Highly purified steviol glycosides

Abstract

Methods for preparing highly purified steviol glycosides, specifically steviolmonoside, steviolmonoside A, steviolbioside, steviolbioside A, steviolbioside B, rubusoside, stevioside, stevioside A (rebaudioside KA), stevioside B, stevioside C, rebaudioside E, rebaudioside E2, rebaudioside E3, and rebaudioside AM, are described. The methods involve utilizing enzyme preparations and recombinant microorganisms to convert various starting compositions to the target steviol glycosides. The highly purified rebaudiosides are useful as non-caloric sweeteners, flavor enhancers, sweetness enhancers, and anti-foaming agents in edible and chewable compositions, such as beverages, confectioneries, bakery products, cookies, and chewing gum.

Description

[Technical Field]

[0001] The present invention relates to methods for preparing compositions containing steviol glycosides, including highly purified steviol glycoside compositions.

[0002] Background of the Invention 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 reduced-calorie products, including food and beverages.High-intensity sweeteners do not cause glycemic reactions, making them suitable for use in products aimed at diabetics and other people who are concerned about controlling their carbohydrate intake.

[0003] Steviol glycosides are a group of compounds found in the leaves of Stevia rebaudiana Bertoni, a perennial shrub in the Asteraceae (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, constituting approximately 10%–20% of the total dry weight. On a dry weight basis, the four major glycosides found in stevia leaves generally 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. Although methods for preparing steviol glycosides from Stevia rebaudiana are known, many of these methods are not suitable for commercial use. Thus, a need remains for simple, efficient, and economical methods for preparing compositions comprising steviol glycosides, including highly purified steviol glycoside compositions.

[0004] Summary of the Invention The present invention provides methods 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 the target steviol glycoside. 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.

[0005] The target steviol glycoside can be any steviol glycoside. In one embodiment, the target steviol glycoside is steviolmonoside, steviolmonoside A, steviolbioside, steviolbioside A, steviolbioside B, rubusoside, stevioside, stevioside A (rebaudioside KA), stevioside B, stevioside C, rebaudioside E, rebaudioside E2, rebaudioside E3, rebaudioside AM, or a synthetic steviol glycoside. In one embodiment, the target steviol glycoside is rebaudioside AM. In some preferred embodiments, an enzyme preparation containing one or more enzymes or microbial cells containing one or more enzymes capable of converting a starting composition to a target steviol glycoside is used. The enzymes may be located on the surface and / or inside the cells. The enzyme preparation may be provided in the form of a whole cell suspension, a crude lysate, or purified enzyme(s). The enzyme preparation may be in free form or immobilized on a solid support made of inorganic or organic materials.

[0006] In some embodiments, the microbial cells contain the enzymes and genes encoding them necessary for converting the starting composition to the target steviol glycoside. Accordingly, the present invention also provides methods for preparing a composition comprising a target steviol glycoside by contacting a starting composition comprising an organic substrate with microbial cells containing at least one enzyme capable of converting the starting composition to the target steviol glycoside, thereby producing a medium comprising at least one target steviol glycoside.

[0007] The enzymes required to convert the starting composition to the target steviol glycosides include steviol biosynthetic enzymes, UDP-glucosyltransferases (UGTs), and / or UDP recycling enzymes. In one embodiment, steviol biosynthetic enzymes include mevalonate (MVA) pathway enzymes. In another embodiment, steviol biosynthetic enzymes include non-mevalonate 2-C-methyl-D-erythritol-4-phosphate pathway (MEP / DOXP) enzymes.

[0008] In one aspect, the steviol biosynthetic enzymes include 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 (CMS), and 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase (CMS). MK), 4-diphosphocytidyl-2-C-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, and the like.

[0009] The UDP-glucosyltransferase can be any UDP-glucosyltransferase capable of adding at least one glucose unit to a steviol and / or steviol glycoside substrate to provide the target steviol glycoside. As used below, the term "SuSy_AT" refers to the sucrose synthase having the amino acid sequence "SEQ ID NO:1" described in Example 1, unless otherwise specified. As used hereinafter, the term "UGTSl2" refers to the UDP-glucosyltransferase having the amino acid sequence "SEQ ID NO:2" described in Example 1, unless otherwise specified.

[0010] As used hereinafter, the term "UGT76G1" refers to the UDP-glucosyltransferase having the amino acid sequence "SEQ ID NO:3" described in Example 1, unless otherwise specified. In one aspect, the steviol biosynthetic enzymes and UDP-glucosyltransferase are produced in a microbial cell. The microbial cell may be, for example, E. coli, Saccharomyces sp., Aspergillus sp., Pichia sp., Bacillus sp., Yarrowia sp., etc. In another aspect, the UDP-glucosyltransferase is synthesized.

[0011] In one aspect, the UDP-glucosyltransferase is selected from the group including UGT74G1, UGT85C2, UGT76G1, UGT91D2, UGTSI2, EUGT11, and UGTs having substantial (greater than 85%, greater than 86%, greater than 87%, greater than 88%, greater than 89%, greater than 90%, greater than 91%, greater than 92%, greater than 93%, greater than 94%, greater than 95%, greater than 96%, greater than 97%, greater than 98%, greater than 99%) amino acid sequence identity to these polypeptides, and isolated nucleic acid molecules encoding these UGTs.

[0012] In one aspect, the steviol biosynthetic enzymes, UGTs, and UDP-glucose recycling system are present in a single microorganism (microbial cell), which may be, for example, E. coli, Saccharomyces sp., Aspergillus sp., Pichia sp., Bacillus sp., or Yarrowia sp.

[0013] In one aspect, 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 C13 to provide a target steviol glycoside having an -O-glucose beta-glucopyranoside glycosidic linkage at C13. In a particular aspect, the UDP-glucosyltransferase is UGT85C2 or a UGT having greater than 85% amino acid sequence identity to UGT85C2.

[0014] 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 provide a target steviol glycoside having a -COO-glucose beta-glucopyranoside glycosidic linkage at C19. In a particular embodiment, the UDP-glucosyltransferase is UGT74G1 or a UGT having greater than 85% amino acid sequence identity to UGT74G1.

[0015] In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to the existing glucose at C19 of any starting steviol glycoside to give a target steviol glycoside with at least one additional glucose and at least one beta-1→2 glucopyranoside glycosidic bond(s) in the newly generated glycosidic bond(s). In a particular embodiment, the UDP-glucosyltransferase is UGTS12 or a UGT having greater than 85% amino acid sequence identity to UGTS12. In another particular embodiment, the UDP-glucosyltransferase is EUGT11 or a UGT having greater than 85% amino acid sequence identity to EUGT11. In yet another particular embodiment, the UDP-glucosyltransferase is UGT91D2 or a UGT having greater than 85% amino acid sequence identity to UGT91D2.

[0016] In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase that can add at least one glucose unit to the existing glucose at C19 of any starting steviol glycoside to give a target steviol glycoside with at least one additional glucose having at least one beta-1→3 glucopyranoside glycosidic bond(s) in the glycosidic bond(s) of the newly generated bond. In a particular embodiment, the UDP-glucosyltransferase is UGT76G1 or a UGT having greater than 85% amino acid sequence identity to UGT76G1.

[0017] In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to the existing glucose at C13 of any starting steviol glycoside to give a target steviol glycoside with at least one additional glucose and at least one beta-1→2 glucopyranoside glycosidic bond(s) in the newly generated glycosidic bond(s). In a particular embodiment, the UDP-glucosyltransferase is UGTS12 or a UGT having greater than 85% amino acid sequence identity to UGTS12. In another particular embodiment, the UDP-glucosyltransferase is EUGT11 or a UGT having greater than 85% amino acid sequence identity to EUGT11. In yet another particular embodiment, the UDP-glucosyltransferase is UGT91D2 or a UGT having greater than 85% amino acid sequence identity to UGT91D2.

[0018] In one embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to steviol to produce steviolmonoside. In a particular embodiment, the UDP-glucosyltransferase is UGT85C2, or a UGT having greater than 85% amino acid sequence identity to UGT85C2, or a UGT having greater than 85% amino acid sequence identity to UGT85C2.

[0019] In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to steviol to produce steviolmonoside A. In a particular embodiment, the UDP-glucosyltransferase is UGT74G1 or a UGT having greater than 85% amino acid sequence identity to UGT74G1. In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase that can add at least one glucose unit to steviolmonoside A to produce steviolmonoside B. In a particular embodiment, the UDP-glucosyltransferase is UGT76G1 or a UGT having greater than 85% amino acid sequence identity to UGT76G1.

[0020] In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to steviolmonoside A to produce steviolbioside A. In a particular embodiment, the UDP-glucosyltransferase is UGTS12 or a UGT having greater than 85% amino acid sequence identity to UGTS12. In another particular embodiment, the UDP-glucosyltransferase is EUGT11 or a UGT having greater than 85% amino acid sequence identity to EUGT11. In yet another particular embodiment, the UDP-glucosyltransferase is UGT91D2 or a UGT having greater than 85% amino acid sequence identity to UGT91D2.

[0021] In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to steviolmonoside A to produce rubusoside. In a particular embodiment, the UDP-glucosyltransferase is UGT85C2 or a UGT having greater than 85% amino acid sequence identity to UGT85C2.

[0022] In another aspect, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to steviolmonoside to produce rubusoside. In a particular aspect, the UDP-glucosyltransferase is UGT74G1 or a UGT having greater than 85% amino acid sequence identity to UGT74G1. In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to a steviolmonoside to produce steviolbioside. In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to steviolbioside B to produce stevioside B. In a particular embodiment, the UDP-glucosyltransferase is UGT85C2 or a UGT having greater than 85% amino acid sequence identity to UGT85C2.

[0023] In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to steviolbioside B to produce stevioside C. In a particular embodiment, the UDP-glucosyltransferase is UGTS12 or a UGT having greater than 85% amino acid sequence identity to UGTS12. In another particular embodiment, the UDP-glucosyltransferase is EUGT11 or a UGT having greater than 85% amino acid sequence identity to EUGT11. In yet another particular embodiment, the UDP-glucosyltransferase is UGT91D2 or a UGT having greater than 85% amino acid sequence identity to UGT91D2.

[0024] In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to steviolbioside A to produce stevioside A. In a particular embodiment, the UDP-glucosyltransferase is UGT85C2 or a UGT having greater than 85% amino acid sequence identity to UGT85C2. In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase that can add at least one glucose unit to steviolbioside A to produce stevioside C. In a particular embodiment, the UDP-glucosyltransferase is UGT76G1 or a UGT having greater than 85% amino acid sequence identity to UGT76G1.

[0025] In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rubusoside to produce stevioside B. In a particular embodiment, the UDP-glucosyltransferase is UGT76G1 or a UGT having greater than 85% amino acid sequence identity to UGT76G1.

[0026] In another aspect, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rubusoside to produce stevioside A (rebaudioside KA). In a particular aspect, the UDP-glucosyltransferase is UGTS12 or a UGT having greater than 85% amino acid sequence identity to UGTS12. In another particular aspect, the UDP-glucosyltransferase is EUGT11 or a UGT having greater than 85% amino acid sequence identity to EUGT11. In yet another particular aspect, the UDP-glucosyltransferase is UGT91D2 or a UGT having greater than 85% amino acid sequence identity to UGT91D2. In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rubusoside to produce stevioside.

[0027] In another aspect, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to steviolbioside to produce stevioside. In a particular aspect, the UDP-glucosyltransferase is UGT74G1 or a UGT having greater than 85% amino acid sequence identity to UGT74G1.

[0028] In another aspect, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to stevioside B to produce rebaudioside E3. In a particular aspect, the UDP-glucosyltransferase is UGTS12 or a UGT having greater than 85% amino acid sequence identity to UGTS12. In another particular aspect, the UDP-glucosyltransferase is EUGT11 or a UGT having greater than 85% amino acid sequence identity to EUGT11. In yet another particular aspect, the UDP-glucosyltransferase is UGT91D2 or a UGT having greater than 85% amino acid sequence identity to UGT91D2.

[0029] In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to stevioside B to produce rebaudioside E2. In another aspect, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to stevioside A (rebaudioside KA) to produce rebaudioside E3. In a particular aspect, the UDP-glucosyltransferase is UGT76G1 or a UGT having greater than 85% amino acid sequence identity to UGT76G1. In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to stevioside A (rebaudioside KA) to produce rebaudioside E.

[0030] In another aspect, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to stevioside C to produce rebaudioside E3. In a particular aspect, the UDP-glucosyltransferase is UGT85C2 or a UGT having greater than 85% amino acid sequence identity to UGT85C2.

[0031] In another aspect, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to stevioside to produce rebaudioside E2. In a particular aspect, the UDP-glucosyltransferase is UGT76G1 or a UGT having greater than 85% amino acid sequence identity to UGT76G1.

[0032] In another aspect, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to stevioside to produce rebaudioside E. In a particular aspect, the UDP-glucosyltransferase is UGTS12 or a UGT having greater than 85% amino acid sequence identity to UGTS12. In another particular aspect, the UDP-glucosyltransferase is EUGT11 or a UGT having greater than 85% amino acid sequence identity to EUGT11. In yet another particular aspect, the UDP-glucosyltransferase is UGT91D2 or a UGT having greater than 85% amino acid sequence identity to UGT91D2. In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase that can add at least one glucose unit to rebaudioside E3 to produce rebaudioside AM.

[0033] In another aspect, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside E2 to produce rebaudioside AM. In a particular aspect, the UDP-glucosyltransferase is UGTS12 or a UGT having greater than 85% amino acid sequence identity to UGTS12. In another particular aspect, the UDP-glucosyltransferase is EUGT11 or a UGT having greater than 85% amino acid sequence identity to EUGT11. In yet another particular aspect, the UDP-glucosyltransferase is UGT91D2 or a UGT having greater than 85% amino acid sequence identity to UGT91D2.

[0034] In another aspect, the UDP-glucosyltransferase is any UDP-glucosyltransferase that can add at least one glucose unit to rebaudioside E to produce rebaudioside AM. In a particular aspect, the UDP-glucosyltransferase is UGT76G1 or a UGT having greater than 85% amino acid sequence identity to UGT76G1.

[0035] Optionally, the methods of the invention further include using two or more UGTs on the starting composition to provide target steviol glycoside(s) with two or more glucose units than the starting composition. In certain embodiments, the UDP-glucosyltransferase is UGT74G1, UGT85C2, UGT76G1, UGTSI2, EUGT11, and / or UGT91D2, or any UGT having greater than 85% amino acid sequence identity with UGT74G1, UGT85C2, UGT76G1, UGTSI2, EUGT11, and / or UGT91D2 that can add two or more glucose units to the starting composition to provide steviol glycoside(s) with two or more glucose units than the starting composition, or any combination thereof.

[0036] In one aspect, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding a total of two glucose units to stevioside to produce rebaudioside AM. In a particular aspect, the UDP-glucosyltransferase is selected from UGTS12, EUGT11, UGT91D2, UGT76G1, or any UGT having greater than 85% amino acid sequence identity with UGTS12, EUGT11, UGT91D2, UGT76G1, or any combination thereof. In another particular aspect, the UDP-glucosyltransferase is UGTS12 and UGT76G1.

[0037] Optionally, the method further comprises recycling UDP to provide UDP-glucose. In one aspect, the method comprises recycling UDP by providing a recycling catalyst and a recycling substrate, such that steviol and / or steviol glycoside substrates are biotransformed to target steviol glycosides using catalytic amounts of a UDP-glucosyltransferase and UDP-glucose. In one embodiment, the recycling catalyst is sucrose synthase SuSy_At or a sucrose synthase having greater than 85% amino acid sequence identity to SuSy_At. In one embodiment, the recycling substrate is sucrose.

[0038] Optionally, the methods of the present invention further include the use of transglycosidases that use oligosaccharides or polysaccharides as sugar donors to modify recipient target steviol glycoside molecules. Non-limiting examples include cyclodextrin glycosyltransferase (CGTase), fructofuranosidase, amylase, saccharase, glucosucrase, beta-fructosidase, beta-fructosidase, SucraseIn some embodiments, glucose and sugar(s) other than glucose, including but not limited to fructose, xylose, rhamnose, arabinose, deoxyglucose, and galactose, are transferred to the recipient target steviol glycoside. In one embodiment, the recipient steviol glycoside is rebaudioside AM.

[0039] Optionally, the methods of the present invention further include separating the target steviol glycoside from the culture medium to obtain a highly purified target steviol glycoside composition. The target steviol glycoside can be separated by at least one suitable method, such as, for example, crystallization, membrane separation, centrifugation, extraction, chromatographic separation, or a combination of such methods. In one aspect, the target steviol glycoside can be produced within the microorganism. In another aspect, the target steviol glycoside can be secreted into the culture medium. In another aspect, the released steviol glycoside can be continuously removed from the culture medium. In yet another aspect, the target steviol glycoside is isolated after the conversion reaction is complete.

[0040] 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 highly purified 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. The target steviol glycoside can be in any polymorphic or amorphous form, including hydrates, solvates, anhydrates, or combinations thereof.

[0041] The purified target steviol glycosides can be used in consumer products as sweeteners, flavor modifiers, flavor modifying agents, and / or anti-foaming agents. Suitable consumer products include, but are not limited to, foods, beverages, pharmaceutical compositions, tobacco products, nutritional compositions, oral hygiene compositions, and cosmetic compositions. [Brief explanation of the drawings]

[0042] [Figure 1] FIG. 1 shows the chemical structure of rebaudioside AM. [Figure 2] FIG. 2 shows pathways for producing rebaudioside AM and various steviol glycosides from steviol. [Figure 3] FIG. 3 shows the biocatalytic production of rebaudioside AM from stevioside using the enzymes UGTSI2 and UGT76G1 and the concomitant recycling of UDP to UDP-glucose via sucrose synthase SuSy_At. [Figure 4] FIG. 4 shows the biocatalytic production of rebaudioside AM from rebaudioside E using the enzyme UGT76G1 and the concomitant recycling of UDP to UDP-glucose via sucrose synthase SuSy_At. [Figure 5] Figure 5 shows the HPLC chromatogram of stevioside. The peak at a retention time of 25.992 minutes corresponds to stevioside. [Figure 6] Figure 6 shows the HPLC chromatogram of the products of the biocatalytic production of rebaudioside AM from stevioside. The peak at a retention time of 10.636 minutes corresponds to rebaudioside AM. [Figure 7] 7 shows the HPLC chromatogram of rebaudioside E. The peak at a retention time of 10.835 minutes corresponds to rebaudioside E. [Figure 8]8 shows the HPLC chromatogram of the products of the biocatalytic production of rebaudioside AM from rebaudioside E. The peaks at retention times of 10.936 minutes and 11.442 minutes correspond to rebaudioside E and rebaudioside AM, respectively. [Figure 9] 9 shows the HPLC chromatogram of rebaudioside AM after purification by methanol crystallization. The peak at a retention time of 10.336 minutes corresponds to rebaudioside AM. [Figure 10] FIG. 10 shows the H NMR spectrum of rebaudioside AM (500 MHz, pyridine-d5). [Figure 11] FIG. 11 shows the HSQC spectrum of rebaudioside AM (500 MHz, pyridine-d5). [Figure 12] FIG. 12 shows the H,H COSY spectrum of rebaudioside AM (500 MHz, pyridine-d5). [Figure 13] FIG. 13 shows the HMBC spectrum of rebaudioside AM (500 MHz, pyridine-d5). [Figure 14] FIG. 14 shows the HSQC-TOCSY spectrum of rebaudioside AM (500 MHz, pyridine-d5). [Figure 15a] Figures 15a and 15b show the LC chromatogram and mass spectrum of rebaudioside AM, respectively. [Figure 15b] Figures 15a and 15b show the LC chromatogram and mass spectrum of rebaudioside AM, respectively.

[0043] Detailed Description The present invention provides methods 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 the target steviol glycoside.

[0044] One object of the present invention is to provide an efficient biocatalytic method for preparing target steviol glycosides, particularly steviolmonoside, steviolmonoside A, steviolbioside, steviolbioside A, steviolbioside B, rubusoside, stevioside, stevioside A (rebaudioside KA), stevioside B, stevioside C, rebaudioside E, rebaudioside E2, rebaudioside E3, rebaudioside AM, or synthetic steviol glycosides, from a variety of starting compositions. As used herein, the abbreviation "reb" refers to "rebaudioside." Both terms have the same meaning and may be used interchangeably.

[0045] As used herein, "biocatalyst" or "biocatalysis" refers to the use of natural or genetically engineered biocatalysts, such as enzymes, or cells, including microorganisms, containing one or more enzymes, capable of single- or multi-step chemical conversions of organic compounds. Biocatalytic processes include fermentation, biosynthesis, bioconversion, and biotransformation processes. Both isolated enzyme and whole-cell biocatalytic methods are known in the art. Biocatalytic protein enzymes can be naturally occurring proteins or recombinant proteins.

[0046] As used herein, the term "steviol glycoside(s)" refers to glycosides of steviol, including, but not limited to, naturally occurring steviol glycosides, such as steviolmonoside, steviolmonoside A, steviolbioside, steviolbioside A, steviolbioside B, rubusoside, stevioside, stevioside A (rebaudioside KA), stevioside B, stevioside C, rebaudioside E, rebaudioside E2, rebaudioside E3, rebaudioside AM, synthetic steviol glycosides, such as enzymatically glycosylated steviol glycosides, and combinations thereof.

[0047] Starting Composition As used herein, "starting composition" refers to any composition (typically an aqueous solution) that includes one or more organic compounds that contain at least one carbon atom. In one aspect, the starting composition is selected from the group consisting of steviol, steviol glycosides, polyols, and various carbohydrates.

[0048] The steviol glycoside of the starting composition is selected from the group consisting of steviolmonoside, steviolmonoside A, steviolbioside, steviolbioside A, steviolbioside B, rubusoside, stevioside, stevioside A (rebaudioside KA), stevioside B, stevioside C, rebaudioside E, rebaudioside E2, rebaudioside E3, or other glycosides of steviol occurring in the Stevia rebaudiana plant, synthetic steviol glycosides, e.g., enzymatically glycosylated steviol glycosides, and combinations thereof. In one embodiment, the starting composition is steviol. In another embodiment, the steviol glycoside of the starting composition is a steviol monoside. In yet another embodiment, the steviol glycoside of the starting composition is steviolmonoside A.

[0049] In yet another aspect, the steviol glycoside of the starting composition is rubusoside. In yet another embodiment, the steviol glycoside of the starting composition is steviolbioside. In yet another embodiment, the steviol glycoside of the starting composition is steviolbioside A. In yet another embodiment, the steviol glycoside of the starting composition is steviolbioside B.

[0050] In yet another embodiment, the steviol glycoside of the starting composition is stevioside. In yet another embodiment, the steviol glycoside of the starting composition is stevioside A, also known as rebaudioside KA. In yet another embodiment, the steviol glycoside of the starting composition is stevioside B. In yet another embodiment, the steviol glycoside of the starting composition is stevioside C.

[0051] In another embodiment, the steviol glycoside of the starting composition is rebaudioside E. In another embodiment, the steviol glycoside of the starting composition is rebaudioside E2. In another embodiment, the steviol glycoside of the starting composition is rebaudioside E3.

[0052] The term "polyol" refers to a molecule containing two or more hydroxyl groups. Polyols may be diols, triols, or tetraols, containing 2, 3, and 4 hydroxyl groups, respectively. Polyols may also contain more than four hydroxyl groups, such as pentaols, hexaols, and heptaols, containing 5, 6, or 7 hydroxyl groups, respectively. Furthermore, polyols may also be sugar alcohols, polyhydric alcohols, or polyhydric alcohols 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 isomalto-oligosaccharides, reduced xylo-oligosaccharides, reduced gentio-oligosaccharides, reduced maltitol syrup, reduced glucose syrup, hydrogenated starch hydrolysates, polyglycitols, and sugar alcohols, or any other carbohydrate that can be reduced.

[0053] The term "carbohydrate" refers to a carbohydrate having the general formula (CHO) where n is 3 to 30. nThe term "carbohydrate" refers to aldehyde or ketone compounds substituted with multiple hydroxyl groups, as well as oligomers and polymers thereof. In addition, the carbohydrates of the present invention may be substituted or deoxygenated at one or more positions. As used herein, carbohydrates include unmodified carbohydrates, carbohydrate derivatives, substituted carbohydrates, and modified carbohydrates. As used herein, the phrases "carbohydrate derivative," "substituted carbohydrate," and "modified carbohydrate" 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 may optionally be deoxygenated at any corresponding C-position and / or substituted with one or more moieties such as hydrogen, halogen, haloalkyl, carboxyl, acyl, acyloxy, amino, amido, carboxyl derivative, alkylamino, dialkylamino, arylamino, alkoxy, aryloxy, nitro, cyano, sulfo, mercapto, imino, sulfonyl, sulfenyl, sulfinyl, sulfamoyl, carboalkoxy, carboxamido, phosphonyl, phosphinyl, phosphoryl, phosphino, thioester, thioether, oximino, hydrazino, carbamyl, phospho, phosphonate, or any other viable functional group, provided that the carbohydrate derivative or substituted carbohydrate functions to improve the sweetness of the sweetener composition.

[0054] Examples of carbohydrates that may be used in accordance with the present invention are tagatose, trehalose, galactose, rhamnose, various cyclodextrins, cyclic oligosaccharides, various types of maltodextrins, dextran, sucrose, glucose, ribulose, fructose, threose, arabinose, xylose, lyxose, allose, altrose, mannose, idose, lactose, maltose, invert sugar, isotrehalose, neotrehalose, isomaltulose, erythrose, deoxyribose, gulose, idose, talose, erythrulose, xylulose, psicose, turanose, cellobiose, amylopectin, glucosamine, mannosamine, fucose, glucuronic acid, gluconic acid, glucono-lactone, abequose, galactosamine, sugar beet oligosaccharides, isomaltose, Examples of oligosaccharides include, but are not limited to, 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 syrups, coupling sugars, and soybean oligosaccharides. Additionally, carbohydrates as used herein may be in either the D or L configuration.

[0055] The starting composition may be synthetic or purified (partially or completely), commercially available or prepared. In one embodiment, the starting composition is glycerol. In another embodiment, the starting composition is glucose. In yet another embodiment, the starting composition is sucrose.

[0056] In yet another embodiment, the starting composition is a starch. In another embodiment, the starting composition is maltodextrin. In yet another embodiment, the starting composition is cellulose.

[0057] In yet another embodiment, the starting composition is amylose. The organic compound(s) of the starting composition serve as substrate(s) for the production of the target steviol glycoside(s), as described herein.

[0058] Targeted Steviol Glycosides The target steviol glycoside of the present method can be any steviol glycoside that can be prepared by the methods disclosed herein. In one embodiment, the target steviol glycoside is selected from the group consisting of steviolmonoside, steviolmonoside A, steviolbioside, steviolbioside A, steviolbioside B, rubusoside, stevioside, stevioside A (rebaudioside KA), stevioside B, stevioside C, rebaudioside E, rebaudioside E2, rebaudioside E3, rebaudioside AM, or other glycosides of steviol occurring in the Stevia rebaudiana plant, synthetic steviol glycosides, e.g., enzymatically glycosylated steviol glycosides, and combinations thereof.

[0059] In one aspect, the target steviol glycoside is a steviolmonoside. In another embodiment, the target steviol glycoside is steviolmonoside A. In another embodiment, the target steviol glycoside is steviolbioside. In another embodiment, the target steviol glycoside is steviolbioside A. In another embodiment, the target steviol glycoside is steviolbioside B. In another aspect, the target steviol glycoside is rubusoside. In another embodiment, the target steviol glycoside is stevioside. In one embodiment, the target steviol glycoside is stevioside A (rebaudioside KA). In another embodiment, the target steviol glycoside is stevioside B.

[0060] In another embodiment, the target steviol glycoside is stevioside C. In another embodiment, the target steviol glycoside is rebaudioside E. In another embodiment, the target steviol glycoside is rebaudioside E2. In another embodiment, the target steviol glycoside is rebaudioside E3. In another embodiment, the target steviol glycoside is rebaudioside AM.

[0061] The target steviol glycoside can be in any polymorphic or amorphous form, including hydrates, solvates, anhydrates, or combinations thereof. In one aspect, the invention is a biocatalytic process for the production of steviolmonoside. In one aspect, the present invention is a biocatalytic process for the production of steviolmonoside A. In one aspect, the invention is a biocatalytic process for the production of steviolbioside. In one aspect, the present invention is a biocatalytic process for the production of steviolbioside A.

[0062] In one aspect, the invention is a biocatalytic process for the production of steviolbioside B. In one aspect, the invention is a biocatalytic process for the production of rubusoside. In one aspect, the present invention is a biocatalytic process for the production of stevioside. In one aspect, the invention is a biocatalytic process for the production of stevioside A (rebaudioside KA). In one aspect, the present invention is a biocatalytic process for the production of stevioside B.

[0063] In one aspect, the present invention is a biocatalytic process for the production of stevioside C. In one aspect, the present invention is a biocatalytic process for the production of rebaudioside E. In one aspect, the present invention is a biocatalytic process for the production of rebaudioside E2. In one aspect, the invention is a biocatalytic process for the production of rebaudioside E3.

[0064] In one aspect, the present invention is a biocatalytic process for the production of rebaudioside AM. In certain aspects, the present invention provides a biocatalytic process for producing rebaudioside AM from a starting composition comprising stevioside and UDP-glucose. In another specific embodiment, the present invention provides a biocatalytic process for producing rebaudioside AM from a starting composition comprising rebaudioside E and UDP-glucose. Optionally, the methods of the present invention further comprise separating the target steviol glycoside from the culture medium to obtain a highly purified 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.

[0065] In certain aspects, the methods described herein result in highly purified target steviol glycoside compositions. As used herein, the term "highly purified" refers to a composition having greater than about 80% target steviol glycoside by weight on an anhydrous (dry) basis. In one aspect, a highly purified target steviol glycoside composition contains greater than about 90% target steviol glycoside by weight on an anhydrous (dry) basis, e.g., greater than about 91%, greater than about 92%, greater than about 93%, greater than about 94%, greater than about 95%, greater than about 96%, greater than about 97%, greater than about 98%, or greater than about 99% target steviol glycoside content by dry weight.

[0066] In one embodiment, when the target steviol glycoside is reb AM, the methods described herein result in a composition having a reb AM content of greater than about 90% by weight on a dry weight basis. In another specific embodiment, when the target steviol glycoside is reb AM, the methods described herein result in a composition comprising a reb AM content of greater than about 95% by weight on a dry weight basis.

[0067] Microbial and Enzyme Preparations In one aspect of the invention, a microorganism (microbial cell) and / or enzyme preparation is contacted with a medium containing a starting composition to produce a target steviol glycoside. The enzymes may be provided in the form of a whole cell suspension, a crude lysate, a purified enzyme, or a combination thereof. In one aspect, the biocatalyst is a purified enzyme capable of converting the starting composition to the target steviol glycoside. In another aspect, the biocatalyst is a crude lysate containing at least one enzyme capable of converting the starting composition to the target steviol glycoside. In yet another aspect, the biocatalyst is a whole cell suspension containing at least one enzyme capable of converting the starting composition to the target steviol glycoside.

[0068] In another aspect, the biocatalyst is one or more microbial cells that contain an enzyme(s) capable of converting the starting composition to the target steviol glycoside. The enzyme(s) can be located on the surface of the cell, inside the cell, or both on the surface and inside the cell. Suitable enzymes for converting the starting composition to the target steviol glycosides include, but are not limited to, steviol biosynthetic enzymes and UDP-glucosyltransferases (UGTs). Optionally, they may include a UDP recycling enzyme(s). In one embodiment, steviol biosynthetic enzymes include mevalonate (MVA) pathway enzymes. In another embodiment, steviol biosynthetic enzymes include non-mevalonate 2-C-methyl-D-erythritol-4-phosphate pathway (MEP / DOXP) enzymes.

[0069] In one aspect, the steviol biosynthetic enzymes include 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), 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, and the like. The UDP-glucosyltransferase can be any UDP-glucosyltransferase capable of adding at least one glucose unit to a steviol and / or steviol glycoside substrate to provide the target steviol glycoside.

[0070] In one aspect, the steviol biosynthetic enzymes and UDP-glucosyltransferase are produced in a microbial cell. The microbial cell may be, for example, E. coli, Saccharomyces sp., Aspergillus sp., Pichia sp., Bacillus sp., Yarrowia sp., etc. In another aspect, the UDP-glucosyltransferase is synthesized.

[0071] In one aspect, the UDP-glucosyltransferase is selected from the group including UGT74G1, UGT85C2, UGT76G1, UGT91D2, UGTSI2, EUGT11, and UGTs having substantial (greater than 85%, greater than 86%, greater than 87%, greater than 88%, greater than 89%, greater than 90%, greater than 91%, greater than 92%, greater than 93%, greater than 94%, greater than 95%, greater than 96%, greater than 97%, greater than 98%, greater than 99%) amino acid sequence identity to these polypeptides, and isolated nucleic acid molecules encoding these UGTs.

[0072] In one aspect, the steviol biosynthetic enzymes, UGTs, and UDP-glucose recycling system are present in a single microorganism (microbial cell), which may be, for example, E. coli, Saccharomyces sp., Aspergillus sp., Pichia sp., Bacillus sp., or Yarrowia sp.

[0073] In one aspect, 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 C13 to provide a target steviol glycoside having an -O-glucose beta-glucopyranoside glycosidic linkage at C13. In a particular aspect, the UDP-glucosyltransferase is UGT85C2 or a UGT having greater than 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 steviol or any starting steviol glycoside having a -COOH functional group at C19 to provide a target steviol glycoside having a -COO-glucose beta-glucopyranoside glycosidic linkage at C19. In a particular embodiment, the UDP-glucosyltransferase is UGT74G1 or a UGT having greater than 85% amino acid sequence identity to UGT74G1.

[0075] In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to the existing glucose at C19 of any starting steviol glycoside to give a target steviol glycoside with at least one additional glucose and at least one beta-1→2 glucopyranoside glycosidic bond(s) in the newly generated glycosidic bond(s). In a particular embodiment, the UDP-glucosyltransferase is UGTS12 or a UGT having greater than 85% amino acid sequence identity to UGTS12. In another particular embodiment, the UDP-glucosyltransferase is EUGT11 or a UGT having greater than 85% amino acid sequence identity to EUGT11. In yet another particular embodiment, the UDP-glucosyltransferase is UGT91D2 or a UGT having greater than 85% amino acid sequence identity to UGT91D2.

[0076] In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase that can add at least one glucose unit to the existing glucose at C19 of any starting steviol glycoside to give a target steviol glycoside with at least one additional glucose having at least one beta-1→3 glucopyranoside glycosidic bond(s) in the glycosidic bond(s) of the newly generated bond. In a particular embodiment, the UDP-glucosyltransferase is UGT76G1 or a UGT having greater than 85% amino acid sequence identity to UGT76G1.

[0077] In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to the existing glucose at C13 of any starting steviol glycoside to give a target steviol glycoside with at least one additional glucose and at least one beta-1→2 glucopyranoside glycosidic bond(s) in the newly generated glycosidic bond(s). In a particular embodiment, the UDP-glucosyltransferase is UGTS12 or a UGT having greater than 85% amino acid sequence identity to UGTS12. In another particular embodiment, the UDP-glucosyltransferase is EUGT11 or a UGT having greater than 85% amino acid sequence identity to EUGT11. In yet another particular embodiment, the UDP-glucosyltransferase is UGT91D2 or a UGT having greater than 85% amino acid sequence identity to UGT91D2.

[0078] In one embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to steviol to produce steviolmonoside. In a particular embodiment, the UDP-glucosyltransferase is UGT85C2, or a UGT having greater than 85% amino acid sequence identity to UGT85C2, or a UGT having greater than 85% amino acid sequence identity to UGT85C2.

[0079] In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to steviol to produce steviolmonoside A. In a particular embodiment, the UDP-glucosyltransferase is UGT74G1 or a UGT having greater than 85% amino acid sequence identity to UGT74G1.

[0080] In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase that can add at least one glucose unit to steviolmonoside A to produce steviolmonoside B. In a particular embodiment, the UDP-glucosyltransferase is UGT76G1 or a UGT having greater than 85% amino acid sequence identity to UGT76G1.

[0081] In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to steviolmonoside A to produce steviolbioside A. In a particular embodiment, the UDP-glucosyltransferase is UGTS12 or a UGT having greater than 85% amino acid sequence identity to UGTS12. In another particular embodiment, the UDP-glucosyltransferase is EUGT11 or a UGT having greater than 85% amino acid sequence identity to EUGT11. In yet another particular embodiment, the UDP-glucosyltransferase is UGT91D2 or a UGT having greater than 85% amino acid sequence identity to UGT91D2. In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to steviolmonoside A to produce rubusoside. In a particular embodiment, the UDP-glucosyltransferase is UGT85C2 or a UGT having greater than 85% amino acid sequence identity to UGT85C2.

[0082] In another aspect, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to steviolmonoside to produce rubusoside. In a particular aspect, the UDP-glucosyltransferase is UGT74G1 or a UGT having greater than 85% amino acid sequence identity to UGT74G1. In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to a steviolmonoside to produce steviolbioside.

[0083] In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to steviolbioside B to produce stevioside B. In a particular embodiment, the UDP-glucosyltransferase is UGT85C2 or a UGT having greater than 85% amino acid sequence identity to UGT85C2. In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to steviolbioside B to produce stevioside C. In a particular embodiment, the UDP-glucosyltransferase is UGTS12 or a UGT having greater than 85% amino acid sequence identity to UGTS12. In another particular embodiment, the UDP-glucosyltransferase is EUGT11 or a UGT having greater than 85% amino acid sequence identity to EUGT11. In yet another particular embodiment, the UDP-glucosyltransferase is UGT91D2 or a UGT having greater than 85% amino acid sequence identity to UGT91D2.

[0084] In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to steviolbioside A to produce stevioside A. In a particular embodiment, the UDP-glucosyltransferase is UGT85C2 or a UGT having greater than 85% amino acid sequence identity to UGT85C2. In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase that can add at least one glucose unit to steviolbioside A to produce stevioside C. In a particular embodiment, the UDP-glucosyltransferase is UGT76G1 or a UGT having greater than 85% amino acid sequence identity to UGT76G1.

[0085] In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rubusoside to produce stevioside B. In a particular embodiment, the UDP-glucosyltransferase is UGT76G1 or a UGT having greater than 85% amino acid sequence identity to UGT76G1.

[0086] In another aspect, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rubusoside to produce stevioside A (rebaudioside KA). In a particular aspect, the UDP-glucosyltransferase is UGTS12 or a UGT having greater than 85% amino acid sequence identity to UGTS12. In another particular aspect, the UDP-glucosyltransferase is EUGT11 or a UGT having greater than 85% amino acid sequence identity to EUGT11. In yet another particular aspect, the UDP-glucosyltransferase is UGT91D2 or a UGT having greater than 85% amino acid sequence identity to UGT91D2. In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rubusoside to produce stevioside.

[0087] In another aspect, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to steviolbioside to produce stevioside. In a particular aspect, the UDP-glucosyltransferase is UGT74G1 or a UGT having greater than 85% amino acid sequence identity to UGT74G1.

[0088] In another aspect, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to stevioside B to produce rebaudioside E3. In a particular aspect, the UDP-glucosyltransferase is UGTS12 or a UGT having greater than 85% amino acid sequence identity to UGTS12. In another particular aspect, the UDP-glucosyltransferase is EUGT11 or a UGT having greater than 85% amino acid sequence identity to EUGT11. In yet another particular aspect, the UDP-glucosyltransferase is UGT91D2 or a UGT having greater than 85% amino acid sequence identity to UGT91D2. In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to stevioside B to produce rebaudioside E2.

[0089] In another aspect, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to stevioside A (rebaudioside KA) to produce rebaudioside E3. In a particular aspect, the UDP-glucosyltransferase is UGT76G1 or a UGT having greater than 85% amino acid sequence identity to UGT76G1. In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to stevioside A (rebaudioside KA) to produce rebaudioside E.

[0090] In another aspect, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to stevioside C to produce rebaudioside E3. In a particular aspect, the UDP-glucosyltransferase is UGT85C2 or a UGT having greater than 85% amino acid sequence identity to UGT85C2.

[0091] In another aspect, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to stevioside to produce rebaudioside E2. In a particular aspect, the UDP-glucosyltransferase is UGT76G1 or a UGT having greater than 85% amino acid sequence identity to UGT76G1.

[0092] In another aspect, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to stevioside to produce rebaudioside E. In a particular aspect, the UDP-glucosyltransferase is UGTS12 or a UGT having greater than 85% amino acid sequence identity to UGTS12. In another particular aspect, the UDP-glucosyltransferase is EUGT11 or a UGT having greater than 85% amino acid sequence identity to EUGT11. In yet another particular aspect, the UDP-glucosyltransferase is UGT91D2 or a UGT having greater than 85% amino acid sequence identity to UGT91D2. In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase that can add at least one glucose unit to rebaudioside E3 to produce rebaudioside AM.

[0093] In another aspect, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside E2 to produce rebaudioside AM. In a particular aspect, the UDP-glucosyltransferase is UGTS12 or a UGT having greater than 85% amino acid sequence identity to UGTS12. In another particular aspect, the UDP-glucosyltransferase is EUGT11 or a UGT having greater than 85% amino acid sequence identity to EUGT11. In yet another particular aspect, the UDP-glucosyltransferase is UGT91D2 or a UGT having greater than 85% amino acid sequence identity to UGT91D2.

[0094] In another aspect, the UDP-glucosyltransferase is any UDP-glucosyltransferase that can add at least one glucose unit to rebaudioside E to produce rebaudioside AM. In a particular aspect, the UDP-glucosyltransferase is UGT76G1 or a UGT having greater than 85% amino acid sequence identity to UGT76G1.

[0095] Optionally, the methods of the invention further include using two or more UGTs on the starting composition to provide target steviol glycoside(s) with two or more glucose units than the starting composition. In certain embodiments, the UDP-glucosyltransferase is UGT74G1, UGT85C2, UGT76G1, UGTSI2, EUGT11, and / or UGT91D2, or any UGT having greater than 85% amino acid sequence identity with UGT74G1, UGT85C2, UGT76G1, UGTSI2, EUGT11, and / or UGT91D2 that can add two or more glucose units to the starting composition to provide steviol glycoside(s) with two or more glucose units than the starting composition, or any combination thereof.

[0096] In one aspect, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding a total of two glucose units to stevioside to produce rebaudioside AM. In a particular aspect, the UDP-glucosyltransferase is selected from UGTS12, EUGT11, UGT91D2, UGT76G1, or any UGT having greater than 85% amino acid sequence identity with UGTS12, EUGT11, UGT91D2, UGT76G1, or any combination thereof. In another particular aspect, the UDP-glucosyltransferase is UGTS12 and UGT76G1.

[0097] Optionally, the method further comprises recycling UDP to provide UDP-glucose. In one aspect, the method comprises recycling UDP by providing a recycling catalyst and a recycling substrate, such that the steviol and / or steviol glycoside substrate is biotransformed to the target steviol glycoside using a catalytic amount of a UDP-glucosyltransferase and UDP-glucose. The UDP recycling enzyme can be sucrose synthase SuSy_At or a sucrose synthase having greater than 85% amino acid sequence identity to SuSy_At, and the recycling substrate can be sucrose.

[0098] Optionally, the methods of the present invention further include the use of transglycosidases that use oligosaccharides or polysaccharides as sugar donors to modify recipient target steviol glycoside molecules. Non-limiting examples include cyclodextrin glycosyltransferase (CGTase), fructofuranosidase, amylase, saccharase, glucosucrase, beta-fructosidase, beta-fructosidase, Sucrase In some embodiments, glucose and sugar(s) other than glucose, including but not limited to fructose, xylose, rhamnose, arabinose, deoxyglucose, and galactose, are transferred to the recipient target steviol glycoside. In one embodiment, the recipient steviol glycoside is rebaudioside AM.

[0099] In another aspect, the UDP-glucosyltransferase capable of adding at least one glucose unit to the starting composition steviol glycoside has greater than 85% amino acid sequence identity to a UGT selected from the following list of GenInfo identifier numbers, preferably from the group shown in Tables 1 and 2. [Table 1-1] [Table 1-2] [Table 1-3] [Table 2-1] [Table 2-2] [Table 2-3] [Table 3]

[0100] One aspect of the present invention is a microbial cell containing an enzyme, i.e., an enzyme capable of converting a starting composition into a target steviol glycoside. Accordingly, some embodiments of the present method include contacting a microorganism with a medium containing the starting composition to obtain a medium containing at least one target steviol glycoside. The microorganism can be any microorganism that possesses the enzyme(s) necessary to convert the starting composition into the target steviol glycoside(s). These enzymes are encoded within the genome of the microorganism. Suitable microorganisms include, but are not limited to, E. coli, Saccharomyces sp., Aspergillus sp., Pichia sp., Bacillus sp., Yarrowia sp., and the like. In one aspect, the microorganisms are free when contacted with the starting composition.

[0101] In another embodiment, the microorganisms are immobilized when contacted with the starting composition. For example, the microorganisms may be immobilized on a solid support made of inorganic or organic materials. Non-limiting examples of solid supports suitable for immobilizing microorganisms include derivatized cellulose or glass, ceramics, metal oxides, or membranes. The microorganisms may be immobilized on the solid support by, for example, covalent bonding, adsorption, cross-linking, entrapment, or encapsulation. In yet another embodiment, the enzymes capable of converting the starting composition into the target steviol glycosides are secreted from the microorganism into the reaction medium. The target steviol glycoside is optionally purified. Purification of the target steviol glycoside from the reaction medium can be achieved by at least one suitable method to obtain a highly purified 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.

[0102] use In particular, the highly purified target glycoside(s) obtained according to the present invention, in particular steviolmonoside, steviolmonoside A, steviolbioside, steviolbioside A, steviolbioside B, rubusoside, stevioside, stevioside A (rebaudioside KA), stevioside B, stevioside C, rebaudioside E, rebaudioside E2, rebaudioside E3 and / or rebaudioside AM, can be used "as is" or in combination with other sweeteners, flavors, food ingredients and combinations thereof.

[0103] Non-limiting examples of flavors include, but are not limited to, lime, lemon, orange, fruit, banana, grape, pear, pineapple, mango, berry, bitter almond, cola, cinnamon, sugar, cotton candy, vanilla, and combinations thereof. Non-limiting examples of other food ingredients include, but are not limited to, acidulants, organic acids and amino acids, colorants, fillers, modified starches, gums, texturizers, preservatives, caffeine, antioxidants, emulsifiers, stabilizers, thickeners, gelling agents, and combinations thereof.

[0104] In particular, the highly purified target glycoside(s) obtained according to the present invention, particularly steviolmonoside, steviolmonoside A, steviolbioside, steviolbioside A, steviolbioside B, rubusoside, stevioside, stevioside A (rebaudioside KA), stevioside B, stevioside C, rebaudioside E, rebaudioside E2, rebaudioside E3, and / or rebaudioside AM, may be prepared in various polymorphic forms, including, but not limited to, hydrates, solvates, anhydrates, amorphous forms, and combinations thereof.

[0105] In particular, the highly purified target glycoside(s) obtained according to the present invention, in particular steviolmonoside, steviolmonoside A, steviolbioside, steviolbioside A, steviolbioside B, rubusoside, stevioside, stevioside A (rebaudioside KA), stevioside B, stevioside C, rebaudioside E, rebaudioside E2, rebaudioside E3 and / or rebaudioside AM, may be incorporated as high-intensity natural sweeteners into foods, beverages, pharmaceutical compositions, cosmetics, chewing gum, tabletop products, cereals, dairy products, toothpaste and other oral compositions, etc.

[0106] The highly purified target glycoside(s) obtained according to the present invention, in particular steviolmonoside, steviolmonoside A, steviolbioside, steviolbioside A, steviolbioside B, rubusoside, stevioside, stevioside A (rebaudioside KA), stevioside B, stevioside C, rebaudioside E, rebaudioside E2, rebaudioside E3 and / or rebaudioside AM, may be used as a sweetening compound as the sole sweetener or may be used in combination with rebaudioside A, rebaudioside A2, rebaudioside A3, rebaudioside B, rebaudioside C, rebaudioside E, rebaudioside E4, 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 E3, rebaudioside E19, rebaudioside E16, rebaudioside E17, rebaudioside E18, rebaudioside E19, rebaudioside E21, rebaudioside E19, rebaudioside E22, rebaudioside E19, rebaudioside E19, rebaudioside E23, rebaudioside E19, rebaudioside E24, rebaudioside E19, rebaudioside E25, rebaudioside Rebaudioside B, Rebaudioside C, Rebaudioside C2, Rebaudioside D, Rebaudioside D2, Rebaudioside F, Rebaudioside F2, Rebaudioside F3, Rebaudioside G, Rebaudioside H, Rebaudioside I, Rebaudioside I2, Rebaudioside I3, Rebaudioside J, Rebaudioside K, Rebaudioside K2, Rebaudioside L, Rebaudioside M, Rebaudioside M2, Rebaudioside N, Rebaudioside O, Rebaudioside O2, Rebaudioside Q, Rebaudioside Q2, Rebaudioside Q3, Rebaudioside R, Rebaudioside S , Rebaudioside T, Rebaudioside T1, Rebaudioside U, Rebaudioside U2, Rebaudioside V, Rebaudioside W, Rebaudioside W2, Rebaudioside W3, Rebaudioside Y, Rebaudioside Z1, Rebaudioside Z2, Dulcoside A, Dulcoside C, Stevioside D, Stevioside E, Stevioside E2, Stevioside F, Mogrosides, Brazzein, Neohesperidin Dihydrochalcone, Glycyrrhizic Acid and Its Salts, Thaumatin, Perillartine, Pernandulcin, Mucuroziosides, Bayunoside, Phlomisoside-I, Dimethyl- Hexahydrofluorene-dicarboxylic acid, abrusosides, periandrin, carnosiflosides, cyclocaryosides, pterocaryosides, polypodoside A, brazilin, hernandulcin, phyllodulcin, glycyphyllin, phloridzin, trilobatin, dihydroflavonol, dihydroquercetin-3-acetate, neoastribin, trans-cinnamaldehyde, monatin and its salts, celigine A, hematoxylin, monellin, osladin, pterocaryoside A, pterocaryoside B, mabinlin, pentadin, miraculin, curculin,It may be used in conjunction with at least one naturally occurring high-intensity sweetener, such as neoculin, chlorogenic acid, cynarin, Luo Han Guo sweetener, mogroside V, siamenoside, and combinations thereof.

[0107] In certain embodiments, steviolmonoside, steviolmonoside A, steviolbioside, steviolbioside A, steviolbioside B, rubusoside, stevioside, stevioside A (rebaudioside KA), stevioside B, stevioside C, rebaudioside E, rebaudioside E2, rebaudioside E3, and / or rebaudioside Rebaudioside AM is a derivative of rebaudioside A, rebaudioside A2, rebaudioside A3, rebaudioside B, rebaudioside C, rebaudioside C2, rebaudioside D, rebaudioside D2, rebaudioside F, rebaudioside F2, rebaudioside F3, rebaudioside G, rebaudioside H, rebaudioside I, rebaudioside I2, rebaudioside Rebaudioside I3, Rebaudioside J, Rebaudioside K, Rebaudioside K2, Rebaudioside L, Rebaudioside M, Rebaudioside M2, Rebaudioside N, Rebaudioside O, Rebaudioside O2, Rebaudioside Q, Rebaudioside Q2, Rebaudioside Q3, Rebaudioside R, Rebaudioside S, Rebaudioside T, Rebaudioside The sweetener composition may be used in a sweetener composition comprising a compound selected from the group consisting of T1, rebaudioside U, rebaudioside U2, rebaudioside V, rebaudioside W, rebaudioside W2, rebaudioside W3, rebaudioside Y, rebaudioside Z1, rebaudioside Z2, dulcoside A, dulcoside C, stevioside D, stevioside E, stevioside E2, stevioside F, NSF-02, mogroside V, Luo Han Guo, allulose, allose, D-tagatose, erythritol, and combinations thereof.

[0108] The highly purified target glycoside(s), particularly steviolmonoside, steviolmonoside A, steviolbioside, steviolbioside A, steviolbioside B, rubusoside, stevioside, stevioside A (rebaudioside KA), stevioside B, stevioside C, rebaudioside E, rebaudioside E2, rebaudioside E3, and / or rebaudioside AM, 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.

[0109] Additionally, highly purified target steviol glycoside(s), particularly steviolmonoside, steviolmonoside A, steviolbioside, steviolbioside A, steviolbioside B, rubusoside, stevioside, stevioside A (rebaudioside KA), stevioside B, stevioside C, rebaudioside E, rebaudioside E2, rebaudioside E3, and / or rebaudioside AM, may be used in combination with a natural sweetener reducer, such as gymnemic acid, phodulcin, digiphin, lactisol, etc. Steviolmonoside, steviolmonoside A, steviolbioside, steviolbioside A, steviolbioside B, rubusoside, stevioside, stevioside A (rebaudioside KA), stevioside B, stevioside C, rebaudioside E, rebaudioside E2, rebaudioside E3, and / or rebaudioside AM may also be combined with various umami taste enhancers. Steviolmonoside, steviolmonoside A, steviolbioside, steviolbioside A, steviolbioside B, rubusoside, stevioside, stevioside A (rebaudioside KA), stevioside B, stevioside C, rebaudioside E, rebaudioside E2, rebaudioside E3, and / or rebaudioside AM can be mixed with umami and sweet amino acids such as glutamic acid, aspartic acid, glycine, alanine, threonine, proline, serine, glutamate, lysine, tryptophan, and combinations thereof.

[0110] The highly purified target steviol glycoside(s), particularly steviolmonoside, steviolmonoside A, steviolbioside, steviolbioside A, steviolbioside B, rubusoside, stevioside, stevioside A (rebaudioside KA), stevioside B, stevioside C, rebaudioside E, rebaudioside E2, rebaudioside E3, and / or rebaudioside AM, may 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, flavorings and flavoring ingredients, astringent compounds, proteins or protein hydrolysates, surfactants, emulsifiers, flavonoids, alcohols, polymers, and combinations thereof.

[0111] The highly purified target steviol glycoside(s), particularly steviolmonoside, steviolmonoside A, steviolbioside, steviolbioside A, steviolbioside B, rubusoside, stevioside, stevioside A (rebaudioside KA), stevioside B, stevioside C, rebaudioside E, rebaudioside E2, rebaudioside E3, and / or rebaudioside AM, may be combined with a polyol or sugar alcohol. The term "polyol" refers to a molecule containing two or more hydroxyl groups. Polyols may be diols, triols, or tetraols, containing 2, 3, and 4 hydroxyl groups, respectively. Polyols may also contain more than four hydroxyl groups, such as pentaols, hexaols, and heptaols, containing 5, 6, or 7 hydroxyl groups, respectively. Furthermore, the polyol may also be a sugar alcohol, a polyhydric alcohol, or a polyhydric alcohol, which is a reduced form of a carbohydrate, where the carbonyl group (aldehyde or ketone, reducing sugar) is reduced to a primary or secondary hydroxyl group. Examples of polyols include, but are not limited to, erythritol, maltitol, mannitol, sorbitol, lactitol, xylitol, inositol, isomalt, propylene glycol, glycerol, threitol, galactitol, hydrogenated isomaltulose, reduced isomalto-oligosaccharides, reduced xylo-oligosaccharides, reduced gentio-oligosaccharides, reduced maltose syrup, reduced glucose syrup, hydrogenated starch hydrolysates, polyglycitols, and sugar alcohols, or any other carbohydrate that can be reduced and does not adversely affect the taste of the sweetener composition.

[0112] The highly purified target steviol glycoside(s), particularly steviolmonoside, steviolmonoside A, steviolbioside, steviolbioside A, steviolbioside B, rubusoside, stevioside, stevioside A (rebaudioside KA), stevioside B, stevioside C, rebaudioside E, rebaudioside E2, rebaudioside E3, and / or rebaudioside AM, may be combined with a low-calorie sweetener such as, for example, D-tagatose, L-sugars, L-sorbose, L-arabinose, and combinations thereof.

[0113] The highly purified target steviol glycoside(s), particularly steviolmonoside, steviolmonoside A, steviolbioside, steviolbioside A, steviolbioside B, rubusoside, stevioside, stevioside A (rebaudioside KA), stevioside B, stevioside C, rebaudioside E, rebaudioside E2, rebaudioside E3, and / or rebaudioside AM, may also be combined with various carbohydrates. The term "carbohydrate" generally refers to a carbohydrate having the general formula (CHO) where n is 3 to 30. nThe term "carbohydrate" refers to aldehyde or ketone compounds substituted with multiple hydroxyl groups, as well as oligomers and polymers thereof. In addition, the carbohydrates of the present invention may be substituted or deoxygenated at one or more positions. As used herein, carbohydrates include unmodified carbohydrates, carbohydrate derivatives, substituted carbohydrates, and modified carbohydrates. As used herein, the phrases "carbohydrate derivative," "substituted carbohydrate," and "modified carbohydrate" 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 may optionally be deoxygenated at any corresponding C-position and / or substituted with one or more moieties such as hydrogen, halogen, haloalkyl, carboxyl, acyl, acyloxy, amino, amido, carboxyl derivative, alkylamino, dialkylamino, arylamino, alkoxy, aryloxy, nitro, cyano, sulfo, mercapto, imino, sulfonyl, sulfenyl, sulfinyl, sulfamoyl, carboalkoxy, carboxamido, phosphonyl, phosphinyl, phosphoryl, phosphino, thioester, thioether, oximino, hydrazino, carbamyl, phospho, phosphonate, or any other viable functional group, provided that the carbohydrate derivative or substituted carbohydrate functions to improve the sweetness of the sweetener composition.

[0114] 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, allose, altrose, mannose, idose, lactose, maltose, invert sugar, isotrehalose, neotrehalose, isomaltulose, erythrose, deoxyribose, gulose, idose, talose, erythrulose, xylulose, psicose, turanose, cellobiose, amylopectin, glucosamine, mannosamine, fucose, glucuronic acid, gluconic acid, glucono-lactone, abequase, galactosamine, sugar beet oligosaccharides, and the like. Sugars, 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.), maltotetraose Examples of carbohydrates include, but are not limited to, maltool, maltotriol, malto-oligosaccharides (such as maltotriose, maltotetraose, maltopentaose, maltohexaose, and maltoheptaose), starch, inulin, inulo-oligosaccharides, lactulose, melibiose, raffinose, ribose, isomerized liquid sugars such as high fructose corn syrups, coupling sugars, and soybean oligosaccharides. Furthermore, carbohydrates used herein may be in either the D- or L-configuration.

[0115] In particular, the highly purified target steviol glycoside(s) obtained according to the present invention, in particular steviolmonoside, steviolmonoside A, steviolbioside, steviolbioside A, steviolbioside B, rubusoside, stevioside, stevioside A (rebaudioside KA), stevioside B, stevioside C, rebaudioside E, rebaudioside E2, rebaudioside E3 and / or rebaudioside AM, can be used in combination with various bioactive 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-lowering, anti-inflammatory, etc. Exemplary functional ingredients are provided in WO2013 / 096420, the contents of which are incorporated herein by reference.

[0116] In particular, the highly purified target steviol glycoside(s) obtained according to the present invention, in particular steviolmonoside, steviolmonoside A, steviolbioside, steviolbioside A, steviolbioside B, rubusoside, stevioside, stevioside A (rebaudioside KA), stevioside B, stevioside C, rebaudioside E, rebaudioside E2, rebaudioside E3, and / or rebaudioside AM, may be applied as high-intensity sweeteners to produce zero-calorie, low-calorie, or diabetic beverages and foods with improved taste characteristics, and may be used in beverages, foods, pharmaceuticals, and other products where sugar cannot be used. Furthermore, highly purified target steviol glycosides, particularly steviolmonoside A, steviolbioside, steviolbioside A, steviolbioside B, rubusoside, stevioside, stevioside A (rebaudioside KA), stevioside B, stevioside C, rebaudioside E, rebaudioside E2, rebaudioside E3, and / or rebaudioside AM, can be used as sweeteners in beverages, foods, and other products for human consumption, as well as in animal feeds and livestock feeds with improved properties.

[0117] In particular, the highly purified target steviol glycoside(s) obtained according to the present invention, in particular steviolmonoside, steviolmonoside A, steviolbioside, steviolbioside A, steviolbioside B, rubusoside, stevioside, stevioside A (rebaudioside KA), stevioside B, stevioside C, rebaudioside E, rebaudioside E2, rebaudioside E3, and / or rebaudioside AM, may be applied as flavor modifiers to produce zero-calorie, low-calorie, or diabetic beverages and foods with altered flavors. When used as flavor modifiers, or flavors with modifying properties (FMPs), the highly purified target steviol glycosides are used in consumer products at below detection levels of the flavor modifier or FMP. Flavor modifiers or FMPs do not impart a detectable taste or flavor of their own to a consumer product, but instead serve to modify the detection of the taste and / or flavor of other ingredients in the consumer product by the consumer. One example of taste and flavor modification is sweetness enhancement, where the flavor modifier or FMP itself does not contribute to the sweetness of the consumer product, but enhances the quality of the sweetness experienced by the consumer.

[0118] Examples of consumer products in which the highly purified target steviol glycoside(s), particularly steviolmonoside A, steviolbioside, steviolbioside A, steviolbioside B, rubusoside, stevioside, stevioside A (rebaudioside KA), stevioside B, stevioside C, rebaudioside E, rebaudioside E2, rebaudioside E3, and / or rebaudioside AM, may be used as flavor modifiers or flavors with modifying properties include alcoholic beverages such as vodka, wine, beer, liqueurs, sake, etc.; natural juices; soft drinks; carbonated soft drinks; diet drinks; zero-calorie drinks; low-calorie beverages and foods; yogurt drinks; instant juices; instant coffee; powders These include, but are not limited to, instant beverages of various types, canned goods, syrups, soy sauce, soy sauce, vinegar, dressings, mayonnaise, ketchup, curry, soups, instant bouillon, soy sauce powder, vinegar powder, biscuits, rice crackers, crackers, bread, chocolate, caramel, candy, chewing gum, jelly, pudding, preserved fruits and vegetables, fresh cream, jams, marmalade, flower paste, milk powder, ice cream, sherbet, bottled vegetables and fruits, canned boiled beans, meat and food products in sweet sauces, agricultural vegetable foods, seafood, ham, sausages, fish ham, fish sausages, fish paste, fried fish products, dried seafood products, frozen foods, preserved seaweed, preserved meat, tobacco, pharmaceuticals, and many others. Essentially, it can have unlimited uses.

[0119] In particular, the highly purified target steviol glycoside(s) obtained according to the present invention, in particular steviolmonoside, steviolmonoside A, steviolbioside, steviolbioside A, steviolbioside B, rubusoside, stevioside, stevioside A (rebaudioside KA), stevioside B, stevioside C, rebaudioside E, rebaudioside E2, rebaudioside E3 and / or rebaudioside AM, may be applied as an antifoaming agent to produce zero-calorie, low-calorie, or diabetic beverages and foods.

[0120] Examples of consumer products in which the highly purified target steviol glycoside(s), particularly steviolmonoside A, steviolbioside, steviolbioside A, steviolbioside B, rubusoside, stevioside, stevioside A (rebaudioside KA), stevioside B, stevioside C, rebaudioside E, rebaudioside E2, rebaudioside E3, and / or rebaudioside AM, may be used as sweetener compounds include alcoholic beverages such as vodka, wine, beer, liqueurs, sake, etc.; natural juices; soft drinks; carbonated soft drinks; diet drinks; zero-calorie drinks; low-calorie beverages and foods; yogurt drinks; instant juices; instant coffee; powdered instant coffee; These include, but are not limited to, canned beverages, canned goods, syrup, soy sauce, soy sauce, vinegar, dressings, mayonnaise, ketchup, curry, soup, instant bouillon, soy sauce powder, vinegar powder, biscuits, rice crackers, crackers, bread, chocolate, caramel, candy, chewing gum, jelly, pudding, preserved fruits and vegetables, fresh cream, jam, marmalade, flower paste, powdered milk, ice cream, sherbet, bottled vegetables and fruits, canned boiled beans, meat and foods cooked in sweet sauces, agricultural vegetable foods, seafood, ham, sausage, fish ham, fish sausage, fish paste, fried fish products, dried seafood products, frozen foods, preserved seaweed, preserved meat, tobacco, pharmaceuticals, and many others. Essentially, it can have unlimited uses. Conventional methods such as mixing, kneading, dissolving, soaking, permeating, infusing, sprinkling, spraying, injecting, and other methods may be used in the manufacture of products such as food, beverages, pharmaceuticals, cosmetics, tableware, and chewing gum.

[0121] Furthermore, the highly purified target steviol glycoside(s), steviolmonoside A, steviolbioside, steviolbioside A, steviolbioside B, rubusoside, stevioside, stevioside A (rebaudioside KA), stevioside B, stevioside C, rebaudioside E, rebaudioside E2, rebaudioside E3, and / or rebaudioside AM obtained by the present invention may be used in dry or liquid form.

[0122] The highly purified target steviol glycoside can be added before or after the heat treatment of the food product. The amount of the highly purified target steviol glycoside, particularly steviolmonoside A, steviolbioside, steviolbioside A, steviolbioside B, rubusoside, stevioside, stevioside A (rebaudioside KA), stevioside B, stevioside C, rebaudioside E, rebaudioside E2, rebaudioside E3, and / or rebaudioside AM, depends on the intended use. As noted above, it can be added alone or in combination with other compounds.

[0123] The present invention is also directed to sweetness enhancement in beverages using steviolmonoside A, steviolbioside, steviolbioside A, steviolbioside B, rubusoside, stevioside, stevioside A (rebaudioside KA), stevioside B, stevioside C, rebaudioside E, rebaudioside E2, rebaudioside E3, and / or rebaudioside AM. Accordingly, the present invention provides a beverage comprising a sweetener and steviolmonoside A, steviolbioside, steviolbioside A, steviolbioside B, rubusoside, stevioside, stevioside A (rebaudioside KA), stevioside B, stevioside C, rebaudioside E, rebaudioside E2, rebaudioside E3, and / or rebaudioside AM as a sweetness enhancer. wherein steviolmonoside A, steviolbioside, steviolbioside A, steviolbioside B, rubusoside, stevioside, stevioside A (rebaudioside KA), stevioside B, stevioside C, rebaudioside E, rebaudioside E2, rebaudioside E3, and / or rebaudioside AM are present at concentrations below their respective sweetness perception thresholds.

[0124] 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."

[0125] 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 the human palate, typically about 1.0% sucrose equivalent (1.0% SE). Generally, a sweetness enhancer may enhance or intensify the sweetness of a sweetener without providing any noticeable sweetness itself when present at or below the sweetness perception threshold concentration of a given sweetness enhancer; however, a sweetness enhancer may provide a sweetness itself at concentrations above their sweetness perception threshold concentration. The sweetness perception threshold concentration is specific to a particular enhancer and may vary depending 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 providing about 1.0% sucrose equivalent is considered to be the sweetness perception threshold.

[0126] In some embodiments, the sweetener is present in the beverage in an amount of from about 0.5% to about 12% by weight, 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%, about 5.0%, about 5.5%, about 6.0%, about 6.5%, about 7.0%, about 7.5%, about 8.0%, about 8.5%, about 9.0%, about 9.5%, about 10.0%, about 10.5%, about 11.0%, about 11.5%, or about 12.0% by weight.

[0127] In certain embodiments, the sweetener 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, about 3% to about 7% by weight, or about 4% to about 6% by weight. In certain embodiments, the sweetener is present in the beverage in an amount of about 0.5% to about 8% by weight. In certain embodiments, the sweetener is present in the beverage in an amount of about 2% to about 8% by weight.

[0128] In one embodiment, the sweetener is a conventional calorie sweetener. Suitable sweeteners include, but are not limited to, sucrose, fructose, glucose, high fructose corn syrup, and high fructose starch syrup. In another embodiment, the sweetener is erythritol.

[0129] 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. It is contemplated that the sweetener may be used alone or in combination with other sweeteners. In one embodiment, the rare sugar is D-allose. In a more particular embodiment, the D-allose is present in the beverage in an amount of from about 0.5% to about 10% by weight, such as from about 2% to about 8% by weight.

[0130] In another embodiment, the rare sugar is D-psicose. In a more particular embodiment, the D-psicose is present in the beverage in an amount of from about 0.5% to about 10% by weight, such as from about 2% to about 8% by weight. In yet another embodiment, the rare sugar is D-ribose. In a more particular embodiment, the D-ribose is present in the beverage in an amount of from about 0.5% to about 10% by weight, such as from about 2% to about 8% by weight. In yet another embodiment, the rare sugar is D-tagatose. In a more particular embodiment, D-tagatose is present in the beverage in an amount of about 0.5% to about 10% by weight, such as about 2% to about 8% by weight.

[0131] In further embodiments, the rare sugar is L-glucose. In more particular embodiments, the L-glucose is present in the beverage in an amount of from about 0.5% to about 10% by weight, such as from about 2% to about 8% by weight. In one embodiment, the rare sugar is L-fucose. In a more particular embodiment, the L-fucose is present in the beverage in an amount of from about 0.5% to about 10% by weight, such as from about 2% to about 8% by weight. In another embodiment, the rare sugar is L-arabinose. In a more particular embodiment, the L-arabinose is present in the beverage in an amount of from about 0.5% to about 10% by weight, such as from about 2% to about 8% by weight.

[0132] In yet another embodiment, the rare sugar is D-turanose. In a more particular embodiment, the D-turanose is present in the beverage in an amount of from about 0.5% to about 10% by weight, such as from about 2% to about 8% by weight. In yet another embodiment, the rare sugar is D-leucrose. In a more particular embodiment, the D-leucrose is present in the beverage in an amount of from about 0.5% to about 10% by weight, such as from about 2% to about 8% by weight. The addition of a sweetness enhancer at a concentration below the 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 any sweetness enhancer. Further, the sweetness may 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.

[0133] Accordingly, the present invention also provides a beverage comprising a sweetener, and a sweetener enhancer selected from steviolmonoside A, steviolbioside, steviolbioside A, steviolbioside B, rubusoside, stevioside, stevioside A (rebaudioside KA), stevioside B, stevioside C, rebaudioside E, rebaudioside E2, rebaudioside E3 and / or rebaudioside AM, or combinations thereof. Also provided is a method for enhancing the sweetness of a beverage containing a sweetener, comprising adding steviolmonoside A, steviolbioside, steviolbioside A, steviolbioside B, rubusoside, stevioside, stevioside A (rebaudioside KA), stevioside B, stevioside C, rebaudioside E, rebaudioside E2, rebaudioside E3, and / or rebaudioside AM to a beverage containing a sweetener, wherein the beverage is present at a concentration below the sweetness perception threshold.

[0134] The addition of steviolmonoside A, steviolbioside, steviolbioside A, steviolbioside B, rubusoside, stevioside, stevioside A (rebaudioside KA), stevioside B, stevioside C, rebaudioside E, rebaudioside E2, rebaudioside E3, and / or rebaudioside AM to a sweetened beverage at concentrations below the sweetness perception threshold may increase the detected sucrose equivalent 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%.

[0135] The following examples illustrate preferred embodiments of the present invention for the preparation of highly purified target steviol glycoside(s), particularly steviolmonoside A, steviolbioside, steviolbioside A, steviolbioside B, rubusoside, stevioside, stevioside A (rebaudioside KA), stevioside B, stevioside C, rebaudioside E, rebaudioside E2, rebaudioside E3, and / or rebaudioside AM. It will be understood that the materials, ratios, conditions, and procedures described in the examples are illustrative only and are not intended to limit the invention.

[0136] example (Example 1) Protein sequences of engineered enzymes used in biocatalytic processes SEQ ID NO:1: >SuSy_At, variant PM1-54-2-E05 (modified sucrose synthase; source of WT gene: Arabidopsis thaliana)

number

number

number

[0137] (Example 2) Expression and preparation of 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 (a derivative of pRSF-1b, Novagen). The resulting plasmid was used to transform E. coli BL21(DE3) cells.

[0138] Cells were grown at 37°C in ZYM505 medium (F. William Studier, Protein Expression and Purification 41 (2005) 207-234) supplemented with kanamycin (50 mg / L). Gene expression was induced with IPTG (0.2 mM) during the logarithmic phase and continued at 30°C, 200 rpm for 16-18 hours. Cells were harvested by centrifugation (3220 × g, 20 min, 4 °C) and resuspended in cell lysis buffer (100 mM Tris-HCl pH 7.0; 2 mM MgCl, DNA nuclease 20 U / mL, lysozyme 0.5 mg / mL) to an optical density of 200 (OD at 600 nm (OD )). 600 )). Cells were then disrupted by sonication, and the crude extract was separated from cell debris by centrifugation (18,000 × g, 40 min, 4 °C). The supernatant was filter-sterilized through a 0.2 μm filter and diluted 50:50 with distilled water to obtain the enzyme activity preparation.

[0139] For the enzymatic activity preparation of SuSy_At, unit activity is defined as follows: 1 mU of SuSy_At converts 1 nmol of sucrose to fructose in 1 min. The reaction conditions for the assay were 30°C, 50 mM potassium phosphate buffer pH 7.0, 400 mM sucrose at t0, 3 mM MgCl2, and 15 mM uridine diphosphate (UDP).

[0140] (Example 3) Expression and preparation 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 (a derivative of pRSF-1b, Novagen). The resulting plasmid was used to transform E. coli BL21(DE3) cells. Cells were grown at 37°C in ZYM505 medium (F. William Studier, Protein Expression and Purification 41 (2005) 207-234) supplemented with kanamycin (50 mg / L). Gene expression was induced with IPTG (0.1 mM) during the logarithmic phase and continued at 30°C, 200 rpm for 16-18 hours.

[0141] Cells were harvested by centrifugation (3220 × g, 20 min, 4 °C) and resuspended in cell lysis buffer (100 mM Tris-HCl pH 7.0; 2 mM MgCl, DNA nuclease 20 U / mL, lysozyme 0.5 mg / mL) to an optical density of 200 (OD at 600 nm (OD )). 600 )). Cells were then disrupted by sonication, and the crude extract was separated from cell debris by centrifugation (18,000 × g, 40 min, 4 °C). The supernatant was filter-sterilized through a 0.2 μm filter and diluted 50:50 with 1 M sucrose solution to obtain the enzyme activity preparation.

[0142] For the enzymatic activity preparation of UGTSl2, unit activity is defined as follows: 1 mU of UGTSl2 converts 1 nmol of rebaudioside A (Reb A) to rebaudioside D (Reb D) in 1 minute. The reaction conditions for the assay were 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.

[0143] (Example 4) Expression and Preparation 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 (a derivative of pRSF-1b, Novagen). The resulting plasmid was used to transform E. coli BL21(DE3) cells. Cells were grown at 37°C in ZYM505 medium (F. William Studier, Protein Expression and Purification 41 (2005) 207-234) supplemented with kanamycin (50 mg / L). Gene expression was induced with IPTG (0.1 mM) during the logarithmic phase and continued at 30°C, 200 rpm for 16-18 hours.

[0144] Cells were harvested by centrifugation (3220 × g, 20 min, 4 °C) and resuspended in cell lysis buffer (100 mM Tris-HCl pH 7.0; 2 mM MgCl, DNA nuclease 20 U / mL, lysozyme 0.5 mg / mL) to an optical density of 200 (OD at 600 nm (OD )). 600 )). Cells were then disrupted by sonication, and the crude extract was separated from cell debris by centrifugation (18,000 × g, 40 min, 4 °C). The supernatant was filter-sterilized through a 0.2 μm filter and diluted 50:50 with 1 M sucrose solution to obtain the enzyme activity preparation. For the enzymatic activity preparation of UGT76G1, unit activity is defined as follows: 1 mU of UGT76G1 converts 1 nmol of rebaudioside D (RebD) to rebaudioside M (RebM) in 1 minute. The assay reaction conditions were 30°C, 50 mM potassium phosphate buffer pH 7.0, 10 mM Reb A at t0, 500 mM sucrose, 3 mM MgCl, 0.25 mM uridine diphosphate (UDP), and 3 U / mL SuSy_At.

[0145] (Example 5) Synthesis of Rebaudioside AM from Stevioside in a One-Pot Reaction by Simultaneous Addition of UGTsl2, SuSy_At, and UGT76G1 Rebaudioside AM (reb Am) was synthesized directly from stevioside in a one-pot reaction ( FIG. 3 ) using three enzymes: UGTSI2 (variant of SEQ ID NO: 2), SuSy_At (variant of SEQ ID NO: 1), and UGT76G1 (variant of SEQ ID NO: 3) (see Examples 1, 2, 3, and 4). The final reaction solution contained 105 U / L UGTSI2, 405 U / L SuSy_At, 3 U / L UGT76G1, 5 mM stevioside, 0.25 mM uridine diphosphate (UDP), 1 M sucrose, 4 mM MgCl, and potassium phosphate buffer (pH 6.6). First, 207 mL of distilled water was mixed with 0.24 g of MgCl₂·6H₂O, 103 g of sucrose, 9.9 mL of 1.5 M potassium phosphate buffer (pH 6.6), and 15 g of stevioside. After dissolving the components, the temperature was adjusted to 45°C, and UGTSI₂, SuSy_At, UGT76G1, and 39 mg of UDP were added. The reaction mixture was incubated on a shaker at 45°C for 24 hours. An additional 39 mg of UDP was added at 8 and 18 hours. At several time points, the contents of reb AM, reb E, stevioside, reb M, reb B, steviolbioside, and reb I were analyzed by HPLC.

[0146] For analysis, the reaction mixture was adjusted to pH 5.5 using 17% H3PO4 and then inactivated by boiling for 10 minutes. The resulting sample was filtered, and the filtrate was diluted 10-fold and used as the sample for HPLC analysis. The HPLC assay was performed on an Agilent HP 1200 HPLC system consisting of a pump, column thermostat, autosampler, UV detector with background correction, and data acquisition system. Analytes were separated using an Agilent Poroshell 120 SB-C18, 4.6 mm x 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.

[0147] 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. The total run time was 45 min. The column temperature was maintained at 40 °C. The injection volume was 5 μL. Rebaudioside species were detected by UV at 210 nm.

[0148] Table 3 shows the conversion of stevioside to the identified rebaudioside species (area percentage) for each time point. Chromatograms of stevioside and the reaction mixture after 24 hours are shown in Figures 5 and 6, respectively. One skilled in the art will appreciate that retention times may vary from time to time due to solvent and / or instrument changes. [Table 4]

[0149] (Example 6) Synthesis of Rebaudioside AM from Rebaudioside E in a Simultaneous One-Pot Reaction of SuSy_At and UGT76G1 Rebaudioside AM (reb AM) was synthesized directly from rebaudioside E (reb E) in a one-pot reaction (Figure 4) using two enzymes: SuSy_At (variant of SEQ ID NO: 1) and UGT76G1 (variant of SEQ ID NO: 3) (see Examples 1, 2, and 4). The final reaction solution contained 405 U / L SuSy_At, 3 U / L UGT76G1, 5 mM reb E, 0.25 mM uridine diphosphate (UDP), 1 M sucrose, 4 mM MgCl 6H O, and potassium phosphate buffer (pH 6.6). First, 37 mL of distilled water was mixed with 40.3 mg of MgCl , 17.12 g of sucrose, 1.65 mL of 1.5 M potassium phosphate buffer (pH 6.6), and 5.04 g of reb E. After dissolving the components, the temperature was adjusted to 45°C, and SuSy_At, UGT76G1, and 6.5 mg of UDP were added. The reaction mixture was incubated on a shaker at 45°C for 24 hours. An additional 6.5 mg of UDP was added at 8 and 18 hours. At several time points, the contents of reb AM, reb E, stevioside, reb A, reb M, reb B, and steviolbioside were analyzed by HPLC.

[0150] For analysis, the reaction mixture was adjusted to pH 5.5 using 17% H3PO4 and then inactivated by boiling for 10 minutes. The resulting sample was filtered, and the filtrate was diluted 10-fold and used as the sample for HPLC analysis. The HPLC assay was performed on an Agilent HP 1200 HPLC system consisting of a pump, column thermostat, autosampler, UV detector with background correction, and data acquisition system. Analytes were separated using an Agilent Poroshell 120 SB-C18, 4.6 mm x 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.

[0151] 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. The total run time was 45 min. The column temperature was maintained at 40 °C. The injection volume was 5 μL. Rebaudioside species were detected by UV at 210 nm.

[0152] Table 4 shows the conversion of reb E to the identified rebaudioside species (area percentage) for each time point. Chromatograms of reb E and the reaction mixture after 24 hours are shown in Figures 7 and 8, respectively. One skilled in the art will understand that retention times may vary over time due to solvent and / or instrument changes. [Table 5]

[0153] (Example 7) Purification of Rebaudioside AM After 24 hours, the reaction mixture of Example 5 was inactivated by adjusting the pH to 5.5 with H3PO4 and then boiled for 10 minutes. After boiling, the reaction mixture was filtered and diluted to 5% solids with RO water. The diluted solution was passed through a 1 L column packed with YWD03 macroporous adsorption resin (Cangzhou Yuanwei, China). The adsorbed steviol glycosides were eluted with 5 L of 70% ethanol. The resulting eluate was evaporated to dryness to obtain 16 g of dry powder, which was dissolved in 80 mL of 70% methanol. The solution was allowed to crystallize at 20 °C for 3 days. The crystals were isolated by filtration and dried in a vacuum oven at 80 °C for 18 hours to yield 10.4 g of pure reb AM crystals with a purity of 95.92% as determined by HPLC assay. A chromatogram of reb AM is shown in Figure 9. Those skilled in the art will appreciate that retention times may occasionally vary due to changes in solvent and / or equipment.

[0154] (Example 8) Structural elucidation of rebaudioside AM NMR experiments were performed on a Bruker 500 MHz spectrometer with samples dissolved in pyridine-d5. In addition to the signal from the sample, δ C 123.5, 135.5, 149.9 ppm and δ H Signals from pyridine-d5 were observed at 7.19, 7.55, and 8.71 ppm.

[0155] of Rebaudioside AM in Pyridine-d5 1 The H-NMR spectrum reveals the excellent quality of the sample (see Figure 10). HSQC (see Figure 11) shows the presence of an exomethylene group in the sugar region, with long-range coupling to C-15 observable in H,H-COSY (Figure 12). Other deep-field signals of quaternary carbons (C-13, C-16, and C-19) are detected by HMBC (Figure 13). Correlation of signals in HSQC, HMBC, and H,H-COSY reveals the presence of steviol glycosides with the following aglycone structure: [ka]

[0156] Correlation of the HSQC and HMBC signals reveals five anomeric signals. The coupling constants of the anomeric protons of approximately 8 Hz and the broad signals of their sugar bonds allow the identification of these five sugars as β-D-glucopyranosides. Observation of the anomeric protons combined with HSQC and HMBC reveals correlations between the sugar linkage and the aglycone. The sugar sequence assignments were confirmed using a combination of HSQC-TOCSY (Figure 14) and HSQC.

[0157] The above NMR experiments were applied to assign proton and carbon chemical shifts, major coupling constants, and major HMBC correlations (see Table 5). [Table 6-1] [Table 6-2] [Table 6-3]

[0158] Correlation of all NMR data points to rebaudioside AM with five β-D-glucopyranose units attached to the steviol aglycone, as shown in the following chemical structure: [ka]

[0159] The chemical formula of Rebaudioside AM is C 50 H 80 O 28 , which corresponds to a calculated monoisotopic molecular weight of 1128.5. For LCMS analysis, rebaudioside AM was dissolved in methanol and analyzed using a Shimadzu Nexera 2020 UFLC LCMS instrument on a Cortecs UPLC C18 1.6 μm, 50 × 2.1 mm column. The observed LCMS (negative ESI mode) results of 1127.3 (see Figures 15a and 15b, respectively) are consistent with rebaudioside AM, and the ion (M−H) was identified. - It corresponds to.

Claims

1. The following formula: 【Chemical 1】 1. A method for producing rebaudioside AM having the formula: a. providing a starting composition comprising rebaudioside E; b. providing a biocatalyst selected from the group consisting of an enzyme preparation, a cell, or a microorganism, said biocatalyst comprising at least one enzyme that is a UDP glycosyltransferase (UGT) of SEQ ID NO: 3 or has a sequence at least 96% identical thereto, and that is capable of converting said starting composition to rebaudioside AM; c. contacting the biocatalyst with a medium containing the starting composition to produce a medium containing rebaudioside AM; d. Separating the rebaudioside AM from the culture medium to obtain a highly purified rebaudioside AM composition. wherein the rebaudioside AM content in the highly purified rebaudioside AM composition is greater than 80% by weight, preferably greater than 90% by weight, and most preferably greater than 95% by weight on a dry weight basis.

2. 2. The method of claim 1, wherein the microorganism is selected from the group consisting of E. coli, Saccharomyces sp., Aspergillus sp., Pichia sp., Bacillus sp., and Yarrowia sp.

3. At least one enzyme is a steviol biosynthetic enzyme, a UDP glucosyltransferase, a UDP glucose recycling enzyme, a mevalonate (MVA) pathway enzyme, a 2-C-methyl-D-erythritol-4-phosphate pathway (MEP / DOXP) enzyme, a geranylgeranyl diphosphate synthase, a copalyl diphosphate synthase, a kaurene synthase, a kaurene oxidase, a kaurenoic acid 13-hydroxylase (KAH), a steviol synthetase, a deoxyxylulose 5-phosphate synthase (DXS), a D-1-deoxyxylulose 5-phosphate reductoisomerase (DXR), a 4-diphosphocytidyl-2-C-methyl-D-erythritol synthase (CMS), a 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase (CMK), a 4-diphosphocytidyl-2-C-methyl-D-erythritol 10. The method of claim 1, further selected from the group consisting of ritol 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, UGT85C2, UGT91D2, EUGT11, UGTS12, or a mutant of SEQ ID NO: 2 having greater than 95% amino acid sequence identity, greater than 96% amino acid sequence identity, greater than 97% amino acid sequence identity, greater than 98% amino acid sequence identity, or greater than 99% amino acid sequence identity; and combinations thereof.

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