High purity steviol glycosides

Abstract

The present application relates to high-purity steviol glycosides. The present application describes methods for preparing highly purified steviol glycosides, particularly rebaudioside A, D and M. These methods include utilizing recombinant microorganisms that convert different starting compositions into target steviol glycosides. In addition, novel steviol glycosides reb D2 and reb M2 are disclosed, as are methods for preparing them. Highly purified rebaudioside can be used as a non-caloric sweetener in edible and chewable compositions such as any beverage, cake, baked product, biscuit and chewing gum.

Description

[0001] This application is a divisional application of an application filed on May 28, 2014, with application number 201480040130.6 and invention name “High-purity steviol glycosides”.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims U.S. Provisional Patent Application No. 61 / 827,922 filed May 28, 2013; U.S. Provisional Patent Application No. 61 / 843,544 filed July 8, 2013; U.S. Provisional Patent Application No. 61 / 861,528 filed August 2, 2013; U.S. Provisional Patent Application No. 61 / 881,166 filed September 23, 2013; U.S. Provisional Patent Application No. 61 / 885,084 filed October 1, 2013; The present invention claims priority to U.S. Provisional Patent Application No. 61 / 904,751 filed on November 15, 2013; U.S. Provisional Patent Application No. 61 / 913,482 filed on December 9, 2013; U.S. Provisional Patent Application No. 61 / 921,635 filed on December 30, 2013; U.S. Provisional Patent Application No. 61 / 925,329 filed on January 9, 2014; and U.S. Provisional Patent Application No. 61 / 939,855 filed on February 14, 2014. The priority documents cited above are fully incorporated herein in their entirety. Technical Field

[0004] The present invention relates to a biocatalytic method for preparing a composition containing steviol glycosides, wherein the composition includes a highly purified steviol glycoside composition. The present invention also relates to novel steviol glycosides, a method for isolating the steviol glycosides, and the use of these novel steviol glycosides. Background of the Invention

[0005] High intensity sweeteners have a sweetness level that is many times higher than the sweetness level of sucrose. They are essentially caloric free and are commonly used in diet and low calorie products, including food and beverages. High intensity sweeteners do not trigger a glycemic response, making them suitable for use in products for diabetics and other populations interested in controlling their carbohydrate intake.

[0006] Steviol glycosides are a class of compounds found in the leaves of Stevia rebaudiana Bertoni, a perennial shrub of the Asteraceae (Compositae) family native to certain areas of South America. Their structure is characterized by a single base, steviol, differing by the presence of carbohydrate residues at positions C13 and C19. They accumulate in the leaves of Stevia, constituting approximately 10%-20% of the total dry weight. 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%), based on dry weight. Other known steviol glycosides include rebaudioside B, C, D, E, F, and M, rebaudioside, and rubusoside.

[0007] Although methods for preparing steviol glycosides from Stevia are known, most of these methods are not suitable for commercial use.

[0008] Therefore, there remains a need for simple, efficient and economical methods for preparing compositions comprising steviol glycosides, including highly purified steviol glycoside compositions.

[0009] Additionally, there remains a need for novel steviol glycosides and methods for their preparation and isolation. SUMMARY OF THE INVENTION

[0010] The present invention provides a biocatalytic method for preparing a composition comprising a target steviol glycoside, the biocatalytic method comprising contacting a medium containing a starting composition with a biocatalyst, the starting composition comprising an organic compound containing at least one carbon atom, thereby producing a composition comprising a target steviol glycoside.

[0011] In another embodiment, the present invention provides a method for producing a target steviol glycoside, the method comprising contacting an organic compound with at least one enzyme selected from a steviol biosynthetic enzyme and a UDP-glycosyltransferase, thereby producing a composition comprising a target steviol glycoside.

[0012] The organic compound comprising at least one carbon atom is a substrate for bioconversion. In one embodiment, the organic compound is selected from the group consisting of a polyol or a sugar alcohol or various carbohydrates. In another embodiment, the organic compound is at least one steviol glycoside.

[0013] The target steviol glycoside can be any steviol glycoside that is not the same as the substrate steviol glycoside. In one embodiment, the target steviol glycoside is stevioside, steviol diglycoside, sweet leaf rubusoside, dulcoside B, dulcoside A, rebaudioside B, rebaudioside G, stevioside, rebaudioside C, rebaudioside F, rebaudioside A, rebaudioside I, rebaudioside E, rebaudioside H, rebaudioside L, rebaudioside K, rebaudioside J, rebaudioside M, rebaudioside M2, rebaudioside D, rebaudioside D2, rebaudioside N, rebaudioside O or a synthetic steviol glycoside.

[0014] In one embodiment, the target steviol glycoside is stevioside.

[0015] In another embodiment, the target steviol glycoside is rebaudioside A.

[0016] In yet another embodiment, the target steviol glycoside is rebaudioside D.

[0017] In yet another embodiment, the target steviol glycoside is Rebaudioside M (also known as Rebaudioside X).

[0018] In a preferred embodiment, the biocatalyst is an enzyme or a cell comprising one or more enzymes that can convert an organic compound into a target steviol glycoside. The biocatalyst can be located on the surface and / or inside the cell. The biocatalyst can be provided in the form of a whole cell suspension, a crude lysate, or one or more purified enzymes. The biocatalyst can be in free form or fixed to a solid support made of an inorganic or organic material.

[0019] In some embodiments, the microorganism comprises one or more necessary biocatalysts for converting the organic compound into the target steviol glycoside. Therefore, the present invention also provides a biocatalytic method for preparing a composition comprising a target steviol glycoside by contacting a starting composition comprising an organic compound with a microorganism comprising at least one enzyme capable of converting the organic compound into a target steviol glycoside, thereby producing a medium comprising at least one target steviol glycoside.

[0020] The enzymes necessary for converting the organic compound to the target steviol glycosides include steviol biosynthetic enzymes, UDP-glycosyltransferases (UGTs) and / or UDP-cycling enzymes.

[0021] In one embodiment, the steviol biosynthetic enzyme is selected from the group consisting of geranylgeranyl diphosphate synthase, copalyl diphosphate synthase, kaurene synthase, kaurene oxidase, kaurenoic acid 13-hydroxylase (KAH), steviol synthase, deoxyxylulose 5-phosphate synthase (DXS), D-1-deoxyxylulose 5-phosphate reductoisomerase (DXR), 4-diphosphocytidine-2-C-methyl-D-erythritol synthase (CMS), 4-diphosphocytidine-2-C-methyl-D -erythritol kinase (CMK), 4-diphosphocytidine-2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (MCS), l-hydroxy-2-methyl-2(E)-butenyl 4-diphosphate synthase (HDS), l-hydroxy-2-methyl-2(E)-butenyl 4-diphosphate reductase (HDR), acetoacetyl-CoA thiolase, truncated HMG-CoA reductase, mevalonate kinase, phosphomevalonate kinase, mevalonate pyrophosphate decarboxylase and cytochrome P450 reductase.

[0022] The UDP-glucosyltransferase can be any UDP-glucosyltransferase that is capable of adding at least one glucose unit to a steviol and / or steviol glycoside substrate to provide a target steviol glycoside.

[0023] In one embodiment, the steviol biosynthetic enzyme and UDP-glucosyltransferase are produced in a microorganism. The microorganism can be, for example, Escherichia coli, Saccharomyces species, Aspergillus species, Pichia species, Bacillus species, Yarrowia species, etc. In another embodiment, UDP-glucosyltransferase is synthesized.

[0024] In one embodiment, the UDP-glucosyltransferase is selected from the group consisting of UGT74G1, UGT85C2, UGT76G1, UGT91D2, UGTSL, UGTSL_Sc, UGTSL2 (GI No. 460410132 Version XP_004250485.1), UGTLB, GI No. 460409128 (UGTSL) Version XP_004249992.1, GI No. 460410132 (UGTSL) Version XP_004249992.1, No. 115454819 version NP_001051010.1, GI No. 187373030, version ACD03249.1, GI No. 222619587 version EEE55719.1, GI No. 297795735 version XP_002865752.1 or EUGT11, and UGTs having substantial (>85%) identity to these polypeptides, as well as UGTs having isolated nucleic acid molecules encoding these UGTs.

[0025] In one embodiment, the steviol biosynthetic enzyme, UGT and UDP-glucose cycle system are present in a microorganism. The microorganism can be, for example, Escherichia coli, Saccharomyces species, Aspergillus species, Pichia species, Bacillus species, Yarrowia species.

[0026] In one embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rubusoside to form stevioside. In a specific embodiment, the UDP-glucosyltransferase is UGT91D2.

[0027] In one embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to stevioside to form rebaudioside A. In a specific embodiment, the UDP-glucosyltransferase is UGT76G1.

[0028] In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside A to form rebaudioside D. In a specific embodiment, the UDP-glucosyltransferase is UGT91D2. In another embodiment, the UGT is an improved variant of UGT91D2 with higher activity and / or selectivity generated by directed evolution.

[0029] In yet another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside D to form rebaudioside M. In a specific embodiment, the UDP-glucosyltransferase is UGT76G1. In another embodiment, the UGT is an improved variant of UGT76G1 produced by directed evolution with higher activity and / or selectivity.

[0030] Optionally, the method of the present invention also includes recycling UDP to provide UDP-glucose. In one embodiment, the method includes recycling UDP by providing a recycling catalyst and a recycling substrate, thereby a catalytic amount of UDP-glucosyltransferase and UDP-glucose can be used to carry out the bioconversion of steviol glycoside substrate to target steviol glycoside.

[0031] In one embodiment, the recycling catalyst is sucrose synthase.

[0032] In one embodiment, the recycled substrate is sucrose.

[0033] Optionally, the method of the present invention also includes separating the target steviol glycoside from the medium to provide a highly purified target steviol glycoside composition. The target steviol glycoside can be separated by at least one appropriate method such as crystallization, separation by membrane, centrifugation, extraction, chromatography or a combination of such methods.

[0034] In one embodiment, the target steviol glycoside can be produced in a microorganism. In another embodiment, the target steviol glycoside can be secreted into a medium. In another embodiment, the released steviol glycoside can be continuously removed from the medium. In another embodiment, the target steviol glycoside is separated after the reaction is completed.

[0035] In one embodiment, separation produces a composition comprising more than about 80% of the target steviol glycoside by dry weight, i.e., a highly purified steviol glycoside composition. In another embodiment, separation produces a composition comprising more than about 90% of the target steviol glycoside by weight. In a specific embodiment, the composition comprises more than about 95% of the target steviol glycoside by weight. In other embodiments, the composition comprises more than about 99% of the target steviol glycoside by weight.

[0036] The target steviol glycoside may be in any polymorphic form or amorphous form, including hydrates, solvates, anhydrous forms, or combinations thereof.

[0037] The purified target steviol glycosides can be used as sweeteners in consumable products. Suitable consumable products include, but are not limited to, foods, beverages, pharmaceutical compositions, tobacco products, nutraceutical compositions, oral hygiene compositions, and cosmetic compositions.

[0038] The present invention also provides steviol glycosides rebaudioside D2 (reb D2, isomer of rebaudioside D) and rebaudioside M2 ​​(reb M2, isomer of rebaudioside M). In one embodiment, isolated and purified reb D2 is provided. In another embodiment, isolated and purified reb M2 is provided. Reb D2 and reb M2 can also be presented in the form of any consumable product disclosed herein. In a specific embodiment, a beverage comprising reb D2 and / or reb M2 is provided.

[0039] Also provided herein are methods for preparing reb D2 and reb M2. Both are formed during the bioconversion process from reb A to reb D. Reb M2 is believed to be formed from reb D2 in situ bioconversion.

[0040] In one embodiment, the present invention is a method for preparing a composition comprising reb D2, the method comprising: (a) contacting a starting composition comprising reb A with an enzyme capable of converting reb A into reb D2, UDP-glucose, and optionally a UDP-glucose cycle enzyme to produce a composition comprising reb D2, and (b) isolating the composition comprising reb D2.

[0041] In another embodiment, the present invention is a method for preparing a composition comprising reb M2, the method comprising (a) contacting a starting composition comprising reb D2 with an enzyme capable of converting reb D2 into reb M2, UDP-glucose, and optionally a UDP-glucose cycle enzyme to produce a composition comprising reb M2, and (b) isolating the composition comprising reb M2.

[0042] Another method for preparing a composition comprising reb M2 comprises (a) contacting a starting composition comprising reb A with an enzyme capable of converting reb A into reb D2, UDP-glucose, and optionally a UDP-glucose cycle enzyme to produce a composition comprising reb D2, (b) optionally, isolating the composition comprising reb D2, (c) contacting the composition comprising reb D2 with an enzyme capable of converting reb D2 into reb M2, UDP-glucose, and optionally a UDP-glucose cycle enzyme to produce a composition comprising reb M2, and (d) isolating the composition comprising reb M2.

[0043] The composition may be further purified to provide reb D2 or reb M2 having a purity greater than about 95% on a dry weight basis.

[0044] This application provides the following:

[0045] 1). A method for producing a target steviol glycoside, the method comprising the following steps:

[0046] a. providing a starting composition comprising an organic compound having at least one carbon atom;

[0047] b. providing a microorganism containing at least one enzyme selected from steviol biosynthetic enzyme, UDP-glycosyltransferase and optionally providing UDP-glucose cycle enzyme;

[0048] c. contacting the microorganism with a medium containing the starting composition to produce a medium comprising at least one target steviol glycoside.

[0049] 2). A method for producing a target steviol glycoside, the method comprising the following steps:

[0050] a. providing a starting composition comprising an organic compound having at least one carbon atom;

[0051] b. providing a biocatalyst comprising at least one enzyme selected from steviol biosynthetic enzymes, UDP-glycosyltransferases, and optionally UDP-glucose cycle enzymes;

[0052] c. contacting the biocatalyst with a medium containing the starting composition to produce a medium comprising at least one target steviol glycoside.

[0053] 3). The method described in 1) or 2) further comprises the following steps:

[0054] d. purifying the target steviol glycoside from the medium to provide a highly purified target steviol glycoside composition.

[0055] 4). The method as described in 1) or 2), wherein the organic compound is selected from the group consisting of polyols, carbohydrates, steviol glycosides and combinations thereof.

[0056] 5). The method as described in 1) or 2), wherein the substance is glucose and the method is a fermentation method.

[0057] 6). The method according to 1) or 2), wherein the starting composition comprises steviol glycosides.

[0058] 7). The method as described in 1), wherein the microorganism is selected from the group consisting of Escherichia coli, Saccharomyces spp., Aspergillus spp., Pichia spp., Bacillus spp., and Yarrowia spp.

[0059] 8). The method as described in 2), wherein the biocatalyst is an enzyme, or a cell comprising one or more enzymes, which can convert the organic compound into target steviol glycoside.

[0060] 9). The method of 1) or 2), wherein the target steviol glycoside is selected from the group consisting of stevioside, reb A, reb D, reb D2, reb E, reb M, reb M2 and mixtures thereof.

[0061] 10). The method of 1) or 2), wherein the enzyme is selected from the group consisting of: geranylgeranyl diphosphate synthase, copalyl diphosphate synthase, kaurene synthase, kaurene oxidase, kaurenoic acid 13-hydroxylase (KAH), steviol synthase, deoxyxylulose 5-phosphate synthase (DXS), D-1-deoxyxylulose 5-phosphate reductoisomerase (DXR), 4-diphosphocytidine-2-C-methyl-D-erythritol synthase (CMS), 4-diphosphocytidine-2-C-methyl-D-erythritol kinase (CMK), 4-diphosphocytidine-2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (MCS), 1-hydroxy-2-methyl-2(E)-butenyl 4-diphosphate Phosphate 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, UGT91D2, UGTSL2, UGT76G1, or UGT76G1 containing one or more point mutations selected from S42A, F46I, I190L, S274G, I295M, K303G, F314S, K316R, K393R, V394I, I407V, N409K, N409R, Q425E, Q432E, S447A and S456L.

[0062] 11). The method of 3), wherein the highly purified target steviol glycoside composition has a target steviol glycoside purity of more than about 95% by dry weight.

[0063] 12). The method of 3), wherein the highly purified target steviol glycoside composition has a target steviol glycoside purity of more than about 99% by dry weight.

[0064] 13). The method of 11), wherein the target steviol glycoside is selected from stevioside, reb A, reb E, reb D, reb D2, reb M, and reb M2.

[0065] 14). The method according to 11), wherein the target steviol glycoside is reb M.

[0066] 15). A highly purified target steviol glycoside composition prepared according to the method described in 3), wherein the target steviol glycoside is polymorphic.

[0067] 16). The highly purified target steviol glycoside composition as described in 15), wherein the target steviol glycoside is reb D or reb M.

[0068] 17). A consumable product comprising a highly purified target glycoside composition produced by the method described in 3), wherein the product is selected from the group consisting of: food, beverage, pharmaceutical composition, tobacco product, nutritional product composition, oral hygiene composition, and cosmetic composition.

[0069] 18). A consumable product comprising a highly purified target steviol glycoside composition produced by the method described in 3), wherein the product is selected from the group consisting of food, beverages, pharmaceutical compositions, tobacco products, nutritional product compositions, oral hygiene compositions, and cosmetic compositions, and wherein the target steviol glycoside is reb D.

[0070] 19). A consumable product comprising a highly purified target steviol glycoside composition produced by the method described in 3), wherein the product is selected from the group consisting of food, beverages, pharmaceutical compositions, tobacco products, nutritional product compositions, oral hygiene compositions, and cosmetic compositions, and wherein the target steviol glycoside is reb M.

[0071] 20). Isolated and highly purified reb D2 having the following structure:

[0072]

[0073] 21). A consumable product comprising isolated and highly purified reb D2.

[0074] 22) A consumable product as described in 21), wherein the consumable product is selected from the group consisting of: beverages; natural juices; refreshing drinks; carbonated soft drinks; diet drinks; zero-calorie drinks; calorie-reducing drinks and foods; yogurt drinks; instant juices; instant coffee; powdered instant drinks; canned products; syrups; fermented soybean paste; soy sauce; vinegar; condiments; mayonnaise; ketchup; curry foods; soups; instant broth; soy sauce powder; vinegar powder; various types of biscuits; rice crackers; crackers; bread; chocolate; caramel; candy; chewing gum; jelly; pudding; preserved fruits and pickles; fresh cream; jam; orange marmalade; sugar flower paste; milk powder; ice cream; sorbet; vegetables and fruits packaged in bottles; canned boiled beans; meat and foods cooked in sweet sauce; agricultural vegetable foods; seafood; ham; sausages; fish ham; fish sausages; fish sauce; fried fish products; dried seafood; frozen foods; pickled seaweed; pickled meat; tobacco and medicines.

[0075] 23). A beverage comprising isolated and highly purified reb D2.

[0076] 24). A method for preparing reb D2, the method comprising:

[0077] a. contacting the starting composition comprising reb A with an enzyme selected from the group consisting of an enzyme capable of converting reb A into reb D2, UDP-glucose, and optionally a UDP-glucose cycle enzyme to produce a composition comprising reb D2; and

[0078] b. isolating, and optionally purifying, the composition comprising reb D2.

[0079] 25). The method of 24), wherein reb D2 has a purity of more than about 95% by dry weight.

[0080] 26). An isolated and highly purified reb M2 having the following structure:

[0081]

[0082] 27). A consumable product comprising isolated and highly purified reb M2.

[0083] 28) A consumable product as described in 27), wherein the consumable product is selected from the group consisting of: beverages; natural juices; refreshing drinks; carbonated soft drinks; diet drinks; zero-calorie drinks; calorie-reducing drinks and foods; yogurt drinks; instant juices; instant coffee; powdered instant drinks; canned products; syrups; fermented soybean paste; soy sauce; vinegar; condiments; mayonnaise; ketchup; curry foods; soups; instant broth; soy sauce powder; vinegar powder; various types of biscuits; rice crackers; crackers; bread; chocolate; caramel; candy; chewing gum; jelly; pudding; preserved fruits and pickles; fresh cream; jam; orange marmalade; sugar flower paste; milk powder; ice cream; sorbet; vegetables and fruits packaged in bottles; canned boiled beans; meat and foods cooked in sweet sauce; agricultural vegetable foods; seafood; ham; sausages; fish ham; fish sausages; fish sauce; fried fish products; dried seafood; frozen foods; pickled seaweed; pickled meat; tobacco and medicines.

[0084] 29). A beverage comprising isolated and highly purified reb M2.

[0085] 30). A method for preparing reb M2, the method comprising:

[0086] a. contacting the starting composition comprising reb D2 with an enzyme selected from the group consisting of an enzyme capable of converting reb D2 into reb M2, UDP-glucose, and optionally a UDP-glucose cycle enzyme to produce a composition comprising reb M2; and

[0087] b. isolating, and optionally purifying, the composition comprising reb M2.

[0088] 31). The method of 30), wherein reb M2 has a purity of more than about 95% by dry weight.

[0089] 32). The consumable product as described in 21) further comprises at least one additive selected from the group consisting of carbohydrates, polyols, amino acids and their corresponding salts, polyamino acids and their corresponding salts, sugar acids and their corresponding salts, nucleotides, organic acids, inorganic acids, organic salts including organic acid salts and organic base salts, inorganic salts, bitter compounds, flavoring agents and flavoring ingredients, astringent compounds, proteins or protein hydrolysates, surfactants, emulsifiers, flavonoids, alcohols, polymers and combinations thereof.

[0090] 33). The consumable product as described in 21) further comprises at least one functional ingredient selected from the group consisting of saponins, antioxidants, dietary fiber sources, fatty acids, vitamins, glucosamine, minerals, preservatives, hydration agents, probiotics, prebiotics, weight management agents, osteoporosis management agents, phytoestrogens, long-chain saturated fatty alcohols, plant sterols and combinations thereof.

[0091] 34). The consumable product as described in 21) further comprises a compound selected from the group consisting of stevia, stevia extract, stevia glycosides, stevia glycosides, dulcoside A, dulcoside B, rebaudioside B, rebaudioside G, stevioside, rebaudioside C, rebaudioside F, rebaudioside A, rebaudioside I, rebaudioside E, rebaudioside H, rebaudioside L, rebaudioside K, rebaudioside J, rebaudioside M, rebaudioside M2, rebaudioside D, rebaudioside D2, rebaudioside N or rebaudioside O, or other steviol glycosides found in stevia, mogroside, brazin, neohesperidin dihydrochalcone, glycyrrhizic acid and its salts, thaumatin, perilla frutescens, pernandulcin, soapberry sesquiterpene glycosides, white ginseng glycosides, scutellaria glycoside-I, dimethyl-hexahydrofluorene-dicarboxylic acid, abrin, glycyrrhizin, scutellaria glycosides, cyclocarya glycosides, butterfly card glycosides, polyboroside A, brazilin, helan sweet essence, phyllotoxin, smilax glabra glycosides, phloridzin, trilobat glycosides, dihydroflavonol, dihydroquercetin-3-acetate, new astilbin, trans-cinnamaldehyde, monatin and its salts, pteridin A, hematoxylin, lecithin, Polypodium vulgare, terebin A, terebin B, mabinin, pantatin, miracle fruit protein, curculigo protein, curculigo protein, chlorogenic acid, cynarin, monk fruit sweetener, mogroside V, mogroside VI, Momordica glabra saponin, simonoside, NSF-02, NSF-03, NSF-04, psicose, allose, D-tagatose, erythritol and combinations thereof.

[0092] 35). A tabletop sweetener composition comprising isolated and purified reb D2.

[0093] 36). The consumable product as described in 27) further includes at least one additive selected from the group consisting of carbohydrates, polyols, amino acids and their corresponding salts, polyamino acids and their corresponding salts, sugar acids and their corresponding salts, nucleotides, organic acids, inorganic acids, organic salts including organic acid salts and organic base salts, inorganic salts, bitter compounds, flavoring agents and flavoring ingredients, astringent compounds, proteins or protein hydrolysates, surfactants, emulsifiers, flavonoids, alcohols, polymers and combinations thereof.

[0094] 37). The consumable product as described in 27) further comprises at least one functional ingredient selected from the group consisting of saponins, antioxidants, dietary fiber sources, fatty acids, vitamins, glucosamine, minerals, preservatives, hydration agents, probiotics, prebiotics, weight management agents, osteoporosis management agents, phytoestrogens, long-chain saturated fatty alcohols, plant sterols and combinations thereof.

[0095] 38). The consumable product as described in 27), further comprising a compound selected from the group consisting of stevia, stevia extract, stevia glycoside, stevia glycoside, dulcoside A, dulcoside B, rebaudioside B, rebaudioside G, stevioside, rebaudioside C, rebaudioside F, rebaudioside A, rebaudioside I, rebaudioside E, rebaudioside H, rebaudioside L, rebaudioside K, rebaudioside J, rebaudioside M, rebaudioside M2, rebaudioside D, rebaudioside D2, rebaudioside N or rebaudioside O, or other steviol glycosides found in stevia, mogroside, brazin, neohesperidin dihydrochalcone, glycyrrhizic acid and its salts, thaumatin, perilla, pernandulcin, Soapberry sesquiterpene glycosides, Baiyun ginsenoside, scutellaria glycoside-I, dimethyl-hexahydrofluorene-dicarboxylic acid, abrin, Brazilian glycyrrhizin, meat flower snow bile glycoside, cyclocarya paliurus glycoside, butterfly card glycoside, polybodoside A, Brazilian wood, Helan sweet essence, phyllotoxin, smilax glabra glycoside, phloridzin, triloba glycoside, dihydroflavonol, dihydroquercetin-3-acetate, new astilbin, trans-cinnamaldehyde, monatin and its salts, Pterostilbene A, hematoxylin, oleoside, polypodium stevioside, tertoside A, tertoside B, mabinin, pantatin, miracle fruit protein, curculigo sweet protein, broad-leaved curculigo sweet protein, chlorogenic acid, cynarin, monk fruit sweetener, mogroside V, mogroside VI, Momordica chinensis saponin, simonoside, NSF-02, NSF-03, NSF-04Reb A, Reb B, Reb D, NSF-02, mogroside V, monk fruit, psicose, allose, D-tagatose, erythritol and combinations thereof.

[0096] 39). A tabletop sweetener composition comprising isolated and purified reb M2.

[0097] 40). A method for enhancing the sweetness of a beverage containing a sweetener, the method comprising:

[0098] a.) providing a beverage comprising a sweetener; and

[0099] b.) adding a sweetness enhancer selected from reb D2, reb M2 or a combination thereof,

[0100] Wherein reb D2 and / or reb M2 are present at a concentration at or below the sweet taste recognition threshold. BRIEF DESCRIPTION OF THE DRAWINGS

[0101] The accompanying drawings are included to provide a further understanding of the present invention. These drawings illustrate embodiments of the present invention and together with the description serve to explain the principle of embodiments of the present invention.

[0102] Figure 1 Biocatalytic production of reb M from stevioside is shown.

[0103] Figure 2 Biocatalytic production of reb A from stevioside using the enzyme UGT76G1 is shown, along with concomitant recycling of UDP to UDP-glucose via sucrose synthase.

[0104] Figure 3 HPLC chromatogram showing the products of the biocatalytic production of reb M from reb D, as described in detail in Example 14. The peak with a retention time of 24.165 minutes corresponds to unreacted reb D. The peak with a retention time of 31.325 minutes corresponds to reb M.

[0105] Figure 4 Shown is the conversion of stevioside to reb A catalyzed by UGT76G1.

[0106] Figure 5 Shown is the conversion of reb D to reb M catalyzed by UGT76G1.

[0107] Figure 6LC-MS analysis of a semi-synthetic steviol glycoside mixture is shown, lot number CB-2977-106, showing TIC (A), MS (B) at the peak at 1.8 minutes, MS (C) at the reb M2 peak at 4.1 minutes, MS (D) at the reb D peak at 6.0 minutes, MS (E) at the reb D2 peak at 7.7 minutes, MS (F) at the peak at 9.4 minutes, MS (G) at the rebaudioside A peak at 15.2 minutes, MS (H) at the peak at 16.5 minutes, and MS (I) at the peak at 18.3 minutes.

[0108] Figure 7 Shown are HPLC analyses of a semisynthetic steviol glycoside mixture, batch number CB-2977-106 (A), isolated reb M2 (B), isolated reb D (C), and isolated reb D2 (D).

[0109] Figure 8 Display reb D2 1 H NMR spectrum (500 MHz, pyridine-d5).

[0110] Fig. 9 Display reb D2 13 C NMR spectrum (125 MHz, pyridine-d5).

[0111] Fig.10 Display reb D2 13 Extension of C NMR spectrum (125 MHz, pyridine-d5).

[0112] Fig.11 Display reb D2 1 H- 1 H COSY spectrum (500 MHz, pyridine-d5).

[0113] Fig.12 The HSQC-DEPT spectrum of reb D2 is shown (500 MHz, pyridine-d5).

[0114] Fig.13 The HMBC spectrum of reb D2 is shown.

[0115] Fig.14 Shown is the expansion of the HMBC spectrum of reb D2 (500 MHz, pyridine-d5).

[0116] Fig.15 The NOESY spectrum of reb D2 is shown.

[0117] Fig.16 The reb D2 obtained after about 46 hours 1H NMR spectrum (500 MHz, pyridine-d5).

[0118] Fig.17 The reb D2 obtained after about 46 hours 1 Extension of the H NMR spectrum (500 MHz, pyridine-d5).

[0119] Fig.18 Show reb M2 1 H NMR spectrum (500 MHz, D2O).

[0120] Fig.19 Show reb M2 13 C NMR spectrum (125 MHz, D2O / TSP).

[0121] Fig. 20 Show reb M2 13 Extension of C NMR spectrum (125 MHz, D2O / TSP).

[0122] Fig.21 Show reb M2 1 H- 1 H COSY spectrum (500 MHz, D2O).

[0123] Fig. 22 The HSQC-DEPT spectrum (500 MHz, D2O) of reb M2 is shown.

[0124] Fig.23 The HMBC spectrum (500 MHz, D2O) of reb M2 is shown.

[0125] Fig.24 Shown is the expansion of the HMBC spectrum of reb M2 (500 MHz, D2O). Detailed description

[0126] The present invention provides a biocatalytic method for preparing a composition containing a target steviol glycoside, the biocatalytic method comprising contacting a medium containing a starting composition with a biocatalyst, the starting composition comprising an organic compound containing at least one carbon atom, thereby producing a medium containing a target steviol glycoside.

[0127] In another embodiment, the present invention provides a method for producing a target steviol glycoside, the method comprising contacting an organic compound with at least one enzyme selected from a steviol biosynthetic enzyme and a UDP-glycosyltransferase, thereby producing a composition comprising a target steviol glycoside.

[0128] The object of the present invention is to provide an efficient biocatalytic method for preparing steviol glycosides, especially stevioside, reb E, reb A, reb D, reb D2, reb M, and reb M2, from different organic compounds in a starting composition.

[0129] As used herein, "biocatalysis" or "biocatalytic" refers to the use of natural or genetically engineered biocatalysts, such as enzymes, or cells containing one or more enzymes capable of performing single-step or multi-step chemical transformations of organic compounds. Biocatalytic processes include fermentation, biosynthesis, and bioconversion processes. Both isolated enzyme biocatalysis and whole-cell biocatalysis are known in the art. The biocatalyst protease may be a naturally occurring or recombinant protein.

[0130] As used herein, the term "one or more steviol glycosides" refers to glycosides of steviol, including, but not limited to, naturally occurring steviol glycosides, such as steviol monoside, steviol diside, rubusoside, dulcoside B, dulcoside A, rebaudioside B, rebaudioside G, stevioside, rebaudioside C, rebaudioside F, rebaudioside A, rebaudioside I, rebaudioside E, rebaudioside H, rebaudioside L, rebaudioside K, rebaudioside J, rebaudioside M, rebaudioside M2, rebaudioside D, rebaudioside D2, rebaudioside N, rebaudioside O, synthetic steviol glycosides such as enzymatically glucosylated steviol glycosides, and combinations thereof.

[0131] Starting composition

[0132] As used herein, "starting compound" refers to any composition (usually an aqueous solution) containing one or more organic compounds comprising at least one carbon atom.

[0133] In one embodiment, the organic compound is selected from the group consisting of polyols and various carbohydrates. Such organic compounds, and starting compositions comprising the organic compounds, are particularly useful when the present method is a fermentation method.

[0134] The term "polyol" refers to a molecule containing more than one hydroxyl group. Polyols can be diols, triols or tetraols containing 2,3 and 4 hydroxyls respectively. Polyols can also contain more than four hydroxyls, for example, amyl alcohol, hexanol, heptanol etc. containing 5,6 or 7 hydroxyls respectively. In addition, polyols can also be sugar alcohols, polyols or polymeric alcohols, which are reduced forms of carbohydrates, in which carbonyls (aldehydes or ketones, reducing sugars) have been reduced to primary alcohol hydroxyls or secondary alcohol hydroxyls. The example of polyols includes, but is not limited to, erythritol, maltitol, mannitol, sorbitol, lactitol, xylitol, inositol, isomalt, propylene glycol, glycerine, threitol, galactitol, hydrogenated isomaltulose, reduced isomaltose oligosaccharides, reduced xylo-oligosaccharides, reduced gentio-oligosaccharides, reduced maltose syrup, reduced glucose syrup, hydrogenated starch hydrolysate, polysaccharide alcohol and sugar alcohol or any other carbohydrate that can be reduced.

[0135] The term "carbohydrate" refers to an aldehyde or ketone compound substituted with multiple hydroxy groups and having the general formula (CH2O) n , wherein n is 3-30, and their oligomers and polymers. In addition, the carbohydrates of the present invention may be substituted or deoxygenated at one or more positions. Carbohydrates as used herein include unmodified carbohydrates, carbohydrate derivatives, substituted carbohydrates, and modified carbohydrates. As used herein, the phrases "carbohydrate derivatives," "substituted carbohydrates," and "modified carbohydrates" are synonymous. Modified carbohydrates mean any carbohydrate in which at least one atom has been added, removed, or substituted or a combination thereof. Therefore, 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 may be substituted with one or more moieties, such as hydrogen, halogen, haloalkyl, carboxyl, acyl, acyloxy, amino, amide, carboxyl derivatives, alkylamino, dialkylamino, arylamino, alkoxy, aryloxy, nitro, cyano, sulfo, sulfhydryl, imino, sulfonyl, sulfenyl, sulfenyl, sulfinyl, sulfamoyl, alkoxycarbonyl, carboxamido, phosphonyl, phosphinyl, phosphoryl, phosphino, thioester, thioether, oxime, hydrazine, carbamoyl, phospho, phosphonato, or any other reactive functional group, as long as the carbohydrate derivative or substituted carbohydrate is capable of improving the sweet taste of the sweetener composition.

[0136] Examples of carbohydrates that can be used according to the present invention include, but are not limited to, tagatose, trehalose, galactose, rhamnose, various cyclodextrins, cyclic oligosaccharides, various types of maltodextrins, dextran, sucrose, glucose, ribulose, fructose, threose, arabinose, xylose, lyxose, allose, altrose, mannose, idose, lactose, maltose, invert sugar, isotrehalose, neotrehalose, isomaltulose, erythrose, deoxyribose, gulose, idose, talose, erythrulose, xylulose, psicose, turanose, cellobiose, amylopectin, glucosamine, mannosamine, fucose, glucuronic acid, gluconic acid, gluconolactone, abequose, galactosamine, beet oligosaccharides, isomaltooligosaccharides (isomaltose, isomaltulose, The term "carbohydrate" refers to oligosaccharides (such as maltotriose, panose, etc.), oligoxylose (xylotriose, xylobiose, etc.), xylose terminal oligosaccharides (xylo-terminated oligosaccharides), gentiooligosaccharides (gentiobiose, gentiotriose, gentiotetraose, etc.), sorbose, niger oligosaccharides, palatinose oligosaccharides, fructo-oligomers (kestose, mold erythraldehyde, etc.), malt tetraol (maltotetraol), maltotriol (maltotriol), malto-oligosaccharides (maltotriose, maltotetraose, maltopentaose, maltohexaose, maltoheptaose, etc.), starch, inulin, inulin oligosaccharides, lactulose, melibiose, raffinose, ribose, isomerized liquid sugars such as high fructose corn syrup, coupling sugars and soybean oligosaccharides. In addition, the carbohydrates used herein can be D or L configuration.

[0137] The starting compounds may be synthetic or purified (partially or completely), commercially available or prepared.

[0138] In one embodiment, the organic compound is glycerol. In another embodiment, the starting composition comprises glycerol.

[0139] In one embodiment, the organic compound is glucose. In another embodiment, the starting composition comprises glucose.

[0140] In one embodiment, the organic compound is sucrose. In another embodiment, the starting composition comprises sucrose.

[0141] In one embodiment, the organic compound is starch. In another embodiment, the starting composition comprises starch.

[0142] In one embodiment, the organic compound is maltodextrin. In another embodiment, the starting composition comprises maltodextrin.

[0143] The one or more organic compounds serve as one or more substrates for producing one or more target steviol glycosides, as described herein.

[0144] The method of the present invention also provides a method for biocatalytically converting a steviol glycoside into another steviol glycoside. Accordingly, in some embodiments, the organic compound is a steviol glycoside, including but not limited to stevioside, stevioside, rubusoside, dulcoside B, dulcoside A, rebaudioside B, rebaudioside G, stevioside, rebaudioside C, rebaudioside F, rebaudioside A, rebaudioside I, rebaudioside E, rebaudioside H, rebaudioside L, rebaudioside K, rebaudioside J, rebaudioside M, rebaudioside M2, rebaudioside D, rebaudioside D2, rebaudioside N or rebaudioside O, or other steviol glycosides.

[0145] It is noteworthy that the substrate steviol glycoside used for biocatalytic conversion is not the same as the target steviol glycoside, as discussed below. However, the starting composition may contain steviol glycosides other than the substrate steviol glycoside, and in some cases, may contain a certain amount of the target steviol glycoside.

[0146] Target Steviol Glycoside

[0147] The target steviol glycoside of the present invention can be any steviol glycoside that can be prepared by the method disclosed herein. In one embodiment, the target steviol glycoside is selected from the group consisting of stevioside, stevioside, rubusoside, dulcoside B, dulcoside A, rebaudioside B, rebaudioside G, stevioside, rebaudioside C, rebaudioside F, rebaudioside A, rebaudioside I, rebaudioside E, rebaudioside H, rebaudioside L, rebaudioside K, rebaudioside J, rebaudioside M, rebaudioside M2, rebaudioside D, rebaudioside D2, rebaudioside N or rebaudioside O, or other steviol glycosides.

[0148] In one embodiment, the target steviol glycoside is stevioside. In another embodiment, the target steviol glycoside is reb A. In yet another embodiment, the target steviol glycoside is reb E. In yet another embodiment, the target steviol glycoside is reb D. In yet another embodiment, the target steviol glycoside is reb D2. In an additional embodiment, the target steviol glycoside is reb M. In yet another embodiment, the target steviol glycoside is reb M2.

[0149] The target steviol glycoside may be in any polymorphic form or amorphous form, including hydrates, solvates, anhydrous forms, or combinations thereof.

[0150] In one embodiment, the invention is a biocatalytic process for producing reb A. In a more specific embodiment, the invention is a fermentation process for producing reb A from a starting composition comprising, for example, glucose.

[0151] In another embodiment, the present invention is a biocatalytic process for producing reb E. In a more specific embodiment, the present invention is a fermentation process for producing reb E from a starting composition comprising, for example, glucose.

[0152] In yet another embodiment, the present invention is a biocatalytic method for producing reb D. In a more specific embodiment, the present invention is a fermentation method for producing reb D from a starting composition comprising, for example, glucose.

[0153] In yet another embodiment, the present invention is a biocatalytic method for producing reb D2. In a more specific embodiment, the present invention is a fermentation method for producing reb D2 from a starting composition comprising, for example, glucose.

[0154] In an additional embodiment, the invention is a biocatalytic process for producing reb M. In a more specific embodiment, the invention is a fermentation process for producing reb M from a starting composition comprising, for example, glucose.

[0155] In yet another embodiment, the invention is a biocatalytic method for producing reb M2. In a more specific embodiment, the invention is a fermentation method for producing reb M2 from a starting composition comprising, for example, glucose.

[0156] In another further embodiment, the present invention is a biocatalytic method for producing stevioside. In a more specific embodiment, the present invention is a fermentation method for producing stevioside from a starting composition comprising, for example, glucose.

[0157] Optionally, the method of the present invention further comprises separating the target steviol glycoside from the medium to provide a highly purified target steviol glycoside composition. The target steviol glycoside can be separated by any appropriate method such as crystallization, separation by membrane, centrifugation, extraction, chromatography or a combination of such methods.

[0158] In a specific embodiment, the method described herein produces a highly purified target steviol glycoside composition. As used herein, the term "highly purified" refers to a composition having a target steviol glycoside content of more than about 80% by dry weight. In one embodiment, a highly purified target steviol glycoside composition comprises a target steviol glycoside content of more than about 90% by dry weight, such as, for example, more than about 91%, more than about 92%, more than about 93%, more than about 94%, more than about 95%, more than about 96%, more than about 97%, more than about 98% or more than about 99% by dry weight.

[0159] In one embodiment, when the target steviol glycoside is reb M, the methods described herein provide a composition having a reb M content of more than about 90% by dry weight. In another specific embodiment, when the target steviol glycoside is reb M, the methods described herein provide a composition comprising a reb M content of more than about 95% by dry weight.

[0160] In another embodiment, when the target steviol glycoside is reb M2, the methods described herein provide compositions having a reb M2 content of more than about 90% by dry weight. In another specific embodiment, when the target steviol glycoside is reb M2, the methods described herein provide compositions comprising a reb M2 content of more than about 95% by dry weight.

[0161] In yet another embodiment, when the target steviol glycoside is reb D, the methods described herein provide compositions having a reb D content of more than about 90% by dry weight. In another specific embodiment, when the target steviol glycoside is reb D, the methods described herein provide compositions having a reb D content of more than about 95% by dry weight.

[0162] In yet another embodiment, when the target steviol glycoside is reb D2, the methods described herein provide compositions having a reb D2 content exceeding about 90% by dry weight. In another specific embodiment, when the target steviol glycoside is reb D2, the methods described herein provide compositions comprising a reb D2 content exceeding about 95% by dry weight.

[0163] In a further embodiment, when the target steviol glycoside is reb A, the methods described herein provide a composition comprising more than about 90% reb A content by dry weight. In another specific embodiment, when the target steviol glycoside is reb A, the methods described herein provide a composition comprising more than about 95% reb A content by dry weight.

[0164] In yet another embodiment, when the target steviol glycoside is reb E, the methods described herein provide compositions comprising more than about 90% reb E content by dry weight. In another specific embodiment, when the target steviol glycoside is reb E, the methods described herein provide compositions comprising more than about 95% reb E content by dry weight.

[0165] In yet another further embodiment, when the target steviol glycoside is stevioside, the methods described herein provide compositions comprising a stevioside content of greater than about 90% by dry weight. In another specific embodiment, when the target steviol glycoside is stevioside, the methods described herein provide compositions comprising a stevioside content of greater than about 95% by dry weight.

[0166] Microorganisms and biocatalysts

[0167] In one embodiment of the present invention, the biocatalyst is contacted with a medium containing a starting composition to produce a target steviol glycoside. In certain embodiments, the biocatalyst is an enzyme, or a cell comprising one or more enzymes, that is capable of converting an organic compound into a target steviol glycoside.

[0168] In one embodiment, the biocatalyst is an enzyme capable of converting an organic compound into a target steviol glycoside. The enzyme may be provided in the form of a whole cell suspension, a crude lysate, a purified enzyme, or a combination thereof. In one embodiment, the biocatalyst is a purified enzyme capable of converting an organic compound into a target steviol glycoside. In another embodiment, the biocatalyst is a crude lysate comprising at least one enzyme capable of converting an organic compound into a target steviol glycoside. In yet another embodiment, the biocatalyst is a whole cell suspension comprising at least one enzyme capable of converting an organic compound into a target steviol glycoside.

[0169] In another embodiment, the biocatalyst is one or more cells comprising an enzyme capable of converting an organic compound into a target steviol glycoside. The enzyme can be located on the cell surface, inside the cell, or both on the cell surface and inside the cell.

[0170] In one embodiment, one biocatalyst enzyme is used in each conversion of the organic compound to the target steviol glycoside. In another embodiment, two or more biocatalyst enzymes are used in each conversion.

[0171] Suitable enzymes for converting organic compounds to target steviol glycosides include, but are not limited to, steviol biosynthetic enzymes and UDP-glycosyltransferases (UGTs).

[0172] In one embodiment, the steviol biosynthetic enzymes include mevalonate (MVA) pathway enzymes.

[0173] In another embodiment, the steviol biosynthetic enzymes include non-mevalonate 2-C-methyl-D-erythritol-4-phosphate pathway (MEP / DOXP) enzymes.

[0174] In one embodiment, the steviol biosynthetic enzyme is selected from the group consisting of geranylgeranyl diphosphate synthase, copalyl diphosphate synthase, kaurene synthase, kaurene oxidase, kaurenoic acid 13-hydroxylase (KAH), steviol synthase, deoxyxylulose 5-phosphate synthase (DXS), D-1-deoxyxylulose 5-phosphate reductoisomerase (DXR), 4-diphosphocytidine-2-C-methyl-D-erythritol synthase (CMS), 4-diphosphocytidine-2-C-methyl-D -erythritol kinase (CMK), 4-diphosphocytidine-2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (MCS), l-hydroxy-2-methyl-2(E)-butenyl 4-diphosphate synthase (HDS), l-hydroxy-2-methyl-2(E)-butenyl 4-diphosphate reductase (HDR), acetoacetyl-CoA thiolase, truncated HMG-CoA reductase, mevalonate kinase, phosphomevalonate kinase, mevalonate pyrophosphate decarboxylase and cytochrome P450 reductase.

[0175] The UDP-glucosyltransferase can be any UDP-glucosyltransferase that is capable of adding at least one glucose unit to a steviol and / or steviol glycoside substrate to provide a target steviol glycoside.

[0176] In one embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rubusoside thereby producing stevioside. The UDP-glucosyltransferase can be, for example, UGT91D2.

[0177] In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rubusoside to produce rebaudioside E. The UDP-glucosyltransferase can be, for example, UGTSL, UGTSL2, or UGTLB.

[0178] In yet another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside E, thereby producing rebaudioside D. The UDP-glucosyltransferase can be, for example, UGT76G1.

[0179] In yet another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to stevioside, thereby producing rebaudioside A. The UDP-glucosyltransferase can be, for example, UGT76G1.

[0180] In a further embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside A, thereby producing rebaudioside D and / or rebaudioside D2 and / or rebaudioside M2. The UDP-glucosyltransferase can be, for example, UGT91D2, UGTSL2, UGTLB or EUGT11.

[0181] In another embodiment, the UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside A is selected from the following list of GenInfo identification numbers, preferably from the group presented in Table 1, and more preferably from the group presented in Table 2.

[0182]

[0183]

[0184]

[0185]

[0186]

[0187] Table 1

[0188]

[0189]

[0190]

[0191]

[0192]

[0193] Table 2

[0194]

[0195]

[0196] In yet another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside D to form rebaudioside M and / or rebaudioside M2. The UDP-glucosyltransferase can be, for example, UGT76G1. In a preferred embodiment, the conversion rate is at least greater than 50%, for example, greater than 60%, greater than 70%, greater than 80% or greater than 90%.

[0197] The UGT76G1 enzyme may also contain one or more point mutations that are beneficial for converting rebaudioside D to rebaudioside M. Suitable mutations include, for example, S42A, F46I, I190L, S274G, I295M, K303G, F314S, K316R, K393R, V394I, I407V, N409K, N409R, Q425E, Q432E, S447A, and S456L. In a preferred embodiment, the use of a UGT76G1 containing such one or more point mutations results in at least about a 5% increase in rebaudioside M conversion compared to the use of an unmutated UGT76G1 under the same conditions (where the results are normalized). In preferred embodiments, the conversion to rebaudioside M is increased from about 5% to about 50%, such as, for example, from about 10% to about 50%, from about 20% to about 50%, from about 30% to about 50% or about 40% to about 50%.

[0198] In some embodiments, the microorganism comprises an enzyme of the present invention, i.e., an enzyme capable of converting an organic compound into a target steviol glycoside. Accordingly, some embodiments of the methods of the present invention include contacting the microorganism with a medium containing a starting composition to provide a medium comprising at least one target steviol glycoside.

[0199] The microorganism can be any microorganism that has one or more necessary enzymes for converting an organic compound into one or more target steviol glycosides. These enzymes are encoded in the genome of the microorganism.

[0200] Suitable microorganisms include, but are not limited to, Escherichia coli, Saccharomyces spp., Aspergillus spp., Pichia spp., Bacillus spp., Yarrowia spp., and the like.

[0201] In one embodiment, the microorganisms are free when contacted with the starting composition.

[0202] In another embodiment, the microorganism is fixed when contacted with the starting composition. For example, the microorganism can be fixed to a solid support made of an inorganic or organic material. Non-limiting examples of solid supports suitable for fixing microorganisms include, derived cellulose or glass, ceramics, metal oxides or films. The microorganism can be fixed to the solid support, for example, by covalent attachment, adsorption, crosslinking, capture or encapsulation.

[0203] In yet another embodiment, an enzyme capable of converting an organic compound to a target steviol glycoside is secreted from the microorganism and into the reaction medium.

[0204] The starting composition / organic compound is contacted with a biocatalyst or microorganism in an aqueous medium comprising water and, for example, various components selected from carbon sources, energy sources, nitrogen sources, trace elements, vitamins, nucleosides, nucleoside phosphates, nucleoside diphosphates, nucleoside triphosphates, organic and inorganic salts, organic and mineral acids, bases, etc. Carbon sources include glycerol, glucose, carbon dioxide, carbonate, bicarbonate. Nitrogen sources may include nitrates, nitrites, amino acids, peptides, peptones, or proteins.

[0205] In a specific embodiment, the medium comprises a buffer. Suitable buffers include, but are not limited to, PIPES buffer, acetate buffer, and phosphate buffer. In a specific embodiment, the medium comprises a phosphate buffer.

[0206] In one embodiment, the medium may further include an organic solvent.

[0207] Optionally, the methods of the present invention further comprise recycling UDP to provide UDP-glucose. Accordingly, these methods comprise concomitantly recycling UDP by providing a recycling catalyst (i.e., a biocatalyst capable of overproducing UDP-glucose) and a recycling substrate, thereby enabling the conversion of a substrate steviol glycoside to a target steviol glycoside using a catalytic amount of UDP-glucosyltransferase and UDP-glucose ( Figure 2 ).

[0208] In one embodiment, the UDP-glucose recycling catalyst is sucrose synthase.

[0209] In one embodiment, the recycled substrate is sucrose.

[0210] Optionally, the target steviol glycoside is purified from the resulting composition. The target steviol glycoside can be purified from the reaction medium by at least one suitable method to provide a highly purified target steviol glycoside composition. Suitable methods include crystallization, separation by membrane, centrifugation, extraction (liquid or solid phase), chromatographic separation, HPLC (preparative or analytical) or a combination of such methods.

[0211] In one embodiment, the present invention provides a method for producing stevioside by (a) contacting a medium containing a starting composition comprising rubusoside with UGT91D2 and UDP-glucose.

[0212] In a more specific embodiment, the present invention provides a catalytic method for producing stevioside by (a) contacting a medium containing a starting composition comprising rubusoside with a catalytic amount of UGT91D2 and UDP-glucose, and (b) providing a sucrose synthase and sucrose recycling UDP-glucose.

[0213] In another specific embodiment, the present invention provides a fermentation method for producing stevioside by (a) contacting a medium containing a starting composition comprising glucose with a microorganism comprising at least one enzyme capable of converting glucose into stevioside.

[0214] In yet another specific embodiment, the present invention provides a fermentation method for producing stevioside by (a) contacting a medium containing a starting composition comprising glucose with a microorganism comprising at least one enzyme capable of converting glucose to stevioside and (b) providing UDP-glucose, sucrose synthase, and sucrose recycling UDP-glucose.

[0215] The above method may further comprise purifying stevioside from the medium to provide a composition comprising highly purified stevioside.

[0216] In another embodiment, the present invention provides a method for producing rebaudioside E by (a) contacting a medium containing a starting composition comprising rubusoside with UGTSL2 and UDP-glucose.

[0217] In a more specific embodiment, the present invention provides a catalytic method for producing rebaudioside E by (a) contacting a medium containing a starting composition comprising rubusoside with a catalytic amount of UGTSL2 and UDP-glucose, and (b) providing sucrose synthase and sucrose recycling UDP-glucose.

[0218] In another specific embodiment, the present invention provides a fermentation method for producing rebaudioside E by (a) contacting a medium containing a starting composition comprising glucose with a microorganism comprising at least one enzyme capable of converting glucose into rebaudioside E.

[0219] In yet another specific embodiment, the present invention provides a fermentation method for producing rebaudioside E by (a) contacting a medium containing a starting composition comprising glucose with a microorganism comprising at least one enzyme capable of converting glucose to rebaudioside E and (b) providing UDP-glucose, sucrose synthase, and sucrose recycling UDP-glucose.

[0220] The above method may further comprise purifying rebaudioside E from the medium to provide a composition comprising highly purified rebaudioside E.

[0221] In yet another embodiment, the present invention provides a method for producing rebaudioside D by (a) contacting a medium containing a starting composition comprising rebaudioside E with UGT76G1 and UDP-glucose.

[0222] In a more specific embodiment, the present invention provides a catalytic method for producing rebaudioside D by (a) contacting a medium containing a starting composition comprising rebaudioside E with a catalytic amount of UGT76G1 and UDP-glucose, and (b) providing a sucrose synthase and a sucrose recycling UDP-glucose.

[0223] In another specific embodiment, the present invention provides a fermentation method for producing rebaudioside D by (a) contacting a medium containing a starting composition comprising glucose with a microorganism comprising at least one enzyme capable of converting glucose into rebaudioside D.

[0224] In yet another specific embodiment, the present invention provides a fermentation method for producing rebaudioside D by (a) contacting a medium containing a starting composition comprising glucose with a microorganism comprising at least one enzyme capable of converting glucose to rebaudioside D and (b) providing UDP-glucose, sucrose synthase, and sucrose recycling UDP-glucose.

[0225] The above method may further comprise purifying rebaudioside D from the medium to provide a composition comprising highly purified rebaudioside D.

[0226] In yet another embodiment, the present invention provides a method for producing rebaudioside A by (a) contacting a medium containing a starting composition comprising stevioside with UGT76G1 and UDP-glucose.

[0227] In a more specific embodiment, the present invention provides a catalytic method for producing rebaudioside A by (a) contacting a medium containing a starting composition comprising stevioside with a catalytic amount of UGT76G1 and UDP-glucose, and (b) providing a sucrose synthase and a sucrose recycling UDP-glucose.

[0228] In another specific embodiment, the present invention provides a fermentation method for producing rebaudioside A by (a) contacting a medium containing a starting composition comprising glucose with a microorganism comprising at least one enzyme capable of converting glucose to rebaudioside A.

[0229] In yet another specific embodiment, the present invention provides a fermentation method for producing rebaudioside A by (a) contacting a medium containing a starting composition comprising glucose with a microorganism comprising at least one enzyme capable of converting glucose to rebaudioside A and (b) providing UDP-glucose, sucrose synthase, and sucrose recycling UDP-glucose.

[0230] The above method may further comprise purifying rebaudioside A from the medium to provide a composition comprising highly purified rebaudioside A.

[0231] In yet further embodiments, the present invention provides methods for producing rebaudioside D, rebaudioside D2 and / or rebaudioside M2 ​​by (a) contacting a medium containing a starting composition comprising rebaudioside A with UGT91D2, UGTSL2 or EUGT11 and UDP-glucose.

[0232] In a more specific embodiment, the present invention provides a catalytic method for producing rebaudioside D, rebaudioside D2 and / or rebaudioside M2 ​​by (a) contacting a medium containing a starting composition comprising rebaudioside A with a catalytic amount of UGT91D2, UGTSL2 or EUGT11, and UDP-glucose, and (b) providing sucrose synthase and sucrose recycling UDP-glucose.

[0233] In another specific embodiment, the present invention provides a fermentation method for producing rebaudioside D, rebaudioside D2 and / or rebaudioside M2 ​​by (a) contacting a medium containing a starting composition comprising glucose with a microorganism comprising at least one enzyme capable of converting glucose into rebaudioside D, rebaudioside D2 and / or rebaudioside M2.

[0234] In yet another specific embodiment, the present invention provides a fermentation method for producing rebaudioside D, rebaudioside D2 and / or rebaudioside M2 ​​by (a) contacting a medium containing a starting composition comprising glucose with a microorganism comprising at least one enzyme capable of converting glucose to rebaudioside D, rebaudioside D2 and / or rebaudioside M2 ​​and (b) providing UDP-glucose, sucrose synthase and sucrose recycling UDP-glucose.

[0235] The above method may further comprise separating rebaudioside D, rebaudioside D2 and / or rebaudioside M2 ​​from the medium to provide a composition comprising highly purified rebaudioside D, rebaudioside D2 and / or rebaudioside M2.

[0236] In yet another embodiment, the present invention provides a method for producing rebaudioside M by (a) contacting a medium containing a starting composition comprising rebaudioside D with UGT76G1 and UDP-glucose.

[0237] In a more specific embodiment, the present invention provides a catalytic method for producing rebaudioside M by (a) contacting a medium containing a starting composition comprising rebaudioside D with a catalytic amount of UGT76G1 and UDP-glucose, and (b) providing a sucrose synthase and a sucrose recycling UDP-glucose.

[0238] In another specific embodiment, the present invention provides a fermentation method for producing rebaudioside M by (a) contacting a medium containing a starting composition comprising glucose with a microorganism comprising at least one enzyme capable of converting glucose into rebaudioside M.

[0239] In yet another specific embodiment, the present invention provides a fermentation method for producing rebaudioside M by (a) contacting a medium containing a starting composition comprising glucose with a microorganism comprising at least one enzyme capable of converting glucose to rebaudioside M and (b) providing UDP-glucose, sucrose synthase, and sucrose recycling UDP-glucose.

[0240] The above method may further comprise separating rebaudioside M from the medium to provide a composition comprising highly purified rebaudioside M.

[0241] In some embodiments, multiple biocatalytic steps are performed sequentially to convert, for example, (i) rebaudioside A to rebaudioside D, and then (ii) rebaudioside D to rebaudioside M, or (i) stevioside to rebaudioside A, and then (ii) rebaudioside A to rebaudioside D, and then (iii) rebaudioside D to rebaudioside M, or (i) rubusoside to stevioside, and then (ii) stevioside to rebaudioside A, and then (iii) rebaudioside A to rebaudioside D, and then (iv) rebaudioside D to rebaudioside M.

[0242] Alternatively, fermentation and biocatalysis steps can be used in sequence. For example, a starting composition comprising glucose can first be fermented with a microorganism containing at least one enzyme that can convert glucose into a target steviol glycoside. The target steviol glycoside (now becoming the starting material for the next bioconversion purpose) can then be contacted with a biocatalyst that can convert it into the next target steviol glycoside.

[0243] Between each transformation, the target steviol glycoside may optionally be separated from the medium before contacting the steviol glycoside (which now becomes the starting steviol glycoside for the purpose of the next biotransformation) with the next biocatalyst.

[0244] Compounds and methods

[0245] The present invention also provides isolated and highly purified reb D2. Reb D2 is an isomer of reb D and has the following structure:

[0246]

[0247] 13-[(2-O-β-D-glucopyranosyl-3-O-β-D-glucopyranosyl-β-D-glucopyranosyl)oxy] ent-kaur-16-ene-19-oic acid-[(6-O-β-D-glucopyranosyl-β-D-glucopyranosyl) ester]

[0248] In another embodiment, the invention provides reb D2 having a purity of greater than about 95% by dry weight, such as, for example, greater than about 96% by weight, greater than about 97% by weight, greater than about 98% by weight, or greater than about 99% by weight.

[0249] In yet another embodiment, the invention provides reb D2 having a purity of greater than about 95% by weight, such as, for example, greater than about 96% by weight, greater than about 97% by weight, greater than about 98% by weight, or greater than about 99% by weight in a steviol glycoside mixture.

[0250] The present invention also provides a composition comprising reb D2.

[0251] The present invention also provides a method for preparing reb D2, the method comprising:

[0252] a. contacting the starting composition comprising reb A with an enzyme capable of converting reb A into reb D2, UDP-glucose, and optionally a UDP-glucose cycle enzyme to produce a composition comprising reb D2; and

[0253] b. Isolating the composition comprising reb D2.

[0254] In some embodiments, the enzyme capable of converting reb A into reb D2 is a UDP-glucosyltransferase, such as, for example, UGT91D2, UGTSL, UGTSL_Sc, UGTSL2 (GI No. 460410132 Version XP_004250485.1), GI No. 460409128 (UGTSL) Version XP_004249992.1, GI No. 115454819 Version NP_001051010.1, GI No. 187373030, Version ACD03249.1, GI No. 222619587 Version EEE55719.1, GI No. 297795735 Version XP_002865752.1 or EUGT11.

[0255] In one embodiment, the enzyme may be provided in the form of one or more cells containing the enzyme.

[0256] In other embodiments, the enzyme can be provided as a whole cell suspension, a crude lysate, a purified enzyme, or a combination thereof. In one embodiment, the enzyme is a purified enzyme. In another embodiment, the enzyme is provided as a crude lysate. In yet another embodiment, the enzyme is provided as a whole cell suspension.

[0257] An enzyme capable of converting reb A to reb D2 may be immobilized.

[0258] In another embodiment, the enzyme is provided in a microorganism.

[0259] In one embodiment, the microorganisms are free when contacted with the starting composition.

[0260] In another embodiment, the microorganism is fixed when contacted with the starting composition. For example, the microorganism can be fixed to a solid support made of an inorganic or organic material. Non-limiting examples of solid supports suitable for fixing microorganisms include, derived cellulose or glass, ceramics, metal oxides or films. The microorganism can be fixed to the solid support, for example, by covalent attachment, adsorption, crosslinking, capture or encapsulation.

[0261] Suitable microorganisms include, but are not limited to, Escherichia coli, Saccharomyces spp., Aspergillus spp., Pichia spp., Bacillus spp., Yarrowia spp., and the like.

[0262] In yet another embodiment, the enzyme is secreted from the microorganism and into the reaction medium.

[0263] The starting composition is contacted with the enzyme or microorganism in an aqueous medium comprising water and various components, wherein the components are selected from the group consisting of carbon sources, energy sources, nitrogen sources, trace elements, vitamins, nucleosides, nucleoside phosphates, nucleoside diphosphates, nucleoside triphosphates, organic and inorganic salts, organic and mineral acids, alkalis, etc. The carbon source comprises glycerol, glucose, carbon dioxide, carbonate, bicarbonate. The nitrogen source may comprise nitrate, nitrite, amino acid, peptide, peptone, or protein.

[0264] In a specific embodiment, the medium comprises a buffer. Suitable buffers include, but are not limited to, PIPES buffer, acetate buffer, and phosphate buffer. In a specific embodiment, the medium comprises a phosphate buffer.

[0265] In one embodiment, the medium may further include an organic solvent.

[0266] In a specific embodiment, the enzyme is a UDP-glucosyltransferase capable of converting reb A to reb D2.

[0267] In a more specific embodiment, the enzyme is selected from UGT91D2, UGTSL, UGTSL_Sc, UGTSL2 (GI No. 460410132 version XP_004250485.1), GI No. 460409128 (UGTSL) version XP_004249992.1, GI No. 115454819 version NP_001051010.1, GI No. 187373030, version ACD03249.1, GI No. 222619587 version EEE55719.1, GI No. 297795735 version XP_002865752.1 or EUGT11 and UGTs having substantial (>85%) sequence identity with these enzymes.

[0268] In yet more specific embodiments, the enzyme is UGTSL2 or an improved variant thereof produced by directed evolution and having higher activity.

[0269] In one embodiment, reb D2 is continuously removed from the medium while the conversion progresses. In yet another embodiment, reb D2 is separated from the reaction medium after the reaction is completed and optionally purified.

[0270] Reb D2 can be separated from the reaction medium by any suitable method to provide a composition comprising reb D2. Suitable methods include, but are not limited to, cracking, crystallization, separation by membrane, centrifugation, extraction (liquid or solid phase), chromatographic separation, HPLC (preparative or analytical) or a combination of such methods. In a specific embodiment, separation can be achieved by cracking and centrifugation.

[0271] In some embodiments, the separation may result in a reb D2 purity of less than about 95% by dry weight, and the composition may contain, for example, steviol glycosides and / or residual reaction products. The composition comprising reb D2 may be further purified to provide highly purified reb D2, i.e., reb D2 having a purity of greater than about 95% by dry weight. In some embodiments, the compositions comprising reb D2 may be further purified to provide reb D2 having a purity of greater than about 96%, greater than about 97%, greater than about 98%, or greater than about 99% by dry weight.

[0272] Purification can be affected by any means known to those skilled in the art, including but not limited to, crystallization, separation by membrane, centrifugation, extraction (liquid or solid phase), chromatographic separation, HPLC (preparative or analytical) or a combination of such methods. In a specific embodiment, HPLC is used to purify reb D2. In a more specific embodiment, semi-preparative HPLC is used to purify reb D2.

[0273] For example, a two-step semi-preparative HPLC purification can be used. The first step utilizes a C18 column with a mobile phase containing A (25% MeCN in water) and B (30% MeCN in water) with the following gradient:

[0274] Time (min) ％A ％B 0.0-5.0 100 0 20 20 80 25 20 80 30 100 0

[0275] The second step utilized the same column and conditions, but only with an isocratic mobile phase: 20% MeCN in water.

[0276] Those skilled in the art will recognize that the specific column, mobile phase, injection volume and other HPLC parameters may vary.

[0277] The present invention provides isolated and highly purified reb M2. Reb M2 is an isomer of reb M and has the following structure:

[0278]

[0279] (13-[(2-O-β-D-glucopyranosyl-3-O-β-D-glucopyranosyl-β-D-glucopyranosyl)oxy] ent-kaur-16-ene-19-oic acid-[(2-O-β-D-glucopyranosyl-6-O-β-D-glucopyranosyl-β-D-glucopyranosyl) ester])

[0280] In another embodiment, the invention provides reb M2 having a purity of greater than about 95% by dry weight, such as, for example, greater than about 96% by weight, greater than about 97% by weight, greater than about 98% by weight, or greater than about 99% by weight.

[0281] In yet another embodiment, the invention provides reb M2 having a purity of greater than about 95% by weight, such as, for example, greater than about 96% by weight, greater than about 97% by weight, greater than about 98% by weight, or greater than about 99% by weight in a steviol glycoside mixture.

[0282] In yet another embodiment, the invention provides reb M2 having a purity of greater than about 95% by weight, such as, for example, greater than about 96% by weight, greater than about 97% by weight, greater than about 98% by weight, or greater than about 99% by weight in stevia extract.

[0283] The present invention also provides a composition comprising reb M2.

[0284] It has been found that reb M2 is produced during the bioconversion of reb A to reb D. As described above, the bioconversion of reb A to reb D also produces reb D2. Accordingly, the present invention also provides a method for preparing reb M2, the method comprising:

[0285] a. contacting a starting composition comprising reb A and / or reb D2 with an enzyme capable of converting reb A and / or reb D2 into rebM2, UDP-glucose, and optionally a UDP-glucose cycle enzyme to produce a composition comprising reb M2; and

[0286] b. Isolating the composition comprising reb M2.

[0287] Without wishing to be bound by theory, it is currently believed that the pathway begins with the conversion of reb A to reb D2, followed by the conversion of reb D2 to reb M2. Accordingly, the present invention provides a method for preparing reb M2, the method comprising:

[0288] a. contacting the starting composition comprising reb D2 with an enzyme capable of converting reb D2 into reb M2, UDP-glucose, and optionally a UDP-glucose cycle enzyme to produce a composition comprising reb M2; and

[0289] b. Isolating the composition comprising reb M2.

[0290] In yet another embodiment, the method for preparing reb M2 comprises:

[0291] a. contacting a starting composition comprising reb A with an enzyme capable of converting reb A into reb D2, UDP-glucose, and optionally a UDP-glucose cycle enzyme to produce a composition comprising reb D2;

[0292] b. Optionally, separating the composition comprising reb D2;

[0293] c. contacting a composition comprising reb D2 with an enzyme capable of converting reb D2 into reb M2, UDP-glucose, and optionally a UDP-glucose cycle enzyme to produce a composition comprising reb M2; and

[0294] d. Isolating the composition comprising reb M2.

[0295] The enzyme can be a UDP-glucosyltransferase, such as, for example, UGT91D2, UGTSL, UGTSL_Sc, UGTSL2 (GI No. 460410132 Version XP_004250485.1), GI No. 460409128 (UGTSL) Version XP_004249992.1, GI No. 115454819 Version NP_001051010.1, GI No. 187373030, Version ACD03249.1, GI No. 222619587 Version EEE55719.1, GI No. 297795735 Version XP_002865752.1 or EUGT11.

[0296] In one embodiment, the enzyme may be provided in the form of one or more cells containing the enzyme.

[0297] In other embodiments, the enzyme can be provided as a whole cell suspension, a crude lysate, a purified enzyme, or a combination thereof. In one embodiment, the enzyme is a purified enzyme. In another embodiment, the enzyme is provided as a crude lysate. In yet another embodiment, the enzyme is provided as a whole cell suspension.

[0298] In some embodiments, the enzyme may be immobilized.

[0299] In another embodiment, the enzyme is provided in a microorganism.

[0300] In one embodiment, the microorganisms are free when contacted with the starting composition.

[0301] In another embodiment, the microorganism is fixed when contacted with the starting composition. For example, the microorganism can be fixed to a solid support made of an inorganic or organic material. Non-limiting examples of solid supports suitable for fixing microorganisms include, derived cellulose or glass, ceramics, metal oxides or films. The microorganism can be fixed to the solid support, for example, by covalent attachment, adsorption, crosslinking, capture or encapsulation.

[0302] Suitable microorganisms include, but are not limited to, Escherichia coli, Saccharomyces spp., Aspergillus spp., Pichia spp., Bacillus spp., Yarrowia spp., and the like.

[0303] In yet another embodiment, the enzyme is secreted from the microorganism and into the reaction medium.

[0304] The starting composition is contacted with the enzyme or microorganism in an aqueous medium comprising water and various components, wherein the components are selected from the group consisting of carbon sources, energy sources, nitrogen sources, trace elements, vitamins, nucleosides, nucleoside phosphates, nucleoside diphosphates, nucleoside triphosphates, organic and inorganic salts, organic and mineral acids, alkalis, etc. The carbon source comprises glycerol, glucose, carbon dioxide, carbonate, bicarbonate. The nitrogen source may comprise nitrate, nitrite, amino acid, peptide, peptone, or protein.

[0305] In a specific embodiment, the medium comprises a buffer. Suitable buffers include, but are not limited to, PIPES buffer, acetate buffer, and phosphate buffer. In a specific embodiment, the medium comprises a phosphate buffer.

[0306] In one embodiment, the medium may further include an organic solvent.

[0307] In a specific embodiment, the enzyme is a UDP-glucosyltransferase capable of converting reb A and / or reb D2 into reb M2 and is contained in E. coli.

[0308] In a more specific embodiment, the enzyme is selected from UGT91D2, UGTSL, UGTSL_Sc, UGTSL2 (GI No. 460410132 version XP_004250485.1), GI No. 460409128 (UGTSL) version XP_004249992.1, GI No. 115454819 version NP_001051010.1, GI No. 187373030, version ACD03249.1, GI No. 222619587 version EEE55719.1, GI No. 297795735 version XP_002865752.1 or EUGT11.

[0309] In yet more specific embodiments, the enzyme is UGTSL2 or an improved variant thereof produced by directed evolution and having higher activity.

[0310] In one embodiment, reb M2 can be continuously removed from the medium while the conversion progresses. In yet another embodiment, reb M2 is separated from the reaction medium after the reaction is completed and optionally purified.

[0311] Reb M2 can be separated from the reaction medium by any suitable method to provide a composition comprising Reb M2. Suitable methods include, but are not limited to, cracking, crystallization, separation by membrane, centrifugation, extraction (liquid or solid phase), chromatographic separation, HPLC (preparative or analytical) or a combination of such methods. In a specific embodiment, separation can be achieved by cracking and centrifugation.

[0312] In some embodiments, the separation can result in a reb M2 purity of less than about 95% by dry weight, and the composition can contain, for example, steviol glycosides and / or residual reaction products.

[0313] The composition comprising reb M2 can be further purified to provide highly purified reb M2, i.e., reb M2 having a purity of greater than about 95% by dry weight. In some embodiments, the composition comprising reb M2 can be further purified to provide reb M2 having a purity of greater than about 96%, greater than about 97%, greater than about 98%, or greater than about 99% by dry weight.

[0314] Purification can be affected by any means known to those skilled in the art, including but not limited to, crystallization, separation by membrane, centrifugation, extraction (liquid or solid phase), chromatographic separation, HPLC (preparative or analytical) or a combination of such methods. In a specific embodiment, HPLC is used to purify reb M2. In a more specific embodiment, semi-preparative HPLC is used to purify reb M2.

[0315] For example, a two-step semi-preparative HPLC purification can be used. The first step utilizes a C18 column with a mobile phase containing A (25% MeCN in water) and B (30% MeCN in water) with the following gradient:

[0316] Time (min) ％A ％B 0.0-5.0 100 0 20 20 80 25 20 80 30 100 0

[0317] The second step utilized the same column and conditions, but only with an isocratic mobile phase: 20% MeCN in water.

[0318] Those skilled in the art will recognize that the specific column, mobile phase, injection volume and other HPLC parameters may vary.

[0319] Purified steviol glycosides prepared according to the present invention can be used in a variety of consumable products, including but not limited to foods, beverages, pharmaceutical compositions, tobacco products, nutraceutical compositions, oral hygiene compositions, and cosmetic compositions.

[0320] The high-purity reb M obtained in the present invention has a molecular weight of 1291.29 and a molecular formula of C 56 H 90 O 33 , CAS registration number 1220616-44-3, and its structure is presented in Figure 1 It is in the form of a white, tasteless powder. When compared to a 10% sucrose solution, the compound is about 200 times sweeter than sugar.

[0321] Other properties of the pure reb M compound include a melting point of 249°C-250°C and a [α] D 25 Specific rotation of -19.0° (C=1.0) The solubility of reb M in water is about 0.3%, and increases with increasing temperature.

[0322] Reb M is soluble in dilute solutions of methanol, ethanol, n-propanol, and isopropanol. However, it is insoluble in acetone, benzene, chloroform, and ether.

[0323] Reb M obtained according to the present invention is heat stable and pH stable.

[0324] One or more highly purified target glycosides obtained according to the present invention, especially reb D, reb D2, reb M and / or reb M2, can be used "as is" or in combination with other sweeteners, flavors and food ingredients and combinations thereof.

[0325] Non-limiting examples of flavors include, but are not limited to, lime, lemon, citrus, fruit, banana, grape, pear, pineapple, mango, berry, almond, cola, cinnamon, sugar, marshmallow, vanilla, and combinations thereof.

[0326] Non-limiting examples of other food ingredients include, but are not limited to, acidulants, organic acids and amino acids, colorants, bulking agents, modified starches, gums, texturizers, preservatives, antioxidants, emulsifiers, stabilizers, thickeners, gelling agents, and combinations thereof.

[0327] One or more highly purified target glycosides obtained according to the present invention, particularly reb D, reb D2, reb M and / or reb M2, can be prepared in various polymorphic forms, including but not limited to hydrates, solvates, anhydrous forms, amorphous forms and combinations thereof.

[0328] One or more highly purified target steviol glycosides obtained according to the present invention, particularly reb D, reb D2, reb M and / or reb M2, can be incorporated into food, beverages, pharmaceutical compositions, cosmetics, chewing gum, table top products, cereals, dairy products, toothpaste and other oral compositions as high intensity natural sweeteners.

[0329] One or more highly purified target steviol glycosides, particularly reb D, reb D2, reb M and / or reb M2 as sweetening compounds, can be used as the sole sweetener, or can be used together with each other, or with at least one other naturally occurring high intensity sweetener, such as stevia, stevia extract, steviol monoglycoside, steviol diglycoside, stevioside, reb A, reb B, reb C, reb E, reb F, reb G, reb I, reb E, reb H, reb L, reb K, reb J, reb N, reb O, stevioside, dulcoside A, dulcoside B, rubusoside, or other steviol glycosides found in stevia, mogroside, brazzein, neohesperidin dihydrochalcone, glycyrrhizic acid and its salts, thaumatin, perilla, pernandulcin, sapindus mukorossi sesquiterpene glycosides, baiyun ginsenoside, scutellaria glycoside-I, dimethyl-hexahydrofluorene-dicarboxylic acid, abrusoside, glycyrrhizin, carnosifloside ), cyclocaryoside, pterocaryoside, polypodoside A, brazilin, hernandulcin, phillodulcin, smilaxoside, phloridzin, trilobatin, dihydroflavonol, dihydroquercetin-3-acetate, neoastilibin, trans-cinnamaldehyde, monatin and its salts, selligueain A, A), hematoxylin, monellin, oleander, pterocarp A, pterocarp B, mabinin, pentadin, miraculin, curculin, neoculin, chlorogenic acid, cynarin, monk fruit sweetener, mogroside V, mogroside VI, grosmomomside, simone, or other mogrosides found in Siraitia grosvenorii, and combinations thereof.

[0330] In a specific embodiment, reb D2 and / or reb M2 can be used with a sweetener composition comprising a compound selected from the group consisting of reb A, reb B, reb D, NSF-02, mogroside V, Luo Han Guo, psicose, allose, D-tagatose, erythritol, and combinations thereof.

[0331] One or more highly purified target steviol glycosides, particularly reb D, reb D2, reb M and / or reb M2, can also be used in combination with synthetic high-intensity sweeteners, such as sucralose, acesulfame potassium, aspartame, alitame, saccharin, neohesperidin dihydrochalcone, cyclamate, neotame, glycin, sodium p-nitrophenylureidopropionate (suosan), advantame, their salts, and combinations thereof.

[0332] Moreover, one or more highly purified target steviol glycosides, particularly reb D, reb D2, reb M and / or reb M2, can be used in combination with natural sweetener inhibitors, such as gymnemic acid, ziziphin, ziziphin, lactisole, etc. reb D, reb D2, reb M and / or reb M2 can also be combined with various umami enhancers. reb D, reb D2, reb M and / or reb M2 can be mixed with umami and sweet amino acids, such as glutamate, aspartic acid, glycine, alanine, threonine, proline, serine, glutamic acid, lysine, tryptophan and combinations thereof.

[0333] One or more highly purified target steviol glycosides, particularly reb D, reb D2, reb M, can be used in combination with one or more additives selected from the group consisting of carbohydrates, polyols, amino acids and their corresponding salts, polyamino acids and their corresponding salts, sugar acids and their corresponding salts, nucleotides, organic acids, inorganic acids, organic salts (including organic acid salts and organic base salts), inorganic salts, bitter compounds, flavoring agents and flavoring ingredients, astringent compounds, proteins or protein hydrolysates, surfactants, emulsifiers, flavonoids, alcohols, polymers and combinations thereof.

[0334] One or more highly purified target steviol glycosides, particularly reb D, reb D2, reb M and / or reb M2, can be combined with polyols or sugar alcohols. The term "polyol" refers to a molecule containing more than one hydroxyl group. Polyols can be diols, triols or tetraols containing 2,3 and 4 hydroxyls respectively. Polyols can also contain more than four hydroxyls, for example, amyl alcohol, hexanol, heptanol or the like containing 5,6 or 7 hydroxyls respectively. In addition, polyols can also be sugar alcohols, polyols or polymeric alcohols, which are reduced forms of carbohydrates, in which carbonyls (aldehydes or ketones, reducing sugars) have been reduced to primary alcohol hydroxyls or secondary alcohol hydroxyls. 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 isomalt-oligosaccharides, reduced xylo-oligosaccharides, reduced gentio-oligosaccharides, reduced maltose syrup, reduced glucose syrup, hydrogenated starch hydrolysates, polysaccharide alcohols and sugar alcohols or any other carbohydrate that can be reduced that does not adversely affect the taste of the sweetener composition.

[0335] One or more highly purified target steviol glycosides, particularly reb D, reb D2, reb M and / or reb M2, can be combined with reduced-calorie sweeteners such as, for example, D-tagatose, L-glucose, L-sorbose, L-arabinose, and combinations thereof.

[0336] One or more highly purified target steviol glycosides, particularly reb D, reb D2, reb M and / or reb M2, can also be combined with various carbohydrates. The term "carbohydrate" generally refers to an aldehyde or ketone compound substituted with multiple hydroxy groups, having the general formula (CH2O) n, wherein n is 3-30, and their oligomers and polymers. In addition, the carbohydrates of the present invention may be substituted or deoxygenated at one or more positions. Carbohydrates as used herein include unmodified carbohydrates, carbohydrate derivatives, substituted carbohydrates, and modified carbohydrates. As used herein, the phrases "carbohydrate derivatives," "substituted carbohydrates," and "modified carbohydrates" are synonymous. Modified carbohydrates mean any carbohydrate in which at least one atom has been added, removed, or substituted or a combination thereof. Therefore, 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 may be substituted with one or more moieties, such as hydrogen, halogen, haloalkyl, carboxyl, acyl, acyloxy, amino, amide, carboxyl derivatives, alkylamino, dialkylamino, arylamino, alkoxy, aryloxy, nitro, cyano, sulfo, sulfhydryl, imino, sulfonyl, sulfenyl, sulfenyl, sulfinyl, sulfamoyl, alkoxycarbonyl, carboxamido, phosphonyl, phosphinyl, phosphoryl, phosphino, thioester, thioether, oxime, hydrazine, carbamoyl, phospho, phosphonato, or any other reactive functional group, as long as the carbohydrate derivative or substituted carbohydrate is capable of improving the sweet taste of the sweetener composition.

[0337] Examples of carbohydrates that can be used according to the present invention include, but are not limited to, psicose, turanose, allose, tagatose, trehalose, galactose, rhamnose, various cyclodextrins, cyclic oligosaccharides, various types of maltodextrins, dextran, sucrose, glucose, ribulose, fructose, threose, arabinose, xylose, lyxose, allose, altrose, mannose, idose, lactose, maltose, invert sugar, isotrehalose, neotrehalose, isomaltulose, erythrose, deoxyribose, gulose, idose, talose, erythrulose, xylulose, psicose, turanose, cellobiose, amylopectin, glucosamine, mannosamine, fucose, glucuronic acid, gluconic acid, gluconolactone, abequose, galactosamine, beet oligosaccharides, isomaltose oligosaccharides (isomalto oligosaccharides), and oligosaccharides. The carbohydrates used herein include oligosaccharides (maltose, isomaltotriose, panose, etc.), oligoxylose (xylotriose, xylobiose, etc.), xylose terminal oligosaccharides (xylo-terminated oligosaccharides), gentiooligosaccharides (gentiobiose, gentiotriose, gentiotetraose, etc.), sorbose, niger oligosaccharides, palatinose oligosaccharides, fructo-oligomers (kestose, mold erythralose, etc.), malt tetraol (maltotetraol), maltotriol (maltotriol), malto-oligosaccharides (maltotriose, maltotetraose, maltopentaose, maltohexaose, maltoheptaose, etc.), starch, inulin, inulin oligosaccharides, lactulose, melibiose, raffinose, ribose, isomerized liquid sugars such as high fructose corn syrup, coupling sugars and soybean oligosaccharides. In addition, the carbohydrates used herein can be D or L configuration.

[0338] One or more highly purified target steviol glycosides obtained according to the present invention, particularly reb D, reb D2, reb M and / or reb M2, can be used in combination with various physiologically active substances or functional ingredients. Functional ingredients are generally divided into multiple categories, such as carotenoids, dietary fiber, fatty acids, saponins, antioxidants, nutraceuticals, flavonoids, isothiocyanates, phenols, phytosterols and stanols (phytosterols and phytostanols); polyols; prebiotics, probiotics; phytoestrogens; soy protein; sulfides / thiols; amino acids; proteins; vitamins; and minerals. Functional ingredients can also be classified based on their health benefits, such as cardiovascular, cholesterol-lowering, and anti-inflammatory benefits. Exemplary functional ingredients are provided in WO 2013 / 096420, the contents of which are hereby incorporated by reference.

[0339] One or more highly purified target steviol glycosides obtained according to the present invention, particularly reb D, reb D2, reb M and / or reb M2, can be used as high-intensity sweeteners for producing zero-calorie, reduced-calorie or diabetic beverages and foods (with improved taste characteristics). It can also be used for beverages, foods, medicines and other products in which sugar cannot be used. In addition, one or more highly purified target steviol glycosides, particularly reb D, reb D2, reb M and / or reb M2, can also be used as sweeteners, not only for beverages, foods and other products specifically for human consumption, but also for animal feed and fodder with improved characteristics.

[0340] Examples of consumable products in which one or more highly purified target steviol glycosides, particularly reb D, reb D2, reb M and / or reb M2, can be used as sweetening compounds include, but are not limited to, alcoholic beverages such as vodka, wine, beer, liquor and sake, etc.; natural juices; energy drinks; carbonated soft drinks; diet drinks; zero-calorie drinks; reduced-calorie drinks and foods; yogurt drinks; instant juices; instant coffee; powdered instant beverages; canned products; syrups; fermented soybean paste; soy sauce; vinegar; condiments; mayonnaise; ketchup; curry foods; soups; instant broths; bouillon); soy sauce powder; vinegar powder; biscuits of various types; rice crackers; crackers; bread; chocolate; caramel; candy; chewing gum; jelly; pudding; preserved fruit and pickles; fresh cream; jam; marmalade; flower paste; milk powder; ice cream; sorbet; vegetables and fruits packaged in bottles; canned and boiled beans; meat and food cooked in sweet sauces; agricultural vegetable food; seafood; ham; sausage; fish ham; fish sausage; fish sauce; fried fish products; dried seafood; frozen food; preserved seaweed; cured meat; tobacco; medicine; etc. In principle it can have unlimited applications.

[0341] In the manufacture of products such as food, beverages, medicines, cosmetics, tabletop products and chewing gum, conventional methods such as mixing, kneading, dissolving, pickling, infiltration, percolation, spraying, atomizing, pouring and the like can be used.

[0342] Moreover, one or more highly purified target steviol glycosides obtained in the present invention, particularly reb D, reb D2, reb M and / or reb M2, can be used in dry form or liquid form. In one embodiment, a tabletop sweetener comprising reb D2 is provided. In another embodiment, a tabletop sweetener comprising reb M2 is provided.

[0343] Highly purified target steviol glycosides can be added before or after food heat treatment. The amount of one or more highly purified target steviol glycosides, particularly reb D, reb D2, reb M and / or reb M2, depends on the purpose of use. As mentioned above, it can be added separately, or added in combination with other compounds.

[0344] The present invention is also directed to using reb D2 and / or reb M2 to enhance sweetness in beverages. Accordingly, the present invention provides beverages comprising sweeteners and reb D2 and / or reb M2 as sweetness enhancers, wherein reb D2 and / or reb M2 exist at a concentration below their corresponding sweetness recognition threshold or the threshold.

[0345] 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 enhancer," "sweetness amplifier," and "sweetness intensifier."

[0346] The term "sweetness recognition threshold concentration" as generally used herein is the lowest known concentration of sweet compounds that can be perceived by the human taste buds, typically about 1.0% sucrose equivalent (1.0% SE). Generally, when present at or below the sweetness recognition threshold concentration of a given sweetness enhancer, the sweetness enhancer can enhance or intensify the sweetness of the sweetener without providing any significant sweetness by itself; however, when the sweetness enhancer is above their sweetness recognition threshold concentration, they can provide sweetness by themselves. The sweetness recognition threshold concentration is specific to a particular enhancer and can vary based on the beverage matrix. The sweetness recognition threshold concentration can be easily determined by taste testing of a given enhancer in a given beverage matrix until the increasing concentration is greater than 1.0% sucrose equivalent. The concentration providing about 1.0% sucrose equivalent is considered as the sweetness recognition threshold.

[0347] In some embodiments, the sweetener is present in the beverage in an amount from about 0.5% to about 12% by weight, such as, for example, about 1.0% by weight, about 1.5% by weight, about 2.0% by weight, about 2.5% by weight, about 3.0% by weight, about 3.5% by weight, about 4.0% by weight, about 4.5% by weight, about 5.0% by weight, about 5.5% by weight, about 6.0% by weight, about 6.5% by weight, about 7.0% by weight, about 7.5% by weight, about 8.0% by weight, about 8.5% by weight, about 9.0% by weight, about 9.5% by weight, about 10.0% by weight, about 10.5% by weight, about 11.0% by weight, about 11.5% by weight, or about 12.0% by weight.

[0348] In a specific embodiment, the sweetener is present in the beverage in an amount of from 0.5% to about 10% by weight, such as, for example, from about 2% to about 8%, from about 3% to about 7% or from about 4% to about 6%. In a specific embodiment, the sweetener is present in the beverage in an amount of from about 0.5% to about 8% by weight. In another specific embodiment, the sweetener is present in the beverage in an amount of from about 2% to about 8% by weight.

[0349] In one embodiment, the sweetener is a traditional caloric sweetener. Suitable sweeteners include, but are not limited to, sucrose, fructose, glucose, high fructose corn syrup, and high fructose starch syrup.

[0350] In another embodiment, the sweetener is erythritol.

[0351] In another embodiment, the sweetener is a rare sugar. Suitable rare sugars include, but are not limited to, D-allose, D-psicose, L-ribose, D-tagatose, L-glucose, L-fucose, L-arabinose (arbinose), D-turanose, D-leucrose (D-leucrose) and combinations thereof.

[0352] It is contemplated that the sweetener may be used alone or in combination with other sweeteners.

[0353] In one embodiment, the rare sugar is D-allose. In a more specific embodiment, D-allose is present in the beverage in an amount of about 0.5% to about 10% by weight, such as, for example, from about 2% to 8%.

[0354] In another embodiment, the rare sugar is D-psicose.In a more specific embodiment, D-psicose is present in the beverage in an amount of about 0.5% to about 10% by weight, such as, for example, from about 2% to 8%.

[0355] In yet another embodiment, the rare sugar is D-ribose.In a more specific embodiment, D-ribose is present in the beverage in an amount of about 0.5% to about 10% by weight, such as, for example, from about 2% to 8%.

[0356] In yet another embodiment, the rare sugar is D-tagatose.In a more specific embodiment, D-tagatose is present in the beverage in an amount of about 0.5% to about 10% by weight, such as, for example, from about 2% to 8%.

[0357] In a further embodiment, the rare sugar is L-glucose.In a more specific embodiment, L-glucose is present in the beverage in an amount of about 0.5% to about 10% by weight, such as, for example, from about 2% to 8%.

[0358] In one embodiment, the rare sugar is L-fucose. In a more specific embodiment, L-fucose is present in the beverage in an amount of about 0.5% to about 10% by weight, such as, for example, from about 2% to 8%.

[0359] In another embodiment, the rare sugar is L-arabinose.In a more specific embodiment, L-arabinose is present in the beverage in an amount of about 0.5% to about 10% by weight, such as, for example, from about 2% to 8%.

[0360] In yet another embodiment, the rare sugar is D-turanose. In a more specific embodiment, D-turanose is present in the beverage in an amount of about 0.5% to about 10% by weight, such as, for example, from about 2% to 8%.

[0361] In yet another embodiment, the rare sugar is D-leucrose.In a more specific embodiment, D-leucrose is present in the beverage in an amount of about 0.5% to about 10% by weight, such as, for example, from about 2% to 8%.

[0362] Adding a sweetness enhancer at a concentration at or below its sweetness recognition threshold increases the detected sucrose equivalent of a beverage comprising the sweetener and the sweetness enhancer compared to a corresponding beverage in the absence of the sweetness enhancer. Moreover, in the absence of any sweetener, the sweetness can be increased by an amount greater than the detectable sweetness of a solution containing the same concentration of at least one sweetness enhancer.

[0363] Accordingly, the present invention also provides a method for enhancing the sweetness of a beverage containing a sweetener, the method comprising providing a beverage containing a sweetener and adding a sweetness enhancer selected from reb D2, reb M2 or a combination thereof, wherein reb D2 and reb M2 are present at a concentration at or below their sweetness recognition threshold.

[0364] Adding reb D2 and / or reb M2 to a beverage containing a sweetener at a concentration at or below its sweetness recognition threshold can increase the detected sucrose equivalent by from about 1.0% to about 5.0%, such as, for example, about 1.0%, about 1.5%, about 2.0%, about 2.5%, about 3.0%, about 3.5%, about 4.0%, about 4.5% or about 5.0%.

[0365] The following examples show preferred embodiments of the present invention for preparing one or more highly purified target steviol glycosides, particularly reb D, reb D2, reb M and / or reb M2. It should be understood that the present invention is not limited to the materials, ratios, conditions and procedures in these examples, which are merely illustrative.

[0366] Example 1

[0367] In vivo production of UGT76G1

[0368] NcoI and NdeI restriction sites were added to the original nucleic acid sequence described in Genbank Accession No. AAR06912.1. After codon optimization, the following nucleic acid sequence was obtained:

[0369]

[0370] After synthesizing gene and this gene is subcloned into pET30A+ carrier utilizing NdeI and XhoI cloning site, UGT76G1_pET30a+ plasmid is introduced into intestinal bacteria Bl21 (DE3) and intestinal bacteria EC100 by electroporation.The cell that obtains is cultivated in culture dish under the situation that kanamycin exists, and selects appropriate bacterium colony, and allows it to grow in liquid LB substratum (Erenmayer flask).Glycerol is added in suspension as cryoprotectant and aliquots of 400 μ L are stored at-20 ℃ and at-80 ℃.

[0371] A stock aliquot of E. coli BL21 (DE3) containing the pET30A+_UGT76G1 plasmid was thawed and added to 30 mL of LBGKP medium (20 g / L Luria Broth Lennox; 50 mM PIPES buffer pH 7.00; 50 mM phosphate buffer pH 7.00; 2.5 g / L glucose and 50 mg / L kanamycin). This culture was continued at 30° C. for 8 h with shaking at 135 rpm.

[0372] The production medium contained 60 g / L overnight rapid expression TB medium (Novagen), 10 g / L glycerol and 50 mg / L kanamycin. The medium was stirred at 20°C while taking samples to measure OD and pH. The culture produced significant growth and achieved a good OD. After 40 hours, the cells were harvested by centrifugation and frozen to produce a cell wet weight of 12.7 g.

[0373] Lysis was performed by adding Bugbuster master mix (Novagen), and the lysate was recovered by centrifugation and kept frozen. Activity test was performed on the thawed lysate.

[0374] Example 2

[0375] In vitro production of UGT76G1

[0376] Use the S30 T7 high yield protein expression system kit from Promega. 4 μg of UGT76G1_pET30a+ plasmid from Escherichia coli EC100 was mixed with 80 μL of S30 premix plus, and 72 μL of S30 T7 extract was added. Nuclease-free water was added to obtain a total volume of 200 μL, and the resulting solution was incubated at 30°C for 2h. 180 μL was used for catalytic test reactions.

[0377] Example 3

[0378] In vitro production of UGT91D2

[0379] NcoI and NdeI restriction sites were added to the original nucleic acid sequence described in Genbank Accession No. ACE87855.1. After codon optimization, the following nucleic acid sequence was obtained:

[0380]

[0381] After synthesizing gene and this gene is subcloned into pET30A+ carrier utilizing NcoI and XhoI cloning site, UGT91D2_pET30a+ plasmid is introduced into Escherichia coli EC100 by electroporation.The cell that obtains is cultivated in the presence of kanamycin, and appropriate bacterium colony is selected and allowed to grow in liquid LB substratum (Erenmayer flask).Glycerol is added in suspension as cryoprotectant and aliquots of 400 μ L are stored at-20 ℃ and at-80 ℃.

[0382] The S30 T7 High Yield Protein Expression System Kit from Promega was used for in vitro synthesis of proteins.

[0383] 4 μg of UGT91D2_pET30a+ plasmid was mixed with 80 μL of S30 premix plus, and 72 μL of S30T7 extract was added. Nuclease-free water was added to obtain a total volume of 200 μL, and the resulting solution was incubated at 30°C for 2 h. 5 μL was used for SDS-PAGE analysis, and the remaining 45 μL was used in the catalytic test reaction.

[0384] Example 4

[0385] Catalytic reactions with in vivo generated UGT76G1

[0386] The total reaction volume was 5.0 mL with the following composition: 50 mM sodium phosphate buffer pH 7.2, 3 mM MgCl2, 2.5 mM UDP-glucose, 0.5 mM stevioside and 500 μL of UGT76G1 thawed lysate. The reaction was run at 135 rpm on an orbital shaker at 30 ° C. For each sample, 460 μL of the reaction mixture was quenched with 40 μL of 2N H2SO4 and 420 μL of methanol / water (6 / 4). The sample was immediately centrifuged and maintained at 10 ° C and then analyzed by HPLC (CAD). HPLC indicated that stevioside was almost completely converted to rebaudioside A ( Figure 4 ).

[0387] Example 5

[0388] Catalytic reactions with in vitro produced UGT91D2

[0389] The total reaction volume was 0.5 mL with the following composition: 50 mM sodium phosphate buffer pH 7.2, 3 mM MgCl2, 3.8 mM UDP-glucose, 0.1 mM rebaudioside A and 180 μL of in vitro produced UGT91D2. The reaction was run at 135 rpm on an orbital shaker at 30 ° C. For each sample, 450 μL of the reaction mixture was quenched with 45 μL of 2N H2SO4 and 405 μL of 60% MeOH. After centrifugation, the supernatant was analyzed by HPLC (CAD). HPLC indicated that 4.7% of rebaudioside A was converted to rebaudioside D after 120 h.

[0390] Example 6

[0391] Catalytic reactions with in vitro generated UGT76G1

[0392] The total reaction volume was 2 mL with the following composition: 50 mM sodium phosphate buffer pH 7.2, 3 mM MgCl2, 3.8 mM UDP-glucose, 0.5 mM rebaudioside D and 180 μL of in vitro produced UGT76G1. The reaction was run at 135 rpm on an orbital shaker at 30 ° C. For each sample, 400 μL of the reaction mixture was quenched with 40 μL of 2N H2SO4 and 360 μL of 60% MeOH. After centrifugation, the supernatant was analyzed by HPLC (CAD). HPLC indicated that 80% of rebaudioside D was converted to rebaudioside M ( Figure 5 ).

[0393] For Examples 7 to 12, the following abbreviations are used:

[0394] LBGKP medium: 20g / L Luria Broth Lennox; 50mM PIPES buffer pH 7.00; 50mM phosphate buffer pH 7.00; 2.5g / L glucose and 50mg / L kanamycin or ampicillin LB medium: (20g / L Luria Broth Lennox)

[0395] Example 7

[0396] Preparation and activity of UGT76G1 produced by pET30a+ plasmid and BL21(DE3) expression strain

[0397] The pET30a+_UGT76G1 plasmid was transformed into the BL21 (DE3) expression strain (Lucigen E. EXPRESS electrocompetent cells). The obtained cells were grown on LB agar medium in a petri dish in the presence of kanamycin. Suitable colonies were selected and grown in liquid LBGKP medium containing kanamycin. Glycerol was added and aliquots of 400 μL were stored at -20°C and at -80°C.

[0398] The storage aliquot was thawed and added to 30mL of LBGKP culture medium. The culture was carried out for 8h at 30°C shaking, and then used to inoculate 400mL of the production medium containing 60g / L of "Overnight Rapid Expression TB Culture Medium" (Novagen, reference 71491-5), 10g / L of glycerol and 50mg / L of kanamycin. The culture medium was stirred at 20°C, and samples were taken to measure OD (600nm) and pH simultaneously. After 40 hours, cells were harvested by centrifugation and frozen. The wet weight of the cells obtained was 10.58g.

[0399] 3.24 g of the obtained precipitate were lysed by adding 8.1 mL of "Bugbuster Master Mix" (Novagen, reference 71456) and 3.5 mL of water. The lysate was recovered by centrifugation and kept frozen.

[0400] Example 8

[0401] Preparation and activity of UGT76G1 produced by pET30a+ plasmid and Tuner (DE3) expression strain

[0402] The pET30a+_UGT76G1 plasmid was transformed into the Tuner (DE3) expression strain (Novagen Tuner tm The obtained cells were grown on LB agar medium in a petri dish in the presence of kanamycin. Suitable colonies were selected and grown in liquid LBGKP medium containing kanamycin). Glycerol was added and aliquots of 400 μL were stored at -20°C and at -80°C.

[0403] The storage aliquot was thawed and added to 100 mL of LB medium containing 50 mg / L kanamycin. This culture was continued for 15 h at 30 ° C with shaking. 4.4 mL of this culture was used to inoculate 200 mL of production medium containing LB. The medium was stirred at 37 ° C until an OD (600 nm) of 0.9 was obtained, followed by the addition of 400 μL of 100 mM IPTG solution, and the medium was stirred at 30 ° C for 4 hours. The cells were harvested by centrifugation and frozen. The wet weight of the cells obtained was 1.38 g.

[0404] The obtained pellet was lysed by adding 4.9 mL of "Bugbuster Master Mix" (Novagen, reference 71456) and 2.1 mL of water. The lysate was recovered by centrifugation and kept frozen.

[0405] Example 9

[0406] Preparation and activity of UGT76G1 produced by pMAL plasmid and BL21 expression strain

[0407] After the synthetic UGT76G1 gene was subcloned into the pMAL plasmid using the Nde1 and Sal1 cloning sites, the pMAL_UGT76G1 plasmid was transformed into a BL21 expression strain (New England Biolabs BL21 competent E. coli) by heat shock treatment. The obtained cells were grown on LB agar medium in a culture dish in the presence of ampicillin. Suitable colonies were selected and grown in liquid LBGKP medium containing ampicillin). Glycerol was added and aliquots of 400 μL were stored at -20°C and at -80°C.

[0408] The storage aliquot was thawed and added to 30 mL of LBGKP medium. The culture was carried out for 8 h at 30 ° C with shaking, and then used to inoculate 400 mL of a production medium containing 60 g / L of "Overnight Rapid Expression TB Medium" (Novagen, reference 71491-5), 10 g / L of glycerol and 50 mg / L of ampicillin. The medium was stirred at 20 ° C, and samples were taken to measure OD and pH. After 40 hours, the cells were harvested by centrifugation and frozen. The wet weight of the cells obtained was 5.86 g.

[0409] 2.74 g of the obtained precipitate were lysed by adding 9.6 mL of "Bugbuster Master Mix" (Novagen, reference 71456) and 4.1 mL of water. The lysate was recovered by centrifugation and kept frozen.

[0410] Example 10

[0411] Preparation and activity of UGT76G1 produced by pMAL plasmid and ArcticExpress expression strain

[0412] By heat shock treatment, pMAL_UGT76G1 plasmid is transformed into ArticExpress expression strain (Agilent (Agilent) ArcticExpress competent cells). The cells obtained are grown on LB agar medium in a culture dish in the presence of ampicillin and geneticin. Select suitable bacterium colonies, and grow them in the liquid LBGKP medium containing ampicillin and geneticin. Add glycerol and store 400 μ L of aliquots at -20 ℃ and at -80 ℃.

[0413] The storage aliquot is thawed and added to 30mL of LBGKP culture medium (containing ampicillin and geneticin). Under 30 ℃ of shaking, the culture is carried out for 8h, and then used to inoculate 400mL of the production culture medium containing "overnight rapid expression TB culture medium" (Novagen company, reference 71491-5) of 60g / L, 10g / L of glycerol and 50mg / L of ampicillin. The culture medium is stirred at 12 ℃, and the sample is taken to measure OD (600nm) and pH simultaneously. After 68 hours, the cells are harvested by centrifugation, and they are frozen. The wet weight of the cells obtained is 8.96g.

[0414] 2.47 g of the obtained precipitate were lysed by adding 8.73 mL of "Bugbuster Master Mix" (Novagen, reference 71456) and 3.79 mL of water. The lysate was recovered by centrifugation and kept frozen.

[0415] Example 11

[0416] Preparation and activity of UGT76G1 produced by pCOLDIII plasmid and ArcticExpress expression strain

[0417] After using Nde1 and Xho1 cloning sites to subclone the synthetic UGT76G1 gene into the pCOLDIII plasmid, the pCOLDIII_UGT76G1 plasmid was transformed into the ArcticExpress expression strain (Agilent ArcticExpress competent cells) by heat shock treatment. The cells obtained were grown on LB agar medium in a culture dish in the presence of ampicillin and geneticin. Suitable bacterium colonies were selected and grown in the liquid LBGKP medium containing ampicillin and geneticin. Glycerol was added and aliquots of 400 μL were stored at -20 °C and -80 °C.

[0418] The storage aliquot is thawed and added in the LBGKP substratum (containing ampicillin and geneticin) of 30mL.Under 30 ℃ of shakings, this cultivation is carried out for 8h, and it is used to inoculate the production substratum of " overnight fast expression TB substratum " (Novagen company, reference 71491-5) containing 60g / L, the glycerol of 10g / L and the kanamycin of 50mg / L of 400mL subsequently.This substratum is stirred at 12 ℃, and sample is taken and measured OD (600nm) and pH simultaneously.After 63 hours, by centrifugal harvesting cell, and it is frozen.The wet weight of the cell that obtains is 6.54g.

[0419] 2.81 g of the obtained precipitate were lysed by adding 9.8 mL of "Bugbuster Master Mix" (Novagen, reference 71456) and 4.2 mL of water. The lysate was recovered by centrifugation and kept frozen.

[0420] Example 12

[0421] Preparation and activity of UGT76G1 produced by pCOLDIII plasmid and Origami2(DE3) expression strain

[0422] The pCOLDIII_UGT76G1 plasmid was transformed into Origami2 (DE3) expression strain (Novagen OrigamiTM2 (DE3) competent cells) by heat shock treatment. The obtained cells were grown on LB agar medium in a culture dish in the presence of ampicillin. Suitable bacterium colonies were selected and grown in liquid LBGKP medium containing ampicillin. Glycerol was added and aliquots of 400 μL were stored at -20 °C and -80 °C.

[0423] The storage aliquot was thawed and added to 30mL of LBGKP culture medium (containing ampicillin). The culture was carried out for 8h at 30°C of shaking, and then used to inoculate 400mL of the production medium of "overnight rapid expression TB culture medium" (Novagen, reference 71491-5) containing 60g / L, 10g / L of glycerol and 50mg / L of kanamycin. The culture medium was stirred at 12°C, and samples were taken to measure OD (600nm) and pH simultaneously. After 68 hours, cells were harvested by centrifugation, and were frozen. The wet weight of the cells obtained was 2.53g.

[0424] 1.71 g of the obtained precipitate were lysed by adding 6.0 mL of "Bugbuster Master Mix" (Novagen, reference 71456) and 1.9 mL of water. The lysate was recovered by centrifugation and kept frozen.

[0425] Example 13

[0426] Activity assay

[0427] Activity testing for the conversion of stevioside to rebaudioside A and rebaudioside D to rebaudioside M was performed at a 5 mL scale using 500 μL of thawed lysate using 0.5 mM substrate, 2.5 mM UDP-glucose and 3 mM MgCl in 50 mM sodium phosphate buffer at pH 7.2. Samples were taken and analyzed by HPLC. The results of the different preparations of UGT76G1 are summarized in the table below.

[0428]

[0429]

[0430] *Notes

[0431] The conversion activity of stevioside and rebaudioside M is mentioned per mL of lysate. 1 U is equivalent to the conversion of 1 μmol of substrate in 1 hour at 30°C and pH 7.2.

[0432] Example 14

[0433] 50 mL scale reaction for the conversion of rebaudioside D to rebaudioside M

[0434] The conversion of rebaudioside D to baudioside M on a 50 mL scale was carried out using 5 mL of the lysate of Example 12. The reaction medium consisted of 50 mM sodium phosphate buffer pH 7.2, 3 mM MgCl2, 2.5 mM UDP-glucose and 0.5 mM rebaudioside D. After this reaction was carried out at 30 ° C with shaking for 90 hours, 50 mL of ethanol was added, and the resulting mixture was stirred for 1 hour at -20 ° C. After centrifugation at 5000 g for 10 minutes, the supernatant was purified via ultrafiltration (Vivaflow MWCO 30000). 78 mL of permeate was obtained, and 9 mL of the retentate was diluted with 9 mL of ethanol and ultrafiltration (Vivaflow MWCO30000) was performed again. Another 14 mL of filtrate was obtained, which was combined with the first permeate. The combined permeate was concentrated at 30 ° C under reduced pressure until 32 mL of a clear solution was obtained.

[0435] The HPLC trace of the product mixture is shown in Figure 3HPLC was performed on an Agilent 1200 series equipped with a binary pump, autosampler, and thermostated column oven. The method was isocratic with a mobile phase consisting of 70% water (0.1% formic acid): 30% acetonitrile. The flow rate was 0.1 μL / min. The column used was a Phenomenex Prodigy 5μODS(3)100A; 250 x 2 mm. The column temperature was maintained at 40°C. The injection volume was 20-40 μl.

[0436] Isolate the material eluting at 31.325 minutes. 1 Comparison of H NMR and HRMS with known reb M standards confirmed that the substance was reb M.

[0437] Example 15

[0438] Preparation of UGT91D2 using pMAL plasmid and BL21 expression strain

[0439] After the synthetic UGT91D2 gene was subcloned into the pMAL plasmid using the Nde1 and Sal1 cloning sites, the pMAL_UGT91D2 plasmid was transformed into a BL21 expression strain (New England Biolabs BL21 competent Escherichia coli) by heat shock treatment. The obtained cells were grown on LB agar medium in a culture dish in the presence of ampicillin. Suitable colonies were selected and grown in liquid LBGKP medium containing ampicillin). Glycerol was added and aliquots of 400 μL were stored at -20°C and -80°C.

[0440] The storage aliquot was thawed and added to 30 mL of LBGKP medium. The culture was carried out for 8 h at 30 ° C with shaking, and then used to inoculate 400 mL of a production medium containing 60 g / L of "Overnight Rapid Expression TB Medium" (Novagen, reference 71491-5), 10 g / L of glycerol and 50 mg / L of ampicillin. The medium was stirred at 20 ° C, and samples were taken to measure OD and pH. After 40 hours, the cells were harvested by centrifugation and frozen. The wet weight of the cells obtained was 12.32 g.

[0441] 2.18 g of the obtained precipitate were lysed by adding 7.7 mL of "Bugbuster Master Mix" (Novagen, reference 71456) and 3.2 mL of water. The lysate was recovered by centrifugation and used directly for the activity test.

[0442] Example 16

[0443] Preparation of UGT91D2 using pMAL plasmid and ArcticExpress expression strain

[0444] By heat shock treatment, pMAL_UGT91D2 plasmid is transformed into ArcticExpress expression strain (Agilent ArcticExpress competent cells).The cells obtained are grown on LB agar medium in a culture dish in the presence of ampicillin and geneticin.Select suitable bacterium colonies, and grow them in the liquid LBGKP medium containing ampicillin and geneticin.Add glycerol and store 400 μ L of aliquots at -20 ℃ and at -80 ℃.

[0445] The storage aliquot is thawed and added in the LBGKP substratum (containing ampicillin and geneticin) of 30mL.Under 30 ℃ of shakings, this cultivation is carried out for 8h, and it is used to inoculate the production substratum of " overnight fast expression TB substratum " (Novagen company, reference 71491-5) containing 60g / L, the glycerol of 10g / L and the ampicillin of 50mg / L of 400mL subsequently.This substratum is stirred 16 hours at 20 ℃, then stirred 50 hours in addition at 12 ℃, and sample is measured OD (600nm) and pH simultaneously.By centrifugal harvesting cell, and it is frozen.The wet weight of the cell that obtains is 15.77g.

[0446] 2.57 g of the obtained precipitate were lysed by adding 9.0 mL of "Bugbuster Master Mix" (Novagen, reference 71456) and 3.8 mL of water. The lysate was recovered by centrifugation and used directly for the activity test.

[0447] Example 17

[0448] Preparation of UGT91D2 using pET30a+ plasmid and Tuner (DE3) expression strain

[0449] The pET30a+_UGT91D2 plasmid was transformed into the Tuner (DE3) expression strain (Novagen Tuner tm The cells were grown on LB agar medium in a culture dish in the presence of kanamycin. Suitable colonies were selected and grown in liquid LBGKP medium (containing kanamycin). Glycerol was added and aliquots of 400 μL were stored at -20°C and -80°C.

[0450] The storage aliquot was thawed and added to 100 mL of LB medium containing 50 mg / L kanamycin. This culture was continued for 15 h at 30 ° C with shaking. 6.2 mL of this culture was used to inoculate 500 mL of production medium containing LB. The medium was stirred at 37 ° C until an OD (600 nm) of 0.9 was obtained, followed by the addition of 500 μ L of 100 mM IPTG solution (IPTG concentration in the medium was 100 μ M), and the medium was stirred at 30 ° C for 4 hours, harvested by centrifugation, and frozen. The wet weight of the cells obtained was 4.02 g.

[0451] 1.92 g of the obtained precipitate were lysed by adding 6.8 mL of "Bugbuster Master Mix" (Novagen, reference 71456) and 2.8 mL of water. The lysate was recovered by centrifugation and directly subjected to the activity test.

[0452] Example 18

[0453] Preparation of UGT91D2 using pET30a+ plasmid and ArcticExpress expression strain

[0454] By heat shock treatment, pET30a+_UGT91D2 plasmid is transformed into ArcticExpress (DE3) expression strain (Agilent ArcticExpress competent cells).The cell obtained is grown on LB agar medium in culture dish in the presence of kanamycin and geneticin.Select suitable bacterium colony, and make it grow in the liquid LBGKP medium containing kanamycin and geneticin.Add glycerol and the aliquot of 400 μ L is stored at -20 ℃ and at -80 ℃.

[0455] The storage aliquot is thawed and added in the LBGKP substratum (containing kanamycin and geneticin) of 30mL.Under 30 ℃ of shakings, this cultivation is carried out for 8h, and it is used to inoculate the production substratum of " overnight fast expression TB substratum " (Novagen company, reference 71491-5) containing 60g / L, the glycerol of 10g / L and the ampicillin of 50mg / L of 400mL subsequently.This substratum is stirred 16 hours at 20 ℃, then stirred 50 hours in addition at 12 ℃, and sample is measured OD (600nm) and pH simultaneously.After 60 hours, by centrifugal harvesting cell, and it is frozen.The wet weight of the cell that obtains is 16.07g.

[0456] 3.24 g of the precipitate obtained were lysed by adding 11.4 mL of "Bugbuster Master Mix" (Novagen, reference 71456) and 4.8 mL of water. The lysate was recovered by centrifugation and used directly for the activity test.

[0457] Example 19

[0458] Activity assay of in vivo preparations of UGT91D2

[0459] Activity testing for the conversion of rubusoside to stevioside was performed on a 5 mL scale using 1000 μL of lysate using 0.5 mM substrate, 2.5 mM UDP-glucose and 3 mM MgCl in 50 mM sodium phosphate buffer at pH 7.2. Samples were obtained and analyzed by HPLC. The results of different preparations of UGT91D2 are summarized in the table below.

[0460]

[0461] *Note: Activity is given per mL of lysate. 1 U is equivalent to 1 μmol of substrate converted in 1 hour at 30°C and pH 7.2.

[0462] Example 20

[0463] Other enzymes for the conversion of rebaudioside A to rebaudioside D

[0464] The genes of the following UDP-glucosyltransferases were identified from public databases, synthesized by DNA2.0, and subsequently subcloned into pET30a+ vector.

[0465]

[0466] The amino acid sequence is as follows:

[0467] >gi|115454819|ref|NP_001051010.1|Os03g0702500[Japonica rice subspecies]

[0468] MDDAHSSQSPLHVVIFPWLAFGHLLPCLDLAERLAARGHRVSFVSTPRNLARLPPVRPELAELVDLVALPLPRVDGLPDGAEATSDVPFDKFELHRKAFDGLAAPFSAFLDTACAGGKRPDWVLADLMHHWVALASQERGVPCAMILPCSAAVVASSAPPTESSADQREAIVRSMGTAAPSFEAKRATEEFATEGASGVSIMTRYSLTLQRSKLVAMRSCPELEPGAFTILTRFYGKPVVPFGLLPPRPDGARGVSKNGKHDAIMQWLDAQPAKSVVYVALGSEAPMSADLLRELAHGLDLAGTRFLWAMRKPAGVDADSVLPAGFLGRTGERGLVTTRWAPQVSILAHAAVCAFLTHCGWGSVVEGLQFGHPLIMLPILGDQGPNARILEGRKLGVAVPRNDEDGSFDRGGVAGAVRAVVVEEEGKTFFANARKLQEIVADREREERCIDEFVQHLTSWNELKNNSDGQYP(SEQ ID NO:3).

[0469] >gi|187373030|gb|ACD03249.1|UDP - glycosyltransferase [Avena strigosa]

[0470] MAVKDEQQSPLHILLFPFLAPGHLIPIADMAALFASRGVRCTILTTPVNAAIIRSAVDRANDAFRGSDCPAIDISVVPFPDVGLPPGVENGNALTSPADRLKFFQAVAELREPFDRFLADNHPDAVVSDSFFHWSTDAAAEHGVPRLGFLGSSMFAGSCNESTLHNNPLETAADDPDALVSLPGLPHRVELRRSQMMDPKKRPDHWALLESVNAADQKSFGEVFNSFHELEPDYVEHYQTTLGRRTWLVGPVALASKDMAGRGSTSARSPDADSCLRWLDTKQPGSVVYVSFGTLIRFSPAELHELARGLDLSGKNFVWVLGRAGPDSSEWMPQGFADLITPRGDRGFIIRGWAPQMLILNHRALGGFVTHCGWNSTLESVSAGVPMVTWPRFADQFQNEKLIVEVLKVGVSIGAKDYGSGIENHDVIRGEVIAESIGKLMGSSEESDAIQRKAKDLGAEARSAVENGGSSYNDVGRLMDELMARRSSVKVGEDIIPTNDGL(SEQ ID NO:4).

[0471] >gi|460409128|ref|XP_004249992.1|Predicted: anthocyanin-3-O-glucosyltransferase 2-O-glucuronosyltransferase-like [Solanum lycopersicum]

[0472] MSPKLHKELFFHSLYKKTRSNHTMATLKVLMFPFLAYGHISPYLNVAKKLADRGFLIYFCSTPINLKSTIEKIPEKYADSIHLIELHLPELPQLPPHYHTTNGLPPNLNQVLQKALKMSKPNFSKILQNLKPDLVIYDILQRWAKHVANEQNIPAVKLLTSGAAVFSYFFNVLKKPGVEFPFPGIYLRKIEQVRLSEMMSKSDKEKELEDDDDDDDLLVDGNMQIMLMSTSRTIEAKYIDFCTALTNWKVVPVGPPVQDLITNDVDDMELIDWLGTKDENSTVFVSFGSEYFLSKEDMEEVAFALELSNVNFIWVARFPKGEERNLEDALPKGFLERIGERGRVLDKFAPQPRILNHPSTGGFISHCGWNSAMESIDFGVPIIAMPMHLDQPMNARLIVELGVAVEIVRDDDGKIHRGEIAETLKGVITGKTGEKLRAKVRDISKNLKTIRDEEMDAAAEELIQLCRNGN(SEQ ID NO:5).

[0473] >gi|222619587|gb|EEE55719.1|Hypothetical protein OsJ_04191 [Oryza sativa subsp. japonica]

[0474] MHVVMLPWLAFGHILPFAEFAKRVARQGHRVTLFSTPRNTRRLIDVPPSLAGRIRVVDIPLPRVEHLPEHAEATIDLPSNDLRPYLRRAYDEAFSRELSRLLQETGPSRPDWVLADY AAYWAPAAASRHGVPCAFLSLFGAAALCFFGPAETLQGRGPYAKTEPAHLTAVPEYVPFPTTVAFRGNEARELFKPSLIPDESGVSESYRFSQSIEGCQLVAVRSNQEFEPEWLELL GELYQKPVIPIGMFPPPPPQDVAGHEETLRWLDRQEPNSVVYAAFGSEVKLTAEQLQRIALGLEASELPFIWAFRAPPDAGDGDGLPGGFKERVNGRGVVCRGWVPQVKFLAHASVG GFLTHAGWNSIAEGLANGVRLVLLPLMFEQGLNARQLAEKKVAVEVARDEDDGSFAANDIVDALRRVMVGEEGDEFGVKVKELAKVFGDDEVNDRYVRDFLKCLSEYKMQRQG(SEQ ID NO:6).

[0475] >gi|297795735|ref|XP_002865752.1|UDP-glucuronylase / UDP-glucosyltransferase family protein [Arabidopsis thaliana subsp. lyrata]

[0476] MDDKKEEVMHIAMFPWLAMGHLLPFLRLSKLLAQKGHKISFISTPRNILRLPKLPSNLSSSITFVSFPLPSISGLPPSSESSMDVPYNKQQSLKAAFDLLQPPLTEFLRLSSPDWIIYDYASHWLPSIA KELGISKAFFSLFNAATLCFMMGPSSSLIEESRSTPEDFTVVPPWVPFKSTIVFRYHEVSRYVEKTDEDVTGVSDSVRFGYTIDGSDAVFVRSCPEFEPEWFSLLQDLYRKPVFPIGFLPPVIEDDDDDTT WVRIKEWLDKQRVNSVVYVSLGTEASLRREELTELALGLEKSETPFFWVLRNEPQIPDGFEERVKGRGMVHVGWVPQVKILSHESVGGFLTHCGWNSVVEGIGFGKVPIFLPVLNEQGLNTRLLQGKGLG VEVLRDERDGSFGSDSVADSVRLVMIDDAGEEIREKVKLMKGLFGNMDENIRYVDELVGFMRNDESSQLKEEEEEDDCSDDQSSEVSSETDEKELNLDLKEEKRRISVYKSLSSEFDDYVANEKMG(SEQ IDNO:7).

[0477] The tested plasmids are received in microtiter plates containing the plasmid as a lyophilized solid in each individual well.

[0478] Plasmid suspension. 24 μL of ultrapure sterile water was added to each well, and the microtiter plate was shaken at room temperature for 30 minutes. Subsequently, the plate was incubated at 4°C for 1 hour. The contents of each well were further mixed by pipetting up and down. 1 μL of the suspension was used for plasmid quantification by Qubit2.0 analysis. The amount of plasmid measured was:

[0479] Microtiter plates Location Internal Standard [Plasmid] ng / μL C908201 A1 S115N01 A1 32.8 C908201 G2 S115N01 G2 41.0 C908201 A7 S115N05 A7 56.6 C912666 E1 S115N06 E1 64.0 C912666 C2 S115N06 C2 31.4

[0480] Transform competent cells with the plasmid. Take aliquots of chemically competent EC100 cells from a refrigerator at -80°C and store them on ice. Thaw the cells on ice for 10 minutes. Add 10 μL of the dilution of the above-mentioned plasmid solution to a 1.5 mL sterile microtube (in order to transform each cell with 50 pg of DNA) and store on ice. Add 100 μL of chemically competent cells to each microtube. After incubating the chemically competent cell plasmid mixture on ice for 20 min, heat shock was performed at 42°C for 30 seconds.

[0481] Further incubation was carried out on ice for 2 minutes. 300 μL of SOC culture medium was added to each microtube, and the resulting mixture was transferred to a 15 mL sterile tube. After incubation at 37°C for 1 hour under 135 rpm shaking, the mixture was coated on a solid Luria broth medium containing kanamycin 50 μg / mL. The culture dish was incubated at 37°C for 16 hours.

[0482] Preparation of stock solutions in glycerol and purification of plasmids . Add 10 mL of Luria broth medium containing 50 μg / mL kanamycin to a 50 mL sterile centrifuge tube (Falcon Tube). Inoculate the medium with the colony isolated from the above culture dish, and allow the culture to incubate at 37°C with shaking at 135 rpm for 16 hours.

[0483] Add 600 μL of culture to a 1.5 mL sterile microtube containing 300 μL of 60% sterile glycerol solution. Store the stock solution at -80 °C.

[0484] The remainder of the culture was centrifuged at 5,525 g for 10 minutes at 10° C., and after removing the supernatant, the pellet was stored on ice. The resulting plasmid was purified according to the Qiagen Qiaprep Spin Miniprep Kit (reference: 27106) and the plasmid yield was measured at 260 nm. The plasmid solution was stored at 4° C. The amount of plasmid was determined as follows:

[0485] Microtiter plates Location Internal Standard Test [Plasmid] ng / μL C908201 A1 S115N01 A1 115.7 C908201 G2 S115N01 G2 120.4 C908201 A7 S115N05 A7 293.8 C912666 E1 S115N06 E1 126.1 C912666 C2 S115N06 C2 98.8

[0486] In vitro expression of enzymes 18 μL of plasmid solution (containing approximately 1.5 μg of plasmid) was used for in vitro expression according to the Promega S30T7 High-Yield Protein Expression System (Reference: L1110) kit. The expression medium was generated as follows:

[0487] S30 Premix Plus T7 S30 Extract total test 30μL 27μL 57μL refer to 20μL 18μL 38μL

[0488] The prepared expression medium mixture was added to the plasmid solution, and the solution was incubated at 30°C for 3 hours while mixing the mixture every 45 minutes. 5 μL of the mixture was frozen, and the remainder was used for the catalytic test of the conversion of rebaudioside A to rebaudioside D.

[0489] Catalytic assay for the conversion of rebaudioside A to rebaudioside D.The reaction mixture of 430 μ L containing 0.5 mM rebaudioside A, 3 mM MgCl , 50 mM phosphate buffer (pH 7.2) and 2.5 mM UDP-glucose is added to the sterile microtube of 1.5 mL. The enzyme expression culture medium of 52 μ L is added, and the resulting mixture is allowed to react at 30 ℃ for 24 hours. After 2 hours, 16 hours and 24 hours, 125 μ L of sample is taken, and it is added to 115 μ L of 60% methanol and 10 μ L of 2N H 2 SO 4. The sample of quenching is centrifuged at room temperature for 2 minutes at 18,000 g. 200 μ L is transferred to HPLC bottle and analyzed.

[0490] HPLC analysis The HPLC assay was performed as follows:

[0491] equipment

[0492]

[0493] Instrument conditions

[0494]

[0495] Mobile phase gradient program

[0496] Time (min) % of water containing 0.04% acetic acid Methanol% 0 40 60 8 25 75 10 25 75 11 40 60 15 40 60

[0497] Enzyme S115N05 A7 had the highest activity (about 22.4%) for the conversion of Reb A to Reb D. At least three enzymes produced significant amounts of an unknown glycoside (labeled as Reb UNK; subsequently identified as reb D2) along with reb D. The unknown peak at about 4.5 minutes was subsequently identified as reb M2.

[0498] The HPLC assay results are provided below:

[0499]

[0500] Example 21

[0501] Activity of EUGT11 produced in vitro

[0502] The EUGT11 gene described in WO / 2013 / 022989A2 was synthesized by DNA2.0 and subsequently subcloned into the pET30a+ vector.

[0503]

[0504] The amino acid sequence is as follows:

[0505] >gi|41469452|gb|AAS07253.1|Hypothetical UDP-glucuronyl and UDP-glucosyltransferase [Oryza sativa subsp. japonica] EUGT11 enzyme from patent application WO / 2013 / 022989 A2

[0506] (SEQ ID NO:8).

[0507] The tested plasmids were received in microtiter plates containing the plasmids as lyophilized solids in individual wells.

[0508] Plasmid suspension 24 μL of ultrapure sterile water was added to the wells, and the microtiter plate was shaken for 30 minutes at room temperature. Subsequently, the plate was incubated at 4°C for 1 hour. The contents of the wells were further mixed by pipetting up and down. 1 μL of the suspension was used for plasmid quantification by Qubit2.0 analysis. The amount of plasmid was determined as follows:

[0509] Microtiter plates Location Internal Standard Test [Plasmid] ng / μL C912666 G4 S115N08 G4 19.2

[0510] Transform competent cells with the plasmid. Take aliquots of chemically competent EC100 cells from a refrigerator at -80°C and store them on ice. Thaw the cells on ice for 10 minutes. Add 10 μL of the dilution of the above-mentioned plasmid solution to a 1.5mL sterile microtube (in order to transform each cell with 50pg of DNA) and store on ice. Add 100 μL of chemically competent cells to the microtube. After incubating the chemically competent cells / plasmid mixture on ice for 20 minutes, heat shock was performed at 42°C for 30 seconds.

[0511] Further incubation was carried out on ice for 2 minutes. 300 μL of SOC culture medium was added to the microtube, and the resulting mixture was transferred to a 15 mL sterile tube. After incubation at 37°C for 1 hour under 135 rpm shaking, the mixture was spread on a solid Luria broth medium containing kanamycin 50 μg / mL. The culture dish was incubated at 37°C for 16 hours.

[0512] Preparation of stock solutions in glycerol and purification of plasmids. To a 50 mL sterile Falcon Tube, 10 mL of Luria broth medium containing 50 μg / mL kanamycin was added. The medium was inoculated with the colony isolated from the above culture dish, and the culture was allowed to incubate at 37° C. with shaking at 135 rpm for 16 hours.

[0513] Add 600 μL of culture to a 1.5 mL sterile microtube containing 300 μL of 60% sterile glycerol solution. Store the stock solution at -80 °C.

[0514] The remainder of the culture was centrifuged at 5,525 g for 10 minutes at 10° C., and after removing the supernatant, the pellet was stored on ice. The resulting plasmid was purified according to the Qiagen Qiaprep Spin Miniprep Kit (reference: 27106) and the plasmid yield was measured at 260 nm. The plasmid solution was stored at 4° C. The amount of plasmid was determined as follows:

[0515] Microtiter plates Location Internal Standard Test [Plasmid] ng / μL C912666 G4 S115N08 G4 38.4

[0516] In vitro expression of EUGT1118 μL of the diluted plasmid solution (containing approximately 1.5 μg of plasmid) was used for in vitro expression according to the Promega S30 T7 High-Yield Protein Expression System (Reference: L1110) kit. The expression medium was generated as follows:

[0517] S30 Premix Plus T7 S30 Extract DNA template total test 30μL 27μL 18μL (about 1.5μg) 75μL refer to 20μL 18μL 12μL (about 1.0μg) 50μL

[0518] The prepared expression medium mixture was added to the plasmid solution, and the solution was incubated at 30°C for 3 hours while mixing the mixture every 45 minutes. 5 μL of the mixture was frozen, and the remainder was used for the catalytic test of the conversion of rebaudioside A to rebaudioside D.

[0519] Catalytic assay for the conversion of rebaudioside A to rebaudioside D. The reaction mixture of 430 μ L containing 0.5 mM rebaudioside A, 3 mM MgCl , 50 mM phosphate buffer (pH 7.2) and 2.5 mM UDP-glucose is added to the sterile microtube of 1.5 mL. The enzyme expression culture medium of 52 μ L is added, and the resulting mixture is allowed to react at 30 ℃ for 24 hours. After 2 hours, 16 hours and 24 hours, 125 μ L of sample is taken, and it is added to 115 μ L of 60% methanol and 10 μ L of 2N H 2 SO 4. The sample of quenching is centrifuged at room temperature for 2 minutes at 18,000 g. 200 μ L is transferred to HPLC bottle and analyzed.

[0520] HPLC analysis. HPLC assay was performed as described in Example 20.

[0521] The HPLC assay results are provided below:

[0522] Compound Retention time Integral(area) Rebaudioside D 5.797 54,654,810 Rebaudioside A 9.157 633,926,835 total 688,581,645

[0523] Example 22

[0524] Enzyme production in vivo

[0525] The enzyme described in Example 20 was produced in vivo.

[0526] The pET30A+ vector containing the gene corresponding to the enzyme was introduced into Escherichia coli BL21 (DE3) by heat shock. The cells obtained were cultured in a culture dish in the presence of kanamycin, and appropriate bacterium colonies were selected and grown in liquid LB medium (Erlenmayer flask). Glycerol was added to the suspension as a cryoprotectant and aliquots of 400 μL were stored at -20°C and -80°C.

[0527] A stock aliquot of E. coli BL21 (DE3) containing the pET30A+_UGT plasmid was thawed and added to 30 mL of LBGKP medium (20 g / L Luria Broth Lennox; 50 mM PIPES buffer pH 7.00; 50 mM phosphate buffer pH 7.00; 2.5 g / L glucose and 50 mg / L kanamycin). This culture was performed at 30° C. with shaking at 135 rpm for 8 hours.

[0528] The production medium contains 60g / L overnight fast expression TB medium (Novagen company), 10g / L glycerol and 50mg / L kanamycin. Pre-culture is added in this medium of 400mL and the solution is stirred at 20 ℃, and sample is taken to measure OD and pH simultaneously. Culture produces significant growth and obtains good OD. After 40 hours, by centrifugal harvesting cell, and it is frozen. Mention the productive rate of following cell wet weight (CWW) hereinafter.

[0529] GI number Version CWW 115454819 NP_001051010.1 9.2g 187373030 ACD03249.1 7.4g 460409128 XP_004249992.1 6.8g 222619587 EEE55719.1 7.5g 297795735 XP_002865752.1 8.8g

[0530] Lysis was performed by adding Bugbuster Master Mix (Novagen), and the lysate was recovered by centrifugation and used fresh.

[0531] Activity assay. Activity assays for conversion of rebaudioside A were performed at a 5 mL scale using 1,000 μL of thawed lysate using 0.5 mM substrate, 2.5 mM UDP-glucose, and 3 mM MgCl2 in 50 mM sodium phosphate buffer, pH 7.2. Samples were taken and analyzed by HPLC.

[0532] HPLC analysis. HPLC assay was performed as described in Example 20.

[0533] The results for the different enzymes are provided below.

[0534] GI number Version Conversion after 45 hours Reb D selectivity 115454819 NP_001051010.1 1.1％ 100％ 187373030 ACD03249.1 0.8％ 100％ 460409128 XP_004249992.1 62.1％ 43.6％ 222619587 EEE55719.1 2.9％ Reb D not detected 297795735 XP_002865752.1 0.0％ Reb D not detected

[0535] HPLC analysis also showed two unknown peaks. The peak at about 4.5 minutes was subsequently identified as reb M2. The peak at about 7.6 minutes was subsequently identified as reb D2.

[0536] Example 23

[0537] Identification of glycosides

[0538] In addition, the reaction mixture of GI No. 460409128 representing Example 20 (hereinafter S115N05A7) and Example 22 (hereinafter S129N04) was determined by LC-MS to identify unknown glycosides. An Agilent 1200 series HPLC system equipped with a binary pump (G1312B), an autosampler (G1367D), a thermostatted column box (G1316B), a DAD detector (G1315C) connected to an Agilent 6110A MSD and coupled to the "LC / MSD Chemstation" software was used.

[0539] Instrument conditions

[0540]

[0541]

[0542] Mobile phase gradient program

[0543] Time (min) A(%): Formic acid 0.1% B(%): acetonitrile 0 75 25 8.5 75 25 10.0 71 29 16.5 70 30

[0544] The compound observed at 3.5 minutes on the LCMS system corresponds to the unknown peak at about 4.5 minutes in Examples 20 and 22. The LCMS data indicated that this compound has six glycoside residues (C 56 H 90 O 33 ), and found to be an isomeric form of reb M, namely reb M2 (see discussion in Example 40).

[0545] The compound observed at 7.6 minutes on the LCMS system corresponds to the unknown peak at about 7.6 minutes in Examples 20 and 22. The LCMS data indicated that this compound has five glycoside residues (C 50 H 80 O 28 ), and was found to be an isomeric form of reb D, namely reb D2 (see discussion in Example 39). The ratios of these compounds are provided below.

[0546]

[0547] Example 24

[0548] Identification of glycosides

[0549] Additionally, the reaction mixture of GI No. 460409128 representing Example 22 (hereinafter S129N04) was assayed by LC-MS along with Stevia leaf extract "MLD1" produced by PureCircle Sdn Bhd (Malaysia) to determine the presence of S129N04 glycosides in nature.

[0550] The measurement showed that the compound (C 56 H 90 O 33 ; subsequently confirmed to be reb M2), and the compound observed at 7.6 minutes on the LCMS system in Example 23 (C 50 H 80 O 28 ; rebUNK; subsequently confirmed to be reb D2) exists in stevia plant extracts.

[0551] Example 25

[0552] Conversion of rebaudioside E to rebaudioside D

[0553] The total reaction volume was 5.0 mL with the following composition: 100 mM potassium phosphate buffer pH 7.5, 3 mM MgCl2, 2.5 mM UDP-glucose, 0.5 mM rebaudioside E and 500 μL of thawed UGT76G1 lysate, the UGT76G1 gene was cloned into the pET30a+ vector and expressed in E. coli BL21 (DE3). The reaction was carried out at 30°C on an orbital shaker at 135 rpm. For sampling, 300 μL of the reaction mixture was quenched with 30 μL of 2N H2SO4 and 270 μL of methanol / water (6 / 4). The sample was immediately centrifuged and maintained at 10°C before analysis by HPLC (CAD detection). The following reaction curve corresponding to the complete conversion of rebaudioside E to rebaudioside D was obtained.

[0554] Example 26

[0555] Directed evolution of UGT76G1 for conversion of rebaudioside D to rebaudioside M

[0556] Starting from the amino acid sequence of UGT76G1 as described in Genbank (AAR06912.1), different mutations at various amino acid positions were identified that could alter the activity of the enzyme for the conversion of rebaudioside D (Reb D) to rebaudioside M (Reb M). TMThe list of the sudden change of strategic design is used for synthesizing 96 variant genes subsequently, and these variant genes contain 3, 4 or 5 in these sudden changes of codon optimization for expression in intestinal bacteria.These genes are subcloned in pET30a+ plasmid and are used for transforming intestinal bacteria BL21 (DE3) chemical competent cells.The cell that obtains is grown on solid LB agar medium in culture dish under the existence of kanamycin.Select suitable bacterium colony, and make it grow in the liquid LB medium in test tube.Glycerol is added in suspension as cryoprotectant and the aliquot of 400 μ L is stored at-20 ℃ and at-80 ℃.

[0557] These stock aliquots of E. coli BL21 (DE3) containing the pET30a+_UGT76G1var plasmid were thawed and added to LBGKP medium (20 g / L Luria Broth Lennox; 50 mM PIPES buffer pH 7.00; 50 mM phosphate buffer pH 7.00; 2.5 g / L glucose and 50 mg / L kanamycin). This culture was carried out in a 96 microtiter plate at 135 rpm at 30° C. with shaking for 8 h.

[0558] 50 μL of the above culture was used to inoculate 60 g / L overnight rapid expression TB medium (Overnight Express TM Instant TB medium) 3.95 mL of production medium with 10 g / L glycerol and 50 mg / L kanamycin. The resulting culture was stirred at 20° C. in a 48-deep well plate. The culture produced significant growth and obtained a good OD (600 nm; 1 cm). After 44 hours, the cells were harvested by centrifugation and frozen.

[0559] By adding Master Mix Lysis was performed and the lysate was recovered by centrifugation. Activity was tested with 100 μL of fresh lysate added to a solution of rebaudioside D (final concentration 0.5 mM), MgCl2 (final concentration 3 mM) and UDP-glucose (final concentration 2.5 mM) in 50 mM phosphate buffer (pH 7.2).

[0560] The reaction was allowed to proceed at 30°C and samples were taken after 2, 4, 7 and 24 hours. To determine the conversion and initial rate by HPLC (CAD detection), the analytical method described above for the conversion of rebaudioside D to rebaudioside M was used. The results are depicted in the table below.

[0561]

[0562]

[0563]

[0564]

[0565]

[0566] *Mutations are annotated as follows: original amino acid-position-new amino acid: e.g. alanine to glycine mutation at position 33 is annotated as A33G.

[0567] Example 27

[0568] In vivo production of UGTSL2

[0569] UGTSL2 (GI_460410132 / XP_004250485.1) amino acid sequence:

[0570] MATNLRVLMFPWLAYGHISPFLNIAKQLADRGFLIYLCSTRINLESIIKKIPEKYADSIHLIELQLPELPELPPHYHTTNGLPPHLNPTLHKALKMSKPNFSRILQNLKPD LLIYDVLQPWAEHVANEQNIPAGKLLTSCAAVFSYFFSFRKNPGVEFPFPAIHLPEVEKVKIREILAKEPEEGGRLDEGNKQMMLMCTSRTIEAKYIDYCTELCNWKVVPVG PPFQDLITNDADNKELIDWLGTKHENSTVFVSFGSEYFLSKEDMEEVAFALELSNVNFIWVARFPKGEERNLEDALPKGFLERIGERGRVLDKFAPQPRILNHPSTGGFIS HCGWNSAMESIDFGVPIIAMPIHNDQPINAKLMVELGVAVEIVRDDDGKIHRGEIAETLKSVVTGETGEILRAKVREISKNLKSIRDEEMDAVAEELIQLCRNSNKSK(SEQ IDNO:9).

[0571] The pET30A+ vector containing the UGTSL2 gene was introduced into Escherichia coli Bl21 (DE3) by heat shock. The cells obtained were cultured in a culture dish in the presence of kanamycin, and appropriate bacterium colonies were selected and grown in liquid LB medium (Erlenmayer flask). Glycerol was added to the suspension as a cryoprotectant and 400 μL aliquots were stored at -20 °C and -80 °C.

[0572] A stock aliquot of E. coli BL21 (DE3) containing the pET30A+_UGTSL2 plasmid was thawed and added to 30 mL of LBGKP medium (20 g / L Luria Broth Lennox; 50 mM PIPES buffer pH 7.00; 50 mM phosphate buffer pH 7.00; 2.5 g / L glucose and 50 mg / L kanamycin). This culture was continued at 30° C. for 8 h with shaking at 135 rpm.

[0573] The production medium contains 60 g / L overnight fast expression TB medium (Novagen), 10 g / L glycerol and 50 mg / L kanamycin. The pre-culture is added to 200 mL of this medium and the solution is stirred at 20 ° C while taking samples to measure OD and pH. The culture produces significant growth and obtains a good OD. After 40 hours, the cells are harvested by centrifugation and frozen to obtain a cell wet weight of 6.22 g.

[0574] Lysis was performed by adding Bugbuster Master Mix (Novagen) to 1.4 g of cells, and the lysate was recovered by centrifugation and used freshly.

[0575] Example 28

[0576] Determination of the activity of UGTSL and UGTSL2 in converting stevioside to rebaudioside E

[0577] UGTSL was prepared according to Example 22, and UGTSL2 was prepared according to Example 27.

[0578] Using 0.5mM substrate, 2.5mM UDP-glucose and 3mM MgCl2 in 50mM sodium phosphate buffer at pH 7.2, 600μL of lysate was used to perform activity testing on the conversion of stevioside on a 3mL scale. Samples were obtained and analyzed by HPLC. HPLC analysis. HPLC determination was performed as described in Example 20.

[0579] The results and corresponding chromatograms for the different enzymes are provided below.

[0580]

[0581] Notes: 1 Based on the initial concentration of stevioside

[0582] Example 29

[0583] Determination of the activity of converting rubusoside to rebaudioside E using UGTSL and UGTSL2

[0584] UGTSL was prepared according to Example 22, and UGTSL2 was prepared according to Example 27.

[0585] Activity testing for the conversion of rubusoside was performed at a 3 mL scale using 600 μL of lysate using 0.5 mM substrate, 2.5 mM UDP-glucose and 3 mM MgCl2 in 50 mM sodium phosphate buffer, pH 7.2. Samples were taken and analyzed by HPLC. The HPLC assay was performed as described in Example 20.

[0586] The results and corresponding chromatograms for the different enzymes are provided below.

[0587]

[0588] Notes: 1 Based on the initial concentration of rubusoside

[0589] Example 30

[0590] Determination of the activity of UGTSL2 in converting rebaudioside A to rebaudioside D

[0591] UGTSL2 was prepared according to Example 27.

[0592] Activity testing for the conversion of rebaudioside A was performed at a 3 mL scale using 60 μL of lysate using 0.5 mM substrate, 2.5 mM UDP-glucose and 3 mM MgCl2 in 50 mM sodium phosphate buffer at pH 7.2. Samples were taken and analyzed by HPLC. HPLC assays were performed as described in Example 20.

[0593] The results after 23 hours of reaction are provided below.

[0594]

[0595] Notes: 1 Based on the initial concentration of rebaudioside A

[0596] Example 31

[0597] Identification of glycosides

[0598] The reaction mixture prepared according to Example 30 and incubated for 45 hours, together with Stevia leaf extract "MLD1" produced by PureCircle Sdn Bhd (Malaysia), was analyzed by LC-MS to determine the presence of the unknown glycosides formed (about 4.5 minutes, about 6.7 minutes, about 7.0 minutes, about 7.3 minutes and about 7.7 minutes) in nature.

[0599] An Agilent 1200 series HPLC system equipped with a binary pump (G1312B), autosampler (G1367D), thermostatted column compartment (G1316B), DAD detector (G1315C) connected to an Agilent 6110A MSD and interfaced with "LC / MSD Chemstation" software was used.

[0600] Instrument conditions

[0601]

[0602] Mobile phase gradient program

[0603] Time (min) A(%): Formic acid 0.1% B(%): acetonitrile 0 75 25 30 75 25 33 68 32 75 68 32

[0604] The measurement showed that the compound observed at 11.77 minutes on the LC-MS system was different from the compound observed at 3.5 minutes in Example 23 (C 56 H 90 O 33 ; subsequently confirmed to be reb M2) is the same, and the compound observed at 26.64 minutes is the same as the compound observed at 7.6 minutes in Example 23 (C 50 H 80 O 28 ; reb UNK; subsequently confirmed to be reb D2) is the same. Other isomers of reb M were observed at 13.96 minutes, and another isomeric form of reb D was observed at 25.06 minutes. All observed compounds are present in stevia plant extracts.

[0605] Example 32

[0606] In vivo preparation and activity assay of UGTLB

[0607] UGTLB (GI_209954733 / BAG80557.1) amino acid sequence

[0608] MGTEVTVHKNTLRVLMFPWLAYGHISPFLNVAKKLVDRGFLIYLCSTAINLKSTIKKIPEKYSDSIQLIELHLPELPELPPHYHTTNGLPPHLNHTLQKALKMSKPNFSKILQN LKPDLVIYDLLQQWAEGVANEQNIPAVKLLTSGAAVLSYFFNLVKKPGVEFPFPAIYLRKNELEKMSELLAQSAKDKEEPDGVDPFADGNMQVMLMSTSRIIEAKYIDYFSGLSNW KVVPVGPPVQDPIADDADEMELIDWLGKKDENSTVFVSFGSEYFLSKEDREEIAFGLELSNVNFIWVARFPKGEEQNLEDALPKGFLERIGDRGRVLDKFAPQPRILNHPSTGG FISHCGWNSVMESVDFGVPIIAMPIHLDQPMNARLIVELGVAVEIVRDDYGKIHREEIAEILKDVIAGKSGENLKAKMRDISKNLKSIRDEEMDTAAEELIQLCKNSPKLK(SEQ ID NO:10).

[0609] The pET30A+ carrier containing the UGTLB gene is introduced into Escherichia coli Bl21 (DE3) by heat shock. The cells obtained are cultured in a culture dish in the presence of kanamycin, and appropriate bacterium colonies are selected and grown in liquid LB medium (Erlenmayer flask). Glycerol is added to the suspension as a cryoprotectant and 400 μ L of aliquots are stored at -20 °C and -80 °C.

[0610] A stock aliquot of E. coli BL21 (DE3) containing the pET30A+_UGTLB plasmid was thawed and added to 30 mL of LBGKP medium (20 g / L Luria Broth Lennox; 50 mM PIPES buffer pH 7.00; 50 mM phosphate buffer pH 7.00; 2.5 g / L glucose and 50 mg / L kanamycin). This culture was carried out at 30° C. with shaking at 135 rpm for 8 h.

[0611] The production medium contains 60 g / L overnight fast expression TB medium (Novagen), 10 g / L glycerol and 50 mg / L kanamycin. The pre-culture is added to 200 mL of this medium and the solution is stirred at 20 ° C while taking samples to measure OD and pH. The culture produces significant growth and obtains a good OD. After 40 hours, the cells are harvested by centrifugation and frozen to obtain a cell wet weight of 5.7 g.

[0612] Lysis was performed by adding 6 mL of Bugbuster master mix (Novagen) to 1.2 g of cells, and the lysate was recovered by centrifugation and used freshly.

[0613] Determination of the activity of UGTLB in converting stevioside to rebaudioside E

[0614] Using 0.5mM substrate, 2.5mM UDP-glucose and 3mM MgCl in 50mM sodium phosphate buffer at pH 7.2, 600 μL of lysate were used to perform activity tests on the conversion of stevioside on a 3mL scale. Samples were taken and analyzed by HPLC according to the method of Example 20. The results are provided below.

[0615]

[0616] Notes: 1 Based on the initial concentration of stevioside

[0617] Determination of the activity of converting rubusoside into rebaudioside E using UGTLB

[0618] Activity testing for the conversion of rubusoside was performed at a 3 mL scale using 0.5 mM substrate, 2.5 mM UDP-glucose and 3 mM MgCl2 (using 600 μL of lysate) in 50 mM sodium phosphate buffer at pH 7.2. Samples were taken and analyzed by HPLC according to the method of Example 20. The results are provided below.

[0619]

[0620] Notes: 1 Based on the initial concentration of rubusoside

[0621] Determination of the activity of UGTLB in converting rebaudioside A to rebaudioside D

[0622] Activity testing for the conversion of rebaudioside A was performed at a 3 mL scale using 600 μL of lysate using 0.5 mM substrate, 2.5 mM UDP-glucose and 3 mM MgCl in 50 mM sodium phosphate buffer at pH 7.2. Samples were taken and analyzed by HPLC according to the method of Example 20. The results after 23 hours of reaction are provided below.

[0623]

[0624] Notes: 1 Based on the initial concentration of rebaudioside A

[0625] Example 33

[0626] Determination of the reaction products of rubusoside and stevioside converted by UGTSL, UGTSL2, and UGTLB

[0627] The conversion of stevioside with UGTSL and UGTSL2 was performed in a manner similar to Example 28, and the conversion of rubusoside with UGTSL and UGTSL2 was performed similar to Example 29. The conversion of rubusoside and stevioside with UGTLB was performed similar to Example 32.

[0628] The reaction mixture was analyzed by LCMS to confirm all reaction products.

[0629] Rubusoside conversion products

[0630]

[0631] Stevioside conversion products

[0632]

[0633] It can be seen that in the rubusoside conversion products, in addition to stevioside, reb E and reb D, there are at least 3 additional compounds with a molecular weight of 804. The retention times of these compounds do not match those of reb B (also known to have the same molecular weight as stevioside).

[0634] Among the stevioside conversion products, in addition to reb E and reb D, there are at least three additional compounds with a molecular weight of 966. The retention times of these compounds do not match those of reb A (also known to have the same molecular weight as reb E).

[0635] Example 34

[0636] In vivo production of UGT76G1 in Saccharomyces cerevisiae

[0637] UGT76G1[Stevia](gi_37993653 / gb_AAR06912.1)

[0638] MENKTETTVRRRRRIILFPVPFQGHINPILQLANVLYSKGFSITIFHTNFNKPKTSNYPHFTFRFILDNDPQDERISNLPTHGPLAGMRIPIINEHGADELRRELELLMLASEED EEVSCLITDALWYFAQSVADSLNLRRLVLMTSSLFNFHAHVSLPQFDELGYLDPDDKTRLEEQASGFPMLKVKDIKSAYSNWQILKEILGKMIKQTKASSGVIWNSFKELEESELE TVIREIPAPSFLIPLPKHLTASSSSLLDHDRTVFQWLDQQPPSSVLYVSFGSTSEVDEKDFLEIARGLVDSKQSFLWVVRPGFVKGSTWVEPLPDGFLGERGRIVKWVPQQEVLA HGAIGAFWTHSGWNSTLESVCEGVPMIFSDFGLDQPLNARYMSDVLKVGVYLENGWERGEIANAIRRRVMVDEEGEYIRQNARVLKQKADVSLMKGGSSYESLESLVSYISSL(SEQ ID NO:11).

[0639] The above amino acid sequence was codon optimized for expression in Saccharomyces cerevisiae. In addition, the yeast consensus sequence AACACA was added before the ATG start codon. The synthetic gene was subcloned into the pYES2 vector using Hind III and Xba I restriction sites. The pYES2_UGT76G1_Sc vector was used to transform chemically competent Saccharomyces cerevisiae INVSc1 cells (Invitrogen).

[0640] Cells were grown on solid synthetic minimal medium containing 2% glucose lacking uracil, and single colonies were picked and grown on liquid synthetic minimal medium lacking uracil (SC-U containing 2% glucose). After centrifugation, cells were suspended with SC-U (containing 2% glucose) and 60% glycerol / water. Aliquots were stored at -80°C, and one aliquot was used to start culturing in SC-U (containing 2% glucose) for 43 hours at 30°C. A portion of this culture was centrifuged and suspended in induction medium (SC-U containing 2% galactose) for 19 hours at 30°C.

[0641] Cells were harvested by centrifugation and washed with five volumes of CelLytic TM Y cell lysis reagent (Sigma) was used for lysis. The lysate was directly used for activity test (UGT76G1_Sc).

[0642] Example 35

[0643] Determination of the activity of UGT76G1_Sc for the conversion of rebaudioside D to rebaudioside M

[0644] UGT76G1_Sc was prepared according to Example 34. Activity testing for conversion of rebaudioside D was performed at a 3 mL scale using 200 μL of lysate using 0.5 mM substrate, 2.5 mM UDP-glucose, and 3 mM MgCl2 in 50 mM sodium phosphate buffer at pH 7.2. Samples were taken and analyzed by HPLC according to the method of Example 20. The results are shown below.

[0645]

[0646]

[0647] Notes: 1 Based on the initial concentration of rebaudioside D

[0648] Example 36

[0649] In vivo production of UGTSL in Saccharomyces cerevisiae

[0650] UGTSL[tomato](gi_460409128 / XP_004249992.1

[0651] MSPKLHKELFFHSLYKKTRSNHTMATLKVLMFPFLAYGHISPYLNVAKKLADRGFLIYFCSTPINLKSTIEKIPEKYADSIHLIELHLPELPQLPPHYHTTNGLPPNLNQVLQKALKM SKPNFSKILQNLKPDLVIYDILQRWAKHVANEQNIPAVKLLTSGAAVFSYFFNVLKKPGVEFPFPGIYLRKIEQVRLSEMMSKSDKEKELEDDDDDDLLVDGNMQIMLMSTSRTIEAK YIDFCTALTNWKVVPVGPPVQDLITNDVDDMELIDWLGTKDENSTVFVSFGSEYFLSKEDMEEVAFALELSNVNFIWVARFPKGEERNLEDALPKGFLERIGERGRVLDKFAPQPRIL NHPSTGGFISHCGWNSAMESIDFGVPIIAMPMHLDQPMNARLIVELGVAVEIVRDDDGKIHRGEIAETLKGVITGKTGEKLRAKVRDISKNLKTIRDEEMDAAAEELIQLCRNGN(SEQ ID NO:12).

[0652] The above amino acid sequence was codon optimized for expression in Saccharomyces cerevisiae. In addition, the yeast consensus sequence AACACA was added before the ATG start codon. The synthetic gene was subcloned into the pYES2 vector using Hind III and Xba I restriction sites. The pYES2_UGTSL_Sc vector was used to transform chemically competent Saccharomyces cerevisiae INVSc1 cells (Invitrogen).

[0653] Cells were grown on solid synthetic minimal medium lacking uracil, containing 2% glucose, and single colonies were picked and grown on liquid synthetic minimal medium lacking uracil (SC-U containing 2% glucose). After centrifugation, cells were suspended with SC-U (containing 2% glucose) and 60% glycerol / water. Aliquots were stored at -80°C, and one aliquot was used to start culturing in SC-U (containing 2% glucose) for 43 hours at 30°C. A portion of this culture was centrifuged and suspended in induction medium (SC-U containing 2% galactose) for 19 hours at 30°C.

[0654] Cells were harvested by centrifugation and lysed with five volumes of CelLyticTMY cell lysis reagent (Sigma). The lysate was used directly for activity assay (UGTSL_Sc).

[0655] Example 37

[0656] Determination of the activity of UGTSL_Sc for the conversion of rebaudioside A to rebaudioside D

[0657] UGTSL_Sc was prepared according to Example 36. Activity testing for conversion of rebaudioside A was performed at a 2 mL scale using 200 μL of lysate using 0.5 mM substrate, 2.5 mM UDP-glucose, and 3 mM MgCl in 50 mM sodium phosphate buffer at pH 7.2. Samples were taken and analyzed by HPLC according to the method of Example 20. The results are provided below.

[0658] Enzyme internal standard <![CDATA[Rebaudioside A conversion 1 (Reaction time)]]> <![CDATA[Rebaudioside D selectivity 1 > UGTSL_Sc 46％(4h) 42％

[0659] Notes: 1 Based on the initial concentration of rebaudioside A

[0660] Example 38

[0661] Isolation of Rebaudioside M

[0662] The amount of the product mixture of Example 14 was not large enough to be separated by preparative HPLC methods. Accordingly, analytical HPLC with a series of injections was used to separate the components of the mixture. Separation was performed according to the method described in Example 14 above to provide Figure 3 The HPLC trace contains two parts of two main peaks: part A (retention time 24.165 minutes) and part B (retention time 31.325 minutes).

[0663] The retention time of fraction A coincided with reb D, indicating unreacted starting material from the bioconversion reaction.

[0664] The retention time of the purified fraction B was consistent with that of reb M, indicating successful biotransformation from reb D. The identity of the material collected in fraction B was confirmed to be reb M by co-injection of the purified fraction B and reb M standards (available from PureCircle). Both fraction B and reb M standards were found to be eluted at the same retention time ( Figure 7 ), indicating that part B is reb M.

[0665] The identity of fraction B was also independently confirmed by NMR and HRMS as reb M. For sampling, fraction B was concentrated under a rotary evaporator, freeze-dried, and dried at 40° C. for 40 hours.

[0666] The NMR sample was dissolved in deuterated pyridine (C5D5N), and a standard pulse sequence was used to obtain a spectrum on a Varian UnityPlus 600MHz instrument. The NMR spectrum of part B was compared with the NMR spectrum of reb M. The overlap of two spectra showed the consistency of part B and reb M. The table designated by the NMR of reb M is shown below:

[0667] For rebaudioside M in C5D5N 1 H and 13 C NMR spectral data a-c

[0668]

[0669]

[0670]

[0671]

[0672] a Designation based on linkage between COSY, HMQC and HMBC; b Chemical shift values ​​are expressed as δ (ppm); c The coupling constants are expressed in Hz.

[0673] HRMS was generated with a Waters Premier quadrupole time-of-flight (Q-TOF) mass spectrometer equipped with an electrospray ionization source operating in positive ion mode. Samples were dissolved in methanol and eluted in 2:2:1 methanol:acetonitrile:water and introduced via infusion using an onboard syringe pump. The [M+Na] + The adduct confirmed the presence of reb M, which corresponds to the molecular formula C 56 H 90 O 33 .

[0674] Example 39

[0675] Isolation and characterization of Reb D2

[0676] Crude reaction sample. Sample lot number CB-2977-106 was prepared for isolation according to Example 22 using UGTSL (GI#460409128).

[0677] HPLC analysis.Preliminary HPLC analysis of samples was performed using a Waters 2695 Alliance system according to the following method: Pheromone Synergi Hydro-RP, 4.6×250 mm, 4 μm (p / n 00G-4375-E0); column temperature: 55° C.; mobile phase A: 0.0284% ammonium acetate (NH4OAc) and 0.0116% acetic acid (HOAc) in water; mobile phase B: acetonitrile (MeCN); flow rate: 1.0 mL / min; injection volume: 10 μL. Detection was by UV (210 nm) and CAD. Gradient:

[0678]

[0679]

[0680] Analysis of semi-preparative purified fractions was performed as follows: Waters Atlantis dC18, 4.6×100 mm, 5 μm (p / n 186001340); Mobile phase A: 25% MeCN in water; Mobile phase B: 30% MeCN in water; Flow rate: 1.0 mL / min; Injection volume: 10 μL. Detection was performed by CAD.

[0681] gradient:

[0682] Time (min) ％A ％B 0.0-5.0 100 0 20 20 80 25 20 80 30 100 0

[0683] LC-MS. Preliminary analysis of the semisynthetic steviol glycoside mixture was performed on a Waters automated purification HPLC / MS system with a Waters 3100 mass detector operating in negative ion mode. The following method was used for analysis of the samples: Pheromone Synergi Hydro-RP, 4.6×250 mm, 4 μm (p / n 00G-4375-E0); column temperature: 55° C.; mobile phase A: 0.0284% NH4OAc and 0.0116% HOAc in water; mobile phase B: acetonitrile; flow rate: 1.0 mL / min; injection volume: 10 μL. Detection was performed by UV (210 nm), and MSD (-ESI m / z 500-2000). Gradient conditions were as listed above.

[0684] Separated by HPLC.Purification was performed in two steps. The first method for semi-preparative purification is summarized below. Column: Waters Atlantis dC18, 30×100 mm, 5 μm (p / n 186001375); Mobile phase A: 25% MeCN in water; Mobile phase B: 30% MeCN in water; Flow rate: 45 mL / min; Injection load: 160 mg dissolved in 20 mL of water. Detection was performed by UV (205 nm).

[0685] gradient:

[0686]

[0687]

[0688] The second purification used the same column and conditions, but with an isocratic mobile phase: 20% MeCN in water.

[0689] Purified from natural extracts. Purification was performed in three steps. The first method used for preparative purification is summarized as follows. Main process: Waters Symmetry C18, 50×250 mm, 7 μm (p / n WAT248000); Isocratic mobile phase: 50% methanol (MeOH) in water with 0.05% HOAc; Flow rate: 85 mL / min; Injection load: 6 g crude extract dissolved in 50 mL mobile phase. Detection was performed by UV (210 nm). After elution of the target analyte, the column was rinsed with 85% MeOH in water.

[0690] Secondary process: Waters Symmetry Shield RP18, 50×250 mm, 7 μm (p / n WAT248000); Isocratic mobile phase: 20% MeCN in water; Flow rate: 100 mL / min; Injection load: 0.5 g main part dissolved in 30 mL water. Detection was by UV (210 nm).

[0691] Three-stage process: Waters Symmetry Shield RP18, 50×250 mm, 7 μm (p / n WAT248000); isocratic mobile phase: 20% MeCN in water; flow rate: 100 mL / min; injection load: 0.5 g of secondary fraction dissolved in 30 mL of water. Detection was by UV (210 nm).

[0692] MS and MS / MS.MS and MS / MS data were generated using a Waters QT equipped with a Premier mass spectrometer with an electrospray ionization source. Samples were analyzed by negative ESI. Samples were diluted 50 times with H2O: acetonitrile (1:1) and introduced via perfusion using an onboard syringe pump. Samples were diluted to produce good s / n, which occurred at approximately 0.01 mg / mL concentration.

[0693] NMR. Samples were prepared by dissolving 1-2 mg in 150 μL of pyridine-d5, and NMR data were acquired on a Bruker Avance 500 MHz instrument with a 2.5 mm trans-detection probe. 1 H NMR spectra are characterized by residual solvent signals (δ H 8.74 and δ C 150.35) for reference.

[0694] Results and discussion

[0695] Isolation and purification. The steviol glycoside mixture of batch number CB-2977-106 prepared with UGTSL (GI#460409128) according to Example 22 was separated. The material was analyzed by LC-MS using the above method and the results are provided in Figure 6 The target peak of interest is the peak at 7.7 minutes in the TIC chromatogram. The mass spectrum of this peak provides [MH] at m / z 1127.6 - ions. The provided sample was initially processed with a single injection (160 mg) using the first process conditions provided above. This method fractionated the material into a mixture of 'polar' and 'non-polar' glycosides. The 'polar' mixture was then reprocessed using the second step conditions above. A compound with a purity of >99% (CAD, AUC) was isolated from the semi-preparative collection. After purification, the combined fractions were concentrated by rotary evaporation at 35°C and lyophilized. Approximately 1-2 mg was obtained for characterization.

[0696] Mass spectrometry. The ESI-TOF mass spectrum obtained from the perfusion sample showed [MH] at m / z 1127.4709. - ion. [MH] - The mass and molecular formula of the ion C 50 H 80 O 28 Very good agreement (for C 50 H 79 O 28 Calculated: 1127.4758, error: -4.3 ppm). MS data confirmed a nominal mass of 1128 Daltons with a molecular formula of C50 H 80 O 28 .

[0697] MS / MS spectrum (select [MH] at m / z 1127.5) - The ions were fragmented) indicating the loss of two glucose units and the subsequent loss of three glucose moieties at m / z 641.3187, 479.2655 and 317.2065.

[0698] NMR spectroscopy. A series of NMR experiments were performed, including 1 H NMR ( Figure 8 ), 13 C NMR ( Fig. 9 and 10 ), 1 H- 1 H COSY( Fig.11 )、HSQC-DEPT( Fig.12 )、HMBC( Fig.13 and 14 )、NOESY( Fig.15 ) and 1D-TOCSY to allow assignment of the compound. 1 In H NMR, the H The end group resonance at 5.04 is shown in the original spectrum ( Figure 8 ) is masked by the solvent (HOD).

[0699] 1 H. 1 H- 1 H COSY, 1 H- 13 C HSQC-DEPT and 1 H- 13 C HMBC NMR data indicate that the central core of this glycoside is a diterpene. 1 H and 1 H- 13 The presence of five anomeric protons observed in the C HSQC-DEPT spectrum confirmed the presence of a pentasaccharide unit in the structure. 1 H- 13 δ in C HSQC-DEPT spectra C Methylene at 69.9 13 C resonance indicates the presence of a 1→6 sugar bond in the structure. 1 H- 13 C HMBC and 1D-TOCSY correlations assigned the linkage of sugar units.

[0700] From in δ H The methyl proton at 1.29 to the C The HMBC correlation of the carbonyl at 177.7 allowed the assignment of the tertiary methyl group (C-18) as well as one of the C-19s and provided a starting point for the assignment of the remaining aglycones. C Additional HMBC correlations of carbons at 38.9, 45.0, and 57.8 allowed assignment of C-3, C-4, and C-5. 1 H- 13 Analysis of C HSQC-DEPT data shows that C The carbon at 38.9 is a methylene group and at δ C The carbons at 57.8 are methines, which are designated C-3 and C-5, respectively. This leaves C The carbon at 45.0, which did not show a correlation in the HSQC-DEPT spectrum, was assigned as a quaternary carbon, C-4. The HSQC-DEPT data were used to assign H 0.98 and 2j.36) and C-5 (δ H 1.04) 1 H chemical shift. One of the H-3 protons (δ H 0.98) and in δ H The COSY correlation between the protons at 1.43 allowed assignment of one of the H-2 protons, which in turn showed a bond with the proton at δ H Then, based on additional COSY and HSQC-DEPT correlations, the remaining 1 H and 13 C chemical shifts are assigned and summarized in the table below.

[0701] 1 H and 13 C NMR (500 and 125 MHz, pyridine-d5), assignment of Reb D2.

[0702]

[0703]

[0704] In δ H The other tertiary methyl singlet observed at 1.30 showed HMBC correlations with C-1 and C-5 and was assigned to C-20. The methyl proton showed correlations with the quaternary carbons (δ C 40.3) and methine carbon (δ C54.5) of the other HMBC association. Then, at H-5 (δ H 1.04) and in δ H The COSY correlation between the protons at 1.92 and 2.43 allowed the assignment of the H-6 proton, which in turn showed a bond with the proton at δ H The correlation of the protons at 1.22 and 1.30 was then determined from the HSQC-DEPT data for C-6 (δ C 22.7) and C-7 (δ C 42.2) 13 C chemical shift. H 0.88) and in δ H The COSY correlation between the protons at 1.65 and 1.69 allowed the assignment of the H-11 proton, which in turn showed a correlation with the proton assigned to H-12 at δ H The COSY correlations for the protons at 1.99 and 2.25 were then used to assign C-11 (δ C 21.1) and C-12 (δ C 37.5). From the H-12 proton (δ H 2.25) to δ C HMBC correlations for carbons at 87.1 and 154.7 allowed the assignment of C-13 and C-16, respectively. H The olefinic protons observed at 5.01 and 5.64 showed HMBC correlations to C-13 and were assigned to C-17 (δ C 105.2). The olefinic proton H-17 and the methine proton H-9 show similarity to the proton assigned to C-15 at δ C HMBC correlation of carbon at 48.3. Then, from H-9 to C Additional HMBC correlations at the methylene carbon at 44.5 allowed the assignment of C-14. Using the HSQC-DEPT data, the H 1.80 and 2.65) and C-15 (δ H 1.31 and 2.04) 1 H chemical shift.

[0705] The relative stereochemistry of the central diterpene core is specified using the association observed in the NOESY spectrum. In this NOESY spectrum, the NOE association observed between H-14 and H-20 shows that H-14 and H-20 are on the same face of the ring. Similarly, at H-9 and H-5; NOE associations are observed between H-9 and H-18 and H-5 and H-18, but NOE associations are not observed between H-9 and H-14, showing that compared with H-14 and H-20, H-5, H-9 and H-18 are located on the opposite face of the ring. These data show that the relative stereochemistry in the central core is retained during the glycosylation step.

[0706] The key HMBC and COSY correlations used to assign aglycone regions are provided below:

[0707]

[0708] 1 H- 13 Analysis of the C HSQC-DEPT data confirmed the presence of five anomeric protons. Three of these anomeric protons are 1 δ in H NMR spectra H 6.02(δ C 96.1)、5.57(δ C 105.3), and 5.34(δ C 105.3) is well resolved. H 5.04(δ C 105.6) and 5.07(δ C 98.7) observed at 1 The remaining two terminal protons in the H spectrum that are masked by the solvent (HOD) resonance are 1 H- 13 C HSQC-DEPT data were used for identification. H The anomeric proton observed at 6.02 showed an HMBC correlation with C-19, indicating that it corresponds to Glc I Similarly, in δ H The anomeric proton observed at 5.07 showed an HMBC correlation with C-13, allowing it to be assigned as Glc II terminal protons.

[0709] Glc I The terminal proton (δ H 6.02) shows that H The proton at 4.07 (assigned as Glc I H-2) COSY correlation, which is H The proton at 4.07 further shows the sameH The proton at 4.22 (Glc I H-3) COSY correlation, which is H The proton at 4.22 shows the same H The proton at 4.12 (Glc I COSY correlation of H-4) was performed. Due to data overlap, the COSY spectrum did not allow assignment of H-5 or H-6 protons. Therefore, Glc I A series of 1D-TOCSY experiments were performed with several different mixing times using selective irradiation of the terminal protons. I In addition to the assignments of H-2 to H-4, the 1D-TOCSY data also show that H 4.04 is designated as Glc I The proton of H-5 and the H 4.68 is designated as Glc I The latter proton was also used in 1D-TOCSY experiments. Selective irradiation of H-6 at several different mixing times also confirmed the Glc I H-1 to H-5 and H-6 (δ H 4.30). 1 H- 13 C HSQC-DEPT data to determine the Glc I C-2(δ C 74.2), C-3(δ C 79.1), C-4(δ C 72.1), C-5(δ C 78.5), and C-6 (δ C 69.9) 13 C chemical shift assignments to complete the Glc I In addition, 1 H- 13 δ in C HSQC-DEPT spectra C Methylene at 69.9 13 The presence of C resonance indicates that Glc I 1→6 sugar bonds.

[0710] Of the four remaining unspecified glucose fractions, based on 1 H- 13 C HSQC-DEPT, HMBC, and 1D-TOCSY correlations, one was assigned to Glc I The substituent at C-6 of CGlc at 69.9 I The methylene 13 The relative downfield shift of the C resonance indicates that Glc I 1→6 sugar bonds. H The anomeric proton observed at 5.04 shows similarity to Glc I C-6 was HMBC-linked and designated as Glc V Similarly, Glc I The methylene protons of Glc V The HMBC correlation of the terminal carbon of Glc I With Glc V The presence of 1→6 sugar bonds between Glc V The anomeric protons show similar H The proton at 4.00 (assigned as Glc V H-2) COSY correlation, which is H The proton at 4.00 further shows the same H The proton at 4.22 (Glc V Due to data overlap, this COSY spectrum does not allow for the Glc V COSY correlation of H-3 Glc V However, in the HMBC spectrum, Glc V H-3 showed V C-5(δ C 78.9). In the HSQC-DEPT spectrum, Glc V C-5 shows similar H 3.89(Glc V H-5). V H-5 showed similar H COSY correlations of 4.21, 4.37, and 4.48. In the HSQC-DEPT spectrum, δ H 4.21 shows that C 71.4(Glc V H-4) and δ H 4.37 and 4.48 show similarities to δ C 63.1 and were designated as Glc V H-6a and H-6b. Use 1 H- 13 C HSQC-DEPT data to determine the Glc V C-2(δ C 75.7) and C-3 (δC 79.1) 13 C chemical shift assignments to complete the Glc V Designation.

[0711] Regarding the glycoside at C-19 1 H and 13 A summary of the C chemical shifts is shown in the following table:

[0712] 1 H and 13 C NMR (500 and 125 MHz, pyridine-d5), assignment of reb D2 C-19 glycoside.

[0713]

[0714]

[0715] 1 H and 13 The C value can be in position Glc1-3, Glc V -3 and Glc IV -3 interchangeably.

[0716] A summary of the key HMBC, COSY, and 1D-TOCSY correlations used to assign the C-19 glycosidic region is provided below.

[0717]

[0718] In a similar manner, Glc II Designation of Glc II The terminal proton (δ H 5.07) shows that H The proton at 4.37 (assigned as Glc II H-2) COSY correlation, which is H The proton at 4.37 further shows the same H The proton at 4.18 (Glc II H-3) COSY correlation. The latter protons show a similar H The proton at 3.88 (Glc II H-4) additional association, which in δ H The proton at 3.88 also shows a similar H The proton at 3.79 (Glc II COSY correlation of H-5). Glc II H-5 also showed II H-6 proton (δ H4.08 and 4.46). Using HSQC-DEPT data, the II C-2(δ C 81.3), C-3(δ C 88.4), C-4(δ C 71.1), C-5(δ C 77.9), and C-6 (δ C 63.2) 13 Assignment of C chemical shifts. II H-3 to C-2 and C-4 and also from Glc II HMBC correlations of H-4 to C-2 and C-5 confirmed the assignments made above. II H-4 and Glc II Additional HMBC association at C-6 further supports completion of the Glc II Designation.

[0719] Two of the remaining unassigned glucose moieties were assigned to Glc based on HMBC correlations. II Substituents at C-2 and C-3. H The anomeric proton observed at 5.57 shows similarity to Glc II C-2 was HMBC associated and designated as Glc III The terminal proton of H The anomeric proton observed at 5.34 shows similarity to Glc II C-3 was HMBC associated and designated as Glc IV It was also observed that the anomeric proton from Glc II H-2 to Glc III The terminal carbon and the II H-3 to Glc IV The mutual HMBC correlation of the terminal carbons.

[0720] Glc III (δ H 5.57) shows the same anomeric proton as in δ H The residue at 4.19 is designated as Glc III COSY correlation of the proton at H-2. The COSY spectrum did not allow assignment of the protons at H-3 to H-6 due to data overlap. Therefore, Glc III A series of 1D-TOCSY experiments were performed with several different mixing times using selective irradiation of the anomeric protons. III In addition to the assignment of H-2, the 1D-TOCSY data also show that H4.24(Glc III H-3), δ H 4.27(Glc III H-4), and δ H 3.94(Glc III Once H-4 is assigned using 1D-TOCSY data, H-6 is assigned using COSY correlations from H-4 to H-5 and then to H-6. In the COSY spectrum, Glc III H-4 showed III H-5 association, the Glc III H-5 further showed a corresponding III Delta of H-6a and H-6b H The COSY correlations of 4.41 and 4.50 are then used 1 H- 13 C HSQC-DEPT correlation to determine the Glc III C-2(δ C 76.8), C-3(δ C 78.9), C-4(δ C 72.4), C-5(δ C 78.8), and C-6 (δ C 63.5) 13 C chemical shift, thus completing the Glc III Designation.

[0721] Glc IV (δ H 5.34) shows the same anomeric proton as in δ H The one at 4.06 is designated as Glc IV COSY correlation of the proton at H-2. The COSY spectrum did not allow assignment of the protons at H-3 to H-6 due to data overlap. Therefore, Glc IV A series of 1D-TOCSY experiments were performed with several different mixing times using selective irradiation of the anomeric protons. IV In addition to the assignment of H-2, the 1D-TOCSY data show that H 4.22(Glc IV H-3), δ H 4.18(Glc IV H-4), and δ H 4.10(Glc IVOnce H-4 is assigned using 1D-TOCSY data, H-6 is assigned using COSY correlations from H-4 to H-5 and then to H-6. In the COSY spectrum, Glc IV H-4 showed IV H-5 association, the Glc IV H-5 further shows a corresponding H 4.32 and 4.58 (Glc IV H-6a and H-6b). Then use 1 H- 13 C HSQC-DEPT correlation to determine the Glc IV C-2(δ C 75.8), C-3(δ C 78.9), C-4(δ C 72.0), C-5(δ C 79.3), and C-6 (δ C 62.9) 13 C chemical shift, thus completing the Glc IV Designation.

[0722] For H 6.02(d,J=8.1Hz), 5.57(d,J=7.6Hz), 5.34(d,J=7.9Hz) and δ H Large coupling constants were observed for the anomeric protons of the glucose moiety at 5.04 (d, J = 8.1 Hz), suggesting their β-orientation. H The remaining anomeric proton at 5.07 is masked by the solvent resonance (HDO), whose coupling constant (J = about 8 Hz) evident from the ID TOCSY data also indicates a β-orientation.

[0723] Regarding the glycoside at C-13 1 H and 13 A summary of the C chemical shifts is shown in the following table:

[0724] 1 H and 13 C NMR (500 and 125 MHz, pyridine-d5), assignment of Reb D2 C-13 glycoside.

[0725]

[0726] *The anomeric protons are masked by solvent (HDO) resonances, coupling constants were obtained from 1D-TOCSY data.

[0727] #1 H and13 The C value can be in Glc1-3, Glc V -3 and Glc IV -3 interchangeably.

[0728] A summary of the key HMBC, COSY, and 1D-TOCSY correlations used to assign the C-13 glycosidic region is provided below:

[0729]

[0730] The chemical name of the compound is 13-[(2-O-β-D-glucopyranosyl-3-O-β-D-glucopyranosyl-β-D-glucopyranosyl)oxy] ent-kaur-16-ene-19-oic acid-[(6-O-β-D-glucopyranosyl-β-D-glucopyranosyl) ester] (rebaudioside D2 or reb D2). The compound is an isomer of rebaudioside D.

[0731] Example 40

[0732] Isolation and characterization of Reb M2

[0733] Crude reaction sample. Sample lot number CB-2977-106 was prepared for isolation according to Example 22 using UGTSL (GI#460409128).

[0734] HPLC analysis. Preliminary HPLC analysis was performed using a Waters 2695 Alliance system according to the following method: Phenomenon Synergi Hydro-RP, 4.6×250 mm, 4 μm (p / n 00G-4375-E0); column temperature: 55° C.; mobile phase A: 0.0284% NH4OAc and 0.0116% HOAc in water; mobile phase B: acetonitrile (MeCN); flow rate: 1.0 mL / min; injection volume: 10 μL. Detection was performed by UV (210 nm) and CAD.

[0735] gradient:

[0736]

[0737]

[0738] Analysis of semi-preparative purified fractions was performed as follows: Waters Atlantis dC18, 4.6×100 mm, 5 μm (p / n 186001340); Mobile phase A: 25% MeCN in water; Mobile phase B: 30% MeCN in water; Flow rate: 1.0 mL / min; Injection volume: 10 μL. Detection was performed by CAD.

[0739] gradient:

[0740] Time (min) ％A ％B 0.0-8.5 75 25 10.0 71 29 16.5 70 30 18.5-24.5 66 34 26.5-29.0 48 52 31-37 30 70 38 75 25

[0741] LC-MS. Preliminary analysis of the semisynthetic steviol glycoside mixture was performed on a Waters automated purification HPLC / MS system with a Waters 3100 mass detector operating in negative ion mode. The following method was used for analysis of the samples: Pheromone Synergi Hydro-RP, 4.6×250 mm, 4 μm (p / n 00G-4375-E0); column temperature: 55° C.; mobile phase A: 0.0284% NH4OAc and 0.0116% HOAc in water; mobile phase B: MeCN; flow rate: 1.0 mL / min; injection volume: 10 μL. Detection was performed by UV (210 nm), and MSD (-ESI m / z 500-2000). Gradient conditions were as listed above.

[0742] Separated by HPLC. Purification was performed in two steps. The first method for semi-preparative purification is summarized below. Column: Waters Atlantis dC18, 30×100 mm, 5 μm (p / n 186001375); Mobile phase A: 25% MeCN in water; Mobile phase B: 30% MeCN in water; Flow rate: 45 mL / min; Injection load: 160 mg dissolved in 20 mL of water. Detection was performed by UV (205 nm).

[0743] gradient:

[0744]

[0745]

[0746] The second purification used the same column and conditions, but with an isocratic mobile phase: 20% MeCN in water.

[0747] MS and MS / MS.MS and MS / MS data were generated using a Waters QT equipped with a Premier mass spectrometer with an electrospray ionization source. Samples were analyzed by negative ESI. Samples were diluted 50 times with H2O:MeCN (1:1) and introduced via perfusion using an onboard syringe pump. Samples were diluted to produce good s / n, which occurred at approximately 0.01 mg / mL concentration.

[0748] NMR. Samples were prepared by dissolving approximately 1.0 mg in 150 μL of D2O, and NMR data were acquired on a Bruker Avance 500 MHz instrument with a 2.5 mm trans-detection probe. 1 H NMR and 13 C NMR spectra were referenced to the residual solvent signal HDO (δ H 4.79ppm) and TSP(δ C 0.00ppm).

[0749] Results and discussion

[0750] Isolation and purification. The steviol glycoside mixture of batch number CB-2977-106 prepared with UGTSL (GI#460409128) according to Example 22 was separated. The material was analyzed by LC-MS using the above method ( Figure 6 ). The target peak of interest is the peak at 4.1 minutes in the TIC chromatogram. The mass spectrum of this peak provides [MH] at m / z 1289.7 - ions. The sample provided was initially processed with a single injection (160 mg) using the first process conditions provided above. This method fractionated the material into a 'polar' and 'non-polar' glycoside mixture. The 'polar' mixture was then reprocessed using the second step conditions provided above. A peak with a purity of> 99% (CAD, AUC) was isolated from the semi-preparative collection. After purification, the fractions were concentrated by rotary evaporation at 35°C and lyophilized. Approximately 1 mg was obtained.

[0751] Mass spectrometry. The ESI-TOF mass spectrum obtained by perfusing a sample of CC-00300 showed [MH] at m / z 1289.5266. - ion. [MH] - The mass of the ion is consistent with the expected molecular formula C for reb M2 56 H 90 O 33 Very good agreement (for C 56 H 89 O 33Calculated: 1289.5286, error: -1.6 ppm). MS data confirmed that CC-00300 had a nominal mass of 1290 Daltons and a molecular formula of C 56 H 90 O 33 .

[0752] MS / MS spectrum (select [MH] at m / z 1289.5) - ions were fragmented) indicating the loss of three glucose units at m / z 803.3688 and the subsequent loss of three glucose moieties at m / z 641.3165, 479.2633 and 317.2082.

[0753] NMR spectroscopy. A series of NMR experiments were performed, including 1 H NMR ( Fig.18 ), 13 C NMR ( Fig.19 and 20 ), 1 H- 1 HCOSY( Fig.21 )、HSQC-DEPT( Fig. 22 )、HMBC( Fig.23 and 24 ), and 1D-TOCSY to allow the assignment of reb M2.

[0754] 1 H. 1 H- 1 H COSY, 1 H- 13 C HSQC-DEPT and 1 H- 13 C HMBC NMR data indicate that the central core of this glycoside is a diterpene. 1 H and 1 H- 13 The presence of six anomeric protons observed in the C HSQC-DEPT spectrum confirmed the hexasaccharide unit in the structure. 1 H- 13 δ in C HSQC-DEPT spectra C Methylene at 70.9 13 C resonance indicates the presence of a 1→6 sugar bond in the structure. 1 H- 13 C HMBC and 1D-TOCSY correlations assigned the linkage of sugar units.

[0755] From in δ H The methyl proton at 1.29 to the CThe HMBC correlation of the carbonyl at 181.5 allowed the assignment of the tertiary methyl group (C-18) as well as one of the C-19s and provided a starting point for the assignment of the remaining aglycones. C Additional HMBC correlations of carbons at 39.8, 43.7, and 59.2 allowed assignment of C3, C4, and C5. 1 H- 13 Analysis of C HSQC-DEPT data shows that C The carbon at 39.8 is a methylene group and at δ C The carbons at 59.2 are methines, which are designated C-3 and C-5, respectively. This leaves C The carbon at 43.7, which did not show a correlation in the HSQC-DEPT spectrum, was assigned as a quaternary carbon, C-4. The HSQC-DEPT data were used to assign H 1.16 and 2.28) and C-5 (δ H 1.24) 1 H chemical shift. One of the H-3 protons (δ H 1.16) and in δ H The COSY correlation between the protons at 1.49 allowed the assignment of one of the H-2 protons, which in turn showed a bond with the proton at δ H Then, based on additional COSY and HSQC-DEPT correlations, the remaining 1 H and 13 C chemical shifts are assigned and summarized in the table below.

[0756] Reb M2 aglycone 1 H NMR (500 MHz, D2O) and 13 C NMR (125 MHz, D2O / TSP) assigned.

[0757]

[0758]

[0759] In δ H The other tertiary methyl singlet observed at 0.92 showed HMBC correlations with C-1 and C-5 and was assigned to C-20. The methyl proton showed correlations with the quaternary carbons (δ C 42.4) and methine (δ C 55.5) of the other HMBC association. Then, at H-5 (δ H 1.24) and in δ HThe COSY correlation between the protons at 1.73 and 1.94 allowed the assignment of the H-6 proton, which in turn showed a bond with the proton at δ H The correlation of the protons at 1.49 and 1.56 was then determined from the HSQC-DEPT data for C-6 (δ C 24.4) and C-7 (δ C 44.2) 13 C chemical shift. H 1.09) and in δ H The COSY correlation between the protons at 1.66 and 1.70 allowed the assignment of the H-11 proton, which in turn showed a correlation with the proton assigned to H-12 at δ H COSY correlations for protons at 1.60 and 2.00. HSQC-DEPT data were then used to assign C-11 (δ C 22.6) and C-12 (δ C 39.9). H The olefinic protons observed at 4.98 and 5.16 show similarities to the C-13 (δ C 90.9) and was assigned to C-17 (δ C 107.0). The olefinic proton H-17 shows a similarity to that assigned to C-15 at δ C HMBC correlation of carbon at 49.4. Then, from H-9 to C Additional HMBC correlations at the methylene carbon at 46.9 allowed the assignment of C-14. Using the HSQC-DEPT data, the H 1.53 and 2.21) and C-15 (δ H 2.15 and 2.18) 1 H chemical shift.

[0760] A summary of the key HMBC and COSY correlations used to assign aglycone regions is provided below:

[0761]

[0762] 1 H- 13 Analysis of the C HSQC-DEPT data confirmed the presence of six anomeric protons. Three of these anomeric protons are 1 δ in H NMR spectra H 5.65(δ C 95.5), 4.92(δ C 104.9), and 4.50(δ C105.7) is well resolved. H 4.85(δ C 98.4), 4.84(δ C 105.0), and 4.83(δ C The remaining three anomeric protons observed at 105.3) are 1 The H spectrum overlaps with the residual solvent resonance. H The anomeric proton observed at 5.65 showed an HMBC correlation with C-19, indicating that it corresponds to Glc I Similarly, in δ H The anomeric proton observed at 4.85 showed an HMBC correlation with C-13, allowing it to be assigned as Glc II terminal protons.

[0763] Glc I The terminal proton (δ H 5.65) shows that H The proton at 3.96 (assigned as Glc I H-2) COSY correlation, which is H The proton at 3.96 further shows the same H The proton at 3.89 (Glc I H-3) COSY correlation, which is H The proton at 3.89 shows the same H The proton at 3.71 (Glc I COSY correlation of H-4) was performed. Due to data overlap, the COSY spectrum did not allow assignment of H-5 or H-6 protons. Therefore, Glc I A series of 1D-TOCSY experiments were performed with several different mixing times using selective irradiation of the terminal protons. I In addition to the assignments of H-2 to H-4, the 1D-TOCSY data also show that H 3.73 is designated as Glc I The proton of H-5 and the H 4.15 is designated as Glc I The latter proton was also used in 1D-TOCSY experiments. Selective irradiation of H-6 at several different mixing times also confirmed the Glc I H-1 to H-5 and H-6 (δ H 4.00) of the remaining methylene protons. 1 H- 13 C HSQC-DEPT data to determine the GlcI C-2(δ C 80.5), C-3(δ C 79.0), C-4(δ C 71.5), C-5(δ C 79.0), and C-6 (δ C 70.9) 13 C chemical shift assignments to complete the Glc I In addition, 1 H- 13 δ in C HSQC-DEPT spectra C Methylene at 70.9 13 The presence of C resonance indicates that Glc I 1→6 sugar bonds.

[0764] Two of the unassigned glucose moieties were assigned to Glc based on HMBC correlations I Substituents at C-2 and C-6. H The anomeric proton observed at 4.83 shows similarity to Glc I C-2 was HMBC associated and designated as Glc V The terminal proton of H The anomeric proton observed at 4.50 shows similarity to Glc I C-6 was HMBC-linked and designated as Glc VI It was also observed that the anomeric proton from Glc I H-2 to Glc V The terminal carbon and the I H-6 to Glc VI The mutual HMBC correlation of the terminal carbons.

[0765] Glc V (δ H 4.83) shows the same anomeric proton as in δ H The one at 3.32 is designated as Glc V COSY correlation of H-2 protons. Glc V H-2 further showed the same H The proton at 3.51 (Glc V H-3) COSY correlation. The latter protons show a similar H The proton at 3.38 (Glc V H-4) also showed an additional association with H The proton at 3.55 (Glc V H-5) COSY correlation, and GlcV H-5 further showed the V H-6 proton (δ H 3.76 and 3.97). Using HSQC-DEPT data, the V C-2(δ C 78.5), C-3(δ C 78.7), C-4(δ C 72.9), C-5(δ C 78.8), and C-6 (δ C 63.6) 13 Assignment of C chemical shifts. V H-3 to C-2 and C-4 and also from Glc V HMBC correlations of H-4 to C-3 and C-6 confirmed the assignments made above, completing the Glc V Designation.

[0766] based on 1 H- 13 C HSQC-DEPT and HMBC correlations, another glucose moiety was assigned as the Glc I The substituent at C-6 of C Glc at 70.9 I The methylene 13 The relative downfield shift of the C resonance indicates that Glc I 1→6 sugar bonds. H The anomeric proton observed at 4.50 shows similarity to Glc I C-6 was HMBC-linked and designated as Glc VI Similarly, Glc I The methylene protons of Glc VI The HMBC correlation of the terminal carbon of Glc I With Glc VI The presence of 1→6 sugar bonds between Glc VI The anomeric protons show similar H The proton at 3.33 (assigned as Glc VI H-2) COSY correlation, which is H The proton at 3.33 further shows the same H The proton at 3.49 (Glc VI Due to data overlap, the COSY spectrum does not allow for the identification of Glc based on COSY correlation. VAssignment of H-4 to H-6 protons. Therefore, using Glc VI A series of 1D-TOCSY experiments were performed with different mixing times by selective irradiation of the terminal protons. VI In addition to the assignment of H-2 to H-3, the 1D-TOCSY data also show that H 3.45(Glc VI H-4) and δ H 3.48(Glc VI H-5) and is assigned to Glc VI The H-6 proton in δ H Protons at 3.92 and 3.94. Use 1 H- 13 C HSQC-DEPT data to determine the Glc VI C-2(δ C 78.1), C-3(δ C 78.6), C-4(δ C 72.3), C-5(δ C 78.8), and C-6 (δ C 64.1) 13 C chemical shift assignments to complete the Glc VI Designation.

[0767] Regarding the glycoside at C-19 1 H and 13 A summary of the C chemical shifts is found in the following table:

[0768] Reb M2 glycosides 1 H NMR (500 MHz, D2O) and 13 C NMR (125 MHz, D2O / TSP) assigned.

[0769]

[0770] * 1 H and 13 The C value can be compared with the Glc IV -1 swap.

[0771] A summary of the key HMBC, COSY, and 1D-TOCSY correlations used to assign the C-19 glycosidic region is provided below:

[0772] Reb M2 glycosides 1 H NMR (500 MHz, D2O) and 13 C NMR (125 MHz, D2O / TSP) assigned.

[0773]

[0774] In a similar manner, Glc II Designation of Glc II The terminal proton (δ H 4.85) shows that H The proton at 3.75 (assigned as Glc II H-2) COSY correlation, which is H The proton at 3.75 further shows the same H The proton at 3.98 (Glc II H-3) COSY correlation. The latter protons show a similar H The proton at 3.54 (Glc II H-4) also showed an additional association with H The proton at 3.96 (Glc II COSY correlation of H-5). Glc II H-5 also showed II H-6 proton (δ H 3.77 and 3.45). Using HSQC-DEPT data, the II C-2(δ C 81.7), C-3(δ C 88.0), C-4(δ C 71.3), C-5(δ C 80.5), and C-6 (δ C 63.6) 13 Assignment of C chemical shifts. II H-3 to C-2 and C-4 and also from Glc II HMBC correlations of H-4 to C-3 and C-6 confirmed the assignments made above, completing the Glc II Designation.

[0775] Two of the remaining unassigned glucose moieties were assigned to Glc based on HMBC correlations. II Substituents at C-2 and C-3. H The anomeric proton observed at 4.92 shows similarity to Glc II C-2 was HMBC associated and designated as Glc III The terminal proton of H The anomeric proton observed at 4.84 shows similarity to Glc II C-3 was HMBC associated and designated as GlcIV It is also observed that in Glc II H-2 and Glc III Between the terminal carbons and between Glc II H-3 and Glc IV The mutual HMBC correlation between the terminal carbons.

[0776] Glc III (δ H 4.92) shows the same anomeric proton as in δ H The one at 3.32 is designated as Glc III COSY correlation of the proton at H-2. The COSY spectrum did not allow assignment of the protons at H-3 to H-6 due to data overlap. Therefore, Glc III A series of 1D-TOCSY experiments were performed with different mixing times by selective irradiation of the terminal protons. III In addition to the assignment of H-2, the 1D-TOCSY data also show that H 3.51(Glc III H-3), δ H 3.26(Glc III H-4), and δ H 3.44(Glc III Once H-4 is assigned using 1D-TOCSY data, H-6 is assigned using COSY correlations from H-4 to H-5 and then to H-6. In the COSY spectrum, Glc III H-4 showed III H-5 association, the Glc III H-5 further showed a corresponding III Delta of H-6a and H-6b H The COSY correlations are 3.94 and 3.75. Then use 1 H- 13 CHSQC-DEPT correlation to determine the Glc III C-2(δ C 76.3), C-3(δ C 78.8), C-4(δ C 73.3), C-5(δ C 78.8), and C-6 (δ C 64.4) 13 C chemical shift, thus completing the Glc III Designation.

[0777] Shows that HCOSY correlation of protons at 3.41 for Glc IV The terminal proton (δ H 4.84) ​​was designated as Glc IV H-2, the Glc IV H-2 further showed the same H The proton at 3.46 (Glc IV H-3) COSY correlation. The latter protons show a similar H The proton at 3.45 (Glc IV H-4) additional association, which in δ H The proton at 3.45 also shows a similar H The proton at 3.75 (Glc IV COSY correlation of H-5). Glc IV H-5 also showed IV H-6 proton (δ H 3.55 and 3.78). Using HSQC-DEPT data, the COSY correlations for Glc IV C-2(δ C 76.1), C-3(δ C 78.8), C-4(δ C 72.5), C-5(δ C 81.7), and C-6 (δ C 65.8) 13 Assignment of C chemical shifts. IV H-3 to C-4 and C-5 and also from Glc IV HMBC correlations of H-4 to C-3 and C-6 confirmed the assignments made above, completing the Glc IV Designation.

[0778] Regarding the glycoside at C-13 1 H and 13 A summary of the C chemical shifts is found in the following table:

[0779] Reb M2 glycosides 1 H NMR (500 MHz, D2O) and 13 C NMR (125 MHz, D2O / TSP) assigned.

[0780]

[0781] A summary of the key HMBC, COSY, and 1D-TOCSY correlations used to assign the C-13 glycosidic region is provided below:

[0782]

[0783] NMR and MS analysis allowed for full specification of its structure shown below. The chemical name of the compound is 13-[(2-O-β-D-glucopyranosyl-3-O-β-D-glucopyranosyl-β-D-glucopyranosyl)oxy] ent-kaur-16-ene-19-oic acid-[(2-O-β-D-glucopyranosyl-6-O-β-D-glucopyranosyl-β-D-glucopyranosyl) ester] (rebaudioside M2 ​​or reb M2). The compound is an isomer of rebaudioside M.

[0784]

[0785] Example 41

[0786] Directed evolution of UGT76G1 for conversion of rebaudioside D to rebaudioside M (round 2)

[0787] The most active clone from the first round of directed evolution of UGT76G1 (see Example 26, UGT76G1var94 containing mutations Q266E_P272A_R334K_G348P_L379G) was selected as the baseline clone for the second round. A list of 53 mutations was established, which contained different positive mutations from the first round of identification and new mutations obtained by DNA2.0 ProteinGPStm strategy. This mutation list was subsequently used to design 92 variant genes, each of which contained 3 different mutations. After codon optimization for expression in E. coli, these genes were synthesized, subcloned into pET30a+ plasmids and used to transform E. coli BL21 (DE3) chemically competent cells. The cells obtained were grown on solid LB agar medium in a culture dish in the presence of kanamycin. Suitable bacterium colonies were selected and grown in liquid LB medium in a test tube. Glycerol was added to the suspension as a cryoprotectant and aliquots of 400 μL were stored at -20°C and at -80°C.

[0788] These stock aliquots of E. coli BL21 (DE3) containing the pET30a+_UGT76G1var plasmid were thawed and added to LBGKP medium (20 g / L Luria Broth Lennox; 50 mM PIPES buffer pH 7.00; 50 mM phosphate buffer pH 7.00; 2.5 g / L glucose and 50 mg / L kanamycin). This culture was carried out in a 96 microtiter plate at 30°C with shaking for 8 h.

[0789] 50 μL of the above culture was used to inoculate 60 g / L overnight rapid expression TB medium (Overnight Express TM Instant TB medium) 3.95 mL of production medium with 10 g / L glycerol and 50 mg / L kanamycin. The resulting culture was stirred in a 48 deep well plate at 20°C. The culture produced significant growth and obtained a good OD (600 nm). After 44 hours, the cells were harvested by centrifugation and frozen.

[0790] By adding Master Mix Lysis was performed and the lysate was recovered by centrifugation. Activity was tested with 100 μL of fresh lysate added to a solution of rebaudioside D (final concentration 0.5 mM), MgCl2 (final concentration 3 mM) and UDP-glucose (final concentration 2.5 mM) in 50 mM phosphate buffer (pH 7.2).

[0791] The reaction was carried out at 30°C and samples were taken after 2, 4, 7 and 24 hours. In order to determine the conversion and initial rate by HPLC (CAD detection), the above-mentioned analytical method for the conversion of rebaudioside D to rebaudioside M was used. The experiment was carried out in parallel with the baseline clone round 1-Var94. After 22h, it was converted. And the initial rate for this baseline clone was defined as 100%, and the normalized conversion rate and initial rate for the second round of cloning are depicted in the following table:

[0792]

[0793]

[0794]

[0795]

[0796]

[0797] *Mutations are annotated as follows: Reference gene - original amino acid - position - new amino acid: For example, for variant 94 from the first round of directed evolution of UGT76G1, the mutation of alanine to glycine at position 33 is annotated as Round 1-Var94(A33G)

[0798] Modeling of these results allowed a ranking of the effect of each mutation to be obtained. The following mutations were determined to be beneficial for activity: S42A, F46I, I190L, S274G, I295M, K303G, F314S, K316R, K393R, V394I, I407V, N409K, N409R, Q425E, Q432E, S447A, S456L.

[0799] Example 42

[0800] In vivo production of AtSUS

[0801] AtSUS

[0802] >gi|79328294|ref|NP_001031915.1|Sucrose synthase 1[Arabidopsis thaliana]

[0803] MANAERMITRVHSQRERLNETLVSERNEVLALLSRVEAKGKGILQQNQIIAEFEALPEQTRKKLEGGPFFDLLKSTQEAIVLPPWVALAVRPRPGVWEYLRVNLHALVVEELQPAEFLHFKEELVDGVKNGNFTLELDFEPFNASIPRPTLHKYIGNGVDFLNRHLSAKLFHDKESLLPLLKFLRLHSHQGKNLMLSEKIQNLNTLQHTLRKAEEYLAELKSETLYEEFEAKFEEIGLERGWGDNAERVLDMIRLLLDLLEAPDPCTLETFLGRVPMVFNVVILSPHGYFAQDNVLGYPDTGGQVVYILDQVRALEIEMLQRIKQQGLNIKPRILILTRLLPDAVGTTCGERLERVYDSEYCDILRVPFRTEKGIVRKWISRFEVWPYLETYTEDAAVELSKELNGKPDLIIGNYSDGNLVASLLAHKLGVTQCTIAHALEKTKYPDSDIYWKKLDDKYHFSCQFTADIFAMNHTDFIITSTFQEIAGSKETVGQYESHTAFTLPGLYRVVHGIDVFDPKFNIVSPGADMSIYFPYTEEKRRLTKFHSEIEELLYSDVENKEHLCVLKDKKKPILFTMARLDRVKNLSGLVEWYGKNTRLRELANLVVVGGDRRKESKDNEEKAEMKKMYDLIEEYKLNGQFRWISSQMDRVRNGELYRYICDTKGAFVQPALYEAFGLTVVEAMTCGLPTFATCKGGPAEIIVHGKSGFHIDPYHGDQAADTLADFFTKCKEDPSHWDEISKGGLQRIEEKYTWQIYSQRLLTLTGVYGFWKHVSNLDRLEARRYLEMFYALKYRPLAQAVPLAQDD(SEQ ID NO:13)。

[0804] The synthesized AtSuS gene was codon optimized for expression in E. coli and subcloned into pET30a+ plasmid using NdeI and XhoI restriction sites. The pET30A+ vector containing the AtSUS gene was used to transform electrocompetent E. coli Bl21 (DE3) cells. The obtained cells were cultured in a culture dish in the presence of kanamycin, and appropriate colonies were selected and grown in liquid LB medium (Erlenmayer flask). Glycerol was added to the suspension as a cryoprotectant and aliquots of 400 μL were stored at -20°C and at -80°C.

[0805] A stock aliquot of E. coli BL21 (DE3) containing the pET30A+_AtSUS plasmid was thawed and added to 30 mL of LBGKP medium (20 g / L Luria Broth Lennox; 50 mM PIPES buffer pH 7.00; 50 mM phosphate buffer pH 7.00; 2.5 g / L glucose and 50 mg / L kanamycin). This culture was carried out at 30° C. with shaking at 135 rpm for 8 h.

[0806] The production medium contains 60 g / L overnight fast expression TB medium (Novagen), 10 g / L glycerol and 50 mg / L kanamycin. The pre-culture is added to 800 mL of this medium and the solution is stirred at 20 ° C while taking samples to measure OD and pH. The culture produces significant growth and obtains a good OD. After 40 hours, the cells are harvested by centrifugation and frozen to obtain a cell wet weight of 30.1 g.

[0807] Lysis was performed by Fastprep (MP Biomedicals, Lysis Matrix B, Speed ​​6.0, 3 x 40 sec) with a cell suspension of 200 mg of cells in 1.0 mL of 50 mM Tris buffer (pH 7.5). The lysate was recovered by centrifugation and used fresh.

[0808] Example 43

[0809] Conversion of rebaudioside A to rebaudioside M using UGTSL2, UGT76G1-R1-F12, and AtSUS with in situ produced UDP-glucose

[0810] The reaction was carried out on a 1 mL scale using 100 mM sucrose, 3 mM MgCl2, 0.25 mM UDP and 0.5 mM rebaudioside A in potassium phosphate buffer (50 mM final concentration, pH 7.5). The reaction was started by adding 15 μL of UGTSL2 (see Example 27) lysate (2 U / mL), 150 μL of UGT76G1var94 (see Example 26) (2.5 U / mL) and 15 μL of AtSUS (see Example 42) (400 U / mL). After the reaction, 125 μL of sample was quenched with 10 μL of 2N H2SO4 and 115 μL of 60% methanol, followed by HPLC. After 21 hours of reaction time, 68% of rebaudioside M and 26% of rebaudioside M2 ​​were obtained. Sequence Listing <110> The Coca-Cola Company PureCircle Sdn Bhd Prakash, Indra Bunders, Cynthia Soni, Pankaj Markosyan, Avetik Cyrille, Jarrin Badie, Aurelien ter Halle, Robert <120> High purity steviol glycosides <130> 12600.105134 <150> US 61 / 827,922 <151> 2013-05-28 <150> US 61 / 843,544 <151> 2013-07-28 <150> US 61 / 861,528 <151> 2013-08-02 <150> US 61 / 881,166 <151> 2013-09-23 <150> US 61 / 885,084 <151> 2013-10-01 <150> US 61 / 904,751 <151> November 15, 2013 <150> US 61 / 913,482 <151> December 9, 2013 <150> US 61 / 921,635 <151> December 30, 2013 <150> US 61 / 925,329 <151> January 9, 2014 <150> US 61 / 939,855 <151> February 14, 2014 <160> 13 <170> PatentIn Version 3.5 <210> 1 <211> 1397 <212> DNA <213> Stevia <400> 1 ccatggccca tatggaaaac aaaaccgaaa ccaccgttcg tcgtcgtcgc cgtattattc 60 tgtttccggt tccgtttcag ggtcatatta atccgattct gcagctggca aatgtgctgt 120 atagcaaagg ttttagcatt accatttttc ataccaattt taacaaaccg aaaaccagca 180 attatccgca ttttaccttt cgctttattc tggataatga tccgcaggat gaacgcatta 240 gcaatctgcc gacacatggt ccgctggcag gtatgcgtat tccgattatt aacgaacatg 300 gtgcagatga actgcgtcgt gaactggaac tgctgatgct ggcaagcgaa gaagatgaag 360 aagttagctg tctgattacc gatgcactgt ggtattttgc acagagcgtt gcagatagcc 420 tgaatctgcg tcgtctggtt ctgatgacca gcagcctgtt taactttcat gcacatgtta 480 gcctgccgca gtttgatgaa ctgggttatc tggatccgga tgataaaacc cgtctggaag 540 aacaggcaag cggttttccg atgctgaaag tgaaagatat caaaagcgcc tatagcaatt 600 ggcagattct gaaagaaatt ctgggcaaaa tgattaaaca gaccaaagca agcagcggtg 660 ttatttggaa tagctttaaa gaactggaag aaagcgaact ggaaaccgtg attcgtgaaa 720 ttccggcacc gagctttctg attccgctgc cgaaacatct gaccgcaagc agcagcagcc 780 tgctggatca tgatcgtacc gtttttcagt ggctggatca gcagcctccg agcagcgttc 840 tgtatgttag ctttggtagc accagcgaag ttgatgaaaa agattttctg gaaattgccc 900 gtggtctggt tgatagcaaa cagagctttc tgtgggttgt tcgtccgggt tttgttaaag 960 gtagcacctg ggttgaaccg ctgccggatg gttttctggg tgaacgtggt cgtattgtta 1020 aatgggttcc gcagcaagaa gttctggcac acggcgcaat tggtgcattt tggacccata 1080 gcggttggaa tagcaccctg gaaagcgttt gtgaaggtgt tccgatgatt tttagcgatt 1140 ttggtctgga tcagccgctg aatgcacgtt atatgagtga tgttctgaaa gtgggtgtgt 1200 atctggaaaa tggttgggaa cgtggtgaaa ttgcaaatgc aattcgtcgt gttatggtgg 1260 atgaagaagg tgaatatatt cgtcagaatg cccgtgttct gaaacagaaa gcagatgtta 1320 gcctgatgaa aggtggtagc agctatgaaa gcctggaaag tctggttagc tatattagca 1380 gcctgtaata actcgag 1397 <210> 2 <211> 1442 <212> DNA <213> Stevia rebaudiana <400> 2 ccatggcaca tatggcaacc agcgatagca ttgttgatga tcgtaaacag ctgcatgttg 60 caacctttcc gtggctggca tttggtcata ttctgccgta tctgcagctg agcaaactga 120 ttgcagaaaa aggtcataaa gtgagctttc tgagcaccac ccgtaatatt cagcgtctga 180 gcagccatat tagtccgctg attaatgttg ttcagctgac cctgcctcgt gttcaagaac 240 tgccggaaga tgccgaagca accaccgatg ttcatccgga agatattccg tatctgaaaa 300 aagcaagtga tggtctgcag ccggaagtta cccgttttct ggaacagcat agtccggatt 360 ggatcatcta tgattatacc cattattggc tgccgagcat tgcagcaagc ctgggtatta 420 gccgtgcaca ttttagcgtt accaccccgt gggcaattgc atatatgggt ccgagcgcag 480 atgcaatgat taatggtagt gatggtcgta ccaccgttga agatctgacc acccctccga 540 aatggtttcc gtttccgacc aaagtttgtt ggcgtaaaca tgatctggca cgtctggttc 600 cgtataaagc accgggtatt agtgatggtt atcgtatggg tctggttctg aaaggtagcg 660 attgtctgct gagcaaatgc tatcatgaat ttggcaccca gtggctgccg ctgctggaaa 720 ccctgcatca ggttccggtt gttccggtgg gtctgctgcc tccggaagtt ccgggtgatg 780 aaaaagatga aacctgggtt agcatcaaaa aatggctgga tggtaaacag aaaggtagcg 840 tggtttatgt tgcactgggt agcgaagttc tggttagcca gaccgaagtt gttgaactgg 900 cactgggtct ggaactgagc ggtctgccgt ttgtttgggc atatcgtaaa ccgaaaggtc 960 cggcaaaaag cgatagcgtt gaactgccgg atggttttgt tgaacgtacc cgtgatcgtg 1020 gtctggtttg gaccagctgg gcacctcagc tgcgtattct gagccatgaa agcgtttgtg 1080 gttttctgac ccattgtggt agcggtagca ttgtggaagg tctgatgttt ggtcatccgc 1140 tgattatgct gccgattttt ggtgatcagc cgctgaatgc acgtctgctg gaagataaac 1200 aggttggtat tgaaattccg cgtaatgaag aagatggttg cctgaccaaa gaaagcgttg 1260 cacgtagcct gcgtagcgtt gttgttgaaa aagaaggcga aatctataaa gccaatgcac 1320 gtgaactgag caaaatctat aatgatacca aagtggaaaa agaatatgtg agccagttcg 1380 tggattatct ggaaaaaaac acccgtgcag ttgccattga tcacgaaagc taatgactcg 1440 ag 1442 <210> 3 <211> 472 <212> PRT <213> Oryza sativa <400> 3 Met Asp Asp Ala His Ser Ser Gln Ser Pro Leu His Val Val Ile Phe 1 5 10 15 Pro Trp Leu Ala Phe Gly His Leu Leu Pro Cys Leu Asp Leu Ala Glu 20 25 30 Arg Leu Ala Ala Arg Gly His Arg Val Ser Phe Val Ser Thr Pro Arg 35 40 45 Asn Leu Ala Arg Leu Pro Pro Val Arg Pro Glu Leu Ala Glu Leu Val 50 55 60 Asp Leu Val Ala Leu Pro Leu Pro Arg Val Asp Gly Leu Pro Asp Gly 65 70 75 80 Ala Glu Ala Thr Ser Asp Val Pro Phe Asp Lys Phe Glu Leu His Arg 85 90 95 Lys Ala Phe Asp Gly Leu Ala Ala Pro Phe Ser Ala Phe Leu Asp Thr 100 105 110 Ala Cys Ala Gly Gly Lys Arg Pro Asp Trp Val Leu Ala Asp Leu Met 115 120 125 His His Trp Val Ala Leu Ala Ser Gln Glu Arg Gly Val Pro Cys Ala 130 135 140 Met Ile Leu Pro Cys Ser Ala Ala Val Val Ala Ser Ser Ala Pro Pro 145 150 155 160 Thr Glu Ser Ser Ala Asp Gln Arg Glu Ala Ile Val Arg Ser Met Gly 165 170 175 Thr Ala Ala Pro Ser Phe Glu Ala Lys Arg Ala Thr Glu Glu Phe Ala 180 185 190 Thr Glu Gly Ala Ser Gly Val Ser Ile Met Thr Arg Tyr Ser Leu Thr 195 200 205 Leu Gln Arg Ser Lys Leu Val Ala Met Arg Ser Cys Pro Glu Leu Glu 210 215 220 Pro Gly Ala Phe Thr Ile Leu Thr Arg Phe Tyr Gly Lys Pro Val Val 225 230 235 240 Pro Phe Gly Leu Leu Pro Pro Arg Pro Asp Gly Ala Arg Gly Val Ser 245 250 255 Lys Asn Gly Lys His Asp Ala Ile Met Gln Trp Leu Asp Ala Gln Pro 260 265 270 Ala Lys Ser Val Val Tyr Val Ala Leu Gly Ser Glu Ala Pro Met Ser 275 280 285 Ala Asp Leu Leu Arg Glu Leu Ala His Gly Leu Asp Leu Ala Gly Thr 290 295 300 Arg Phe Leu Trp Ala Met Arg Lys Pro Ala Gly Val Asp Ala Asp Ser 305 310 315 320 Val Leu Pro Ala Gly Phe Leu Gly Arg Thr Gly Glu Arg Gly Leu Val 325 330 335 Thr Thr Arg Trp Ala Pro Gln Val Ser Ile Leu Ala His Ala Ala Val 340 345 350 Cys Ala Phe Leu Thr His Cys Gly Trp Gly Ser Val Val Glu Gly Leu 355 360 365 Gln Phe Gly His Pro Leu Ile Met Leu Pro Ile Leu Gly Asp Gln Gly 370 375 380 Pro Asn Ala Arg Ile Leu Glu Gly Arg Lys Leu Gly Val Ala Val Pro 385 390 395 400 Arg Asn Asp Glu Asp Gly Ser Phe Asp Arg Gly Gly Val Ala Gly Ala 405 410 415 Val Arg Ala Val Val Val Glu Glu Glu Gly Lys Thr Phe Phe Ala Asn 420 425 430 Ala Arg Lys Leu Gln Glu Ile Val Ala Asp Arg Glu Arg Glu Glu Arg 435 440 445 Cys Ile Asp Glu Phe Val Gln His Leu Thr Ser Trp Asn Glu Leu Lys 450 455 460 Asn Asn Ser Asp Gly Gln Tyr Pro 465 470 <210> 4 <211> 502 <212> PRT <213> Avena strigosa <400> 4 Met Ala Val Lys Asp Glu Gln Gln Ser Pro Leu His Ile Leu Leu Phe 1 5 10 15 Pro Phe Leu Ala Pro Gly His Leu Ile Pro Ile Ala Asp Met Ala Ala 20 25 30 Leu Phe Ala Ser Arg Gly Val Arg Cys Thr Ile Leu Thr Thr Pro Val 35 40 45 Asn Ala Ala Ile Ile Arg Ser Ala Val Asp Arg Ala Asn Asp Ala Phe 50 55 60 Arg Gly Ser Asp Cys Pro Ala Ile Asp Ile Ser Val Val Pro Phe Pro 65 70 75 80 Asp Val Gly Leu Pro Pro Gly Val Glu Asn Gly Asn Ala Leu Thr Ser 85 90 95 Pro Ala Asp Arg Leu Lys Phe Phe Gln Ala Val Ala Glu Leu Arg Glu 100 105 110 Pro Phe Asp Arg Phe Leu Ala Asp Asn His Pro Asp Ala Val Val Ser 115 120 125 Asp Ser Phe Phe His Trp Ser Thr Asp Ala Ala Ala Glu His Gly Val 130 135 140 Pro Arg Leu Gly Phe Leu Gly Ser Ser Met Phe Ala Gly Ser Cys Asn 145 150 155 160 Glu Ser Thr Leu His Asn Asn Pro Leu Glu Thr Ala Ala Asp Asp Pro 165 170 175 Asp Ala Leu Val Ser Leu Pro Gly Leu Pro His Arg Val Glu Leu Arg 180 185 190 Arg Ser Gln Met Met Asp Pro Lys Lys Arg Pro Asp His Trp Ala Leu 195 200 205 Leu Glu Ser Val Asn Ala Ala Asp Gln Lys Ser Phe Gly Glu Val Phe 210 215 220 Asn Ser Phe His Glu Leu Glu Pro Asp Tyr Val Glu His Tyr Gln Thr 225 230 235 240 Thr Leu Gly Arg Arg Thr Trp Leu Val Gly Pro Val Ala Leu Ala Ser 245 250 255 Lys Asp Met Ala Gly Arg Gly Ser Thr Ser Ala Arg Ser Pro Asp Ala 260 265 270 Asp Ser Cys Leu Arg Trp Leu Asp Thr Lys Gln Pro Gly Ser Val Val 275 280 285 Tyr Val Ser Phe Gly Thr Leu Ile Arg Phe Ser Pro Ala Glu Leu His 290 295 300 Glu Leu Ala Arg Gly Leu Asp Leu Ser Gly Lys Asn Phe Val Trp Val 305 310 315 320 Leu Gly Arg Ala Gly Pro Asp Ser Ser Glu Trp Met Pro Gln Gly Phe 325 330 335 Ala Asp Leu Ile Thr Pro Arg Gly Asp Arg Gly Phe Ile Ile Arg Gly 340 345 350 Trp Ala Pro Gln Met Leu Ile Leu Asn His Arg Ala Leu Gly Gly Phe 355 360 365 Val Thr His Cys Gly Trp Asn Ser Thr Leu Glu Ser Val Ser Ala Gly 370 375 380 Val Pro Met Val Thr Trp Pro Arg Phe Ala Asp Gln Phe Gln Asn Glu 385 390 395 400 Lys Leu Ile Val Glu Val Leu Lys Val Gly Val Ser Ile Gly Ala Lys 405 410 415 Asp Tyr Gly Ser Gly Ile Glu Asn His Asp Val Ile Arg Gly Glu Val 420 425 430 Ile Ala Glu Ser Ile Gly Lys Leu Met Gly Ser Ser Glu Glu Ser Asp 435 440 445 Ala Ile Gln Arg Lys Ala Lys Asp Leu Gly Ala Glu Ala Arg Ser Ala 450 455 460 Val Glu Asn Gly Gly Ser Ser Tyr Asn Asp Val Gly Arg Leu Met Asp 465 470 475 480 Glu Leu Met Ala Arg Arg Ser Ser Val Lys Val Gly Glu Asp Ile Ile 485 490 495 Pro Thr Asn Asp Gly Leu 500 <210> 5 <211> 470 <212> PRT <213> Tomato <400> 5 Met Ser Pro Lys Leu His Lys Glu Leu Phe Phe His Ser Leu Tyr Lys 1 5 10 15 Lys Thr Arg Ser Asn His Thr Met Ala Thr Leu Lys Val Leu Met Phe 20 25 30 Pro Phe Leu Ala Tyr Gly His Ile Ser Pro Tyr Leu Asn Val Ala Lys 35 40 45 Lys Leu Ala Asp Arg Gly Phe Leu Ile Tyr Phe Cys Ser Thr Pro Ile 50 55 60 Asn Leu Lys Ser Thr Ile Glu Lys Ile Pro Glu Lys Tyr Ala Asp Ser 65 70 75 80 Ile His Leu Ile Glu Leu His Leu Pro Glu Leu Pro Gln Leu Pro Pro 85 90 95 His Tyr His Thr Thr Asn Gly Leu Pro Pro Asn Leu Asn Gln Val Leu 100 105 110 Gln Lys Ala Leu Lys Met Ser Lys Pro Asn Phe Ser Lys Ile Leu Gln 115 120 125 Asn Leu Lys Pro Asp Leu Val Ile Tyr Asp Ile Leu Gln Arg Trp Ala 130 135 140 Lys His Val Ala Asn Glu Gln Asn Ile Pro Ala Val Lys Leu Leu Thr 145 150 155 160 Ser Gly Ala Ala Val Phe Ser Tyr Phe Phe Asn Val Leu Lys Lys Pro 165 170 175 Gly Val Glu Phe Pro Phe Pro Gly Ile Tyr Leu Arg Lys Ile Glu Gln 180 185 190 Val Arg Leu Ser Glu Met Met Ser Lys Ser Asp Lys Glu Lys Glu Leu 195 200 205 Glu Asp Asp Asp Asp Asp Asp Asp Leu Leu Val Asp Gly Asn Met Gln 210 215 220 Ile Met Leu Met Ser Thr Ser Arg Thr Ile Glu Ala Lys Tyr Ile Asp 225 230 235 240 Phe Cys Thr Ala Leu Thr Asn Trp Lys Val Val Pro Val Gly Pro Pro 245 250 255 Val Gln Asp Leu Ile Thr Asn Asp Val Asp Asp Met Glu Leu Ile Asp 260 265 270 Trp Leu Gly Thr Lys Asp Glu Asn Ser Thr Val Phe Val Ser Phe Gly 275 280 285 Ser Glu Tyr Phe Leu Ser Lys Glu Asp Met Glu Glu Val Ala Phe Ala 290 295 300 Leu Glu Leu Ser Asn Val Asn Phe Ile Trp Val Ala Arg Phe Pro Lys 305 310 315 320 Gly Glu Glu Arg Asn Leu Glu Asp Ala Leu Pro Lys Gly Phe Leu Glu 325 330 335 Arg Ile Gly Glu Arg Gly Arg Val Leu Asp Lys Phe Ala Pro Gln Pro 340 345 350 Arg Ile Leu Asn His Pro Ser Thr Gly Gly Phe Ile Ser His Cys Gly 355 360 365 Trp Asn Ser Ala Met Glu Ser Ile Asp Phe Gly Val Pro Ile Ile Ala 370 375 380 Met Pro Met His Leu Asp Gln Pro Met Asn Ala Arg Leu Ile Val Glu 385 390 395 400 Leu Gly Val Ala Val Glu Ile Val Arg Asp Asp Asp Gly Lys Ile His 405 410 415 Arg Gly Glu Ile Ala Glu Thr Leu Lys Gly Val Ile Thr Gly Lys Thr 420 425 430 Gly Glu Lys Leu Arg Ala Lys Val Arg Asp Ile Ser Lys Asn Leu Lys 435 440 445 Thr Ile Arg Asp Glu Glu Met Asp Ala Ala Ala Glu Glu Leu Ile Gln 450 455 460 Leu Cys Arg Asn Gly Asn 465 470 <210> 6 <211> 464 <212> PRT <213> Rice <400> 6 Met His Val Val Met Leu Pro Trp Leu Ala Phe Gly His Ile Leu Pro 1 5 10 15 Phe Ala Glu Phe Ala Lys Arg Val Ala Arg Gln Gly His Arg Val Thr 20 25 30 Leu Phe Ser Thr Pro Arg Asn Thr Arg Arg Leu Ile Asp Val Pro Pro 35 40 45 Ser Leu Ala Gly Arg Ile Arg Val Val Asp Ile Pro Leu Pro Arg Val 50 55 60 Glu His Leu Pro Glu His Ala Glu Ala Thr Ile Asp Leu Pro Ser Asn 65 70 75 80 Asp Leu Arg Pro Tyr Leu Arg Arg Ala Tyr Asp Glu Ala Phe Ser Arg 85 90 95 Glu Leu Ser Arg Leu Leu Gln Glu Thr Gly Pro Ser Arg Pro Asp Trp 100 105 110 Val Leu Ala Asp Tyr Ala Ala Tyr Trp Ala Pro Ala Ala Ala Ser Arg 115 120 125 His Gly Val Pro Cys Ala Phe Leu Ser Leu Phe Gly Ala Ala Ala Leu 130 135 140 Cys Phe Phe Gly Pro Ala Glu Thr Leu Gln Gly Arg Gly Pro Tyr Ala 145 150 155 160 Lys Thr Glu Pro Ala His Leu Thr Ala Val Pro Glu Tyr Val Pro Phe 165 170 175 Pro Thr Thr Val Ala Phe Arg Gly Asn Glu Ala Arg Glu Leu Phe Lys 180 185 190 Pro Ser Leu Ile Pro Asp Glu Ser Gly Val Ser Glu Ser Tyr Arg Phe 195 200 205 Ser Gln Ser Ile Glu Gly Cys Gln Leu Val Ala Val Arg Ser Asn Gln 210 215 220 Glu Phe Glu Pro Glu Trp Leu Glu Leu Leu Gly Glu Leu Tyr Gln Lys 225 230 235 240 Pro Val Ile Pro Ile Gly Met Phe Pro Pro Pro Pro Pro Gln Asp Val 245 250 255 Ala Gly His Glu Glu Thr Leu Arg Trp Leu Asp Arg Gln Glu Pro Asn 260 265 270 Ser Val Val Tyr Ala Ala Phe Gly Ser Glu Val Lys Leu Thr Ala Glu 275 280 285 Gln Leu Gln Arg Ile Ala Leu Gly Leu Glu Ala Ser Glu Leu Pro Phe 290 295 300 Ile Trp Ala Phe Arg Ala Pro Pro Asp Ala Gly Asp Gly Asp Gly Leu 305 310 315 320 Pro Gly Gly Phe Lys Glu Arg Val Asn Gly Arg Gly Val Val Cys Arg 325 330 335 Gly Trp Val Pro Gln Val Lys Phe Leu Ala His Ala Ser Val Gly Gly 340 345 350 Phe Leu Thr His Ala Gly Trp Asn Ser Ile Ala Glu Gly Leu Ala Asn 355 360 365 Gly Val Arg Leu Val Leu Leu Pro Leu Met Phe Glu Gln Gly Leu Asn 370 375 380 Ala Arg Gln Leu Ala Glu Lys Lys Val Ala Val Glu Val Ala Arg Asp 385 390 395 400 Glu Asp Asp Gly Ser Phe Ala Ala Asn Asp Ile Val Asp Ala Leu Arg 405 410 415 Arg Val Met Val Gly Glu Glu Gly Asp Glu Phe Gly Val Lys Val Lys 420 425 430 Glu Leu Ala Lys Val Phe Gly Asp Asp Glu Val Asn Asp Arg Tyr Val 435 440 445 Arg Asp Phe Leu Lys Cys Leu Ser Glu Tyr Lys Met Gln Arg Gln Gly 450 455 460 <210> 7 <211> 515 <212> PRT <213> Arabidopsis lyrata <400> 7 Met Asp Asp Lys Lys Glu Glu Val Met His Ile Ala Met Phe Pro Trp 1 5 10 15 Leu Ala Met Gly His Leu Leu Pro Phe Leu Arg Leu Ser Lys Leu Leu 20 25 30 Ala Gln Lys Gly His Lys Ile Ser Phe Ile Ser Thr Pro Arg Asn Ile 35 40 45 Leu Arg Leu Pro Lys Leu Pro Ser Asn Leu Ser Ser Ser Ile Thr Phe 50 55 60 Val Ser Phe Pro Leu Pro Ser Ile Ser Gly Leu Pro Pro Ser Ser Glu 65 70 75 80 Ser Ser Met Asp Val Pro Tyr Asn Lys Gln Gln Ser Leu Lys Ala Ala 85 90 95 Phe Asp Leu Leu Gln Pro Pro Leu Thr Glu Phe Leu Arg Leu Ser Ser 100 105 110 Pro Asp Trp Ile Ile Tyr Asp Tyr Ala Ser His Trp Leu Pro Ser Ile 115 120 125 Ala Lys Glu Leu Gly Ile Ser Lys Ala Phe Phe Ser Leu Phe Asn Ala 130 135 140 Ala Thr Leu Cys Phe Met Gly Pro Ser Ser Ser Leu Ile Glu Glu Ser 145 150 155 160 Arg Ser Thr Pro Glu Asp Phe Thr Val Val Pro Pro Trp Val Pro Phe 165 170 175 Lys Ser Thr Ile Val Phe Arg Tyr His Glu Val Ser Arg Tyr Val Glu 180 185 190 Lys Thr Asp Glu Asp Val Thr Gly Val Ser Asp Ser Val Arg Phe Gly 195 200 205 Tyr Thr Ile Asp Gly Ser Asp Ala Val Phe Val Arg Ser Cys Pro Glu 210 215 220 Phe Glu Pro Glu Trp Phe Ser Leu Leu Gln Asp Leu Tyr Arg Lys Pro 225 230 235 240 Val Phe Pro Ile Gly Phe Leu Pro Pro Val Ile Glu Asp Asp Asp Asp 245 250 255 Asp Thr Thr Trp Val Arg Ile Lys Glu Trp Leu Asp Lys Gln Arg Val 260 265 270 Asn Ser Val Val Tyr Val Ser Leu Gly Thr Glu Ala Ser Leu Arg Arg 275 280 285 Glu Glu Leu Thr Glu Leu Ala Leu Gly Leu Glu Lys Ser Glu Thr Pro 290 295 300 Phe Phe Trp Val Leu Arg Asn Glu Pro Gln Ile Pro Asp Gly Phe Glu 305 310 315 320 Glu Arg Val Lys Gly Arg Gly Met Val His Val Gly Trp Val Pro Gln 325 330 335 Val Lys Ile Leu Ser His Glu Ser Val Gly Gly Phe Leu Thr His Cys 340 345 350 Gly Trp Asn Ser Val Val Glu Gly Ile Gly Phe Gly Lys Val Pro Ile 355 360 365 Phe Leu Pro Val Leu Asn Glu Gln Gly Leu Asn Thr Arg Leu Leu Gln 370 375 380 Gly Lys Gly Leu Gly Val Glu Val Leu Arg Asp Glu Arg Asp Gly Ser 385 390 395 400 Phe Gly Ser Asp Ser Val Ala Asp Ser Val Arg Leu Val Met Ile Asp 405 410 415 Asp Ala Gly Glu Glu Ile Arg Glu Lys Val Lys Leu Met Lys Gly Leu 420 425 430 Phe Gly Asn Met Asp Glu Asn Ile Arg Tyr Val Asp Glu Leu Val Gly 435 440 445 Phe Met Arg Asn Asp Glu Ser Ser Gln Leu Lys Glu Glu Glu Glu Glu 450 455 460 Asp Asp Cys Ser Asp Asp Gln Ser Ser Glu Val Ser Ser Glu Thr Asp 465 470 475 480 Glu Lys Glu Leu Asn Leu Asp Leu Lys Glu Glu Lys Arg Arg Ile Ser 485 490 495 Val Tyr Lys Ser Leu Ser Ser Glu Phe Asp Asp Tyr Val Ala Asn Glu 500 505 510 Lys Met Gly 515 <210> 8 <211> 772 <212> PRT <213> Rice <400> 8 Met His Val Val Ile Cys Pro Leu Leu Ala Phe Gly His Leu Leu Pro 1 5 10 15 Cys Leu Asp Leu Ala Gln Arg Leu Ala Cys Gly His Arg Val Ser Phe 20 25 30 Val Ser Thr Pro Arg Asn Ile Ser Arg Leu Pro Pro Val Arg Pro Ser 35 40 45 Leu Ala Pro Leu Val Ser Phe Val Ala Leu Pro Leu Pro Arg Val Glu 50 55 60 Gly Leu Pro Asn Gly Ala Glu Ser Thr His Asn Val Pro His Asp Arg 65 70 75 80 Pro Asp Met Val Glu Leu His Leu Arg Ala Phe Asp Gly Leu Ala Ala 85 90 95 Pro Phe Ser Glu Phe Leu Gly Thr Ala Cys Ala Asp Trp Val Met Pro 100 105 110 Thr Ser Ser Ala Pro Arg Gln Thr Leu Ser Ser Asn Ile His Arg Asn 115 120 125 Ser Ser Arg Pro Gly Thr Pro Ala Pro Ser Gly Arg Leu Leu Cys Pro 130 135 140 Ile Thr Pro His Ser Asn Thr Leu Glu Arg Ala Ala Glu Lys Leu Val 145 150 155 160 Arg Ser Ser Arg Gln Asn Ala Arg Ala Arg Ser Leu Leu Ala Phe Thr 165 170 175 Ser Pro Pro Leu Pro Tyr Arg Asp Val Phe Arg Ser Leu Leu Gly Leu 180 185 190 Gln Met Gly Arg Lys Gln Leu Asn Ile Ala His Glu Thr Asn Gly Arg 195 200 205 Arg Thr Gly Thr Leu Pro Leu Asn Leu Cys Arg Trp Met Trp Lys Gln 210 215 220 Arg Arg Cys Gly Lys Leu Arg Pro Ser Asp Val Glu Phe Asn Thr Ser 225 230 235 240 Arg Ser Asn Glu Ala Ile Ser Pro Ile Gly Ala Ser Leu Val Asn Leu 245 250 255 Gln Ser Ile Gln Ser Pro Asn Pro Arg Ala Val Leu Pro Ile Ala Ser 260 265 270 Ser Gly Val Arg Ala Val Phe Ile Gly Arg Ala Arg Thr Ser Thr Pro 275 280 285 Thr Pro Pro His Ala Lys Pro Ala Arg Ser Ala Ala Pro Arg Ala His 290 295 300 Arg Pro Pro Ser Ser Val Met Asp Ser Gly Tyr Ser Ser Ser Tyr Ala 305 310 315 320 Ala Ala Ala Gly Met His Val Val Ile Cys Pro Trp Leu Ala Phe Gly 325 330 335 His Leu Leu Pro Cys Leu Asp Leu Ala Gln Arg Leu Ala Ser Arg Gly 340 345 350 His Arg Val Ser Phe Val Ser Thr Pro Arg Asn Ile Ser Arg Leu Pro 355 360 365 Pro Val Arg Pro Ala Leu Ala Pro Leu Val Ala Phe Val Ala Leu Pro 370 375 380 Leu Pro Arg Val Glu Gly Leu Pro Asp Gly Ala Glu Ser Thr Asn Asp 385 390 395 400 Val Pro His Asp Arg Pro Asp Met Val Glu Leu His Arg Arg Ala Phe 405 410 415 Asp Gly Leu Ala Ala Pro Phe Ser Glu Phe Leu Gly Thr Ala Cys Ala 420 425 430 Asp Trp Val Ile Val Asp Val Phe His His Trp Ala Ala Ala Ala Ala 435 440 445 Leu Glu His Lys Val Pro Cys Ala Met Met Leu Leu Gly Ser Ala His 450 455 460 Met Ile Ala Ser Ile Ala Asp Arg Arg Leu Glu Arg Ala Glu Thr Glu 465 470 475 480 Ser Pro Ala Ala Ala Gly Gln Gly Arg Pro Ala Ala Ala Pro Thr Phe 485 490 495 Glu Val Ala Arg Met Lys Leu Ile Arg Thr Lys Gly Ser Ser Gly Met 500 505 510 Ser Leu Ala Glu Arg Phe Ser Leu Thr Leu Ser Arg Ser Ser Leu Val 515 520 525 Val Gly Arg Ser Cys Val Glu Phe Glu Pro Glu Thr Val Pro Leu Leu 530 535 540 Ser Thr Leu Arg Gly Lys Pro Ile Thr Phe Leu Gly Leu Met Pro Pro 545 550 555 560 Leu His Glu Gly Arg Arg Glu Asp Gly Glu Asp Ala Thr Val Arg Trp 565 570 575 Leu Asp Ala Gln Pro Ala Lys Ser Val Val Tyr Val Ala Leu Gly Ser 580 585 590 Glu Val Pro Leu Gly Val Glu Lys Val His Glu Leu Ala Leu Gly Leu 595 600 605 Glu Leu Ala Gly Thr Arg Phe Leu Trp Ala Leu Arg Lys Pro Thr Gly 610 615 620 Val Ser Asp Ala Asp Leu Leu Pro Ala Gly Phe Glu Glu Arg Thr Arg 625 630 635 640 Gly Arg Gly Val Val Ala Thr Arg Trp Val Pro Gln Met Ser Ile Leu 645 650 655 Ala His Ala Ala Val Gly Ala Phe Leu Thr His Cys Gly Trp Asn Ser 660 665 670 Thr Ile Glu Gly Leu Met Phe Gly His Pro Leu Ile Met Leu Pro Ile 675 680 685 Phe Gly Asp Gln Gly Pro Asn Ala Arg Leu Ile Glu Ala Lys Asn Ala 690 695 700 Gly Leu Gln Val Ala Arg Asn Asp Gly Asp Gly Ser Phe Asp Arg Glu 705 710 715 720 Gly Val Ala Ala Ala Ile Arg Ala Val Ala Val Glu Glu Glu Ser Ser 725 730 735 Lys Val Phe Gln Ala Lys Ala Lys Lys Leu Gln Glu Ile Val Ala Asp 740 745 750 Met Ala Cys His Glu Arg Tyr Ile Asp Gly Phe Ile Gln Gln Leu Arg 755 760 765 Ser Tyr Lys Asp 770 <210> 9 <211> 442 <212> PRT <213> Tomato <400> 9 Met Ala Thr Asn Leu Arg Val Leu Met Phe Pro Trp Leu Ala Tyr Gly 1 5 10 15 His Ile Ser Pro Phe Leu Asn Ile Ala Lys Gln Leu Ala Asp Arg Gly 20 25 30 Phe Leu Ile Tyr Leu Cys Ser Thr Arg Ile Asn Leu Glu Ser Ile Ile 35 40 45 Lys Lys Ile Pro Glu Lys Tyr Ala Asp Ser Ile His Leu Ile Glu Leu 50 55 60 Gln Leu Pro Glu Leu Pro Glu Leu Pro Pro His Tyr His Thr Thr Asn 65 70 75 80 Gly Leu Pro Pro His Leu Asn Pro Thr Leu His Lys Ala Leu Lys Met 85 90 95 Ser Lys Pro Asn Phe Ser Arg Ile Leu Gln Asn Leu Lys Pro Asp Leu 100 105 110 Leu Ile Tyr Asp Val Leu Gln Pro Trp Ala Glu His Val Ala Asn Glu 115 120 125 Gln Asn Ile Pro Ala Gly Lys Leu Leu Thr Ser Cys Ala Ala Val Phe 130 135 140 Ser Tyr Phe Phe Ser Phe Arg Lys Asn Pro Gly Val Glu Phe Pro Phe 145 150 155 160 Pro Ala Ile His Leu Pro Glu Val Glu Lys Val Lys Ile Arg Glu Ile 165 170 175 Leu Ala Lys Glu Pro Glu Glu Gly Gly Arg Leu Asp Glu Gly Asn Lys 180 185 190 Gln Met Met Leu Met Cys Thr Ser Arg Thr Ile Glu Ala Lys Tyr Ile 195 200 205 Asp Tyr Cys Thr Glu Leu Cys Asn Trp Lys Val Val Pro Val Gly Pro 210 215 220 Pro Phe Gln Asp Leu Ile Thr Asn Asp Ala Asp Asn Lys Glu Leu Ile 225 230 235 240 Asp Trp Leu Gly Thr Lys His Glu Asn Ser Thr Val Phe Val Ser Phe 245 250 255 Gly Ser Glu Tyr Phe Leu Ser Lys Glu Asp Met Glu Glu Val Ala Phe 260 265 270 Ala Leu Glu Leu Ser Asn Val Asn Phe Ile Trp Val Ala Arg Phe Pro 275 280 285 Lys Gly Glu Glu Arg Asn Leu Glu Asp Ala Leu Pro Lys Gly Phe Leu 290 295 300 Glu Arg Ile Gly Glu Arg Gly Arg Val Leu Asp Lys Phe Ala Pro Gln 305 310 315 320 Pro Arg Ile Leu Asn His Pro Ser Thr Gly Gly Phe Ile Ser His Cys 325 330 335 Gly Trp Asn Ser Ala Met Glu Ser Ile Asp Phe Gly Val Pro Ile Ile 340 345 350 Ala Met Pro Ile His Asn Asp Gln Pro Ile Asn Ala Lys Leu Met Val 355 360 365 Glu Leu Gly Val Ala Val Glu Ile Val Arg Asp Asp Asp Gly Lys Ile 370 375 380 His Arg Gly Glu Ile Ala Glu Thr Leu Lys Ser Val Val Thr Gly Glu 385 390 395 400 Thr Gly Glu Ile Leu Arg Ala Lys Val Arg Glu Ile Ser Lys Asn Leu 405 410 415 Lys Ser Ile Arg Asp Glu Glu Met Asp Ala Val Ala Glu Glu Leu Ile 420 425 430 Gln Leu Cys Arg Asn Ser Asn Lys Ser Lys 435 440 <210> 10 <211> 454 <212> PRT <213> Lycium barbarum <400> 10 Met Gly Thr Glu Val Thr Val His Lys Asn Thr Leu Arg Val Leu Met 1 5 10 15 Phe Pro Trp Leu Ala Tyr Gly His Ile Ser Pro Phe Leu Asn Val Ala 20 25 30 Lys Lys Leu Val Asp Arg Gly Phe Leu Ile Tyr Leu Cys Ser Thr Ala 35 40 45 Ile Asn Leu Lys Ser Thr Ile Lys Lys Ile Pro Glu Lys Tyr Ser Asp 50 55 60 Ser Ile Gln Leu Ile Glu Leu His Leu Pro Glu Leu Pro Glu Leu Pro 65 70 75 80 Pro His Tyr His Thr Thr Asn Gly Leu Pro Pro His Leu Asn His Thr 85 90 95 Leu Gln Lys Ala Leu Lys Met Ser Lys Pro Asn Phe Ser Lys Ile Leu 100 105 110 Gln Asn Leu Lys Pro Asp Leu Val Ile Tyr Asp Leu Leu Gln Gln Trp 115 120 125 Ala Glu Gly Val Ala Asn Glu Gln Asn Ile Pro Ala Val Lys Leu Leu 130 135 140 Thr Ser Gly Ala Ala Val Leu Ser Tyr Phe Phe Asn Leu Val Lys Lys 145 150 155 160 Pro Gly Val Glu Phe Pro Phe Pro Ala Ile Tyr Leu Arg Lys Asn Glu 165 170 175 Leu Glu Lys Met Ser Glu Leu Leu Ala Gln Ser Ala Lys Asp Lys Glu 180 185 190 Pro Asp Gly Val Asp Pro Phe Ala Asp Gly Asn Met Gln Val Met Leu 195 200 205 Met Ser Thr Ser Arg Ile Ile Glu Ala Lys Tyr Ile Asp Tyr Phe Ser 210 215 220 Gly Leu Ser Asn Trp Lys Val Val Pro Val Gly Pro Pro Val Gln Asp 225 230 235 240 Pro Ile Ala Asp Asp Ala Asp Glu Met Glu Leu Ile Asp Trp Leu Gly 245 250 255 Lys Lys Asp Glu Asn Ser Thr Val Phe Val Ser Phe Gly Ser Glu Tyr 260 265 270 Phe Leu Ser Lys Glu Asp Arg Glu Glu Ile Ala Phe Gly Leu Glu Leu 275 280 285 Ser Asn Val Asn Phe Ile Trp Val Ala Arg Phe Pro Lys Gly Glu Glu 290 295 300 Gln Asn Leu Glu Asp Ala Leu Pro Lys Gly Phe Leu Glu Arg Ile Gly 305 310 315 320 Asp Arg Gly Arg Val Leu Asp Lys Phe Ala Pro Gln Pro Arg Ile Leu 325 330 335 Asn His Pro Ser Thr Gly Gly Phe Ile Ser His Cys Gly Trp Asn Ser 340 345 350 Val Met Glu Ser Val Asp Phe Gly Val Pro Ile Ile Ala Met Pro Ile 355 360 365 His Leu Asp Gln Pro Met Asn Ala Arg Leu Ile Val Glu Leu Gly Val 370 375 380 Ala Val Glu Ile Val Arg Asp Asp Tyr Gly Lys Ile His Arg Glu Glu 385 390 395 400 Ile Ala Glu Ile Leu Lys Asp Val Ile Ala Gly Lys Ser Gly Glu Asn 405 410 415 Leu Lys Ala Lys Met Arg Asp Ile Ser Lys Asn Leu Lys Ser Ile Arg 420 425 430 Asp Glu Glu Met Asp Thr Ala Ala Glu Glu Leu Ile Gln Leu Cys Lys 435 440 445 Asn Ser Pro Lys Leu Lys 450 <210> 11 <211> 458 <212> PRT <213> Stevia <400> 11 Met Glu Asn Lys Thr Glu Thr Thr Val Arg Arg Arg Arg Arg Ile Ile 1 5 10 15 Leu Phe Pro Val Pro Phe Gln Gly His Ile Asn Pro Ile Leu Gln Leu 20 25 30 Ala Asn Val Leu Tyr Ser Lys Gly Phe Ser Ile Thr Ile Phe His Thr 35 40 45 Asn Phe Asn Lys Pro Lys Thr Ser Asn Tyr Pro His Phe Thr Phe Arg 50 55 60 Phe Ile Leu Asp Asn Asp Pro Gln Asp Glu Arg Ile Ser Asn Leu Pro 65 70 75 80 Thr His Gly Pro Leu Ala Gly Met Arg Ile Pro Ile Ile Asn Glu His 85 90 95 Gly Ala Asp Glu Leu Arg Arg Glu Leu Glu Leu Leu Met Leu Ala Ser 100 105 110 Glu Glu Asp Glu Glu Val Ser Cys Leu Ile Thr Asp Ala Leu Trp Tyr 115 120 125 Phe Ala Gln Ser Val Ala Asp Ser Leu Asn Leu Arg Arg Leu Val Leu 130 135 140 Met Thr Ser Ser Leu Phe Asn Phe His Ala His Val Ser Leu Pro Gln 145 150 155 160 Phe Asp Glu Leu Gly Tyr Leu Asp Pro Asp Asp Lys Thr Arg Leu Glu 165 170 175 Glu Gln Ala Ser Gly Phe Pro Met Leu Lys Val Lys Asp Ile Lys Ser 180 185 190 Ala Tyr Ser Asn Trp Gln Ile Leu Lys Glu Ile Leu Gly Lys Met Ile 195 200 205 Lys Gln Thr Lys Ala Ser Ser Gly Val Ile Trp Asn Ser Phe Lys Glu 210 215 220 Leu Glu Glu Ser Glu Leu Glu Thr Val Ile Arg Glu Ile Pro Ala Pro 225 230 235 240 Ser Phe Leu Ile Pro Leu Pro Lys His Leu Thr Ala Ser Ser Ser Ser 245 250 255 Leu Leu Asp His Asp Arg Thr Val Phe Gln Trp Leu Asp Gln Gln Pro 260 265 270 Pro Ser Ser Val Leu Tyr Val Ser Phe Gly Ser Thr Ser Glu Val Asp 275 280 285 Glu Lys Asp Phe Leu Glu Ile Ala Arg Gly Leu Val Asp Ser Lys Gln 290 295 300 Ser Phe Leu Trp Val Val Arg Pro Gly Phe Val Lys Gly Ser Thr Trp 305 310 315 320 Val Glu Pro Leu Pro Asp Gly Phe Leu Gly Glu Arg Gly Arg Ile Val 325 330 335 Lys Trp Val Pro Gln Gln Glu Val Leu Ala His Gly Ala Ile Gly Ala 340 345 350 Phe Trp Thr His Ser Gly Trp Asn Ser Thr Leu Glu Ser Val Cys Glu 355 360 365 Gly Val Pro Met Ile Phe Ser Asp Phe Gly Leu Asp Gln Pro Leu Asn 370 375 380 Ala Arg Tyr Met Ser Asp Val Leu Lys Val Gly Val Tyr Leu Glu Asn 385 390 395 400 Gly Trp Glu Arg Gly Glu Ile Ala Asn Ala Ile Arg Arg Val Met Val 405 410 415 Asp Glu Glu Gly Glu Tyr Ile Arg Gln Asn Ala Arg Val Leu Lys Gln 420 425 430 Lys Ala Asp Val Ser Leu Met Lys Gly Gly Ser Ser Tyr Glu Ser Leu 435 440 445 Glu Ser Leu Val Ser Tyr Ile Ser Ser Leu 450 455 <210> 12 <211> 470 <212> PRT <213> Tomato <400> 12 Met Ser Pro Lys Leu His Lys Glu Leu Phe Phe His Ser Leu Tyr Lys 1 5 10 15 Lys Thr Arg Ser Asn His Thr Met Ala Thr Leu Lys Val Leu Met Phe 20 25 30 Pro Phe Leu Ala Tyr Gly His Ile Ser Pro Tyr Leu Asn Val Ala Lys 35 40 45 Lys Leu Ala Asp Arg Gly Phe Leu Ile Tyr Phe Cys Ser Thr Pro Ile 50 55 60 Asn Leu Lys Ser Thr Ile Glu Lys Ile Pro Glu Lys Tyr Ala Asp Ser 65 70 75 80 Ile His Leu Ile Glu Leu His Leu Pro Glu Leu Pro Gln Leu Pro Pro 85 90 95 His Tyr His Thr Thr Asn Gly Leu Pro Pro Asn Leu Asn Gln Val Leu 100 105 110 Gln Lys Ala Leu Lys Met Ser Lys Pro Asn Phe Ser Lys Ile Leu Gln 115 120 125 Asn Leu Lys Pro Asp Leu Val Ile Tyr Asp Ile Leu Gln Arg Trp Ala 130 135 140 Lys His Val Ala Asn Glu Gln Asn Ile Pro Ala Val Lys Leu Leu Thr 145 150 155 160 Ser Gly Ala Ala Val Phe Ser Tyr Phe Phe Asn Val Leu Lys Lys Pro 165 170 175 Gly Val Glu Phe Pro Phe Pro Gly Ile Tyr Leu Arg Lys Ile Glu Gln 180 185 190 Val Arg Leu Ser Glu Met Met Ser Lys Ser Asp Lys Glu Lys Glu Leu 195 200 205 Glu Asp Asp Asp Asp Asp Asp Asp Leu Leu Val Asp Gly Asn Met Gln 210 215 220 Ile Met Leu Met Ser Thr Ser Arg Thr Ile Glu Ala Lys Tyr Ile Asp 225 230 235 240 Phe Cys Thr Ala Leu Thr Asn Trp Lys Val Val Pro Val Gly Pro Pro 245 250 255 Val Gln Asp Leu Ile Thr Asn Asp Val Asp Asp Met Glu Leu Ile Asp 260 265 270 Trp Leu Gly Thr Lys Asp Glu Asn Ser Thr Val Phe Val Ser Phe Gly 275 280 285 Ser Glu Tyr Phe Leu Ser Lys Glu Asp Met Glu Glu Val Ala Phe Ala 290 295 300 Leu Glu Leu Ser Asn Val Asn Phe Ile Trp Val Ala Arg Phe Pro Lys 305 310 315 320 Gly Glu Glu Arg Asn Leu Glu Asp Ala Leu Pro Lys Gly Phe Leu Glu 325 330 335 Arg Ile Gly Glu Arg Gly Arg Val Leu Asp Lys Phe Ala Pro Gln Pro 340 345 350 Arg Ile Leu Asn His Pro Ser Thr Gly Gly Phe Ile Ser His Cys Gly 355 360 365 Trp Asn Ser Ala Met Glu Ser Ile Asp Phe Gly Val Pro Ile Ile Ala 370 375 380 Methionine - Proline - Methionine - Histidine - Leucine - Asparagine - Glutamine - Proline - Methionine - Asparagine - Alanine - Arginine - Leucine - Isoleucine - Valine - Glutamic acid 385 390 395 400 Leucine - Glycine - Valine - Alanine - Valine - Glutamic acid - Isoleucine - Valine - Arginine - Aspartic acid - Aspartic acid - Aspartic acid - Glycine - Lysine - Isoleucine - Histidine 405 410 415 Arginine - Glycine - Glutamic acid - Isoleucine - Alanine - Glutamic acid - Threonine - Leucine - Lysine - Glycine - Valine - Isoleucine - Threonine - Glycine - Lysine - Threonine 420 425 430 Glycine - Glutamic acid - Lysine - Leucine - Arginine - Alanine - Lysine - Valine - Arginine - Aspartic acid - Isoleucine - Serine - Lysine - Asparagine - Leucine - Lysine 435 440 445 Threonine - Isoleucine - Arginine - Aspartic acid - Glutamic acid - Methionine - Aspartic acid - Alanine - Alanine - Alanine - Glutamic acid - Glutamic acid - Leucine - Isoleucine - Glutamine 450 455 460 Leucine - Cysteine - Arginine - Asparagine - Glycine - Asparagine 465 470 <210> 13 <211> 808 <212> PRT <213> Arabidopsis thaliana <400> 13 Methionine - Alanine - Asparagine - Alanine - Glutamic acid - Arginine - Methionine - Isoleucine - Threonine - Arginine - Valine - Histidine - Serine - Glutamine - Arginine - Glutamic acid 1 5 10 15 Arginine - Leucine - Asparagine - Glutamic acid - Threonine - Leucine - Valine - Serine - Glutamic acid - Arginine - Asparagine - Glutamic acid - Valine - Leucine - Alanine - Leucine 20 25 30 Leucine - Serine - Arginine - Valine - Glutamic acid - Alanine - Lysine - Glycine - Lysine - Glycine - Isoleucine - Leucine - Glutamine - Glutamine - Asparagine - Glutamine 35 40 45 Ile Ile Ala Glu Phe Glu Ala Leu Pro Glu Gln Thr Arg Lys Lys Leu 50 55 60 Glu Gly Gly Pro Phe Phe Asp Leu Leu Lys Ser Thr Gln Glu Ala Ile 65 70 75 80 Val Leu Pro Pro Trp Val Ala Leu Ala Val Arg Pro Arg Pro Gly Val 85 90 95 Trp Glu Tyr Leu Arg Val Asn Leu His Ala Leu Val Val Glu Glu Leu 100 105 110 Gln Pro Ala Glu Phe Leu His Phe Lys Glu Glu Leu Val Asp Gly Val 115 120 125 Lys Asn Gly Asn Phe Thr Leu Glu Leu Asp Phe Glu Pro Phe Asn Ala 130 135 140 Ser Ile Pro Arg Pro Thr Leu His Lys Tyr Ile Gly Asn Gly Val Asp 145 150 155 160 Phe Leu Asn Arg His Leu Ser Ala Lys Leu Phe His Asp Lys Glu Ser 165 170 175 Leu Leu Pro Leu Leu Lys Phe Leu Arg Leu His Ser His Gln Gly Lys 180 185 190 Asn Leu Met Leu Ser Glu Lys Ile Gln Asn Leu Asn Thr Leu Gln His 195 200 205 Thr Leu Arg Lys Ala Glu Glu Tyr Leu Ala Glu Leu Lys Ser Glu Thr 210 215 220 Leu Tyr Glu Glu Phe Glu Ala Lys Phe Glu Glu Ile Gly Leu Glu Arg 225 230 235 240 Gly Trp Gly Asp Asn Ala Glu Arg Val Leu Asp Met Ile Arg Leu Leu 245 250 255 Leu Asp Leu Leu Glu Ala Pro Asp Pro Cys Thr Leu Glu Thr Phe Leu 260 265 270 Gly Arg Val Pro Met Val Phe Asn Val Val Ile Leu Ser Pro His Gly 275 280 285 Tyr Phe Ala Gln Asp Asn Val Leu Gly Tyr Pro Asp Thr Gly Gly Gln 290 295 300 Val Val Tyr Ile Leu Asp Gln Val Arg Ala Leu Glu Ile Glu Met Leu 305 310 315 320 Gln Arg Ile Lys Gln Gln Gly Leu Asn Ile Lys Pro Arg Ile Leu Ile 325 330 335 Leu Thr Arg Leu Leu Pro Asp Ala Val Gly Thr Thr Cys Gly Glu Arg 340 345 350 Leu Glu Arg Val Tyr Asp Ser Glu Tyr Cys Asp Ile Leu Arg Val Pro 355 360 365 Phe Arg Thr Glu Lys Gly Ile Val Arg Lys Trp Ile Ser Arg Phe Glu 370 375 380 Val Trp Pro Tyr Leu Glu Thr Tyr Thr Glu Asp Ala Ala Val Glu Leu 385 390 395 400 Ser Lys Glu Leu Asn Gly Lys Pro Asp Leu Ile Ile Gly Asn Tyr Ser 405 410 415 Asp Gly Asn Leu Val Ala Ser Leu Leu Ala His Lys Leu Gly Val Thr 420 425 430 Gln Cys Thr Ile Ala His Ala Leu Glu Lys Thr Lys Tyr Pro Asp Ser 435 440 445 Asp Ile Tyr Trp Lys Lys Leu Asp Asp Lys Tyr His Phe Ser Cys Gln 450 455 460 Phe Thr Ala Asp Ile Phe Ala Met Asn His Thr Asp Phe Ile Ile Thr 465 470 475 480 Ser Thr Phe Gln Glu Ile Ala Gly Ser Lys Glu Thr Val Gly Gln Tyr 485 490 495 Glu Ser His Thr Ala Phe Thr Leu Pro Gly Leu Tyr Arg Val Val His 500 505 510 Gly Ile Asp Val Phe Asp Pro Lys Phe Asn Ile Val Ser Pro Gly Ala 515 520 525 Asp Met Ser Ile Tyr Phe Pro Tyr Thr Glu Glu Lys Arg Arg Leu Thr 530 535 540 Lys Phe His Ser Glu Ile Glu Glu Leu Leu Tyr Ser Asp Val Glu Asn 545 550 555 560 Lys Glu His Leu Cys Val Leu Lys Asp Lys Lys Lys Pro Ile Leu Phe 565 570 575 Thr Met Ala Arg Leu Asp Arg Val Lys Asn Leu Ser Gly Leu Val Glu 580 585 590 Trp Tyr Gly Lys Asn Thr Arg Leu Arg Glu Leu Ala Asn Leu Val Val 595 600 605 Val Gly Gly Asp Arg Arg Lys Glu Ser Lys Asp Asn Glu Glu Lys Ala 610 615 620 Glu Met Lys Lys Met Tyr Asp Leu Ile Glu Glu Tyr Lys Leu Asn Gly 625 630 635 640 Gln Phe Arg Trp Ile Ser Ser Gln Met Asp Arg Val Arg Asn Gly Glu 645 650 655 Leu Tyr Arg Tyr Ile Cys Asp Thr Lys Gly Ala Phe Val Gln Pro Ala 660 665 670 Leu Tyr Glu Ala Phe Gly Leu Thr Val Val Glu Ala Met Thr Cys Gly 675 680 685 Leu Pro Thr Phe Ala Thr Cys Lys Gly Gly Pro Ala Glu Ile Ile Val 690 695 700 His Gly Lys Ser Gly Phe His Ile Asp Pro Tyr His Gly Asp Gln Ala 705 710 715 720 Ala Asp Thr Leu Ala Asp Phe Phe Thr Lys Cys Lys Glu Asp Pro Ser 725 730 735 His Trp Asp Glu Ile Ser Lys Gly Gly Leu Gln Arg Ile Glu Glu Lys 740 745 750 Tyr Thr Trp Gln Ile Tyr Ser Gln Arg Leu Leu Thr Leu Thr Gly Val 755 760 765 Tyr Gly Phe Trp Lys His Val Ser Asn Leu Asp Arg Leu Glu Ala Arg 770 775 780 Arg Tyr Leu Glu Met Phe Tyr Ala Leu Lys Tyr Arg Pro Leu Ala Gln 785 790 795 800 Ala Val Pro Leu Ala Gln Asp Asp 805

Claims

1. An isolated and purified reb D2 having the following structure and having a reb D2 content of more than 90% by dry weight:

2. A consumable product comprising isolated and purified reb D2, wherein the purified reb D2 has a reb D2 content of more than 90% by dry weight, and wherein reb D2 has the following structure:

3. The consumable product of claim 2, wherein the consumable product is selected from the group consisting of: Beverages; reduced-calorie foods; canned products; condiments; soups; biscuits of all types; bread; chocolate; caramel; candy; jelly; pudding; fresh cream; jam; confectionery; milk powder; meat and food cooked in sweet sauces; agricultural vegetable foods; seafood; ham; sausages; frozen foods; cured meats; tobacco and medicines.

4. The consumable product of claim 2, wherein the consumable product is selected from the group consisting of: Natural juices; instant juices; ice cream and sorbet.

5. The consumable product of claim 2, wherein the consumable product is selected from the group consisting of: Refreshing drinks; Diet drinks; Yogurt drinks; powdered instant drinks; syrups; fermented soybean paste; soy sauce; vinegar; mayonnaise; ketchup; curry products; instant broth; soy sauce powder; vinegar powder; rice crackers; crackers; chewing gum; preserved fruits and pickles; marmalade; packaged vegetables and fruits in bottles; canned boiled beans; fish ham; fish sausages; fish sauce; fried fish products; dried seafood and pickled seaweed.

6. The consumable product of claim 2, wherein the consumable product is selected from the group consisting of: Carbonated soft drinks; zero-calorie beverages; Reduced-calorie drinks and instant coffee.

7. The consumable product of claim 2, further comprising at least one additive selected from the group consisting of: Carbohydrates, nucleotides, organic acids, inorganic acids, organic salts including organic acid salts and organic base salts, inorganic salts, flavoring agents and flavoring ingredients, surfactants, flavonoids, polymers and combinations thereof.

8. The consumable product of claim 2, further comprising at least one additive selected from the group consisting of: Bitter compounds, astringent compounds and emulsifiers.

9. The consumable product of claim 2, further comprising protein or protein hydrolysate.

10. The consumable product of claim 2, further comprising alcohol.

11. The consumable product of claim 2, further comprising at least one additive selected from the group consisting of: Polyols, amino acids and their corresponding salts, polyamino acids and their corresponding salts, sugar acids and their corresponding salts, and combinations thereof.

12. The consumable product of claim 2, further comprising at least one functional ingredient selected from the group consisting of: Saponins, antioxidants, fatty acids, vitamins, glucosamine, minerals, preservatives, hydration agents, probiotics, prebiotics, weight management agents, osteoporosis management agents, phytoestrogens, long chain saturated fatty alcohols, plant sterols, and combinations thereof.

13. The consumable product of claim 2, further comprising a compound selected from the group consisting of: Stevia, stevia extract, monk fruit sweetener, and combinations thereof.

14. The consumable product of claim 2, further comprising a compound selected from the group consisting of: Stevioside, stevioside, mogroside and their combination.

15. The consumable product of claim 2, further comprising a compound selected from the group consisting of: Rubus strychnifolius glycosides, dulcoside A, dulcoside B, rebaudioside B, rebaudioside G, stevioside, rebaudioside C, rebaudioside F, rebaudioside A, rebaudioside I, rebaudioside E, rebaudioside H, rebaudioside L, rebaudioside K, rebaudioside J, rebaudioside M, rebaudioside M2, rebaudioside D, rebaudioside D2, rebaudioside N, rebaudioside O, brazil, neohesperidin dihydrochalcone, glycyrrhizic acid and its salts, thaumatin, perilla frutescens, soapberry sesquiterpene glycosides, baiyun ginsenoside, scutellaria glycoside-I, dimethyl-hexahydrofluorene-dicarboxylic acid, abrin, Brazilian glycyrrhizin, meat flower Snow bile glycoside, cyclocarya paliurus glycoside, butterfly card glycoside, polyboroside A, brazilin, helan sweet essence, phyllotoxin, sarsaparilla glycoside, phloridzin, trilobatin, dihydroflavonol, dihydroquercetin-3-acetate, neoastilbin, trans-cinnamaldehyde, monatin and its salts, pteridin A, hematoxylin, oleoside, polypodium stevioside, butterfly card glycoside A, butterfly card glycoside B, mabinan sweet protein, pantatin, miracle fruit protein, curculigo sweet protein, broad-leaved curculigo sweet protein, chlorogenic acid, cynarin, mogroside V, mogroside VI, light fruit wood turtle saponin, simonoside, psicose, allose, D-tagatose, erythritol and combinations thereof.

16. A beverage comprising isolated and purified reb D2, wherein the purified reb D2 has a reb D2 content of more than 90% by dry weight, and wherein reb D2 has the following structure:

17. A method for enhancing the sweetness of a beverage comprising a sweetener, the method comprising: a.) providing a beverage comprising a sweetener; and b.) adding a sweetness enhancer including reb D2, wherein reb D2 is present at a concentration at or below the sweet taste recognition threshold, and wherein reb D2 has the following structure:

Images