High-purity steviol glycosides

Abstract

This application describes a method for preparing highly purified steviol glycosides, particularly rebaudine glycosides A, D, and M. The method involves converting various starting compositions into target steviol glycosides using recombinant microorganisms. Furthermore, novel steviol glycosides reb D2, reb M2, and reb I, and methods for their preparation are disclosed. The highly purified rebaudine glycosides can be used as calorie-free sweeteners in edible and chewable compositions such as any beverage, confectionery, baked goods, biscuits, and chewing gum.

Description

[0001] This application is a divisional application of Chinese patent application No. 201580061214.2 (PCT / US2015 / 046354), filed on August 21, 2015, entitled "High Purity Steviosides". Technical Field

[0002] This invention relates to a biocatalytic method for preparing compositions comprising steviol glycosides, said compositions comprising highly purified steviol glycoside compositions. The invention also relates to novel steviol glycosides, methods for their isolation, and uses of these novel steviol glycosides. Background Technology

[0003] High-intensity sweeteners have a sweetness level many times higher than that of sucrose. They are essentially calorie-free and are commonly used in low-calorie and reduced-calorie products, including foods and beverages. High-intensity sweeteners do not cause a glycemic response, making them suitable for products targeting diabetics and others interested in controlling their carbohydrate intake.

[0004] Steviosides are a class of compounds found in the leaves of stevia (Stevia rebaudiana Bertoni), a perennial shrub of the Asteraceae family (Compositae) native to certain regions of South America. They are structurally characterized by the monobasic steviol, varying depending on the presence of carbohydrate residues at positions C13 and C19. They accumulate in stevia leaves, constituting approximately 10%–20% of the total dry weight. Based on dry weight, the four main glycosides typically found in stevia leaves include steviol glycoside (9.1%), rebaudioside A (3.8%), rebaudioside C (0.6–1.0%), and durqueside A (0.3%). Other known steviolsides include rebaudiosides B, C, D, E, F, and M, steviolbioside, and rubusoside.

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

[0006] Therefore, there remains a need for simple, effective and economical methods for preparing compositions containing steviol glycosides, which include highly purified steviol glycoside compositions.

[0007] In addition, there remains a need for new steviol glycosides and their preparation and separation methods. Summary of the Invention

[0008] The present invention provides a biocatalytic method for preparing compositions containing steviol glycosides by contacting a starting composition containing an organic substrate with microorganisms and / or a biocatalyst, thereby producing a composition containing steviol glycosides.

[0009] The starting composition comprises an organic compound. In one embodiment, the starting composition is selected from polyols and various carbohydrates.

[0010] The target steviol glycoside can be any steviol glycoside. In one embodiment, the target steviol glycoside is a steviol monoglucoside, a steviol diglucoside, raspberry glycoside, durqueside B, durqueside A, rebaudiside B, rebaudiside G, styracifolioside, rebaudiside C, rebaudiside F, rebaudiside A, rebaudiside I, rebaudiside E, rebaudiside H, rebaudiside L, rebaudiside K, rebaudiside J, rebaudiside M, rebaudiside M2, rebaudiside D, rebaudiside D2, rebaudiside N, rebaudiside O, or a synthetic steviol glycoside.

[0011] In one implementation, the target steviol glycoside is a chamomile glycoside.

[0012] In another embodiment, the target steviol glycoside is rebaudioside A.

[0013] In yet another embodiment, the target steviol glycoside is rebaudioside D.

[0014] In yet another embodiment, the target steviol glycoside is rebaudioside M.

[0015] The microorganism can be any microorganism containing at least one biocatalyst suitable for converting the starting composition into target steviol glycosides.

[0016] Biocatalysts can be located on the surface of microorganisms and / or inside microorganisms.

[0017] Biocatalysts include steviol biosynthetic enzymes and UDP-glucosyltransferases (UGT), or variants thereof with more than 75% amino acid sequence identity.

[0018] In one embodiment, the steviol biosynthetic enzyme includes a mevalonate (MVA) pathway enzyme.

[0019] In another embodiment, the stevia biosynthetic enzyme includes a non-methoxyvalerate-2-C-methyl-D-erythritol-4-phosphate pathway (MEP / DOXP) enzyme.

[0020] In one embodiment, the stevia biosynthetic enzyme is selected from geranyl geranyl diphosphate synthase, copalyl diphosphate synthase, kauriene synthase, kauriene oxidase, isokaurienoic acid 13-hydroxylase (KAH), stevia synthase, deoxyxylulose 5-phosphate synthase (DXS), D-1-deoxyxylulose 5-phosphate reductase (DXR), cytidyl-2-C-methyl-D-erythritol synthase (CMS), and cytidyl-2-C-methyl-D-erythritol synthase. Methyl-D-erythritol kinase (CMK), cytidine-2-C-methyl-D-erythritol 2,4-cyclic diphosphate synthase (MCS), 1-hydroxy-2-methyl-2(E)-butenyl-4-bisphosphate synthase (HDS), 1-hydroxy-2-methyl-2(E)-butenyl-4-bisphosphate reductase (HDR), acetyl-CoA thiolysis enzyme, truncated HMG-CoA reductase, mevalonate kinase, phosphate mevalonate kinase, mevalonate pyrophosphate decarboxylase, cytochrome P450 reductase, etc.

[0021] UDP-glucosyltransferase can be any UDP-glucosyltransferase capable of adding at least one glucose monomer to steviol and / or steviol glycoside substrates to provide target steviol glycosides.

[0022] The microorganism can be any suitable microorganism. In one embodiment, the microorganism can be, for example, *Escherichia coli*, *Saccharomyces sp.*, *Aspergillus sp.*, *Pichia sp.*, *Bacillus sp.*, or *Yarrowia sp.*. In another embodiment, the UDP-glucosyltransferase is synthetic.

[0023] In one embodiment, the UDP-glucosyltransferase is selected from UGT74G1, UGT85C2, UGT76G1, UGT91D2, or a variant thereof having more than 75% amino acid sequence identity.

[0024] In one embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose monomer to raspberry glycoside to form chamomile glycoside. In a particular embodiment, the UDP-glucosyltransferase is UGT91D2 or a variant of UGT91D2 having more than 75% amino acid sequence identity with UGT91D2.

[0025] In one embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose monomer to hedyotis diffusa to form rebaudioside A. In a particular embodiment, the UDP-glucosyltransferase is UGT76G1 or a UGT76G1 variant having more than 75% amino acid sequence identity with UGT76G1.

[0026] In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose monomer to rebaudioside A to form rebaudioside D. In a particular embodiment, the UDP-glucosyltransferase is UGT91D2 or a UGT91D2 variant having more than 75% amino acid sequence identity with UGT91D2.

[0027] In yet another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose monomer to rebaudioside D to form rebaudioside M. In a particular embodiment, the UDP-glucosyltransferase is UGT76G1 or a UGT76G1 variant having more than 75% amino acid sequence identity with UGT76G1.

[0028] In yet another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose monomer to rebaudioside I to form rebaudioside M. In a particular embodiment, the UDP-glucosyltransferase is UGTSL or a UGTSL variant having more than 75% amino acid sequence identity with UGTSL.

[0029] In yet another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose monomer to rebaudioside E to form rebaudioside M. In a particular embodiment, the UDP-glucosyltransferase is UGT76G1 or a UGT76G1 variant having more than 75% amino acid sequence identity with UGT76G1.

[0030] Optionally, the method of the present invention further includes recycling UDP to provide UDP-glucose. In one embodiment, the method includes recycling UDP by providing a recycling catalyst and a recycling substrate, such that the biotransformation of the steviol glycoside substrate into the target steviol glycoside is carried out using catalytic amounts of UDP-glucosyltransferase and UDP-glucose. Figure 3 ).

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

[0032] In one implementation, the recycling substrate is sucrose.

[0033] Optionally, the method of the present invention further includes purifying the composition containing steviol glycosides. The composition containing steviol glycosides can be purified by any suitable method, such as, for example, crystallization, membrane separation, centrifugation, extraction, chromatographic separation, or a combination of these methods.

[0034] In one embodiment, purification yields a composition comprising more than about 80% by weight of steviol glycosides on an anhydrous basis. In another embodiment, purification yields a composition comprising more than about 90% by weight of steviol glycosides. In a particular embodiment, the composition comprises more than about 95% by weight of steviol glycosides.

[0035] Steviosides can be in any polycrystalline or amorphous form, including hydrolysates, solvates, anhydrous forms, or combinations thereof.

[0036] The present invention also provides consumer products comprising compositions prepared by the disclosed methods. Suitable consumer products include, but are not limited to, food, beverage, pharmaceutical compositions, tobacco products, nutritional supplement compositions, oral hygiene compositions, and cosmetic compositions.

[0037] This invention also provides novel steviol glycosides reb D2 and reb M2, which are isomers of reb D and reb M, respectively. 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 are also present in any of the consumer products disclosed herein. In a particular embodiment, a beverage comprising reb D2 and / or reb M2 is provided.

[0038] This article also provides methods for preparing reb D2 and reb M2. Both are formed during the biotransformation of reb A to reb D. It is believed that reb M2 is formed from the in situ biotransformation of reb D2.

[0039] This article also provides a method for selectively hydrolyzing the 1,6-β-glycosidic bonds in reb D2 and / or reb M2 using enzymes with β-1,6-glucosidase activity.

[0040] In one embodiment, for selectively hydrolyzing the 1,6-β-glycosidic bonds in reb D2 and / or reb M2, at least one enzyme is selected from glycosidases (NC-IUBMB EC 3.2.1.), glucosidases, dextranases, Isolase (011410; National Enzyme Company, USA), Aromase (GLY0151441; Amano Enzyme, Japan), naringinase (NAH0550102; Amano Enzyme, Japan), cellulases (e.g., cellulases from Trichoderma reesei ATCC 26921; Sigma C2730), cellobiases (e.g., cellobiases from Aspergillus niger; Sigma C6105), Viscozyme L (Sigma V2010), etc.

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

[0042] In another embodiment, the present invention is a method for preparing a composition containing reb M, comprising: (a) contacting a starting composition containing reb D with an enzyme capable of converting reb D to reb M, UDP-glucose, and optionally a UDP-glucose recycling enzyme to produce a composition containing reb M, and (b) isolating the composition containing reb M.

[0043] In another embodiment, the present invention is a method for preparing a composition comprising reb M, comprising: (a) contacting a starting composition comprising reb A with an enzyme capable of converting reb A to reb D, UDP-glucose, and optionally a UDP-glucose recycling enzyme to produce a composition comprising reb D; and (b) optionally, isolating the composition comprising reb D; (c) contacting the composition comprising reb D with an enzyme capable of converting reb D to reb M, UDP-glucose, and optionally a UDP-glucose recycling enzyme to produce a composition comprising reb M; and (d) isolating the composition comprising reb M.

[0044] The composition can be further purified to provide reb D or reb M with a purity of more than about 95% of the total amount on a dry basis. Attached Figure Description

[0045] The accompanying drawings are included to provide a further understanding of the invention. The drawings illustrate embodiments of the invention and, together with the description, serve to explain the principles of those embodiments.

[0046] Figure 1 The structure of reb M is shown.

[0047] Figure 2 The biocatalytic production of reb M from hedyotis diffusa glycosides was demonstrated.

[0048] Figure 3 The biocatalytic production of reb A from chamomile glycosides was demonstrated using the enzyme UGT76G1 and the accompanying recycling of UDP to UDP glucose via sucrose synthase.

[0049] Figure 4 The IR spectrum of reb M is shown.

[0050] Figure 5 The HPLC chromatogram of the product from reb D to reb M via biocatalysis, as detailed in Example 14, is shown. The peak with a residence time of 24.165 min corresponds to untreated reb D. The peak with a residence time of 31.325 min corresponds to reb M.

[0051] Figure 6 The HPLC chromatogram of purified reb M generated from reb D via biocatalysis is shown.

[0052] Figure 7 The HPLC chromatogram of the reb M standard is shown.

[0053] Figure 8 The HPLC chromatograms of co-injection of reb M standard and reb M purified from the biotransformation of reb D are shown.

[0054] Figure 9 The images show reb M standards and reb M purified from reb D biosynthesis. 1 Overlap of H NMR spectra.

[0055] Figure 10 The HRMS spectrum of reb M purified after biocatalytic generation from reb D is shown.

[0056] Figure 11The LC-MS analysis of the semi-synthetic steviol glycoside mixture is shown, lot number CB-2977-106, showing the MS values ​​of TIC (A), the peak at 1.8 min (B), the reb M2 peak at 4.1 min (C), the reb D peak at 6.0 min (D), the reb D2 peak at 7.7 min (E), the peak at 9.4 min (F), the rebaudioside A peak at 15.2 min (G), the peak at 16.5 min (H), and the peak at 18.3 min (I).

[0057] Figure 12 Traces of a semi-synthetic steviol glycoside mixture were shown, batch number CB-2977-106. The chromatographic grid lines were uneven because the detector was recalibrated 14 minutes after injection.

[0058] Figure 13 The HPLC analysis of the semi-synthetic steviol glycoside mixture is shown, lot number CB-2977-106(A), isolated reb M2(B), isolated reb D(C) and isolated reb D2(D).

[0059] Figure 14 The reb D2 was displayed 1 1H NMR spectrum (500MHz, pyridine-d5).

[0060] Figure 15 The reb D2 was displayed 13 C10 NMR spectrum (125 MHz, pyridine-d5).

[0061] Figure 16 The reb D2 was displayed 13 Development of C10 NMR spectrum (125 MHz, pyridine-d5).

[0062] Figure 17 The reb D2 was displayed 1 H- 1 H COSY spectrum (500MHz, pyridine-d5).

[0063] Figure 18 The HSQC-DEPT spectrum of reb D2 (500 MHz, pyridine-d5) is shown.

[0064] Figure 19 The HMBC spectrum of reb D2 is shown.

[0065] Figure 20 The HMBC spectrum (500 MHz, pyridine-d5) of reb D2 is shown in the expanded image.

[0066] Figure 21Showing reb M2 1 1H NMR spectrum (500MHz, D2O).

[0067] Figure 22 Showing reb M2 13 C NMR spectrum (500MHz, D2O / TSP).

[0068] Figure 23 Showing reb M2 13 Development of C NMR spectrum (125MHz, D2O / TSP).

[0069] Figure 24 Showing reb M2 1 H- 1 H COSY spectrum (500MHz, D2O).

[0070] Figure 25 The HSQC-DEPT spectrum of reb M2 is shown (500 MHz, D2O).

[0071] Figure 26 The HMBC spectrum of reb M2 (500 MHz, D2O) is shown.

[0072] Figure 27 The expanded HMBC spectrum (500 MHz, D2O) of reb M2 is shown.

[0073] Figure 28 The HPLC chromatograms for the analysis performed in Example 47 are shown.

[0074] Figure 29 The HPLC chromatograms for the analysis performed in Example 47 are shown.

[0075] Figure 30 The LC-CAD analysis performed in Example 47 is shown.

[0076] Figure 31 The ESI-TOF mass spectrometry as described in Example 47 is displayed.

[0077] Figure 32 The mass spectrometry was displayed as described in Example 47.

[0078] Figure 33 The MS / MS spectrum as described in Example 47 is displayed.

[0079] Figure 34 The MS / MS spectrum as described in Example 47 is displayed.

[0080] Figure 35 The following is shown as described in Example 471 Results of H NMR.

[0081] Figure 36 The following is shown as described in Example 47 1 Results of H NMR.

[0082] Figure 37 The following is shown as described in Example 47 1 Results of H NMR.

[0083] Figure 38 The following is shown as described in Example 47 13 Results of C NMR.

[0084] Figure 39 The following is shown as described in Example 47 13 Results of C NMR.

[0085] Figure 40 The following is shown as described in Example 47 1 H- 1 Results of H COSY.

[0086] Figure 41 The results of HSQC-DEPT as described in Example 47 are shown.

[0087] Figure 42 The results of HMBC as described in Example 47 are shown.

[0088] Figure 43 The results of HMBC as described in Example 47 are shown.

[0089] Figure 44 The results of NOESY as described in Example 47 are shown.

[0090] Figure 45 The results of NOESY as described in Example 47 are shown.

[0091] Figure 46 The results of 1D TOCSY as described in Example 47 are shown.

[0092] Figure 47 The results of 1D TOCSY as described in Example 47 are shown.

[0093] Figure 48 The results of 1D TOCSY as described in Example 47 are shown.

[0094] Figure 49 The results of 1D TOCSY as described in Example 47 are shown.

[0095] Figure 50 The results of 1D TOCSY as described in Example 47 are shown.

[0096] Figure 51 The HPLC (CAD) chromatogram shows the conversion of chamomile glycoside to rebaudioside A.

[0097] Figure 52 The HPLC (CAD) chromatogram shows the conversion of rebaudioside D to rebaudioside M.

[0098] Figure 53a -e shows the HPLC chromatogram, which displays the HPLC test results of Example 20.

[0099] Figure 54 The HPLC chromatogram is shown, which displays the HPLC test results of Example 21.

[0100] Figure 55a -e shows the HPLC chromatogram, which displays the HPLC test results of Example 22.

[0101] Figure 56a -b shows the HPLC chromatogram, which displays the HPLC test results of Example 23.

[0102] Figure 57a -b shows the LC-MS chromatogram, which displays the LC-MS test results of Example 24.

[0103] Figure 58 A figure is shown, illustrating the reaction characteristics of Example 25.

[0104] Figure 59a -b shows the HPLC chromatogram, which displays the HPLC test results of Example 28.

[0105] Figure 60a -b shows the HPLC chromatogram, which displays the HPLC test results of Example 29.

[0106] Figure 61 The HPLC chromatogram is shown, which displays the HPLC test results of Example 30.

[0107] Figure 62 The LS-MS chromatogram is shown, which displays the LS-MS test results of Example 31.

[0108] Figure 63a -c shows the HPLC chromatogram, which displays the HPLC test results of Example 32.

[0109] Figure 64The HPLC chromatogram is shown, which displays the HPLC test results of Example 35.

[0110] Figure 65 The HPLC chromatogram is shown, which displays the HPLC test results of Example 37.

[0111] Figure 66 The figure shown illustrates the HPLC results of Example 43.

[0112] Figure 67 shows a graph illustrating the reaction characteristics of Example 46.

[0113] Figure 68a -f indicates the reaction characteristics of Example 49.

[0114] Figure 69a -c shows the graph, which displays the HPLC results of Example 50.

[0115] Figure 70a -d shows the reaction characteristic diagram of Example 51.

[0116] Figure 71 A reaction characteristic diagram of Example 52 is shown.

[0117] Figure 72a A reaction characteristic diagram of Example 54 is shown.

[0118] Figure 72b The HPLC chromatogram is shown, which illustrates the HPLC analysis of Example 54.

[0119] Figure 73a A reaction characteristic diagram of Example 55 is shown.

[0120] Figure 73b The HPLC chromatogram is shown, which illustrates the HPLC analysis of Example 55.

[0121] Figure 74a A reaction characteristic diagram of Example 56 is shown.

[0122] Figure 74b The HPLC chromatogram is shown, which illustrates the HPLC analysis of Example 56.

[0123] Figure 75a A reaction characteristic diagram of Example 57 is shown.

[0124] Figure 75b The HPLC chromatogram is shown, which illustrates the HPLC analysis of Example 57.

[0125] Figure 76a A reaction characteristic diagram of Example 58 is shown.

[0126] Figure 76b The HPLC chromatogram is shown, which illustrates the HPLC analysis of Example 58. Invention Details

[0127] The present invention provides a biocatalytic method for preparing compositions containing steviol glycosides by contacting a starting composition containing an organic substrate with microorganisms, thereby producing a composition containing steviol glycosides.

[0128] One object of the present invention is to provide an efficient biocatalytic method for preparing steviol glycosides from starting compositions, particularly reb E, reb A, reb D, reb D2, reb M and reb M2.

[0129] As used herein, "biocatalysis" or "biocatalytic" refers to the chemical transformation of organic compounds using natural or genetically engineered biocatalysts, such as cells or proteases, in one or more steps. Biocatalysis includes fermentation, biosynthesis, and biotransformation methods. Isolated enzymes and whole-cell biocatalysis methods are known in the art. Biocatalyst proteases can be naturally occurring proteins or recombinant proteins.

[0130] All sequences listed herein, including any nucleic acid or amino acid sequence, including variants having >75%, >80%, >90%, >95%, >96%, >97%, >98%, or >99% sequence identity with the nucleic acid or amino acid sequence described herein.

[0131] As used herein, the term "stevioside" refers to glycosides of steviol, including but not limited to naturally occurring steviol glycosides such as steviol monoglycosides, steviol diglycosides, raspberry glycosides, durqueside B, durqueside A, rebaudioside B, rebaudioside G, styraxoside, rebaudioside C, rebaudioside F, rebaudioside A, rebaudioside I, rebaudioside E, rebaudioside H, rebaudioside L, rebaudioside K, rebaudioside J, rebaudioside M, rebaudioside D, rebaudioside M2, rebaudioside D2, rebaudioside N, and rebaudioside O; synthetic steviol glycosides such as enzymatically glycosylated steviol glycosides; and combinations thereof.

[0132] Chemical structure of steviol and its glycosides

[0133]

[0134]

[0135] (Glc = glucose)

[0136] Starting Composition

[0137] As used herein, “starting composition” means any composition (typically an aqueous solution) containing one or more organic compounds comprising at least one carbon atom.

[0138] In one embodiment, the starting composition is selected from polyols and various carbohydrates.

[0139] 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 hydroxyl groups, respectively. Polyols can also contain more than four hydroxyl groups, such as pentanol, hexanetanol, and heptanol, which contain 5, 6, or 7 hydroxyl groups, respectively. Additionally, polyols can also be sugar alcohols, polyhydric alcohols, or polyalcohols, which are reduced forms of carbohydrates in which the carbonyl group (aldehyde or ketone, reducing sugar) has been reduced to a primary or tertiary hydroxyl group. Examples of polyols include, but are not limited to, erythritol, maltitol, mannitol, sorbitol, lactitol, xylitol, xylitol, isomaltulitol, propylene glycol, glycerol, threitol, galactitol, hydrogenated isomaltulose, reduced isomalt oligosaccharides, reduced xylose oligosaccharides, reduced gentian oligosaccharides, reduced maltose syrup, reduced glucose syrup, hydrogenated starch hydrolysate, polyglycitol and sugar alcohols or any other carbohydrate that can be reduced.

[0140] The term "carbohydrate" refers to the general formula (CH2O). nAldehydes or ketones substituted with multiple hydroxyl groups (where n is 3-30), and their oligomers and polymers. The carbohydrates of the present invention may further be substituted or deoxygenated at one or more sites. As used herein, carbohydrates include unmodified carbohydrates, carbohydrate derivatives, substituted carbohydrates, and modified carbohydrates. As used herein, the phrases “carbohydrate derivative,” “substituted carbohydrate,” and “modified carbohydrate” are synonymous. A modified carbohydrate refers to any carbohydrate in which at least one atom or a combination thereof has been added, removed, or substituted. Thus, carbohydrate derivatives or substituted carbohydrates include substituted and unsubstituted monosaccharides, disaccharides, oligosaccharides, and polysaccharides. The carbohydrate derivative or substituted carbohydrate may optionally be deoxygenated at any corresponding C-position and / or substituted with one or more moieties such as hydrogen, halogen, haloalkyl, carboxyl, acyl, acyloxy, amino, amide, carboxyl derivative, alkylamino, dialkylamino, arylamino, alkoxy, aryloxy, nitro, cyano, sulfonyl, mercapto, imino, sulfonyl, thio, sulfinyl, aminosulfonyl, alkoxycarbonyl, formamide, phosphono, phosphinic, phosphoryl, phosphono, thioester, thioether, oxime, hydrazine, carbamoyl, phosphoric acid, phosphonato, or any other feasible functional group, provided that the carbohydrate derivative or substituted carbohydrate contributes to improving the sweetness of the sweetener composition.

[0141] 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, lysolose, allose, azulose, mannose, idulose, lactose, maltose, invert sugar, isotrehalose, neotrehalose, isomaltulose, erythrose, deoxyribose, gulose, idulose, tarose, erythritolose, xylulose, allulose, mesobiose, cellobiose, amylopectin, glucosamine, mannosamine, fucose, glucuronic acid, gluconic acid, gluconic acid-lactone, and abirate. Cosmos, galactosamine, beet oligosaccharides, isomaltose oligosaccharides (isomaltose, isomalttriose, panose, etc.), xylooligosaccharides (xylotriose, xylobiose, etc.), xylose-terminated oligosaccharides, gentian oligosaccharides (gentiobiose, gentiotriose, gentiotetraose, etc.), sorbitol, Aspergillus niger oligosaccharides, paraginose oligosaccharides, fructose oligosaccharides (sucrose triose, fungal tetraose, etc.), maltitol, maltitol, maltose oligosaccharides (malttriose, maltite, maltpentose, maltohexose, maltoheptose, etc.), starch, inulin, inulin-oligosaccharides, lactulose, melibiose, melibiose, ribose, isomerized liquid sugars (such as high-fructose corn syrup), conjugated sugars, and soybean oligosaccharides. Additionally, the carbohydrates used in this article can be D- or L-configured.

[0142] The starting composition may be synthetic or purified (partial or complete), commercially available or prepared.

[0143] In one embodiment, the starting composition is glycerol.

[0144] In another embodiment, the starting composition is glucose.

[0145] In yet another embodiment, the starting composition is sucrose.

[0146] In yet another embodiment, the starting composition is starch.

[0147] In another embodiment, the starting composition is maltodextrin.

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

[0149] As described herein, the organic compounds in the starting composition are used as substrates for the production of target steviol glycosides.

[0150] Stevioside

[0151] The target steviol glycoside of the present invention can be any steviol glycoside prepared by the methods disclosed herein. In one embodiment, the target steviol glycoside is selected from steviol monoglycoside, steviol diglycoside, raspberry glycoside, durqueside B, durqueside A, rebaudioside B, rebaudioside G, styraxoside, 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 other steviol glycosides.

[0152] In one embodiment, the target steviol glycoside is reb A. In 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 a further embodiment, the target steviol glycoside is reb M. In yet another embodiment, the target steviol glycoside is reb M2.

[0153] Steviosides can be in any polymeric or amorphous form, including hydrolysates, solvates, anhydrous forms, or combinations thereof.

[0154] In one embodiment, the present invention is a biocatalytic method for producing reb D.

[0155] In yet another embodiment, the present invention is a biocatalytic method for producing reb D2.

[0156] In yet another embodiment, the present invention is a biocatalytic method for producing reb M.

[0157] In further embodiments, the present invention is a biocatalytic method for producing reb M2.

[0158] In one embodiment, the present invention is a biocatalytic method for producing reb I.

[0159] In yet another embodiment, the present invention is a biocatalytic method for producing reb E.

[0160] Optionally, the method of the present invention further includes the separation of steviol glycosides from the starting composition. Steviol glycosides can be separated by any suitable method, such as, for example, crystallization, membrane separation, centrifugation, extraction, chromatographic separation, or a combination of these methods.

[0161] In certain embodiments, the methods described herein yield highly purified steviol glycoside compositions. As used herein, the term "highly purified" refers to a composition having more than about 80% by weight of steviol glycosides on an anhydrous basis. In one embodiment, the highly purified steviol glycoside composition contains more than about 90% by weight of steviol glycosides on an anhydrous basis, 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% steviol glycoside content on a dry basis.

[0162] In one embodiment, when the target steviol glycoside is reb M, the method described herein provides a composition having a reb M content of more than about 90% by weight on a dry basis. In another specific embodiment, when the target steviol glycoside is reb M, the method described herein provides a composition containing a reb M content of more than about 95% by weight on a dry basis.

[0163] In another embodiment, when the target steviol glycoside is reb M2, the method described herein provides a composition having a reb M2 content of more than about 90% by weight on a dry basis. In another specific embodiment, when the target steviol glycoside is reb M2, the method described herein provides a composition containing a reb M2 content of more than about 95% by weight on a dry basis.

[0164] In yet another embodiment, when the target steviol glycoside is reb D, the methods described herein provide a composition with a reb D content of more than about 90% by weight on a dry basis. In yet another specific embodiment, when the target steviol glycoside is reb D, the methods described herein provide a composition containing more than about 95% by weight on a dry basis.

[0165] In yet another embodiment, when the target steviol glycoside is reb D2, the methods described herein provide a composition with a reb D2 content of more than about 90% by weight on a dry basis. In yet another specific embodiment, when the target steviol glycoside is reb D2, the methods described herein provide a composition containing more than about 95% by weight on a dry basis.

[0166] In a further embodiment, when the target steviol glycoside is reb A, the methods described herein provide a composition with a reb A content of more than about 90% by weight on a dry basis. In another specific embodiment, when the target steviol glycoside is reb A, the methods described herein provide a composition containing more than about 95% by weight on a dry basis.

[0167] In a further embodiment, when the target steviol glycoside is reb E, the methods described herein provide a composition with a reb E content of more than about 90% by weight on a dry basis. In another specific embodiment, when the target steviol glycoside is reb E, the methods described herein provide a composition containing a reb E content of more than about 95% by weight on a dry basis.

[0168] In one embodiment, when the target steviol glycoside is reb I, the method described herein provides a composition with a reb I content of more than about 90% by weight on a dry basis. In another specific embodiment, when the target steviol glycoside is reb I, the method described herein provides a composition containing a reb I content of more than about 95% by weight on a dry basis.

[0169] In a further embodiment, when the target steviol glycoside is hedyotis diffusa glycoside, the method described herein provides a composition having a hedyotis diffusa glycoside content of more than about 90% by weight on a dry basis. In another specific embodiment, when the target steviol glycoside is hedyotis diffusa glycoside, the method described herein provides a composition having a hedyotis diffusa glycoside content of more than about 95% by weight on a dry basis.

[0170] microorganism

[0171] In one embodiment of the invention, microorganisms are contacted with a starting composition to produce a composition comprising target steviol glycosides. The microorganisms can be any microorganism having a biocatalyst suitable for converting the starting composition into target steviol glycosides. These biocatalysts are encoded within the genome of the microorganism.

[0172] In one implementation, the microorganism may be, for example, Escherichia coli, Saccharomyces sp., Aspergillus sp., Pichia sp., Bacillus sp., Yarrowia sp., etc.

[0173] Biocatalysts can be located on the surface of microbial cells and / or inside microbial cells.

[0174] A biocatalyst can be isolated from microorganisms and used to convert the starting composition into target steviol glycosides. This separation can be achieved by any means known in the art, including but not limited to lysis of microbial cells, centrifugation, and filtration.

[0175] Biocatalysts (extracellular enzymes) can be extracted from microorganisms and used to convert the starting composition into target steviol glycosides.

[0176] In one embodiment, the biocatalyst is steviol biosynthetic enzyme and UDP-glycosyltransferase (UGT), or a variant thereof having more than 75% amino acid sequence identity.

[0177] Stevia biosynthesis can be any steviol biosynthetic enzyme, or a variant thereof with more than 75% amino acid sequence identity.

[0178] In one embodiment, the steviol biosynthetic enzyme includes a mevalonate (MVA) pathway enzyme, or a variant thereof having more than 75% amino acid sequence identity.

[0179] In another embodiment, the stevia biosynthetic enzyme includes a non-methoxyvalerate 2-C-methyl-D-erythritol-4-phosphate pathway (MEP / DOXP) enzyme, or a variant thereof having more than 75% amino acid sequence identity.

[0180] In one embodiment, the stevioside biosynthetic enzyme is selected from geraniol geraniol diphosphate synthase, cobazin diphosphate synthase, kauriene synthase, kauriene oxidase, isokaurienoic acid 13-hydroxylase (KAH), stevioside synthase, deoxyxylulose 5-phosphate synthase (DXS), D-1-deoxyxylulose 5-phosphate reductase (DXR), cytidine-2-C-methyl-D-erythritol 4-bisphosphate synthase (CMS), cytidine-2-C-methyl-D-erythritol kinase (CMK), and 4-diphosphate kinase. Cytidine-2-C-methyl-D-erythritol 2,4-cyclic diphosphate synthase (MCS), 1-hydroxy-2-methyl-2(E)-butenyl-4-bisphosphate synthase (HDS), 1-hydroxy-2-methyl-2(E)-butenyl-4-bisphosphate reductase (HDR), acetyl-CoA thiolysis enzyme, truncated HMG-CoA reductase, mevalonate kinase, phosphate mevalonate kinase, mevalonate pyrophosphate decarboxylase, cytochrome P450 reductase, etc., or variants thereof with more than 75% amino acid sequence identity.

[0181] UDP-glucosyltransferase can be any UDP-glucosyltransferase capable of adding at least one glucose monomer to a steviol and / or steviol glycoside substrate to provide a target steviol glycoside.

[0182] In one embodiment, the microorganisms are free. In another embodiment, the microorganisms are immobilized. For example, the microorganisms may be immobilized on a solid support made from inorganic or organic materials. Non-limiting examples of solid supports suitable for immobilizing microorganisms include derived cellulose or glass, ceramics, metal oxides, or membranes. For example, microorganisms may be immobilized on a solid support by covalent bonding, adsorption, cross-linking, embedding, or encapsulation.

[0183] In one embodiment, the microorganisms are in an aqueous medium comprising water and various components selected from carbon sources, energy sources, nitrogen sources, trace elements, vitamins, nucleosides, bisphosphates, triphosphates, organic and inorganic salts, organic and mineral acids, bases, etc. Carbon sources include glycerol, glucose, carbon dioxide, carbonates, and bicarbonates. Nitrogen sources may include nitrates, nitrites, amino acids, peptides, peptones, or proteins.

[0184] In a particular embodiment, the medium comprises a buffer. Suitable buffers include, but are not limited to, PIPES buffer, acetate buffer, and phosphate buffer. In a particular embodiment, the medium comprises a phosphate buffer.

[0185] In one embodiment, the medium may further include an organic solvent.

[0186] In one embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose monomer to raspberry glycoside, thereby producing raspberry glycoside. The UDP-glucosyltransferase may be, for example, UGT91D2 or a variant of UGT91D2 having more than 75% amino acid sequence identity with UGT91D2.

[0187] In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose monomer to raspberry glycoside, thereby producing lebodiin E. The UDP-glucosyltransferase may be, for example, UGTSL2 or a variant of UGTSL2 having more than 75% amino acid sequence identity with UGTSL2.

[0188] In yet another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose monomer to rebaudioside E, thereby producing rebaudioside D. The UDP-glucosyltransferase may be, for example, UGT76G1 or a UGT76G1 variant having more than 75% amino acid sequence identity with UGT76G1.

[0189] In yet another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose monomer to hedyotis diffusa glycoside, thereby producing rebaudioside A. The UDP-glucosyltransferase may be, for example, UGT76G1 or a UGT76G1 variant having more than 75% amino acid sequence identity with UGT76G1.

[0190] In yet another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose monomer to rebaudioside A, thereby producing rebaudioside D and / or rebaudioside D2 and / or rebaudioside M2. The UDP-glucosyltransferase may be, for example, UGT91D2 or UGTSL2 or a variant thereof having greater than 75% amino acid sequence identity with UGT91D2 or UGTSL2.

[0191] In yet another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose monomer to rebaudioside I, thereby producing rebaudioside M. The UDP-glucosyltransferase may be, for example, UGTSL or a UGTSL variant having more than 75% amino acid sequence identity with UGTSL.

[0192] In another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose monomer to rebaudioside E, thereby producing rebaudioside M. The UDP-glucosyltransferase may be, for example, UGT76G1 or a UGT76G1 variant having more than 75% amino acid sequence identity with UGT76G1.

[0193] In another embodiment, the UDP-glucosyltransferase is capable of adding at least one glucose monomer to produce a target steviol glycoside having more than 75% amino acid sequence identity with at least one enzyme selected from the GenInfo identification number list below, preferably from the group presented in Table 1, and more preferably from the group presented in Table 2.

[0194]

[0195]

[0196]

[0197]

[0198] Table 1

[0199]

[0200]

[0201]

[0202] Table 2

[0203] GI number Login ID source 460409128 XP.004249992.1 tomato 460386018 XP.004238697.1 tomato 460409134 XP.004249995.1 tomato 460410132 XP.004250485.1 tomato 460410130 XP.004250484.1 tomato 460410128 XP.004250483.1 tomato 460378310 XP.004234916.1 tomato 209954733 BAG80557.1 Goji berries 209954725 BAG80553.1 Goji berries

[0204] In yet another embodiment, the UDP-glucosyltransferase is any UDP-glucosyltransferase capable of adding at least one glucose monomer to rebaudioside D, thereby producing rebaudioside M and / or rebaudioside M2. The UDP-glucosyltransferase may be, for example, UGT76G1 or a UGT76G1 variant having more than 75% amino acid sequence identity with UGT76G1.

[0205] Optionally, the method of the present invention further includes recycling UDP to provide UDP-glucose. In one embodiment, the method includes recycling UDP by providing a recycling catalyst, i.e., a biocatalyst capable of producing excess UDP-glucose, and recycling the substrate such that the substrate steviol glycoside is converted to the target steviol glycoside using a catalytic amount of UDP-glucosyltransferase and UDP-glucose. Figure 3 ).

[0206] In one implementation, the UDP-glucose recycling catalyst is sucrose synthase.

[0207] In one implementation, the recycling substrate is sucrose.

[0208] Optionally, the method of the present invention further comprises hydrolyzing the 1,6-β-glycosidic bonds in reb D2 and / or reb M2. In one embodiment, the method comprises hydrolyzing the 1,6-β-glycosidic bonds in reb D2 and / or reb M2 by providing a β-glucosidase.

[0209] In one embodiment, β-glucosidase is provided together with the UDP-recycled biocatalyst and UGT to minimize the content of reb D2 and / or reb M2 in the final reaction mixture and maximize the yield of reb M.

[0210] In a particular embodiment, in order to minimize the reb D2 and / or reb M2 content in the final reaction mixture and maximize the yield of reb M, a β-glucosidase is provided together with the UDP-recycled catalysts UGT76G1 and UGTSL2 or variants thereof having more than 75% amino acid sequence identity with UGT76G1 or UGTSL2.

[0211] Optionally, steviosides can be purified from the resulting composition. Purification of steviosides from the reaction medium can be achieved by any suitable method to provide a highly purified stevioside composition. Suitable methods include crystallization, membrane separation, centrifugation, extraction (liquid or solid phase), chromatographic separation, HPLC (preparative or analytical), or a combination of these methods.

[0212] Compounds and methods

[0213] This invention also provides isolated and highly purified reb D2. Reb D2 is an isomer of reb D and has the following structure:

[0214]

[0215] 13-[(2-O-β-D-glucopyranosyl-3-O-β-D-glucopyranosyl-β-D-glucopyranosyl)oxy] equivalent to ent-kaur-16-en-19-oic acid-[(6-O-β-D-glucopyranosyl-β-D-glucopyranosyl) ester]

[0216] In another embodiment, the present invention provides reb D2 based on an anhydrous basis having a purity of more than about 95% by weight, such as, for example, more than about 96% by weight, more than about 97% by weight, more than about 98% by weight, or more than about 99% by weight.

[0217] In yet another embodiment, the present invention provides reb D2 with a purity of more than about 95% by weight in a mixture of steviol glycosides, such as, for example, more than about 96% by weight, more than about 97% by weight, more than about 98% by weight, or more than about 99% by weight.

[0218] The present invention also provides compositions comprising reb D2.

[0219] In one embodiment, the present invention provides a method for preparing reb D2, comprising:

[0220] a. Contacting a starting composition containing reb A with an enzyme capable of converting reb A to reb D2, UDP-glucose, and optionally a UDP-glucose recycling enzyme to produce a composition containing reb D2; and

[0221] b. Separate the composition containing reb D2.

[0222] In some implementations, the enzyme capable of converting reb A to 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.

[0223] The enzyme that can convert reb A into reb D2 can be provided either as a fixed enzyme or as a recombinant microorganism.

[0224] In one embodiment, the enzyme is immobilized. In another embodiment, the enzyme is provided in the form of recombinant microorganisms.

[0225] In one embodiment, the microorganisms are free. In another embodiment, the microorganisms are immobilized. For example, the microorganisms may be immobilized on a solid support made of inorganic or organic material. Non-limiting examples of solid supports suitable for immobilizing microorganisms include derived cellulose or glass, ceramics, metal oxides, or membranes. Microorganisms may be immobilized on the solid support, for example, by covalent bonding, adsorption, cross-linking, embedding, or encapsulation.

[0226] Suitable microorganisms include, but are not limited to, Escherichia coli, yeast, Aspergillus, Pichia pastoris, Bacillus, and Yersinia.

[0227] In one embodiment, the microorganisms are in an aqueous medium comprising water and various components selected from carbon sources, energy sources, nitrogen sources, trace elements, vitamins, nucleosides, bisphosphates, triphosphates, organic and inorganic salts, organic and mineral acids, bases, etc. Carbon sources include glycerol, glucose, carbon dioxide, carbonates, and bicarbonates. Nitrogen sources may include nitrates, nitrites, amino acids, peptides, peptones, or proteins.

[0228] In a particular embodiment, the medium comprises a buffer. Suitable buffers include, but are not limited to, PIPES buffer, acetate buffer, and phosphate buffer. In a particular embodiment, the medium comprises a phosphate buffer.

[0229] In one embodiment, the medium may further include an organic solvent.

[0230] In a particular embodiment, the enzyme is a UDP-glucosyltransferase capable of converting reb A to reb D2 and is contained in Escherichia coli.

[0231] In a more specific embodiment, the enzyme is selected from UGT91D2, UGTSL, UGTSL_Sc, UGTSL2 (GINo. 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 is contained in Escherichia coli.

[0232] In a more specific embodiment, the enzyme is UGTSL2 and is contained in Escherichia coli.

[0233] Reb D2 can be separated from the reaction medium by any suitable method to provide a composition containing reb D2. Suitable methods include, but are not limited to, pyrolysis, crystallization, membrane separation, centrifugation, extraction (liquid or solid phase), chromatographic separation, HPLC (preparative or analytical), or combinations of these methods. In a particular embodiment, separation can be achieved by pyrolysis and centrifugation.

[0234] In some embodiments, the separation can yield a reb D2 purity of less than about 95% by weight on an anhydrous basis, and the composition may contain, for example, steviol glycosides and / or residual reaction products. The composition containing reb D2 can be further purified to provide highly purified reb D2, i.e., reb D2 with a purity greater than about 95% by weight on an anhydrous basis. In some embodiments, the composition containing reb D2 can be further purified to provide reb D2 with a purity greater than about 96%, greater than about 97%, greater than about 98%, or greater than about 99% by weight on an anhydrous basis.

[0235] Purification can be achieved by any means known to those skilled in the art, including but not limited to crystallization, membrane separation, centrifugation, extraction (liquid or solid phase), chromatographic separation, HPLC (preparative or analytical), or combinations of these methods. In a particular embodiment, HPLC is used to purify reb D2. In a more particular embodiment, semi-preparative HPLC is used to purify reb D2.

[0236] For example, a two-and-a-half-step 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), the mobile phase having the following gradient:

[0237] Time (minutes) ％A ％B 0.0-5.0 100 0 20 20 80 25 20 80 30 100 0

[0238] The second step uses the same column and conditions, but only the isocratic moving phase: 20% MeCN in water.

[0239] Those skilled in the art will recognize that specific column, mobile phase, injection volume, and other HPLC parameters can be varied.

[0240] In one embodiment, the present invention provides isolated and highly purified reb M2. reb M2 is an isomer of reb M and has the following structure:

[0241]

[0242] (13-[(2-O-β-D-glucopyranosyl-3-O-β-D-glucopyranosyl-β-D-glucopyranosyl)oxy] equivalent to 16-kaurene-19-acid [(2-O-β-D-glucopyranosyl-6-O-β-D-glucopyranosyl-β-D-glucopyranosyl) ester])

[0243] In another embodiment, the present invention provides reb M2 based on anhydrous base having a purity of more than about 95% by weight, such as, for example, more than about 96% by weight, more than about 97% by weight, more than about 98% by weight, or more than about 99% by weight.

[0244] In yet another embodiment, the present invention provides reb M2 with a purity of more than about 95% by weight in a mixture of steviol glycosides, such as, for example, more than about 96% by weight, more than about 97% by weight, more than about 98% by weight, or more than about 99% by weight.

[0245] In yet another embodiment, the present invention provides reb M2 in stevia extract having a purity of more than about 95% by weight, such as, for example, more than about 96% by weight, more than about 97% by weight, more than about 98% by weight, or more than about 99% by weight.

[0246] The present invention also provides compositions comprising reb M2.

[0247] It has been discovered that reb M2 is generated during the bioconversion of reb A to reb D. As mentioned above, the bioconversion of reb A to reb D also produces reb D2. Therefore, in one embodiment, the present invention provides a method for preparing reb M2, comprising:

[0248] a. Contacting a starting composition containing reb A and / or reb D2 with an enzyme capable of converting reb A and / or reb D2 to rebM2, UDP-glucose, and optionally a UDP-glucose recycling enzyme to produce a composition containing reb M2; and

[0249] b. Separate the composition containing reb M2.

[0250] To avoid being bound by theory, the current understanding is that the pathway begins with the conversion of reb A to reb D2, followed by the conversion of reb D2 to reb M2. Therefore, in one embodiment, this invention provides a method for preparing reb M2, comprising:

[0251] a. Contacting a starting composition containing reb D2 with an enzyme capable of converting reb D2 to reb M2, UDP-glucose, and optionally a UDP-glucose recycling enzyme to produce a composition containing reb M2; and

[0252] b. Separate the composition containing reb M2.

[0253] In yet another embodiment, the method for preparing reb M2 includes:

[0254] a. Contact a starting composition containing reb A with an enzyme capable of converting reb A to reb D2, UDP-glucose, and optionally a UDP-glucose recycling enzyme to produce a composition containing reb D2.

[0255] b. Optionally, isolate the composition containing reb D2;

[0256] c. Contacting the starting composition containing reb D2 with an enzyme capable of converting reb D2 to reb M2, UDP-glucose, and optionally a UDP-glucose recycling enzyme to produce a composition containing reb M2; and

[0257] d. Separate the composition containing reb M2.

[0258] The enzyme can be a UDP-glucosyltransferase, such as, for example, UGT91D2, UGTSL, UGTSL_Sc, UGTSL2 (GINo. 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.

[0259] Enzymes can be immobilized or in recombinant microorganisms.

[0260] In one embodiment, the enzyme is immobilized. In another embodiment, the enzyme is in recombinant microorganisms.

[0261] In one embodiment, the microorganisms are free. In another embodiment, the microorganisms are immobilized. For example, the microorganisms may be immobilized on a solid support made of inorganic or organic material. Non-limiting examples of solid supports suitable for immobilizing microorganisms include derived cellulose or glass, ceramics, metal oxides, or membranes. Microorganisms may be immobilized on the solid support, for example, by covalent bonding, adsorption, cross-linking, embedding, or encapsulation.

[0262] Suitable microorganisms include, but are not limited to, Escherichia coli, yeast, Aspergillus, Pichia pastoris, Bacillus, and Yersinia.

[0263] In one embodiment, the microorganisms are in an aqueous medium comprising water and various components selected from carbon sources, energy sources, nitrogen sources, trace elements, vitamins, nucleosides, bisphosphates, triphosphates, organic and inorganic salts, organic and mineral acids, bases, etc. Carbon sources include glycerol, glucose, carbon dioxide, carbonates, and bicarbonates. Nitrogen sources may include nitrates, nitrites, amino acids, peptides, peptones, or proteins.

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

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

[0266] In a particular embodiment, the enzyme is a UDP-glucosyltransferase capable of converting reb A and / or reb D2 into reb M2 and is contained in Escherichia coli.

[0267] In a more specific embodiment, the enzyme is selected from UGT91D2, UGTSL, UGTSL_Sc, UGTSL2 (GINo. 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 is contained in Escherichia coli.

[0268] In a more specific embodiment, the enzyme is UGTSL2 and is contained in Escherichia coli.

[0269] Reb M2 can be separated from the reaction medium by any suitable method to provide a composition containing reb M2. Suitable methods include, but are not limited to, pyrolysis, crystallization, membrane separation, centrifugation, extraction (liquid or solid phase), chromatographic separation, HPLC (preparative or analytical), or combinations of these methods. In a particular embodiment, separation can be achieved by pyrolysis and centrifugation.

[0270] In some embodiments, the separation can yield a reb M2 purity of less than about 95% by weight on an anhydrous basis, and the composition may contain, for example, steviol glycosides and / or residual reaction products.

[0271] The composition containing reb M2 can be further purified to provide highly purified reb M2, i.e., reb M2 with a weight purity greater than about 95% based on an anhydrous basis. In some embodiments, the composition containing reb M2 can be further purified to provide reb M2 with a weight purity greater than about 96%, greater than about 97%, greater than about 98%, or greater than about 99% based on anhydrous basis.

[0272] Purification can be achieved by any means known to those skilled in the art, including but not limited to crystallization, membrane separation, centrifugation, extraction (liquid or solid phase), chromatographic separation, HPLC (preparative or analytical), or combinations of these methods. In a particular embodiment, HPLC is used to purify reb M2. In a more particular embodiment, semi-preparative HPLC is used to purify reb M2.

[0273] For example, a two-and-a-half-step 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), the mobile phase having the following gradient:

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

[0275] The second step uses the same column and conditions, but only the isocratic moving phase: 20% MeCN in water.

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

[0277] The purified steviol glycosides prepared according to the present invention can be used in various consumer products, including but not limited to food, beverages, pharmaceutical compositions, tobacco products, nutritional supplement compositions, oral hygiene compositions, and cosmetic compositions.

[0278] The high-purity reb M obtained by this invention has a molecular weight of 1291.29 and a C0.05. 56 H 90 O 33 The molecular formula, CAS registration number 1220616-44-3, and the structure shown are as follows: Figure 1 It is a white and odorless powder. When compared to a 10% sucrose solution, the compound is approximately 200 times sweeter than sugar. Infrared absorption spectra are shown in... Figure 4 middle.

[0279] Other properties of the pure reb M compound include a melting point of 249-250 °C and a [α] content in 50% ethanol. D 25 Specific rotation of -19.0° (C = 1.0). The solubility of reb M in water is approximately 0.3%, and increases with increasing temperature.

[0280] reb M is soluble in dilute solutions of methanol, ethanol, n-propanol, and isopropanol. However, it is insoluble in acetone, benzene, chloroform, and ether.

[0281] The reb M obtained according to the present invention is thermally and pH stable.

[0282] The highly purified target glycosides obtained according to the present invention, particularly reb D, reb D2, reb M and / or reb M2, can be used "as is" or in combination with other sweeteners, flavorings and food ingredients.

[0283] Non-limiting examples of flavorings include lime, lemon, orange, fruit, banana, grape, pear, pineapple, mango, bitter almond, cola, cinnamon, sugar, marshmallow and vanilla flavoring.

[0284] Other non-limiting examples of food ingredients include flavorings, acidifiers, organic acids and amino acids, colorings, fillers, modified starches, gums, texture agents, preservatives, antioxidants, emulsifiers, stabilizers, thickeners and gelling agents.

[0285] The highly purified target glycosides obtained according to the present invention, particularly reb D, reb D2, reb M and / or reb M2, can be formulated into various polymeric forms, including but not limited to, hydrolysates, solvates, anhydrous, amorphous forms and / or mixtures thereof.

[0286] The highly purified reb D, reb D2, reb M and / or reb M2 obtained according to the present invention can be incorporated as high-intensity natural sweeteners into foods, beverages, pharmaceutical compositions, cosmetics, chewing gum, tableware, cereals, dairy products, toothpaste and other oral compositions.

[0287] 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 in combination with other naturally occurring high-intensity sweeteners such as steviol glycoside, reb A, reb B, reb C, reb D, reb E, reb F, steviol diglycoside, durqueside A, raspberry glycoside, mogroside, sweet protein, neohesperidin dihydrochalcone, glycyrrhizic acid and its salts, sematrandini, perilla syrup, pernandulcin, mukurozioside, scutellarin, perilla syrup-I, dimethyl-hexahydrofluorene-dicarboxylic acid, abrusoside, brassica oleracea oleifera, carnosifloside, pterocaryoside, and polypodoside. A. Brazilian rosehip, hernandulcin, phillodulcin, smilax china, phlorizin, trifolin, dihydroflavonol, dihydroquercetin-3-acetate, neooastilibin, trans-cinnamaldehyde, monadine and its salt, selligueain A, hematoxylin, monetin, pterocaryoside A, pterocaryoside B, arecoside, pentadine, flavor-modifying glycoprotein, curculigoside, neoculin, chlorogenic acid, cinnarizine, monk fruit sweetener, mogroside V, sarcoside, etc.

[0288] In certain embodiments, reb D2 and / or reb M2 may be used with a sweetener composition comprising a compound selected from reb A, reb B, reb D, NSF-02, mogroside V, erythritol, and combinations thereof.

[0289] Highly purified steviosides, 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, alitane, saccharin, hesperidin dihydrochalcone, cyclohexylsulfamic acid salt, neotame, gluconol, suosan advantame, and its salts.

[0290] Furthermore, highly purified steviol glycosides, particularly reb D, reb D2, reb M, and / or reb M2, can be used in combination with natural sweetener inhibitors such as gymnonic acid, aspartame, 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, glutamate, lysine, and tryptophan.

[0291] Highly purified reb D, reb D2, and reb M glycosides can be used in combination with one or more additives selected from 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 flavor components, astringent compounds, proteins or protein hydrolysates, surfactants, emulsifiers, flavonoids, alcohols, polymers, and combinations thereof.

[0292] Highly purified reb D, reb D2, reb M, and / or reb M2 can be used in combination 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, each containing 2, 3, or 4 hydroxyl groups. Polyols can also contain more than four hydroxyl groups, such as pentanol, hexanol, heptanol, etc., each containing 5, 6, or 7 hydroxyl groups. Additionally, polyols can also be sugar alcohols, polyols, or polyols in the reduced form of carbohydrates, where the carbonyl group (aldehyde or ketone, reducing sugar) has been reduced to a primary or secondary hydroxyl group. Examples of polyols include, but are not limited to, erythritol, maltitol, mannitol, sorbitol, lactitol, xylitol, inositol, isomaltitol, propylene glycol, glycerol, threitol, galactitol, hydrogenated isomaltulose, reduced isomaltose oligosaccharides, reduced xylose oligosaccharides, reduced gentian oligosaccharides, reduced maltose syrup, reduced glucose syrup, hydrogenated starch hydrolysate, polyols and sugar alcohols, or any other carbohydrate that can be reduced without adversely affecting the flavor of the sweetener composition.

[0293] Highly purified target steviol glycosides, especially reb D, reb D2, reb M and / or reb M2, can be combined with calorie-reducing sweeteners such as D-tagatose, L-sugar, L-sorbose, L-arabinose, etc.

[0294] Highly purified target steviol glycosides, particularly reb D, reb D2, reb M, and / or reb M2, can also be conjugated with various carbohydrates. The term "carbohydrate" generally refers to the general formula (CH2O). n Aldehydes or ketones substituted with multiple hydroxyl groups (where n is 3-30), and their oligomers and polymers. The carbohydrates of the present invention may be further substituted or deoxygenated at one or more positions. The carbohydrates used herein include unmodified carbohydrates, carbohydrate derivatives, substituted carbohydrates, and modified carbohydrates. The phrases “carbohydrate derivative,” “substituted carbohydrate,” and “modified carbohydrate” used herein are synonymous. A modified carbohydrate refers to any carbohydrate in which at least one atom has been added, removed, or substituted, or a combination thereof. Thus, carbohydrate derivatives or substituted carbohydrates include substituted and unsubstituted monosaccharides, disaccharides, oligosaccharides, and polysaccharides. The carbohydrate or substituted carbohydrate may optionally be deoxygenated at any corresponding C-position and / or substituted with one or more moieties such as hydrogen, halogen, haloalkyl, carboxyl, acyl, acyloxy, amino, amide, carboxyl derivative, alkylamino, dialkylamino, arylamino, alkoxy, aryloxy, nitro, cyano, sulfonyl, mercapto, imino, sulfonyl, thio, sulfinyl, aminosulfonyl, alkoxycarbonyl, formamide, phosphono, phosphinic, phosphoryl, phosphoester, thioether, oxime, hydrazine, carbamoyl, phosphorus, phosphonic acid, or any other feasible functional group, provided that the carbohydrate derivative or substituted carbohydrate contributes to improving the sweetness of the sweetener composition.

[0295] Examples of carbohydrates that can be used according to the present invention include, but are not limited to, allulose, maltodextrose, allose, tagatose, trehalose, galactose, rhamnose, various cyclodextrins, cyclic oligosaccharides, various types of maltodextrins, dextran, sucrose, glucose, ribulose, fructose, threose, arabinose, xylose, lysolose, allose, azoose, mannose, idoleose, lactose, maltose, invert sugar, isotrehalose, neotrehalose, isomaltulose, and erythritol. Deoxyribose, gulose, idulose, tarose, erythritolose, xylulose, allulose, mesobiose, cellobiose, amylopectin, glucosamine, mannosamine, fucose, glucuronic acid, gluconic acid, gluconic acid-lactone, abigolose, galactosamine, beet oligosaccharides, isomaltose oligosaccharides (isomaltose, isomalttriose, panose, etc.), xylooligosaccharides (xylotriose, xylobiose, etc.), xylose-terminated oligosaccharides. Oligosaccharides, gentian oligosaccharides (gentiobiose, gentiotriose, gentiotetraose, etc.), sorbitol, Aspergillus niger oligosaccharides, paraginose oligosaccharides, fructosaccharides (sucrose triose, fungal tetraose, etc.), maltetrol, maltitol, maltoliposides (malttriose, maltetrol, maltpentose, malthexose, maltoheptose, etc.), starch, inulin, inulo-oligosaccharides, lactulose, melibiose, melibiose, ribose, isomerized liquid sugars (such as high-fructose corn syrup), conjugated sugars, and soybean oligosaccharides. Additionally, the carbohydrates used in this article can be D- or L-configured.

[0296] The 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 a variety of physiologically active substances or functional ingredients. Functional ingredients are generally classified into categories such as carotenoids, dietary fiber, fatty acids, saponins, antioxidants, nutritional supplements, flavonoids, isothiocyanates, phenols, plant sterols and stanols; 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 effects. Exemplary functional ingredients are provided in WO2013 / 096420, the contents of which are hereby incorporated by reference.

[0297] The highly purified reb D, reb D2, reb M and / or reb M2 obtained according to the present invention can be used as high-intensity sweeteners to produce zero-calorie, low-calorie, or diabetic beverages and foods with improved taste characteristics. They can also be used in beverages, foods, pharmaceuticals, and other products in which sugar cannot be used. Furthermore, the highly purified reb D, reb D2, reb M and / or reb M2 can be used not only as sweeteners in beverages, foods, and other products intended for human consumption, but also in animal feed and forage with improved characteristics.

[0298] Examples of consumer products in which highly purified 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, liqueurs, and sake; natural juices; soft drinks; carbonated soft drinks; low-calorie beverages; zero-calorie beverages; reduced-calorie beverages and foods; yogurt drinks; instant juices; instant coffee; powdered instant beverages; canned products; syrups; fermented soy sauce; soy sauce; vinegar; salad dressing; mayonnaise; ketchup; curry; soup; instant broth; powdered soy sauce; powdered vinegar; and various other types of... Cookies; rice crackers; crackers; bread; chocolate; caramel; candy; chewing gum; jelly; pudding; dried fruit and pickled vegetables; whipped cream; jam; orange marmalade; flower jam; milk powder; ice cream; fruit juice sorbet; bottled vegetables and fruits; canned and boiled beans; meat and food boiled in sweet sauces; agricultural vegetable products; seafood; ham; sausage; fish ham; fish sausage; fish sauce; fried fish products; dried seafood; frozen food; pickled seaweed; cured meat; tomatoes; medicines; etc. In principle, its applications are virtually limitless.

[0299] In the manufacturing process of products such as food, beverages, pharmaceuticals, cosmetics, tableware, and chewing gum, conventional methods such as mixing, kneading, dissolving, acid soaking, permeation, filtration, spraying, atomizing, filling, and other methods can be used.

[0300] Furthermore, the highly purified reb D, reb D2, reb M and / or reb M2 obtained according to the present invention can be used in dry or liquid form. In one embodiment, a tabletop sweetener containing reb D2 is provided. In another embodiment, a tabletop sweetener containing reb M2 is provided.

[0301] Highly purified rebsteviosides can be added before or after food heat treatment. The amount of highly purified rebsteviosides (especially reb D, reb D2, reb M, and / or reb M2) depends on the intended use. As discussed above, they can be added alone or in combination with other compounds.

[0302] The present invention also relates to using reb D2 to enhance the sweetness of beverages. The present invention also relates to including reb M2 to enhance the sweetness of beverages. Therefore, the present invention provides beverages comprising a sweetener and reb D2 and / or reb M2 as a sweetness enhancer, wherein reb D2 and / or reb M2 are present at concentrations equal to or below their respective sweetness perception thresholds.

[0303] As used herein, the term "sweetness enhancer" refers to a compound that can enhance or strengthen the perception of sweetness in a composition, such as a beverage. The term "sweetness enhancer" is synonymous with the terms "sweetness synergist," "sweetness enhancer," "sweetness increaser," and "sweetness fortifier."

[0304] As used herein, the term "sweetness recognition threshold concentration" is the lowest known concentration of a sweet compound that is perceptible to the human palate, typically about 1.0% sucrose equivalent (1.0% SE). Generally, a sweetener can enhance or intensify the sweetness of a given sweetener at a concentration equal to or below its sweetness recognition threshold concentration without providing any perceptible sweetness itself; however, a sweetener can provide sweetness at concentrations above its sweetness recognition threshold concentration. The sweetness recognition threshold concentration is specific to a particular sweetener and can vary based on the beverage matrix. It can be readily determined by tasting incremental concentrations of a given sweetener until a concentration above 1.0% sucrose equivalent is detected in a given beverage matrix. A concentration providing approximately 1.0% sucrose equivalent is considered the sweetness recognition threshold.

[0305] In some embodiments, the sweetener is present in the beverage in amounts 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.

[0306] In a particular embodiment, the sweetener is present in the beverage in an amount of about 0.5% to about 10%, such as, for example, about 2% to about 8%, about 3% to about 7%, or about 4% to about 6% by weight. In another particular embodiment, the sweetener is present in the beverage in an amount of about 0.5% to about 8% by weight.

[0307] In one implementation, the sweetener is a conventional calorie sweetener. Suitable sweeteners include, but are not limited to, sucrose, fructose, glucose, high-fructose corn syrup, and high-fructose starch syrup.

[0308] In another implementation, the sweetener is erythritol.

[0309] In yet another embodiment, the sweetener is a rare sugar. Suitable rare sugars include, but are not limited to, D-allose, D-allulose, L-ribose, D-tagatose, L-glucose, L-fructose, L-arabinose, D-minobiose, D-leuconitose, and combinations thereof.

[0310] The sweetener can be used alone or in combination with other sweeteners.

[0311] In one embodiment, the rare sugar is D-allose. In a more particular embodiment, D-allose is present in the beverage in an amount of about 0.5% to about 10% by weight, such as, for example, about 2% to about 8%.

[0312] In another embodiment, the rare sugar is D-allulose. In a more particular embodiment, D-allulose is present in the beverage in an amount of about 0.5% to about 10% by weight, such as, for example, about 2% to about 8%.

[0313] In yet another embodiment, the rare sugar is D-ribose. In a more particular embodiment, D-ribose is present in the beverage in an amount of about 0.5% to about 10% by weight, such as, for example, about 2% to about 8%.

[0314] In yet another embodiment, the rare sugar is D-tagatin. In a more particular embodiment, D-tagatin is present in the beverage in an amount of about 0.5% to about 10% by weight, such as, for example, about 2% to about 8%.

[0315] In a further embodiment, the rare sugar is L-glucose. In a more particular embodiment, L-glucose is present in the beverage in an amount of about 0.5% to about 10% by weight, such as, for example, about 2% to about 8%.

[0316] In one embodiment, the rare sugar is L-fructose. In a more particular embodiment, L-fructose is present in the beverage in an amount of about 0.5% to about 10% by weight, such as, for example, about 2% to about 8%.

[0317] In another embodiment, the rare sugar is L-arabinose. In a more particular embodiment, L-arabinose is present in the beverage in an amount of about 0.5% to about 10% by weight, such as, for example, about 2% to about 8%.

[0318] In yet another embodiment, the rare sugar is D-minobiose. In a more particular embodiment, D-minobiose is present in the beverage in an amount of about 0.5% to about 10% by weight, such as, for example, about 2% to about 8%.

[0319] In yet another embodiment, the rare sugar is D-leucobacterium disaccharide. In a more particular embodiment, D-leucobacterium disaccharide is present in the beverage in an amount of about 0.5% to about 10% by weight, such as, for example, about 2% to about 8%.

[0320] Compared to a corresponding beverage without a sweetener, adding a sweetener at a concentration equal to or below its sweetness recognition threshold increases the detectable sucrose equivalent in the beverage containing both sweetener and sweetener. Furthermore, in the absence of any sweetener, sweetness can be increased by exceeding the amount of detectable sweetness in a solution containing at least one sweetener at the same concentration.

[0321] Therefore, the present invention also provides a method for enhancing the sweetness of a beverage containing a sweetener, comprising providing a beverage containing a sweetener and adding a sweetener selected from reb D2, reb M2 or a combination thereof, wherein reb D2 and reb M2 are present at a concentration equal to or below their sweetness recognition threshold.

[0322] Adding reb D2 and / or reb M2 at concentrations equal to or below the sweetness recognition threshold to a beverage containing sweeteners can increase the detected sucrose equivalent from 1.0% to approximately 5.0%, such as, for example, approximately 1.0%, approximately 1.5%, approximately 2.0%, approximately 2.5%, approximately 3.0%, approximately 3.5%, approximately 4.0%, approximately 4.5%, or approximately 5.0%.

[0323] The following examples illustrate preferred embodiments of the present invention for the preparation of highly purified target steviol glycosides (particularly, reb D, reb D2, reb M, and / or reb M2). It should be understood that the invention is not limited to the materials, proportions, conditions, and procedures listed in the examples, and are merely illustrative.

[0324] Example 1

[0325] UGT76G1 produced in vivo

[0326] The NcoI and NdeI restriction sites were added to the initial nucleic acid sequence described in Genbank accession number AAR06912.1. After codon optimization, the following nucleic acid sequence was obtained:

[0327]

[0328] After synthesizing the gene using the NdeI and XhoI cloning sites and subcloning it into the pET30A+ vector, the UGT76G1_pET30a+ plasmid was introduced into *E. coli* B121(DE3) and *E. coli* EC100 via electroporation. The resulting cells were grown in Piper dishes containing kanamycin, and suitable clones were selected for growth in liquid LB medium (Eragonal flasks). Glycerol was added to the suspension as a cryoprotectant, and 400 μL aliquots were stored at -20°C and -80°C.

[0329] A storage 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). The culture was incubated at 135 rpm and 30°C with shaking for 8 hours.

[0330] The production medium contained 60 g / L overnight expression TB medium (Novagen), 10 g / L glycerol, and 50 mg / L kanamycin. The medium was stirred at 20°C while OD and pH were measured. The culture showed significant growth and good OD. After 40 hours, cells were collected by centrifugation and frozen, yielding a wet cell weight of 12.7 g.

[0331] Lysis was performed by adding Bugbuster Master mixture (Novagen), and the lysate was collected by centrifugation and kept frozen. Activity testing was conducted using the thawed lysate.

[0332] Example 2

[0333] In vitro production of UGT76G1

[0334] The Promega S30 T7 high-yield expression system kit was used. 4 μg of UGT76G1_pET30a+ plasmid from *E. 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 2 h. 180 μL was used in the catalytic assay reaction.

[0335] Example 3

[0336] In vitro production of UGT91D2

[0337] The NcoI and NdeI restriction sides were added to the initial nucleic acid sequence as described in Genbank accession number ACE87855.1. After codon optimization, the following nucleic acid sequence was obtained:

[0338]

[0339] After synthesizing the gene using the NcoI and XhoI cloning sites and subcloning it into pET30A+, UGT91D2_pET30a+ was introduced into *E. coli* EC100 cells via electroporation. The resulting cells were grown in the presence of kanamycin, and suitable clones were selected for growth in liquid LB medium (Eragonal flasks). Glycerol was added to the suspension as a cryoprotectant, and 400 μL aliquots were stored at -20°C and -80°C.

[0340] The Promega S30 T7 high-yield protein expression system kit was used for in vitro protein synthesis.

[0341] 4 μg of UGT91D2_pET30a+ plasmid 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 2 hours. 5 μL was used for SDS-PAGE analysis, and the remaining 45 μL was used for the catalytic assay.

[0342] Example 4

[0343] Catalytic reaction using in vivo generated UGT76G1

[0344] 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 limonin, and 500 μL of lysate from UGT76G1. The reaction was run at 30 °C on an orbital oscillator at 135 rpm. 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 samples were immediately centrifuged and kept at 10 °C before analysis by HPLC (CAD). HPLC showed that limonin was almost completely converted to lebodiin A, as... Figure 51 As shown in the image.

[0345] Example 5

[0346] Catalytic reaction using in vitro generated UGT91D2

[0347] 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 generated UGT91D2. The reaction was run at 30 °C on an orbital oscillator at 135 rpm. 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 showed that 4.7% of rebaudioside A was converted to rebaudioside D after 120 hours.

[0348] Example 6

[0349] Catalytic reaction using in vitro generated UGT76G1

[0350] The total reaction volume was 2 mL, containing the following components: 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 generated UGT76G1. The reaction was run at 30 °C on an orbital oscillator at 135 rpm. 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 showed that after 120 hours, 80% of rebaudioside D was converted to rebaudioside M, as... Figure 52 As shown in the image.

[0351] For Examples 7 to 12, the following abbreviations were used:

[0352] LBGKP medium20 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 or ampicillin

[0353] LB medium (20g / L Luria broth Lennox)

[0354] Example 7

[0355] Preparation and activity of UGT76G1 prepared by pET30a+ plasmid and BL21(DE3) expression strain

[0356] The pET30a+_UGT76G1 plasmid was transformed into the BL21(DE3) expression strain (Lucigen E). EXPRESS electrocompetent cells were obtained. Cells were grown on LB agar in Piper dishes in the presence of kanamycin. Suitable clones were selected and grown in liquid LBGKP medium containing kanamycin. Glycerol was added, and 400 μL aliquots were stored at -20°C and -80°C.

[0357] The stored aliquots were thawed and added to 30 mL of LBGKP medium. The culture was incubated at 30°C with shaking for 8 hours and then used to inoculate 400 mL of production medium containing 60 g / L "overnight expression pre-prepared TB medium" (Novagen, reference 71491-5), 10 g / L glycerol, and 50 mg / L kanamycin. The medium was stirred at 20°C while sampling to measure OD (600 nm) and pH. After 40 hours, cells were collected by centrifugation and frozen. The obtained cell wet weight was 10.58 g.

[0358] The 3.24 g precipitate was lysed by adding 8.1 mL of "Bugbuster Master Mixture" (Novagen, reference 71456) and 3.5 mL of water. The lysate was collected by centrifugation and kept frozen.

[0359] Example 8

[0360] Preparation and activity of UGT76G1 prepared by pET30a+ plasmid and Turner(DE3) expression strain

[0361] The pET30a+_UGT76G1 plasmid was transformed into the Turner(DE3) expression strain (NovagenTuner) through heat shock treatment. tm(DE3) competent cells. The obtained cells were grown on LB agar in Piper dishes in the presence of kanamycin. Suitable clones were selected and grown in liquid LBGKP medium containing kanamycin. Glycerol was added, and 400 μL aliquots were stored at -20°C and -80°C.

[0362] The stored aliquots were thawed and added to 100 mL of LB medium containing 50 mg / L kanamycin. The culture was incubated at 30°C with shaking for 15 hours. 4.4 mL of this culture was then inoculated into 200 mL of production medium containing LB. The medium was stirred at 37°C until an OD of 0.9 (600 nm) was obtained. Subsequently, 400 μL of 100 mM IPTG solution was added, and the medium was stirred at 30°C for 4 hours. Cells were collected by centrifugation and frozen. The obtained cell wet weight was 1.38 g.

[0363] The precipitate was lysed by adding 4.9 mL of "Bugbuster Master Mixture" (Novagen, reference 71456) and 2.1 mL of water. The lysate was collected by centrifugation and kept frozen.

[0364] Example 9

[0365] Preparation and activity of UGT76G1 prepared by pMAL plasmid and BL21 expression strain

[0366] Using the Nde1 and Sal1 cloning sites, the synthesized UGT76G1 gene was subcloned into the pMAL plasmid. The pMAL_UGT76G1 plasmid was then transformed into the BL21 expression strain (New England Biolabs BL21 competent Escherichia coli) after heat shock treatment. The resulting cells were grown on LB agar in Piper dishes in the presence of ampicillin. Suitable clones were selected and grown in liquid LBGKP medium containing ampicillin. Glycerol was added, and 400 μL aliquots were stored at -20°C and -80°C.

[0367] The stored aliquots were thawed and added to 30 mL of LBGKP medium. This culture was incubated at 30°C with shaking for 8 hours and then used to inoculate 400 mL of production medium containing 60 g / L "overnight expression pre-prepared TB medium" (Novagen, reference 71491-5), 10 g / L glycerol, and 50 mg / L ampicillin. The medium was stirred at 20°C while sampling to measure OD and pH. After 40 hours, cells were collected by centrifugation and frozen. The obtained cell wet weight was 5.86 g.

[0368] The 2.74 g precipitate was lysed by adding 9.6 mL of "Bugbuster Master Mixture" (Novagen, reference 71456) and 4.1 mL of water. The lysis product was collected by centrifugation and kept frozen.

[0369] Example 10

[0370] Preparation and activity of UGT76G1 prepared by pMAL plasmid and Arctic Express expression strain

[0371] The pMAL_UGT76G1 plasmid was transformed into ArticExpress expression strains (Agilent ArcticExpress competent cells) via heat shock treatment. The resulting cells were grown on LB agar in Piper dishes in the presence of ampicillin and genimycin. Suitable clones were selected and grown in liquid LBGKP medium containing ampicillin and genimycin. Glycerol was added, and 400 μL aliquots were stored at -20°C and -80°C.

[0372] The stored aliquots were thawed and added to 30 mL of LBGKP medium (containing ampicillin and genistein). This culture was incubated at 30°C with shaking for 8 hours and then used to inoculate 400 mL of production medium containing 60 g / L "overnight expression pre-prepared TB medium" (Novagen, reference 71491-5), 10 g / L glycerol, and 50 mg / L ampicillin. The medium was stirred at 12°C while sampling to measure OD (600 nm) and pH. After 68 hours, cells were collected by centrifugation and frozen. The obtained cell wet weight was 8.96 g.

[0373] The 2.47 g precipitate was lysed by adding 8.73 mL of "Bugbuster Master Mixture" (Novagen, reference 71456) and 3.79 mL of water. The lysis product was collected by centrifugation and kept frozen.

[0374] Example 11

[0375] Preparation and activity of UGT76G1 prepared by pCOLDIII plasmid and ArcticExpress expression strain

[0376] Using the Nde1 and Xho1 cloning sites, the synthesized UGT76G1 gene was subcloned into the pCOLDIII plasmid. The pCOLDIII_UGT76G1 plasmid was then transformed into ArcticExpress expression strains (Agilent ArcticExpress competent cells) after heat shock treatment. The resulting cells were grown on LB agar in Piper dishes in the presence of ampicillin and genimycin. Suitable clones were selected and grown in liquid LBGKP medium containing ampicillin and genimycin. Glycerol was added, and 400 μL aliquots were stored at -20°C and -80°C.

[0377] The stored aliquots were thawed and added to 30 mL of LBGKP medium (containing ampicillin and genistein). This culture was incubated at 30°C with shaking for 8 hours and then used to inoculate 400 mL of production medium containing 60 g / L "overnight expression pre-prepared TB medium" (Novagen, reference 71491-5), 10 g / L glycerol, and 50 mg / L kanamycin. The medium was stirred at 12°C while sampling to measure OD (600 nm) and pH. After 63 hours, cells were collected by centrifugation and frozen. The obtained cell wet weight was 6.54 g.

[0378] The 2.81 g precipitate was lysed by adding 9.8 mL of "Bugbuster Master Mixture" (Novagen, reference 71456) and 4.2 mL of water. The lysis product was collected by centrifugation and kept frozen.

[0379] Example 12

[0380] Preparation and activity of UGT76G1 prepared by pCOLDIII plasmid and Origami2(DE3) expression strain

[0381] The pCOLDIII_UGT76G1 plasmid was transformed into the Origami2(DE3) expression strain (Novagen Origami) through heat shock treatment. TM 2(DE3) competent cells). The obtained cells were grown on LB agar in Piper dishes in the presence of ampicillin. Suitable clones were selected and grown in liquid LBGKP medium containing ampicillin. Glycerol was added, and 400 μL aliquots were stored at -20°C and -80°C.

[0382] The stored aliquots were thawed and added to 30 mL of LBGKP medium (containing ampicillin). This culture was incubated at 30°C with shaking for 8 h, and then used to inoculate 400 mL of production medium containing 60 g / L "overnight expression pre-prepared TB medium" (Novagen, reference 71491-5), 10 g / L glycerol, and 50 mg / L kanamycin. The medium was stirred at 12°C while sampling to measure OD (600 nm) and pH. After 68 hours, cells were collected by centrifugation and frozen. The obtained cell wet weight was 2.53 g.

[0383] The 1.71 g precipitate was lysed by adding 6.0 mL of "Bugbuster Master Mixture" (Novagen, reference 71456) and 1.9 mL of water. The lysis product was collected by centrifugation and kept frozen.

[0384] Example 13

[0385] Activity determination

[0386] Using 0.5 mM substrate, 2.5 mM UDP-glucose, and 3 mM MgCl2 in 50 mM pH 7.2 sodium phosphate buffer, and 500 μL of thawed lysis product, activity tests were performed on a 5 mL scale for the conversion of daunoside to rebaunoside A and rebaunoside D to rebaunoside M. Samples were taken and analyzed by HPLC. Results for different UGT76G1 preparations are summarized in the table below.

[0387]

[0388] *Notes

[0389] The conversion activities of limonin and rebaudioside M are described per mL of lysis product. At 30°C and pH 7.2, 1 U converts 1 μmol of substrate in 1 hour.

[0390] Example 14

[0391] 50 mL scale reaction for the conversion of rebaudioside D to rebaudioside M

[0392] Five mL of the lysis product from Example 12 was used to convert rebaudioside D to rebaudioside M in a 50 mL scale. 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 shaking at 30°C for 90 hours, 50 mL of ethanol was added, and the resulting mixture was shaken at -20°C for 1 hour. After centrifugation at 5000 g for 10 minutes, the supernatant was purified by ultrafiltration (Vivaflow MWCO 30000). 78 mL of permeate was obtained, and 9 mL of the retentate was diluted with 9 mL of ethanol and subjected to ultrafiltration again (Vivaflow MWCO 30000). Another 14 mL of filtrate was obtained and combined with the first permeate. The combined permeate was concentrated under reduced pressure at 30°C until 32 mL of clear solution was obtained.

[0393] HPLC traces of the product mixture were observed in Figure 5 HPLC was performed on an Agilent 1200 series system equipped with a dual-pump, autosampler, and a temperature-controlled column chamber. The method was isocratic, using a mobile phase 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 × 2 mm. The column temperature was maintained at 40 °C. The injection volume was 20–40 μL.

[0394] Example 15

[0395] Preparation of UGT91D2 using pMAL plasmid and BL21 expression strain

[0396] Using the Nde1 and Sal1 cloning sites, the synthesized UGT91D2 gene was subcloned into the pMAL plasmid. The pMAL_UGT91D2 plasmid was then transformed into the BL21 expression strain (New England Biolabs BL21 competent Escherichia coli) after heat shock treatment. The resulting cells were grown on LB agar in Piper dishes in the presence of ampicillin. Suitable clones were selected and grown in liquid LBGKP medium containing ampicillin. Glycerol was added, and 400 μL aliquots were stored at -20°C and -80°C.

[0397] The stored aliquots were thawed and added to 30 mL of LBGKP medium. The culture was incubated at 30°C with shaking for 8 hours and then used to inoculate 400 mL of production medium containing 60 g / L "overnight expression pre-prepared TB medium" (Novagen, reference 71491-5), 10 g / L glycerol, and 50 mg / L ampicillin. The medium was stirred at 20°C while sampling to measure OD and pH. After 40 hours, cells were collected by centrifugation and frozen. The obtained cell wet weight was 12.32 g.

[0398] The 2.18 g precipitate was lysed by adding 7.7 mL of "Bugbuster Master Mixture" (Novagen, reference 71456) and 3.2 mL of water. The lysate was collected by centrifugation and used directly for activity testing.

[0399] Example 16

[0400] Preparation of UGT91D2 using pMAL plasmid and ArcticExpress expression strain

[0401] The pMAL_UGT91D2 plasmid was transformed into ArcticExpress expression strains (Agilent ArcticExpress competent cells) via heat shock treatment. The resulting cells were grown on LB agar in Piper dishes in the presence of ampicillin and genimycin. Suitable clones were selected and grown in liquid LBGKP medium containing ampicillin and genimycin. Glycerol was added, and 400 μL aliquots were stored at -20°C and -80°C.

[0402] The stored aliquots were thawed and added to 30 mL of LBGKP medium (containing ampicillin and genistein). This culture was incubated at 30°C with shaking for 8 hours and then used to inoculate 400 mL of production medium containing 60 g / L "overnight expression pre-prepared TB medium" (Novagen, reference 71491-5), 10 g / L glycerol, and 50 mg / L ampicillin. The medium was stirred at 20°C for 16 hours, followed by stirring at 12°C for another 50 hours, while simultaneously measuring OD (600 nm) and pH. Cells were collected by centrifugation and frozen. The obtained cell wet weight was 15.77 g.

[0403] The 2.57 g precipitate was lysed by adding 9.0 mL of "Bugbuster Master Mixture" (Novagen, reference 71456) and 3.8 mL of water. The lysate was collected by centrifugation and used directly for activity testing.

[0404] Example 17

[0405] Preparation of UGT91D2 using pET30a+ plasmid and Turner(DE3) expression strain

[0406] The pET30a+_UGT91D2 plasmid was transformed into the Tuner(DE3) expression strain (NovagenTuner) through heat shock treatment. tm (DE3) competent cells. The obtained cells were grown on LB agar in Piper dishes in the presence of kanamycin. Suitable clones were selected and grown in liquid LBGKP medium containing kanamycin. Glycerol was added, and 400 μL aliquots were stored at -20°C and -80°C.

[0407] The stored aliquots were thawed and added to 100 mL of LB medium containing 50 mg / L kanamycin. The culture was incubated at 30°C with shaking for 15 hours. 6.2 mL of this culture was then inoculated into 500 mL of production medium containing LB. The medium was stirred at 37°C until an OD of 0.9 (600 nm) was obtained. Subsequently, 500 μL of 100 mM IPTG solution was added (IPTG concentration in the medium was 100 μM), and the medium was stirred at 30°C for 4 hours. Cells were collected by centrifugation and frozen. The wet weight of the obtained cells was 4.02 g.

[0408] The 1.92 g precipitate was lysed by adding 6.8 mL of "Bugbuster Master Mixture" (Novagen, reference 71456) and 2.8 mL of water. The lysate was collected by centrifugation and used directly for activity testing.

[0409] Example 18

[0410] Preparation of UGT91D2 using pET30a+ plasmid and ArcticExpress expression strain: The pET30a+_UGT91D2 plasmid was transformed into ArcticExpress(DE3) expression strain (Agilent ArcticExpress competent cells) via heat shock treatment. The resulting cells were grown on LB agar in Piper's dishes in the presence of ampicillin and genimycin. Suitable clones were selected and grown in liquid LBGKP medium containing ampicillin and genimycin. Glycerol was added, and 400 μL aliquots were stored at -20°C and -80°C.

[0411] The stored aliquots were thawed and added to 30 mL of LBGKP medium (containing ampicillin and genistein). This culture was incubated at 30°C with shaking for 8 hours and then used to inoculate 400 mL of production medium containing 60 g / L "overnight expression pre-prepared TB medium" (Novagen, reference 71491-5), 10 g / L glycerol, and 50 mg / L ampicillin. The medium was stirred at 20°C for 16 hours, followed by stirring at 12°C for another 50 hours, while simultaneously measuring OD (600 nm) and pH. After 60 hours, cells were collected by centrifugation and frozen. The obtained cell wet weight was 16.07 g.

[0412] The 3.24 g precipitate was lysed by adding 11.4 mL of "Bugbuster Master Mixture" (Novagen, reference 71456) and 4.8 mL of water. The lysate was collected by centrifugation and used directly for activity testing.

[0413] Example 19

[0414] Activity assay of UGT91D2 in vivo preparation

[0415] Using 0.5 mM substrate, 2.5 mM UDP-glucose, and 3 mM MgCl2 in 50 mM sodium phosphate buffer (pH 7.2), 1000 μL of the lysis product was used to convert raspberry glycoside to hedyotis diffusa glycoside, and activity tests were performed on a 5 mL scale. Samples were taken and analyzed by HPLC. Results for different UGT91D2 preparations are summarized in the table below.

[0416]

[0417] *Note: Activity is mentioned per mL of lysis product. At 30°C and pH 7.2, 1 U will convert 1 μmol of substrate in 1 hour.

[0418] Example 20

[0419] Other enzymes used in the conversion of rebodiosyl A to rebodiosyl D

[0420] The following UDP-glucosyltransferase genes were identified from public databases, synthesized using DNA2.0, and subsequently subcloned in the pET30a+ vector.

[0421] Microtiter plate Location Gene name Internal Reference RebA is converted to RebD C908201 A1 gi115454819_NP_001051010.1 S115N01 A1 Active C908201 G2 gi187373030_ACD03249.1 S115N01 G2 Active C908201 A7 gi460409128_XP_004249992.1 S115N05 A7 Active C912666 E1 gi222619587_EEE55719.1 S115N06 E1 Active C912666 C2 gi297795735_XP_002865752.1 S115N06 C2 Active

[0422] The amino acid sequence is as follows:

[0423] >gi|115454819|ref|NP_001051010.1|Os03g0702500[Oryza sativa Japonica Group]

[0424]

[0425] >gi|187373030|gb|ACD03249.1|UDP-glucosyltransferase[Avena strigosa]

[0426]

[0427] >gi|460409128|ref|XP_004249992.1|PREDICTED: Anthocyanin-3-O-glucoside 2-O-glucuronosyltransferase-like[Solanum lycopersicum]

[0428]

[0429] >gi|222619587|gb|EEE55719.1|Hypothetical protein OsJ_04191[Oryza sativa Japonica Group]

[0430]

[0431] >gi|297795735|ref|XP_002865752.1|UDP-glucuronosyl / UDP-glucosyltransferase family protein[Arabidopsis lyrata subsp. lyrata]

[0432]

[0433] Receive the plasmid to be tested in a microtiter plate containing the plasmid, which is in each separate well as a lyophilized solid.

[0434] Plasmid suspension To each well, add 24 μL of ultrapure sterile water, and shake the microtiter plate at room temperature for 30 minutes. Subsequently, incubate the plate at 4 °C for 1 hour. Further mix the contents of each well by pipetting up and down. Use 1 μL of the suspension for plasmid quantification by Qubit 2.0 analysis. The measured plasmid content is:

[0435] Microtiter plate Location Internal Reference [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

[0436] Transform competent cells with plasmidsRemove aliquots of chemicompetent EC100 cells from a -80°C freezer and store on ice. Thaw the cells on ice for 10 minutes. Add 10 μL of the diluted plasmid solution to a 1.5 mL sterile microtube (enough to transform each cell with 50 pg DNA) and store on ice. Add 100 μL of chemicompetent cells to each microtube. After incubating the chemicompetent cell-plasmid mixture on ice for 20 minutes, perform a heat shock at 42°C for 30 seconds.

[0437] Incubate on ice for another 2 minutes. Add 300 μL of SOC medium to each microtube and transfer the resulting mixture to a sterile 15 mL test tube. Incubate at 37°C for 1 hour with shaking at 135 rpm, then spread the mixture onto solid Luria broth containing 50 μg / mL kanamycin. Incubate the Piper dishes at 37°C for 16 hours.

[0438] Preparation of stock solution in glycerol and purification of plasmids Add 10 mL of Luria broth containing 50 μg / mL kanamycin to a 50 mL sterile Falcon tube. Inoculate the culture medium with clones isolated from the above Piper dishes and incubate the culture at 37°C for 16 hours with shaking at 135 rpm.

[0439] 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.

[0440] The remaining culture was centrifuged at 5,525 g for 10 minutes at 10°C, and the supernatant was removed. The precipitate was stored on ice. The resulting plasmid was purified using 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 plasmid content was determined as follows:

[0441] Microtiter plate Location Internal reference for testing [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

[0442] In vitro expression of enzymes According to the Promega S30 T7 high-yield protein expression system kit (reference: L1110), 18 μL of plasmid solution (containing approximately 1.5 μg of plasmid) was used for in vitro expression. The expression medium was prepared as follows:

[0443]

[0444] The prepared expression medium mixture was added to the plasmid solution and incubated at 30°C for 3 hours, mixing the mixture every 45 minutes. 5 μL of the mixture was frozen, while the remainder was used for a catalytic assay targeting the conversion of rebaudioside A to rebaudioside D.

[0445] Catalytic assay for the conversion of rebaudioside A to rebaudioside D 430 μL of a reaction mixture containing 0.5 mM rebaudioside A, 3 mM MgCl2, 50 mM phosphate buffer (pH 7.2), and 2.5 mM UDP-glucose was added to a 1.5 mL sterile microtube. 52 μL of enzyme expression medium was added, and the resulting mixture was incubated at 30 °C for 24 h. At 2 h, 16 h, and 24 h, 125 μL of the sample was collected and added to 115 μL of 60% methanol and 10 μL of 2N H2SO4. The quenched sample was centrifuged at 18,000 g for 2 min at RT. 200 μL was transferred to an HPLC vial for analysis.

[0446] HPLC analysis The following HPLC tests were performed:

[0447] Device

[0448]

[0449] Instrument conditions

[0450] Column temperature 55℃ Detection UV 205nm; BW 400nm CAD Inspection Analysis duration 15 minutes Injected volume 10μL Flow rate 1mL / min

[0451] Moving phase gradient scheme

[0452] Time (minutes) Water containing 0.04% acetic acid Methanol % 0 40 60 8 25 75 10 25 75 11 40 60 15 40 60

[0453] The following provides the HPLC test results and displays them. Figure 53a -e in:

[0454]

[0455] The enzyme S115N05 A7 exhibits the highest activity (approximately 22.4%) for the conversion of Reb A to Reb D.

[0456] At least three enzymes produced significant amounts of the unknown glycoside (labeled Reb UNK; later identified as reb D2) along with reb D.

[0457] Example 21

[0458] Activity of EUGT11 produced in vitro

[0459] The EUGT11 gene described in patent application WO / 2013 / 022989A2 was synthesized using DNA2.0 and subsequently subcloned in the pET30a+ vector.

[0460]

[0461] The amino acid sequence is as follows:

[0462] >gi|41469452|gb|AAS07253.1|Proposed UDP-glucuronide and UDP-glucosyltransferase [Oryza sativa Japonica Group] EUGT11 enzyme from patent application WO / 2013 / 022989A2

[0463]

[0464] The plasmids to be tested are received in a microtiter plate containing the plasmids, the plasmids being lyophilized solids in each separate well.

[0465] Plasmid suspension Add 24 μL of ultrapure sterile water to each well and shake the microtiter plate at room temperature for 30 minutes. Then, incubate the plate at 4°C for 1 hour. Further mix the contents of each well by pipetting up and down. Quantify the plasmid using 1 μL of the suspension via Qubit 2.0 analysis. The determined plasmid concentration is:

[0466] Microtiter plate Location Internal reference of the experiment [Plasmid] ng / μL C912666 G4 S115N08 G4 19.2

[0467] Transform competent cells with plasmids Remove aliquots of chemicompetent EC100 cells from a -80°C freezer and store on ice. Thaw the cells on ice for 10 minutes. Add 10 μL of the above plasmid solution dilution to a 1.5 mL sterile microtube (enough to transform each cell with 50 pg DNA) and store on ice. Add 100 μL of chemicompetent cells to each microtube. After incubating the chemicompetent cell / plasmid mixture on ice for 20 minutes, perform a heat shock at 42°C for 30 seconds.

[0468] Incubate on ice for another 2 minutes. Add 300 μL of SOC medium to each microtube and transfer the resulting mixture to a sterile 15 mL test tube. Incubate at 37°C for 1 hour with shaking at 135 rpm, then spread the mixture onto solid Luria broth containing 50 μg / mL kanamycin. Incubate the Piper dishes at 37°C for 16 hours.

[0469] Preparation of stock solution in glycerol and purification of plasmidsAdd 10 mL of Luria broth containing 50 μg / mL kanamycin to a 50 mL sterile Falcon tube. Inoculate the culture medium with clones isolated from the above Piper dishes and incubate the culture at 37°C for 16 hours with shaking at 135 rpm.

[0470] 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.

[0471] The remaining culture was centrifuged at 5,525 g for 10 minutes at 10°C, and the supernatant was removed. The precipitate was stored on ice. The resulting plasmid was purified using 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 plasmid content was determined as follows:

[0472] Microtiter plate Location Internal reference for testing [Plasmid] ng / μL C912666 G4 S115N08 G4 38.4

[0473] In vitro expression of EUGT11 According to the Promega S30 T7 high-yield protein expression system kit (reference: L1110), 18 μL of diluted plasmid solution (containing approximately 1.5 μg of plasmid) was used for in vitro expression. The expression medium was generated as follows:

[0474]

[0475] The prepared expression medium mixture was added to the plasmid solution and incubated at 30°C for 3 hours, mixing the mixture every 45 minutes. 5 μL of the mixture was frozen, while the remainder was used for a catalytic assay targeting the conversion of rebaudioside A to rebaudioside D.

[0476] Catalytic assay for the conversion of rebaudioside A to rebaudioside D Add 430 μL of a reaction mixture containing 0.5 mM rebaudioside A, 3 mM MgCl2, 50 mM phosphate buffer (pH 7.2), and 2.5 mM UDP-glucose to a 1.5 mL sterile microtube. Add 52 μL of enzyme expression medium and incubate the mixture at 30 °C for 24 h. After 2 h, 16 h, and 24 h, remove 125 μL of sample and add 115 μL of 60% methanol and 10 μL of 2N H2SO4. Centrifuge the quenched sample at 18,000 g for 2 min at RT. Transfer 200 μL to an HPLC vial for analysis.

[0477] HPLC analysis Perform HPLC testing as described in Example 20.

[0478] HPLC test results showed Figure 54 middle.

[0479] Example 22

[0480] In vivo production of enzymes

[0481] The enzyme described in Example 20 was produced in vivo.

[0482] The pET30A+ vector containing the gene corresponding to the enzyme was introduced into *E. coli* BL21(DE3) via heat shock. Cells were grown in Piper dishes in the presence of kanamycin, and suitable clones were selected for growth in liquid LB medium (Eragonal flasks). Glycerol was added to the suspension as a cryoprotectant, and 400 μL aliquots were stored at -20°C and -80°C.

[0483] A storage 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). The culture was incubated at 135 rpm and 30°C with shaking for 8 hours.

[0484] The production medium contained 60 g / L overnight expression TB medium (Novagen), 10 g / L glycerol, and 50 mg / L kanamycin. The pre-culture was added to 400 mL of this medium, and the solution was stirred at 20°C while sampling to measure OD and pH. The culture showed significant growth and good OD. After 40 hours, cells were collected by centrifugation and frozen. The following refers to the cell wet weight (CWW) yield.

[0485] 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

[0486] The lysis was carried out by adding Bugbuster Master mixture (Novagen), the lysis products were collected by centrifugation and used fresh.

[0487] Activity determination Using 0.5 mM substrate, 2.5 mM UDP-glucose, and 3 mM MgCl2 in 50 mM pH 7.2 sodium phosphate buffer, 1,000 μL of thawed lysis product was used to convert rebaudioside A, and activity was tested on a 5 mL scale. Samples were taken and analyzed by HPLC.

[0488] HPLC analysis HPLC tests were performed as described in Example 20.

[0489] The results for different enzymes are provided below and shown in Figure 55a -e.

[0490] GI number Version Transformation 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

[0491] Example 23

[0492] Identification of Glycosides

[0493] The reaction mixtures representative of GI No.460409128, particularly the sample "12400S115N05A7 T24h 130627ABA" of Example 20 (hereinafter referred to as S115N05A7) and the sample "12400S129N04 T45h 130712ABA" of Example 22 (hereinafter referred to as S129N04), were additionally tested by LC-MS to identify the unknown glycosides. An Agilent 1200 series HPLC system was used, which was equipped with a binary pump (G1312B), an autosampler (G1367D), a thermostatted column compartment (G1316B), a DAD detector (C1315C), connected to an Agilent 6110A MSD, and was coupled with the "LC / MSD Chemstation" software.

[0494] Instrument conditions

[0495]

[0496] Moving phase gradient scheme

[0497] Time (minutes) A (%): Formic acid 0.1% B (%): Acetonitrile 0 75 25 8.5 75 25 10.0 71 29 16.5 70 30

[0498] The compounds observed on the LCMS system at 3.5 minutes corresponded to the compound "unknown @4.508" in the sample "S115N05A7" (Example 20) and the compound "unknown @RT4.526" in the sample "S129N04" (Example 22). The LCMS data indicated that this compound had six glucoside residues (C 56 H 90 O 33 ) in its structure and was found to be an isomer of reb M, named reb M2 (for discussion, see Example 40).

[0499] The compounds observed on LCMS at 7.6 minutes correspond to "reb UNK" in sample "S115N05A7" (Example 20) and compound "reb UNK" in sample "S129N04" (Example 22). LCMS data indicate that "rebUNK" has five glucoside residues (C50, C60, C7 ... 50 H 80 O 28 Furthermore, it was discovered to be an isomer of reb D, named rebD2 (see Example 39 for discussion). The proportions of these compounds and LCMS chromatograms are provided below.

[0500]

[0501] Example 24

[0502] Identification of glycosides

[0503] Along with the stevia leaf extract “MLD1” produced by PureCircle Sdn Bhd (Malaysia), the reaction mixture representing GI No. 460409128, particularly the sample “12400S129N04T45h 130712ABA” (hereinafter referred to as S129N04) of Example 22, was further tested by LC-MS to determine the naturally occurring S129N04 glycosides.

[0504] Figure 57a The tests in -b indicate that the compound (C) observed in Example 23 on a 3.5-minute LCMS system... 56 H 90 O 33 ; later confirmed to be reb M2) and the compound observed on the LCMS system in Example 23 at 7.6 minutes (C 50 H 80 O 28 ;reb UNK; later confirmed to be reb D2) appears in stevia plant extracts.

[0505] Example 25

[0506] Rebaddiglycoside E is converted to rebaddiglycoside D

[0507] 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 lysed product of UGT76G1 (the UGT76G1 gene was cloned in the pET30a+ vector and expressed in *E. coli* BL21(DE3)). The reaction was run at 30 °C on an orbital oscillator 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 kept at 10 °C before analysis by HPLC (CAD detection). Figure 58 The reaction characteristics shown correspond to the complete conversion of rebaudioside E to rebaudioside D.

[0508] Example 26

[0509] Directed evolution of UGT76G1 for converting rebaudioside D to rebaudioside M

[0510] Starting with the amino acid sequence of UGT76G1 described in GenBank (AAR06912.1), different mutations at various amino acid positions were identified, which could alter the activity of the enzyme that converts rebaudioside D (Reb D) to rebaudioside M (Reb M). This was achieved through DNA2.0 ProteinGPS. TM A mutation list designed using the strategy was then used to synthesize 96 variant genes containing 3, 4, or 5 of these mutations, which were codon-optimized for expression in *E. coli*. The genes were subcloned in the pET30a+ plasmid and used for transformation of *E. coli* BL21(DE3) chemocompetent cells. The resulting cells were grown in solid LB medium in Piper dishes in the presence of kanamycin. Suitable clones were selected and grown in liquid LB medium in test tubes. Glycerol was added to the suspension as a cryoprotectant, and 400 μL aliquots were stored at -20°C and -80°C.

[0511] These stored 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). The culture was incubated at 135 rpm and 30 °C with shaking in 96 μL titration plates for 8 hours.

[0512] Inoculate 3.95 mL of Overnight Express solution with 50 μL of the above culture. TM Prepared TB culture medium Production medium containing 10 g / L glycerol and 50 mg / L kanamycin. The resulting culture was stirred at 20°C in 48-well plates. The culture showed significant growth and achieved a good OD (600 nm; 1 cm). After 44 hours, cells were collected by centrifugation and frozen.

[0513] By Master Mixture Lysis was performed by adding the lysed cells, and the lysate was collected by centrifugation. Activity was tested using 100 μL of fresh lysate, which was added to a solution of lebodiin 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).

[0514] The reaction was run at 30°C, and samples were taken at 2, 4, 7, and 24 hours. The conversion and initial rates were determined by HPLC (CAD detection) using the analytical method described above for the conversion of rebaudioside D to rebaudioside M. The results are shown in the table below.

[0515]

[0516]

[0517]

[0518] *Mutations are annotated as follows: Initial amino acid-position-new amino acid: For example, the mutation of alanine at position 33 to glycine is annotated as A33G.

[0519] Example 27

[0520] UGTSL2 produced in vivo

[0521] UGTSL2 (GI_460410132 / XP_004250485.1) amino acid sequence:

[0522]

[0523] The pET30A+ vector containing the UGTSL2 gene was introduced into Escherichia coli Bl21(DE3) by heat shock. The obtained cells were grown on Petri dishes in the presence of kanamycin, and appropriate clones were selected and grown in liquid LBG medium (conical flask). Glycerol, which serves as a cryoprotectant, was added to the suspension and 400 μL aliquots were stored at -20 °C and -80 °C.

[0524] A stored aliquot of Escherichia 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 shaken at 135 rpm and 30 °C for 8 hours.

[0525] The production medium contained 60 g / L of pre-prepared TB medium (Novagen) for overnight expression, 10 g / L glycerol and 50 mg / L kanamycin. The pre-culture was added to 200 mL of this medium, and the solution was stirred at 20 °C while samples were taken to measure OD and pH. The culture showed significant growth and a good OD was obtained. After 40 hours, the cells were collected by centrifugation and frozen, obtaining 6.22 g of wet cell weight.

[0526] 1.4 g of cells were lysed by adding Bugbuster Master Mix (Novagen), and the lysate was collected by centrifugation and used fresh.

[0527] Example 28

[0528] The determination of the activity of converting stevioside to rebaudioside E using UGTSL and UGTSL2. UGTSL was prepared according to Example 22 and UGTSL2 was prepared according to Example 27.

[0529] Using 0.5 mM substrate, 2.5 mM UDP-glucose and 3 mM MgCl2 in 50 mM sodium phosphate buffer pH 7.2, 600 μL of the lysate was used for the conversion of stevioside, and the activity test was carried out on a 3 mL scale. Samples were taken and analyzed by HPLC.

[0530] HPLC analysis. The HPLC test was carried out as described in Example 20.

[0531] The results for different enzymes and the corresponding chromatograms are provided below and shown in Figure 59a -b.

[0532]

[0533] Note: 1 Based on the initial concentration of steviolbioside

[0534] Example 29

[0535] Determination of the activity of converting rubusoside to rebaudioside E using UGTSL and UGTSL2

[0536] UGTSL was prepared according to Example 22, and UGTSL2 was prepared according to Example 27.

[0537] Using 0.5 mM substrate, 2.5 mM UDP - glucose and 3 mM MgCl2 in 50 mM sodium phosphate buffer at pH 7.2, 600 μL of the lysate was used for the conversion of rubusoside, and the activity test was carried out on a 3 mL scale. Samples were taken and analyzed by HPLC. The HPLC test was carried out as described in Example 20.

[0538] The following provides the results for different enzymes and the corresponding chromatograms and are shown in Figure 60a -b.

[0539]

[0540] Note: 1 Based on the initial concentration of rubusoside

[0541] Example 30

[0542] Determination of the activity of converting rebaudioside A to rebaudioside D using UGTSL2

[0543] UGTSL2 was prepared according to Example 27.

[0544] Using 0.5 mM substrate, 2.5 mM UDP - glucose and 3 mM MgCl2 in 50 mM sodium phosphate buffer at pH 7.2, 60 μL of the lysate was used for the conversion of rebaudioside A, and the activity test was carried out on a 3 mL scale. Samples were taken and analyzed by HPLC. The HPLC test was carried out as described in Example 20.

[0545] The following provides the results after a 23 - hour reaction and the corresponding chromatograms and are shown in Figure 61 -c.

[0546]

[0547] Note: 1 Based on the initial concentration of rebaudioside A

[0548] Example 31

[0549] Identification of Hedyotis diffusa

[0550] The presence of naturally formed glycosides was determined by LC-MS analysis of 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).

[0551] An Agilent 1200 series HPLC system was used, which is equipped with a dual pump (G1312B), an autosampler (G1367D), a temperature-controlled column (G1316B), and a DAD detector (G1315C), connected to an Agilent 6110A MSD and used with the “LC / MSDChemstation” software.

[0552] Instrument conditions

[0553]

[0554] Moving phase gradient scheme

[0555] Time (minutes) A (%): Formic acid 0.1% B (%): Acetonitrile 0 75 25 30 75 25 33 68 32 75 68 32

[0556] Figure 62 The tests shown indicate that the compound observed on the 11.77-minute LC-MS system is identical to the compound observed on the 3.5-minute system in Example 23 (C). 56 H 90 O 33 ; later confirmed to be reb M2), the compound observed at 26.64 minutes was the same as the compound observed at 7.6 minutes in Example 23 (C 50 H 80 O 28 (reb UNK; later confirmed to be reb D2). Other isomers of reb X were observed at 13.96 minutes, and another isomer of reb D was observed at 25.06 minutes. All observed compounds were present in the stevia extract.

[0557] Example 32

[0558] In vivo preparation and activity assay of UGTLB

[0559] UGTLB(GI_209954733 / BAG80557.1) amino acid sequence

[0560]

[0561] The pET30A+ vector containing the UGTLB gene was introduced into *E. coli* Bl21(DE3) via heat shock. Cells were grown in Piper dishes in the presence of kanamycin, and suitable clones were selected for growth in liquid LB medium (Eragonal flasks). Glycerol was added to the suspension as a cryoprotectant, and 400 μL aliquots were stored at -20°C and -80°C.

[0562] A storage 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). The culture was incubated at 135 rpm and 30°C with shaking for 8 hours.

[0563] The production medium contained 60 g / L overnight expression TB medium (Novagen), 10 g / L glycerol, and 50 mg / L kanamycin. The pre-culture was added to 200 mL of this medium, and the solution was stirred at 20°C while OD and pH were measured. The culture showed significant growth and good OD. After 40 hours, cells were collected by centrifugation and frozen to obtain a wet cell weight of 5.7 g.

[0564] 1.2 g of cells were lysed by adding 6 mL of Bugbuster Master mixture (Novagen), and the lysate was collected by centrifugation and used fresh.

[0565] Determination of the activity of converting hedyotis diffusa glycoside to lebodiin E using UGTLB

[0566] Using 0.5 mM substrate, 2.5 mM UDP-glucose, and 3 mM MgCl2 in 50 mM pH 7.2 sodium phosphate buffer, 600 μL of the lysis product was used for the conversion of hedyotis diffusa glycosides, and activity tests were performed on a 3 mL scale. Samples were taken and analyzed by HPLC. The corresponding chromatograms were plotted on... Figure 63a middle.

[0567]

[0568] Note: 1 Based on the initial concentration of hedyotis diffusin

[0569] Determination of the activity of raspberry glycoside to lebodiin E by UGTLB

[0570] Using 0.5 mM substrate, 2.5 mM UDP-glucose, and 3 mM MgCl2 in 50 mM pH 7.2 sodium phosphate buffer, 600 μL of the lysis product was used for the conversion of raspberry glycosides, and activity tests were performed on a 3 mL scale. Samples were taken and analyzed by HPLC. The corresponding chromatograms were plotted on... Figure 63b middle.

[0571]

[0572] Note: 1 Based on the initial concentration of raspberry glycoside

[0573] Determination of the activity of rebaudioside A to rebaudioside D by UGTLB

[0574] Using 0.5 mM substrate, 2.5 mM UDP-glucose, and 3 mM MgCl2 in 50 mM pH 7.2 sodium phosphate buffer, 600 μL of the lysis product was used for the conversion of rebaudioside A, and activity tests were performed on a 3 mL scale. Samples were taken and analyzed by HPLC. The corresponding chromatograms after 23 hours of reaction were plotted on... Figure 63c middle.

[0575]

[0576] Note: 1 Based on the initial concentration of lebodiin A

[0577] Example 33

[0578] Determination of reaction products of raspberry and calendula glycoside conversion using UGTSL, UGTSL2 and UGTLB

[0579] The conversion of hedyotis diffusin with UGTSL and UGTSL2 was performed in a manner similar to that in Example 28, and the conversion of scutellarin with UGTSL and UGTSL2 was performed in a manner similar to that in Example 29. The conversion of scutellarin with UGTSL and hedyotis diffusin with UGTSLB was performed in a manner similar to that in Example 32.

[0580] The reaction mixture was analyzed by LCMS to determine all reaction products.

[0581] Rubus glycoside conversion products

[0582]

[0583] Hedyotis diffusa glycoside conversion products

[0584]

[0585] As can be seen from the raspberry glycoside conversion products, besides rutin, Reb E, and Reb D, there are at least three other compounds with a molecular weight of 804. The residence times of these compounds do not match those of Reb B, which is known to have the same molecular weight as rutin. Because these compounds have the same molecular weight as rutin, these new steviol glycosides can be considered isomers of rutin. On the other hand, in the rutin conversion products, besides Reb E and Reb D, there are at least three other compounds with a molecular weight of 966. The residence times of these compounds do not match those of Reb A, which is known to have the same molecular weight as Reb E. Because these compounds have the same molecular weight as Reb A and Reb E, these new steviol glycosides can be considered isomers of Reb A (Reb E).

[0586] Example 34

[0587] In vivo production of UGT76G1 in Saccharomyces cerevisiae

[0588] UGT76G1[Stevia](gi_37993653 / gb_AAR06912.1)

[0589]

[0590] The above amino acid sequence was codon-optimized for expression in *Saccharomyces cerevisiae*. Furthermore, the yeast-common sequence AACACA was added before the ATG start codon. The gene was subcloned and synthesized in 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).

[0591] Cells were grown on solid synthetic minimal medium (SMR) containing 2% glucose and lacking uracil, and single clones were selected and grown in liquid synthetic minimal medium (SC-U containing 2% glucose) lacking uracil. After centrifugation, the cells were resuspended in SC-U (containing 2% glucose) and 60% glycerol / water. Aliquots were stored at -80°C, and one aliquot was used to initiate a culture in SC-U (containing 2% glucose) at 30°C for 43 hours. A portion of this culture was centrifuged and resuspended in incubation medium (SC-U containing 2% galactose) at 30°C for 19 hours and 30 minutes.

[0592] Cells were obtained by centrifugation and analyzed using five volumes of CelLytic... TM Y-cell lysis reagent (Sigma) was used. The lysis products were then used directly for activity testing (UGT76G1_Sc).

[0593] Example 35

[0594] UGT76G1_Sc is used for the determination of the activity of rebaudioside D to rebaudioside M.

[0595] UGT76G1_Sc was prepared according to Example 34.

[0596] Using 0.5 mM substrate, 2.5 mM UDP-glucose, and 3 mM MgCl2 in 50 mM pH 7.2 sodium phosphate buffer, and with 200 μL of lysis product, rebaudioside D was converted, and activity was tested on a 2 mL scale. Samples were taken and analyzed by HPLC. The corresponding chromatograms were plotted on... Figure 64 middle.

[0597] Enzyme internal reference <![CDATA[Rebaudioside D conversion 1 (Reaction time)]]> <![CDATA[Rebaudioside M selectivity 1 > UGT76G1_Sc 85% (21 hours) 100％

[0598] Note: 1 Based on the initial concentration of lebodiin D

[0599] Example 36

[0600] UGTSL produced in Saccharomyces cerevisiae

[0601] UGTSL[Tomato](gi_460409128 / XP_004249992.1)

[0602]

[0603] The above amino acid sequence was codon-optimized for expression in *Saccharomyces cerevisiae*. Furthermore, the yeast-common sequence AACACA was added before the ATG start codon. The gene was subcloned and synthesized in the pYES2 vector using Hind III and Xba I restriction sites. The pYES2_UGTSL_Sc vector was then used to transform chemically competent *Saccharomyces cerevisiae* INVSC1 cells (Invitrogen).

[0604] Cells were grown on solid synthetic minimal medium (SMR) containing 2% glucose and lacking uracil, and single clones were selected and grown in liquid synthetic minimal medium (SC-U containing 2% glucose) lacking uracil. After centrifugation, the cells were resuspended in SC-U (containing 2% glucose) and 60% glycerol / water. Aliquots were stored at -80°C, and one aliquot was used to initiate a culture in SC-U (containing 2% glucose) at 30°C for 43 hours. A portion of this culture was centrifuged and resuspended in incubation medium (SC-U containing 2% galactose) at 30°C for 19 hours and 30 minutes.

[0605] Cells were obtained by centrifugation and analyzed using five volumes of CelLytic... TM Y-cell lysis reagent (Sigma) was used. The lysis products were then used directly for activity testing (UGTSL_Sc).

[0606] Example 37

[0607] UGTSL_Sc is used to determine the activity of rebaudioside A to rebaudioside D.

[0608] UGTSL_Sc was prepared according to Example 36. Using 0.5 mM substrate, 2.5 mM UDP-glucose, and 3 mM MgCl2 in 50 mM sodium phosphate buffer (pH 7.2), 200 μL of the lysis product was used for the conversion of rebaudioside A, and activity was tested on a 2 mL scale. Samples were taken and analyzed by HPLC. The corresponding chromatograms were plotted on... Figure 65 middle.

[0609] Enzyme internal reference <![CDATA[Rebaudioside A conversion 1 (Reaction time)]]> <![CDATA[Rebaudioside D selectivity 1 > UGTSL_Sc 46％(4h.) 42％

[0610] Note: 1 Based on the initial concentration of lebodiin A

[0611] Example 38

[0612] Isolation of Lebodiin M

[0613] The amount of the product mixture in Example 14 was insufficient for separation by preparative HPLC. Therefore, a series of injection analytical HPLC analyses were used to separate the components of the mixture. Separation was performed according to the method described in Example 14 above to provide a product corresponding to... Figure 5 The two main peaks in the HPLC traces are fraction A (residence time 24.165 min) and fraction B (residence time 31.325 min).

[0614] The residence time of fraction A is consistent with that of reb D, indicating that it comes from unreacted starting material from the biotransformation reaction.

[0615] Residence time of purified fraction B ( Figure 6 Consistent with reb M, this indicates successful biotransformation from reb D. The purified fraction B was co-injected with the reb M standard (obtained from PureCircle). Figure 7 The HPLC traces of the reb M standard shown in the image were used to confirm the nature of the material collected as reb M in fraction B. It was found that fraction B and the reb M standard eluted at the same residence time. Figure 8 ), indicating that the level B is reb M.

[0616] The properties of fraction B as reb M were also separately confirmed by NMR and HRMS. For sampling, fraction B was concentrated in a rotary evaporator, freeze-dried, and then dried at 40°C for 40 hours.

[0617] The NMR sample was dissolved in deuterated pyridine (C5D5N) and mass spectra were acquired using a Varian Unity Plus 600MHz instrument with standard pulse sequence. The NMR spectrum of fraction B was compared with the NMR spectrum of reb M. The overlap of the two spectra ( Figure 9 The peaks of fraction B show good agreement with those of reb M. The following is a table showing the NMR distribution of reb M:

[0618] C5D5N a-c Zhonglaibaodi glycoside M 1 H and 13 C NMR spectral data

[0619]

[0620]

[0621]

[0622] a Allocation is based on COSY, HMQC, and HMBC associations; b Chemical shift values ​​are expressed as δ (ppm); c The coupling constant is expressed in Hz.

[0623] HRMS was generated using a Waters Premier quadrupole time-of-flight (Q-TOF) mass spectrometer equipped with an electrospray ionization source operating in positive ion mode. Figure 10 The sample was dissolved in methanol and eluted in a 2:2:1 methanol:acetonitrile:water solution, and then introduced via infusion using an in-chamber syringe pump. [M+Na] was measured at m / z 1313.5265. + The adduct confirmed the existence of reb M, which corresponds to C. 56 H 90 O 33 The molecular formula.

[0624]

[0625] Rebadinoside D Rebadinoside M

[0626] Chemical formula: C 50 H 80 O 28 Chemical formula: C 56 H 90 O 33

[0627] Molecular weight: 1128 Molecular weight: 1290

[0628] Example 39

[0629] Isolation and characterization of Reb D2

[0630] crude reaction sample According to Example 22, a sample of batch number CB-2977-106 for separation was prepared using UGTSL (GI#460409128).

[0631] HPLC analysis Preliminary HPLC analysis was performed using a Waters 2695 Alliance system with the following methods: Phenomenex 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 performed by UV (210 nm) and CAD.

[0632] gradient:

[0633] Time (minutes) ％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

[0634] The semi-preparative purified fraction was analyzed using the following method: 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 using CAD.

[0635] gradient:

[0636] Time (minutes) ％A ％B 0.0-5.0 100 0 20 20 80 25 20 80 30 100 0

[0637] LC-MSPreliminary analysis of the semi-synthetic steviol glycoside mixture was performed using a Waters 3100 mass analyzer operating in negative ion mode on a Waters AutoPurification HPLC / MS system. The samples were analyzed using the following methods: Phenomenex 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 are listed above.

[0638] Separation by HPLC Purification was performed in two steps. The first method used 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 loading: 160 mg dissolved in 20 mL of water. Detection was performed by UV (205 nm).

[0639] Time (minutes) ％A ％B 0.0-5.0 100 0 20 20 80 25 20 80 30 100 0

[0640] The second purification used the same column and conditions, but with an isocratic mobile phase: 20% MeCN in water.

[0641] Purification from natural extracts Purification was performed in three steps. The first method used for preparative purification is summarized below. Main procedure: Waters Symmetry C18, 50×250 mm The column was 7 μm (p / n WAT 248000); isocratic mobile phase: 50% methanol (MeOH) in water containing 0.05% HOAc; flow rate: 85 mL / min; injection loading: 6 g of crude extract dissolved in 50 mL of mobile phase. Detection was performed by UV (210 nm). After elution of the target analyte, the column was washed with 85% MeOH in water.

[0642] The second method: Waters Symmetry Shield RP18, 50×250mm, 7μm (p / n WAT248000); isocratic mobile phase: 20% MeCN in water; flow rate: 100mL / min; injection loading: 0.5g of the first fraction dissolved in 30mL of water. Detection was performed by UV (210nm).

[0643] The third method: Waters Symmetry Shield RP18, 50×250mm, 7μm (p / n WAT248000); isocratic mobile phase: 20% MeCN in water; flow rate: 100mL / min; injection loading: 0.5g of the second fraction dissolved in 30mL of water. Detection was performed by UV (210nm).

[0644] MS and MS / MS MS and MS / MS data were generated using a Waters QT of Premier mass spectrometer equipped with an electrospray ionization source. Samples were analyzed by negative ESI. Samples were diluted 50-fold with H₂O:acetonitrile (1:1) and introduced via infusion using an in-chamber syringe pump. Samples were diluted to produce a good S / N, which appeared at an appropriate concentration of 0.01 mg / mL.

[0645] 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 inverse detection probe. 1 1H NMR spectrum referenced residual solvent signal (for pyridine-d5, δ H 8.74 and δ C 150.35).

[0646] Results and discussion

[0647] Separation and purification The steviol glycoside mixture of batch number CB-2977-106, prepared according to Example 22 using UGTSL (GI#460409128), was separated. The material was analyzed by LC-MS using the method described above, and the results are provided. Figure 11 The target peak of interest is at 7.7 min in the TIC chromatogram. The mass spectrum of this peak provides [MH] at m / z 1127.6. - The provided sample was initially prepared using the first method conditions described above with a single injection (160 mg). This method separates the material into a mixture of “polar” and “nonpolar” glycosides. The “polar” mixture was then processed again using the second step conditions described above. Semi-preparative HPLC traces are provided. Figure 12 From this semi-preparative set, compounds with purities >99% (CAD, AUC) were isolated. Fractional analysis was provided. Figure 13 After purification, the combined fractions were concentrated by rotary evaporation at 35°C and then lyophilized. Approximately 1–2 mg was obtained for characterization.

[0648] mass spectrometryThe ESI-TOF mass spectra obtained by perfusion of the sample showed a [MH] value of m / z 1127.4709. - [MH] - The mass of an ion and its molecular formula C 50 H 80 O 28 Very consistent (calculated as C) 50 H 79 O 28 :1127.4758, error: -4.3ppm). MS data confirmed the presence of the molecular formula C 50 H 80 O 28 The nominal mass is 1128 Daltons.

[0649] MS / MS spectrum (select [MH] at m / z 1127.5) - The ions (for splitting) indicate the loss of two glucose monomers and the sequential loss of three glucose moieties at m / z 641.3187, 479.2655 and 317.2065.

[0650] NMR spectroscopy A series of actions were carried out, including... 1 H NMR ( Figure 14 ), 13 C NMR ( Figure 15 and 16 ), 1 H- 1 H COSY( Figure 17 ), HSQC-DEPT Figure 18 ), HMBC ( Figure 19 and 20 NMR experiments with 1D-TOCSY were performed to allow for the partitioning of the compounds.

[0651] 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 nucleus of the glycoside is a diterpenoid. 1 H and 1 H- 13 The presence of five angioprotons observed in the C HSQC-DEPT spectrum confirms the presence of five sugar monomers in the structure. 1 H- 13 δ in C HSQC-DEPT spectrum C Methylene at 69.9 13C-resonance indicates the presence of 1→6 sugar bonds in the structure. Using 1 H- 13 C HMBC and 1D-TOCSY correlate the allocation of bonds to sugar monomers.

[0652] From δ H 1.29 methyl protons and δ C The HMBC association of the carbonyl group at 177.7 allows for the allocation of one of the tertiary methyl groups (C-18) and C-19 and provides a starting point for the allocation of the remaining glycosidic ligand. The methyl proton (H-18) and δ C Other HMBC associations of carbon at 38.9, 45.0, and 57.8 allow for the allocation of C-3, C-4, and C-5. 1 H- 13 Analysis of C HSQC-DEPT data indicates that δ C The carbon at 38.9 is a methylene group, while δ C Carbon 57.8 is a methine, which is assigned to C-3 and C-5. The remaining δ C The carbon at 45.0, which showed no correlation in the HSQC-DEPT spectrum, was assigned as a quaternary carbon, C-4. C-3 (δ) was assigned using HSQC-DEPT data. 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 δ H The COSY correlation between protons at 1.43 allows for the allocation of one of the H-2 protons, which in turn exhibits a correlation with the δ proton allocated as C-1. H The correlation of 0.75 protons was then used to allocate the remaining protons for C-1 and C-2 based on other COSY and HSQC-DEPT correlations. 1 H and 13 C chemical shifts are summarized in the table below.

[0653] 1 H and 13 Partitioning of C10 NMR (500 and 125 MHz, pyridine-d5), Reb D2

[0654]

[0655]

[0656] In δ H Other tertiary methyl singlets observed at 1.30 showed association with HMBCs at C-1 and C-5 and were assigned to C-20. The methyl protons showed association with quaternary carbons (δ¹⁰). C40.3) and methylene carbon (δ C 54.5 (which were assigned as C-10 and C-9 respectively) additional HMBC associations. H-5 (δ H 1.04) and δ H The COSY correlation between 1.92 and 2.43 subsequently allowed for the allocation of H-6 protons, which in turn showed a correlation with the allocation of δ protons as C-7. H The correlation between protons at 1.22 and 1.30 was then determined from HSQC-DEPT data for C-6 (δ C 22.7) and C-7(δ) C 42.2) 13 C chemical shift. H-9(δ) H 0.88) and δ H The COSY correlation between protons of 1.65 and 1.69 allows for the allocation of H-11 protons, which in turn exhibits a δ correlation with the allocation of H-12 protons. H COSY correlations for protons at 1.99 and 2.25. HSQC-DEPT data were then used to assign C-11 (δ... C 21.1) and C-12 (δ C 37.5). From H-12 protons (δ) H 2.25) and δ C The HMBC associations of carbons at 87.1 and 154.7 allow for the allocation of C-13 and C-16, respectively. (In δ...) H The olefin protons observed at 5.01 and 5.64 showed an association with the HMBC at C-13 and were assigned to C-17 (via HSQC-DEPT, δ C 105.2). The olefin proton H-17 and methine proton H-9 show a δ-symmetric relationship with the partition of C-15. C HMBC association of 48.3% of carbon. From H-9 and δ C The additional HMBC association of the 44.5 methylene carbon subsequently allowed for the allocation of C-14. C-14 (δ) was allocated using HSQC-DEPT data. H 1.80 and 2.65) and C-15 (δ H 1.31 and 2.04) 1 H chemical shift.

[0657] The following provides key HMBC and COSY associations for allocating glycoside ligand regions:

[0658]

[0659] 1 H- 13Analysis of the C HSQC-DEPT data confirmed the presence of five anomeric protons. Three of the anomeric protons were in... 1 δ in HNMR spectrum H 6.02(δ C 96.1), 5.57 (δ) C 105.3) and 5.34 (δ) C At 105.3), it was sufficiently distinguishable. Through 1 H- 13 C HSQC-DEPT data identified the δ H 5.04(δ C 105.6) and 5.07 (δ) C The remaining two anomeric protons observed in 98.7) 1 The 1H spectrum was masked by solvent (HOD) resonance. In the δ spectrum... H The observed anomaly protons at 6.02 showed an association with the HMBC of C-19, suggesting that it corresponds to Glc. I Anisotropic protons. Similarly, in δ H 5.07 Anomalies were observed to show HMBC association with C-13, allowing them to be assigned as Glc. II Anisotropic protons.

[0660] Glc I Anisotropic protons (δ) H 6.02) shows the difference between being assigned as Glc I δ of H-2 H COSY correlation of protons at 4.07, Glc I H-2 then showed a similarity to δ H 4.22 (Glc) I COSY correlation of H-3) protons, Glc I H-3 showed a correlation with δ H 4.12 (Glc) I The COSY correlation of H-4 protons was used. Due to data overlap, the COSY spectrum did not allow for the partitioning of H-5 or H-6 protons. Therefore, several different mixing times were used, with Glc... I Selective irradiation with anomeric protons was performed, followed by a series of 1D-TOCSY experiments. These experiments confirmed the effectiveness of targeting Glc... I The allocation from H-2 to H-4, as shown by 1D-TOCSY data, is Glc. I H-5's δ H 4.04 protons and their distribution as Glc I One of the H-6 protons, δ H4.68 protons. The latter proton was also used in the 1D-TOCSY experiment. Selective irradiation with H-6 using several different mixing times also confirmed the presence of Glc. I The allocation of H-1 to H-5 and H-6 (δ H The remaining methylene protons (4.30). Using 1 H- 13 C HSQC-DEPT data determined the efficacy of 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 The allocation of C chemical shifts to complete Glc I The allocation. Furthermore. 1 H- 13 δ in C HSQC-DEPT spectrum C 69.9% methylene 13 The presence of C resonance indicates the presence of Glc in the structure. I The 1→6 sugar bond.

[0661] Of the four remaining unallocated glucose fractions, one is based on 1 H- 13 C HSQC-DEPT, HMBC, and 1D-TOCSY associations were assigned as Glc I The C-6 substituent. δ in the HSQC-DEPT spectrum. C 69.9% methylene 13 The relative low-field displacement of C resonance indicates that Glc I The 1→6 sugar bond. δ H The anomeric protons observed at 5.04 showed similarities to Glc I C-6's HMBC is associated and assigned to Glc. V Anomalies. Similarly, Glc I The methylene proton indicates that it is related to Glc V The HMBC association of the anomeric carbon confirms that Glc I and Glc V The presence of 1→6 sugar bonds between Glc V Anisotope protons showed distribution as Glc V δ of H-2 H COSY correlation of protons at 4.00, Glc V H-2 then showed a similarity to δ H 4.22 (Glc)V COSY correlation of protons (H-3). Due to data overlap, the COSY spectrum did not allow for Glc-based correlation. V H-3 COSY association to allocate Glc V H-4. However, in the HMBC spectrum, Glc V H-3 shows similarities to Glc V C-5(δ C The association of 78.9). In HSQC-DEPT, Glc V C-5 shows a correlation with δ H 3.89 (Glc) V The association with H-5). Glc V H-5 showed a correlation with δ H COSY correlations at 4.21, 4.37, and 4.48. In the HSQC-DEPT spectrum, δ... H 4.21 shows the relationship with δ C 71.4 (Glc) V The correlation between H-4) and δ H 4.37 and 4.48 show correlation with δ C The association of 63.1 and the assignment of Glc respectively V H-6a and H-6b. Use 1 H- 13 C HSQC-DEPT data determined the target for Glc V C-2(δ C 75.7) and C-3(δ C 79.1) 13 The allocation of C chemical shifts to complete Glc V The allocation.

[0662] Glycosides targeting C-19 1 H and 13 An overview of C chemical shifts is shown in the table below:

[0663] 1 H and 13 Partitioning of C10 NMR (500 and 125 MHz, pyridine-d5), reb D2 C-19 glycosides

[0664]

[0665] *The anomeric protons were masked by solvent (HDO) resonance. Therefore, the coupling constant could not be determined.

[0666] #1 H and 13 The C value is at positions Glc1-3, Glc V -3 and GlcIV -3 can be interchanged.

[0667] The following provides an overview of the key HMBC, COSY, and 1D-TOCSY associations used for the allocation of C-19 glycosides.

[0668]

[0669] Glc was performed in a similar manner II The allocation of Glc. II Anisotropic protons (δ) H 5.07) shows the allocation as Glc II δ of H-2 H COSY correlation of protons at 4.37, Glc II H-2 then showed a similarity to δ H 4.18 (Glc) II The COSY correlation of the H-3) proton. This latter proton shows a correlation with δ. H 3.88 (Glc) II Other associations of the H-4 proton also show a correlation with δ H 3.79 (Glc) II COSY correlation of H-5 protons. Glc II The H-5 also showed similarities to Glc II H-6 protons (δ) H The COSY associations for Glc were 4.08 and 4.46. The results were determined using HSQC-DEPT data. 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 The distribution of C chemical shifts. From Glc II H-3 to C-2 and C-4, as well as from Glc II The HMBC associations from H-4 to C-2 and C-5 confirm the above allocations. II H-4 to Glc II Other HMBC associations of C-6 further support the completion of Glc II The allocation.

[0670] Based on HMBC association, the remaining two unallocated glucose fractions were allocated as Glc. II The substituents at C-2 and C-3. In δ HThe observed anisotopes at 5.57 show similarities to Glc. II C-2's HMBC association and was assigned as Glc III Anisotropic protons. In δ H 5.34 The observed anomeric protons showed similarities to Glc II C-3's HMBC association and assigned as Glc IV Anomalies were observed from Glc. II H-2 and Glc III Angiocarbons and those from Glc II H-3 and Glc IV The mutual HMBC correlation of the anterior carbons.

[0671] Glc III Anisotope (δ) H 5.57) shows the allocation as Glc III δ of H-2 H 4.19 COSY correlation of protons. Due to data overlap, the COSY spectrum did not allow for the partitioning of H-3 to H-6 protons. Therefore, several different mixing times were used, with Glc... III Selective irradiation with anomeric protons was performed, followed by a series of 1D-TOCSY experiments. These experiments confirmed the effectiveness of targeting Glc... III H-2 allocation, 1D-TOCSY data show δ H 4.24 (Glc) III H-3), δ H 4.27 (Glc) III H-4) and δ H 3.94 (Glc) III The protons of H-5). Once H-4 is assigned using 1D-TOCSY data, the COSY correlations from H-4 with H-5 and then with H-6 are used to assign H-6. In the COSY spectrum, Glc III H-4 showed similarities to Glc III The connection between H-5 and Glc, in turn, reveals their respective links. III δ of H-6a and H-6b H COSY associations in 4.41 and 4.50. Then use... 1 H- 13 C HSQC-DEPT association determination for 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 to complete Glc III The allocation.

[0672] Glc IV (δ H The anoprotons of 5.34) showed a distribution similar to that of Glc. IV δ of H-2 H The COSY correlation of protons was 4.06. Due to data overlap, the COSY spectrum did not allow for the partitioning of H-3 to H-6 protons. Therefore, several different mixing times were used, with Glc... IV Selective irradiation with anomeric protons was performed, followed by a series of 1D-TOCSY experiments. These experiments confirmed the effectiveness of targeting Glc... IV H-2 allocation, 1D-TOCSY data show δ H 4.22 (Glc) IV H-3), δ H 4.18 (Glc) IV H-4) and δ H 4.10 (Glc) IV The protons of H-5). Once H-4 is assigned using 1D-TOCSY data, the COSY correlations from H-4 with H-5 and then with H-6 are used to assign H-6. In the COSY spectrum, Glc IV H-4 showed similarities to Glc IV The connection between H-5 and Glc, in turn, reveals their respective links. IV δ of H-6a and H-6b H COSY associations for 4.32 and 4.58. Then use... 1 H- 13 C HSQC-DEPT association determination for 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 to complete Glc IV The allocation.

[0673] Targeting C-13 glycosides 1 H and 13 An overview of C chemical shifts is shown in the table below:

[0674] 1 H and 13Partitioning of Reb D2 C-13 glycosides by C NMR (500 and 125 MHz, pyridine-d5)

[0675]

[0676]

[0677] The following provides an overview of the key HMBC, COSY, and 1D-TOCSY associations used for the allocation of C-13 glycosides:

[0678]

[0679] NMR and MS analyses allowed for the complete allocation of the 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]equivalent-16-kaurene-19-acid-[(6-O-β-D-glucopyranosyl-β-D-glucopyranosyl)ester] (rebaudioside D2 or reb D2). The compound is an isomer of rebaudioside D.

[0680]

[0681] Example 40

[0682] Isolation and characterization of Reb M2

[0683] crude reaction sample According to Example 22, a sample of batch number CB-2977-106 for separation was prepared using UGTSL (GI#460409128).

[0684] HPLC analysis Preliminary HPLC analysis was performed using a Waters 2695 Alliance system with the following methods: Phenomenex 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 performed by UV (210 nm) and CAD.

[0685] gradient:

[0686] Time (minutes) ％A ％B 0.0-5.0 100 0 20 20 80 25 20 80 30 100 0

[0687] The semi-preparative purified fraction was analyzed using the following method: 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 using CAD.

[0688] gradient:

[0689] Time (minutes) ％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

[0690] LC-MS Preliminary analysis of the semi-synthetic steviol glycoside mixture was performed using a Waters 3100 mass analyzer operating in negative ion mode on a Waters AutoPurification HPLC / MS system. The samples were analyzed using the following methods: Phenomenex 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 are listed above.

[0691] Separation by HPLC Purification was performed in two steps. The first method used 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 loading: 160 mg dissolved in 20 mL of water. Detection was performed by UV (205 nm).

[0692] gradient:

[0693] Time (minutes) ％A ％B 0.0-5.0 100 0 20 20 80 25 20 80 30 100 0

[0694] The second purification used the same column and conditions, but with an isocratic mobile phase: 20% MeCN in water.

[0695] MS and MS / MSMS and MS / MS data were generated using a Waters QT of Premier mass spectrometer equipped with an electrospray ionization source. Samples were analyzed by negative ESI. Samples were diluted 50-fold with H₂O:MeCN (1:1) and introduced via infusion using an in-chamber syringe pump. Samples were diluted to produce a good S / N, which appeared at an appropriate concentration of 0.01 mg / mL.

[0696] NMR The sample was prepared by dissolving ~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 inverse detection probe. 1 H NMR and 13 The C NMR spectra were referenced to the residual solvent signal HDO(δ) H 4.79ppm) and TSP (δ C 0.00ppm).

[0697] Results and discussion

[0698] Separation and purification The mixture of steviol glycosides, batch number CB-2977-106, prepared according to Example 22 using UGTSL (GI#460409128), was separated. The above method was used ( Figure 11 The material was analyzed by LC-MS. The target peak of interest was observed at 4.1 min in the TIC chromatogram. The mass spectrum of this peak provided [MH] m / z 1289.7. - The sample provided was initially prepared using the first method conditions described above with a single injection (160 mg). This method separates the material into a mixture of “polar” and “nonpolar” glycosides. The “polar” mixture was then processed again using the second step conditions described above. Semi-preparative HPLC traces are shown. Figure 12 From this semi-preparative set, peaks with purity >99% were separated (CAD, AUC). Fractional analysis is provided... Figure 13 After purification, the combined fractions were concentrated by rotary evaporation at 35°C and then lyophilized. Approximately 1 mg was obtained.

[0699] mass spectrometry ESI-TOF mass spectra obtained by perfusing sample CC-00300 showed a [MH] value of m / z 1289.5266. - [MH] - The mass of the ion is similar to the expected molecular formula C of reb M2. 56 H 90 O 33 Very consistent (calculated as C) 56 H89 O 33 :1289.5286, error: -1.6ppm). MS data confirmed that CC-00300 has the molecular formula C 56 H 90 O 33 The nominal mass is 1290 Daltons.

[0700] MS / MS spectrum (select [MH] at m / z 1289.5) - The ions (for splitting) indicate the loss of three glucose monomers at m / z 803.3688 and the sequential loss of three glucose moieties at m / z 641.3165, 479.2633 and 317.2082.

[0701] NMR spectroscopy A series of actions were carried out, including... 1 H NMR ( Figure 21 ), 13 C NMR ( Figure 22 and 23 ), 1 H- 1 H COSY( Figure 24 ), HSQC-DEPT Figure 25 ), HMBC ( Figure 26 and 27 NMR experiments with 1D-TOCSY were conducted to allow for the distribution of reb M2.

[0702] 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 nucleus of the glycoside is a diterpenoid. 1 H and 1 H- 13 The presence of six angioprotons observed in the C HSQC-DEPT spectrum confirms the presence of six sugar monomers in the structure. 1 H- 13 δ in C HSQC-DEPT spectrum C Methylene at 70.9 13 C-resonance indicates the presence of 1→6 sugar bonds in the structure. Using 1 H- 13 C HMBC and 1D-TOCSY correlate the allocation of bonds to sugar monomers.

[0703] From δ H 1.29 methyl protons and δC The HMBC association of the carbonyl group at 181.5 allows for the allocation of one of the tertiary methyl groups (C-18) and C-19 and provides a starting point for the allocation of the remaining glycosidic ligand. The methyl proton (H-18) and δ C The additional HMBC associations of carbon at 39.8, 43.7, and 59.2 allow for the allocation of C-3, C-4, and C-5. 1 H and 1 H- 13 Analysis of C HSQC-DEPT data indicates that δ C The carbon at 39.8 is a methylene group, while δ C Carbon 59.2 is a methine, which is assigned to C-3 and C-5. The remaining δ C The carbon at 43.7, which showed no correlation in the HSQC-DEPT spectrum, was assigned as a quaternary carbon, C-4. C-3 (δ) was assigned using HSQC-DEPT data. 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 δ H The COSY correlation between protons at 1.49 allows for the allocation of one of the H-2 protons, which in turn exhibits a correlation with the δ proton allocated as C-1. H The proton correlation was 0.92. The remaining protons were then allocated for C-1 and C-2 based on other COSY and HSQC-DEPT correlations. 1 H and 13 C chemical shifts are summarized in the table below.

[0704] 1 H NMR (500MHz, D2O) and 13 C10 NMR (125 MHz, D2O / TSP), partitioning of Reb M2 glycoside ligands

[0705]

[0706]

[0707] In δ H Other tertiary methyl singlets observed at 0.92 showed association with HMBCs at C-1 and C-5 and were assigned to C-20. The methyl protons showed association with quaternary carbons (δ¹²). C 42.4) and methine (δ) C 55.5)(which are respectively assigned as C-10 and C-9) additional HMBC associations. H-5(δ H 1.24) and δ HThe COSY correlation between 1.73 and 1.94 subsequently allowed for the allocation of H-6 protons, which in turn showed a correlation with the allocation of δ protons as C-7. H The correlation between protons at 1.49 and 1.56 was then determined from HSQC-DEPT data for C-6 (δ C 24.4) and C-7 (δ) C 44.2) 13 C chemical shift. H-9(δ) H 1.09) and δ H The COSY correlation between protons at 1.66 and 1.70 allows for the allocation of H-11 protons, which in turn exhibits a δ correlation with the allocation of H-12 protons. H COSY correlations of 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). In δ H The olefin protons observed at 4.98 and 5.16 show a correlation with C-13 (δ) C 90.9) HMBC associated and assigned as C-17 (via HSQC-DEPT, δ C 107.0). The olefin proton H-17 shows a δ-peptide distribution similar to that of C-15. C HMBC correlation of 49.4% carbon. From H-9 and δ C The additional HMBC association of the 46.9 methylene carbon subsequently allowed for the allocation of C-14. C-14 (δ) was allocated using HSQC-DEPT data. H 1.53 and 2.21) and C-15 (δ H 2.15 and 2.18) 1 H chemical shift.

[0708] The following provides an overview of the key HMBC and COSY associations used for allocating glycoside ligand regions:

[0709]

[0710] 1 H- 13 Analysis of the C HSQC-DEPT data confirmed the presence of six anomeric protons. Three of the anomeric protons were in... 1 δ in HNMR spectrum H 5.65(δ C 95.5), 4.92 (δ) C 104.9) and 4.50 (δ) C Sufficient resolution was achieved at 105.7). At δ H 4.85(δC 98.4), 4.84 (δ) C 105.0) and 4.83 (δ C 105.3) The remaining three anotrons observed were 1 The residual solvent resonances in the H spectrum overlapped. In the δ... H The observed anomaly protons at 5.65 showed an association with the HMBC of C-19, suggesting that it corresponds to Glc. I Anisotropic protons. Similarly, in δ H 4.85 Anomalies were observed to show HMBC association with C-13, allowing them to be assigned as Glc. II Anisotropic protons.

[0711] Glc I Anisotropic protons (δ) H 5.65) shows the assignment as Glc I δ of H-2 H COSY correlation of 3.96 for protons, Glc I H-2 then showed a similarity to δ H 3.89 (Glc) I COSY correlation of H-3) protons, Glc I H-3 showed a correlation with δ H 3.71 (Glc) I The COSY correlation of H-4 protons was used. Due to data overlap, the COSY spectrum did not allow for the partitioning of H-5 or H-6 protons. Therefore, several different mixing times were used, with Glc... I Selective irradiation with anomeric protons was performed, followed by a series of 1D-TOCSY experiments. These experiments confirmed the effectiveness of targeting Glc... I The allocation from H-2 to H-4, as shown by 1D-TOCSY data, is Glc. I H-5's δ H 3.73 protons and their distribution as Glc I One of the H-6 protons, the δ H 4.15 protons. The latter proton was also used in the 1D-TOCSY experiment. Selective irradiation with H-6 using several different mixing times also confirmed the presence of Glc. I The allocation of H-1 to H-5 and H-6 (δ) H The remaining methylene protons (4.00). Using 1 H- 13 C HSQC-DEPT data determined the target for Glc I 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 The allocation of C chemical shifts to complete Glc I The allocation. Furthermore. 1 H- 13 δ in C HSQC-DEPT spectrum C 70.9% methylene 13 The presence of C resonance indicates the presence of Glc in the structure. I The 1→6 sugar bond.

[0712] Two unassigned glucose fractions were assigned as Glc based on HMBC association. I Substituents at C-2 and C-6. δ H 4.83 The observed anomeric protons showed similarities to Glc I C-2's HMBC is associated and assigned to Glc. V Anisotropic protons. δ H 4.50 The observed anomeric protons showed similarities to Glc I C-6's HMBC is associated and assigned to Glc. VI Anomalies were observed from Glc. I H-2 and Glc V Anisoprotons and those from Glc I H-6 and Glc VI The mutual HMBC correlation of anomeric protons.

[0713] Glc V Anisotope (δ) H 4.83) shows the allocation as Glc V δ of H-2 H COSY correlation of protons at 3.32. Glc V H-2 then showed a similarity to δ H 3.51 (Glc) V The COSY correlation of the H-3) proton. This latter proton shows a correlation with δ. H 3.38 (Glc) V Other associations of H-4 with protons. H-4 also shows association with δ H 3.55 (Glc) V The COSY association of H-5) and Glc V The H-5 then displayed its connection to Glc V H-6 protons (δ) H The COSY associations for Glc were determined using HSQC-DEPT data (3.76 and 3.97).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 The distribution of C chemical shifts. From Glc V H-3, C-2, and C-4, as well as those from Glc V The HMBC associations of H-4 with C-3 and C-6 confirm the above allocations to complete the Glc V The allocation.

[0714] based on 1 H- 13 C HSQC-DEPT and HMBC are associated, allocating another glucose fraction as Glc I The C-6 substituent. δ in the HSQC-DEPT spectrum. C 70.9 Glc I methylene 13 The relative low-field displacement of C resonance indicates that Glc I The 1→6 sugar bond. In the δ H 4.50 The observed anomeric protons showed similarities to Glc I C-6's HMBC association and was assigned as Glc VI Anomalies. Similarly, Glc I The methylene protons show a similarity to Glc VI The HMBC association of the anomeric carbon, and this confirms the Glc I With Glc VI The presence of 1→6 sugar bonds between Glc VI Anisotope protons showed distribution as Glc VI δ of H-2 H COSY correlation of protons at 3.33, Glc VI H-2 then showed a similarity to δ H 3.49 protons (Glc) VI COSY correlation (H-3). Due to data overlap, the COSY spectrum did not allow for the assignment of Glc based on COSY correlation. V H-4 to H-6. Therefore, several different mixing times are used, employing Glc VI Selective irradiation with anomeric protons was performed, followed by a series of 1D-TOCSY experiments. These experiments confirmed the effectiveness of targeting Glc... VI The allocation from H-2 to H-3, 1D-TOCSY data showed δ H3.45 (Glc) VI H-4) and δ H 3.48 (Glc) VI The protons of H-5 and their distribution as Glc VI H-6 proton δ H Protons at 3.92 and 3.94. (Using...) 1 H- 13 C HSQC-DEPT data determined the target for 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 The allocation of C chemical shifts to complete Glc VI The allocation.

[0715] Glycosides targeting C-19 1 H and 13 An overview of C chemical shifts is shown in the table below:

[0716] H NMR (500 MHz, D2O) of Reb M2 glycoside and 13 C NMR (125MHz, D2O / TSP) allocation

[0717]

[0718]

[0719] * 1 H and 13 The C value can be compared with the Glc value in the table below. IV -1 swap.

[0720] The following provides an overview of the key HMBC, COSY, and 1D-TOCSY associations used to allocate the C-19 glycoside region:

[0721] Reb M2 glycoside 1 H NMR (500MHz, D2O) and 13 C NMR (125MHz, D2O / TSP)

[0722] distribute

[0723]

[0724]

[0725] Glc was performed in a similar manner II The allocation of Glc. II Anisotropic protons (δ) H 4.85) shows the result of assigning Glc II δ of H-2 H COSY correlation of protons at 3.75, Glc II H-2 then showed a similarity to δ H 3.98 protons (Glc) II The COSY correlation of H-3) was observed. This latter proton showed a correlation with δ. H 3.54 protons (Glc) II Other associations of H-4). H-4 also showed associations with δ. H 3.96 protons (Glc) II COSY association of H-5). Glc II The H-5 also showed similarities to Glc II H-6 protons (δ) H The COSY associations for Glc were 3.77 and 3.45. The correlations were determined using HSQC-DEPT data. 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 The distribution of C chemical shifts. From Glc II H-3, C-2, and C-4, as well as those from Glc II The HMBC associations of H-4 with C-3 and C-6 confirm the above allocations to complete the Glc II The allocation.

[0726] The remaining two unallocated glucose fractions were allocated as Glc based on HMBC association. II The substituents at C-2 and C-3. In δ H 4.92 The observed anomeric protons showed similarities to Glc II C-2's HMBC association and was assigned as Glc III Anisotropic protons. In δ H 4.84 The observed anomeric protons showed similarities to Glc II C-3's HMBC association and assigned as Glc IV Anisotropic protons were also observed. Glc was also observed. II H-2 and Glc III Between anodic carbons and GlcII H-3 and Glc IV HMBC correlations between anomeric carbons.

[0727] Glc III Anisotope (δ) H 4.92) shows the allocation as Glc III δ of H-2 H The COSY correlation of protons at 3.32 was not allowed due to data overlap in the COSY spectrum, which did not permit the partitioning of H-3 to H-6 protons. Therefore, several different mixing times were used, employing Glc... III Selective irradiation with anomeric protons was performed, followed by a series of 1D-TOCSY experiments. These experiments confirmed the effectiveness of targeting Glc... III The allocation of H-2, 1D-TOCSY data showed δ H 3.51 (Glc) III H-3), δ H 3.26 (Glc) III H-4) and δ H 3.44 (Glc) III The protons of H-5). Once H-4 has been assigned using 1D-TOCSY data, the COSY correlation from H-4 with H-5 and then with H-6 is used to assign H-6. In the COSY spectrum, Glc III H-4 showed similarities to Glc III H-5 association, Glc III H-5 then showed differences from δ H Glc 3.94 and 3.75 III H-6a and Glc III COSY association of H-6b. Using... 1 H- 13 C HSQC-DEPT association identified the target 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 to complete Glc III The allocation.

[0728] Showing the relationship with δ H 3.41 proton COSY-related Glc IV (δ H The anomeric protons of 4.84 were assigned as GlcIV H-2, in turn, shows a similarity to δ H 3.46 protons (Glc) IV The COSY correlation of H-3) was observed. This latter proton showed a correlation with δ. H 3.45 protons (Glc) IV Other associations with H-4, Glc IV H-4 also showed similarities to δ H 3.75 protons (Glc) IV COSY association of H-5). Glc IV The H-5 also showed similarities to Glc IV H-6 protons (δ) H The COSY associations for Glc were 3.55 and 3.78. The correlations were determined using HSQC-DEPT data. IV C-2(δ C 76.1), C-3(δ) C 78.8), C4(δ) C 72.5), C-5(δ) C 81.7) and C-6 (δ) C 65.8) 13 The distribution of C chemical shifts. From Glc IV H-3, C-4, and C-5, as well as those from Glc IV The HMBC associations of H-4 with C-3 and C-6 confirm the above allocations to complete the Glc IV The allocation.

[0729] Targeting C-13 glycosides 1 H and 13 An overview of C chemical shifts is shown in the table below:

[0730] Reb M2 glycoside 1 H NMR (500MHz, D2O) and 13 C NMR

[0731] (125MHz, D2O / TSP) allocation

[0732]

[0733]

[0734] The following provides an overview of the key HMBC, COSY, and 1D-TOCSY associations used to allocate the C-13 glycoside region:

[0735]

[0736] NMR and MS analyses allowed for the complete allocation of the 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]equivalent-16-kaurene-19-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.

[0737]

[0738] Example 41

[0739] UGT76G1 was used for directed evolution (round 2) to convert rebaudioside D into rebaudioside X.

[0740] The most active clone from the first round of directed evolution of UGT76G1 (see Example 26, UGT76G1var94 containing the mutation Q266E_P272A_R334K_G348P_L379G) was selected as the baseline clone for the second round. A list of 53 mutations was established, containing positive mutations identified from different rounds and novel mutations obtained through the DNA2.0 ProteinGPStm strategy. This mutation list was then used to design 92 variant genes, each containing 3 different mutations. After optimization for expression codons in *E. coli*, the genes were synthesized, subcloned in the pET30a+ plasmid, and used for transformation of *E. coli* BL21(DE3) chemocompetent cells. The resulting cells were grown on solid LB medium in Piper dishes in the presence of kanamycin. Suitable clones were selected and grown in liquid LB medium in test tubes. Glycerol was added to the suspension as a cryoprotectant, and 400 μL aliquots were stored at -20°C and -80°C.

[0741] The stored aliquots of *E. coli* BL21(DE3) containing the pET30a+_UTG76G1var 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). The culture was incubated at 30°C with shaking on 96 μL microplates for 8 hours.

[0742] Inoculate 3.95 mL of Overnight Express solution with 50 μL of the above culture. TM Prepared TB culture medium Production medium containing 10 g / L glycerol and 50 mg / L kanamycin. The resulting culture was stirred at 20°C in 48-well plates. The culture showed significant growth and achieved a good OD (600 nm). After 44 hours, cells were collected by centrifugation and frozen.

[0743] By Master Mixture Lysis was performed by adding the lysed cells and collecting the lysate by centrifugation. Activity was tested using 100 μL of fresh lysate in a solution containing 50 mM phosphate buffer (pH 7.2) with rebaudioside D (final concentration 0.5 mM), MgCl2 (final concentration 3 mM), and UDP-glucose (final concentration 2.5 mM).

[0744] The reaction was run at 30°C, and samples were taken at 2, 4, 7, and 24 hours. The conversion and initial rates were determined by HPLC (CAD detection) using the analytical method described above for the conversion of rebaudioside D to rebaudioside X. Experiments were performed in parallel with the baseline clone, round 1 - Var94. The conversion and initial rates at 22 hours for this baseline clone were defined as 100%, and the normalized conversion and initial rates for the second-round clone are plotted in the table below:

[0745]

[0746]

[0747]

[0748]

[0749] *Mutations are annotated as follows: Refer to gene-initial amino acid-position-new amino acid: For example, for variant 94 from the first round of directed evolution of UGT76G1, the alanine at position 33 is mutated to glycine, annotated as Round1-Var94(A33G).

[0750] Modeling these results allows for the grading of the effects of each mutation. The following mutations were identified as beneficial to activity: S42A, F46I, I190L, S274G, I295M, K303G, F314S, K316R, K393R, V394I, I407V, N409K, N409R, Q425E, Q432E, S447A, and S456L.

[0751] Example 42

[0752] AtSUS in vivo production

[0753] AtSUS

[0754] >gi|79328294|ref|NP_001031915.1|Sucrose synthase 1 [Arabidopsis thaliana]

[0755]

[0756] The AtSUS synthetic gene was optimized for expression codons in *E. coli* and subcloned in the pET30a+ plasmid using NdeI and XhoI restriction sites. The pET30A+ vector containing AtSUS was used to transform electrocompetent *E. coli* Bl21(DE3) cells. Cells were grown in Piper dishes in the presence of kanamycin, and suitable clones were selected for growth in liquid LB medium (Eragonal flasks). Glycerol was added to the suspension as a cryoprotectant, and 400 μL aliquots were stored at -20°C and -80°C.

[0757] Melt the stored aliquots of *E. coli* BL21(DE3) containing the pET30A+_AtSUS plasmid and add 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). Incubate the culture at 135 rpm and 30°C with shaking for 8 hours.

[0758] The production medium contained 60 g / L overnight expression TB medium (Novagen), 10 g / L glycerol, and 50 mg / L kanamycin. The pre-culture was added to 800 mL of this medium, and the solution was stirred at 20°C while samples were taken to measure OD and pH. The culture showed good growth and good OD. After 40 h, cells were collected by centrifugation and frozen to obtain a wet cell weight of 30.1 g.

[0759] Cell suspension of 200 mg cells in 1.0 mL of 50 mM Tris buffer, pH 7.5, was lysed using Fastprep (MP Biomedicals, Lysis Matrix B, speed 6.0, 3 × 40 sec). Lysis products were collected by centrifugation and used fresh.

[0760] Example 43

[0761] Using UGTSL2, UGT76G1-R1-F12, and AtSUS, lebodiin A was converted to lebodiin X using in-situ prepared UDP-glucose.

[0762] The reaction was carried out in 1 mL increments using potassium phosphate buffer (50 mM final concentration, pH 7.5) containing 100 mM sucrose, 3 mM MgCl2, 0.25 mM UDP, and 0.5 mM rebaudioside A. The reaction was initiated by adding 15 μL of UGTSL2 (see Example 27) cleavage product (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). The reaction was followed by HPLC after quenching 125 μL of the sample with 10 μL of 2N H2SO4 and 115 μL of 60% methanol. After a 21-hour reaction time, 68% rebaudioside X and 26% rebaudioside M2 ​​were obtained, as shown in the figure. Figure 66 As shown in the image.

[0763] Example 44

[0764] UGT76G1 was used for directed evolution (round 3) to convert rebaudioside D to rebaudioside X. The most active clone from the second round of UGT76G1 directed evolution (see Example 41, round 2 UGT76G1var66 containing the mutation S42A_F46I_I407V) was selected as the baseline clone for round 3. A mutation list of 56 was established, containing positive mutations identified from different rounds and 30 new mutations obtained through the DNA2.0 ProteinGPStm strategy. This mutation list was then used to design 92 variant genes, each containing 3 or 4 different mutations. After optimization for expression codons in E. coli, the genes were synthesized, subcloned in pET30a+ plasmids, and used for transformation of E. coli BL21(DE3) chemocompetent cells. The resulting cells were grown on solid LB medium in Piper dishes in the presence of kanamycin. Suitable clones were selected and grown in liquid LB medium in test tubes. Glycerol, used as a cryoprotectant, was added to the suspension and 400 μL aliquots were stored at -20 °C and -80 °C.

[0765] The stored 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). The culture was incubated at 30°C with shaking on 96 μL microplates for 8 hours.

[0766] Inoculate 3.95 mL of Overnight Express solution with 50 μL of the above culture. TMPrepared TB culture medium Production medium containing 10 g / L glycerol and 50 mg / L kanamycin. The resulting culture was stirred at 20°C in 48-well plates. The culture showed significant growth and achieved a good OD (600 nm). After 44 hours, cells were collected by centrifugation and frozen.

[0767] By Master Mixture Lysis was performed by adding the lysed cells and collecting the lysate by centrifugation. Activity was tested using 100 μL of fresh lysate in a solution containing 50 mM phosphate buffer (pH 7.2) with rebaudioside D (final concentration 0.5 mM), MgCl2 (final concentration 3 mM), and UDP-glucose (final concentration 2.5 mM).

[0768] The reaction was run at 30°C, and samples were taken at 1, 2, 4, 6, and 22 hours. The conversion and initial rates were determined by HPLC (CAD detection) using the analytical method described above for the conversion of rebaudioside D to rebaudioside X. Experiments were performed in parallel with the baseline clone, round 2 – Var66. The conversion and initial rates at 22 hours for this baseline clone were defined as 100%, and the normalized conversion and initial rates for the round 3 clone are depicted in the table below:

[0769]

[0770]

[0771]

[0772] *Mutations are annotated as follows: Refer to gene-initial amino acid-position-new amino acid: For example, for variant 66 from the second round of directed evolution of UGT76G1, the isoleucine at position 190 is mutated to leucine, annotated as round 2-Var66(I190L).

[0773] Modeling these results allows for the grading of the effects of each mutation. The following mutations were identified as beneficial to activity: I46L, I295M, S119A, S274G, K334R, F314S, K303G, K316R, K393R, I190L, Q425E, Q432E, N138G, V394I, F182L, V407I, A272P, V264C, E449D, and A352G.

[0774] Example 45

[0775] UGTSL2 was used for directed evolution to convert rebaudioside A into rebaudioside D (Round 1).

[0776] Starting with the natural enzyme UGTSL2 (GI_460410132), a list of 60 mutations was established, containing positive mutations identified in the first round and novel mutations obtained through the DNA2.0 ProteinGPStm strategy. This mutation list was then used to design 92 variant genes, each containing three different mutations. After optimization for expression codons in *E. coli*, the genes were synthesized, subcloned in the pET30a+ plasmid, and used for transformation of *E. coli* BL21(DE3) chemocompetent cells. The resulting cells were grown on solid LB medium in Piper dishes in the presence of kanamycin. Suitable clones were selected and grown in liquid LB medium in test tubes. Glycerol was added to the suspension as a cryoprotectant, and 400 μL aliquots were stored at -20°C and -80°C.

[0777] The stored aliquots of *E. coli* BL21(DE3) containing the pET30a+_UGTSL2var 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). The culture was incubated at 30°C with shaking on 96 μL microtiter plates for 8 hours.

[0778] Inoculate 3.95 mL of Overnight Express solution with 50 μL of the above culture. TM Prepared TB culture medium Production medium containing 10 g / L glycerol and 50 mg / L kanamycin. The resulting culture was stirred at 20°C in 48-well plates. The culture showed significant growth and achieved a good OD (600 nm). After 44 hours, cells were collected by centrifugation and frozen.

[0779] By Master Mixture Lysis was performed by adding the lysed cells and collecting the lysate by centrifugation. Activity was tested using 100 μL of fresh lysate in a solution containing 50 mM phosphate buffer (pH 7.2) with rebaudioside D (final concentration 0.5 mM), MgCl2 (final concentration 3 mM), and UDP-glucose (final concentration 2.5 mM).

[0780] The reaction was run at 30°C and samples were taken at 2, 4, 6, and 22 hours. The conversion and initial rate were determined by HPLC (CAD detection) using the analytical method described above for the conversion of rebaudioside A to rebaudioside D. Experiments were performed in parallel with the baseline clone UGTSL2. The initial rate for this baseline clone was defined as 100%. As an indicator of clone specificity, the content of rebaudioside M2 ​​was determined at 100% UDP-glucose conversion, and the content of rebaudioside D2 was determined at 50% UDP-glucose conversion. The UDP-glucose conversion was defined as: ([Reb D] / [Reb A]0) + ([Reb D2] / [Reb A]0) + 2*([Reb M2] / [Reb A]0).

[0781] The table below describes the normalized initial rate, rebaudioside M2 ​​content, and rebaudioside D2 content at 100% UDP-glucose conversion and 50% UDP-glucose conversion.

[0782]

[0783]

[0784]

[0785] *Mutations are annotated as follows: Refer to gene-initial amino acid-position-new amino acid: For example, for UGTSL2, the isoleucine at position 240 is mutated to leucine, annotated as UGTSL2(I240L).

[0786] Modeling these results allowed for the grading of the effects of each mutation. The following mutations were identified as beneficial to activity: L276A, T392A, Q27R, N278G, T329V, A341V, I333L, G387E, H247P, M354L, A285V, V270I, N325S, I240L, F253Y, A285L, and I352V.

[0787] The following mutations were identified as beneficial for lower lebaudidine M2 formation: Q27R, N325S, G387E, I333L, H247P, T329I, R312L, T199S, E259G, S334T, I131V, A285L, I389L, L393V, V254L, N339S, I345L, and T245R.

[0788] Example 46

[0789] Rebaudioside A was converted to Rebaudioside I using UGT76G1.

[0790] The reaction was carried out using UGT76G1-R1-F12 (also known as UGT76G1var94) (see Example 26).

[0791] The total reaction volume was 40 mL, containing the following components: 50 mM sodium phosphate buffer (pH 7.5), 3 mM MgCl2, 2.5 mM UDP-glucose, 0.5 mM rebaudioside A, and 4 mL of UGT76G1-1-F12 lysis product (2.5 U / mL). The reaction was run at 30 °C on an orbital oscillator at 135 rpm. For sampling, 125 μL of the reaction mixture was quenched with 10 μL of 2N H2SO4 and 115 μL of methanol / water (7 / 3). The sample was immediately centrifuged and maintained at 10 °C before LC-MS analysis. An Agilent 1200 series HPLC system was used, equipped with a dual pump (G1312B), an autosampler (G1367D), a temperature-controlled column (G1316B), and a DAD detector (C1315C), connected to an Agilent 6110A MSD and used with LC / MSD Chemstation software.

[0792] Instrument conditions

[0793]

[0794] Moving phase gradient scheme

[0795] Time (minutes) A (%): Formic acid 0.1% B (%): Acetonitrile 0 76 24 8.5 76 24 10.0 71 29 16.5 70 30

[0796] Obtained Figure 67a The reaction characteristics shown in the image are:

[0797] After 42 hours of reaction, 20 mL of the reaction mixture was rapidly cooled with 20 mL of ethanol and used for structural analysis.

[0798] Similarly, for the conversion of rebaudine A to rebaudine I, the optimal clones of UGT76G1 from the second round (UGT76G1-R2-B9, identified above as "round 2-Var66", see Example 41) and the third round (UGT76G1-R3-G3, identified above as "round 3-Var21", see Example 44) of directed evolution, as well as the natural UGT76G1 (see Example 26), were tested, and the optimal clones were determined. Figure 67b The activity shown in the image.

[0799] Example 47

[0800] Isolation and characterization of RebI

[0801] crude reaction sampleAccording to Example 46, a sample of batch number CB-2977-198 for separation was prepared using UGT76G1.

[0802] HPLC analysis Preliminary HPLC analysis was performed using a Waters 2695 Alliance system with the following methods: Phenomenex 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% acetic acid 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.

[0803] gradient:

[0804] Time (minutes) ％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

[0805] Separation by HPLC .

[0806] Purification was performed using a Waters Atlantis dC18 (30×100 mm, 5 μm, p / n 186001375) column with an isocratic mobile phase of 80:20 water / MeCN. The flow rate was maintained at 45 mL / min and the injection loading was 180 mg. The detector wavelength was set to 210 nm.

[0807] Fractional analysis was performed using a Waters Atlantis dC18 column (4.6 × 150 mm, 5 μm, p / n 186001342); mobile phase A: water; mobile phase B: MeCN; flow rate: 1 mL / min; isocratic mobile phase conditions: 75:25 A / B, duration 30 min.

[0808] MS and MS / MS MS and MS / MS data were generated using a Waters QT Micro mass spectrometer equipped with an electrospray ionization source. Samples were separated by negative ESI. Samples were diluted to 0.25 mg / mL with H₂O:MeCN (1:1) and introduced via flow injection for MS data acquisition. Further dilution to 0.01 mg / mL yielded a good s / n ratio for MS / MS tuning and was acquired via direct infusion. The collision energy was set to 60 V to obtain MS / MS data, which exhibited enhanced fragment ion peaks due to the nature of the molecules.

[0809] NMRSamples were prepared by dissolving ~1.0 mg in 180 μL of pyridine-d5+ TMS, and NMR data were acquired on a Bruker Avance 500 MHz instrument using either a 2.5 mm reverse probe or a 5 mm broadband probe. At the Rensselaer Polytechnic Institute, 13C and HMBC NMR data were acquired using Bruker Avance 600 MHz and 800 MHz instruments with 5 mm cryogenic probes, respectively. 1 H and 13 C NMR spectrum referenced TMS resonance (δ) H 0.00ppm and δ C 0.0ppm).

[0810] Reb I was separated using a semi-synthetic steviol glycoside mixture with batch number CB-2977-198. The material was analyzed by HPLC as described above. The residence time was approximately 17 min (t). R A Reb I peak was observed, such as... Figure 28 As shown in the image.

[0811] Results and discussion

[0812] As described above, the reb I peak was separated from the crude reaction product and displayed in [the following text is missing from the original] Figure 29 The separated fractions were combined and lyophilized. As confirmed by LC-CAD using the above method, the purity of the final product was 91%. Figure 30 Approximately 1 mg of RebI was provided for spectroscopic and spectral analysis.

[0813] mass spectrometry ESI-TOF mass spectra obtained by perfusing a sample with reb I showed a [MH] value of m / z 1127.4741. - ( Figure 31 ). [MH] - The mass of the ion is expected to be C of the molecular formula of reb I. 50 H 79 O 28 Very consistent (calculated as C) 50 H 79 O 28 :1127.4758, error: -1.5ppm)( Figure 32 MS data confirmed that reb I has the molecular formula C. 50 H 80 O 28 The nominal mass is 1128 Daltons.

[0814] MS / MS spectrum of reb I (selected m / z 1127.4 [MH]) - The ions (used for splitting) indicated the loss of two sugar monomers at m / z 803.5301; however, using a collision energy of 30 V did not reveal any other splitting. Figure 33 When applying higher collision energy (60V) ( Figure 34 No parent ion was observed, but the sequential loss of three sugar monomers at m / z 641.4488, 479.3897 and 317.3023 was observed at m / z 803.5301.

[0815] NMR spectroscopy A series of actions were carried out, including... 1 H NMR ( Figures 35-37 ), 13 C NMR ( Figures 38-39 ), 1 H- 1 H COSY( Figure 40 ), HSQC-DEPT Figure 41 ), HMBC ( Figures 42-43 NOESY Figures 44-45 ) and 1D-TOCSY ( Figures 46-50 NMR experiments were conducted to allow for the distribution of reb I.

[0816] reb I obtained at 300K 1 In the H NMR spectrum ( Figure 35 One of the anomeric protons was completely masked by water resonance. Therefore, the sample was obtained at a relatively low temperature (292 K). 1 H NMR spectroscopy, to remove water resonance, and at that temperature, sufficient to distinguish anomeric protons ( Figures 36-37 Therefore, all other NMR data for reb I were obtained at 292K.

[0817] 1D and 2D NMR data indicate that the central nucleus of the glycoside is a diterpenoid. (Source: δ) H 1.22 methyl protons and δ C The HMBC association of the carbonyl group at 176.9 allows for the allocation of one of the tertiary methyl groups (C-18) and C-19, and provides a starting point for the allocation of the remaining glycosidic ligand. The methyl proton (H-18) and δ C Other HMBC associations of carbon at 38.5, 44.0, and 57.2 allow for the allocation of C-3, C-4, and C-5. 1 H- 13 Analysis of C HSQC-DEPT data indicates that δ C The 38.5 carbon atom is a methylene group, while the δ... CCarbon 57.2 is a methine, which is assigned to C-3 and C-5. The remaining δ C The carbon at 44.0, which showed no correlation in the HSQC-DEPT spectrum, was assigned as a quaternary carbon, C-4. C-3 (δ) was assigned using HSQC-DEPT data. H 1.02 and 2.35) and C-5 (δ H 1.03) 1 H chemical shift. One of the H-3 protons (δ) H 1.02) and δ H The COSY correlation between protons at 1.44 allows for the allocation of one of the H-2 protons, which in turn exhibits a correlation with the δ proton allocated as H-1. H The proton correlation was 0.74. The remaining protons were then allocated for C-1 and C-2 based on other COSY and HSQC-DEPT correlations. 1 H and 13 C chemical shifts are summarized in the table below.

[0818] 1 H and 13 Partitioning of lebodiin I glycoside ligands by C NMR (500 and 150 MHz, pyridine-d5)

[0819]

[0820] In δ H Other tertiary methyl singlets observed in 1.26 showed association with HMBCs at C-1 and C-5 and were assigned to H-20. The methyl protons showed association with quaternary carbons (δ¹²). C 39.8) and methylene carbon (δ C 54.1) (which are respectively assigned as C-10 and C-9) additional HMBC associations. H-5 (δ H 1.03) and δ H The COSY correlation between 1.90 and 2.33 subsequently allowed for the allocation of H-6 protons, which in turn showed a correlation with the allocation of H-7 δ protons. H The correlation between protons at 1.29 and 1.31 was then determined from HSQC-DEPT data for C-6 (δ C 22.2) and C-7(δ) C 41.7) 13 C chemical shift. H-9(δ) H 0.88) and δ H The COSY correlation between protons at 1.67 and 1.70 allows for the allocation of H-11 protons, which in turn exhibits a δ correlation with the allocation of H-12 protons. HCOSY correlations for protons at 1.98 and 2.28. HSQC-DEPT data were then used to assign C-11 (δ... C 20.5) and C-12 (δ C 37.3). In δ H The olefin protons observed at 5.02 and 5.67 show a correlation with δ C The quaternary carbon of 86.7 (C-13) is HMBC associated and assigned to H-17 (via HSQC-DEPT, δ C 104.8). The methine proton H-9 shows δ-ions assigned to C-8, C-14 and C-15, respectively. C HMBC correlations for carbons at 42.3, 44.3, and 47.6. C-14 (δ) was assigned using HSQC-DEPT data. H 1.78 and 2.59) and C-15 (δ H 2.04) 1 H chemical shifts. Further HMBC associations from H-9 with C-11 and H-12 with C-9 confirm the above-described allocation. From H-14 with δ... C The HMBC association observed at 154.0 quaternary carbon allows for the allocation of C-16 to complete the allocation of the central nucleus.

[0821] The correlations observed in the NOESY spectra were used to assess the relative stereochemistry of the partition center diterpene nucleus. In the NOESY spectra, a NOE correlation was observed between H-14 and H-20, indicating that H-14 and H-20 are located on the same plane of the ring. Similarly, NOE correlations were observed between H-9 and H-5, and between H-5 and H-18. No NOE correlation was observed between H-9 and H-14. Therefore, the NOESY data indicate that H-5, H-9, and H-18 are located on opposite planes of the ring compared to H-14 and H-20, as presented in the figure below. These data therefore suggest that the relative stereochemistry in the central nucleus is preserved during the glycosylation step.

[0822] For reb I 1 H- 13 Analysis using C HSQC-DEPT confirmed the existence of five anomeric protons. Spectra of all five anomeric protons obtained at 292 K showed a δ-particle size distribution. H 6.14(δ C 95.3), 5.57 (δ) C 104.6), 5.38 (δ) C 104.7), 5.29 (δ) C 105.0) and 5.06 (δ) CAll five anomeric protons were resolvable at 98.0 Hz. Furthermore, all five anomeric protons exhibited large coupling (7.7 Hz–8.2 Hz), indicating a β-structure. In the δ... H The anoprotons observed in 6.14 showed an association with the HMBC of C-19, indicating that they correspond to Glc. I Anisotropic protons. Similarly, in δ H The observed anomalies in 5.06 showed an HMBC association with C-13, allowing them to be assigned as Glc. II Anisotropic protons.

[0823] Glc I Anisotropic protons (δ) H 6.14) shows the assignment as Glc I δ of H-2 H 4.18 COSY correlation of protons. Due to data overlap, the COSY spectrum did not allow for the assignment of H-3 or H-4. Therefore, several different mixing times were used, with Glc... I Selective irradiation with anomeric protons was used to conduct a series of 1D TOCSY experiments. Figure 46 In addition to confirming the targeting of Glc I The TOCSY data shows that the H-2 allocation was assigned to H-3, H-4, and H-5 respectively, with δ values... H Protons at 4.27, 4.25, and 3.93. In the TOCSY spectrum, δ H 4.37 The observed protons were assigned as Glc I One of the H-6 protons. Based on H-5 and δ H The COSY association in 4.27 was assigned δ. H 4.27 Other H-6 methylene protons. Allocations for Glc were performed using HSQC-DEPT data. I C-2(δ C 72.5), C-3(δ) C 89.4), C-4(δ) C 69.2), C-5(δ) C 78.2-78.8) and C-6 (δ C 61.7) 13 C chemical shifts. HMBC associations from H-1 with C-3 and H-4 with C-6 further confirm the above-described allocation to complete the Glc chemical shift. I The allocation.

[0824] Of the four remaining unallocated glucose fractions, one is allocated as Glc based on HMBC association. I The substituent at C-3. In δ HThe anoprotons observed on 5.29 showed similarities to Glc I C-3's HMBC association and assigned as Glc V Anomalies were observed from Glc. I H-3 and Glc V The mutual HMBC correlation of the anterior carbons.

[0825] The table below shows the glycosides targeting C-19. 1 H and 13 Overview of C chemical shift:

[0826] Lebodiin I C-19 glycoside 1 H and 13 C10 NMR (500 and 150 MHz, pyridine-d5) partitioning

[0827]

[0828]

[0829] Five carbon resonances (78.16, 78.47, 78.50, 78.55 and 78.77) in the 78.2-78.8 range, therefore the chemical shifts cannot be clearly assigned.

[0830] The following provides an overview of the key HMBC and COSY associations used to allocate the C-19 glycoside region.

[0831]

[0832] Glc V Anisotope (δ) H 5.29) shows the assignment as Glc V δ of H-2 H The COSY correlation of protons was 4.04. Subsequently, HSQC-DEPT data were used to assign Glc. V C-2(δ C (75.3 or 75.5). Due to data overlap, the COSY spectrum did not allow for the distribution of the remaining protons. Therefore, several different mixing times were used, employing Glc... V Selective irradiation with anomeric protons was used to conduct a series of 1D TOCSY experiments. Figure 47 In addition to confirming the targeting of Glc V H-2 allocation, TOCSY data allows for the allocation of Glc V H-3(δ H 4.27), H-4 (δ) H 4.12) and H-5 (δ) H4.05). In the TOCSY spectrum, δ H 4.56 The observed protons were assigned as Glc V One of the H-6 protons. Based on H-5 and δ H The COSY association in 4.26 was assigned δ. H 4.26 Other H-6 methylene protons. Allocations for Glc were performed using HSQC-DEPT data. V C-3(δ C 78.2-78.6), C-4(δ) C 71.5 or 71.6), C-5 (δ) C 78.5 or 78.6) and C-6 (δ C (62.3 or 62.4) 13 C chemical shift to complete Glc V The allocation.

[0833] Glc was performed in a similar manner II The allocation of Glc. II Anisotropic protons (δ) H 5.06) shows the assignment as Glc II δ of H-2 H The COSY correlation of the proton at 4.34, in turn, shows a correlation with δ H 4.20 (Glc) II COSY correlation of H-3) protons, Glc II H-3 showed a correlation with δ H 3.97 (Glc) II Other associations of H-4) protons, Glc II H-4 also showed similarities to δ H 3.80 (Glc) II The COSY association of protons with H-5 was shown. H-5 exhibited a correlation with δ protons partitioned as H-6. H Other COSY correlations of protons at 4.18 and 4.49. Several different mixing times were also used, with Glc II Selective irradiation with anomeric protons was used to conduct a series of 1D TOCSY experiments. Figure 48 TOCSY data confirmed the above proton allocation. Regarding Glc... II C-2(δ C 80.2), C-3(δ) C 87.5), C-4 (δ) C 70.1), C-5 (δ) C 77.6) and C-6(δ) C 62.5) 13The C chemical shift assignments are based on HSQC-DEPT data. (Source: Glc) II H-3, C-2, and C-4, as well as those from Glc II The HMBC associations of H-4 with C-3, C-5, and C-6 confirm the above allocations to complete the Glc II The allocation.

[0834] Based on HMBC association, the remaining two unallocated glucose fractions were allocated as Glc. II The substituents at C-2 and C-3. In δ H The observed anisotopes at 5.57 show similarities to Glc. II C-2's HMBC association and was assigned as Glc III Anisotropic protons. In δ H The anoprotons observed at 5.38 showed similarities to Glc II C-3's HMBC association and assigned as Glc IV Anomalies were observed from Glc. II H-2 and Glc III Angiocarbons and those from Glc II H-3 and Glc IV The mutual HMBC correlation of the anterior carbons.

[0835] Glc III Anisotope (δ) H 5.57) shows the allocation as Glc III δ of H-2 H 4.21 Proton COSY correlation. Then, HSQC-DEPT data are used to assign Glc. III C-2(δ C 76.3). Due to data overlap, the COSY spectrum did not allow for the distribution of the remaining protons. Therefore, several different mixing times were used, employing Glc... III Selective irradiation with anomeric protons was used to conduct a series of 1D TOCSY experiments. Figure 49 In addition to confirming the targeting of Glc III H-2 allocation, TOCSY data allows for the allocation of Glc III H-3(δ H 4.27), H-4 (δ) H 4.25) and H-5 (δ H 3.94). In the TOCSY spectrum, δ H 4.41 and δ H 4.53 The observed protons were assigned as Glc IIIH-6 protons. Allocation of C-3(δ) protons was performed using HSQC-DEPT data. C 78.2-78.6), C-4(δ) C 72.1), C-5(δ) C 78.2-78.8) and C-6 (δ C 63.1) 13 C chemical shift. From H-5 and δ C The HMBC correlation of carbon at 63.1 further confirms the presence of Glc. III The allocation of C-6 is to complete Glc III The allocation.

[0836] Glc IV Anisotope (δ) H 5.38) shows the assignment as Glc IV δ of H-2 H The COSY correlation of protons was 4.01. Then, HSQC-DEPT data was used to assign Glc. IV C-2(δ C (75.3 or 75.5). Due to data overlap, the COSY spectrum did not allow for the distribution of the remaining protons. Therefore, several different mixing times were used, employing Glc... IV Selective irradiation with anomeric protons was used to conduct a series of 1D TOCSY experiments. Figure 50 In addition to confirming the targeting of Glc IV The allocation of H-2, 1D TOCSY data allows for the allocation of H-3 (δ). H 4.28), H-4 (δ) H 4.11), H-5 (δ) H 4.13) and H-6 (δ) H 4.25 and 4.58). δ H The 4.25 proton also showed similarities to δ H The COSY association at 4.58 confirmed these proton terms H-6. Allocations for C-3 (δ) were assigned using HSQC-DEPT data. C 78.2-78.6), C-4(δ) C 72.1), C-5(δ) C 78.2-78.6) and C-6 (δ C 62.3 or 62.4) 13 C chemical shift. HMBC associations from H-4 with C-6 and H-5 with C-1 further confirm Glc IV The allocation of C-6 is to complete Glc IV The allocation.

[0837] The following shows the discovered glycosides targeting C-13. 1 H and 13 Overview of C chemical shift:

[0838] Lebodiin I C-13 glycoside 1 H and 13 C10 NMR (500 and 150 MHz, pyridine-d5) partitioning

[0839]

[0840]

[0841] Five carbon resonances (78.16, 78.47, 78.50, 78.55 and 78.77) in the 78.2-78.8 range, therefore the chemical shifts cannot be clearly assigned.

[0842] The following provides an overview of the key HMBC and COSY associations used to allocate the C-13 glycoside region.

[0843]

[0844] NMR and MS analyses of rebaudioside I and reb I allowed for the full allocation of the structure, as shown below. The compound is named 13-[(2-O-β-D-glucopyranosyl-3-O-β-D-glucopyranosyl)-β-D-glucopyranosyl)oxy]equivalent-16-kaurene-19-acid [(3-O-β-D-glucopyranosyl)-β-D-glucopyranosyl) ester].

[0845]

[0846] Example 48

[0847] UGTSL2 was used for directed evolution to convert rebaudioside A into rebaudioside D (round 2).

[0848] Using the natural enzyme UGTSL2 (GI_460410132) as a baseline, a list of 23 mutations was established, including positive mutations from the first round of identification targeting different activities (Example 45) and mutations identified via DNA2.0 ProteinGPS. TMThe strategy yielded novel mutations. This mutation list was then used to design 46 variant genes, each containing three different mutations. After optimization for expression codons in *E. coli*, the genes were synthesized, subcloned in the pET30a+ plasmid, and used for transformation of *E. coli* BL21(DE3) chemocompetent cells. The resulting cells were grown on solid LB medium in Piper dishes in the presence of kanamycin. Suitable clones were selected and grown in liquid LB medium in test tubes. Glycerol was added to the suspension as a cryoprotectant, and 400 μL aliquots were stored at -20°C and -80°C.

[0849] The stored aliquots of *E. coli* BL21(DE3) containing the pET30a+_UGTSL2var 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). The culture was incubated at 30°C with shaking on 96 μL microtiter plates for 8 hours.

[0850] Inoculate 3.95 mL of Overnight Express solution with 50 μL of the above culture. TM Prepared TB culture medium Production medium containing 10 g / L glycerol and 50 mg / L kanamycin. The resulting culture was stirred at 20°C in 48-well plates. The culture showed significant growth and achieved a good OD (600 nm). After 44 hours, cells were collected by centrifugation and frozen.

[0851] By Master Mixture The lysed cells were added to induce lysis, and the lysis products were collected by centrifugation.

[0852] To measure the activity of the variant in converting rebaudioside A to rebaudioside D, 100 μL of fresh lysis product was added to a solution of rebaudioside A (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. The reaction was run at 30 °C, and samples were taken at 2, 4, 6, and 22 hours. The initial rate was determined by HPLC analysis (CAD) following the analytical method described above for the conversion of rebaudioside A to rebaudioside D.

[0853] In parallel, for the most active clone, 100 μL of fresh lysis product was 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. The reaction was run at 30 °C, and samples were taken at 2, 4, 6, and 22 hours. The initial rate was determined by HPLC analysis (CAD detection) after targeting the conversion of rebaudioside D.

[0854] In addition to the new variant, two experiments were performed using the baseline clone UGTSL2. The initial rate of conversion to rebaudioside A or rebaudioside D against this baseline clone was limited to 100%.

[0855] The activity of each clone was defined as normalized activity compared to the baseline clone UGTSL2, while the specificity of each clone was expressed as the ratio between the initial rates of conversion to rebaudioside A and rebaudioside D.

[0856] The normalized rate of rebaudioside A conversion and the ratio between the initial rates of rebaudioside A and rebaudioside D conversion are described in the table below.

[0857]

[0858] *Mutations are annotated as follows: Refer to gene-initial amino acid-position-new amino acid: For example, for UGTSL2, the mutation of isoleucine at position 240 to leucine is annotated as UGTSL2(I240L). Nd indicates not determined.

[0859] Modeling these results allows for a tiered approach to the effect of each mutation. The following mutations are identified as beneficial for activity:

[0860] N325S, G387E, A285V, I 333L, V270I, Q27R, N278G, L393V, S258T, A341V, H247P and T392A.

[0861] The following mutations were identified as beneficial for increasing the ratio between the initial rates of rebaudioside A and rebaudioside D conversion:

[0862] V270I, T392A, T329V, L276A, L393V, A341V and S255C.

[0863] Example 49

[0864] Uses of β-glucosidase in the conversion of rebaudioside M2 ​​to rebaudioside D

[0865] For the hydrolysis of rebaudioside M2, different β-glucosidases were tested. The target was to selectively hydrolyze the (1→6) glycosidic bond to obtain rebaudioside D. The general reaction scheme required is as follows:

[0866]

[0867] First, the selected β-glucosidases were tested on the reference substrate 4-nitrophenyl-β-D-glucopyranoside to determine the activity. Based on the determined activity, the amount of enzyme used was calculated as the units used in the hydrolysis of rebaudioside M2.

[0868] The β-glucosidases tested are described in the following table:

[0869]

[0870] *Isolase (011410; National Enzyme Company, USA); Aromase (GLY0151441; Amano Enzyme, Japan); Naringinase (NAH0550102; Amano Enzyme, Japan), Cellulase from Trichoderma reesei ATCC 26921 (Sigma C2730); Cellobiase from Aspergillus niger (Sigma C6105); Viscozyme L (Sigma V2010)

[0871] The test conditions were as follows:

[0872] The reaction was carried out at 30 °C in a total volume of 10 mL containing 15 mM sodium acetate buffer (pH 4.5) and 1 mM rebaudioside M2. The reaction was initiated by adding the enzyme.

[0873] After 0, 0.5, 1, 1.5, 2, 2.5, 3, and 3.3 hours, 625 μL samples of the reaction mixture were taken and quenched with a mixture of 575 μL 80% methanol and 50 μL 2N H2SO4. The samples were analyzed by HPLC analysis (CAD detection) using the above analytical method.

[0874] The reaction characteristics of these reactions using different β-glucosidases are shown in Figure 68a -f.

[0875] It can be inferred that naringinase and CWD catalyzed the formation of rebaudioside D2 and rebaudioside A, which respectively indicated (1→2) bond glycolysis and (1→6) bond glycolysis. It can be considered that these enzymes are non-selective for the conversion of rebaudioside M2.

[0876] Isolase, cellulase Tr, and cellobiase As exhibit distinct selectivity for the conversion of rebaudioside M2 ​​to rebaudioside D (hydrolysis of the (1→6) glycosidic bond), while Aromase has low overall activity for the conversion of rebaudioside M2.

[0877] Example 50

[0878] The stability of rebaudioside in the presence of Isolase, cellulase Tr, and cellobiase As

[0879] To evaluate the selectivity of Isolase, cellulose Tr, and cellobiase As for rebaudioside M2, rebaudioside A, rebaudioside D, and rebaudioside M were tested as substrates under the following conditions:

[0880] The reaction was carried out for more than 24 hours at 30°C in a total volume of 10 mL containing 15 mM sodium acetate buffer (pH 4.5) and 1 mM rebaudioside A, rebaudioside D, or rebaudioside M. The reaction was initiated by adding an enzyme.

[0881] After incubation at 0, 0.5, 1, 1.5, 2, 2.5, 3, and 3.3 hours, 625 μL of the reaction mixture was obtained and quenched with a mixture of 575 μL of 80% methanol and 50 μL of 2N H2SO4. The samples were analyzed by HPLC.

[0882] Obtained Figure 69a The results shown in -c indicate that no significant conversion of rebaudioside A, rebaudioside D, and rebaudioside M was observed in the presence of Isolase, cellulose Tr, and cellobiase As.

[0883] Example 51

[0884] Four enzymatic reactions used to convert rebaudioside A to rebaudioside M.

[0885] The effects of introducing Isolase, cellulose Tr, or cellobiase As into a one-pot reaction using UGTSL2, UGT76G1-1R-F12, and AtSUS to convert rebaudioside A to rebaudioside M were investigated. The following reaction conditions were used:

[0886]

[0887]

[0888] Results of experiments with and without added β-glucosidase were shown in Figure 70aIn the -d reaction, it can be seen that the addition of cellobiase As blocked the reaction, and the addition of cellulase Tr had no effect on the reaction characteristics. However, the addition of Isolase to the reaction mixture had a positive effect on the quality of rebaudioside M formed in the reaction. An increase of approximately 20% was observed when Isolase was added. Compared with the reaction without the addition of β-glucosidase, the content of rebaudioside M2 ​​was approximately 10% lower and the content of rebaudioside I was approximately 15% lower when Isolase was added to the reaction.

[0889] By optimizing reaction parameters and the content of isolase, it is possible to further improve the yield of Reb M and reduce the content of RebM2 and Reb I.

[0890] Example 52

[0891] Uses of β-glucosidase in the conversion of rebaudioside I to rebaudioside A

[0892] Three β-glucosidases were tested to hydrolyze rebaudioside I to rebaudioside A. The target was the selective hydrolysis of (1→6) glycosidic bonds to obtain rebaudioside D. The required general reaction protocol is as follows:

[0893]

[0894] Selected β-glucosidases were tested against the reference substrate 4-nitrophenyl-β-D-glucopyranoside to determine activity. Based on the determined activity, the amount of enzyme used was calculated as units used in the hydrolysis of rebaudioside I. The tested β-glucosidases are described in the table below:

[0895]

[0896] *Isolase (011410; National Enzyme Company, USA); cellulase from Trichoderma reesei ATCC 26921 (Sigma C2730); cellobiase from Aspergillus niger (Sigma C6105)

[0897] The test conditions were as follows. The reaction was carried out at 30°C in a total volume of 2 mL containing 15 mM sodium acetate buffer (pH 4.5) and 1 mM lebaudoside I. The reaction was initiated by adding an enzyme.

[0898] After 0, 1.5, 2.5, and 18 hours, 125 μL of the reaction mixture was obtained and quenched with a mixture of 115 μL 80% methanol and 10 μL 2N H2SO4. The samples were analyzed by HPLC (CAD detection) using the analytical method described above. The reaction characteristics of different β-glucosidases of rebaudioside I were characterized on... Figure 71 As shown in the diagram.

[0899] It was observed that all three β-glucosidases were converted to rebaudioside I. The only product was rebaudioside A.

[0900] Example 53

[0901] UGTSL2 was used for directed evolution to convert rebaudioside A into rebaudioside D (round 3).

[0902] Using the natural enzyme UGTSL2 (GI_460410132) as a baseline, a list of 13 mutations identified in the second round was prepared (Example 48), and a list was generated using DNA2.0 ProteinGPS. TM A list of 12 new mutations was obtained from the strategy. This mutation list was then used to design 46 variant genes, each containing 1 to 8 different mutations. After optimization for expression codons in *E. coli*, the genes were synthesized, subcloned in the pET30a+ plasmid, and used for transformation of *E. coli* BL21(DE3) chemocompetent cells. The resulting cells were grown on solid LB medium in Piper dishes in the presence of kanamycin. Suitable clones were selected and grown in liquid LB medium in test tubes. Glycerol was added to the suspension as a cryoprotectant, and 400 μL aliquots were stored at -20°C and -80°C.

[0903] The stored aliquots of *E. coli* BL21(DE3) containing the pET30a+_UGTSL2var 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). The culture was incubated at 30°C with shaking on 96 μL microtiter plates for 8 hours.

[0904] Inoculate 3.95 mL of Overnight Express solution with 50 μL of the above culture. TM Prepared TB culture medium Production medium containing 10 g / L glycerol and 50 mg / L kanamycin. The resulting culture was stirred at 20°C in 48-well plates. The culture showed significant growth and achieved a good OD (600 nm). After 44 hours, cells were collected by centrifugation and frozen.

[0905] By Master Mixture The lysed cells were added to induce lysis, and the lysis products were collected by centrifugation.

[0906] To measure the activity of the variant in converting rebaudioside A to rebaudioside D, 100 μL of fresh lysis product was added to a solution of rebaudioside A (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. The reaction was run at 30 °C, and samples were taken at 2, 4, 6, and 22 hours. The initial rate was determined by HPLC analysis (CAD detection) following the analytical method described above for the conversion of rebaudioside A to rebaudioside D.

[0907] In parallel, 100 μL of fresh lysis product was 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). The reaction was run at 30 °C, and samples were taken at 2, 4, 6, and 22 hours. The initial rate was determined for rebaudioside D conversion by HPLC analysis (CAD detection).

[0908] In addition to the new variant used in this round, two experiments were conducted using the baseline clone UGTSL2. The initial rate of conversion to rebaudioside A or rebaudioside D against this baseline clone was limited to 100%.

[0909] The activity of each clone was defined as normalized activity compared to the baseline clone UGTSL2, while the specificity of each clone was expressed as the ratio between the initial rates of conversion to rebaudioside A and rebaudioside D.

[0910] The normalized rate of rebaudioside A conversion and the ratio between the initial rates of rebaudioside A and rebaudioside D conversion are described in the table below.

[0911]

[0912]

[0913] *Mutations are annotated as follows: Refer to gene-initial amino acid-position-new amino acid: For example, for UGTSL2, the mutation of isoleucine at position 240 to leucine is annotated as UGTSL2(I240L). Nd indicates not determined.

[0914] Modeling these results allows for a tiered approach to the effect of each mutation. The following mutations are identified as beneficial for activity:

[0915] N130G, H247P, F253Y, V270I, L276A, A285I, A285V, K301E, A341V, T392A, K408R, I412L.

[0916] The following mutations were identified as beneficial for increasing the ratio between the initial rates of rebaudioside A and rebaudioside D conversion:

[0917] I203L, S255C, I333L, A341V, H357Y, L393V, K408R, I412L.

[0918] Example 54

[0919] One-pot, four-enzyme conversion of rebaudioside A to rebaudioside M

[0920] 10 mL of a solution containing 5.0 mM rebaudioside A, 0.25 mM UDP, 2 mM MgCl2, 100 mM sucrose, 50 mM potassium phosphate buffer (pH 7.5), 2.5 U of UGTSL2-R3-D2 (UGTSL2-R3-var12, see Example 53), 25 U of UGT76G1-R3-G3 (UGT76G1-R3-var21, see Example 44), 25 U of AtSUS, and 5 U of [unclear - likely a specific compound or solution]. The reaction mixture was filtered through a 0.2 μm filter into a sterile flask. The resulting reaction mixture was then gently shaken at 30 °C for 65 hours.

[0921] Under aseptic conditions, 125 μL of the reaction mixture was obtained at regular intervals and quenched with 10 μL of 2N H2SO4 and 765 μL of 50% methanol. After centrifugation, 200 μL of the supernatant was analyzed by HPLC.

[0922] Obtained Figure 72a The reaction characteristics are shown in the figure. HPLC analysis after 48 hours of reaction shows... Figure 72b middle.

[0923] Example 55

[0924] One-pot, four-enzyme conversion of rebaudioside A to rebaudioside M

[0925] 10 mL of a solution containing 10.0 mM rebaudioside A, 0.50 mM UDP, 3 mM MgCl2, 100 mM sucrose, 50 mM potassium phosphate buffer (pH 7.5), 5.0 U UGTSL2-R3-D2 (UGTSL2-R3-var12, see Example 53), 50 U UGT76G1-R3-G3 (UGT76G1-R3-var21, see Example 44), 50 U AtSUS, and 10 U of [unclear - likely a specific compound or ingredient]. The reaction mixture was filtered through a 0.2 μm filter into a sterile flask. The resulting reaction mixture was then gently shaken at 30 °C for 66 hours.

[0926] Under aseptic conditions, 125 μL of the reaction mixture was obtained at regular intervals and quenched with 10 μL of 2N H2SO4 and 765 μL of 50% methanol. After centrifugation, 200 μL of the supernatant was analyzed by HPLC.

[0927] Obtained Figure 73a The reaction characteristics are shown in the figure. HPLC analysis after 48 hours of reaction shows... Figure 73b middle.

[0928] Example 56

[0929] One-pot, four-enzyme conversion of rebaudioside A to rebaudioside M

[0930] 50 mL of a buffer solution containing 10.0 mM rebaudioside A, 0.5 mM UDP, 4 mM MgCl2, 100 mM sucrose, 50 mM potassium phosphate buffer (pH 7.5), 25 U of UGTSL2-R3-D2 (UGTSL2-R3-var12, see Example 53), 250 U of UGT76G1-R3-G3 (UGT76G1-R3-var21, see Example 44), 250 U of AtSUS, and 50 U of [unclear - possibly a specific compound or ingredient]. The reaction mixture was filtered through a 0.2 μm filter into a sterile flask. The resulting reaction mixture was then gently shaken at 35 °C for 95 hours.

[0931] Under aseptic conditions, 125 μL of the reaction mixture was obtained at regular intervals and quenched with 10 μL of 2N H2SO4 and 765 μL of 50% methanol. After centrifugation, 200 μL of the supernatant was analyzed by HPLC.

[0932] At the end of the reaction, the reaction mixture became a fine suspension. Filtration of the suspension and HPLC analysis of the residue and filtrate showed that the Reb M content in the filtrate was 79% and the Reb M content in the solid was 97%.

[0933] Obtained Figure 74aThe reaction characteristics are shown in the figure. HPLC analysis of the reaction mixture after 95 hours shows... Figure 74b middle.

[0934] Example 57

[0935] One-pot, four-enzyme conversion of rebaudioside A to rebaudioside M (UGT76G1 and Isolase added after 6.5 hours).

[0936] The mixture containing rebaudioside A, UDP, MgCl2, sucrose, potassium phosphate buffer (pH 7.5), UGTSL2-R3-D2 (UGTSL2-R3-var12, see Example 53), and AtSUS was filtered into a sterile flask through a 0.2 μm filter. The resulting reaction mixture was gently shaken at 35°C for 6.5 hours. UGT76G1-R3-G3 (UGT76G1-R3-var21, see Example 44) and... The reaction mixture was filtered through a 0.2 μm filter into a sterile flask and gently shaken at 35 °C for 89 hours. The final volume of the reaction mixture was 50 mL, and the final concentrations of the reagents and enzymes were as follows: 10.0 mM rebaudioside A, 0.5 mM UDP, 4 mM MgCl2, 100 mM sucrose, 50 mM potassium phosphate buffer (pH 7.5), 25 U UGTSL2-R3-D2, 250 U UGT76G1-R3-G3, 250 U AtSUS, and 50 U of [unclear - possibly a specific enzyme or ingredient].

[0937] Under aseptic conditions, 125 μL of the reaction mixture was obtained at regular intervals and quenched with 10 μL of 2N H2SO4 and 765 μL of 50% methanol. After centrifugation, 200 μL of the supernatant was analyzed by HPLC.

[0938] Obtained Figure 75a The reaction characteristics are shown in the figure. HPLC analysis of the reaction mixture after 95 hours shows... Figure 75b middle.

[0939] Example 58

[0940] One-pot, four-enzyme conversion of rebaudioside A to rebaudioside M (UGT76G1 and Isolase added after 6.5 hours).

[0941] The mixture containing rebaudioside A, UDP, MgCl2, sucrose, potassium phosphate buffer (pH 7.5), UGTSL2-R3-D2 (UGTSL2-R3-var12, see Example 53), and AtSUS was filtered into a sterile flask through a 0.2 μm filter. The resulting reaction mixture was gently shaken at 35°C for 6.5 hours. UGT76G1-R3-G3 (UGT76G1-R3-var21, see Example 44) and... The reaction mixture was filtered through a 0.2 μm filter into a sterile flask and gently shaken at 35 °C for 89 hours. The final volume of the reaction mixture was 50 mL, and the final concentrations of the reagents and enzymes were as follows: 10.0 mM rebaudioside A, 0.5 mM UDP, 4 mM MgCl2, 100 mM sucrose, 50 mM potassium phosphate buffer (pH 7.5), 25 U UGTSL2-R3-D2, 250 U UGT76G1-R3-G3, 250 U AtSUS, and 25 U of [unclear - possibly a specific enzyme or ingredient].

[0942] Under aseptic conditions, 125 μL of the reaction mixture was obtained at regular intervals and quenched with 10 μL of 2N H2SO4 and 765 μL of 50% methanol. After centrifugation, 200 μL of the supernatant was analyzed by HPLC.

[0943] At the end of the reaction, the reaction mixture became a fine suspension. Filtration of the suspension and HPLC analysis of the residue and filtrate showed that the Reb M content in the filtrate was 81% and the Reb M content in the solids was 98%.

[0944] Obtained Figure 76a The reaction characteristics are shown in the figure. HPLC analysis of the reaction mixture after 95 hours shows... Figure 76b middle.

[0945] Example 59

[0946] UGTSL2 was used for directed evolution to convert rebaudioside A into rebaudioside D (round 4).

[0947] The most active enzyme UGTSL2_round3-var45 from round 3 (see Example 53) was used as the starting point. Five optimal mutations for activity from round 3 were used to form a set of 10 variants, each containing two of these mutations. After codon optimization for expression in *E. coli*, the gene was synthesized, subcloned in the pET30a+ plasmid, and used for transformation of *E. coli* BL21(DE3) chemocompetent cells. The resulting cells were grown on solid LB medium in Piper dishes in the presence of kanamycin. Suitable clones were selected and grown in liquid LB medium in test tubes. Glycerol was added to the suspension as a cryoprotectant, and 400 μL aliquots were stored at -20°C and -80°C.

[0948] The stored aliquots of *E. coli* BL21(DE3) containing the pET30a+_UGTSL2var 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). The culture was incubated at 30°C with shaking on 96 μL microtiter plates for 8 hours.

[0949] Inoculate 3.95 mL of Overnight Express solution with 50 μL of the above culture. TM Prepared TB culture medium Production medium containing 10 g / L glycerol and 50 mg / L kanamycin. The resulting culture was stirred at 20°C in 48-well plates. The culture showed significant growth and achieved a good OD (600 nm). After 44 hours, cells were collected by centrifugation and frozen.

[0950] By Master Mixture Lysis was initiated by adding the lysed cells, and the lysate was collected by centrifugation. The lysate was diluted five-fold with water prior to activity testing.

[0951] To measure the activity of the variant in converting rebaudioside A to rebaudioside D, 100 μL of fresh lysis product was added to a solution of rebaudioside A (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). The reaction was run at 30 °C, and samples were taken at 2, 4, 6, and 22 hours. The activity was determined by HPLC analysis (CAD detection) using the analytical method described above for the conversion of rebaudioside A to rebaudioside D.

[0952] Selectivity for each clone was determined by measuring the amount of rebaudioside M2 ​​formed under 100% UDP-Glc transformation (defined as (2*[Reb M2]+[Reb D]) / ([Reb A]+[Reb D]+[Reb M2]).

[0953] Experiments were conducted in parallel using the baseline clone UGTSL2-Round 3-Var45. The initial rate for this baseline clone was capped at 100%. The relative initial rates for the Round 4 clone and the amounts of rebaudioside M2 ​​formed under 100% UDP-Glc transformation are described in the table below:

[0954]

[0955] *Mutations are annotated as follows: Refer to gene-initial amino acid-position-new amino acid: For example, for UGTSL2, the mutation of isoleucine at position 240 to leucine is annotated as UGTSL2(I240L).

[0956] Example 60

[0957] UGT76G1 was used for directed evolution (round 4) to convert rebaudioside D into rebaudioside X.

[0958] The most active clone from the directed evolution of UGT76G1 in round 3 (see Example 44 Round 3 _UGT76G1var21, containing the mutation: I46L_K303G_K393R) was selected as the baseline clone for round 4. The best-identified mutations from round 3 (S119A, 274G, I295M, F314S, and K334R) were used to form a set of 10 variants, each containing two of these mutations. After codon optimization for expression in *E. coli*, the gene was synthesized, subcloned in the pET30a+ plasmid, and used for transformation of *E. coli* BL21(DE3) chemocompetent cells. The resulting cells were grown on solid LB medium in Piper dishes in the presence of kanamycin. Suitable clones were selected and grown in liquid LB medium in test tubes. Glycerol was added to the suspension as a cryoprotectant, and 400 μL aliquots were stored at -20°C and -80°C.

[0959] The stored aliquots of *E. coli* BL21(DE3) containing the pET30a+_UTG76G1var 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). The culture was incubated at 30°C with shaking on 96 μL microplates for 8 hours.

[0960] Inoculate 3.95 mL of Overnight Express solution with 50 μL of the above culture. TM Prepared TB culture medium Production medium containing 10 g / L glycerol and 50 mg / L kanamycin. The resulting culture was stirred at 20°C in 48-well plates. The culture showed significant growth and achieved a good OD (600 nm). After 44 hours, cells were collected by centrifugation and frozen.

[0961] By Master Mixture Lysis was performed by adding the lysed cells, and the lysate was collected by centrifugation. 100 μL of fresh lysate was added to a solution of lebodiin 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) for activity testing.

[0962] The reaction was run at 30°C, and samples were taken at 1, 2, 4, 6, and 22 hours. The conversion and initial rates were determined by HPLC analysis (CAD detection) using the analytical method described above for the conversion of rebaudioside D to rebaudioside X. Experiments were performed in parallel with the baseline clone, round 3 – Var21. The conversion and initial rates at 22 hours for this baseline clone were defined as 100%, and the normalized conversion and initial rates for the round 4 clone are described in the table below:

[0963]

[0964] *Mutations are annotated as follows: Refer to gene-initial amino acid-position-new amino acid: For example, for variant 1 from the fourth round of directed evolution of UGT76G1, the serine at position 119 is mutated to alanine, annotated as round 3-Var21(S119A).

[0965] It should be understood that the foregoing description and specific embodiments have fully disclosed, illustrated and provided the best mode and principles of the invention, and that those skilled in the art may make changes and additions without departing from the spirit and scope of the invention, which is limited only by the scope of the appended claims. Sequence Listing <110> Spectrum Technology Co., Ltd. <120> High-purity steviol glycosides <130> 39227-52WO <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 <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> Rice <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> Large intestinal bacteria <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 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> 13 <211> 808 <212> PRT <213> Arabidopsis thaliana <400> 13 Met Ala Asn Ala Glu Arg Met Ile Thr Arg Val His Ser Gln Arg Glu 1 5 10 15 Arg Leu Asn Glu Thr Leu Val Ser Glu Arg Asn Glu Val Leu Ala Leu 20 25 30 Leu Ser Arg Val Glu Ala Lys Gly Lys Gly Ile Leu Gln Gln Asn Gln 35 40 45 Ile Ile Ala Glu Phe Glu Ala Leu Pro Glu Gln Thr Arg Lys Lys Leu 50 55 60 Glu Gly Gly Pro Phe Phe Asp Leu Leu Lys Ser Thr Gln Glu Ala Ile 65 70 75 80 Val Leu Pro Pro Trp Val Ala Leu Ala Val Arg Pro Arg Pro Gly Val 85 90 95 Trp Glu Tyr Leu Arg Val Asn Leu His Ala Leu Val Val Glu Glu Leu 100 105 110 Gln Pro Ala Glu Phe Leu His Phe Lys Glu Glu Leu Val Asp Gly Val 115 120 125 Lys Asn Gly Asn Phe Thr Leu Glu Leu Asp Phe Glu Pro Phe Asn Ala 130 135 140 Ser Ile Pro Arg Pro Thr Leu His Lys Tyr Ile Gly Asn Gly Val Asp 145 150 155 160 Phe Leu Asn Arg His Leu Ser Ala Lys Leu Phe His Asp Lys Glu Ser 165 170 175 Leu Leu Pro Leu Leu 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. A method for producing a highly purified steviol glycoside composition, comprising the following steps: a. Provide reb A; b. Provides a UDP-glycosyltransferase and an enzyme with β-glucosidase activity, wherein the UDP-glycosyltransferase is selected from the UGT76G1 variant and the UGTSL2 variant, wherein the UGT76G1 variant is UGT76G1 with the following mutations: S42A, F46I, Q266E, P272A, R334K, G348P, L379G, I407V, I46L, K303G, and K393R, and the UGTSL2 variant is UGTSL2 with the following mutations: S255C, V270I, A285V, A341V, T392A, and I412L, and the enzyme with β-glucosidase activity is an isolase; c. Contact the UDP-glycosyltransferase and the enzyme having β-glucosidase activity with a culture medium containing reb A to produce a composition containing target steviol glycoside, wherein the target steviol glycoside is reb M; and d. Isolate the steviol glycosides from the culture medium to provide a highly purified steviol glycoside composition.

2. The method of claim 1, wherein target steviol glycosides are isolated from the culture medium by crystallization, membrane separation, centrifugation, extraction, chromatographic separation, or a combination of these methods.

3. The method of claim 1, wherein the highly purified steviol glycoside composition comprises steviol glycosides at a dry basis content of more than 95% by weight.

4. The highly purified steviol glycoside composition prepared by the method of claim 1, comprising steviol glycoside contents of more than 95% by weight on a dry basis.

5. The highly purified steviol glycoside composition prepared by the method of claim 1, wherein the steviol glycoside is polycrystalline.

6. A consumer product comprising a highly purified steviol glycoside composition prepared according to the method of claim 1, wherein the product is selected from food, beverage, pharmaceutical composition, tobacco product, nutritional supplement composition, oral hygiene composition, and cosmetic composition.

7. The method of claim 1, wherein the enzyme having β-glucosidase activity is used to hydrolyze reb D2 and / or reb M2.

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