioside B. The peak at 33.979 min corresponds to steviolbioside. [Figure 6] 1 shows the HPLC chromatogram of rebaudioside 2a after purification by HPLC. The peak at retention time 6.261 minutes corresponds to rebaudioside 2a. [Figure 7] FIG. 1 shows the 1H NMR spectrum of rebaudioside 2a (500 MHz, pyridine-d5). [Figure 8]FIG. 1 shows the HSQC spectrum of rebaudioside 2a (500 MHz, pyridine-d5). [Figure 9] FIG. 1 shows the H,H COSY spectrum of rebaudioside 2a (500 MHz, pyridine-d5). [Figure 10] FIG. 1 shows the HMBC spectrum of rebaudioside 2a (500 MHz, pyridine-d5). [Figure 11a] FIG. 1 shows the HSQC-TOCSY spectrum of rebaudioside 2a (500 MHz, pyridine-d5). [Figure 11b] FIG. 1 shows the 1D-NOESY spectrum of rebaudioside 2a (500 MHz, pyridine-d5). [Figure 12a] FIG. 1 shows LC chromatogram of rebaudioside 2a. [Figure 12b] FIG. 1 shows the mass spectrum of rebaudioside 2a. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0131] 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 microbial cells and / or an enzyme preparation, thereby producing a composition comprising a target steviol glycoside.

[0132] One object of the present invention is to obtain target steviol glycosides, in particular steviolmonosides, 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 G, rebaudioside H, rebaudioside I ... 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 or synthetic steviol glycosides.

[0133] Starting Composition As used herein, "starting composition" refers to any composition (generally an aqueous solution) that contains one or more organic compounds that contain at least one carbon atom.

[0134] In one embodiment, the starting composition is selected from the group consisting of steviol, steviol glycosides, polyols, and various carbohydrates.

[0135] The steviol glycoside starting composition can be 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 Stevia The glycosides are selected from the group consisting of other glycosides of steviol occurring in the Glycosyl steviol plant, synthetic steviol glycosides, such as enzymatically glycosylated steviol glycosides, and combinations thereof.

[0136] In one embodiment, the starting composition is steviol.

[0137] In another embodiment, the steviol glycoside of the starting composition is a steviol monoside.

[0138] In yet another embodiment, the steviol glycoside of the starting composition is steviolmonoside A.

[0139] In another embodiment, the steviol glycoside of the starting composition is steviolbioside.

[0140] In another embodiment, the steviol glycoside of the starting composition is steviolbioside D.

[0141] In another embodiment, the steviol glycoside of the starting composition is rubusoside.

[0142] In another embodiment, the steviol glycoside of the starting composition is rubusoside.

[0143] In another embodiment, the steviol glycoside of the starting composition is steviolbioside A.

[0144] In another embodiment, the steviol glycoside of the starting composition is steviolbioside B.

[0145] In another embodiment, the steviol glycoside of the starting composition is rebaudioside B.

[0146] In another embodiment, the steviol glycoside of the starting composition is stevioside.

[0147] In another embodiment, the steviol glycoside of the starting composition is rebaudioside G.

[0148] In another embodiment, the steviol glycoside of the starting composition is stevioside A.

[0149] In another embodiment, the steviol glycoside of the starting composition is stevioside B.

[0150] In another embodiment, the steviol glycoside of the starting composition is stevioside C.

[0151] In another embodiment, the steviol glycoside of the starting composition is rebaudioside A.

[0152] In another embodiment, the steviol glycoside of the starting composition is rebaudioside E.

[0153] In another embodiment, the steviol glycoside of the starting composition is rebaudioside E2.

[0154] In another embodiment, the steviol glycoside of the starting composition is rebaudioside E4.

[0155] In another embodiment, the steviol glycoside of the starting composition is rebaudioside E6.

[0156] In another embodiment, the steviol glycoside of the starting composition is rebaudioside E3.

[0157] In another embodiment, the steviol glycoside of the starting composition is rebaudioside D.

[0158] In another embodiment, the steviol glycoside of the starting composition is rebaudioside I.

[0159] In another embodiment, the steviol glycoside of the starting composition is rebaudioside AM.

[0160] In another embodiment, the steviol glycoside of the starting composition is rebaudioside D7.

[0161] In another embodiment, the steviol glycoside of the starting composition is rebaudioside M.

[0162] In another embodiment, the steviol glycoside of the starting composition is rebaudioside M4.

[0163] The term "polyol" refers to a molecule that contains two or more hydroxyl groups. Polyols may be diols, triols, or tetraols, containing two, three, and four hydroxyl groups, respectively. Polyols may also contain more than four hydroxyl groups, such as pentaols, hexaols, heptaols, containing five, six, or seven hydroxyl groups, respectively. In addition, polyols may also be sugar alcohols, polyhydric alcohols, or polyalcohols that are reduced forms of carbohydrates, in which the carbonyl groups (aldehydes or ketones, reducing sugars) have been reduced to primary or secondary hydroxyl groups. Examples of polyols include, but are not limited to, erythritol, maltitol, mannitol, sorbitol, lactitol, xylitol, inositol, isomalt, propylene glycol, glycerol, threitol, galactitol, hydrogenated isomaltulose, reduced isomaltooligosaccharides, reduced xylooligosaccharides, reduced gentiooligosaccharides, reduced maltose syrup, reduced glucose syrup, hydrogenated starch hydrolysates, polyglycitols and sugar alcohols, or any other carbohydrate that can be reduced.

[0164] The term "carbohydrate" refers to a compound having the general formula (CHO) n(wherein n is 3-30), and oligomers and polymers thereof, as well as aldehyde or ketone compounds substituted with multiple hydroxyl groups. Additionally, the carbohydrates of the present invention may be substituted or deoxygenated at one or more positions. As used herein, carbohydrate encompasses unmodified carbohydrates, carbohydrate derivatives, substituted carbohydrates, and modified carbohydrates. As used herein, the phrases "carbohydrate derivatives", "substituted carbohydrates", and "modified carbohydrates" are synonymous. Modified carbohydrates refer to any carbohydrate in which at least one atom has been added, removed, or replaced, or a combination thereof. Thus, carbohydrate derivatives or substituted carbohydrates include substituted and unsubstituted monosaccharides, disaccharides, oligosaccharides, and polysaccharides. The carbohydrate derivative or substituted carbohydrate can be optionally 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 derivative, alkylamino, dialkylamino, arylamino, alkoxy, aryloxy, nitro, cyano, sulfo, mercapto, imino, sulfonyl, sulfenyl, sulfinyl, sulfamoyl, carboalkoxy, carboxamido, phosphonyl, phosphinyl, phosphoryl, phosphino, thioester, thioether, oxyimino, hydrazino, carbamyl, phospho, phosphonato, or any other possible functional group, if the carbohydrate derivative or substituted carbohydrate functional group functions to enhance the sweetness of the sweetener composition.

[0165] Examples of carbohydrates which may be used according to the invention include 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, gluconolactone, abequose, galactosamine, beet oligosaccharides, Examples of such oligosaccharides include, but are not limited to, isomaltooligosaccharides (isomaltose, isomaltotriose, panose, etc.), xylooligosaccharides (xylotriose, xylobiose, etc.), xyloterminal oligosaccharides, gentiooligosaccharides (gentiobiose, gentiotriose, gentiotetraose, etc.), sorbose, nigerooligosaccharides, palatinose oligosaccharides, fructooligosaccharides (kestose, nystose, etc.), maltotetraol, maltotriol, maltooligosaccharides (maltotriose, maltotetraose, maltopentaose, maltohexaose, maltoheptaose, etc.), starch, inulin, inulooligosaccharides, lactulose, melibiose, raffinose, ribose, isomerized liquid sugars such as high fructose corn syrup, coupling sugars, and soybean oligosaccharides. In addition, carbohydrates, as used herein, may be in either the D- or L-configuration.

[0166] The starting compositions may be synthetic or purified (partially or fully) and may be commercially available or prepared.

[0167] In one embodiment, the starting composition is glycerol.

[0168] In another embodiment, the starting composition is glucose.

[0169] In another embodiment, the starting composition is rhamnose.

[0170] In yet another embodiment, the starting composition is sucrose.

[0171] In yet another embodiment, the starting composition is a starch.

[0172] In another embodiment, the starting composition is maltodextrin.

[0173] In yet another embodiment, the starting composition is cellulose.

[0174] In yet another embodiment, the starting composition is amylose.

[0175] The organic compound(s) of the starting composition serve as substrate(s) for the production of target steviol glycoside(s), as described herein.

[0176] Targeted Steviol Glycosides 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 steviol monoside, steviol monoside A, steviol bioside, steviol bioside D, rubusoside, steviol bioside A, steviol bioside 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 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, SvG7, or other glycosides of steviol occurring in the Stevia rebaudiana plant, synthetic steviol glycosides, e.g., enzymatically glycosylated steviol glycosides, and combinations thereof.

[0177] In one embodiment, the target steviol glycoside is a steviol monoside.

[0178] In another embodiment, the target steviol glycoside is steviolmonoside A.

[0179] In another embodiment, the target steviol glycoside is steviolbioside.

[0180] In another embodiment, the target steviol glycoside is steviolbioside D.

[0181] In another embodiment, the target steviol glycoside is rubusoside.

[0182] In another embodiment, the target steviol glycoside is steviolbioside A.

[0183] In another embodiment, the target steviol glycoside is steviolbioside B.

[0184] In another embodiment, the target steviol glycoside is rebaudioside B.

[0185] In another embodiment, the target steviol glycoside is stevioside.

[0186] In another embodiment, the target steviol glycoside is rebaudioside G.

[0187] In another embodiment, the target steviol glycoside is stevioside A.

[0188] In another embodiment, the target steviol glycoside is stevioside B.

[0189] In another embodiment, the target steviol glycoside is stevioside C.

[0190] In another embodiment, the target steviol glycoside is rebaudioside A.

[0191] In another embodiment, the target steviol glycoside is rebaudioside E.

[0192] In another embodiment, the target steviol glycoside is rebaudioside E2.

[0193] In another embodiment, the target steviol glycoside is rebaudioside E4.

[0194] In another embodiment, the target steviol glycoside is rebaudioside E6.

[0195] In another embodiment, the target steviol glycoside is rebaudioside E3.

[0196] In another embodiment, the target steviol glycoside is rebaudioside D.

[0197] In another embodiment, the target steviol glycoside is rebaudioside I.

[0198] In another embodiment, the target steviol glycoside is rebaudioside AM.

[0199] In another embodiment, the target steviol glycoside is rebaudioside D7.

[0200] In another embodiment, the target steviol glycoside is rebaudioside M.

[0201] In another embodiment, the target steviol glycoside is rebaudioside M4.

[0202] In another embodiment, the target steviol glycoside is rebaudioside 1a.

[0203] In another embodiment, the target steviol glycoside is rebaudioside 1b.

[0204] In another embodiment, the target steviol glycoside is rebaudioside 1c.

[0205] In another embodiment, the target steviol glycoside is rebaudioside 1d.

[0206] In another embodiment, the target steviol glycoside is rebaudioside 1e.

[0207] In another embodiment, the target steviol glycoside is rebaudioside 1f.

[0208] In another embodiment, the target steviol glycoside is rebaudioside 1g.

[0209] In another embodiment, the target steviol glycoside is rebaudioside 1h.

[0210] In another embodiment, the target steviol glycoside is rebaudioside 1i.

[0211] In another embodiment, the target steviol glycoside is rebaudioside 1j.

[0212] In another embodiment, the target steviol glycoside is rebaudioside 1k.

[0213] In another embodiment, the target steviol glycoside is rebaudioside 1l.

[0214] In another embodiment, the target steviol glycoside is rebaudioside 1m.

[0215] In another embodiment, the target steviol glycoside is rebaudioside 1n.

[0216] In another embodiment, the target steviol glycoside is rebaudioside 2a.

[0217] In another embodiment, the target steviol glycoside is SvG7.

[0218] The target steviol glycosides can be in any polymorphic or amorphous form, including hydrates, solvates, anhydrates, or combinations thereof.

[0219] In one embodiment, the present invention is a biocatalytic process for the production of steviolmonoside.

[0220] In one embodiment, the present invention is a biocatalytic process for the production of steviolmonoside A.

[0221] In one embodiment, the invention is a biocatalytic process for the production of steviolbioside.

[0222] In one embodiment, the present invention is a biocatalytic process for the production of steviolbioside D.

[0223] In one embodiment, the present invention is a biocatalytic process for the production of rubusoside.

[0224] In one embodiment, the present invention is a biocatalytic process for the production of steviolbioside A.

[0225] In one embodiment, the present invention is a biocatalytic process for the production of steviolbioside B.

[0226] In one embodiment, the present invention is a biocatalytic process for the production of Rebaudioside B.

[0227] In one embodiment, the present invention is a biocatalytic process for the production of stevioside.

[0228] In one embodiment, the present invention is a biocatalytic process for the production of rebaudioside G.

[0229] In one embodiment, the present invention is a biocatalytic process for the production of stevioside A.

[0230] In one embodiment, the present invention is a biocatalytic process for the production of stevioside B.

[0231] In one embodiment, the present invention is a biocatalytic process for the production of Stevioside C.

[0232] In one embodiment, the present invention is a biocatalytic process for the production of rebaudioside A.

[0233] In one embodiment, the present invention is a biocatalytic process for the production of Rebaudioside E.

[0234] In one embodiment, the present invention is a biocatalytic process for the production of rebaudioside E2.

[0235] In one embodiment, the present invention is a biocatalytic process for the production of rebaudioside E4.

[0236] In one embodiment, the present invention is a biocatalytic process for the production of rebaudioside E6.

[0237] In one embodiment, the present invention is a biocatalytic process for the production of rebaudioside E3.

[0238] In one embodiment, the present invention is a biocatalytic process for the production of Rebaudioside D.

[0239] In one embodiment, the present invention is a biocatalytic process for the production of Rebaudioside I.

[0240] In one embodiment, the present invention is a biocatalytic process for the production of Rebaudioside AM.

[0241] In one embodiment, the present invention is a biocatalytic process for the production of rebaudioside D7.

[0242] In one embodiment, the present invention is a biocatalytic process for the production of rebaudioside E3.

[0243] In one embodiment, the present invention is a biocatalytic process for the production of Rebaudioside M.

[0244] In one embodiment, the present invention is a biocatalytic process for the production of rebaudioside M4.

[0245] In one embodiment, the present invention is a biocatalytic process for the production of rebaudioside 1a.

[0246] In one embodiment, the present invention is a biocatalytic process for the production of rebaudioside 1b.

[0247] In one embodiment, the present invention is a biocatalytic process for the production of rebaudioside 1c.

[0248] In one embodiment, the present invention is a biocatalytic process for the production of rebaudioside 1d.

[0249] In one embodiment, the present invention is a biocatalytic process for the production of rebaudioside 1e.

[0250] In one embodiment, the present invention is a biocatalytic process for the production of rebaudioside 1f.

[0251] In one embodiment, the present invention is a biocatalytic process for the production of 1 g of rebaudioside.

[0252] In one embodiment, the present invention is a biocatalytic process for the production of rebaudioside 1h.

[0253] In one embodiment, the present invention is a biocatalytic process for the production of rebaudioside 1i.

[0254] In one embodiment, the present invention is a biocatalytic process for the production of rebaudioside 1j.

[0255] In one embodiment, the present invention is a biocatalytic process for the production of rebaudioside 1k.

[0256] In one embodiment, the present invention is a biocatalytic process for the production of rebaudioside 1l.

[0257] In one embodiment, the present invention is a biocatalytic process for the production of rebaudioside 1m.

[0258] In one embodiment, the present invention is a biocatalytic process for the production of rebaudioside 1n.

[0259] In one embodiment, the present invention is a biocatalytic process for the production of rebaudioside 2a.

[0260] In one embodiment, the present invention is a biocatalytic process for the production of SvG7.

[0261] In certain embodiments, the present invention provides a biocatalytic process for the production of rebaudioside 1a from a starting composition comprising rebaudioside A and UDP-glucose.

[0262] In certain embodiments, the present invention provides a biocatalytic process for the production of rebaudioside 1b from a starting composition comprising rebaudioside A and UDP-glucose.

[0263] In certain embodiments, the present invention provides a biocatalytic process for the production of rebaudioside 1c from a starting composition comprising rebaudioside A and UDP-glucose.

[0264] In certain embodiments, the present invention provides a biocatalytic process for the production of rebaudioside 1d from a starting composition comprising rebaudioside A and UDP-glucose.

[0265] In certain embodiments, the present invention provides a biocatalytic process for the production of rebaudioside 1e from a starting composition comprising rebaudioside A and UDP-glucose.

[0266] In certain embodiments, the present invention provides a biocatalytic process for the production of rebaudioside 1f from a starting composition comprising rebaudioside A and UDP-glucose.

[0267] In certain embodiments, the present invention provides a biocatalytic process for the production of 1 g of rebaudioside from a starting composition comprising rebaudioside A and UDP-glucose.

[0268] In certain embodiments, the present invention provides a biocatalytic process for the production of rebaudioside 1h from a starting composition comprising rebaudioside A and UDP-glucose.

[0269] In certain embodiments, the present invention provides a biocatalytic process for the production of rebaudioside 1i from a starting composition comprising rebaudioside A and UDP-glucose.

[0270] In certain embodiments, the present invention provides a biocatalytic process for the production of rebaudioside 1j from a starting composition comprising rebaudioside A and UDP-glucose.

[0271] In certain embodiments, the present invention provides a biocatalytic process for the production of rebaudioside 1k from a starting composition comprising rebaudioside A and UDP-glucose.

[0272] In certain embodiments, the present invention provides a biocatalytic process for the production of rebaudioside 1l from a starting composition comprising rebaudioside A and UDP-glucose.

[0273] In certain embodiments, the present invention provides a biocatalytic process for the production of rebaudioside 1m from a starting composition comprising rebaudioside A and UDP-glucose.

[0274] In certain embodiments, the present invention provides a biocatalytic process for the production of rebaudioside 1n from a starting composition comprising rebaudioside A and UDP-glucose.

[0275] In certain embodiments, the present invention provides a biocatalytic process for the production of rebaudioside 2a from a starting composition comprising stevioside and UDP-glucose.

[0276] In certain embodiments, the present invention provides a biocatalytic process for the production of rebaudioside M4 from a starting composition comprising stevioside and UDP-glucose.

[0277] In certain embodiments, the present invention provides a biocatalytic process for the production of rebaudioside 2a from a starting composition comprising rebaudioside AM and UDP-glucose.

[0278] In certain embodiments, the present invention provides a biocatalytic process for the production of rebaudioside M4 from a starting composition comprising rebaudioside AM and UDP-glucose.

[0279] In certain embodiments, the present invention provides a biocatalytic process for the production of rebaudioside 2a from a starting composition comprising rebaudioside M4 and UDP-glucose.

[0280] In certain embodiments, the present invention provides a biocatalytic process for the production of rebaudioside 1a from a starting composition comprising rebaudioside M and UDP-glucose.

[0281] In certain embodiments, the present invention provides a biocatalytic process for the production of rebaudioside 1b from a starting composition comprising rebaudioside M and UDP-glucose.

[0282] In certain embodiments, the present invention provides a biocatalytic process for the production of rebaudioside 1c from a starting composition comprising rebaudioside M and UDP-glucose.

[0283] In certain embodiments, the present invention provides a biocatalytic process for the production of rebaudioside 1d from a starting composition comprising rebaudioside M and UDP-glucose.

[0284] In certain embodiments, the present invention provides a biocatalytic process for the production of rebaudioside 1e from a starting composition comprising rebaudioside M and UDP-glucose.

[0285] In certain embodiments, the present invention provides a biocatalytic process for the production of rebaudioside 1f from a starting composition comprising rebaudioside M and UDP-glucose.

[0286] In certain embodiments, the present invention provides a biocatalytic process for the production of 1 g of rebaudioside from a starting composition comprising rebaudioside M and UDP-glucose.

[0287] In certain embodiments, the present invention provides a biocatalytic process for the production of rebaudioside 1h from a starting composition comprising rebaudioside M and UDP-glucose.

[0288] In certain embodiments, the present invention provides a biocatalytic process for the production of rebaudioside 1i from a starting composition comprising rebaudioside M and UDP-glucose.

[0289] In certain embodiments, the present invention provides a biocatalytic process for the production of rebaudioside 1j from a starting composition comprising rebaudioside M and UDP-glucose.

[0290] In certain embodiments, the present invention provides a biocatalytic process for the production of rebaudioside 1k from a starting composition comprising rebaudioside M and UDP-glucose.

[0291] In certain embodiments, the present invention provides a biocatalytic process for the production of rebaudioside 1l from a starting composition comprising rebaudioside M and UDP-glucose.

[0292] In certain embodiments, the present invention provides a biocatalytic process for the production of rebaudioside 1m from a starting composition comprising rebaudioside M and UDP-glucose.

[0293] In certain embodiments, the present invention provides a biocatalytic process for the production of rebaudioside 1n from a starting composition comprising rebaudioside M and UDP-glucose.

[0294] In certain embodiments, the present invention provides a biocatalytic process for the production of rebaudioside 2a from a starting composition comprising rebaudioside M4 and UDP-glucose.

[0295] In certain embodiments, the present invention provides a biocatalytic process for the production of SvG7 from a starting composition comprising stevioside and UDP-glucose.

[0296] In certain embodiments, the present invention provides a biocatalytic process for the production of SvG7 from a starting composition comprising rebaudioside A and UDP-glucose.

[0297] In certain embodiments, the present invention provides a biocatalytic process for the production of SvG7 from a starting composition comprising stevioside, rebaudioside A and UDP-glucose.

[0298] In certain embodiments, the present invention provides a biocatalytic process for the production of SvG7 from a starting composition comprising Rebaudioside AM and UDP-glucose.

[0299] In certain embodiments, the present invention provides a biocatalytic process for the production of Rebaudioside M from a starting composition comprising Rebaudioside M and UDP-glucose.

[0300] In certain embodiments, the present invention provides a biocatalytic process for the production of SvG7 from a starting composition comprising rebaudioside M4 and UDP-glucose.

[0301] Optionally, the method of the present invention further comprises separating the target steviol glycoside from the culture medium to provide a high purity target steviol glycoside composition. The target steviol glycoside can be separated by any suitable method, such as, for example, crystallization, membrane separation, centrifugation, extraction, chromatographic separation, or a combination of such methods.

[0302] In certain embodiments, the processes described herein result in a high purity target steviol glycoside composition. As used herein, the term "high purity" refers to a composition having greater than about 80% target steviol glycosides by weight on an anhydrous (dry) basis. In one embodiment, a high purity target steviol glycoside composition contains greater than about 90% target steviol glycosides by weight on an anhydrous (dry) basis, such as, for example, a target steviol glycoside content of greater than about 91%, greater than about 92%, greater than about 93%, greater than about 94%, greater than about 95%, greater than about 96%, greater than about 97%, greater than about 98%, or greater than about 99% by weight on a dry basis.

[0303] In one embodiment, when the target steviol glycoside is rebaudioside M4, the process described herein provides a composition having a rebaudioside M4 content of greater than about 90% by weight on a dry basis. In another specific embodiment, when the target steviol glycoside is rebaudioside M4, the process described herein provides a composition comprising a content of greater than about 95% by weight on a dry basis.

[0304] In one embodiment, when the target steviol glycoside is rebaudioside 2a, the process described herein provides a composition having a rebaudioside 2a content of greater than about 90% by weight on a dry basis. In another specific embodiment, when the target steviol glycoside is rebaudioside 2a, the process described herein provides a composition comprising a content of greater than about 95% by weight on a dry basis.

[0305] In one embodiment, when the target steviol glycoside is SvG7, the process described herein provides a composition having an SvG7 content of greater than about 90% by weight on a dry basis. In another specific embodiment, when the target steviol glycoside is SvG7, the process described herein provides a composition comprising an SvG7 content of greater than about 95% by weight on a dry basis.

[0306] Microorganisms and Enzyme Preparations In one embodiment of the invention, a microorganism (microbial cell) and / or an enzyme preparation is contacted with a medium containing a starting composition to produce the target steviol glycosides.

[0307] The enzymes 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 yet 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.

[0308] In another embodiment, the biocatalyst is one or more microbial cells that contain an enzyme(s) capable of converting the starting composition into the target steviol glycosides. The enzymes may be located on the surface of the cells, intracellularly, or both on the surface and intracellularly of the cells.

[0309] Suitable enzymes for converting the starting composition to the target steviol glycosides include, but are not limited to, steviol biosynthetic enzymes, NDP-glucosyltransferase (NGT), ADP-glucosyltransferase (AGT), CDP-glucosyltransferase (CGT), GDP-glucosyltransferase (GGT), TDP-glucosyltransferase (TDP), UDP-glucosyltransferase (UGT). Optionally, the suitable enzymes can 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).

[0310] In one embodiment, steviol biosynthetic enzymes include mevalonate (MVA) pathway enzymes.

[0311] In another embodiment, steviol biosynthetic enzymes include non-mevalonate 2-C-methyl-D-erythritol-4-phosphate pathway (MEP / DOXP) enzymes.

[0312] In one embodiment, the steviol biosynthetic enzymes are geranylgeranyl diphosphate synthase, copalyl diphosphate synthase, kaurene synthase, kaurene oxidase, kaurenoic acid 13-hydroxylase (KAH), steviol synthetase, deoxyxylulose 5-phosphate synthase (DXS), D-1-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, and the like.

[0313] The UDP-glucosyltransferase can be any UDP-glucosyltransferase capable of adding at least one glucose unit to steviol and / or steviol glycoside substrates to provide the target steviol glycoside.

[0314] In one embodiment, the steviol biosynthetic enzymes and UDP-glucosyltransferase are produced in a microbial cell, which may be, for example, E. coli, Saccharomyces sp., Aspergillus sp., Pichia sp., Bacillus sp., Yarrowia sp., etc. In another embodiment, the UDP-glucosyltransferase is synthetic.

[0315] In one embodiment, the UDP-glucosyltransferase is selected from the group including UGT74G1, UGT85C2, UGT76G1, UGT91D2, UGTSI2, EUGT11 and UGTs having significant (>85%, >86%, >87%, >88%, >89%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99%) amino acid sequence identity to these polypeptides, as well as isolated nucleic acid molecules encoding these UGTs.

[0316] In one embodiment, the steviol biosynthetic enzymes, UGTs, and UDP-glucose salvage system are present in one microorganism (microbial cell), which may be, for example, E. coli, Saccharomyces sp., Aspergillus sp., Pichia sp., Bacillus sp., Yarrowia sp.

[0317] 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 having an -OH functional group at C 13, to give a target steviol glycoside having an -O-glucose β-glucopyranoside glycosidic linkage at C 13. In a particular embodiment, the UDP-glucosyltransferase is UGT85C2 or a UGT having >85% amino acid sequence identity with UGT85C2.

[0318] 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 having a -COOH functional group at C19 to give a target steviol glycoside having a -COO-glucose β-glucopyranoside glycosidic linkage at C19. In a particular embodiment, the UDP-glucosyltransferase is UGT74G1 or a UGT having >85% amino acid sequence identity with UGT74G1.

[0319] In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to any existing glucose at the C19 side of any starting steviol glycoside to give a target steviol glycoside with at least one additional glucose having at least one β1→2 glucopyranoside glycosidic bond in the newly formed glycosidic bond. In a particular embodiment, the UDP-glucosyltransferase is UGTSl2 or a UGT having >85% amino acid sequence identity with UGTSl2. In another particular embodiment, the UDP-glucosyltransferase is EUGT11 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.

[0320] 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 having at least one β1→3 glucopyranoside glycosidic bond in the newly formed glycosidic bond. In a particular embodiment, the UDP-glucosyltransferase is UGT76G1 or a UGT having >85% amino acid sequence identity with UGT76G1.

[0321] In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to any existing glucose at the C19 side of any starting steviol glycoside to give a target steviol glycoside with at least one additional glucose having at least one β1→4 glucopyranoside glycosidic bond in the newly formed glycosidic bond. In a particular embodiment, the UDP-glucosyltransferase is UGTSl2 or a UGT having >85% amino acid sequence identity with UGTSl2. In another particular embodiment, the UDP-glucosyltransferase is EUGT11 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 specific embodiment, the UDP-glucosyltransferase is UGT76G1 or a UGT having >85% amino acid sequence identity with UGT76G1.

[0322] In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to any existing glucose at the C19 side of any starting steviol glycoside to give a target steviol glycoside with at least one additional glucose having at least one β1→6 glucopyranoside glycosidic bond in the newly formed glycosidic bond. In a particular embodiment, the UDP-glucosyltransferase is UGTSl2 or a UGT having >85% amino acid sequence identity with UGTSl2. In another particular embodiment, the UDP-glucosyltransferase is EUGT11 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.

[0323] In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to any existing glucose at the C13 side of any starting steviol glycoside to give a target steviol glycoside with at least one additional glucose having at least one β1→2 glucopyranoside glycosidic bond in the newly formed glycosidic bond. In a particular embodiment, the UDP-glucosyltransferase is UGTSl2 or a UGT having >85% amino acid sequence identity with UGTSl2. In another particular embodiment, the UDP-glucosyltransferase is EUGT11 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.

[0324] 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 having at least one β1→3 glucopyranoside glycosidic bond in the newly formed glycosidic bond. In a particular embodiment, the UDP-glucosyltransferase is UGT76G1 or a UGT having >85% amino acid sequence identity with UGT76G1.

[0325] In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to any existing glucose at the C13 side of any starting steviol glycoside to give a target steviol glycoside with at least one additional glucose having at least one β1→4 glucopyranoside glycosidic bond in the newly formed glycosidic bond. In a particular embodiment, the UDP-glucosyltransferase is UGTSl2 or a UGT having >85% amino acid sequence identity with UGTSl2. In another particular embodiment, the UDP-glucosyltransferase is EUGT11 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 specific embodiment, the UDP-glucosyltransferase is UGT76G1 or a UGT having >85% amino acid sequence identity with UGT76G1.

[0326] In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to any existing glucose at the C13 side of any starting steviol glycoside to give a target steviol glycoside with at least one additional glucose having at least one β1→6 glucopyranoside glycosidic bond in the newly formed glycosidic bond. In a particular embodiment, the UDP-glucosyltransferase is UGTSl2 or a UGT having >85% amino acid sequence identity with UGTSl2. In another particular embodiment, the UDP-glucosyltransferase is EUGT11 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.

[0327] In one embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to steviol to form a steviol monoside, hi certain embodiments, the UDP-glucosyltransferase is UGT85C2, or a UGT with >85% amino acid sequence identity with UGT85C2, or a UGT with >85% amino acid sequence identity with UGT85C2.

[0328] 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 UGT74G1 or a UGT having >85% amino acid sequence identity with UGT74G1.

[0329] 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 UGTSl2 or a UGT with >85% amino acid sequence identity to UGTSl2. In another particular embodiment, the UDP-glucosyltransferase is EUGT11 or a UGT with >85% amino acid sequence identity to EUGT11. In yet another particular embodiment, the UDP-glucosyltransferase is UGT91D2 or a UGT with >85% amino acid sequence identity to UGT91D2.

[0330] 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.

[0331] In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to steviolmonoside to form rubusoside, hi a particular embodiment, the UDP-glucosyltransferase is UGT74G1 or a UGT having >85% amino acid sequence identity with UGT74G1.

[0332] 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 with >85% amino acid sequence identity to UGT85C2, or a UGT with >85% amino acid sequence identity to UGT85C2.

[0333] 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 UGTSl2 or a UGT with >85% amino acid sequence identity to UGTSl2. In another particular embodiment, the UDP-glucosyltransferase is EUGT11 or a UGT with >85% amino acid sequence identity to EUGT11. In yet another particular embodiment, the UDP-glucosyltransferase is UGT91D2 or a UGT with >85% amino acid sequence identity to UGT91D2.

[0334] 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 UGT76G1.

[0335] 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 UGT76G1 or a UGT having >85% amino acid sequence identity with UGT76G1.

[0336] In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to steviolbioside to form stevioside, hi a particular embodiment, the UDP-glucosyltransferase is UGT74G1 or a UGT having >85% amino acid sequence identity with UGT74G1.

[0337] 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 UGTSl2 or a UGT with >85% amino acid sequence identity to UGTSl2. In another particular embodiment, the UDP-glucosyltransferase is EUGT11 or a UGT with >85% amino acid sequence identity to EUGT11. In yet another particular embodiment, the UDP-glucosyltransferase is UGT91D2 or a UGT with >85% amino acid sequence identity to UGT91D2.

[0338] 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.

[0339] 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 UGTSl2 or a UGT with >85% amino acid sequence identity to UGTSl2. In another particular embodiment, the UDP-glucosyltransferase is EUGT11 or a UGT with >85% amino acid sequence identity to EUGT11. In yet another particular embodiment, the UDP-glucosyltransferase is UGT91D2 or a UGT with >85% amino acid sequence identity to UGT91D2.

[0340] 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.

[0341] 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 UGTSl2 or a UGT with >85% amino acid sequence identity to UGTSl2. In another particular embodiment, the UDP-glucosyltransferase is EUGT11 or a UGT with >85% amino acid sequence identity to EUGT11. In yet another particular embodiment, the UDP-glucosyltransferase is UGT91D2 or a UGT with >85% amino acid sequence identity to UGT91D2.

[0342] 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.

[0343] 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 particular embodiments, the UDP-glucosyltransferase is UGT85C2, or a UGT with >85% amino acid sequence identity with UGT85C2, or a UGT with >85% amino acid sequence identity with UGT85C2.

[0344] 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.

[0345] 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 with >85% amino acid sequence identity to UGT85C2, or a UGT with >85% amino acid sequence identity to UGT85C2.

[0346] 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 UGTSl2 or a UGT with >85% amino acid sequence identity to UGTSl2. In another particular embodiment, the UDP-glucosyltransferase is EUGT11 or a UGT with >85% amino acid sequence identity to EUGT11. In yet another particular embodiment, the UDP-glucosyltransferase is UGT91D2 or a UGT with >85% amino acid sequence identity to UGT91D2.

[0347] 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.

[0348] 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.

[0349] 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 UGTSl2 or a UGT with >85% amino acid sequence identity to UGTSl2. In another particular embodiment, the UDP-glucosyltransferase is EUGT11 or a UGT with >85% amino acid sequence identity to EUGT11. In yet another particular embodiment, the UDP-glucosyltransferase is UGT91D2 or a UGT with >85% amino acid sequence identity to UGT91D2.

[0350] 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 UGT76G1 or a UGT having >85% amino acid sequence identity with UGT76G1.

[0351] 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 UGTSl2 or a UGT with >85% amino acid sequence identity to UGTSl2. In another particular embodiment, the UDP-glucosyltransferase is EUGT11 or a UGT with >85% amino acid sequence identity to EUGT11. In yet another particular embodiment, the UDP-glucosyltransferase is UGT91D2 or a UGT with >85% amino acid sequence identity to UGT91D2.

[0352] 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 UGTSl2 or a UGT with >85% amino acid sequence identity to UGTSl2. In another particular embodiment, the UDP-glucosyltransferase is EUGT11 or a UGT with >85% amino acid sequence identity to EUGT11. In yet another particular embodiment, the UDP-glucosyltransferase is UGT91D2 or a UGT with >85% amino acid sequence identity to UGT91D2.

[0353] 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.

[0354] 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 UGTSl2 or a UGT with >85% amino acid sequence identity to UGTSl2. In another particular embodiment, the UDP-glucosyltransferase is EUGT11 or a UGT with >85% amino acid sequence identity to EUGT11. In yet another particular embodiment, the UDP-glucosyltransferase is UGT91D2 or a UGT with >85% amino acid sequence identity to UGT91D2.

[0355] 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.

[0356] 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.

[0357] 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 UGTSl2 or a UGT with >85% amino acid sequence identity to UGTSl2. In another particular embodiment, the UDP-glucosyltransferase is EUGT11 or a UGT with >85% amino acid sequence identity to EUGT11. In yet another particular embodiment, the UDP-glucosyltransferase is UGT91D2 or a UGT with >85% amino acid sequence identity to UGT91D2.

[0358] 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.

[0359] 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 UGTSl2 or a UGT with >85% amino acid sequence identity to UGTSl2. In another particular embodiment, the UDP-glucosyltransferase is EUGT11 or a UGT with >85% amino acid sequence identity to EUGT11. In yet another particular embodiment, the UDP-glucosyltransferase is UGT91D2 or a UGT with >85% amino acid sequence identity to UGT91D2.

[0360] 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 with >85% amino acid sequence identity to UGT85C2, or a UGT with >85% amino acid sequence identity to UGT85C2.

[0361] 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 UGTSl2 or a UGT with >85% amino acid sequence identity to UGTSl2. In another particular embodiment, the UDP-glucosyltransferase is EUGT11 or a UGT with >85% amino acid sequence identity to EUGT11. In yet another particular embodiment, the UDP-glucosyltransferase is UGT91D2 or a UGT with >85% amino acid sequence identity to UGT91D2.

[0362] 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.

[0363] In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase that can add 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 to UGT76G1.

[0364] 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.

[0365] 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 to UGT76G1.

[0366] 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 UGTSl2 or a UGT with >85% amino acid sequence identity to UGTSl2. In another particular embodiment, the UDP-glucosyltransferase is EUGT11 or a UGT with >85% amino acid sequence identity to EUGT11. In yet another particular embodiment, the UDP-glucosyltransferase is UGT91D2 or a UGT with >85% amino acid sequence identity to UGT91D2.

[0367] 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 UGTSl2 or a UGT with >85% amino acid sequence identity to UGTSl2. In another particular embodiment, the UDP-glucosyltransferase is EUGT11 or a UGT with >85% amino acid sequence identity to EUGT11. In yet another particular embodiment, the UDP-glucosyltransferase is UGT91D2 or a UGT with >85% amino acid sequence identity to UGT91D2.

[0368] In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase that can add 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.

[0369] 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 UGTSl2 or a UGT with >85% amino acid sequence identity to UGTSl2. In another particular embodiment, the UDP-glucosyltransferase is EUGT11 or a UGT with >85% amino acid sequence identity to EUGT11. In yet another particular embodiment, the UDP-glucosyltransferase is UGT91D2 or a UGT with >85% amino acid sequence identity to UGT91D2.

[0370] 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 UGTSl2 or a UGT with >85% amino acid sequence identity to UGTSl2. In another particular embodiment, the UDP-glucosyltransferase is EUGT11 or a UGT with >85% amino acid sequence identity to EUGT11. In yet another particular embodiment, the UDP-glucosyltransferase is UGT91D2 or a UGT with >85% amino acid sequence identity to UGT91D2.

[0371] 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 UGTSl2 or a UGT with >85% amino acid sequence identity to UGTSl2. In another particular embodiment, the UDP-glucosyltransferase is EUGT11 or a UGT with >85% amino acid sequence identity to EUGT11. In yet another particular embodiment, the UDP-glucosyltransferase is UGT91D2 or a UGT with >85% amino acid sequence identity to UGT91D2.

[0372] In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase that can add 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.

[0373] In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase that can add 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 to UGT76G1.

[0374] 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 UGTSl2 or a UGT with >85% amino acid sequence identity to UGTSl2. In another particular embodiment, the UDP-glucosyltransferase is EUGT11 or a UGT with >85% amino acid sequence identity to EUGT11. In yet another particular embodiment, the UDP-glucosyltransferase is UGT91D2 or a UGT with >85% amino acid sequence identity to UGT91D2.

[0375] In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase that can add 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.

[0376] 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 UGTSl2 or a UGT with >85% amino acid sequence identity to UGTSl2. In another particular embodiment, the UDP-glucosyltransferase is EUGT11 or a UGT with >85% amino acid sequence identity to EUGT11. In yet another particular embodiment, the UDP-glucosyltransferase is UGT91D2 or a UGT with >85% amino acid sequence identity to UGT91D2. In a particular embodiment, the UDP-glucosyltransferase is UGT76G1 or a UGT with >85% amino acid sequence identity to UGT76G1.

[0377] 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 UGTSl2 or a UGT with >85% amino acid sequence identity to UGTSl2. In another particular embodiment, the UDP-glucosyltransferase is EUGT11 or a UGT with >85% amino acid sequence identity to EUGT11. In yet another particular embodiment, the UDP-glucosyltransferase is UGT91D2 or a UGT with >85% amino acid sequence identity to UGT91D2.

[0378] In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside M to form rebaudioside 1a. In a particular embodiment, the UDP-glucosyltransferase is UGTSl2 or a UGT with >85% amino acid sequence identity to UGTSl2. In another particular embodiment, the UDP-glucosyltransferase is EUGT11 or a UGT with >85% amino acid sequence identity to EUGT11. In yet another particular embodiment, the UDP-glucosyltransferase is UGT91D2 or a UGT with >85% amino acid sequence identity to UGT91D2.

[0379] In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside M to form rebaudioside 1b. In a particular embodiment, the UDP-glucosyltransferase is UGT76G1 or a UGT having >85% amino acid sequence identity to UGT76G1.

[0380] In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside M to form rebaudioside 1c. In a particular embodiment, the UDP-glucosyltransferase is UGTSl2 or a UGT with >85% amino acid sequence identity to UGTSl2. In another particular embodiment, the UDP-glucosyltransferase is EUGT11 or a UGT with >85% amino acid sequence identity to EUGT11. In yet another particular embodiment, the UDP-glucosyltransferase is UGT91D2 or a UGT with >85% amino acid sequence identity to UGT91D2.

[0381] In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside M to form rebaudioside 1d. In a particular embodiment, the UDP-glucosyltransferase is UGTSl2 or a UGT with >85% amino acid sequence identity to UGTSl2. In another particular embodiment, the UDP-glucosyltransferase is EUGT11 or a UGT with >85% amino acid sequence identity to EUGT11. In yet another particular embodiment, the UDP-glucosyltransferase is UGT91D2 or a UGT with >85% amino acid sequence identity to UGT91D2.

[0382] In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside M to form rebaudioside 1e. In a particular embodiment, the UDP-glucosyltransferase is UGT76G1 or a UGT having >85% amino acid sequence identity with UGT76G1.

[0383] In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside M to form rebaudioside 1f. In a particular embodiment, the UDP-glucosyltransferase is UGTSl2 or a UGT with >85% amino acid sequence identity to UGTSl2. In another particular embodiment, the UDP-glucosyltransferase is EUGT11 or a UGT with >85% amino acid sequence identity to EUGT11. In yet another particular embodiment, the UDP-glucosyltransferase is UGT91D2 or a UGT with >85% amino acid sequence identity to UGT91D2.

[0384] In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside M to form rebaudioside 1g. In a particular embodiment, the UDP-glucosyltransferase is UGTSl2 or a UGT with >85% amino acid sequence identity to UGTSl2. In another particular embodiment, the UDP-glucosyltransferase is EUGT11 or a UGT with >85% amino acid sequence identity to EUGT11. In yet another particular embodiment, the UDP-glucosyltransferase is UGT91D2 or a UGT with >85% amino acid sequence identity to UGT91D2.

[0385] In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase that can add at least one glucose unit to rebaudioside M to form rebaudioside 1h. In a particular embodiment, the UDP-glucosyltransferase is UGT76G1 or a UGT having >85% amino acid sequence identity to UGT76G1.

[0386] In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside M to form rebaudioside 1i. In a particular embodiment, the UDP-glucosyltransferase is UGTSl2 or a UGT with >85% amino acid sequence identity to UGTSl2. In another particular embodiment, the UDP-glucosyltransferase is EUGT11 or a UGT with >85% amino acid sequence identity to EUGT11. In yet another particular embodiment, the UDP-glucosyltransferase is UGT91D2 or a UGT with >85% amino acid sequence identity to UGT91D2.

[0387] In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside M to form rebaudioside 1j. In a particular embodiment, the UDP-glucosyltransferase is UGTSl2 or a UGT with >85% amino acid sequence identity to UGTSl2. In another particular embodiment, the UDP-glucosyltransferase is EUGT11 or a UGT with >85% amino acid sequence identity to EUGT11. In yet another particular embodiment, the UDP-glucosyltransferase is UGT91D2 or a UGT with >85% amino acid sequence identity to UGT91D2.

[0388] In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside M to form rebaudioside 1k. In a particular embodiment, the UDP-glucosyltransferase is UGT76G1 or a UGT having >85% amino acid sequence identity to UGT76G1.

[0389] In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside M to form rebaudioside 1l. In a particular embodiment, the UDP-glucosyltransferase is UGTSl2 or a UGT with >85% amino acid sequence identity to UGTSl2. In another particular embodiment, the UDP-glucosyltransferase is EUGT11 or a UGT with >85% amino acid sequence identity to EUGT11. In yet another particular embodiment, the UDP-glucosyltransferase is UGT91D2 or a UGT with >85% amino acid sequence identity to UGT91D2.

[0390] In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside M to form rebaudioside 1m. In a particular embodiment, the UDP-glucosyltransferase is UGTSl2 or a UGT with >85% amino acid sequence identity to UGTSl2. In another particular embodiment, the UDP-glucosyltransferase is EUGT11 or a UGT with >85% amino acid sequence identity to EUGT11. In yet another particular embodiment, the UDP-glucosyltransferase is UGT91D2 or a UGT with >85% amino acid sequence identity to UGT91D2.

[0391] In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside M to form rebaudioside 1n. In a particular embodiment, the UDP-glucosyltransferase is UGTSl2 or a UGT with >85% amino acid sequence identity to UGTSl2. In another particular embodiment, the UDP-glucosyltransferase is EUGT11 or a UGT with >85% amino acid sequence identity to EUGT11. In yet another particular embodiment, the UDP-glucosyltransferase is UGT91D2 or a UGT with >85% amino acid sequence identity to UGT91D2.

[0392] 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 UGTSl2 or a UGT with >85% amino acid sequence identity to UGTSl2. In another particular embodiment, the UDP-glucosyltransferase is EUGT11 or a UGT with >85% amino acid sequence identity to EUGT11. In yet another particular embodiment, the UDP-glucosyltransferase is UGT91D2 or a UGT with >85% amino acid sequence identity to UGT91D2. In a particular embodiment, the UDP-glucosyltransferase is UGT76G1 or a UGT with >85% amino acid sequence identity to UGT76G1.

[0393] Optionally, the method further comprises recycling UDP to provide UDP-glucose. In one embodiment, the method comprises recycling UDP by providing a recycled catalyst and a recycled substrate such that bioconversion of steviol and / or steviol glycoside substrates to target steviol glycosides occurs using catalytic amounts of UDP-glucosyltransferase and UDP-glucose.

[0394] In one embodiment, the recycled catalyst is sucrose synthase SuSy_At, or a sucrose synthase having >85% amino acid sequence identity with SuSy_At.

[0395] In one embodiment, the recycling substrate for the UDP-glucose recycling catalyst is sucrose.

[0396] Optionally, the method of the present invention further comprises the use of a transglycosidase that uses an oligosaccharide or polysaccharide as a sugar donor to modify the target acceptor steviol glycoside molecule. Non-limiting examples include cyclodextrin glycosyltransferase (CGTase), fructofuranosidase, amylase, saccharase, glucosucrase, β-h-fructosidase, β-fructosidase, sucrase, fructosyl invertase, alkaline invertase, acid invertase, and fructofuranosidase. In some embodiments, glucose and sugars other than glucose, including but not limited to fructose, xylose, rhamnose, arabinose, deoxyglucose, and galactose, are transferred to the target acceptor steviol glycoside. In one embodiment, the acceptor steviol glycoside is rebaudioside 1a, rebaudioside 1b, rebaudioside 1c, rebaudioside 1d, rebaudioside 1e, rebaudioside 1f, rebaudioside 1g, rebaudioside 1h, rebaudioside 1i, rebaudioside 1j, rebaudioside 1k, rebaudioside 1l, rebaudioside 1m, and / or rebaudioside 1n. In another embodiment, the acceptor steviol glycoside is rebaudioside 2a. In another embodiment, the acceptor steviol glycoside is rebaudioside M4. In another embodiment, the acceptor steviol glycoside is SvG7.

[0397] In another embodiment, the UDP-glucosyltransferase capable of adding at least one glucose unit to the starting composition, a steviol glycoside, has >85% amino acid sequence identity with a UGT selected from the list of GenInfo Identifier Numbers below, preferably from the group presented in Tables 1 and 2.

[0398] [Table 1-1]

[0399] [Table 1-2]

[0400] [Table 1-3]

[0401] [Table 2-1]

[0402] [Table 2-2]

[0403] [Table 3]

[0404] One embodiment of the present invention is a microbial cell that comprises an enzyme, i.e., an enzyme capable of converting a starting composition into a target steviol glycoside. Thus, some embodiments of the method include contacting a microorganism with a medium containing the starting composition to provide a medium that includes at least one target steviol glycoside.

[0405] The microorganism can be any microorganism that has the necessary enzyme(s) to convert the starting composition into the target steviol glycoside(s). These enzymes are encoded within the genome of the microorganism.

[0406] Suitable microorganisms include, but are not limited to, E. coli, Saccharomyces sp., Aspergillus sp., Pichia sp., Bacillus sp., Yarrowia sp., and the like.

[0407] In one embodiment, the microorganisms are free when contacted with the starting composition.

[0408] In another embodiment, the microorganism is immobilized when contacted with the starting composition. For example, the microorganism may be immobilized on a solid support made of inorganic or organic materials. Non-limiting examples of solid supports suitable for immobilizing microorganisms include derivatized cellulose or derivatized glass, ceramics, metal oxides, or membranes. The microorganism may be immobilized on the solid support by, for example, covalent binding, adsorption, cross-linking, entrapment, or encapsulation.

[0409] In yet another embodiment, the enzymes capable of converting the starting composition into the target steviol glycosides are secreted outside the microorganism and into the reaction medium.

[0410] The target steviol glycoside is optionally purified. Purification of the target steviol glycoside from the reaction medium can be accomplished by at least one suitable method to provide a high purity target steviol glycoside composition. Suitable methods include crystallization, membrane separation, centrifugation, extraction (liquid or solid phase), chromatographic separation, HPLC (preparative or analytical), or a combination of such methods.

[0411] use The high purity target glycoside(s) obtained according to the present invention, in particular 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 B, rebaudioside C, rebaudioside D ... B, rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside E, rebaudioside B, rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside E, rebaudioside E, rebaudioside E, rebaudioside E, rebaudioside E, rebaudioside E, rebaudioside E, rebaudioside E, rebaudioside E, rebaudioside E, rebaudioside E, rebaudioside E, rebaudioside E, rebaudioside E, rebaudioside E, rebaudioside E, rebaudioside E, rebaudioside E, rebaudioside E, rebaudioside E, rebaudioside E, rebaudioside E, reba 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 can be used "as is" or in combination with other sweeteners, flavors, food ingredients, and combinations thereof.

[0412] Non-limiting examples of flavorings 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.

[0413] Non-limiting examples of other food ingredients include, but are not limited to, acidulants, organic acids and amino acids, colorants, bulking agents, modified starches, gums, texturizing agents, preservatives, caffeine, antioxidants, emulsifiers, stabilizers, thickeners, gelling agents, and combinations thereof.

[0414] The high purity target glycoside(s) obtained according to the present invention, in particular 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 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 can be prepared in various polymorphic forms, including, but not limited to, hydrates, solvates, anhydrous, amorphous forms, and combinations thereof.

[0415] The high purity target glycoside(s) obtained according to the present invention, in particular 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 G, rebaudioside H, rebaudioside I, rebaudioside AM, rebaudioside D8, rebaudioside I10, rebaudioside I21, rebaudioside I31, rebaudioside I41, rebaudioside I51, rebaudioside I61, rebaudioside I71, rebaudioside I82, rebaudioside I91, rebaudioside I102, rebaudioside I111, rebaudioside I121, rebaudioside I131, rebaudioside I141, rebaudioside I151, rebaudioside I161, rebaudioside I171, rebaudioside I182, rebaudioside I191, rebaudioside I221, rebaudioside I231, rebaudioside I341, rebaudioside I191, rebaudioside I241, rebaudioside I191, rebaudioside I251, rebaudioside I351, rebaudioside I191, rebaudioside I261, rebaudi Oside 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 may be incorporated as high intensity natural sweeteners in foods, beverages, pharmaceutical compositions, cosmetics, chewing gum, tabletop products, cereals, dairy products, toothpaste, and other oral compositions and the like.

[0416] In some embodiments, the high purity target glycosides of the present invention may be, for example, about 0.0001 wt%, about 0.0005 wt%, about 0.001 wt%, about 0.005 wt%, about 0.01 wt%, about 0.05 wt%, about 0.1 wt%, about 0.5 wt%, about 1.0 wt%, about 1.5 wt%, about 2.0 wt%, about 2.5 wt%, about 3.0 wt%, about 3.5 wt%, about 4.0 wt%, about 4.5 wt%, about 5.0 wt%, about 5.5 wt%, about 6.0 wt%, about 7.0 wt%, about 8.0 wt%, about 9.0 wt%, about 10.0 wt%, about 11.0 wt%, about 12.0 wt%, about 13.0 wt%, about 14.0 wt%, about 15.0 wt%, about 16.0 wt%, about 17.0 wt%, about 18.0 wt%, about 19.0 wt%, about 20.0 wt%, about 21.0 wt%, about 22.0 wt%, about 23.0 wt%, about 24.0 wt%, about 25.0 wt%, about 26.0 wt%, about 27.0 wt%, about 28.0 wt%, about 29.0 wt%, about 30.0 wt%, about 31.0 wt%, about 32.0 wt%, about 33.0 wt%, about 34.0 wt%, about 35.0 wt%, about 36.0 wt%, about 37.0 wt%, about 38.0 wt%, about 39.0 wt%, about 40.0 wt%, about 41.0 wt%, about 42.0 wt%, about 43. In some embodiments, the glycerin-containing compound is present in foods, beverages, pharmaceutical compositions, cosmetics, chewing gum, tabletop products, cereals, dairy products, toothpastes, and other oral compositions in an amount of about 0.0001% to about 12% by weight, such as about 6.5% by weight, about 7.0% by weight, about 7.5% by weight, about 8.0% by weight, about 8.5% by weight, about 9.0% by weight, about 9.5% by weight, about 10.0% by weight, about 10.5% by weight, about 11.0% by weight, about 11.5% by weight, or about 12.0% by weight.

[0417] In certain embodiments, the sweetener is present in the beverage in an amount of from about 0.0001% to about 8% by weight, such as, for example, from about 0.0001% to about 0.0005% by weight, from about 0.0005% to about 0.001% by weight, from about 0.001% to about 0.005% by weight, from about 0.005% to about 0.01% by weight, from about 0.01% to about 0.05% by weight, from about 0.05% to about 0.1% by weight, from about 0.1% to about 0.5% by weight, from about 0.5% to about 1% by weight, from about 1% to about 2% by weight, from about 2% to about 3% by weight, from about 3% to about 4% by weight, from about 4% to about 5% by weight, from about 5% to about 6% by weight, from about 6% to about 7% by weight, and from about 7% to about 8% by weight.

[0418] The high purity target glycoside(s) obtained according to the present invention, in particular 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 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, SvG7, and / or combinations thereof may be used as sweetener compounds, or Dulcoside A, Dulcoside B, Dulcoside C, Dulcoside D, Rebaudioside A2, Dulcoside B, Dulcoside C, Dulcoside D, Rebaudioside A3, Dulcoside B4, Dulcoside C, Dulcoside D, Rebaudioside A5, Dulcoside B6, Dulcoside C7, Dulcoside D8, Dulcoside C9, Dulcoside D10, Dulcoside D11, Dulcoside D12, Dulcoside D13, Dulcoside D14, Dulcoside D15, Dulcoside D16, Dulcoside D17, Dulcoside D18, Dulcoside D19, Dulcoside D20, Dulcoside D21, Dulcoside D22, Dulcoside D3, Dulcoside D4, Dulcoside D5, Dulcoside D6, Dulcoside D19, Dulcoside D23, Dulcoside D34, Dulcoside D4, Dulcoside D5, Dulcoside D6, Dulcoside D7, Dulcoside D10, Dulcoside D11, Dulcoside D22, Dulcoside D12, Dulcoside D23, Dul , 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 H4, Rebaudioside H5, Rebaudioside H6, Rebaudioside H7, Rebaudioside H8, Rebaudioside H9, Rebaudioside H10, Rebaudioside H11, Rebaudioside H12, Rebaudioside H13, Rebaudioside H14, Rebaudioside H15, Rebaudioside H16, Rebaudioside H17, Rebaudioside H18, Rebaudioside H19, Rebaudioside H20, Rebaudioside H21, Rebaudioside H22, Rebaudioside H23, Rebaudioside H24, Rebaudioside H25, Rebaudioside H26, Rebaudioside H27, Rebaudioside H28, Rebaudioside H29, Rebaudioside H30, Rebaudioside H31, Rebaudioside H32, Rebaudioside H33, Rebaudioside H34, Rebaudioside H35, Rebaudioside H36, Rebaudioside H37, Rebaudioside H38, Rebaudioside H39, Rebaudioside H41, Re Rebaudioside H6, Rebaudioside I2, Rebaudioside I3, 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 O2, Rebaudioside O3, Rebaudioside O4, Rebaudioside Q, Rebaudioside Q2, Rebaudioside Q3, Rebaudioside R, Rebaudioside S, Rebaudioside T, Rebaudioside T1,Rebaudioside U, Rebaudioside U2, Rebaudioside V, Rebaudioside V2, Rebaudioside V3, Rebaudioside W, Rebaudioside W2, Rebaudioside W3, Rebaudioside Y, Rebaudioside Z1, Rebaudioside Z2, Steviolbioside C, Steviolbioside E, Stevioside D, Stevioside E, Stevioside E2, Stevioside F, Stevioside G, Stevioside H, Mogroside, Brazzein, Neohesperidin Dihydrochalcone, Glycyrrhizic Acid and its Salts, Thaumatin, Perillartine, Pernandulcin, Mukuroziosid, Bayunoside, Phromisoside I, Dimethyl-Hexahydrofluorene-Dicarboxylic Acid, Abrusoside, Periandrin, Carnosifloside , cyclocaryoside, pterocaryoside, polypodoside A, brazilin, hernandulcin, phyllodulcin, glycyphyllin, phloridzin, trilobatin, dihydroflavonol, dihydroquercetin-3-acetate, neoastiribin, trans-cinnamaldehyde, monatin, monatin salts, other indole derivative sweeteners, seligeain A, hematoxylin, monellin, osladin, pterocaryoside A, pterocaryoside B, mabinlin, pentajin, miraculin, curculin, neoculin, chlorogenic acid, cynarin, monk fruit sweetener, mogroside V, siamenoside, sciratose, and combinations thereof.

[0419] In certain embodiments, 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 M 4, 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 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 H4, Rebaudioside H5, Rebaudioside H6, Rebaudioside I2, Rebaudioside I3, Rebaudioside J, Rebaudioside K, Rebaudioside I Udioside K2, Rebaudioside KA, Rebaudioside L, Rebaudioside M2, Rebaudioside M3, Rebaudioside N, Rebaudioside N2, Rebaudioside N3, Rebaudioside N4, Rebaudioside N5, Rebaudioside O, Rebaudioside O2, Rebaudioside O3, Rebaudioside O4, Rebaudioside Q, Rebaudioside Q2, Rebaudioside Q3, Rebaudioside R, Rebaudioside S, Rebaudioside T, Rebaudioside T1, Rebaudioside U, Rebaudioside U2, Rebaudioside V, Rebaudioside V2, Rebaudioside V3,The sweetener composition may include a compound selected from the group consisting of rebaudioside W, rebaudioside W2, rebaudioside W3, rebaudioside Y, rebaudioside Z1, rebaudioside Z2, steviolbioside C, steviolbioside E, stevioside D, stevioside E, stevioside E2, stevioside F, stevioside G, stevioside H, NSF-02, mogroside V, sciratose, monk fruit, allulose, allose, D-tagatose, erythritol, and combinations thereof.

[0420] Highly purified target glycoside(s), especially 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 1a, rebaudioside 1 b, 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 may also be used in combination with synthetic high intensity sweeteners such as sucralose, acesulfame potassium, aspartame, alitame, saccharin, neohesperidin dihydrochalcone, cyclamate, neotame, dulcin, suosan advantame, salts thereof, and combinations thereof.

[0421] Additionally, the present invention provides high purity target steviol glycoside(s), in particular steviol monoside, steviol monoside A, steviol bioside, steviol bioside D, rubusoside, steviol bioside A, steviol bioside 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 B, rebaudioside C, rebaudioside D ... Uudioside 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 can be used in combination with a natural sweetener reducer, such as gymnemic acid, phozurcin, digiphin, lactisol, and the like. 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 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 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 I, Rebaudioside AM, Rebaudioside D7, Rebaudioside M, Rebaudioside M4, Rebaudioside 1a, Rebaudioside 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 can be mixed with umami flavorful amino acids and sweet amino acids, such as glutamic acid, aspartic acid, glycine, alanine, threonine, proline, serine, glutamic acid, lysine, tryptophan, and combinations thereof.

[0422] High purity target steviol glycoside(s), in particular steviol monoside, steviol monoside A, steviol bioside, steviol bioside D, rubusoside, steviol bioside A, steviol bioside B, rebaudioside B, stevioside, rebaudioside G, stevioside A, stevioside B, stevioside C, rebaudioside A, rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside G ... Rebaudioside E, Rebaudioside E2, Rebaudioside E4, Rebaudioside E6, Rebaudioside E3, Rebaudioside D, Rebaudioside I, Rebaudioside AM, Rebaudioside D7, Rebaudioside M, Rebaudioside M4, Rebaudioside 1a, Rebaudioside 1b, Rebaudioside 1c, Rebaudioside 1d, Rebaudioside 1e, Rebaudio Rebaudioside 1f, Rebaudioside 1g, Rebaudioside 1h, Rebaudioside 1i, Rebaudioside 1j, Rebaudioside 1k, Rebaudioside 1l, Rebaudioside 1m, Rebaudioside 1n, Rebaudioside 2a, and / or SvG7 can be used in combination with one or more additives selected from the group consisting of carbohydrates, polyols, amino acids and their corresponding salts, polyamino acids and their corresponding salts, sugar acids and their corresponding salts, nucleotides, organic acids, inorganic acids, organic salts including organic acid salts and organic base salts, inorganic salts, bitter compounds, flavorants and flavor components, astringent compounds, proteins or protein hydrolysates, surfactants, emulsifiers, flavonoids, alcohols, polymers, and combinations thereof.

[0423] Highly purified target steviol glycoside(s), specifically, 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 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 may be combined with a polyol or sugar alcohol. The term "polyol" refers to a molecule that contains two or more hydroxyl groups. The polyol may be a diol, triol, or tetraol, containing two, three, and four hydroxyl groups, respectively. Polyols may also contain more than 5 hydroxyl groups, such as pentaols, hexaols, heptaols, which contain 5, 6, or 7 hydroxyl groups, respectively. In addition, polyols may also be sugar alcohols, polyhydric alcohols, or polyalcohols that are reduced forms of carbohydrates, in which the carbonyl groups (aldehydes or ketones, reducing sugars) are reduced to primary or secondary hydroxyl groups. Examples of polyols include erythritol, maltitol, mannitol, sorbitol, lactitol, xylitol, inositol, isomalt, propylene glycol, glycerol, threitol, galactitol, hydrogenated isomaltulose, reduced isomaltooligosaccharides, reduced xylooligosaccharides, reduced gentiooligosaccharides, reduced maltose syrup, reduced glucose syrup, hydrogenated starch hydrolysates, polyglycitols, and sugar alcohols, or any other carbohydrate that can be reduced without adversely affecting the taste of the sweetener composition.

[0424] High purity target steviol glycoside(s), especially steviol monoside, steviol monoside A, steviol bioside, steviol bioside D, rubusoside, steviol bioside A, steviol bioside 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 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 may be combined with a low calorie sweetener such as, for example, D-tagatose, L-sugar, L-sorbose, L-arabinose, and combinations thereof.

[0425] Highly purified targeted steviol glycoside(s), specifically steviol monoside, steviol monoside A, steviol bioside, steviol bioside D, rubusoside, steviol bioside A, steviol bioside 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 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 may also be combined with various carbohydrates. The term "carbohydrate" generally refers to a carbohydrate having the general formula (CHO) n(wherein n is 3-30), and oligomers and polymers thereof, as well as aldehyde or ketone compounds substituted with multiple hydroxyl groups. Additionally, the carbohydrates of the present invention may be substituted or deoxygenated at one or more positions. As used herein, carbohydrate encompasses unmodified carbohydrates, carbohydrate derivatives, substituted carbohydrates, and modified carbohydrates. As used herein, the phrases "carbohydrate derivatives", "substituted carbohydrates", and "modified carbohydrates" are synonymous. Modified carbohydrates refer to any carbohydrate in which at least one atom has been added, removed, or replaced, or a combination thereof. Thus, carbohydrate derivatives or substituted carbohydrates include substituted and unsubstituted monosaccharides, disaccharides, oligosaccharides, and polysaccharides. The carbohydrate derivative or substituted carbohydrate can be optionally 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 derivative, alkylamino, dialkylamino, arylamino, alkoxy, aryloxy, nitro, cyano, sulfo, mercapto, imino, sulfonyl, sulfenyl, sulfinyl, sulfamoyl, carboalkoxy, carboxamido, phosphonyl, phosphinyl, phosphoryl, phosphino, thioester, thioether, oxyimino, hydrazino, carbamyl, phospho, phosphonato, or any other possible functional group, if the carbohydrate derivative or substituted carbohydrate functional group functions to enhance the sweetness of the sweetener composition.

[0426] Examples of carbohydrates that may be used in accordance with the present invention include 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, gluconolactone, abecoose, galactosamine, beta-glucose, glycerol ... These include, but are not limited to, isomalto-oligosaccharides (isomaltose, isomaltotriose, panose, etc.), xylo-oligosaccharides (xylotriose, xylobiose, etc.), xylo-terminal oligosaccharides, gentio-oligosaccharides (gentiobiose, gentiotriose, gentiotetraose, etc.), sorbose, nigero-oligosaccharides, palatinose oligosaccharides, fructooligosaccharides (kestose, nystose, etc.), maltotetraol, maltotriol, malto-oligosaccharides (maltotriose, maltotetraose, maltopentaose, maltohexaose, maltoheptaose, etc.), starch, inulin, inulo-oligosaccharides, lactulose, melibiose, raffinose, ribose, isomerized liquid sugars such as high fructose corn syrup, coupling sugars, and soybean oligosaccharides. In addition, carbohydrates, as used herein, may be in either the D- or L-configuration.

[0427] The high purity target steviol glycoside(s) obtained according to the present invention, in particular 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 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 can be used in combination with various physiologically active substances or functional ingredients. Functional ingredients are generally classified into categories such as carotenoids, dietary fiber, fatty acids, saponins, antioxidants, dietary supplements, flavonoids, isothiocyanates, phenols, plant sterols and stanols (phytosterols and phytostanols), polyols, prebiotics, probiotics, phytoestrogens, soy protein; sulfides / thiols, amino acids, proteins, vitamins, and minerals. Functional ingredients may also be classified based on health benefits such as cardiovascular, cholesterol-reducing, and anti-inflammatory. Exemplary functional ingredients are provided in WO2013 / 096420, the contents of which are incorporated herein by reference.

[0428] The high purity target steviol glycoside(s) obtained according to the present invention, in particular 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 B, rebaudioside C, rebaudioside D ... 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 may be applied as high-intensity sweeteners to produce zero-calorie, low-calorie or diabetic beverages and food products with improved taste characteristics, and may also be used in beverages, foods, pharmaceuticals, and other products where sugar cannot be used.Additionally, the present invention provides high purity target steviol glycoside(s), in particular steviol monoside, steviol monoside A, steviol bioside, steviol bioside D, rubusoside, steviol bioside A, steviol bioside 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 B8, rebaudioside C9, rebaudioside D10, rebaudioside D11, rebaudioside D12, rebaudioside D13, rebaudioside D14, rebaudioside D15, rebaudioside D16, rebaudioside D17, rebaudioside D18, rebaudioside D19, rebaudioside D20, rebaudioside D21, rebaudioside D22, rebaudioside D23, rebaudioside D24, rebaudioside D25, rebaudioside D26, rebaudioside D27, rebaudioside D28, rebaudioside D29, rebaudioside D30, rebaudioside D31, rebaudioside D32, rebaudioside D33, rebaudioside D34, rebaudioside D35, rebaudioside D36, rebaudioside D37, rebaudioside D38, rebaudioside D39, rebaudioside D40, rebaudioside D41, rebaudioside D42, rebaudioside D43, reba 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 can be used as sweeteners for beverages, foods, and other products intended for human consumption, as well as in animal feed and forage with improved properties.

[0429] The high purity target steviol glycoside(s) obtained according to the present invention, in particular 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 A Rebaudioside M, 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 may be applied as foam suppressants to produce zero calorie, low calorie, or diabetic beverage and food products.

[0430] Highly purified targeted steviol glycoside(s), specifically steviol monoside, steviol monoside A, steviol bioside, steviol bioside D, rubusoside, steviol bioside A, steviol bioside 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 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 Examples of consumer products in which osid 2a and / or SvG7 may be used as sweetener compounds include alcoholic beverages such as vodka, wine, beer, sake, and sake; natural juices, soft drinks, carbonated soft drinks, diet drinks, zero-calorie drinks, low-calorie drinks and foods; yogurt drinks, instant juices, instant coffee, instant drinks in powder form, canned products, syrups, fermented soybean paste, soy sauce, vinegar, dressings, mayonnaise, ketchup, curry, soups, instant bouillon, powdered soy sauce, powdered vinegar, various biscuits, rice crackers, crackers, bread, chocolate, caramel, candy, chewing gum, jellies, puddings, preserved fruits and vegetables, fresh cream, jams, marmalades, flower pastes, milk powders, ice cream, sorbets, bottled vegetables and fruits, canned boiled beans, meat and foods boiled in sweet sauces, agricultural vegetable food products. These include, but are not limited to, seafood, ham, sausage, fish ham, fish sausage, fish paste, fried fish products, dried seafood, frozen food products, preserved seaweed, preserved meat, tobacco products, medicines, and many others. In principle, it can have unlimited uses.

[0431] High purity target steviol glycoside(s), especially steviol monoside, steviol monoside A, steviol bioside, steviol bioside D, rubusoside, steviol bioside A, steviol bioside 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 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 may be used as a flavor modifier or flavoring having modifying properties. Examples of the above include alcoholic beverages such as vodka, wine, beer, sake, and Japanese sake, natural juices, soft drinks, carbonated soft drinks, diet drinks, zero-calorie drinks, low-calorie drinks and foods, yogurt drinks, instant juices, instant coffee, powdered instant drinks, canned products, syrups, fermented soybean paste, soy sauce, vinegar, dressings, mayonnaise, ketchup, curry, soups, instant bouillon, powdered soy sauce, powdered vinegar, various biscuits, rice crackers, crackers, breads, etc. , chocolate, caramel, candy, chewing gum, jelly, pudding, preserved fruits and vegetables, cream, jam, marmalade, flower paste, milk powder, ice cream, sorbet, bottled vegetables and fruits, canned boiled beans, meat and food products cooked in sweet sauce, agricultural vegetable food products, seafood, ham, sausage, fish ham, fish sausage, fish paste, fried fish products, dried seafood, frozen food products, preserved seaweed, preserved meat, tobacco products, medicines, and many others. In principle, it can have non-limiting uses.

[0432] Conventional methods such as mixing, kneading, dissolving, soaking, permeating, infusing, sprinkling, spraying, injecting, and other methods may be used during the manufacture of products such as food, beverages, pharmaceuticals, cosmetics, tabletop products, and chewing gum.

[0433] Further, the high purity target steviol glycoside(s) obtained according to the present invention include steviol monoside, steviol monoside 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 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 may be used in a dry or liquid form.

[0434] The high purity targeted steviol glycosides can be added before or after the heat treatment of the food product. The high purity targeted steviol glycoside(s) can be added before or after the heat treatment of the food product, in particular steviol monoside, steviol monoside A, steviol bioside, steviol bioside D, rubusoside, steviol bioside A, steviol bioside 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 E5, rebaudioside E6, rebaudioside E7, rebaudioside E8, rebaudioside E9, rebaudioside E10, rebaudioside E11, rebaudioside E12, rebaudioside E13, rebaudioside E14, rebaudioside E15, rebaudioside E16, rebaudioside E17, rebaudioside E18, rebaudioside E19, rebaudioside E20, rebaudioside E21, rebaudioside E22, rebaudioside E23, rebaudioside E24, rebaudioside E25, rebaudioside E26, rebaudioside E27, rebaudioside E28, rebaudioside E29, rebaudioside E30, rebaudioside E31, rebaudioside E32, rebaudioside E33, rebaudioside E34, rebaudioside E35, rebaudioside E36, rebaudioside E37, rebaudioside E38, rebaudioside E39, reba The amount of Uudioside I, 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 will depend on the intended use. As noted above, they may be added alone or in combination with other compounds.

[0435] The present invention also provides 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 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 as sweetness enhancers. The purpose of the present invention is to enhance the sweetness of beverages in which the compound is used as a sweetener (steviol enhancer), and the compound is selected from the group consisting of steviolmonoside, steviolbioside 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, and rebaudioside A. Rebaudioside I, 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 present at concentrations at or below their respective sweetness perception thresholds.

[0436] As used herein, the term "sweetness enhancer" refers to a compound that can enhance or intensify the perception of sweetness 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."

[0437] The term "sweetness perception threshold concentration" as generally used herein refers to the lowest known concentration of a sweet compound that can be perceived by human taste, typically about 1.0% sucrose equivalent (1.0% SE). In general, a sweetness enhancer may enhance or intensify the sweetness of a sweetener without itself producing any perceptible sweetness when present below the sweetness perception threshold concentration of a given sweetness enhancer, but the sweetness enhancer itself may produce sweetness at concentrations above its sweetness perception threshold concentration. The sweetness perception threshold concentration is specific to a particular enhancer and may vary based on the beverage matrix. The sweetness perception threshold concentration can be easily determined by taste testing, increasing the concentration of a given enhancer until more than 1.0% sucrose equivalent is detected in a given beverage matrix. The concentration that produces about 1.0% sucrose equivalent is considered to be the sweetness perception threshold.

[0438] In some embodiments, the sweetener may be, for example, about 0.0001%, about 0.0005%, about 0.001%, about 0.005%, about 0.01%, about 0.05%, about 0.1%, about 0.5%, about 1.0%, about 1.5%, about 2.0%, about 2.5%, about 3.0%, about 3.5%, about 4.0%, about 4.5% by weight. , is present in the beverage in an amount of from about 0.0001% to about 12% by weight, such as about 5.0% by weight, about 5.5% by weight, about 6.0% by weight, about 6.5% by weight, about 7.0% by weight, about 7.5% by weight, about 8.0% by weight, about 8.5% by weight, about 9.0% by weight, about 9.5% by weight, about 10.0% by weight, about 10.5% by weight, about 11.0% by weight, about 11.5% by weight, or about 12.0% by weight.

[0439] In certain embodiments, the sweetener may be, for example, from about 0.0001% to about 0.0005% by weight, from about 0.0005% to about 0.001% by weight, from about 0.001% to about 0.005% by weight, from about 0.005% to about 0.01% by weight, from about 0.01% to about 0.05% by weight, from about 0.05% to about 0.1% by weight, from about 0.1% to about 0.5% by weight, from about 0.005% to about 0.01% by weight, from about 0.01% to about 0.05% by weight, from about 0.05% to about 0.1% by weight, from about 0.1% to about 0.5% by weight, from about 0.005% to about 0.01% by weight, from about 0.005% to about 0.01% by weight, from about 0.005% to about 0.01% by weight, from about 0.005% to about 0.1% by weight, from about 0.005% to about 0.1% by weight, from about 0.005% to about 0.005 ... The sweetener is present in the beverage in an amount of about 0.0001% to about 10% by weight, such as about 0.5% to about 1% by weight, about 1% to about 2% by weight, about 2% to about 3% by weight, about 3% to about 4% by weight, about 4% to about 5% by weight, about 5% to about 6% by weight, about 6% to about 7% by weight, about 7% to about 8% by weight, about 8% to about 9% by weight, or about 9% to about 10% by weight. In a particular embodiment, the sweetener is present in the beverage in an amount of about 0.5% to about 10% by weight. In another particular embodiment, the sweetener is present in the beverage in an amount of about 2% to about 8% by weight.

[0440] In one embodiment, the sweetener is a conventional caloric sweetener. Suitable sweeteners include, but are not limited to, sucrose, fructose, glucose, high fructose corn syrup, and high fructose starch syrup.

[0441] In another embodiment, the sweetener is erythritol.

[0442] In yet 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.

[0443] It is contemplated that the sweetener may be used alone or in combination with other sweeteners.

[0444] In one embodiment, the rare sugar is D-allose. In a more specific embodiment, the D-allose is present in the beverage in an amount of about 0.5% to about 10% by weight, such as, for example, about 2% to about 8% by weight.

[0445] In another embodiment, the rare sugar is D-psicose. In a more specific embodiment, D-psicose is present in the beverage in an amount of about 0.5% by weight to about 10% by weight, such as, for example, about 2% by weight to about 8% by weight.

[0446] In yet another embodiment, the rare sugar is D-ribose. In a more specific embodiment, D-ribose is present in the beverage in an amount of about 0.5% to about 10% by weight, such as, for example, about 2% to about 8% by weight.

[0447] In yet another embodiment, the rare sugar is D-tagatose. In a more specific embodiment, D-tagatose is present in the beverage in an amount of about 0.5% by weight to about 10% by weight, such as about 2% by weight to about 8% by weight.

[0448] In a further embodiment, the rare sugar is L-glucose. In a more specific embodiment, the L-glucose is present in the beverage in an amount of about 0.5% to about 10% by weight, such as, for example, about 2% to about 8% by weight.

[0449] In one embodiment, the rare sugar is L-fucose. In a more specific embodiment, L-fucose is present in the beverage in an amount of about 0.5% to about 10% by weight, such as, for example, about 2% to about 8% by weight.

[0450] In another embodiment, the rare sugar is L-arabinose. In a more specific embodiment, the L-arabinose is present in the beverage in an amount of about 0.5% to about 10% by weight, such as, for example, about 2% to about 8% by weight.

[0451] In yet another embodiment, the rare sugar is D-turanose. In a more specific embodiment, D-turanose is present in the beverage in an amount of about 0.5% to about 10% by weight, such as, for example, about 2% to about 8% by weight.

[0452] In yet another embodiment, the rare sugar is D-leucrose. In a more specific embodiment, the D-leucrose is present in the beverage in an amount of about 0.5% to about 10% by weight, such as, for example, about 2% to about 8% by weight.

[0453] The addition of a sweetness enhancer at a concentration below its sweetness perception threshold increases the detected sucrose equivalent of a beverage containing a sweetener and a sweetness enhancer, compared to a corresponding beverage in the absence of the sweetness enhancer. Furthermore, the sweetness can be increased by an amount greater than the detectable sweetness of a solution containing the same concentration of at least one sweetness enhancer in the absence of the sweetener.

[0454] Thus, the present invention also provides a beverage comprising a sweetener, and a method for preparing a beverage comprising the steps of: providing a beverage comprising a sweetener 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 rebaudioside I, 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, or a combination thereof. and adding to a beverage a sweetener 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 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 present at concentrations below the sweet taste perception threshold.

[0455] 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 1a, Rebaudioside 1b, Rebaudio By adding 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 to a sweetener-containing beverage at a concentration below the sweetness perception threshold, the detected sucrose equivalent may be increased by 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%.

[0456] The following examples are directed to the preparation of high purity target steviol glycoside(s), specifically steviol monoside, steviol monoside A, steviol bioside, steviol bioside D, rubusoside, steviol bioside A, steviol bioside 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 E5, rebaudioside E6, rebaudioside E7, rebaudioside E8, rebaudioside E9, rebaudioside E10, rebaudioside E11, rebaudioside E12, rebaudioside E13, rebaudioside E14, rebaudioside E15, rebaudioside E16, rebaudioside E17, rebaudioside E18, rebaudioside E19, rebaudioside E20, rebaudioside E21, rebaudioside E22, rebaudioside E23, rebaudioside E24, rebaudioside E25, rebaudioside E26, rebaudioside E27, rebaudioside E28, rebaudioside E29, rebaudioside E30, rebaudioside E31, rebaudioside E32, rebaudioside E33, rebaudioside E34, rebaudioside E35, rebaudioside E36, rebaudioside E37, rebaudioside E38, rebaudioside E39, rebaudioside E40, rebaudioside E41, rebaudioside E42, rebaudioside E4

[0033] Figure 2 illustrates preferred embodiments of the invention for the preparation of Rebaudioside I, 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. It will be understood that the invention is not limited to the materials, proportions, conditions, and procedures set forth in the examples, which are merely illustrative. EXAMPLES

[0457] Example 1 Protein sequences of engineered enzymes used in biocatalytic processes SEQ ID NO:1: >SuSy_At, variant PM1-54-2-E05 (engineered sucrose synthase; source of wild-type gene: Arabidopsis thaliana) MANAERMITRVHSQERLNETLVSERNEVLALLSRVEAKGKGILQQNQIIAEFEALPEQTRKKLEGGPFFDLLKSTQEAIVLPPWVALAVRPRPGVWEYLRVNLHALVVEELQPAEFLHFKEELVDGVKNGNFTLELDFEPFNASIPRPTLHKYIGNGVDFLNRHLSAKLFHDKESLLPLLDFLRLHSHQGKNLMLSEKIQN LNTLQHTLRKAEEYLAELKSETLYEEFEAKFEEIGLERGWGDNAERVLDMIRLLLDLLEAPDPSTTLETFLGRVPMVFNVVILSPHGYFAQDNVLGYPDTGG QVVYILDQVRALEIEMLQRIKQQGLNIKPRILILTRLLPDAVGTTCGERLERVYDSEYCDILRVPFRTEKGIVRKWISRFEVWPYLETYTEDAAVELSKEL NGKPDLIIGNYSDGNLVASLLAHKLGVTQCTIAHALEKTKYPDSDIYWKKLDDKYHFSCQFTADIFAMNHTDFIITSTFQEIAGSKETVGQYESHTAFTLPGLYRVVHGIDVFDPKFNIVSPGADMSIYFPYTEEKRRLTKFHSEIEELLYSDVENDEHLCVLKDKKKPILFTMARLDRVKNLSGLVEWYGKNTRLRELVNL VVVGGDRKESKDNEEKAEMKKMYDLIEEYKLNGQFRWISSQMDRVRNGELYRYICDTKGAFVQPALYEAFGLTVVEAMTCGLPTFATCKGGPAEIIVHGKSGFHIDPYHGDQAADLLADFFTKCKEDPSHWDEISKGGLQRIEKYTWQIYSQRLLTLTGVYGFWKHVSNLDRLEHRRYLEMFYALKYRPLAQAVPLAQDD

[0458] SEQ ID NO:2: >UGTSl2 variant 0234 (engineered glucosyltransferase; source of wild-type gene: Solanum lycopersicum) MATNLRVLMFPWLAYGHISPFLNIAKQLADRGFLIYLCSTRINLESIIKKIPEKYADSIHLIELQLPELPELPHYHTTNGLPPHLNPTLHKALKMSKPNFSRILQNLKP DLLIYDVLQPWAEHVANEQGIPAGKLLVSCAAVFSYFFSFRKNPGVEFPFPAIHLPEVEKVKIREILAKEPEEGGRLDEGNKQMMLMCTSRTIEAKYIDYCTELCNWKVVP VGPPFQDLITNDADNKELIDWLGTKPENSTVFVSFGSEYFLSKEDMEIAFALEASNVNFIWVVRFPKGEERNLEDALPEGFLERIGERGRVLDKFAPQPRILNHPSTGG FISHCGWNSVMESIDFGVPIIAMPIHNDQPINAKLMVELGVAVEIVRDDDGKIHRGEIAEALKSVVTGETGEILRAKVREISKNLKSIRDEEMDAVAEELIQLCRNNSNKSK

[0459] SEQ ID NO:3: >UGT76G1 variant 0042 (engineered glucosyltransferase; source of wild-type gene: Stevia rebaudiana) MENKTETTVRRRRRIILFPVPFQGHINPILQLANVLYSKGFAITILHTNFNKPKTSNYPHFTFRFILDNDPQDERISNLPTHGPLAGMRIPIINEHGADELRRELELLMLASEE DEEVSCLITDALWYFAQDVADSLNLRRLVLMTSSLFNFHAHVSLPQFDELGYLDPDDKTRLEEQASGFPMLKVKDIKSAYSNWQIGKEILGKMIKQTKASSGVIWNSFKELEESE LETVIREIPAPSFLIPLPKHLTASSSSLLDHDRTVFEWLDQQAPSSVLYVSFGSTSEVDEKDFLEIARGLVDSGQSFLWVVRPGFVKGSTWVEPLPDGFLGERGKIVKWVPQQE VLAHPAIGAFWTHSGWNSTLESVCEGVPMIFSSFGGDQPLNARYMSDVLRVGVYLENGWERGEVVNAIRRVMVDEEGEYIRQNARVLKQKADVSLMKGGSSYESLESLVSYISSL

[0460] Example 2 Expression and formulation of the SuSy_At variant of SEQ ID NO:1 The gene encoding the SuSy_At variant of SEQ ID NO: 1 (Example 1) was cloned into the expression vector pLE1A17 (derivative of pRSF-1b, Novagen). The resulting plasmid was used to transform E. coli BL21(DE3) cells.

[0461] Cells were cultivated in ZYM505 medium (F. William Studier, Protein Expression and Purification 41 (2005) 207-234) supplemented with kanamycin (50 mg / L) at 37 ° C. Gene expression was induced in the logarithmic growth phase with IPTG (0.2 mM) for 16-18 h at 30 ° C and 200 rpm.

[0462] Cells were harvested by centrifugation (3220xg, 20 min, 4°C) and resuspended using cell lysis buffer (100mM Tris-HCl pH 7.0; 2mM MgCl2, DNA nuclease 20U / mL, lysozyme 0.5mg / mL) and incubated at an optical density of 200 (measured at 600nm (OD 600 The cells were then disrupted by sonication and the crude extract was separated from the cell debris by centrifugation (18000xg 40 min, 4°C). The supernatant was sterilized by filtration through a 0.2 μm filter and diluted 50:50 with distilled water to obtain the enzyme activity preparation.

[0463] For the enzyme activity preparation of SuSy_At, the activity in units is defined as follows: 1 mU of SuSy_At turns over 1 nmol of sucrose to fructose per minute. The reaction conditions for the assay are 30° C., 50 mM potassium phosphate buffer pH 7.0, 400 mM sucrose at t0, 3 mM MgCl2, and 15 mM uridine diphosphate (UDP).

[0464] Example 3 Expression and formulation of UGTSl2 variant of SEQ ID NO:2 The gene encoding the UGTSl2 variant of SEQ ID NO: 2 (Example 1) was cloned into the expression vector pLE1A17 (derivative of pRSF-1b, Novagen). The resulting plasmid was used to transform E. coli BL21(DE3) cells.

[0465] Cells were grown in ZYM505 medium (F. William Studier, Protein Expression and Purification 41 (2005) 207-234) supplemented with kanamycin (50 mg / L) at 37 ° C. Gene expression was induced in the logarithmic growth phase with IPTG (0.1 mM) for 16-18 h at 30 ° C and 200 rpm.

[0466] Cells were harvested by centrifugation (3220xg, 20 min, 4°C) and resuspended using cell lysis buffer (100mM Tris-HCl pH 7.0; 2mM MgCl2, DNA nuclease 20U / mL, lysozyme 0.5mg / mL) and incubated at an optical density of 200 (measured at 600nm (OD 600 The cells were then disrupted by sonication and the crude extract was separated from the cell debris by centrifugation (18000xg 40 min, 4°C). The supernatant was sterilized by filtration through a 0.2 μm filter and diluted 50:50 with 1 M sucrose solution to obtain the enzyme active preparation.

[0467] For the enzymatic activity preparation of UGTSl2, the activity in units is defined as follows: 1 mU of UGTSl2 turns over 1 nmol of rebaudioside A (Reb A) to rebaudioside D (Reb D) per minute. The reaction conditions for the assay are 30°C, 50 mM potassium phosphate buffer (pH 7.0, 10 mM Reb A at t0, 500 mM sucrose, 3 mM MgCl2, 0.25 mM uridine diphosphate (UDP), and 3 U / mL SuSy_At.

[0468] Example 4 Expression and formulation of UGT76G1 variant of SEQ ID NO:3 The gene encoding the UGT76G1 variant of SEQ ID NO: 3 (Example 1) was cloned into the expression vector pLE1A17 (derivative of pRSF-1b, Novagen). The resulting plasmid was used to transform E. coli BL21(DE3) cells.

[0469] Cells were grown in ZYM505 medium (F. William Studier, Protein Expression and Purification 41 (2005) 207-234) supplemented with kanamycin (50 mg / L) at 37 ° C. Gene expression was induced in the logarithmic growth phase with IPTG (0.1 mM) for 16-18 h at 30 ° C and 200 rpm.

[0470] Cells were harvested by centrifugation (3220xg, 20 min, 4°C) and resuspended using cell lysis buffer (100mM Tris-HCl pH 7.0; 2mM MgCl2, DNA nuclease 20U / mL, lysozyme 0.5mg / mL) and incubated at an optical density of 200 (measured at 600nm (OD 600 The cells were then disrupted by sonication and the crude extract was separated from the cell debris by centrifugation (18000xg 40 min, 4°C). The supernatant was sterilized by filtration through a 0.2 μm filter and diluted 50:50 with 1 M sucrose solution to obtain the enzyme active preparation.

[0471] For the enzyme activity preparation of UGT76G1, the activity in units is defined as follows: 1 mU of UGT76G1 turns over 1 nmol of rebaudioside D (Reb D) to rebaudioside M (Reb M) per minute. The reaction conditions for the assay are 30° C., 50 mM potassium phosphate buffer pH 7.0, 10 mM Reb D, 500 mM sucrose, 3 mM MgCl2, 0.25 mM uridine diphosphate (UDP) and 3 U / mL SuSy_At at t0.

[0472] Example 5 For the one-pot reaction synthesis of SvG7, UGTSL2, SuSy_At and UGT76G1 are added simultaneously. Various SvG7 molecules were synthesized directly from stevioside (see FIG. 4) in a one-pot reaction using three enzymes (see Examples 1, 2, 3 and 4): UGTSl2 (variant of SEQ ID NO: 2), SuSy_At (variant of SEQ ID NO: 1) and UGT76G1 (variant of SEQ ID NO: 3).

[0473] The final reaction solution contained 348 U / L UGTSl2, 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 MgCl2, and potassium phosphate buffer (pH 6.6). First, 206 mL of distilled water was diluted with 0.24 g of MgCl2. 2· 6H2O, 102 g of sucrose, 9.8 mL of 1.5 M potassium phosphate buffer (pH 6.6) and 15 g of stevioside were mixed. The final volume of the reaction mixture was adjusted to 300 mL.

[0474] After dissolving the components, the temperature was adjusted to 45° C. and UGTSL2, SuSy_At, UGT76G1 and 39 mg of UDP were added. The reaction mixture was incubated in a shaker at 45° C. for 24 h. An additional 39 mg of UDP was added at 12 h, 24 h, and 36 h. The contents of reb 2a and various SvG7 at the end of the reaction (48 h) were analyzed by HPLC.

[0475] Example 6 HPLC analysis For analysis, the biotransformation samples were inactivated by adjusting the reaction mixture to pH 5.5 with 17% H3PO4 and then boiling for 10 min. The resulting samples were filtered and the filtrate was diluted 10-fold and used as the sample for HPLC analysis. The HPLC assays were performed on an Agilent HP 1200 HPLC system consisting of a pump, column thermostat, autosampler, UV detector with background correction, and a data acquisition system. The analytes were separated using an Agilent Prorshell 120 SB-C18, 4.6 mm × 150 mm, 2.7 μm at 40 °C. The mobile phase consisted of two premixes. - Premix 1 containing 75% 10 mM phosphate buffer (pH 2.6) and 25% acetonitrile, and - Premix 2 containing 68% 10 mM phosphate buffer (pH 2.6) and 32% acetonitrile.

[0476] The elution gradient started with Premix 1, changed to 50% Premix 2 at 12.5 min, and changed to 100% Premix 2 at 13 min. Total run time was 45 min. Column temperature was maintained at 40° C. Injection volume was 5 μL. Rebaudioside species were detected by UV at 210 nm.

[0477] Table 3 shows the conversion (area percentage) of stevioside to the specified rebaudioside species at each time point. Chromatograms of the starting stevioside and reaction mixture at 48 hours are shown in Figures 4 and 5, respectively. One of skill in the art will appreciate that retention times may vary in some cases with changes in solvents and / or equipment.

[0478] [Table 4]

[0479] Example 7 Purification of rebaudioside 2a and various SvG7 300 mL of the reaction mixture of Example 5 (after 48 hours) was inactivated by adjusting the pH to pH 5.5 with H3PO4, then boiling for 10 minutes and filtered. The filtrate was loaded onto a column containing 500 mL of YWD03 (Cangzhou Yuanwei, China) resin pre-equilibrated with water. The resin was washed with 2.5 L of water, and the water eluate from this step was discarded.

[0480] Steviol glycosides were eluted from the YWD03 resin column by eluting with 2.5 L of 70% v / v ethanol / water. The effluent from this step was collected and dried under vacuum at 60° C. to give 20 g of dry 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.

[0481] HPLC fractions corresponding to individual compounds from multiple runs were pooled according to retention time. Fractions were lyophilized to dryness.

[0482] [Table 5]

[0483] The purity of the obtained fractions was assessed by the analytical HPLC method described in Example 6. The chromatogram of purified rebaudioside 2a is shown in FIG.

[0484] Example 8 Structural analysis of rebaudioside 2a The samples were dissolved in pyridine-d5 and NMR experiments were carried out on a Bruker 500 MHz spectrometer. Along with the signal from the sample, the signal from pyridine-d5 was observed at δ C 123.5, 135.5, 149.9 ppm and δ H Observed at 7.19, 7.55, and 8.71 ppm. of rebaudioside 2a recorded in pyridine-d5 1 The H-NMR spectrum confirmed the excellent quality of the sample (see FIG. 7). HSQC (see FIG. 8) shows the presence of an exo-methylene group in the sugar region remotely attached to C-15 observable in H,H-COSY (FIG. 9). Other downfield signals of quaternary carbons (C-13, C-16 and C-19) are detected by HMBC (FIG. 10). Correlation of signals in HSQC, HMBC and H,H-COSY reveals the presence of steviol glycosides with the following aglycone structure:

[0485] [ka]

[0486] Correlation between HSQC and HMBC shows the presence of seven anomeric signals marked 1i, 1ii, 1iii, 1iv, 1v, 1vi and 1vii. The coupling constants of the anomeric protons of about 8 Hz, the broad signals of their sugar bonds and the NOE correlations of the anomeric protons allow the identification of these seven sugars as β-D-glucopyranosides.

[0487] The combined data from HSQC and HMBC reveals sugar-sugar and sugar-aglycon linkages. The sugar sequence assignments were confirmed by using a combination of HSQC-TOCSY (Figure 11a) and NOESY (Figure 11b).

[0488] Similarly, the results of the above NMR experiments were used to assign the proton and carbon chemical shifts of the structure of rebaudioside 2a (see Table 5).

[0489] [Table 6-1]

[0490] [Table 6-2]

[0491] [Table 6-3]

[0492] Correlation of all NMR results indicates rebaudioside 2a with seven β-D-glucose attached to the steviol aglycone, as shown in the chemical structure below.

[0493] [ka]

[0494] LCMS (FIGS. 12a and 12b) analysis of rebaudioside 2a revealed [MH] at m / z 1451.6. - ion, C 62 H 100 O 38 The molecular formula was in good agreement with the predicted formula of [C 62 H 99 O 38 ] - Calculated values ​​for monoisotopic ions : 1451.6-). MS data shows that rebaudioside 2a is C 62 H 100 O 38 The molecular formula of the compound is: LCMS analysis was performed under the following conditions listed in Table 6.

[0495] [Table 7]

Claims

1. The following formula: 【Chemistry 1】 wherein the R1 and R2 glycans are defined in the table below. 【Table 1】

2. 10. A method for producing at least one steviol glycoside according to claim 1, comprising: a. providing a starting composition comprising an organic compound having at least one carbon atom; b. Providing an enzyme preparation or a microorganism containing at least one enzyme selected from steviol biosynthesis enzymes, UDP-glucosyltransferases, and optionally UDP-glucose salvage enzymes; and c. contacting the enzyme preparation or the microorganism with a medium containing the starting composition to produce a medium comprising at least one steviol glycoside as described in claim 1.

3. 10. A method for producing at least one steviol glycoside according to claim 1, comprising: a. providing a starting composition comprising an organic compound having at least one carbon atom; b. Providing a biocatalyst comprising at least one enzyme selected from steviol biosynthesis enzymes, UDP-glucosyltransferases, and optionally UDP-glucose salvage enzymes; and c. contacting the biocatalyst with a medium containing the starting composition to produce a medium comprising at least one steviol glycoside as described in claim 1.

4. d. The method of claim 2 or 3, further comprising the step of separating the at least one steviol glycoside of claim 1 from the culture medium to provide a high purity composition of the at least one steviol glycoside of claim 1.

5. 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 I, rebaudioside AM, rebaudioside D7, rebaudioside M, rebaudioside M4, other steviol glycosides, polyols, carbohydrates, and combinations thereof.

6. 3. 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. 4. The method of claim 3, wherein the biocatalyst is an enzyme or a cell containing one or more enzymes capable of converting the starting composition into at least one steviol glycoside as described in claim 1.

8. The enzymes include mevalonate (MVA) pathway enzymes, 2-C-methyl-D-erythritol-4-phosphate pathway (MEP / DOXP) enzymes, geranylgeranyl diphosphate synthase, copalyl diphosphate synthase, kaurene synthase, kaurene oxidase, kaurenoic acid 13-hydroxylase (KAH), steviol synthetase, deoxyxylulose 5-phosphate synthase (DXS), D-1-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 3. The method of claim 2, wherein the IL-10 kinase is selected from the group consisting of 1-methyl-D-erythritol 2,4-cyclodiphosphate synthase (MCS), 1-hydroxy-2-methyl-2(E)-butenyl 4-diphosphate synthase (HDS), 1-hydroxy-2-methyl-2(E)-butenyl 4-diphosphate reductase (HDR), acetoacetyl-CoA thiolase, truncated HMG-CoA reductase, mevalonate kinase, phosphomevalonate kinase, mevalonate pyrophosphate decarboxylase, cytochrome P450 reductase, UGT74G1, UGT85C2, UGT91D2, EUGT11, UGTSl2, UGT76G1, UGlyT91C1, or a mutant variant thereof.

9. 5. The method of claim 4, wherein the content of at least one steviol glycoside in the high purity composition of at least one steviol glycoside described in claim 1 is greater than 95% by weight on a dry weight basis.

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

11. 11. The consumer product of claim 10, wherein the product is selected from the group consisting of beverages, natural juices, soft drinks, carbonated soft drinks, diet drinks, zero calorie beverages, low calorie beverages and foods, yogurt drinks, instant juices, instant coffee, instant beverages in powder form, canned products, syrups, fermented soybean paste, soy sauce, vinegar, dressings, mayonnaise, ketchup, curry, soups, instant bouillon, powdered soy sauce, powdered vinegar, various biscuits, rice crackers, crackers, bread, chocolates, caramels, candies, chewing gums, jellies, puddings, preserved fruits and vegetables, fresh cream, jams, marmalades, flower paste, powdered milk, ice cream, sorbets, bottled vegetables and fruits, canned boiled beans, meat and foods boiled in sweet sauces, agricultural vegetable food products, seafood, ham, sausages, fish ham, fish sausages, fish paste, fried fish products, dried seafood, frozen food products, preserved seaweed, preserved meat, tobacco products and medicines.

12. 11. The consumer product of claim 10, further comprising at least one additive selected from the group consisting of carbohydrates, polyols, amino acids and their corresponding salts, polyamino acids and their corresponding salts, sugar acids and their corresponding salts, nucleotides, organic acids, inorganic acids, organic salts including organic acid salts and organic base salts, inorganic salts, bitter compounds, caffeine, flavorants and flavor components, astringent compounds, proteins or protein hydrolysates, surfactants, emulsifiers, flavonoids, alcohols, polymers, and combinations thereof.

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

14. 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, Reba Udioside 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 I, Rebaudioside I2, Rebaudioside I3, 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 O2, Rebaudio Rebaudioside O3, Rebaudioside O4, Rebaudioside Q, Rebaudioside Q2, Rebaudioside Q3, Rebaudioside R, Rebaudioside S, Rebaudioside T, Rebaudioside T1, Rebaudioside U, Rebaudioside U2, Rebaudioside V, Rebaudioside V2, Rebaudioside V3, Rebaudioside W, Rebaudioside W2, Rebaudioside W3, Rebaudioside Y, Rebaudioside Z1, Rebaudioside Z2, Rebaudioside AM, SvG7, Mogroside V, Sylatose, Allulose, D-Allose, D-Tagatose, Erythritol, Brazzein,Neohesperidin dihydrochalcone, glycyrrhizic acid and its salts, thaumatin, perillartine, pernandulcin, mukuroziosid, bayunoside, phlomisoside I, dimethyl-hexahydrofluorene-dicarboxylic acid, abrusoside, periandrin, carnosifloside, cyclocaryoside, pterocaryoside, polypodoside A, brazilin, hernandulcin, phyllodulcin, glycyphyllin, phloridzin, trilobatin, dihydroflavonol, dihydroquercetin-3-acetate, neoastiribin, trans-cinnamaldehyde, monad tin and its salts, serigeain A, hematoxylin, monellin, osladin, pterocaryoside A, pterocaryoside B, mabinlin, pentasine, miraculin, curculin, neoculin, chlorogenic acid, cynarin, siamenoside, sucralose, acesulfame potassium, aspartame, alitame, saccharin, cyclamate, neotame, dulcin, suosan advantame, gymnemic acid, phodulcin, digiphin, lactisole, glutamic acid, aspartic acid, glycine, alanine, threonine, proline, serine, lysine, tryptophan, Maltitol, mannitol, sorbitol, lactitol, xylitol, inositol, isomalt, propylene glycol, glycerol, threitol, galactitol, hydrogenated isomaltulose, reduced isomaltooligosaccharides, reduced xylooligosaccharides, reduced gentiooligosaccharides, reduced maltose syrup, reduced glucose syrup, hydrogenated starch hydrolysates, polyglycitol, sugar alcohols, L-sugars, L-sorbose, L-arabinose, trehalose, galactose, rhamnose, various cyclodextrins, cyclic oligosaccharides, various types of maltodextrins. Kistrin, 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, gluconolactone, abequase, galactosamine, beet oligosaccharides, isomaltooligosaccharides (isomaltose,11. The consumer product of claim 10, further comprising a compound selected from the group consisting of: isomaltotriose, panose, etc.), xylooligosaccharides (xylotriose, xylobiose, etc.), xyloterminal oligosaccharides, gentiooligosaccharides (gentiobiose, gentiotriose, gentiotetraose, etc.), nigerooligosaccharides, palatinose oligosaccharides, fructooligosaccharides (kestose, nystose, etc.), maltotetraol, maltotriol, maltooligosaccharides (maltotriose, maltotetraose, maltopentaose, maltohexaose, maltoheptaose, etc.), starch, inulin, inulooligosaccharides, lactulose, melibiose, raffinose, isomerized liquid sugars such as high fructose corn syrup, coupling sugars, soybean oligosaccharides, D-psicose, D-ribose, L-glucose, L-fucose, D-turanose, D-leucrose. ,

15. 1. A method for enhancing the sweetness of a beverage or food product containing a sweetener, comprising: a. providing a beverage or food product that includes a sweetener; b. adding a sweetener enhancer comprising at least one steviol glycoside according to claim 1; 10. The method of claim 1, wherein the at least one steviol glycoside is present at a concentration below the sweetness perception threshold.

16. 1. A method for modifying the flavor of a beverage or food product, comprising: a. providing a beverage or food product; b. adding a composition comprising at least one steviol glycoside according to claim 1.

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