WO2014193888A1

WO2014193888A1 - High-purity steviol glycosides

Info

Publication number: WO2014193888A1
Application number: PCT/US2014/039666
Authority: WO
Inventors: Avetik Markosyan; Indra Prakash; Cynthia Bunders; Pankaj SONI; Jarrin CYRILLE; Aurélien BADIE; Robert Ter Halle
Original assignee: Purecircle Sdn Bhd; The Coca-Cola Company
Priority date: 2013-05-28
Filing date: 2014-05-28
Publication date: 2014-12-04
Also published as: EP3004127A4; US10683526B2; US20190040434A1; CN112538512A; US9752174B2; EP3004128A4; CN105492453B; MX2015016362A; EP3004128A1; CN105492453A; US20210254116A1; CN105408339A; US20180100175A1; MX365044B; MX2019005688A; BR112015029684B1; BR112015030082A2; WO2014193934A1; CN105408339B; CA3089783C

Abstract

Methods of preparing highly purified steviol glycosides, particularly rebaudiosides A, D and M are described. The methods include utilizing recombinant microorganisms for converting various staring compositions to target steviol glycosides. In addition, novel steviol glycosides reb D2 and reb M2 are disclosed, as are methods of preparing the same. The highly purified rebaudiosides are useful as non-caloric sweetener in edible and chewable compositions such as any beverages, confectioneries, bakery products, cookies, and chewing gums.

Description

HIGH-PURITY STEVIOL GLYCOSIDES

TECHNICAL FIELD

The present invention relates to a biocatalytic process for preparing compositions comprising steviol glycosides, including highly purified steviol glycoside compositions. The present invention also relates to novel steviol glycosides, methods for isolation of the same and uses for the novel steviol glycosides.

BACKGROUND OF THE INVENTION

High intensity sweeteners possess a sweetness level that is many times greater than the sweetness level of sucrose. They are essentially non-caloric and are commonly used in diet and reduced-calorie products, including foods and beverages. High intensity sweeteners do not elicit a glycemic response, making them suitable for use in products targeted to diabetics and others interested in controlling for their intake of carbohydrates.

Steviol glycosides are a class of compounds found in the leaves of Stevia rebaudiana Bertoni, a perennial shrub of the Asteraceae (Compositae) family native to certain regions of South America. They are characterized structurally by a single base, steviol, differing by the presence of carbohydrate residues at positions CI 3 and CI 9. They accumulate in Stevia leaves, composing approximately 10% - 20% of the total dry weight. On a dry weight basis, the four major glycosides found in the leaves of Stevia typically include stevioside (9.1%), rebaudioside A (3.8%), rebaudioside C (0.6-1.0%) and dulcoside A (0.3%). Other known steviol glycosides include rebaudioside B, C, D, E, F and M, steviolbioside and rubusoside.

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

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

Additionally, there remains a need for novel steviol glycosides and methods of preparing and isolating the same. SUMMARY OF THE INVENTION

The present invention provides a biocatalytic process for preparing a composition comprising a target steviol glycoside by contacting a starting composition comprising an organic substrate with a microorganism and/or biocatalyst, thereby producing a composition comprising a target steviol glycoside.

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

The target steviol glycoside can be any steviol glycoside. In one embodiment, the target steviol glycoside is steviolmonoside, steviolbioside, rubusoside, dulcoside B, dulcoside A, rebaudioside B, rebaudioside G, stevioside, rebaudioside C, rebaudioside F, rebaudioside A, rebaudioside I, rebaudioside E, rebaudioside H, rebaudioside L, rebaudioside K, rebaudioside J rebaudioside , rebaudioside M2, rebaudioside D, rebaudioside D2, rebaudioside N, rebaudioside O or a synthetic steviol glycoside.

In one embodiment, the target steviol glycoside is stevioside.

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

In still another embodiment, the target steviol glycoside is rebaudioside D.

In yet another embodiment, the target steviol glycoside is rebaudioside M (also known as rebaudioside X).

The microorganism can be any microorganism possessing the necessary enzymes for converting the starting composition to target steviol glycosides.

The biocatalysts will comprise at least one enzyme for converting the starting composition to target steviol glycosides.

The biocatalysts can be located on the surface and/or inside the cell of the microorganism or can be secreted out of the microorganism.

The biocatalyst can be whole cell suspension, crude lysate or purified enzymes. The biocatalyst can be in free form or immobilized to a solid support made from inorganic or organic materials.

The enzymes necessary for converting the starting composition to target steviol glycosides include the steviol biosynthesis enzymes, UDP-glycosyltransferases (UGTs) and/or UDP-recycling enzyme.

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

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

In one embodiment the steviol biosynthesis enzymes are selected from the group including geranylgeranyl diphosphate synthase, copalyl diphosphate synthase, kaurene synthase, kaurene oxidase, kaurenoic acid 13-hydroxylase (KAH), steviol synthetase, deoxyxylulose 5 -phosphate synthase (DXS), D-l -deoxyxylulose 5-phosphate reductoisomerase (DXR), 4-diphosphocytidyl-2-C-methyl-D-erythritol synthase (CMS), 4- diphosphocytidyl-2-C-methyl-D-erythritol kinase (CMK), 4-diphosphocytidyl-2-C- methyl-D-erythritol 2,4- cyclodiphosphate synthase (MCS), l-hydroxy-2-methyl-2(E)- butenyl 4-diphosphate synthase (HDS), l-hydroxy-2-methyl-2(E)-butenyl 4-diphosphate reductase (HDR), acetoacetyl-CoA thiolase, truncated HMG-CoA reductase, mevalonate kinase, phosphomevalonate kinase, mevalonate pyrophosphate decarboxylase, cytochrome P450 reductase etc.

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

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

In one embodiment, the UDP-glucosyltransferase is selected from group including UGT74G1, UGT85C2, UGT76G 1, UGT91D2 and UGTs having substantial (>85%) identity to these polypeptides as well as isolated nucleic acid molecules that code for these UGTs.

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

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

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

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

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

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

In one embodiment, the recycling catalyst is sucrose synthase.

In one embodiment, the recycling substrate is sucrose. Optionally, the method of the present invention further comprises separating the target steviol glycoside from the starting composition. The target steviol glycoside can be separated by at least one suitable method, such as, for example, crystallization, separation by membranes, centrifugation, extraction, chromatographic separation or a combination of such methods.

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

In one embodiment, separation produces a composition comprising greater than about 80% by weight of the target steviol glycoside on an anhydrous basis, i.e., a highly purified steviol glycoside composition. In another embodiment, separation produces a composition comprising greater than about 90% by weight of the target steviol glycoside. In particular embodiments, the composition comprises greater than about 95% by weight of the target steviol glycoside. In other embodiments, the composition comprises greater than about 99% by weight of the target steviol glycoside.

The target steviol glycoside can be in any polymorphic or amorphous form, including hydrates, solvates, anhydrous or combinations thereof.

Purified target steviol glycosides can be used in consumable products as a sweetener. Suitable consumer products include, but are not limited to, food, beverages, pharmaceutical compositions, tobacco products, nutraceutical compositions, oral hygiene compositions, and cosmetic compositions.

The present invention also provides novel steviol glycosides rebaudioside D2 (reb D2, isomer of rebaudioside D) and rebaudioside M2 (reb M2, isomer of rebaudioside M) , 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 may also be present in any consumable products disclosed herein. In a particular embodiment, beverages comprising reb D2 and/or reb M2 are provided. Methods of preparing reb D2 and reb M2 are also provided herein. Both are formed during the biotransformation of reb A to reb D. Reb M2 is believed to form from biotransformation of reb D2 in situ.

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

In another embodiment, the present invention is a method for the preparation of a composition comprising reb M2 comprising (a) contacting a starting composition comprising reb D2 with an enzyme capable of transforming reb D2 to reb M2, UDP- glucose, and optionally UDP-glucose recycling enzymes, to produce a composition comprising reb M2, and (b) and isolating the composition comprising reb M2.

A further method for the preparation of a composition comprising reb M2 comprises (a) contacting a starting composition comprising reb A with an enzyme capable of transforming reb A to reb D2, UDP-glucose, and optionally UDP-glucose recycling enzymes, to produce a composition comprising reb D2, (b) optionally, isolating the composition comprising reb D2, (c) contacting the composition comprising reb D2 with an enzyme capable of transforming reb D2 to reb M2, UDP-glucose, and optionally UDP- glucose recycling enzymes to produce a composition comprising reb M2, and (d) isolating the composition comprising reb M2.

The composition can be further purified to provide reb D2 or reb M2 with purities greater than about 95% by weight on a dry basis.

BRIEF DESCRIPTION OF THE DRAWINGS

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 the embodiments of the invention.

FIG. 1 shows the structure of reb M.

FIG. 2 shows the biocatalytic production of reb from stevioside. FIG. 3 shows the biocatalytic production of reb A from stevioside using the enzyme UGT76G1 and concomitant recycling of UDP to UDP glucose via sucrose synthase.

FIG. 4 shows the IR spectrum of reb M.

FIG. 5. shows the HPLC chromatogram of the product of the biocatalytic production of reb M from reb D, as detailed in Example 14. The peak with retention time of 24.165 minutes corresponds to unreacted reb D. The peak with retention time of 31 .325 minutes corresponds to reb M.

FIG. 6. shows the HPLC chromatogram of purified reb M produced by biocatalysis from reb D.

FIG. 7 shows the HPLC chromatogram of a reb M standard.

FIG. 8 shows the HPLC chromatogram of co-injection of a reb M standard and reb M purified from biotransformation from reb D.

FIG. 9 shows an overlay of the Ή NMR spectra of a reb M standard and reb M purified following biosynthesis from reb D.

FIG. 10 shows the HRMS spectrum of reb M purified following biocatalytic production from reb D.

FIG. 1 1 shows LC-MS analysis of semi-synthetic steviol glycoside mixture, Lot number CB-2977-106, showing TIC (A), MS of peak at 1.8 min (B), MS of reb M2 peak at 4.1 min (C), MS of reb D peak at 6.0 min (D), MS of reb D2 peak at 7.7 min (E), MS of peak at 9.4 min (F), MS of rebaudioside Apeak at 15.2 min (G), MS of peak at 16.5 min (H), and MS of peak at 18.3 min (I).

FIG. 12 shows the trace of semi-synthetic steviol glycoside mixture, Lot number CB- 2977-106. Chromatogram gridlines are not homogeneous as the detector was re-calibrated 14 min following injection.

FIG. 13 shows HPLC analysis of semi-synthetic steviol glycoside mixture, Lot number CB-2977-106 (A), Isolated reb M2 (B), isolated reb D (C) and isolated reb D2 (D).

FIG. 14 shows the Ή NMR spectrum of reb D2 (500 MHz, pyridine-d₅). FIG. 15 shows the ¹³C NMR spectrum of reb 1)2(125 MHz, pyridine-d₅).

FIG. 16 shows an expansion of the ¹³C NMR spectrum of reb D2 (125 MHz, pyridine-d₅).

FIG. 17 shows the Ή-Ή COSY Spectrum of reb D2 (500 MHz, pyridine-d₅).

FIG. 18 shows the HSQC-DEPT spectrum of reb £>2(500 MHz, pyridine-d₅).

FIG. 19 shows the HMBC spectrum of reb D2.

FIG. 20 shows an expansion of HMBC spectrum of reb D2 (500 MHz, pyridine-ds).

FIG. 21 shows the Ή NMR spectrum of reb 2(500 MHz, D₂0).

FIG. 22 shows the ^{, 3}C NMR spectrum of reb M2 (125 MHz, D₂0/TSP).

FIG. 23 shows an expansion of the ¹³C NMR spectrum of reb M2 (125 MHz, D₂0/TSP).

FIG. 24 shows the Ή-Ή COSY spectrum of reb M2 (500 MHz, D₂0).

FIG. 25 shows the HSQC-DEPT spectrum of reb 2(500 MHz, D₂0).

FIG. 26 shows the HMBC spectrum of reb M2 (500 MHz, D₂0).

FIG. 27 shows an expansion of HMBC spectrum of reb M2 (500 MHz, D₂0).

FIG. 28 shows another HMBC spectrum of reb M2.

FIG. 29 shows aΉ NMR spectrum of reb M2.

FIG. 30 shows a ¹³C NMR spectrum of reb M2 .

FIG. 31 shows another ¹³C NMR spectrum of reb M2.

FIG. 32 shows aΉ-Ή COSY spectrum of reb M2.

FIG. 33 shows a HSQC-DEPT spectrum of reb M2.

FIG. 34 shows an HMBC spectrum of reb M2.

FIG. 35 shows another HMBC spectrum of reb M2. FIG. 36 shows a ID-TOCSY spectrum of reb M2. FIG. 37 shows a ID-TOCSY spectrum of reb M2. FIG. 38 shows a ID-TOCSY spectrum of reb M2. FIG. 39 shows a ID-TOCSY spectrum of reb M2. FIG. 40 shows an HPLC (CAD) analysis.

FIG. 41 shows an HPLC (CAD) analysis.

FIG. 42 shows an HPLC (CAD) analysis.

FIG. 43 shows an HPLC (CAD) analysis.

FIG. 44 shows an HPLC (CAD) analysis.

FIG. 45 shows an HPLC (CAD) analysis.

FIG. 46 shows an HPLC (CAD) analysis.

FIG. 47 shows an HPLC (CAD) analysis.

FIG. 48 shows an HPLC (CAD) analysis.

FIG. 49 shows an HPLC (CAD) analysis.

FIG. 50 shows an HPLC (CAD) analysis.

FIG. 51 shows an HPLC (CAD) analysis.

FIG. 52 shows an HPLC (CAD) analysis.

FIG. 53 shows an LCMS chromatogram.

FIG. 54 shows an LCMS chromatogram.

FIG. 55 shows an LCMS chromatogram.

FIG. 56 shows an LCMS chromatogram. FIG. 57 shows a reaction profile. FIG. 58 shows an HPLC (CAD) analysis. FIG. 59 shows an HPLC (CAD) analysis. FIG. 60 shows an HPLC (CAD) analysis. FIG. 61 shows an HPLC (CAD) analysis. FIG. 62 shows an HPLC (CAD) analysis. FIG. 63 shows an LCMS chromatogram. FIG. 64 shows an HPLC (CAD) analysis. FIG. 65 shows an HPLC (CAD) analysis. FIG. 66 shows an HPLC (CAD) analysis. FIG. 67 shows an HPLC (CAD) analysis. FIG. 68 shows an HPLC (CAD) analysis. FIG. 69 shows the results of an HPLC analysis.

DETAILED DESCRIPTION

The present invention provides a biocatalytic process for preparing a composition comprising a target steviol glycoside by contacting a starting composition, comprising an organic substrate, with a microorganism and/or biocatalyst, thereby producing a composition comprising a target steviol glycoside.

One object of the invention is to provide an efficient biocatalytic method for preparing steviol glycosides, particularly stevioside, reb E, reb A, reb D, reb D2, reb M, and reb M2 from various starting compositions. As used herein, "biocatalysis" or "biocatalytic" refers to the use of natural or genetically engineered biocatalysts, such as cells, protein enzymes, to perform single or multiple step chemical transformations on organic compounds. Biocatalysis include fermentation, biosynthesis and biotransformation processes. Both, isolated enzyme and whole-cell biocatalysis methods, using biocatalysts in free as well as immobilized forms, are known in the art. Biocatalyst protein enzymes can be naturally occurring or recombinant proteins.

As used herein, the term "steviol glycoside(s)" refers to a glycoside of steviol, including, but not limited to, naturally occurring steviol glycosides, e.g. steviolmonoside, steviolbioside, rubusoside, dulcoside B, dulcoside A, rebaudioside B, rebaudioside G, stevioside, rebaudioside C, rebaudioside F, rebaudioside A, rebaudioside /, rebaudioside E, rebaudioside H, rebaudioside L, rebaudioside K, rebaudioside J rebaudioside , rebaudioside M2, rebaudioside D, rebaudioside D2, rebaudioside N, rebaudioside O, synthetic steviol glycosides, e.g. enzymatically glucosylated steviol glycosides and combinations thereof.

Chemical structures of steviol and its glycosides

Compound Ri R₂

Steviol H H

Steviolmonoside H β-Glc

Steviol monoglucosyl ester β-Glc H

Rubusoside β-Glc β-Glc

Steviolbioside H β-Glc-p-Glc (2→1)

Stevioside β-Glc β-ϋΐο-β-θΐο (2→1)

Rebaudioside A β-Glc β-ϋΐοβ-αο (2→1 ) β-Glc (3→1)

Rebaudioside D β-Glc-p-Glc (2→1) β-ΰΐο-β-θΐο (2→1) β-Glc (3→1 )

Rebaudioside E β-Glc-p-Glc (2→1 ) β-ϋΐοβ-θΐο (2→1)

Rebaudioside M β-σΐο-β-σΐο (2→1) β-αοβ-ϋΐο (2→1) β-Glc (3→1) β-Glc (3→1)

(Glc=glucose)

Starting Composition

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

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

The term "polyol" refers to a molecule that contains more than one hydroxyl group. A polyol may be a diol, triol, or a tetraol which contain 2, 3, and 4 hydroxyl groups, respectively. A polyol also may contain more than four hydroxyl groups, such as a pentaol, hexaol, heptaol, or the like, which contain 5, 6, or 7 hydroxyl groups, respectively. Additionally, a polyol also may be a sugar alcohol, polyhydric alcohol, or polyalcohol which is a reduced form of carbohydrate, wherein the carbonyl group (aldehyde or ketone, reducing sugar) has been reduced to a primary or secondary hydroxyl group. Examples of polyols include, but are not limited to, erythritol, maltitol, mannitol, sorbitol, lactitol, xylitol, inositol, isomalt, propylene glycol, glycerol, threitol, galactitol, hydrogenated isomaltulose, reduced isomalto-oligosaccharides, reduced xylo- oligosaccharides, reduced gentio-oligosaccharides, reduced maltose syrup, reduced glucose syrup, hydrogenated starch hydrolyzates, polyglycitols and sugar alcohols or any other carbohydrates capable of being reduced.

The term "carbohydrate" refers to aldehyde or ketone compounds substituted with multiple hydroxyl groups, of the general formula (CH₂0)_n, wherein n is 3-30, as well as their oligomers and polymers. The carbohydrates of the present invention can, in addition, be substituted or deoxygenated at one or more positions. Carbohydrates, as used herein, encompass unmodified carbohydrates, carbohydrate derivatives, substituted carbohydrates, and modified carbohydrates. As used herein, the phrases "carbohydrate derivatives", "substituted carbohydrate", and "modified carbohydrates" are synonymous. Modified carbohydrate means any carbohydrate wherein at least one atom has been added, removed, or substituted, or combinations thereof. Thus, carbohydrate derivatives or substituted carbohydrates include substituted and unsubstituted monosaccharides, disaccharides, oligosaccharides, and polysaccharides. The carbohydrate derivatives or substituted carbohydrates optionally can be deoxygenated at any corresponding C-position, and/or substituted with one or more moieties such as hydrogen, halogen, haloalkyl, carboxyl, acyl, acyloxy, amino, amido, carboxyl derivatives, alkylamino, dialkylamino, arylamino, alkoxy, aryloxy, nitro, cyano, sulfo, mercapto, imino, sulfonyl, sulfenyl, sulfinyl, sulfamoyl, carboalkoxy, carboxamido, phosphonyl, phosphinyl, phosphoryl, phosphino, thioester, thioether, oximino, hydrazino, carbamyl, phospho, phosphonato, or any other viable functional group provided the carbohydrate derivative or substituted carbohydrate functions to improve the sweet taste of the sweetener composition.

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

The starting composition may be synthetic or purified (partially or entirely), commercially available or prepared.

In one embodiment, the starting composition is glycerol.

In another embodiment, the starting composition is glucose.

In still another embodiment, the starting composition is sucrose.

In yet another embodiment, the starting composition is starch.

In another embodiment, the starting composition is maltodextrin.

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

Target Steviol Glycoside

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

In one embodiment, the target steviol glycoside is stevioside. In another embodiment, the target steviol glycoside is reb A. In still 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 a still further another embodiment, the target steviol glycoside is reb M2.

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

In yet another embodiment, the present invention is a biocatalytic process for the production of reb D2.

In still another embodiment, the present invention is a biocatalytic process for the production of reb M.

In a further embodiment, the present invention is a biocatalytic process for the production of reb M2.

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

In particular embodiments, the process described herein results in a highly purified target steviol glycoside composition. The term "highly purified", as used herein, refers to a composition having greater than about 80% by weight of the target steviol glycoside on an anhydrous basis. In one embodiment, the highly purified target steviol glycoside composition contains greater than about 90% by weight of the target steviol glycoside on an anhydrous basis, such as, for example, greater than about 91%, greater than about 92%, greater than about 93%, greater than about 94%, greater than about 95%, greater than about 96%, greater than about 97%, greater than about 98% or greater than about 99% target steviol glycoside content on a dry basis.

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

In another embodiment, when the target steviol glycoside is reb M2, the process described herein provides a composition having greater than about 90% reb M2 content by weight on a dry basis. In another particular embodiment, when the target steviol glycoside is reb M2, the process described herein provides a composition comprising greater than about 95% reb M2 content by weight on a dry basis.

In yet another embodiment, when the target steviol glycoside is reb D, the process described herein provides a composition greater than about 90% reb D content by weight on a dry basis. In another particular embodiment, when the target steviol glycoside is reb D, the process described herein provides a composition comprising greater than about 99% reb D content by weight on a dry basis.

In still another embodiment, when the target steviol glycoside is reb D2, the process described herein provides a composition greater than about 90% reb D2 content by weight on a dry basis. In another particular embodiment, when the target steviol glycoside is reb D2, the process described herein provides a composition comprising greater than about 95%) reb D2 content by weight on a dry basis.

In a further embodiment, when the target steviol glycoside is reb A, the process described herein provides a composition comprising greater than about 90% reb A content by weight on a dry basis. In another particular embodiment, when the target steviol glycoside is reb A, the process described herein provides a composition comprising greater than about 95% reb A content by weight on a dry basis.

In a still further embodiment, when the target steviol glycoside is reb E, the process described herein provides a composition comprising greater than about 90% reb E content by weight on a dry basis. In another particular embodiment, when the target steviol glycoside is reb E, the process described herein provides a composition comprising greater than about 95% reb E content by weight on a dry basis.

In a still further embodiment, when the target steviol glycoside is reb I, the process described herein provides a composition comprising greater than about 90% reb / content by weight on a dry basis. In another particular embodiment, when the target steviol glycoside is reb /, the process described herein provides a composition comprising greater than about 95% reb / content by weight on a dry basis.

In yet a further embodiment, when the target steviol glycoside is stevioside, the process described herein provides a composition comprising greater than about 90% stevioside content by weight on a dry basis. In another particular embodiment, when the target steviol glycoside is stevioside, the process described herein provides a composition comprising greater than about 95% stevioside content by weight on a dry basis.

Microorganism and biocatalysts

In one embodiment of present invention, a microorganism or biocatalyst is contacted with the starting composition to produce target steviol glycosides. The microorganism can be any microorganism possessing the necessary enzymes for converting the starting composition to target steviol glycosides. These enzymes are encoded within the microorganism's genome.

In one embodiment the microoganism may be, for example, E.coli, Saccharomyces sp., Aspergillus sp., Pichia sp,. Bacillus sp., Yarrowia sp. etc.

The enzymes can be located on the surface and/or inside the cell of the microorganism and/or can be secreted out in the medium by the microorganism.

The biocatalyst comprises at least one enzyme and can be whole cell suspension, crude lysate or purified enzyme.

The enzymes necessary for converting the starting composition to target steviol glycosides include the steviol biosynthesis enzymes and UDP-glycosyltransferases (UGTs). Optionally it may include UDP recycling enzyme(s). The UDP recycling enzyme can be sucrose synthase and the recycling substrate can be sucrose. In one embodiment the steviol biosynthesis enzymes include mevalonate (MVA) pathway enzymes.

In one embodiment the steviol biosynthesis enzymes are selected from the group including geranylgeranyl diphosphate synthase, copalyl diphosphate synthase, kaurene synthase, kaurene oxidase, kaurenoic acid 13-hydroxylase (KAH), steviol synthetase, deoxyxylulose 5 -phosphate synthase (DXS), D-l-deoxyxylulose 5-phosphate reductoisomerase (DXR), 4-diphosphocytidyl-2-C-methyl-D-erythritol synthase (CMS), 4- diphosphocytidyl-2-C-methyl-D-erythritol kinase (CMK), 4-diphosphocytidyl-2-C- methyl-D-erythritol 2,4- cyclodiphosphate synthase (MCS), l-hydroxy-2-methyl-2(E)- butenyl 4-diphosphate synthase (HDS), l-hydroxy-2-methyl-2(E)-butenyl 4-diphosphate reductase (HDR), acetoacetyl-CoA thiolase, truncated HMG-CoA reductase, mevalonate kinase, phosphomevalonate kinase, mevalonate pyrophosphate decarboxylase, cytochrome P450 reductase etc.

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

In one embodiment the microorganism is in aqueous medium, comprising water, and various components selected form group including carbon sources, energy sources, nitrogen sources, microelements, vitamins, nucleosides, nucleoside phosphates, nucleoside diphosphates, nucleoside triphosphates, organic and inorganic salts, organic and mineral acids, bases etc. Carbon sources include glycerol, glucose, carbon dioxide, carbonates, bicarbonates. Nitrogen sources can include nitrates, nitrites, amino acids, peptides, peptones, or proteins.

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

In one embodiment, the medium can also include an organic solvent.

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

In another embodiment, the UDP-glucosyltransferase is any UDP- glucosyltransferase capable of adding at least one glucose unit to rubusoside, thereby producing rebaudioside E. The UDP-glucosyltransferase may be, for example, UGTSL2.

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

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

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

In another embodiment, the UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside A is selected from the following listing of Genlnfo identifier numbers, preferably from the group presented in Table 1 , and more preferably the group presented in Table 2.

397567 30680413 1 15480946 147798902 218193594 225443294

454245 32816174 1 16310259 14781 1764 218193942 225444853

1359905 32816178 1 16310985 147827151 219885307 225449296 1685003 34393978 1 16788066 147836230 222615927 225449700

1685005 37993665 1 16788606 147839909 222619587 225454338

2191 136 37993671 1 16789315 147846163 222623142 225454340

2501497 37993675 1 19394507 147855977 222625633 225454342

291 1049 39104603 1 19640480 148905778 222625635 225454473

4218003 41469414 122209731 148905999 222636620 225454475

4314356 41469452 125526997 148906835 222636621 225458362

13492674 42566366 125534279 148907340 222636628 225461551

13492676 42570280 125534461 148908935 222636629 225461556

15217773 42572855 125540090 148909182 224053242 225461558

15217796 44890129 125541516 148909920 224053386 225469538

15223396 46806235 125545408 148910082 224055535 225469540

15223589 50284482 125547340 148910154 224056138 226316457

15227766 51090402 125547520 148910612 224056160 226492603

15230017 51090594 125554547 148910769 224067918 226494221

1 5231757 52839682 125557592 156138791 224072747 226495389

15234056 56550539 125557593 156138797 224080189 226495945

15234195 62734263 125557608 156138799 224091845 226502400

15234196 62857204 125559566 156138803 224094703 226507980

15238503 62857206 125563266 165972256 224100653 226531 147

15239523 62857210 125571055 168016721 224100657 226532094

15239525 62857212 125579728 171674071 224101569 238477377

15239543 75265643 125588307 171906258 224103105 240254512

15239937 75285934 125589492 183013901 224103633 242032615

15240305 75288884 125599469 183013903 224103637 242032621

15240534 77550661 125601477 186478321 224109218 242038423

15982889 77556148 126635837 187373030 2241 14583 242043290

18086351 82791223 126635845 187373042 2241 16284 242044836

18418378 83778990 126635847 190692175 224120552 242051252

18418380 89953335 126635863 194701936 224121288 242056217

18418382 1 10741436 126635867 195620060 224121296 242056219

19743740 1 10743955 126635883 209954691 224121300 242056663

1991 1201 1 15438196 126635887 209954719 224130358 242059339

20149064 1 15438785 133874210 209954725 224140703 242059341

20260654 1 15441237 133874212 209954733 224143404 242060922

21435782 1 15454819 145358033 210063105 224143406 24206741 1

21553613 1 15456047 147772508 210063107 224144306 242067413

21593514 1 15457492 147776893 212275846 224285244 242076258

22759895 1 15459312 147776894 216296854 225431707 242076396

23955910 1 15464719 147776895 217074506 225435532 242084750

26452040 1 15471069 147786916 218185693 225436321 242091005

28393204 1 15471071 147798900 218187075 225440041 242095206

30679796 1 15474009 147798901 218189427 225441 1 16 242345159

242345161 297724601 326492035 356523945 357140904 359486938

255536859 297725463 326493430 356523957 357165849 359487055

255538228 297728331 326500410 356523959 357165852 359488135

255541676 297738632 326506816 356523961 357168415 359488708

255547075 297745347 326507826 356523963 357437837 359493630

255552620 297745348 326508394 356524387 357442755 359493632

255552622 297795735 326509445 356524403 357442757 359493634 255555343 297796253 32651 1261 356527181 357445729 359493636

255555361 297796257 32651 1866 356533209 357445731 359493815

255555363 297796261 326512412 356533852 357445733 359495856

255555365 297797587 326517673 356534718 357446799 359495858

255555369 297798502 326518800 356535480 357446805 359495869

255555373 297799226 326521 124 356542996 357452779 359495871

255555377 297805988 326525567 356543136 357452781 359497638

255556812 297807499 326525957 356543932 357452783 359807261

255556818 297809125 326526607 356549841 357452787 374256637

255563008 297809127 326527141 356549843 357452789 377655465

255564074 29781 1403 326530093 356554358 357452791 378405177

255564531 297820040 326534036 356554360 357452797 378829085

255572878 297821483 326534312 356558606 357452799 387135070

255577901 297825217 332071 132 356560333 357470367 387135072

255583249 297832276 339715876 356560599 357472193 387135078

255583253 297832280 342306012 356560749 357472195 387135092

255583255 297832518 342306016 356566018 357474295 387135094

255585664 297832520 343457675 356566169 357474493 387135098

255585666 297840825 343457677 356566173 357474497 387135100

255634688 297840827 350534960 356567761 357474499 387135134

255644801 297847402 356498085 356574704 357490035 387135136

255645821 297849372 356499771 356576401 357493567 387135174

255647456 300078590 356499777 356577660 357497139 387135176

255648275 300669727 356499779 3571 14993 357497581 387135184

260279126 302142947 356501328 3571 15447 357497671 387135186

260279128 302142948 356502523 3571 15451 357500579 387135188

261343326 302142950 356503180 3571 15453 357504663 387135190

283132367 302142951 356503184 3571 16080 357504691 387135192

2833621 12 302765302 356503295 3571 16928 357504699 387135194

289188052 302796334 356504436 3571 17461 357504707 387135282

295841350 30281 1470 356504523 3571 17463 357505859 387135284

296088529 302821 107 356504765 3571 17829 357510851 387135294

296090415 302821679 35651 1 1 13 3571 17839 357516975 387135298

296090524 319759260 356515120 357125059 359477003 387135300

296090526 319759266 356517088 357126015 359477998 387135302

297599503 320148814 356520732 357134488 359478043 387135304

297601531 326489963 356522586 357135657 359478286 387135312

29761 1791 326490273 356522588 357138503 359484299 387135314

297722841 326491 131 356522590 357139683 359486936 387135316

387135318 449440433 460376293 460413408 462423864 475546199

387135320 449445896 460378310 460416351 470101924 475556485

387135322 449446454 460380744 462394387 470102280 475559699

387135324 449447657 460381726 462394433 470102858 475578293

387135326 449449002 460382093 462394557 47010421 1 475591753

387135328 449449004 460382095 462395646 470104264 475593742

388493506 449449006 460382754 462395678 470104266 475612072

388495496 449451379 460384935 462396388 470106317 475622476

388498446 449451589 460384937 462396389 470106357 475622507

388499220 449451591 460385076 462396419 4701 15448 475623787

388502176 449451593 460385872 462396542 470130404 482550481 388517521 449453712 460386018 462397507 470131550 482550499

388519407 449453714 460389217 462399998 470136482 482550740

388521413 449453716 460394872 462400798 470136484 482550999

388827901 449453732 460396139 462401217 470136488 482552352

388827903 449457075 460397862 4624021 18 470136492 482554970

388827907 449467555 460397864 462402237 470137933 482555336

388827909 449468742 460398541 462402284 470137937 482555478

388827913 449495638 460403139 462402416 470140422 482556454

393887637 449495736 460403141 462404228 470140426 482557289

393887646 449499880 460403143 462406358 470140908 482558462

393887649 449502786 460403145 462408262 470141232 482558508

393990627 449503471 460405998 462409325 470142008 482558547

397746860 449503473 460407578 462409359 470142010 482561055

397789318 449515857 460407590 462409777 470142012 482561555

413924864 449518643 460409128 46241 1467 470143607 482562795

414590349 449519559 460409134 46241431 1 470143939 482562850

414590661 449522783 460409136 462414416 470145404 482565074

414591 157 449524530 460409459 462414476 473923244 482566269

414879558 449524591 460409461 462415526 4741 14354 482566296

414879559 449528823 460409463 462415603 474143634 482566307

414879560 449528825 460409465 462415731 474202268 482568689

414888074 449534021 460409467 462416307 474299266 482570049

431812559 460365546 460410124 462416920 4743631 19 482570572

449432064 460366882 460410126 462416922 474366157 482575121

449432066 460369823 460410128 462416923 474429346

449433069 460369829 460410130 462416924 475432777

449436944 460369831 460410132 462417401 475473002

449438665 460369833 460410134 462419769 475489790

449438667 460370755 460410213 462420317 47551 1330

449440431 460374714 46041 1200 462423366 475516200

Table 1

GI number Accession Origin

190692175 ACE87855.1 Stevia rebaudiana

41469452 AAS07253.1 Oryza sativa

62857204 BAD95881.1 Ipomoea nil

62857206 BAD95882.1 Ipomoea purperea

56550539 BAD77944.1 Bellis perennis

115454819 NP 001051010.1 Oryza sativa Japonica Group

115459312 NP 001053256.1 Oryza sativa Japonica Group

115471069 NP 001059133.1 Oryza sativa Japonica Group

115471071 NP 001059134.1 Oryza sativa Japonica Group

116310985 CAH67920.1 Oryza sativa Indica Group

116788066 AB 24743.1 Picea sitchensis

122209731 Q2V6J9.1 Fragaria x ananassa

125534461 EAY81009.1 Oryza sativa Indica Group

125559566 EAZ05102.1 Oryza sativa Indica Group

125588307 EAZ28971.1 Oryza sativa Japonica Group 148907340 AB 16806.1 Picea sitchensis

148910082 ABR18123.1 Picea sitchensis

148910612 ABR18376.1 Picea sitchensis

15234195 NP 194486.1 Arabidopsis thaliana

15239523 NP 200210.1 Arabidopsis thaliana

15239937 NP 196793.1 Arabidopsis thaliana

1685005 AAB36653.1 Nicotiana tabacum

183013903 ACC38471.1 Medicago truncatula

186478321 NP 17251 1.3 Arabidopsis thaliana

187373030 ACD03249.1 Avena strigosa

194701936 ACF85052.1 Zea mays

19743740 AAL92461.1 Solanum lycopersicum

212275846 NP 001 131009.1 Zea mays

222619587 EEE55719.1 Oryza sativa Japonica Group

224055535 XP 002298527.1 Populus trichocarpa

224101569 XP 002334266.1 Populus trichocarpa

224120552 XP 002318358.1 Populus trichocarpa

224121288 XP 002330790.1 Populus trichocarpa

225444853 XP 002281094 Vitis vinifera

225454342 XP 002275850.1 Vitis vinifera

225454475 XP 002280923.1 Vitis vinifera

225461556 XP 002285222 Vitis vinifera

225469540 XP 002270294.1 Vitis vinifera

226495389 NP 001 148083.1 Zea mays

226502400 NP 001 147674.1 Zea mays

238477377 ACR43489.1 Triticum aestivum

240254512 NP 565540.4 Arabidopsis thaliana

2501497 Q43716.1 Petunia x hybrida

255555369 XP 002518721.1 Ricinus communis

26452040 BAC43110.1 Arabidopsis thaliana

296088529 CBI37520.3 Vitis vinifera

29761 1791 NP 001067852.2 Oryza sativa Japonica Group

297795735 XP__002865752.1 Arabidopsis lyrata subsp. lyrata

297798502 XP 002867135.1 Arabidopsis lyrata subsp. lyrata

297820040 XP 002877903.1 Arabidopsis lyrata subsp. lyrata

297832276 XP 002884020.1 Arabidopsis lyrata subsp. lyrata

302821 107 XP 002992218.1 Selaginella moellendorffii

30680413 NP 179446.2 Arabidopsis thaliana

319759266 ADV71369.1 Pueraria montana var. I o bat a

326507826 BAJ86656.1 Hordeum vulgare subsp. Vulgare

343457675 AEM37036.1 Brassica rapa subsp. oleifera

350534960 NP 001234680.1 Solanum lycopersicum

356501328 XP 003519477.1 Glycine max

356522586 XP 003529927.1 Glycine max

356535480 XP 003536273.1 Glycine max

357445733 XP 003593144.1 Medicago truncatula

357452783 XP 003596668.1 Medicago truncatula

357474493 XP 003607531.1 Medicago truncatula

357500579 XP 003620578.1 Medicago truncatula

357504691 XP 003622634.1 Medicago truncatula

359477998 XP 003632051.1 Vitis vinifera

359487055 XP 002271587 Vitis vinifera

359495869 XP 003635104.1 Vitis vinifera

387135134 AFJ52948.1 Linum usitatissimum

387135176 AFJ52969.1 Linum usitatissimum

387135192 AFJ52977.1 Linum usitatissimum

387135282 AFJ53022.1 Linum usitatissimum 387135302 AFJ53032.1 Linum usitatissimum

387135312 AFJ53037.1 Linum usitatissimum

388519407 AFK47765.1 Medicago Iruncatula

393887646 AFN26668.1 Barbarea vulgaris subsp. arcuata

414888074 DAA64088.1 Zea mays

42572855 NP 974524.1 Arabidopsis thaliana

449440433 XP 004137989.1 Cucumis sativus

449446454 XP 004140986.1 Cucumis sativus

449449004 XP 004142255.1 Cucumis sativus

449451593 XP 004143546.1 Cucumis sativus

449515857 XP 004164964.1 Cucumis sativus

460382095 XP 004236775.1 Solarium lycopersicum

460409128 XP 004249992.1 Solarium lycopersicum

460409461 XP 004250157.1 Solarium lycopersicum

460409465 XP 004250159.1 Solarium lycopersicum

462396388 EMJ02187.1 Prunus persica

462402118 EMJ07675.1 Prunus persica

462409359 EMJ 14693.1 Prunus persica

462416923 EMJ21660.1 Prunus persica

46806235 BAD 17459.1 Oryza sativa Japonica Group

470104266 XP 004288529.1 Fragaria vesca subsp. vesca

470142008 XP 004306714.1 Fragaria vesca subsp. vesca

475432777 EMT01232.1 Aegilops tauschii

51090402 BAD35324.1 Oryza sativa Japonica Group

Table 2

In yet another embodiment, the UDP-glucosyltransferase is any UDP- glucosyltransferase capable of adding at least one glucose unit to rebaudioside D to form rebaudioside M and/or rebaudioside M2. The UDP-glucosyltransferase may be, for example, UGT76G1.

Optionally, the method of the present invention further comprises recycling UDP to provide UDP-glucose. In one embodiment, the method comprises recycling UDP by providing a recycling catalyst, i.e., a biocatalyst capable of UDP-glucose overproduction, and a recycling substrate, such that the conversion of the substrate steviol glycoside to the target steviol glycoside is carried out using catalytic amounts of UDP-glucosyltransferase and UDP-glucose (FIG. 3).

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

In one embodiment, the recycling substrate is sucrose.

In one embodiment the biocatalyst comprises more than one UDP- gl ucosy transferase .

In embodiment the biocatalyst comprises more than one UDP-glucosyltransferase and UDP-glucose recycling catalyst.

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

Compounds and Methods

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

13-[(2-0-p-D-glucopyranosyI-3-0- -D-glucopyranosyl- -D-glucopyranosyl)oxy] e/7f-kaur-16- en-19-oic acid-[(6-0- -D-glucopyranosyl- -D-glucopyranosyl) ester]

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

In still another embodiment, the present invention provides reb D2 having a purity greater than about 95% by weight in a steviol glycoside mixture, such as, for example, greater than about 96%> by weight, greater than about 97% by weight, greater than about 98%o by weight or greater than about 99% by weight.

The present invention also provides compositions comprising reb D2.

In one embodiment, the present invention provides a method for preparing reb D2 comprising: a. contacting a starting composition comprising reb A with an enzyme capable of transforming reb A to reb D2, UDP-glucose, and optionally UDP-glucose recycling enzymes, to produce a composition comprising reb D2; and b. isolating the composition comprising reb D2. In some embodiments, the enzyme capable of transforming 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. 1 15454819 version NP_001051010.1 , GI No.

187373030, version ACD03249.1. GI No. 222619587 version EEE55719.1 , GI No.

297795735 version XP_002865752.1 or EUGT1 1.

The enzyme capable of transforming reb A to reb D2 can be immobilized or in a recombinant microorganism.

In one embodiment, the enzyme is immobilized. In another embodiment, the enzyme is in a recombinant microorganism.

Suitable microorganisms include, but are not limited to, E.coli, Saccharomyces sp., Aspergillus sp., Pichia sp., Bacillus sp., Yarrow ia sp.

In one embodiment the microorganism is in an aqueous medium, comprising water, and various components selected form group including carbon sources, energy sources, nitrogen sources, microelements, vitamins, nucleosides, nucleoside phosphates, nucleoside diphosphates, nucleoside triphosphates, organic and inorganic salts, organic and mineral acids, bases etc. Carbon sources include glycerol, glucose, carbon dioxide, carbonates, bicarbonates. Nitrogen sources can include nitrates, nitrites, amino acids, peptides, peptones, or proteins.

In a particular embodiment, the medium comprises buffer. Suitable buffers include, but are not limited to, PIPES buffer, acetate buffer and phosphate buffer. In a particular embodiment, the medium comprises phosphate buffer. In one embodiment the medium can also include an organic solvent.

In a particular embodiment, the enzyme is a UDP-glucosyltransferase capable of transforming reb A to reb D2.

In a more particular embodiment, the enzyme is selected from UGT91D2, UGTSL, UGTSL_Sc, UGTSL2 (GI No. 460410132 version XP_004250485.1), GI No. 460409128 (UGTSL) verison XP_004249992.1, GI No. 1 15454819 version NP 001051010.1, GI No. 187373030, version ACD03249.1. GI No. 222619587 version EEE55719.1, GI No. 297795735 version XP_002865752.1 or EUGT1 1 and UGTs having substantial (>85%) sequence identity to these.

In a still more particular embodiment, the enzyme is UGTSL2 or its improved variant produced by directed evolution and having higher activity.

Isolation of reb D2 from the reaction medium can be achieved by any suitable method to provide a composition comprising reb D2. Suitable methods include, but are not limited to, lysis, crystallization, separation by membranes, centrifugation, extraction (liquid or solid phase), chromatographic separation, HPLC (preparative or analytical) or a combination of such methods. In a particular embodiment, isolation can be achieved by lysis and centrifugation.

In some embodiments, isolation may result in a reb D2 purity less than about 95% by weight on an anhydrous basis, and the composition may contain, e.g., steviol glycosides and/or residual reaction products. The composition comprising reb D2 can be further purified to provide highly purified reb D2, i.e. reb D2 having a purity greater than about 95% by weight on an anhydrous basis. In some embodiments, the compositions comprising reb D2 can be further purified to provide reb D2 having 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.

Purification can be affected by any means known to one of skill in the art including, but not limited to, crystallization, separation by membranes, centrifugation, extraction (liquid or solid phase), chromatographic separation, HPLC (preparative or analytical) or a combination of such 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.

For example, a two-step semi-preparative HPLC purification can be used. The first step utilizes a C 18 column with a mobile phase containing A (25% MeCN in water) and B (30% MeCN in water) with the following gradient:

The secondary step utilizes the same column and conditions, but with only an isocratic mobile phase: 20% MeCN in water.

Those of skill in the art will recognize that the particular column, mobile phases, injection volumes and other HPLC parameters can vary.

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

(13-[(2-0- -D-glucopyranosyl-3-0- -D-glucopyranosyl-p-D-glucopyranosyl)oxy] eni-kaur- 16-en-19-oic acid-[(2-0- -D-glucopyranosyl-6-0- -D-glucopyranosyl- -D-glucopyranosyl) ester])

In another embodiment, the present invention provides reb M2 having a purity greater than about 95% by weight on an anhydrous basis, such as, for example, greater than about 96% by weight, greater than about 97% by weight, greater than about 98% by weight or greater than about 99% by weight.

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

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

The present invention also provides compositions comprising reb M2. It has been found that reb M2 is produced during biotransformation of reb A to reb D. As noted above, biotransformation of reb A to reb D also produces reb D2. Accordingly, in one embodiment, the present invention provides a method for preparing reb M2 comprising: a. contacting a starting composition comprising reb A and/or reb D2 with an enzyme capable of transforming reb A and/or reb D2 to reb M2, UDP-glucose, and optionally UDP-glucose recycling enzymes to produce a composition comprising reb M2; and b. isolating a composition comprising reb M2.

Not wishing to be bound by theory, it is currently believed that the pathway begins with transformation of reb A to reb D2, followed by transformation of reb D2 to reb M2. Accordingly, In one embodiment, the present invention provides a method for preparing reb M2 comprising: a. contacting a starting composition comprising reb D2 with an enzyme capable of transforming reb D2 to reb M2, UDP-glucose, and optionally UDP-glucose recycling enzymes to produce a composition comprising reb M2; and b. isolating a composition comprising reb M2.

In yet another embodiment, a method for preparing reb M2 comprises: a. contacting a starting composition comprising reb A with an enzyme capable of transforming reb A to reb D2, UDP-glucose, and optionally UDP-glucose recycling enzymes to produce a composition comprising reb D2; b. optionally, isolating a composition comprising reb D2; c. contacting the composition comprising reb D2 with an enzyme capable of transforming reb D2 to reb M2, UDP-glucose, and optionally UDP-glucose recycling enzymes to produce a composition comprising reb M2; and d. isolating a composition comprising reb M2. The enzyme can be a UDP-glucosyltransferase, such as, for example, UGT91D2, UGTSL, UGTSL_Sc, UGTSL2 (GI No. 460410132 version XPJ)04250485.1), GI No. 460409128 (UGTSL) version XP 004249992.1, GI No. 1 15454819 version NP_001051010.1, GI No. 187373030, version ACD03249.1. GI No. 222619587 version EEE55719.1, GI No. 297795735 version XP_002865752.1 or EUGTl l .

The enzyme can be immobilized or in a recombinant microorganism.

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

In one embodiment the medium can also include an organic solvent. In a particular embodiment, the enzyme is a UDP-glucosyltransferase capable of transforming reb A and/or reb D2 to reb M2 and is contained in E.coli.

In a more particular embodiment, the enzyme is selected from UGT91D2, UGTSL, UGTSL_Sc, UGTSL2 (GI No. 460410132 version XP_004250485.1), GI No. 460409128 (UGTSL) verison XP 004249992.1, GI No. 1 15454819 version NP_001051010.1 , GI No. 187373030, version ACD03249.1. GI No. 222619587 version EEE55719.1, GI No. 297795735 version XP_002865752.1 or EUGTl l .

In one embodiment, the target steviol glycoside reb M2 can be produced within the microorganism. In another embodiment, the target steviol glycoside can be secreted out in the medium. In one another embodiment, the released steviol glycoside can be continuously removed from the medium. In yet another embodiment, the target steviol glycoside is separated after the completion of the reaction.

Isolation of reb M2 from the reaction medium can be achieved by any suitable method to provide a composition comprising reb M2. Suitable methods include, but are not limited to, lysis, crystallization, separation by membranes, centrifugation, extraction (liquid or solid phase), chromatographic separation, HPLC (preparative or analytical) or a combination of such methods. In a particular embodiment, isolation can be achieved by lysis and centrifugation.

In some embodiments, isolation may result in a reb M2 purity less than about 95% by weight on an anhydrous basis, and the composition may contain, e.g., steviol glycosides and/or residual reaction products.

The composition comprising reb M2 can be further purified to provide highly purified reb M2, i.e. reb M2 having a purity greater than about 95% by weight on an anhydrous basis. In some embodiments, the compositions comprising reb M2 can be further purified to provide reb M2 having 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. Purification can be affected by any means known to one of skill in the art including, but not limited to, crystallization, separation by membranes, centrifugation, extraction (liquid or solid phase), chromatographic separation, HPLC (preparative or analytical) or a combination of such 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.

For example, a two-step semi-preparative HPLC purification can be used. The first step utilizes a CI 8 column with a mobile phase containing A (25% MeCN in water) and B (30% MeCN in water) with the following gradient:

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

The high purity reb M obtained in this invention, having a molecular weight of 1291 .29, a molecular formula of C₅₆H₉o0 CAS registry number 1220616-44-3, and the structure presented in FIG. 1, is in the form of a white and odorless powder. The compound is about 200 times sweeter than sugar when compared to a 10% sucrose solution. The infrared absorption spectrum is shown in FIG. 4.

Other properties of the pure reb M compound include a melting point of 249- 250°C, and a specific rotation of [cc]_D ²⁵ -19.0° in 50% ethanol (C=1.0). The solubility of reb M in water is around 0.3%, and increases with an increase in temperature.

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

Reb M obtained in accordance with the present invention is heat and pH-stable.

Highly purified target glycoside(s) particularly, reb D, reb D2, reb M and/or reb M2 obtained according to this invention can be used "as-is" or in combination with at least one sweetener, flavor, food ingredient and/or combination thereof.

Non-limiting examples of flavors include lime, lemon, orange, fruit, banana, grape, pear, pineapple, mango, berry, bitter almond, cola, cinnamon, sugar, cotton candy and vanilla flavors and/or combination thereof.

Non-limiting examples of other food ingredients include at least one selected from flavors, acidulants, organic and amino acids, coloring agents, bulking agents, modified starches, gums, texturizers, preservatives, antioxidants, emulsifiers, stabilizers, thickeners and gelling agents and/or combination thereof.

Highly purified target glycoside(s) particularly, reb D, reb D2, reb M and/or reb M2 obtained according to this invention can be prepared in various polymorphic forms, including but not limited to hydrates, solvates, anhydrous, amorphous forms and/or combination thereof.

Highly purified target steviol glycoside(s), particularly, reb D, reb D2, reb M and/or reb M2 obtained according to this invention may be incorporated as a high intensity natural sweetener in foodstuffs, beverages, pharmaceutical compositions, cosmetics, chewing gums, table top products, cereals, dairy products, toothpastes and other oral cavity compositions, etc. Highly purified target steviol glycoside(s), particularly, reb D, reb D2, reb M and/or reb M2 as a sweetening compound may be employed as the sole sweetener, or it may be used together with at least one naturally occurring high intensity sweeteners such as stevioside, reb A, reb B, reb C, reb D, reb E, reb F, steviolbioside, dulcoside A, rubusoside, mogrosides, brazzein, neohesperidin dihydrochalcone, glycyrrhizic acid and its salts, thaumatin, perillartine, pernandulcin, mukuroziosides, baiyunoside, phlomisoside- I, dimethyl-hexahydrofluorene-dicarboxylic acid, abrusosides, periandrin, carnosiflosides, cyclocarioside, pterocaryosides, polypodoside A, brazilin, hernandulcin, phillodulcin, glycyphyllin, phlorizin, trilobatin, dihydroflavonol, dihydroquercetin-3 -acetate, neoastilibin, irara'-cinnamaldehyde, monatin and its salts, selligueain A, hematoxylin, monellin, osladin, pterocaryoside A, pterocaryoside B, mabinlin, pentadin, miraculin, curculin, neoculin, chlorogenic acid, cynarin, Luo Han Guo sweetener, mogroside V, siamenoside and/or combination thereof.

In a particular embodiment, reb D2 and/or reb M2 can be used together in a sweetener composition comprising a compound selected from the group consisting of reb A, reb B, reb D, NSF-02, Mogroside V, erythritol and/or combinations thereof.

Highly purified target steviol glycoside(s), particularly, reb D, reb D2, reb M and/or reb M2 may also be used in combination with synthetic high intensity sweeteners such as sucralose, potassium acesulfame, aspartame, alitame, saccharin, neohesperidin dihydrochalcone, cyclamate, neotame, dulcin, suosan advantame, salts thereof, and the like.

Moreover, highly purified target steviol glycoside(s), particularly, reb D, reb D2, reb M and/or reb M2 can be used in combination with natural sweetener suppressors such as gymnemic acid, hodulcin, ziziphin, lactisole, and others. Reb D, reb D2, reb M and/or reb M2 may also be combined with various umami taste enhancers. Reb D, reb D2, reb M and/or reb M2 can be mixed with umami tasting and sweet amino acids such as glutamate, aspartic acid, glycine, alanine, threonine, proline, serine, glutamate, lysine and tryptophan.

Highly purified target steviol glycoside(s), particularly, reb D, reb D2, reb M can be used in combination with one or more additive selected from the group consisting of carbohydrates, polyols, amino acids and their corresponding salts, poly-amino acids and their corresponding salts, sugar acids and their corresponding salts, nucleotides, organic acids, inorganic acids, organic salts including organic acid salts and organic base salts, inorganic salts, bitter compounds, flavorants and flavoring ingredients, astringent compounds, proteins or protein hydrolysates, surfactants, emulsifiers, flavonoids, alcohols, polymers and combinations thereof.

Highly purified target steviol glycoside(s), particularly, reb D, reb D2, reb M and/or reb M2 may be combined with polyols or sugar alcohols. The term "polyol" refers to a molecule that contains more than one hydroxyl group. A polyol may be a diol, triol, or a tetraol which contain 2, 3, and 4 hydroxyl groups, respectively. A polyol also may contain more than four hydroxyl groups, such as a pentaol, hexaol, heptaol, or the like, which contain 5, 6, or 7 hydroxyl groups, respectively. Additionally, a polyol also may be a sugar alcohol, polyhydric alcohol, or polyalcohol which is a reduced form of carbohydrate, wherein the carbonyl group (aldehyde or ketone, reducing sugar) has been reduced to a primary or secondary hydroxyl group. Examples of polyols include, but are not limited to, erythritol, maltitol, mannitol, sorbitol, lactitol, xylitol, inositol, isomalt, propylene glycol, glycerol, threitol, galactitol, hydrogenated isomaltulose, reduced isomalto-oligosaccharides, reduced xylo-oligosaccharides, reduced gentio- oligosaccharides, reduced maltose syrup, reduced glucose syrup, hydrogenated starch hydrolyzates, polyglycitols and sugar alcohols or any other carbohydrates capable of being reduced which do not adversely affect the taste of the sweetener composition.

Highly purified target steviol glycoside(s), particularly, reb D, reb D2, reb M and/or reb M2 may be combined with reduced calorie sweeteners such as D-tagatose, allulose, allose, L-sugars, L-sorbose, L-arabinose, and others.

Highly purified target steviol glycoside(s), particularly, reb D, reb D2, reb M and/or reb M2 may also be combined with various carbohydrates. The term

"carbohydrate" generally refers to aldehyde or ketone compounds substituted with multiple hydroxyl groups, of the general formula (CH₂0)_n, wherein n is 3-30, as well as their oligomers and polymers. The carbohydrates of the present invention can, in addition, be substituted or deoxygenated at one or more positions. Carbohydrates, as used herein, encompass unmodified carbohydrates, carbohydrate derivatives, substituted carbohydrates, and modified carbohydrates. As used herein, the phrases "carbohydrate derivatives",

"substituted carbohydrate", and "modified carbohydrates" are synonymous. Modified carbohydrate means any carbohydrate wherein at least one atom has been added, removed, or substituted, or combinations thereof. Thus, carbohydrate derivatives or substituted carbohydrates include substituted and unsubstituted monosaccharides, disaccharides, oligosaccharides, and polysaccharides. The carbohydrate derivatives or substituted carbohydrates optionally can be deoxygenated at any corresponding C-position, and/or substituted with one or more moieties such as hydrogen, halogen, haloalkyl, carboxyl, acyl, acyloxy, amino, amido, carboxyl derivatives, alkylamino, dialkylamino, arylamino, alkoxy, aryloxy, nitro, cyano, sulfo, mercapto, imino, sulfonyl, sulfenyl, sulfinyl, sulfamoyl, carboalkoxy, carboxamido, phosphonyl, phosphinyl, phosphoryl, phosphino, thioester, thioether, oximino, hydrazino, carbamyl, phospho, phosphonato, or any other viable functional group provided the carbohydrate derivative or substituted carbohydrate functions to improve the sweet taste of the sweetener composition.

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

Highly purified target steviol glycoside(s), particularly, reb D, reb D2, reb M and/or reb M2 obtained according to this invention can be used in combination with various physiologically active substances or functional ingredients. Functional ingredients generally are classified into categories such as carotenoids, dietary fiber, fatty acids, saponins, antioxidants, nutraceuticals, flavonoids, isothiocyanates, phenols, plant sterols and stanols (phytosterols and phytostanols); polyols; prebiotics, probiotics; phytoestrogens; soy protein; sulfides/thiols; amino acids; proteins; vitamins; and minerals. Functional ingredients also may be classified based on their health benefits, such as cardiovascular, cholesterol-reducing, and anti-inflammatory. Exemplary functional ingredients are provided in WO2013/096420, the contents of which is hereby incorporated by reference.

Highly purified target steviol glycoside(s), particularly, reb D, reb D2, reb M and/or reb M2 obtained according to this invention may be applied as a high intensity sweetener to produce zero calorie, reduced calorie or diabetic beverages and food products with improved taste characteristics. It may also be used in drinks, foodstuffs, pharmaceuticals, and other products in which sugar cannot be used. In addition, highly purified target steviol glycoside(s), particularly, reb D, reb D2, reb M and/or reb M2 can be used as a sweetener not only for drinks, foodstuffs, and other products dedicated for human consumption, but also in animal feed and fodder with improved characteristics.

Examples of consumable products in which highly purified target steviol glycoside(s), particularly, reb D, reb D2, reb M and/or reb M2 may be used as a sweetening compound include, but are not limited to, alcoholic beverages such as vodka, wine, beer, liquor, and sake, etc.; natural juices; refreshing drinks; carbonated soft drinks; diet drinks; zero calorie drinks; reduced calorie drinks and foods; yogurt drinks; instant juices; instant coffee; powdered types of instant beverages; canned products; syrups; fermented soybean paste; soy sauce; vinegar; dressings; mayonnaise; ketchups; curry; soup; instant bouillon; powdered soy sauce; powdered vinegar; types of biscuits; rice biscuit; crackers; bread; chocolates; caramel; candy; chewing gum; jelly; pudding; preserved fruits and vegetables; fresh cream; jam; marmalade; flower paste; powdered milk; ice cream; sorbet; vegetables and fruits packed in bottles; canned and boiled beans; meat and foods boiled in sweetened sauce; agricultural vegetable food products; seafood; ham; sausage; fish ham; fish sausage; fish paste; deep fried fish products; dried seafood products; frozen food products; preserved seaweed; preserved meat; tobacco; medicinal products; and many others. In principle it can have unlimited applications. During the manufacturing of products such as foodstuffs, drinks, pharmaceuticals, cosmetics, table top products, and chewing gum, the conventional methods such as mixing, kneading, dissolution, pickling, permeation, percolation, sprinkling, atomizing, infusing and other methods may be used.

Moreover, the highly purified target steviol glycoside(s), particularly, reb D, reb D2, reb M and/or reb M2 obtained in this invention may be used in dry or liquid forms. In one embodiment, a tabletop sweetener comprising reb D2 is provided. In another embodiment, a tabletop sweetener comprising reb M2 is provided.

The highly purified target steviol glycoside can be added before or after heat treatment of food products. The amount of the highly purified target steviol glycoside(s), particularly, reb D, reb D2, reb M and/or reb M2 depends on the purpose of usage. As discussed above, it can be added alone or in combination with other compounds.

The present invention is also directed to sweetness enhancement in beverages using reb D2. The present invention is also directed to sweetness enhancement in beverages using reb M2. Accordingly, the present invention provides a beverage comprising a sweetener and reb D2 and/or reb M2 as a sweetness enhancer, wherein reb D2 and/or reb M2 is present in a concentration at or below their respective sweetness recognition thresholds.

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

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

In some embodiments, sweetener is present in the beverage in an amount from about 0.5% 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 1 1.0% by weight, about 1 1.5% by weight or about 12.0% by weight.

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

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

In another embodiment, the sweetener is erythritol.

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

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

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

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

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

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

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

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

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

In yet another embodiment, the rare sugar is D-leucrose. In a more particular embodiment, D-leucrose is present in the beverage in an amount of about 0.5% to about 10%) by weight, such as, for example, from about 2% to about 8%. The addition of the sweetness enhancer at a concentration at or below its sweetness recognition threshold increases the detected sucrose equivalence of the beverage comprising the sweetener and the sweetness enhancer compared to a corresponding beverage in the absence of the sweetness enhancer. Moreover, sweetness can be increased by an amount more than the detectable sweetness of a solution containing the same concentration of the at least one sweetness enhancer in the absence of any sweetener.

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

Addition of reb D2 and/or reb M2 in a concentration at or below the sweetness recognition threshold to a beverage containing a sweetener may increase the detected sucrose equivalence from about 1.0% to about 5.0%, such as, for example, about 1.0%, about 1.5%, about 2.0%, about 2.5%, about 3.0%, about 3.5%, about 4.0%, about 4.5% or about 5.0%.

The following examples illustrate preferred embodiments of the invention for the preparation of highly purified target steviol glycoside(s), particularly, reb D, reb D2, reb M and/or reb M2. It will be understood that the invention is not limited to the materials, proportions, conditions and procedures set forth in the examples, which are only illustrative.

EXAMPLE 1

In-vivo production of UGT76G1

Ncol and Ndel restriction sides were added to the original nucleic sequence as described in Genbank accession no. AAR06912.1. After codon optimization the following nucleic sequence was obtained (SEQ ID 1):

CCATGGCCCATATGGAAAACAAAACCGAAACCACCGTTCGTCGTCGTCGCC

GTATTATTCTGTTTCCGGTTCCGTTTCAGGGTCATATTAATCCGATTCTGCAG

CTGGCAAATGTGCTGTATAGCAAAGGTTTTAGCATTACCATTTTTCATACCAA

TTTTAACAAACCGAAAACCAGCAATTATCCGCATTTTACCTTTCGCTTTATTCT

GGATAATGATCCGCAGGATGAACGCATTAGCAATCTGCCGACACATGGTCCG CTGGCAGGTATGCGTATTCCGATTATTAACGAACATGGTGCAGATGAACTGC

GTCGTGAACTGGAACTGCTGATGCTGGCAAGCGAAGAAGATGAAGAAGTTA

GCTGTCTGATTACCGATGCACTGTGGTATTTTGCACAGAGCGTTGCAGATAG

CCTGAATCTGCGTCGTCTGGTTCTGATGACCAGCAGCCTGTTTAACTTTCAT

GCACATGTTAGCCTGCCGCAGTTTGATGAACTGGGTTATCTGGATCCGGATG

ATAAAACCCGTCTGGAAGAACAGGCAAGCGGTTTTCCGATGCTGAAAGTGAA

AGATATCAAAAGCGCCTATAGCAATTGGCAGATTCTGAAAGAAATTCTGGGC

AAAATGATTAAACAGACCAAAGCAAGCAGCGGTGTTATTTGGAATAGCTTTAA

AGAACTGGAAGAAAGCGAACTGGAAACCGTGATTCGTGAAATTCCGGCACC

GAGCTTTCTGATTCCGCTGCCGAAACATCTGACCGCAAGCAGCAGCAGCCT

GCTGGATCATGATCGTACCGTTTTTCAGTGGCTGGATCAGCAGCCTCCGAGC

AGCGTTCTGTATGTTAGCTTTGGTAGCACCAGCGAAGTTGATGAAAAAGATTT

TCTGGAAATTGCCCGTGGTCTGGTTGATAGCAAACAGAGCTTTCTGTGGGTT

GTTCGTCCGGGTTTTGTTAAAGGTAGCACCTGGGTTGAACCGCTGCCGGAT

GGTTTTCTGGGTGAACGTGGTCGTATTGTTAAATGGGTTCCGCAGCAAGAAG

TTCTGGCACACGGCGCAATTGGTGCATTTTGGACCCATAGCGGTTGGAATAG

CACCCTGGAAAGCGTTTGTGAAGGTGTTCCGATGATTTTTAGCGATTTTGGT

CTGGATCAGCCGCTGAATGCACGTTATATGAGTGATGTTCTGAAAGTGGGTG

TGTATCTGGAAAATGGTTGGGAACGTGGTGAAATTGCAAATGCAATTCGTCG

TGTTATGGTGGATGAAGAAGGTGAATATATTCGTCAGAATGCCCGTGTTCTG

AAACAGAAAGCAGATGTTAGCCTGATGAAAGGTGGTAGCAGCTATGAAAGCC

TGGAAAGTCTGGTTAGCTATATTAGCAGCCTGTAATAACTCGAG

After synthesis of the gene and subcloning into pET30A+ vector using Ndel and Xhol cloning sites, the UGT76Gl_pET30a+ plasmid was introduced in E. coli B121(DE3) and E. coli EC 100 by electroporation. The obtained cells were grown in petri-dishes in the presence of Kanamycin and suitable colonies were selected and allowed to grow in liquid LB medium (erlenmeyer flasks). Glycerol was added to the suspension as cryoprotectant and 400 aliquots were stored at -20 °C and at -80 °C.

The storage aliquots of E. coli BL21(DE3) containing the pET30A+_UGT76Gl plasmid were 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 of Kanamycin). This culture was allowed to shake at 135 rpm at 30 °C for 8 h.

The production medium contained 60 g/L of overnight express instant TB medium (Novagen), 10 g/L of glycerol and 50 mg/L of Kanamycin. The medium was allowed to stir at 20 °C while taking samples to measure the OD and pH. The cultures gave significant growth and a good OD was obtained. After 40 h, the cells were harvested by centrifugation and frozen to yield 12.7 g of cell wet weight. Lysis was performed by addition of Bugbuster Master mix (Novagen) and the lysate was recovered by centrifugation and kept frozen. Activity tests were performed with thawed lysate.

EXAMPLE 2

In-vitro production of UGT76G1

The S30 T7 High Yield Protein expression system kit from Promega was used. 4 μg of UGT76Gl_pET30a+ plasmid from E. coli ECI OO was mixed with 80 μΐ of S30 premix plus and 72 of S30 T7 extract was added. Nuclease-free water was added in order to obtain a total volume of 200 μί, and the resulting solution was incubated for 2 h at 30°C. 180 μL· was used in the catalytic test reaction.

EXAMPLE 3

In-vitro production of UGT91D2

Ncol and Ndel restriction sides were added to the original nucleic sequence as described in Genbank accession no. ACE87855.1. After codon optimization the following nucleic sequence was obtained (SEQ ID 2):

CCATGGCACATATGGCAACCAGCGATAGCATTGTTGATGATCGTAAACAGCT

GCATGTTGCAACCTTTCCGTGGCTGGCATTTGGTCATATTCTGCCGTATCTG

CAGCTGAGCAAACTGATTGCAGAAAAAGGTCATAAAGTGAGCTTTCTGAGCA

CCACCCGTAATATTCAGCGTCTGAGCAGCCATATTAGTCCGCTGATTAATGTT

GTTCAGCTGACCCTGCCTCGTGTTCAAGAACTGCCGGAAGATGCCGAAGCA

ACCACCGATGTTCATCCGGAAGATATTCCGTATCTGAAAAAAGCAAGTGATG

GTCTGCAGCCGGAAGTTACCCGTTTTCTGGAACAGCATAGTCCGGATTGGAT

CATCTATGATTATACCCATTATTGGCTGCCGAGCATTGCAGCAAGCCTGGGT

ATTAGCCGTGCACATTTTAGCGTTACCACCCCGTGGGCAATTGCATATATGG

GTCCGAGCGCAGATGCAATGATTAATGGTAGTGATGGTCGTACCACCGTTGA

AGATCTGACCACCCCTCCGAAATGGTTTCCGTTTCCGACCAAAGTTTGTTGG

CGTAAACATGATCTGGCACGTCTGGTTCCGTATAAAGCACCGGGTATTAGTG

ATGGTTATCGTATGGGTCTGGTTCTGAAAGGTAGCGATTGTCTGCTGAGCAA

ATGCTATCATGAATTTGGCACCCAGTGGCTGCCGCTGCTGGAAACCCTGCAT

CAGGTTCCGGTTGTTCCGGTGGGTCTGCTGCCTCCGGAAGTTCCGGGTGAT

G AAAAAG ATG AAACCTG G GTTAG C ATC AAAAAATG G CTG G ATG G TAAAC AG A

AAGGTAGCGTGGTTTATGTTGCACTGGGTAGCGAAGTTCTGGTTAGCCAGAC

CGAAGTTGTTGAACTGGCACTGGGTCTGGAACTGAGCGGTCTGCCGTTTGTT

TGGGCATATCGTAAACCGAAAGGTCCGGCAAAAAGCGATAGCGTTGAACTG

CCGGATGGTTTTGTTGAACGTACCCGTGATCGTGGTCTGGTTTGGACCAGCT

GGGCACCTCAGCTGCGTATTCTGAGCCATGAAAGCGTTTGTGGTTTTCTGAC

CCATTGTGGTAGCGGTAGCATTGTGGAAGGTCTGATGTTTGGTCATCCGCTG

ATTATGCTGCCGATTTTTGGTGATCAGCCGCTGAATGCACGTCTGCTGGAAG ATAAACAGGTTGGTATTGAAATTCCGCGTAATGAAGAAGATGGTTGCCTGAC

CAAAGAAAGCGTTGCACGTAGCCTGCGTAGCGTTGTTGTTGAAAAAGAAGGC

GAAATCTATAAAGCCAATGCACGTGAACTGAGCAAAATCTATAATGATACCAA

AGTGGAAAAAGAATATGTGAGCCAGTTCGTGGATTATCTGGAAAAAAACACC

CGTGCAGTTGCCATTGATCACGAAAGCTAATGACTCGAG

After synthesis of the gene and subcloning into pET30A+ vector using Ncol and Xhol cloning sites, the UGT91D2_pET30a+ plasmid was introduced into E. coli ECIOO by electroporation. The obtained cells were grown in the presence of Kanamycin and suitable colonies were selected and allowed to grow in liquid LB medium (erlenmeyer flasks). Glycerol was added to the suspension as cryoprotectant and 400 aliquots were stored at -20 °C and at -80 °C.

The S30 T7 High Yield Protein expression system kit from Promega was used for the in-vitro synthesis of the protein.

4 μg of UGT91D2_pET30a+ plasmid was mixed with 80 μΙ> of S30 premix plus and 72 μΐ, of S30 T7 extract was added. Nuclease-free water was added in order to obtain a total volume of 200 μΐ, and the resulting solution was incubated for 2 h at 30°C. 5 μΐ, was used for SDS-page analysis while the remaining 45 μΕ was used in the catalytic test reaction.

EXAMPLE 4

Catalytic reaction with in-vivo produced UGT76G1

The total volume of the reaction was 5.0 mL with the following composition: 50 mM sodium phosphate buffer pH 7.2, 3 mM MgCl₂, 2.5 mM UDP-glucose, 0.5 mM Stevioside and 500 μΐ_^ of UGT76G1 thawed lysate. The reactions were run at 30 °C on an orbitary shaker at 135 rpm. For each sample, 460 μΕ of the reaction mixture was quenched with 40 μΐ, of 2N H₂S0₄ and 420 μΐ, of methanol/water (6/4). The samples were immediately centrifuged and kept at 10 °C before analysis by HPLC (CAD). HPLC indicated almost complete conversion of stevioside to rebaudioside A as seen in Figure 40. UGT76G1 catalyzed transformation of stevioside to Reb A

de

0 5 10 15 20 25

time (h)

EXAMPLE 5

Catalytic reaction with in-vitro produced UGT91D2

The total volume of the reaction 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 of in-vitro produced UGT91D2. The reactions were run at 30 °C on an orbitary shaker at 135 rpm. For each sample, 450 of reaction mixture was quenched with 45 μί of 2N H₂S0₄ and 405 μΐ, of 60% MeOH. After centrifugation, the supernatant was analyzed by HPLC (CAD). HPLC indicated a 4.7% conversion of rebaudioside A to rebaudioside D after 120 h.

EXAMPLE 6

Catalytic reaction with in-vitro produced UGT76G1

The total volume of the reaction was 2 mL with the following composition: 50 mM sodium phosphate buffer pH 7.2, 3 mM MgCl₂, 3.8 mM UDP-glucose, 0.5 mM Rebaudioside D and 180 μΐ_^ of in-vitro produced UGT76G1. The reactions were run at 30 °C on an orbitary shaker at 135 rpm. For each sample, 400 μΥ, of reaction mixture was quenched with 40 μΐ, of 2N H₂S0₄ and 360 μΐ, of 60% MeOH. After centrifugation, the supernatant was analyzed by HPLC (CAD). HPLC indicated 80% conversion of rebaudioside D to rebaudioside M after 120 h as seen in Figure 41. Synthesis of Rebaudioside M from Rebaudioside D/ CAD detection

20 40 60 80 100 120 140

Time (h)

For examples 7 to 12, the following abbreviations were used:

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 of Kanamycin or Ampicillin LB medium: (20 g/L Luria Broth Lennox)

EXAMPLE 7

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

The pET30a+_UGT76Gl plasmid was transformed into BL21(DE3) expression strain (Lucigen E. Cloni® EXPRESS Electrocompetent Cells). The obtained cells were grown on LB Agar medium in petri-dishes in the presence of Kanamycin. Suitable colonies were selected and allowed to grow in liquid LBGKP medium containing Kanamycin. Glycerol was added and 400 aliquots were stored at -20°C and at -80°C.

A storage aliquot was thawed and added to 30 mL of LBGKP medium. This culture was allowed to shake at 30°C for 8 h. and subsequently used to inoculate 400 mL of production medium containing 60 g/L of "Overnight express instant TB medium" (Novagen, reference 71491-5), 10 g/L of glycerol and 50 mg/L of Kanamycin. The medium was allowed to stir at 20°C while taking samples to measure the OD (600 nm) and pH. After 40 h, the cells were harvested by centrifugation and frozen. The obtained cell wet weight was 10.58 g.

3.24 g of obtained pellet was lysed by addition of 8.1 mL of "Bugbuster Master mix" (Novagen, reference 71456) and 3.5 mL of water. The lysate was recovered by centrifugation and kept frozen. EXAMPLE 8

Preparation and activity of UGT76G1 prepared by pET30a+ plasmid and Tuner (DE3) expression strain

The pET30a+_UGT76Gl plasmid was transformed into Tuner (DE3) expression strain (Novagen Tuner^tm (DE3) Competent cells) by heat shock treatment. The obtained cells were grown on LB Agar medium in petri-dishes in the presence of Kanamycin. Suitable colonies were selected and allowed to grow in liquid LBGKP medium containing Kanamycin). Glycerol was added and 400 aliquots were stored at -20°C and at -80°C.

A storage aliquot was thawed and added to 100 mL of LB medium containing 50 mg/L of Kanamycin. This culture allowed to shake at 30°C for 15 h. 4.4 mL of this culture was used to inoculate 200 mL of production medium containing LB. This medium was allowed to stir at 37°C until an OD (600 nm) of 0.9 was obtained, after which 400 μΐ. of a 100 mM IPTG solution was added and the medium was allowed to stir at 30 °C for 4 h. The cells were harvested by centrifugation and frozen. The obtained cell wet weight was 1.38 g.

The obtained pellet was lysed by addition of 4.9 mL of "Bugbuster Master mix" (Novagen, reference 71456) and 2.1 mL of water. The lysate was recovered by centrifugation and kept frozen.

EXAMPLE 9

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

After subcloning the synthetic UGT76G1 gene into the pMAL plasmid using Ndel and Sail cloning sites, the pMAL_UGT76Gl plasmid was transformed into BL21 expression strain (New England Biolabs BL21 Competent E. coli) by heat shock treatment. The obtained cells were grown on LB Agar medium in petri-dishes in the presence of Ampicillin. Suitable colonies were selected and allowed to grow in liquid LBGKP medium containing Ampicillin). Glycerol was added and 400 aliquots were stored at -20°C and at -80°C.

A storage aliquot was thawed and added to 30 mL of LBGKP medium. This culture was allowed to shake at 30°C for 8 h. and subsequently used to inoculate 400 mL of production medium containing 60 g/L of "Overnight express instant TB medium" (Novagen, reference 71491-5), 10 g/L of glycerol and 50 mg/L of Ampicillin. The medium was allowed to stir at 20 °C while taking samples to measure the OD and pH. After 40 h, the cells were harvested by centrifugation and frozen. The obtained cell wet weight was 5.86 g.

2.74 g of obtained pellet was lysed by addition of 9.6 mL of "Bugbuster Master Mix" (Novagen, reference 71456) and 4.1 mL of water. The lysate was recovered by centrifugation and kept frozen.

EXAMPLE 10

Preparation and activity of UGT76G1 prepared by pMAL plasmid and ArcticExpress expression strain

The pMAL_UGT76Gl plasmid was transformed into ArticExpress expression strain (Agilent ArcticExpress competent cells) by heat shock treatment. The obtained cells were grown on LB Agar medium in petri-dishes in the presence of Ampicillin and Geneticin. Suitable colonies were selected and allowed to grow in liquid LBGKP medium containing of Ampicillin and Geneticin. Glycerol was added and 400 aliquots were stored at -20°C and at -80°C.

A storage aliquot was thawed and added to 30 mL of LBGKP medium (containing Ampicillin and Geneticin). This culture was allowed to shake at 30°C for 8 h. and subsequently used to inoculate 400 mL of production medium containing 60 g/L of "Overnight express instant TB medium" (Novagen, reference 71491 -5), 10 g/L of glycerol and 50 mg/L of Ampicillin. The medium was allowed to stir at 12°C while taking samples to measure the OD (600 nm) and pH. After 68 h, the cells were harvested by centrifugation and frozen. The obtained cell wet weight was 8.96 g.

2.47 g of the obtained pellet was lysed by addition of 8.73 mL of "Bugbuster Master Mix" (Novagen, reference 71456) and 3.79 mL of water. The lysate was recovered by centrifugation and kept frozen.

EXAMPLE 11

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

After subcloning the synthetic UGT76G1 gene into the pCOLDIII plasmid using Ndel and Xhol cloning sites, the pCOLDIII_UGT76Gl plasmid was transformed into ArcticExpress expression strain (Agilent ArcticExpress competent cells) by heat shock treatment. The obtained cells were grown on LB Agar medium in petri-dishes in the presence of Ampicillin and Geneticin. Suitable colonies were selected and allowed to grow in liquid LBGKP medium containing Ampicillin and Geneticin. Glycerol was added and 400 aliquots were stored at -20°C and at -80°C.

A storage aliquot was thawed and added to 30 mL of LBGKP medium (containing Ampicillin and Geneticin). This culture was allowed to shake at 30°C for 8 h. and subsequently used to inoculate 400 mL of production medium containing 60 g/L of "Overnight express instant TB medium" (Novagen, reference 71491 -5), 10 g/L of glycerol and 50 mg/L of Kanamycin. The medium was allowed to stir at 12°C while taking samples to measure the OD (600 nm) and pH. After 63 h, the cells were harvested by centrifugation and frozen. The obtained cell wet weight was 6.54 g.

2.81 g of the obtained pellet was lysed by addition of 9.8 mL of "Bugbuster Master Mix" (Novagen, reference 71456) and 4.2 mL of water. The lysate was recovered by centrifugation and kept frozen.

EXAMPLE 12

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

The pCOLDIII_UGT76Gl plasmid was transformed into Origami2 (DE3) expression strain (Novagen Origami™2 (DE3) Competent Cells) by heat shock treatment. The obtained cells were grown on LB Agar medium in petri-dishes in the presence of Ampicillin. Suitable colonies were selected and allowed to grow in liquid LBGKP medium containing Ampicillin. Glycerol was added and 400 μΕ aliquots were stored at - 20°C and at -80°C.

A storage aliquot was thawed and added to 30 mL of LBGKP medium (containing Ampicillin). This culture was allowed to shake at 30°C for 8 h. and subsequently used to inoculate 400 mL of production medium containing 60 g/L of "Overnight express instant TB medium" (Novagen, reference 71491 -5), 10 g/L of glycerol and 50 mg/L of Kanamycin. The medium was allowed to stir at 12 °C while taking samples to measure the OD (600 nm) and pH. After 68 h, the cells were harvested by centrifugation and frozen. The obtained cell wet weight was 2.53 g. 1.71 g of the obtained pellet was lysed by addition of 6.0 mL of "Bugbuster Master mix" (Novagen, reference 71456) and 1.9 mL of water. The lysate was recovered by centrifugation and kept frozen.

EXAMPLE 13

Determination of activity

Activity tests were performed on a 5 mL scale with 500 of thawed lysate for the transformation of Stevioside to Rebaudioside A and Rebaudioside D to Rebaudioside M using 0.5 mM of substrate, 2.5 mM of UDP-Glucose and 3 mM MgCl₂ in 50 mM Sodium Phosphate buffer at pH 7.2. Samples were taken and analyzed by HPLC. The results for the different preparations of UGT76G1 are summarized in the following table.

Example Plasmid Expression Transformation activity*

strain Stevioside to Rebaudioside D

Rebaudioside A to Rebaudioside

M

7 pET30a+ BL21 (DE3) 29 U mL^"1 0.31 U mL^{" 1}

8 pET30a+ Tuner (DE3) 33 U mL^"1 0.40 U mL^"1

9 pMAL BL21 20 U mL^"1 0.15 U mL^"1

10 pMAL ArticExpress 15 U mL^"' 0.25 U mL^"1

1 1 pCOLDIII ArticExpress 15 U mL^{" 1} 0.1 1 U mL^"1

12 pCOLDIII Origami2 (DE3) 37 U mL^"1 0.20 U mL^"1

* Note

The activities for the transformation of Stevioside and Rebaudioside M are mentioned per mL of lysate. 1 U will transform 1 μιηοΐ of substrate in 1 hour at 30 °C and pH 7.2

EXAMPLE 14

50 mL scale reaction for the transformation of Rebaudioside D to Rebaudioside M

5 mL of the lysate of Example 12 was used to transform Rebaudioside D to Rebaudioside M on a 50 mL scale. The reaction medium consisted of 50 mM Sodium Phosphate buffer pH 7.2, 3 mM of MgCl₂, 2.5 mM of UDP-Glucose and 0.5 mM of Rebaudioside D. After allowing the reaction to be shaken at 30°C for 90 h. 50 mL of ethanol was added and the resulting mixture was allowed to stir at -20 °C for 1 h. After centrifugation at 5000 g for 10 min. the supernatant was purified via ultrafiltration (Vivaflow MWCO 30000). 78 mL of permeate was obtained and the 9 mL of retentate was diluted with 9 mL of ethanol and resubjected to Ultrafiltration (Vivaflow MWCO 30000). Another 14 mL of filtrate was obtained, which was combined with the first permeate. The combined permeates were concentrated under reduced pressure at 30°C until 32 mL of a clear solution was obtained.

The HPLC trace of the product mixture is shown in FIG. 5. HPLC was carried out on an Agilent 1200 series equipped with a binary pump, auto sampler, and thermostat column compartment. The method was isocratic, with a mobile phase composed of 70% water (0.1%) formic acid): 30% acetonitrile. The flow rate was 0.1 μί/ηπη. The column used was Phenomenex Prodigy 5μ ODS (3) 100 A; 250x2mm. The column temperature was maintained at 40 °C. The injection volume was 20-40 μΐ. EXAMPLE 15

Preparation of UGT91D2 using pMAL plasmid and BL21 expression strain

After subcloning the synthetic UGT91 D2 gene into the pMAL plasmid using Ndel and Sail cloning sites, the pMAL_UGT91 D2 plasmid was transformed into BL21 expression strain (New England Biolabs BL21 Competent E. coli) by heat shock treatment. The obtained cells were grown on LB Agar medium in petri-dishes in the presence of Ampicillin. Suitable colonies were selected and allowed to grow in liquid LBGKP medium containing Ampicillin). Glycerol was added and 400 μΕ aliquots were stored at -20°C and at -80°C.

A storage aliquot was thawed and added to 30 mL of LBGKP medium. This culture was allowed to shake at 30°C for 8 h. and subsequently used to inoculate 400 mL of production medium containing 60 g/L of "Overnight express instant TB medium" (Novagen, reference 71491-5), 10 g/L of glycerol and 50 mg/L of Ampicillin. The medium was allowed to stir at 20 °C while taking samples to measure the OD and pH. After 40 h, the cells were harvested by centrifugation and frozen. The obtained cell wet weight is 12.32g.

2.18 g of obtained pellet was lysed by addition of 7.7 mL of "Bugbuster Master Mix" (Novagen, reference 71456) and 3.2 mL of water. The lysate was recovered by centrifugation and used directly for activity testing.

EXAMPLE 16

Preparation of UGT91D2 using pMAL plasmid and ArcticExpress expression strain

The pMAL_UGT91D2 plasmid was transformed into ArcticExpress expression strain (Agilent ArcticExpress competent cells) by heat shock treatment. The obtained cells were grown on LB Agar medium in petri-dishes in the presence of Ampicillin and Geneticin. Suitable colonies were selected and allowed to grow in liquid LBGKP medium containing Ampicillin and Geneticin. Glycerol was added and 400 μΐ_^ aliquots were stored at -20°C and at -80°C.

A storage aliquot was thawed and added to 30 mL of LBGKP medium (containing Ampicillin and Geneticin). This culture was allowed to shake at 30°C for 8 h. and subsequently used to inoculate 400 mL of production medium containing 60 g/L of "Overnight express instant TB medium" (Novagen, reference 71491 -5), 10 g/L of glycerol and 50 mg/L of Ampicillin. The medium was allowed to stir at 20°C for 16 h. followed by another 50 h. at 12°C while taking samples to measure the OD (600 nm) and pH. The cells were harvested by centrifugation and frozen. The obtained cell wet weight is 15.77 g.

2.57 g of the obtained pellet was lysed by addition of 9.0 mL of "Bugbuster Master Mix" (Novagen, reference 71456) and 3.8 mL of water. The lysate was recovered by centrifugation and used directly for activity testing.

EXAMPLE 17

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

The pET30a+_UGT91D2 plasmid was transformed into Tuner (DE3) expression strain (Novagen Tuner^tm (DE3) Competent cells) by heat shock treatment. The obtained cells were grown on LB Agar medium in petri-dishes in the presence of Kanamycin. Suitable colonies were selected and allowed to grow in liquid LBGKP medium (containing Kanamycin). Glycerol was added and 400 aliquots were stored at -20°C and at -80°C.

A storage aliquot was thawed and added to 100 mL of LB medium containing 50 mg/L of Kanamycin. This culture allowed to shake at 30°C for 15 h. 6.2 mL of this culture was used to inoculate 500 mL of production medium containing LB. This medium was allowed to stir at 37°C until an OD (600 nm) of 0.9 was obtained after which 500 of a 100 mM IPTG solution was added (IPTG concentration in medium is 100 μΜ) and the medium was allowed to stir at 30 °C for 4 h, the cells were harvested by centrifugation and frozen. The obtained cell wet weight is 4.02 g.

1.92 g of the obtained pellet was lysed by addition of 6.8 mL of "Bugbuster Master mix" (Novagen, reference 71456) and 2.8 mL of water. The lysate was recovered by centrifugation and tested directly for activity.

EXAMPLE 18

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) by heat shock treatment. The obtained cells were grown on LB Agar medium in petri-dishes in the presence of Kanamycin and Geneticin. Suitable colonies were selected and allowed to grow in liquid LBGKP medium containing of Kanamycin and Geneticin. Glycerol was added and 400 μί, aliquots were stored at -20°C and at -80°C.

A storage aliquot was thawed and added to 30 mL of LBGKP medium (containing Kanamycin and Geneticin). This culture was allowed to shake at 30°C for 8 h. and subsequently used to inoculate 400 mL of production medium containing 60 g/L of "Overnight express instant TB medium" (Novagen, reference 71491-5), 10 g/L of glycerol and 50 mg/L of Ampicillin. The medium was allowed to stir at 20°C for 16h. followed by another 50 h. at 12°C while taking samples to measure the OD (600 nm) and pH. After 60 h, the cells were harvested by centrifugation and frozen. The obtained cell wet weight is 16.07 g.

3.24 g of the obtained pellet was lysed by addition of 1 1.4 mL of "Bugbuster Master Mix" (Novagen, reference 71456) and 4.8 mL of water. The lysate was recovered by centrifugation and used directly for activity testing.

EXAMPLE 19

Determination of activity of in-vivo preparations of UGT91D2

Activity tests were performed at 5 mL scale with 1000 of lysate for the transformation of Rubusoside to Stevioside using 0.5 mM of substrate, 2.5 mM of UDP- Glucose and 3 mM MgCl₂ in 50 mM Sodium Phosphate buffer at pH 7.2. Samples were taken and analyzed by HPLC. The results for the different preparations of UGT91D2 are summarized in the following table.

* Note: The activities are mentioned per mL of lysate. 1 U will transform 1 μιηοΐ of substrate in 1 hour at 30 °C and pH 7.2 EXAMPLE 20

Other enzymes for Rebaudioside A to Rebaudioside D conversion

The following genes of UDP-glucosyltransferases were identified from databases, synthesized by DNA2.0 and subsequently subcloned in pET30a+ vector.

The aminoacid sequences are as follows: SEQ ID 3

>gi| l 15454819|ref]NP_001051010.11 Os03g0702500 [Oryza sativa Japonica Group]

MDDAHSSQSPLHVVIFPWLAFGHLLPCLDLAEPvLAARGHRVSFVSTPRNLARLPP

VRPELAELVDLVALPLPRVDGLPDGAEATSDVPFDKFELHRKAFDGLAAPFSAFL

DTACAGGKRPDWVLADLMHHWVALASQERGVPCAMILPCSAAVVASSAPPTESS

ADQREAIVRSMGTAAPSFEAKRATEEFATEGASGVSIMTRYSLTLQRSKLVAMRS

CPELEPGAFTILTRFYGKPVVPFGLLPPRPDGARGVSKNGKHDAIMQWLDAQPAK

SVVYVALGSEAPMSADLLRELAHGLDLAGTRFLWAMRKPAGVDADSVLPAGFL

GRTGERGLVTTRWAPQVSILAHAAVCAFLTHCGWGSVVEGLQFGHPLIMLPILGD

QGPNARILEGRKLGVAVPRNDEDGSFDRGGVAGAVRAVVVEEEGKTFFANARKL

QEIVADREREERCIDEFVQHLTSWNELKNNSDGQYP

SEQ ID 4

>gi| 187373030|gb|ACD03249.11 UDP-glycosyltransferase [Avena strigosa]

MAVKDEQQSPLHILLFPFLAPGHLIPIADMAALFASRGVRCTILTTPVNAAIIRSAV

DRANDAFRGSDCPAIDISVVPFPDVGLPPGVENGNALTSPADRLKFFQAVAELREP

FDRFLADNHPDAVVSDSFFHWSTDAAAEHGVPRLGFLGSSMFAGSCNESTLFIN P

LETAADDPDALVSLPGLPHRVELRRSQMMDPKKRPDHWALLESVNAADQKSFGE

VFNSFHELEPDYVEHYQTTLGRRTWLVGPVALASKDMAGRGSTSARSPDADSCL

R WLDTKQPGS V V Y V SFGTLIRF S PAELHEL ARGLDL SGKNF V W VLGRAGPD S SE WMPQGFADL1TPRGDRGFIIRGWAPQMLILNHRALGGFVTHCGWNSTLESVSAGV PMVTWPRFADQFQNEKLIVEVLKVGVSIGAKDYGSGIENHDVIRGEVIAESIGKLM GSSEESDAIQRKAKDLGAEARSAVENGGSSYNDVGRLMDELMARRSSVKVGEDII PTNDGL

SEQ ID 5

>gi|460409128|ref]XP_004249992.1 | PREDICTED: cyanidin-3-O-glucoside 2-0- glucuronosyltransferase-like [Solarium fycopersicitm]

MSPKLHKELFFHSLYKKTRSNHT ATLKVLMFPFLAYGHISPYLNVAKKLADRGF

LIYFCSTPINLKSTIEKIPEKYADSIHLIELHLPELPQLPPHYHTTNGLPPNLNQVLQK

ALKMSKPNFSKILQNLKPDLVIYDILQRWAKHVANEQNIPAVKLLTSGAAVFSYFF

NVLKKPGVEFPFPGIYLRKIEQVRLSEMMSKSD EKELEDDDDDDDLLVDGNMQI

MLMSTSRTIEAKYIDFCTALTNWKVVPVGPPVQDLITNDVDDMELIDWLGTKDE

NSTVFVSFGSEYFLSKEDMEEVAFALELSNVNFIWVARFPKGEERNLEDALPKGFL

ERIGERGRVLDKFAPQPRILNHPSTGGFISHCGW SAMESIDFGVPIIAMPMHLDQP

MNARLIVELGVAVEIVRDDDGKIHRGEIAETLKGVITGKTGEKLRAKVRDISKNLK

TIRDEEMDAAAEELIQLCRNGN

SEQ ID 6

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

MHVVMLPWLAFGHILPFAEFAKRVARQGHRVTLFSTPRNTRRLIDVPPSLAGRIR

VVDIPLPRVEHLPEHAEATIDLPSNDLRPYLRRAYDEAFSRELSRLLQETGPSRPD

WVLADYAAYWAPAAASRHGVPCAFLSLFGAAALCFFGPAETLQGRGPYAKTEPA

HLTAVPEYVPFPTTVAFRGNEARELFKPSLIPDESGVSESYRFSQSIEGCQLVAVRS

NQEFEPEWLELLGELYQKPVIPIGMFPPPPPQDVAGHEETLRWLDRQEPNSVVYA

AFGSEVKLTAEQLQRIALGLEASELPFIWAFRAPPDAGDGDGLPGGFKERVNGRG

VVCRGWVPQVKFLAHASVGGFLTHAGWNSIAEGLANGVRLVLLPLMFEQGLNA

RQLAEKKVAVEVARDEDDGSFAANDIVDALRRVMVGEEGDEFGVKVKELAKVF

GDDEVNDRYVRDFLKCLSEYKMQRQG SEQ ID 7

>gi|297795735|refjXP_002865752.1 | UDP-glucoronosyl/UDP-glucosyl transferase family protein [Arabidopsis lyrata subsp. lyrata]

MDDKKEEVMHIAMFPWLAMGHLLPFLRLSKLLAQKGHKISFISTPRNILRLPKLPS

NLSSSITFVSFPLPSISGLPPSSESSMDVPY KQQSLKAAFDLLQPPLTEFLRLSSPD

WIIYDYASHWLPSIAKELGISKAFFSLFNAATLCFMGPSSSLIEESRSTPEDFTVVPP

WVPFKSTIVFRYHEVSRYVEKTDEDVTGVSDSVRFGYTIDGSDAVFVRSCPEFEPE

WFSLLQDLYRKPVFPIGFLPPVIEDDDDDTTWVRIKEWLDKQRVNSVVYVSLGTE

ASLRREELTELALGLEKSETPFFWVLRNEPQIPDGFEERVKGRGMVHVGWVPQVK

ILSHESVGGFLTHCGWNSVVEGIGFGKVPIFLPVLNEQGLNTRLLQGKGLGVEVLR

DERDGSFGSDSVADSVRLVMIDDAGEEIREKVKLMKGLFGNMDENIRYVDELVG

FMRNDES SQLKEEEEEDDC SDDQS SE VS SETDEKELNLDLKEEKRRIS VYKSLS SE

FDDYVANEKMG

The tested plasmids were received in a microtiterplate containing a plasmid as freeze-dried solid in each separate well.

Suspension of plasmids. To each well was added 24 μΕ of ultra-pure sterile water and the microtiter plate was shaken for 30 minutes at Room Temperature. Subsequently, the plate was incubated at 4°C for 1 hour. The content of each well were further mixed by pipetting up and down. The plasmid quantification was performed by Qubit2.0 analysis using 1 μΕ of suspension. Determined quantities of plasmids were:

Transformation of competent cells with plasmids. Aliquots of chemically competent EC 100 cells were taken from freezer at -80°C and stored on ice. The cells were allowed to thaw on ice for 10 minutes. 10 μΕ of a dilution of above described plasmid solution was added to a sterile microtube of 1.5 mL (in order to transform each cell with 50 pg of DNA) and stored on ice. 100 μΕ of chemically competent cells was added to each microtube. After incubation of the chemically competent cells plasmid mixtures on ice for 20 min a thermal shock of 30 seconds at 42°C was performed.

Further incubation was performed on ice for 2 minutes. To each microtube 300 of SOC medium was added and the resulting mixture was transferred to a sterile 15 mL tube. After incubate for 1 hour at 37°C while shaking at 135 rpm, the mixture is spread on solid Luria Broth medium containing Kanamycin 50 μg/mL. The petri-dishes are allowed to incubate for 16 hours at 37 °C

Preparation of stock solutions in glycerol and purification of plasmids. To a 50 mL sterile Falcon Tube 10 mL of Luria Broth medium containing 50 μg/mL of Kanamycin was added. The medium was seeded with an isolated colony from the above described Petri dish and the cultures were allowed to incubate for 16 hours at 37°C while shaking at 135 rpm.

To sterile microtube of 1.5 mL containing 300 μL of a 60% sterile glycerol solution, 600 μΐ, of the culture was added. The stock solution was stored at -80°C.

The remainder of the culture was centrifuged at 5,525g for 10 minutes at 10°C and after removal of the supernatant, the pellet was stored on ice. The produced plasmids were purified according to the Qiagen Qiaprep Spin Miniprep kit (ref : 27106) and the plasmid yield was measured at 260 nm. The plasmid solution was stored at 4°C. Plasmid quantities were determined as follows:

In-vitro Expression of enzymes. 18 μΐ, of plasmid solution (containing approximately 1.5 μg of plasmid) was used for in-vitro expression according to the Promega S30 T7 High- Yield Protein Expression System (ref : LI 1 10) kit. The expression medium was produced as follows:

The prepared expression medium mix was added to the plasmid solution and the solution was allowed to incubate at 30°C for 3 hours while mixing the mixture every 45 minutes. 5 μΐ_^ of the mixture was frozen whereas the remainder was used for the catalytic test for the conversion of Rebaudioside A to Rebaudioside D.

Catalytic test for transformation of Rebaudioside A to Rebaudioside D. 430 μΐ, of a reaction mixture containing 0.5 mM Rebaudioside A, 3mM MgCl₂, 50 mM phosphate buffer (pH7.2) and 2.5 mM UDP-glucose was added to a 1.5 mL sterile microtube. 52 of the enzyme expression medium was added and the resulting mixture was allowed to react at 30 °C for 24 hours. 125 μΐ_^ samples were taken after 2 hours, 16 hours and 24 hours and added to a 1 15 μΐ, of 60% methanol and 10 μΐ, of 2 N H2SO4. The quenched sample was centrifuged at 18,000 g for 2 minutes at RT. 200 μΐ, was transferred to an HPLC vial and analyzed.

HPLC Analysis The HPLC assay was performed as follows:

Apparatus

Instrument conditions

Mobile phase gradient program

The HPLC assay results are provided below:

The enzyme SI 15N05 A 7 had the highest activity for Reb A to Reb D conversion (ca. 22.4%)

At least three enzymes produced a significant amount of an unknown glycoside (marked as Reb UNK; later identified as reb D2) along with reb D, as seen in Figures 42- 46.

Sample : 12400 S115N01A1 T24h 130621 ABA

gij 1 15454819|ref]NP_001051010.1 j Os03g0702500 [Oryza sativa Japonica Group]

Filename : 130621 12400 01 1.dat

CAD Ch 1 Results

Compound Retention time Integration (area)

Rebaudioside D 5.797 13,532,277

[email protected] 7.890 7,094,778

Rebaudioside A 9.157 613,483,011

Total 634, 110,066

Sample : 12400 S115N01G2 T24h 130621ABA

>gi| 187373030|gb|ACD03249.11 UDP-glycosyltransferase [A vena strigosa]

Filename : 130621 12400 020.dat

CAD Ch 1 Results

Compound Retention time Integration (area)

Rebaudioside D 5.788 3,547,834

Rebaudioside A 9.148 585,285,463

Total 588,833,297

Sample : 12400 S115N05A7 T24h 130627ABA

>gi|460409128|rei]XP__004249992.1 | PREDICTED: cyanidin-3-O-glucoside 2-0- glucuronosyltransferase-like [Solarium lycopersicum]

Filename : 130627 12400 027.dat

CAD Ch 1 Results

Compound Retention time Integration (area)

[email protected] 4.508 64,361,822

Rebaudioside D 5.761 191,273,935

Rebaudioside UNK 6.685 198,934,644

Rebausioside A 9.106 398,1 15,681

Total 852,686,082

Sample : 12400 S115N06E1 T24h 130627ABA

>gi|222619587jgb|EEE55719.1| hypothetical protein OsJ 04191 [Oryza sativa Japonica Group] Filename : 130627 12400 046.dat

CAD Ch 1 Results

Compound Retention time Integration (area)

Rebaudioside D 5.737 964,715

Rebaudioside UNK 6.685 46,027,361

Rebausioside A 9.1 13 606,312,523

Total 653,304,599 Sample : 12400 S115N06C2 T24h 130627ABA

>gi|297795735|ref|XP_002865752.1 | UDP-glucoronosyl/UDP-glucosyl transferase family protein [Arabidopsis lyrata subsp. lyrata]

Filename : 130627 12400 052.dat

CAD Ch 1 Results

Compound Retention time Integration (area)

Rebaudioside D 5.757 1,852,407

Rebaudioside UNK 6.708 26,033,636

Rebausioside A 9.136 633,014,654

Total 660,900,697

EXAMPLE 21

Activity of in-vitro produced EUGT11

EUGT1 1 gene as was described in the Patent application WO/2013/022989A2 was synthesized by DNA2.0 and subsequently subcloned in pET30a+ vector.

The amino-acid sequence is as follows: SEQ ID 8

>gi|41469452|gb|AAS07253.1 | putative UDP-glucoronosyl and UDP-glucosyl transferase [Oryza sativa Japonica Group] EUGT1 1 enzyme from patent application WO/2013/022989A2

MHVVICPLLAFGHLLPCLDLAQRLACGHRVSFVSTPRNISRLPPVPvPSLAPLVSFVA LPLPRVEGLPNGAESTHNVPHDRPDMVELHLRAFDGLAAPFSEFLGTACADWVM PTSSAPRQTLSSNIHRNSSRPGTPAPSGRLLCPITPHSNTLERAAEKLVRSSRQNAR ARSLLAFTSPPLPYRDVFRSLLGLQMGRKQLNIAHETNGRRTGTLPLNLCRWMW KQ RCGKLRPSDVEFNTSRSNEAISPIGASLVNLQSIQSPNPRAVLPIASSGVRAVFI

GRARTSTPTPPHAKPARSAAPRAHRPPSSVMDSGYSSSYAAAAGMHVVICPWLAF

GHLLPCLDLAQRLASRGHRVSFVSTPRNISRLPPVRPALAPLVAFVALPLPRVEGLP

DGAESTNDVPHDRPDMVELHRRAFDGLAAPFSEFLGTACADWV1VDVFHHWAA

AAALEHK VPC AMMLLG S AHM I A S I ADRRLERAETE SP A AAGQGRP AA A PTFE V A

RMKLIRTKGSSGMSLAERFSLTLSRSSLVVGRSCVEFEPETVPLLSTLRGKPITFLG

LMPPLHEGRREDGEDATVRWLDAQPAKSVVYVALGSEVPLGVEKVHELALGLEL

AGTRFLWALRKPTGVSDADLLPAGFEERTRGRGVVATRWVPQMSILAPIAAVGAF

LTHCGWNSTIEGLMFGHPLIMLPIFGDQGPNARLIEAKNAGLQVARNDGDGSFDR

EGVAAAIRAVAVEEESSKVFQAKAK LQEIVADMACHERYIDGFIQQLRSYKD

The tested plasmid was received in a microtiterplate containing a plasmid as freeze-dried solid in a separate well.

Suspension of plasmid To the well was added 24 μΕ of ultra-pure sterile water and the microtiter plate was shaken for 30 minutes at Room Temperature. Subsequently, the plate was incubated at 4°C for 1 hour. The content of the well was further mixed by pipetting up and down. The plasmid quantification was performed by Qubit2.0 analysis using 1 μί of suspension. Plasmid quantity was determined as follows:

Transformation of competent cells with plasmid. An aliquot of chemically competent EC 100 cells was taken from freezer at -80 °C and stored on ice. The cells were allowed to thaw on ice for 10 minutes. 10 μΕ of a dilution of above described plasmid solution was added to a sterile microtube of 1.5 mL (in order to transform each cell with 50 pg of DNA) and stored on ice. 100 μΐ, of chemically competent cells was added to the microtube. After incubation of the chemically competent cells/plasmid mixture on ice for 20 min a thermal shock of 30 seconds at 42°C was performed.

Further incubation was performed on ice for 2 minutes. To the microtube 300 μΐ_^ of SOC medium was added and the resulting mixture was transferred to a sterile 15 mL tube. After incubate for 1 hour at 37°C while shaking at 135 rpm, the mixture is spread on solid Luria Broth medium containing Kanamycin 50 μg/mL. The Petri dish is allowed to incubate for 16 hours at 37°C. Preparation of stock solutions in glycerol and purification of plasmid. To a 50 mL sterile Falcon Tube 10 mL of Luria Broth medium containing 50 μg/mL of Kanamycin was added. The medium was seeded with an isolated colony from the above described Petri dish and the cultures were allowed to incubate for 16 hours at 37 °C while shaking at 135 rpm.

To sterile microtube of 1.5 mL containing 300 iL of a 60% sterile glycerol solution, 600 iL of the culture was added. The stock solution was stored at -80 °C.

The remainder of the culture was centrifuged at 5,525 g for 10 minutes at 10°C and after removal of the supernatant, the pellet was stored on ice. The produced plasmids were purified according to the Qiagen Qiaprep Spin Miniprep kit (ref : 27106) and the plasmid yield was measured at 260 nm. The plasmid solution was stored at 4°C. Plasmid quantity was determined as follows:

In-vitro Expression of EUGT1 1. 18 of a diluted plasmid solution (containing approximately 1.5 μg of plasmid) was used for in-vitro expression according to the Promega S30 T7 High-Yield Protein Expression System (ref : LI 1 10) kit. The expression medium was produced as follows:

The prepared expression medium mix was added to the plasmid solution and the solution was allowed to incubate at 30°C for 3 hours while mixing the mixture every 45 minutes. 5 μΐ, of the mixture was frozen whereas the remainder was used for the catalytic test for the conversion of Rebaudioside A to Rebaudioside D.

Catalytic test for transformation of Rebaudioside A to Rebaudioside D. 430 μΐ, of a reaction mixture containing 0.5 mM Rebaudioside A, 3mM MgCl₂, 50 mM phosphate buffer (pH7.2) and 2.5 mM UDP-glucose was added to a 1,5 mL sterile microtube. 52 μΐ_^ of the enzyme expression medium was added and the resulting mixture was allowed to react at 30°C for 24 hours. 125 μϊ, samples were taken after 2 hours, 16 hours and 24 hours and added to a 1 15 of 60% methanol and 10 μΐ, of 2 N H₂S0₄. The quenched sample was centrifuged at 18,000 g for 2 minutes at RT. 200 iL was transferred to HPLC vial and analyzed as seen in Figure 47.

HPLC Analysis. The HPLC assay was performed as described in EXAMPLE 20.

The HPLC assay results are provided below:

Sample : 12400 S115N08G4 T24h 130702CJA

>gi 41469452 gb AAS07253.1 putative UDP-glucoronosyl and UDP-glucosyl transferase [Oiyza sativa Japonica Group] (EUGT1 1 enzyme from Patent application WO/2013/022989A2)

Filename : 130702 12400 026.dat

CAD Ch 1 Results

Compound Retention time Integration (area) ebaudioside D 5.797 54,654,810

Rebaudioside A 9.157 633,926,835

Total 688,581,645

EXAMPLE 22

In-vivo production of enzymes

The enzymes described in EXAMPLE 20 were produced in vivo.

The pET30A+ vector containing the gene corresponding to the enzyme was introduced in E. coli BL21(DE3) by heat shock. The obtained cells were grown in Petri dishes in the presence of Kanamycin and suitable colonies were selected and allowed to grow in liquid LB medium (Erlenmeyer flasks). Glycerol was added to the suspension as cryoprotector and 400 iL aliquots were stored at -20°C and at -80°C.

The storage aliquots of E. coli BL21 (DE3) containing the pET30A+_UGT plasmids were 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 of Kanamycine). This culture was allowed to shake at 135 rpm at 30°C for 8hrs. The production medium contained 60 g/L of overnight express instant TB medium (Novagen), 10 g/L of glycerol and 50 mg/L of Kanamycine. The preculture was added to 400 mL of this medium and the solution was allowed to stir at 20°C while taking samples to measure the OD and pH. The cultures gave significant growth and a good OD was obtained. After 40hrs, the cells were harvested by centrifugation and frozen. The following yields of cell wet weights (CWW) are mentioned below.

Lysis was performed by addition of Bugbuster Master mix (Novagen) and the lysate was recovered by centrifugation and used fresh.

Determination of activity. Activity tests were performed at 5 mL scale with 1,000 μί of thawed lysate for the transformation of Rebaudioside A using 0.5 mM of substrate, 2.5 mM of UDP-Glucose and 3 mM MgCl₂ in 50 mM Sodium Phosphate buffer at pH 7.2. Samples were taken and analyzed by HPLC as seen in Figures 48-52.

HPLC Analysis. The HPLC assay was performed as described in EXAMPLE 20.

The results for the different enzymes are provided below.

SAMPLE : 12400 S129N01 T45h 130712ABA

> gi l 15454819 / NP 001051010.1

Filename : 130712 12400 003.dat

CAD Ch 1 Results

Compound Retention time Integration (area )

Rebaudioside D 5.775 3,264,475

[email protected] 6.986 4, 1 10,607

[email protected] 7.330 564,033,104

[email protected] 7.700 328,710,539

[email protected] 8.158 6,344,796

Rebaudioside A 9.135 673,271,863

[email protected] 9.653 616,489,141

Total 2,196,224,525

Sample : 12400 S129N02 T45h 130712ABA

> gi 187373030 / ACD03249.1

Filename : 130712 12400 004.dat

Sample : 12400 S129N04 T45h 130712ABA > gi460409128 / XP_004249992.1 Filename : 130712 12400 006.dat

Sample : 12400 S129N05 T45h 130712ABA > gi222619587 / EEE55719.1

Filename : 130712 12400 007.dat

Sample : 12400 S129N06 T45h 130712ABA

> gi297795735 / XP 002865752.1

Filename : 130712 12400 008.dat

CAD Ch 1 Results

Compound Retention time Integration (area)

[email protected] 6.998 920,620,332

[email protected] 7.696 314,421,575

Rebaudioside A 9.128 688,195,594

[email protected] 9.645 308,115,680

Total 2,231,353, 181

EXAMPLE 23

Identification of glycosides

The reaction mixtures representing GI No. 460409128, particularly the sample "12400 S1 15N05A7 T24h 130627ABA" of EXAMPLE 20 (hereinafter S1 15N05A7), and the sample "12400 S129N04 T45h 130712ABA" of EXAMPLE 22 (hereinafter S129N04) were additionally assayed by LC-MS to identify the unknown glycosides. An Agilent 1200 series HPLC system, equipped with binary pump (G1312B), autosampler (G1367D), thermostatted column compartment (G1316B), DAD detector (G1315C), connected with Agilent 61 10A MSD, and interfaced with "LC/MSD Chemstation" software, was used.

Instrument conditions

Mobile phase gradient program

The compound observed on LCMS system at 3.5min, corresponds to compound "[email protected]" in sample "S 1 15N05A7" (EXAMPLE 20), and compound "[email protected]" in sample "S 129N04" (EXAMPLE 22). The LCMS data suggests that this compound has six glucosidic residues (C56H90O33) in its structure, and was found to be an isomer form of reb M, namely reb M2 (see Example 40 for discussion).

Whereas the compound observed on LCMS system at 7.6min, corresponds with compound "reb UNK in sample "S1 15N05A7" (EXAMPLE 20), and compound "reb UNK" in sample "S 129N04" (EXAMPLE 22), The LCMS data suggests that "reb UNK" has five glucosidic residues (CsoHsoChs) in its structure, and was found to be an isomer form of reb D, namely reb D2 (see Example 39 for discussion). The ratio of these compounds and the LCMS chromatograms are provided below and as shown in Figures 53-54.

Total 2,501,929

EXAMPLE 24

Identification of glycosides

The reaction mixture representing GI No. 460409128, particularly the sample "12400 S 129N04 T45h 130712ABA" of EXAMPLE 22 (hereinafter S129N04) were additionally assayed by LC-MS, as seen in Figures 55-56, along with Stevia rebaudiana Bertoni leaf extract "MLDl" produced by PureCircle Sdn Bhd (Malaysia) to determine the occurrence of S 129N04 glycosides in nature.

ebaudioside UNK 1,129

Samples :

1) 12400 S129N04 T45h 130712ABA

> gi460409128 / XP_004249992.1

2) MLD1 Stevia rebaudiana Bertoni extract

MSP SIM 1,291 Results

Compound MW

Unknown(¾RT3.550 1 ,291

Rebaudioside M 1 ,291

The assay shows that the compound observed on LCMS system at 3.5min, in EXAMPLE 23 (C56H₉₀O33; later confirmed as reb Ml), and the compound observed on LCMS system at 7.6min, in EXAMPLE 23 (C₅oH₈o0₂8_; reb UNK; later confirmed as reb D2) occur in the extract of Stevia rebaudiana Bertoni plant.

EXAMPLE 25

Conversion of Rebaudioside E to Rebaudioside D The total volume of the reaction was 5.0 mL with the following composition: 100 raM potassium phosphate buffer pH 7.5, 3 mM MgCl₂, 2.5 mM UDP-glucose, 0.5 mM Rebaudioside E and 500 μί of UGT76G1 thawed lysate (UGT76G1 gene was cloned in pET30a+ vector and expressed in E. coli BL21 (DE3)). The reactions were run at 30°C on an orbitary shaker at 135 rpm. For sampling 300 of the reaction mixture was quenched with 30 \iL of 2N H₂S0₄ and 270 iL of methanol/water (6/4). The samples were immediately centrifuged and kept at 10°C before analysis by HPLC (CAD detection). The following reaction profile was obtained corresponding to a complete conversion of Rebaudioside E to Rebaudioside D as seen in Figure 57.

Conversion of Rebaudioside E to Rebauc

D by in-vivo produced UGT76G1

EXAMPLE 26

Directed evolution of UGT76G1 for the conversion of Rebaudioside D to Rebaudioside M

Starting from the amino acid sequence of UGT76G1 , as is described in Genbank (AAR06912.1 ), different mutations at various amino acid positions were identified that could alter the activity of the enzyme for the transformation of Rebaudioside D (Reb D) to Rebaudioside M (Reb M). This list of mutations, designed by DNA2.0 ProteinGPS™ strategy, was subsequently used to synthesize 96 variant genes that contained 3, 4 or 5 of these mutations that 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) chemically competent cells. The obtained cells were grown in Petri-dishes on solid LB medium in the presence of Kanamycin. Suitable colonies were selected and allowed to grow in liquid LB medium in tubes. Glycerol was added to the suspension as cryoprotectant and 400 μΐ, aliquots were stored at -20 °C and at -80 °C.

These storage aliquots of E. coli BL21(DE3) containing the pET30a+_UGT76Gl var plasmids 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 of Kanamycine). This culture was allowed to shake in a 96 microtiter plate at 135 rpm at 30°C for 8 h.

3.95 mL of production medium containing 60 g/L of Overnight Express™ Instant TB medium ( ovagen®), 10 g/L of glycerol and 50 mg/L of Kanamycin was inoculated with 50 h of above described culture. In a 48 deepwell plate the resulting culture was allowed to stir at 20°C. The cultures gave significant growth and a good OD (600 nm; 1 cm) was obtained. After 44 h, the cells were harvested by centrifugation and frozen.

Lysis was performed by addition of Bugbuster® Master mix (Novagen®) to the thawed cells and the lysate was recovered by centrifugation. Activity tests were performed with 100 of fresh lysate that was added to a solution of ebaudioside 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 allowed to run at 30°C and samples were taken after 2, 4, 7 and 24 h. to determine conversion and initial rate by HPLC (CAD detection) using the analytical method that was described above for the transformation of Rebaudioside D to Rebaudioside M . The results are depicted in the following table.

mutat on o an a an ne at pos t on 33 to a glyc ne is note as A33G.

EXAMPLE 27

In-vivo production of UGTSL2

SEQ ID 9

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

MATNLRVLMFPWLAYGHISPFLNIAKQLADRGFLIYLCSTRINLESIIKKIPEKYAD

SIHLIELQLPELPELPPHYHTTNGLPPHLNPTLHKALKMSKPNFSRILQNLKPDLLIY

DVLQPWAEHVANEQNIPAGKLLTSCAAVFSYFFSFRKNPGVEFPFPAIHLPEVEKV

KIREILAKEPEEGGRLDEGNKQMMLMCTSRTIEAKYIDYCTELCNWKVVPVGPPF

QDLITNDADNKELIDWLGTKHENSTVFVSFGSEYFLSKEDMEEVAFALELSNVNFI

WVARFPKGEERNLEDALPKGFLERIGERGRVLDKFAPQPRILNHPSTGGFISHCGW

NSAMESIDFGVPIIAMPIHNDQPINAKLMVELGVAVEIVRDDDGKIHRGEIAETLKS

VVTGETGEILRAKVREISKNLKSIRDEEMDAVAEELIQLCRNSNKSK

The pET30A+ vector containing the UGTSL2 gene was introduced in E. coli B121(DE3) by heat shock. The obtained cells were grown in petri-dishes in the presence of Kanamycin and suitable colonies were selected and allowed to grow in liquid LB medium (erlenmeyer flasks). Glycerol was added to the suspension as cryoprotecteur and 400 aliquots were stored at -20°C and at -80°C.

The storage aliquots of E. coli BL21(DE3) containing the pET30A+_UGTSL2 plasmids were 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 of Kanamycin). This culture was allowed to shake at 135 rpm at 30°C for 8 h. The production medium contained 60 g/L of overnight express instant TB medium (Novagen), 10 g/L of glycerol and 50 mg/L of Kanamycin. The preculture was added to 200 mL of this medium and the solution was allowed to stir at 20°C while taking samples to measure the OD and pH. The culture gave significant growth and a good OD was obtained. After 40 h, the cells were harvested by centrifugation and frozen to obtain 6.22 g of cell wet weight.

Lysis was performed on 1.4 g of cells by addition of Bugbuster Master mix (Novagen) and the lysate was recovered by centrifugation and used fresh.

EXAMPLE 28

Determination of activity for Stevioside to Rebaudioside E conversion with UGTSL and UGTSL2

UGTSL was prepared according to EXAMPLE 22, and UGTSL2 was prepared according to EXAMPLE 27.

Activity tests were performed at 3 mL scale with 600 \iL of lysate for the transformation of Stevioside using 0.5 mM of substrate, 2.5 mM of UDP-Glucose and 3 mM MgCl₂ in 50 mM Sodium Phosphate buffer at pH 7.2. Samples were taken and analyzed by HPLC. HPLC Analysis as shown in Figures 58-59. The HPLC assay was performed as described in EXAMPLE 20.

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

Note: Based on initial concentration of Stevioside

SAMPLE : 12400 S 151N33 T22h 130926ABA

Gene references: UGTSL (XP_004249992.1)

Filename : 130926 12400 042.dat

EXAMPLE 29

Determination of activity for Rubusoside to Rebaudioside E conversion with UGTSL and UGTSL2

Activity tests were performed at 3 mL scale with 600 μΕ of lysate for the transformation of Rubusoside using 0.5 mM of substrate, 2.5 mM of UDP-Glucose and 3 mM MgCl₂ in 50 mM Sodium Phosphate buffer at pH 7.2. Samples were taken and analyzed by HPLC as shown in Figures 60-61. The HPLC assay was performed as described in EXAMPLE 20.

Note: Based on initial concentration of Rubusoside

EXAMPLE 30

Determination of activity for Rebaudioside A to Rebaudioside D conversion with UGTSL2

UGTSL2 was prepared according to EXAMPLE 27.

Activity tests were performed at 3 mL scale with 60 iL of lysate for the transformation of Rebaudioside A using 0.5 mM of substrate, 2.5 mM of UDP-Glucose and 3 mM MgCl₂ in 50 mM Sodium Phosphate buffer at pH 7.2. Samples were taken and analyzed by HPLC as shown in Figure 62. The HPLC assay was performed as described in EXAMPLE 20.

The result after 23 h. of reaction and the corresponding chromatogram is provided below.

Note: Based on initial concentration of Rebaudioside A

EXAMPLE 31

Identification of glycosides

The reaction mixtures prepared according to EXAMPLE 30 and incubated for 45hrs was analyzed by LC-MS, along with Sterna rebaudiana Bertoni leaf extract "MLD1" produced by PureCircle Sdn Bhd (Malaysia), to determine the occurrence of formed glycosides in nature.

An Agilent 1200 series HPLC system, equipped with binary pump (G1312B), autosampler (G1367D), thermostatted column compartment (G1316B), DAD detector (G1315C), connected with Agilent 61 10A MSD, and interfaced with "LC/MSD Chemstation" software, was used, and the chromatogram is shown in Figure 63.

Instrument conditions

Mobile phase gradient program

The assay shows that the compound observed on LC-MS system at 1 1.77min is the same as the compound at 3.5min, in EXAMPLE 23 (C56H 0O33; later confirmed as reb M2), and the compound observed at 26.64 min is the same as the compound at 7.6min, in EXAMPLE 23 (C₅oH₈o0₂8; reb UNK; later confirmed as reb D2). Other isomers of reb M were observed at 13.96min and also another isomer form of reb D was observed at 25.06min. All observed compounds occurred in the extract of Stevia rebaudiana Bertoni plant.

EXAMPLE 32

In vivo preparation and activity determination of UGTLB

SEQ ID 10

UGTLB (GI_209954733 / BAG80557.1) amino acid sequence

MGTEVTVHKNTLRVLMFPWLAYGHISPFLNVAKKLVDRGFLIYLCSTAINLKSTIK

KIPEKYSDSIQLIELHLPELPELPPHYHTTNGLPPHLNHTLQKALKMSKPNFSKILQ

NLKPDLVIYDLLQQWAEGVANEQNIPAVKLLTSGAAVLSYFFNLVKKPGVEFPFP

AIYLRKNELEKMSELLAQSAKDKEPDGVDPFADGNMQVMLMSTSRIIEAKYIDYF

SGLSNWKVVPVGPPVQDPIADDADEMELIDWLGKKDENSTVFVSFGSEYFLSKED

REEIAFGLELSNVNFIWVARFPKGEEQNLEDALPKGFLERIGDRGRVLD FAPQPRI

LNHPSTGGFISHCGWNSVMESVDFGVPIIAMPIHLDQPMNARLIVELGVAVEIVRD

DYGKIHREEIAEILKDVIAGKSGENLKAKMRDISKNLKSIRDEEMDTAAEELIQLC

KNSPKLK

The pET30A+ vector containing the UGTLB gene was introduced in E. coli B121(DE3) by heat shock. The obtained cells were grown in petri-dishes in the presence of Kanamycin and suitable colonies were selected and allowed to grow in liquid LB medium (erlenmeyer flasks). Glycerol was added to the suspension as cryoprotecteur and 400 iL aliquots were stored at -20°C and at -80°C.

The storage aliquots of E. coli BL21(DE3) containing the pET30A+_UGTLB plasmids were 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 of Kanamycine). This culture was allowed to shake at 135 rpm at 30°C for 8 h.

The production medium contained 60 g/L of overnight express instant TB medium (Novagen), 10 g/L of glycerol and 50 mg/L of Kanamycine. The preculture was added to 200 mL of this medium and the solution was allowed to stir at 20°C while taking samples to measure the OD and pH. The culture gave significant growth and a good OD was obtained. After 40 h, the cells were harvested by centrifugation and frozen to obtain 5.7 g of cell wet weight.

Lysis was performed on 1 .2 g of cells by addition of 6 mL Bugbuster Master mix (Novagen) and the lysate was recovered by centrifugation and used fresh.

Determination of activity for Stevioside to Rebaudioside E conversion with UGTLB Activity tests were performed at 3 mL scale with 600 μί of lysate for the transformation of Stevioside using 0.5 mM of substrate, 2.5 mM of UDP-Glucose and 3 mM MgCl₂ in 50 mM Sodium Phosphate buffer at pH 7.2. Samples were taken and analyzed by HPLC as shown in Figures 64-66. The corresponding chromatograms are depicted below.

Note: Based on initial concentration of Stevioside

Determination of activity for Rubusoside to Rebaudioside E conversion with UGTLB

Activity tests were performed at 3 mL scale with 600 of lysate for the transformation of Rubusoside using 0.5 mM of substrate, 2.5 mM of UDP-Glucose and 3 mM MgCl₂ in 50 mM Sodium Phosphate buffer at pH 7.2. Samples were taken and analyzed by HPLC.

The corresponding chromatograms are depicted below.

Note: Based on initial concentration of Rubusoside SAMPLE : 12400 S 151N17 T5h 130927ABA

Gene references: UGTLB (BAG80557.1)

Filename : 130927 12400 084.dat

CAD Ch 1 Results

Compound Retention time Integration (area)

Rebaudioside E 5.491 21921232

[email protected] 7.010 9764063

[email protected] 7.295 12510947

[email protected] 7.677 283386906

[email protected] 8.728 402240506

[email protected] 9.630 878990745

Rubusoside 11.227 176000085

Totals 1784814484

Determination of activity for Rebaudioside A to Rebaudioside D conversion with UGTLB Activity tests were performed at 3 mL scale with 600 μΐ, of lysate for the transformation of Rebaudioside A using 0.5 mM of substrate, 2.5 mM of UDP-Glucose and 3 mM MgCl₂ in 50 mM Sodium Phosphate buffer at pH 7.2. Samples were taken and analyzed by

HPLC. The corresponding chromatogram after 23 h. of reaction is depicted below.

Note: Based on initial concentration of Rebaudioside A

SAMPLE : 12400 S154N6 T22h 130926ABA

Gene references: UGTLB (BAG80557.1)

Filename : 130926 12400 015.dat

CAD Ch 1 Results | I Compound Retention time Integration (area)

Unkno [email protected] 4.522 137,916,950

[email protected] 4.903 2,015,271

Rebaudioside D 5.762 59,876,764

[email protected] 6.689 364,185,331

[email protected] 6.973 26,368,965

[email protected] 7.318 110,284, 197

[email protected] 7.689 294,579,799

[email protected] 8.293 7,867,452

[email protected] 8.779 15,928,550

Rebausioside A 9.118 165,602,247

[email protected] 9.642 868,327,712

Totals 2,052,953,238

EXAMPLE 33

Determination of reaction products for Rubusoside and Stevioside conversion with UGTSL, UGTSL2, and UGTLB

Conversion of stevioside with UGTSL and UGTSL2 was conducted in similar manner to Example 28, and the conversion of rubusoside with UGTSL and UGTSL2 was conducted similarly to Example 29. Conversions of rubusoside and stevioside with UGTLB was conducted similarly to Example 32.

The reaction mixtures were analyzed by LCMS to determine all reaction products.

Rubusoside conversion roducts

Stevioside conversion roducts

It can be seen that amongst Rubusoside conversion products, besides Stevioside, Reb E and Reb D, there are at least 3 additional compounds with Molecular Weight of 804. The retention time of these compounds do not match with Reb B which is known to have same Molecular Weight as Stevioside. Since these compounds have same molecular weight with Stevioside it can be assumed that these novel steviol glycosides are isomers of Stevioside. On the other hand amongst Stevioside conversion products, besides Reb E and Reb D, there are at least 3 additional compounds with Molecular Weight of 966. The retention time of these compounds do not match with Reb A which is known to have same Molecular Weight as Reb E. Since these compounds have same molecular weight with Reb A and Reb E it can be assumed that these novel steviol glycosides are isomers of Reb A (Reb E).

EXAMPLE 34

In vivo production of UGT76G1 in S. cerevisiae SEQ ID 1 1

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

MENKTETTVRRRRRIILFPVPFQGHINPILQLANVLYSKGFSITIFHTNFNKPKTSNY

PHFTFRFILDNDPQDERISNLPTHGPLAGMRIPIINEHGADELRRELELLMLASEEDE

EVSCLITDALWYFAQSVADSLNLRRLVLMTSSLFNFHAHVSLPQFDELGYLDPDD

KTRLEEQASGFPMLKVKDIKSAYSNWQILKEILGKMIKQTKASSGVIW SFKELEE

SELETVIREIPAPSFLIPLPKHLTASSSSLLDHDRTVFQWLDQQPPSSVLYVSFGSTS

EVDEKDFLEIARGLVDSKQSFLWVVRPGFVKGSTWVEPLPDGFLGERGRIVKWVP

QQEVLAHGAIGAFWTHSGW STLESVCEGVPM1FSDFGLDQPLNARYMSDVLKV

GVYLENGWERGEIANAIRRVMVDEEGEYIRQNARVLKQKADVSLMKGGSSYESL

ESLVSYISSL

The above mentioned amino acid sequence was codon optimized for expression in S. cerevisiae. Furthermore the yeast consensus sequence AACACA was added before the ATG start codon. The synthetic gene was subcloned in the pYES2 vector using Hind III and Xba I restriction sites. The pYES2_UGT76Gl_Sc vector was used to transform chemically competent S. cerevisiae INVScl cells (Invitrogen).

The cells were grown on a solid synthetic minimal medium containing 2% glucose lacking Uracil and a single colony was picked and allowed to grow in liquid synthetic minimal medium lacking Uracil (SC-U containing 2% glucose). After centrifugation, the cells were suspended with SC-U (containing 2% glucose) and 60% glycerol/water. Aliquots were stored at -80°C and one aliquot was used to start a culture in SC-U (containing 2% glucose) for 43 h at 30°C. Part of this culture was centrifuged and suspended in induction medium (SC-U containing 2% galactose) for 19h30 at 30 °C.

Cells were obtained by centrifugation and lysis with five volumes of CelLytic™ Y Cell Lysis Reagent (Sigma). The lysates were used directly for activity testing (UGT76Gl_Sc).

EXAMPLE 35

Determination of activity of UGT76Gl_Sc for the conversion of Rebaudioside D to Rebaudioside M

UGT76Gl_Sc was prepared according to EXAMPLE 34. Activity tests were performed at 2 mL scale with 200 of lysate for the transformation of Rebaudioside D using 0.5 mM of substrate, 2.5 mM of UDP-Glucose and 3 mM MgCl₂ in 50 mM Sodium Phosphate buffer at pH 7.2. Samples were taken and analyzed by HPLC as shown in Figure 67. The corresponding chromatogram is depicted below.

Note: Based on initial concentration of Rebaudioside D

SAMPLE : 12400 S 169N10 T21h 131119CJA

Gene references: UGT76G1 Sc

Filename : 131122 12400 238.dat

CAD Ch 1 Results

Compound Retention time Integration (area)

Rebaudioside D 5.750 112,094,430

[email protected] 6.235 17,886,043

Rebaudioside M 6.700 616,583,935

Rebaudioside A 9.095 11, 183,884

Unknown® 10.27 10.272 62,863,156

Unknown@l 1.31 11.310 35,839,478

Total 856,450,926

EXAMPLE 36

In vivo production of UGTSL in S. cerevisiae SEQ ID 12

UGTSL [Solanum lycopersicum] (gi_460409128 / XP_004249992.1

MSPKLHKELFFHSLYKKTRSNHTMATLKVLMFPFLAYGHISPYLNVAK LADRGF

LIYFCSTPINLKSTIEKIPEKYADSIHLIELHLPELPQLPPHYHTTNGLPPNLNQVLQK

ALKMSKPNFSKILQNLKPDLVIYDILQRWAKHVANEQNIPAVKLLTSGAAVFSYFF

NVLKKPGVEFPFPGIYLRKIEQVRLSEMMSKSDKEKELEDDDDDDDLLVDGNMQI

MLMSTSRTIEAKYIDFCTALTNWKVVPVGPPVQDLITNDVDDMELIDWLGTKDE

NSTVFVSFGSEYFLSKEDMEEVAFALELSNVNFIWVARFPKGEERNLEDALPKGFL

ERIGERGRVLDKFAPQPRILNHPSTGGFISHCGW SAMESIDFGVPIIAMPMHLDQP

MNARLIVELGVAVEIVRDDDGKIHRGEIAETLKGVITGKTGEKLRAKVRDISKNLK

TIRDEEMDAAAEELIQLCRNGN

The above mentioned amino acid sequence was codon optimized for expression in S. cerevisiae. Furthermore the yeast consensus sequence AACACA was added before the ATG start codon. The synthetic gene was subcloned in the pYES2 vector using Hind Hi and Xba I restriction sites. The pYES2_UGTSL_Sc vector was used to transform chemically competent S. cerevisiae INVScl cells (Invitrogen).

The cells were grown on a solid synthetic minimal medium containing 2% glucose, lacking Uracil and a single colony was picked and allowed to grow in liquid synthetic minimal medium lacking Uracil (SC-U containing 2% glucose). After centrifugation, the cells were suspended with SC-U (containing 2% glucose) and 60% glycerol/water. Aliquots were stored at -80°C and one aliquot was used to start a culture in SC-U (containing 2% glucose) for 43 h at 30°C. Part of this culture was centrifuged and suspended in induction medium (SC-U containing 2% galactose) for 19h30 at 30°C.

Cells were obtained by centrifugation and lysis with five volumes of CelLytic™ Y Cell Lysis Reagent (Sigma). The lysates were used directly for activity testing (UGTSL_Sc).

EXAMPLE 37

Determination of activity of UGTSL Sc for the conversion of Rebaudioside A to Rebaudioside D

UGTSL_Sc was prepared according to EXAMPLE 36. Activity tests were performed at 2 mL scale with 200 iL of lysate for the transformation of Rebaudioside A using 0.5 mM of substrate, 2.5 mM of UDP-Glucose and 3 mM MgCl₂ in 50 mM Sodium Phosphate buffer at pH 7.2. Samples were taken and analyzed by HPLC as shown in Figure 68. The corresponding chromatogram is depicted below.

Note: Based on initial concentration of Rebaudioside A

SAMPLE : 12400 S 169N02 T4h 13 1 1 19CJA

Gene references: UGTSL Sc

Filename : 13 1 122 12400 203.dat

CAD Ch 1 Results

Compound Retention time Integration (area)

[email protected] 4.500 75,046,986

Rebaudioside D 5.73 1 223,409,643

[email protected] 6.658 228,651 ,278

Rebaudioside A 9.084 404,642,305

Unknown® 10.08 10.079 43,992,253

Unknown® 1 1 .21 1 1.21 1 29,776,761

Unknown® 1 1 .90 1 1.905 2, 185,3 16

Total 1 ,007,704,542

EXAMPLE 38

Isolation of Rebaudioside M

The amount of the product mixture of Example 14 was not large enough to separate via preparative HPLC methods. Accordingly, analytical HPLC with a series of injections was used to separate the components of the mixture. Separation was conducted according to the method described above in Example 14 to provide two fractions corresponding to the two main peaks in the HPLC trace of FIG. 5: Fraction A (retention time 24.165 minutes) and Fraction B (retention time 31.325 minutes).

The retention time of Fraction A was consistent with reb D, indicating unreacted starting material from the biotransformation reaction.

The retention time of purified Fraction B (FIG. 6) was consistent with reb M, indicating successful biotransformation from reb D. The identity of the material collected in Fraction B as reb was confirmed by co-injection of purified Fraction B with a reb M standard (available from PureCircle, HPLC trace of reb M standard shown in FIG. 7). Both Fraction B and the reb M standard were found to elute at the same retention time (FIG. 8), indicating Fraction B was reb M.

The identity of Fraction B as reb M was also separately confirmed by NMR and HRMS. For sampling, Fraction B was concentrated under rotary evaporator, freeze dried and dried for 40 h at 40 °C.

The NMR sample was dissolved in deuterated pyridine (C₅D₅N) and spectra were acquired on a Varian Unity Plus 600 MHz instrument using standard pulse sequences. The NMR spectra of Fraction B was compared to the NMR spectra of reb M. An overlay of the two spectra (FIG. 9) showed consistency of peaks of Fraction B with reb M. A table of the NMR assignments for reb is shown below:

1H and "C NMR spectral data for Rebaudioside M in C₅D₅N

1 ' 94.9 6.39 d (8.2)

2' 76.9 4.51 t (8.5)

3' 88.6 5.09 t (8.5)

4' 70.1 4.18 m

5' 78.4 4.13 m

4.20 m

6' 61.8

4.31 m

1 " 96.2 5.46 d (7.1)

2" 81.4 4.13 m

3" 87.9 4.98 t (8.5)

4" 70.4 4.07 t (9.6)

5" 77.7 3.94 m

4.19 m

6" 62.6

4.32 m

Γ' 104.8 5.48 d (7.7)

2'" 75.8 4.15 m

78.6 4.13 m

4'" 73.2 3.98 m

5'" 77.6 3.74 ddd (2.8, 6.4, 9.9)

4.27 m

6'" 64.0

4.51m

1 103.9 5.45 d (7.5)

2"" 75.6 3.98 m

3"" 77.8 4.50 t (7.8)

4"" 71.3 4.14 m

5"" 78.0 3.99 m

4.20 m

6"" 62.1

4.32 m

104.2 5.81 d (7.2)

2""' 75.5 4.20 m

3""' 78.4 4.20 m

4""' 73.6 4.10 m 5""' 77.8 3.90 ddd (2.8, 6.4, 9.9)

4.32 m

6""' 64.0

4.64 d (10.3)

1 ^tt,f,t 104.1 5.31 d (8.0)

2""" 75.5 3.95 m

78.0 4.37 1 (9.1 )

71.1 4.10 m

78.1 3.85 ddd (1.7, 6.1, 9.9)

4.10 m

62.1

4.32 m ^a assignments made on the basis of COSY, HMQC and HMBC correlations; ^b Chemical shift values are in δ (ppm); ^c Coupling constants are in Hz.

HRMS (FIG. 10) was generated with a Waters Premier Quadropole Time-of-Flight (Q-TOF) mass spectrometer equipped with an electrospray ionization source operated in the positive-ion mode. The sample was dissolved in methanol and eluted in 2:2: 1 methanol: acetonitrile: water and introduced via infusion using the onboard syringe pump. The presence of reb was confirmed by a [M+Na]⁺ adduct at m/z 1313.5265, which corresponds to a molecular formula of C56H90O₃₃

EXAMPLE 39

Isolation and Characterization of Reb D2

Crude Reaction Sample. The sample, Lot CB-2977-106, used for isolation, was prepared according to Example 22 with UGTSL (GI #460409128).

HPLC Analysis. Preliminary HPLC analyses of samples were performed using a Waters 2695 Alliance System with the following method: Phenomenex Synergi Hydro-RP, 4.6 ^χ 250 mm, 4 μηι (p/n 00G-4375-E0); Column Temp: 55 °C; Mobile Phase A: 0.0284% ammonium acetate (NH4OAC) and 0.01 16% acetic acid (HOAc) in water; Mobile Phase B: Acetonitrile (MeCN); Flow Rate: 1.0 mL/min; Injection volume: 10 \iL. Detection was by UV (210 nm) and CAD.

Gradient:

Time (min) %A %B

0.0 - 8.5 75 25

10.0 71 29

16.5 70 30

18.5 - 24.5 66 34

26.5 - 29.0 48 52

31 - 37 30 70

38 75 25

semi-preparative purification fractions were performed with the following method: Waters Atlantis dCl 8, 4.6 100 mm, 5 μιη (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 μΐ,.

Detection was by CAD.

Gradient:

LC-MS. Preliminary analysis of the semi-synthetic steviol glycoside mixture was carried out on a Waters AutoPurification HPLC/MS System with a Waters 3100 Mass Detector operating in negative ion mode. Analysis of the sample was performed using the following method: Phenomenex Synergi Hydro-RP, 4.6 ^χ 250 mm, 4 μιη (p/n 00G-4375- E0); Column Temp: 55 °C; Mobile Phase A: 0.0284% NH₄OAc and 0.01 16% HOAc in water; Mobile Phase B: Acetonitrile; Flow Rate: 1.0 mL/min; Injection volume: 10 iL. Detection was by UV (210 nm), and MSD (-ESI m/z 500 - 2000). Gradient conditions were as listed above.

Isolation by HPLC. The purification was performed in two steps. The first method used for the semi-preparative purification is summarized below. Column: Waters Atlantis dC18, 30 x 100 mm, 5 μιη (p/n 186001375); Mobile Phase A: 25% MeCN in water; Mobile Phase B: 30% MeCN in water; Flow Rate: 45 mL/min; Injection load: 160 mg dissolved in 20 mL of water. Detection was by UV (205 nm).

Gradient:

The secondary purification used the same column and conditions, but isocratic mobile phase: 20% MeCN in water.

Purification from Natural Extracts. The purification was performed in three steps. The first method used for the preparative purification is summarized below. Primary Process: Waters Symmetry CI 8, 50 * 250 mm, 7 μιη (p/n WAT248000); Isocratic mobile phase: 50% methanol (MeOH) in water with 0.05% HOAc; Flow Rate: 85 mL/min; Injection load: 6 g crude extract dissolved in 50 mL of mobile phase. Detection was by UV (210 nm). Following the elution of target analytes, the column was flushed with 85% MeOH in water.

Secondary Process: Waters Symmetry Shield RP18, 50 ^χ 250 mm, 7 μιη (p/n WAT248000); Isocratic mobile phase: 20% MeCN in water; Flow Rate: 100 mL/min; Injection load: 0.5 g primary fraction dissolved in 30 mL of water. Detection was by UV (210 nm).

Tertiary Process: Waters Symmetry Shield RP18, 50 ^χ 250 mm, 7 μηι (p/n WAT248000); Isocratic mobile phase: 20% MeCN in water; Flow Rate: 100 mL/min; Injection load: 0.5 g secondaiy fraction dissolved in 30 mL of water. Detection was by UV (210 nm).

MS and MS/MS. MS and MS/MS data were generated with a Waters QT of Premier mass spectrometer equipped with an electrospray ionization source. Samples were analyzed by negative ESI. Samples were diluted with ELC^acetonitrile (1 : 1 ) by 50 fold and introduced via infusion using the onboard syringe pump. The samples were diluted to yield good s/n which occurred at an approximate concentration of 0.01 mg/mL.

NM . The sample was prepared by dissolving 1 - 2 mg in 150 of pyridine-i/₅ and NMR data were acquired on a Bruker Avance 500 MHz instrument with a 2.5 mm inverse detection probe. The Ή NMR spectrum was referenced to the residual solvent signal (5H 8.74 and 5_C 150.35 for pyridine-c 5).

Results and Discussion

Isolation and Purification. Isolation was performed on steviol glycoside mixture, Lot number CB-2977-106, prepared according to Example 22 with UGTSL (GI #460409128) The material was analyzed by LC-MS using the method described above and results are provided in Figure 1 1. The targeted peak of interest was that at 7.7 min in the TIC chromatogram. The mass spectrum of this peak provided a [M-H]^" ion at m/z 1 127.6. The provided sample was preliminarily processed in a single injection (160 mg) using the first method condition provided above. This method fractionated the material into 'polar' and 'non-polar' mixtures of glycosides. The 'polar' mixture was then reprocessed using the second-step conditions above. The semi-preparative HPLC trace is provided in Figure 12. From this semi-preparative collection, the compound was isolated with a purity >99% (CAD, AUC). The fraction analysis is provided in Figure 13. Following the purification, the combined fractions were concentrated by rotary evaporation at 35 °C and lyophilized. Approximately 1 - 2 mg was obtained for characterization.

Mass Spectrometry. The ESI- TOF mass spectrum acquired by infusing a sample showed a [M-H]^" ion at m/z 1 127.4709. The mass of the [M-H]^" ion was in good agreement with the molecular formula C₅oH₈o0₂₈ (calcd for C50H79O28: 1 127.4758, error: -4.3 ppm). The MS data confirmed a nominal mass of 1 128 Daltons with the molecular formula, C50H80O28.

The MS/MS spectrum (selecting the [M-H]^" ion at m/z 1 127.5 for fragmentation) indicated the loss of two glucose units and sequential loss of three glucose moieties at m/z 641.3187, 479.2655 and 317.2065. NMR Spectroscopy. A series of NMR experiments including Ή NMR (Figure 14), ¹³C NMR (Figure 15 and 16), Ή-Ή COSY (Figure 17), HSQC-DEPT (Figure 18), HMBC (Figures 19 and 20), NOESY (Figure 21) and 1 D-TOCSY (Figure 22-26) were performed to allow assignment of the compound. In the 'H NMR acquired after -46 hrs of sample preparation (Figures 27-28), the anomeric resonance at δπ 5.04 is resolved which was obscured by the solvent (HOD) in the original spectrum (Figure 14)

The Ή, Ή-Ή COSY, 1H-¹³C HSQC-DEPT and 'H-^,3C HMBC NMR data indicated that the central core of the glycoside is a diterpene. The presence of five anomeric protons observed in the 'H and 'H-¹³C HSQC-DEPT spectra confirm five sugar units in the structure. The methylene ¹³C resonance at 5c 69.9 in the ^]H-¹³C HSQC-DEPT spectrum indicated the presence of a 1→6 sugar linkage in the structure. The linkages of sugar units were assigned using 'H-¹³C HMBC and 1D-TOCSY correlations.

A HMBC correlation from the methyl protons at δ_Η 1.29 to the carbonyl at 5_C 177.7 allowed assignment of one of the tertiary methyl groups (C-18) as well as C-19 and provided a starting point for the assignment of the rest of the aglycone. Additional HMBC correlations from the methyl protons (H-18) to carbons at 5c 38.9, 45.0, and 57.8 allowed assignment of C-3, C-4, and C-5. Analysis of the 'H-¹³C HSQC-DEPT data indicated that the carbon at 8c 38.9 was a methylene group and the carbon at 5c 57.8 was a methine which were assigned as C-3 and C-5, respectively. This left the carbon at 5c 45.0, which did not show a correlation in the HSQC-DEPT spectrum, to be assigned as the quaternary carbon, C-4. The *H chemical shifts for C-3 (δ_Η 0.98 and 2J.36) and C-5 (δ_Η 1.04) were assigned using the HSQC-DEPT data. A COSY correlation between one of the H-3 protons (5H 0.98) and a proton at 5H 1.43 allowed assignment of one of the H-2 protons which in turn showed a correlation with a proton at δπ 0.75 which was assigned to C-l . The remaining Ή and ¹³C chemical shifts for C-l and C-2 were then assigned on the basis of additional COSY and HSQC-DEPT correlations and are summarized in the table below.

1H and °C NMR (500 and 125 MHz, pyridine-^), Assignments of Reb D2.

Reb D2

Position ¹ C

1 41.3 0.75 t (1 1.0) 1.76 m

19.9 1.43 m

2

2.20 m

38.9 0.98 m

3

2.36 d (12.1)

4 45.0 —

5 57.8 1.04 d (12.5)

22.7 1.92 m

6

2.43 m

42.2 1.22 m

7

1.30 m

8 43.1 —

9 54.5 0.88 brs

10 40.3 —

21.1 1.65 m

1 1

1.69 m

37.5 1.99 m

12

2.25 m

13 87.1 —

44.5 1.80 d (1 1.7)

14

2.65 d (1 1.7)

48.3 1.31 m

15

2.04 brs

154.

16

7

105. 5.01 s

17

2 5.64 s

18 28.8 1.29 s

177.

19

7

20 16.0 1.30 s The other tertiary methyl singlet, observed at δ_Η 1.30 showed HMBC correlations to C-l and C-5 and was assigned as C-20. The methyl protons showed additional HMBC correlations to a quaternary carbon (5c 40.3) and a methine carbon (5c 54.5) which were assigned as C-10 and C-9, respectively. COSY correlations between H-5 (6H 1 -04) and protons at 5H 1.92 and 2.43 then allowed assignment of the H-6 protons which in turn

13 showed correlations to protons at 5H 1.22 and 1.30 which were assigned to C-7. The C chemical shifts for C-6 (5_C 22.7) and C-7 (5_C 42.2) were then determined from the HSQC- DEPT data. COSY correlations between H-9 (δ_Η 0.88) and protons at δ_Η 1.65 and 1 .69 allowed assignment of the H-l 1 protons which in turn showed COSY correlations to protons at δπ 1.99 and 2.25 which were assigned as the H-12 protons. The HSQC-DEPT data was then used to assign C-l 1 (8_C 21.1) and C-12 (5 37.5). HMBC correlations from the H-12 proton (δκ 2.25) to carbons at c 87.1 and 154.7 allowed assignment of C-13 and C-16, respectively. The olefinic protons observed at 5H 5.01 and 5.64 showed HMBC correlations to C-13 and were assigned to C-17 (δο 105.2 via HSQC-DEPT). The olefinic protons H-l 7 and the methine proton H-9 showed HMBC correlations to a carbon at δς 48.3 which was assigned as C-l 5. An additional HMBC correlation from H-9 to a methylene carbon at 5c 44.5 then allowed assignment of C-14. The Ή chemical shifts at C-14 (δ_Η 1.80 and 2.65) and C-15 (δ_Η 1.31 and 2.04) were assigned using the HSQC- DEPT data.

Correlations observed in the NOESY spectrum were used to assign the relative stereochemistry of the central diterpene core. In the NOESY spectrum, NOE correlations were observed between H-14 and H-20 indicating that H-14 and H-20 are on the same face of the rings. Similarly, NOE correlations were observed between H-9 and H-5; H-9 and H- 18 as well as H-5 and H-l 8 but NOE correlations were not observed between H-9 and H- 14 indicating that H-5, H-9 and H-l 8 were on the opposite face of the rings compared to H-14 and H-20 as presented in Figure 21. These data thus indicated that the relative stereochemistry in the central core was retained during the glycosylation step.

The key HMBC and COSY correlations used to assign the aglycone region are provided below:

Analysis of the 'H-¹³C HSQC-DEPT data confirmed the presence of five anomeric protons. Three of the anomeric protons were well resolved at δ_Η 6.02 (5_C 96.1), 5.57 (5_C 105.3), and 5.34 (8_C 105.3) in the Ή NMR spectrum. The remaining two anomeric protons observed at δ_Η 5.04 (5_C 105.6) and 5.07 (5_C 98.7) which were obscured by solvent (HOD) resonance in the Ή spectrum were identified by ^]H-¹³C HSQC-DEPT data. The anomeric proton observed at δ_Η 6.02 showed HMBC correlation to C-19 which indicated that it corresponds to the anomeric proton of Glci. Similarly, the anomeric proton observed at δ_Η 5.07 showed an HMBC correlation to C-13 allowing it to be assigned as the anomeric proton of Glen.

The Glci anomeric proton (δπ 6.02) showed a COSY correlation to a proton at δη 4.07 was assigned as Glcj H-2 which in turn showed a COSY correlation to a proton at δπ 4.22 (Glci H-3) which showed a COSY correlation with a proton at δ_Η 4.12 (Glci H-4). Due to data overlap, the COSY spectrum did not allow assignment of H-5 or the H-6 protons. Therefore, a series of ID-TOCSY experiments were performed using selective irradiation of the Glci anomeric proton with several different mixing times. In addition to confirming the assignments for Glc_f H-2 through H-4, the ID-TOCSY data showed a proton at δ_Η 4.04 assigned as Glci H-5 and a proton at δ_Η 4.68 assigned as one of the Glci H-6 protons. The latter proton was also used for I D-TOCSY experiments. The selective irradiation of H-6 with several different mixing times also confirmed the assignment of Glci H-1 to H-5 as well as the remaining methylene proton of H-6 (5H 4.30). Assignment of the C chemical shifts for Glc, C-2 (5_C 74.2), C-3 (5_C 79.1), C-4 (5_C 72.1), C-5 (6_C 78.5), and C-6 (5_C 69.9) was determined using the 'H-^{, 3}C HSQC-DEPT data to complete the assignment of Glci. Furthermore, the presence of a methylene ¹³C resonance at 5c 69.9 in the ^!H-¹³C HSQC-DEPT spectrum indicated a 1→6 sugar linkage of Glci in the structure.

Out of four remaining unassigned glucose moieties, one was assigned as a substituent at C-6 of Gl¾ on the basis of 'H-¹ C HSQC-DEPT, HMBC, and 1D-TOCSY correlations. The relatively downfield shift of a methylene ^{, 3}C resonance of Glci at 5c 69.9 in the HSQC-DEPT spectrum indicated a 1→6 sugar linkage of Glci. The anomeric proton observed at δ_Η 5.04 showed HMBC correlation to Glci C-6 and was assigned as the anomeric proton of Glc_v. Similarly, methylene protons of Glci showed HMBC correlations to anomeric carbon of Glcy confirming the presence of a 1→6 sugar linkage between Glci and Glcy. The Glcy anomeric proton showed a COSY correlation to a proton at διι 4.00 which was assigned as Glc_v H-2 which in turn showed a COSY correlation to a proton at δ_Η 4.22 (Glc_v H-3). Due to data overlap, the COSY spectrum did not allow assignment of Glc_v H-4 based on the COSY correlation of Glc_v H-3. However, in the HMBC spectrum, Glc_v H-3 showed a correlation to Glc_v C-5 (5_C 78.9). In HSQC-DEPT spectrum, Glc_v C-5 showed a correlation to δ_Η 3.89 (Glc_v H-5). The Glc_v H-5 showed COSY correlations to δ_Η 4.21, 4.37, and 4.48. In the HSQC-DEPT spectrum, δ_Η 4.21 showed a correlation to 5_C 71.4 (Glc_v H-4), while δ_Η 4.37 and 4.48 showed a correlation to δς 63.1 and were assigned to Glc_v H-6a and H-6b, respectively. Assignment of the ¹³C chemical shifts for Glc_v C-2 (δ₀ 75.7) and C-3 (δ₀ 79.1) was determined using the 1H-¹³C HSQC-DEPT data to complete the assignment of Glcy.

A summary of the Ή and ¹³C chemical shifts for the glycoside at C-19 are shown in the following table:

1H and ¹³C NMR (500 and 125 MHz, pyridine-< ₅), Assignments of the reb D2 C-19 glycoside.

and ¹³C values can be exchangeable between positions Glcj-3, Glcy-3 and Glc;

A summary of the key HMBC, COSY, and ID-TOCSY correlations used to assign the C-19 glycoside region are provided below.

'H-¹³C HMBC

Ή-'Η COSY

Ή-Ή spin system based on ID-TOCSY

Assignment of Glen was carried out in a similar manner. The Glen anomeric proton (5H 5.07) showed a COSY correlation to a proton at δπ 4.37, assigned as Glen H-2, which in turn showed a COSY correlation to a proton at δ_Η 4.18 (Glen H-3). This latter proton showed an additional correlation with a proton at 5H 3.88 (Glcn H-4) which also showed a COSY correlation to a proton at δ_Η 3.79 (Glen H-5). Glen H-5 also showed a COSY correlation to Glen H-6 protons (δ_Η 4.08 and 4.46). Assignment of the ¹³C chemical shifts for Glc_n C-2 (5_C 81.3), C-3 (5_C 88.4), C-4 (5_C 71.1), C-5 (5_C 77.9), and C- 6 (5c 63.2) was determined using the HSQC-DEPT data. HMBC correlations from Glcn H-3 to C-2 and C-4 and also from Glc_n H-4 to C-2 and C-5 confirmed the assignments made above. Additional HMBC correlations of Glcn H-4 to Glcn C-6 further support to complete the assignment of Glcn.

Two of the remaining unassigned glucose moieties were assigned as substituents at C-2 and C-3 of Glcn on the basis of HMBC correlations. The anomeric proton observed at 5H 5.57 showed a HMBC correlation to Glcn C-2 and was assigned as the anomeric proton of Glcni. The anomeric proton observed at 5H 5.34 showed a HMBC correlation to Glcn C-3 and was assigned as the anomeric proton of Gicjv- The reciprocal HMBC correlations from Glc H-2 to the anomeric carbon of Glc_ni and from Glcn H-3 to the anomeric carbon of Glciv were also observed.

The anomeric proton of Glcm (δκ 5.57) showed a COSY correlation with a proton at 5H 4.19 which was assigned as Glcm H-2. Due to data overlap, the COSY spectrum did not allow assignment of H-3 to H-6 protons. Therefore, a series of I D-TOCSY experiments were performed using selective irradiation of the Glcm anomeric proton with several different mixing times. In addition to confirming the assignments for Glcm H-2, the ID-TOCSY data showed protons at δ_Η 4.24 (Glc_m H-3), δ_Η 4.27 (Glc_m H-4), and δ_Η 3.94 (Glcm H-5). Once H-4 was assigned using ID-TOCSY data, COSY correlations from H-4 to H-5 and in turn to H-6 were used to assign H-6. In the COSY spectrum, Glcm H-4 showed a correlation to Glcni H-5, which in turn showed COSY correlations to 6H 4.41 and 4.50 of Glc_m H-6a and H-6b, respectively. The ¹³C chemical shifts for Glc_m C-2 (6_C 76.8), C-3 (6c 78.9), C-4 (5_C 72.4), C-5 (5_C 78.8), and C-6 (5_C 63.5) were then determined

1 13

using the Ή- C HSQC-DEPT correlations to complete the assignment of Glcm-

The anomeric proton of Glciv (δ_Η 5.34) showed a COSY correlation with a proton at δ_Η 4.06 which was assigned as Glciv H-2. Due to data overlap, the COSY spectrum did not allow assignment of H-3 to H-6 protons. Therefore, a series of ID-TOCSY

i l l experiments were performed using selective irradiation of the Glciv anomeric proton with several different mixing times. In addition to confirming the assignments for Glcrv H-2, the 1D-TOCSY data showed protons at δ_Η 4.22 (Glc_]V H-3), δ_Η 4.18 (Glciv H-4), and δ_Η 4.10 (Glciv H-5). Once H-4 was assigned using 1D-TOCSY data, COSY correlations from H-4 to H-5 and in turn to H-6 were used to assign H-6. In the COSY spectrum, Glciv H-4 showed a correlation to Glciv H-5, which in turn showed COSY correlations to δπ 4.32 and 4.58, Glciv H-6a and H-6b, respectively. The ¹³C chemical shifts for Glcrv C-2 (6_C 75.8), C-3 (5c 78.9), C-4 (5_C 72.0), C-5 (5_C 79.3), and C-6 (5_C 62.9) were then determined using the ^]H-¹³C HSQC-DEPT correlations to complete the assignment of Glcrv.

The large coupling constants observed for the anomeric protons of the glucose moieties at δ_Η 6.02 (d, J = 8.1 Hz), 5.57 (d, J = 7.6 Hz), 5.34 (d, J = 7.9 Hz) and δ_Η 5.04 (d, J = 8.1 Hz), suggested their β-orientation (Figures 14, 27, and 28). While the remaining anomeric proton at δπ 5.07 was obscured by the solvent resonance (HDO) it's coupling constant (J = ~8 Hz) evident from ID TOCSY data (Figure 24) also indicated β-orientation.

A summary of the Ή and ¹³C chemical shifts for the glycoside at C-13 are shown in the table below:

1H and ¹³C NMR (500 and 125 MHz, pyridine-d₅),

Assignments of the Reb D2 C-13 glycoside.

*Anomeric proton was obscured by solvent (HDO) resonance, coupling constant value obtained from 1D-TOCSY data.

^#1H and ¹³C values can be exchangeable between Glci-3, Glcy-3 and Glciv-3. A summary of the key HMBC, COSY, and ID-TOCSY correlations used to assign the C- 13 glycoside region are provided below:

^ = ¹H-¹³C HMBC

= ¹H-¹H COSY

= ^-Ή spin system based on I D-TOCSY

NMR and MS analyses allowed a full assignment of structure, shown below. The chemical name of the compound is 13-[(2-0-P-D-giucopyranosyl-3-0- β -D- glucopyranosyl- β -D-glucopyranosyl)oxy] e«Z-kaur-16-en-19-oic acid-[(6-<9- β -D- glucopyranosyl- β -D-glucopyranosyl) ester] (rebaudioside D2 or reb D2). The compound is an isomer of rebaudioside D.

EXAMPLE 40

Isolation and Characterization of Reb M2

Crude Reaction Sample. The sample, Lot CB-2977-106, used for isolation was prepared according to Example 22 with UGTSL (GI #460409128).

HPLC Analysis. Preliminary HPLC analyses was performed using a Waters 2695 Alliance System with the following method: Phenomenex Synergi Hydro-RP, 4.6 ^χ 250 mm, 4 μιη (p/n 00G-4375-E0); Column Temp: 55 °C; Mobile Phase A: 0.0284% NH₄OAc and 0.01 16% HOAc in water; Mobile Phase B: Acetonitrile (MeCN); Flow Rate: 1.0 mL/min; Injection volume: 10 iL. Detection was by UV (210 nm) and CAD.

Gradient:

Analyses of semi-preparative purification fractions were performed with the following method: Waters Atlantis dC18, 4.6 ^χ 100 mm, 5 μιη (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 i . Detection was by CAD.

Gradient:

LC-MS. Preliminary analysis of the semi-synthetic steviol glycoside mixture was carried out on a Waters AutoPurification HPLC/MS System with a Waters 3100 Mass Detector operating in negative ion mode. Analysis of the sample was performed using the following method: Phenomenex Synergi Hydro-RP, 4.6 ^χ 250 mm, 4 μιη (p/n 00G-4375- E0); Column Temp: 55 °C; Mobile Phase A: 0.0284% NH₄OAc and 0.01 16% HOAc in water; Mobile Phase B: MeCN; Flow Rate: 1.0 mL/min; Injection volume: 10 \iL. Detection was by UV (210 nm), and MSD (-ESI mlz 500 - 2000). Gradient conditions were as listed above.

Isolation by HPLC. The purification was performed in two steps. The first method used for the semi-preparative purification is summarized below. Column: Waters Atlantis dC18, 30 100 mm, 5 μιη (p/n 186001375); Mobile Phase A: 25% MeCN in water; Mobile Phase B: 30% MeCN in water; Flow Rate: 45 mL/min; Injection load: 160 mg dissolved in 20 mL of water. Detection was by UV (205 nm).

Gradient:

Time (min) %A %B 0.0 - 5.0 100 0

20 20 80

25 20 80

30 100 0

MS and MS/MS. MS and MS/MS data were generated with a Waters QTof Premier mass spectrometer equipped with an electrospray ionization source. Samples were analyzed by negative ESI. Samples were diluted with H₂0:MeCN (1 : 1) by 50 fold and introduced via infusion using the onboard syringe pump. The samples were diluted to yield good s/n which occurred at an approximate concentration of 0.01 mg/mL.

NMR. The sample was prepared by dissolving -1.0 mg in 150 μΐ_^ of D₂0 and NMR data were acquired on a Bruker Avance 500 MHz instrument with a 2.5 mm inverse detection probe. The Ή NMR and ¹³C NMR spectra were referenced to the residual solvent signal HDO (δ_Η 4.79 ppm) and TSP (5_C 0.00 ppm), respectively.

Results and Discussion

Isolation and Purification. Isolation was performed using on a steviol glycoside mixture, Lot number CB-2977-106, prepared according to Example 22 with UGTSL (GI #460409128). The material was analyzed by LC-MS using the method described above (Figure 1 1). The targeted peak of interest was that at 4.1 min in the TIC chromatogram. The mass spectrum of this peak provided a [M-H]^" ion at m/z 1289.7. The provided sample was preliminarily processed in a single injection (160 mg) using the first method condition provided above. This method fractionated the material into 'polar' and 'non-polar' mixtures of glycosides. The 'polar' mixture was then reprocessed using the second-step conditions provided above. The semi-preparative HPLC trace is shown in Figure 12. From this semi-preparative collection, the peak was isolated with a purity >99% (CAD, AUC). The fraction analysis is provided in Figure 13. Following the purification, the fractions were concentrated by rotary evaporation at 35 °C and lyophilized. Approximately 1 mg was obtained. Mass Spectrometry. The ESI- TOF mass spectrum acquired by infusing a sample of CC- 00300 showed a [M-H]^" ion at m/z 1289.5266. The mass of the [M-H]^" ion was in good agreement with the molecular formula C56H90O33 (calcd for C56H89O33 : 1289.5286, error: - 1.6 ppm) expected for reb M2. The MS data confirmed that CC-00300 has a nominal mass of 1290 Daltons with the molecular formula, C56H90O33.

The MS/MS spectrum (selecting the [M-H]^" ion at m/z 1289.5 for fragmentation) indicated the loss of three glucose units at mlz 803.3688 and sequential loss of three glucose moieties at m/z 641.3165, 479.2633 and 317.2082.

NMR Spectroscopy. A series of NMR experiments including Ή NMR (Figure 29), ¹³C NMR (Figure 30 and 31), Ή-Ή COSY (Figure 32), HSQC-DEPT (Figure 33), HMBC (Figures 34 and 35), and 1D-TOCSY (Figure 36-39) were performed to allow assignment of reb M2.

The Ή, 'Η-Ή COSY, 'H-¹³C HSQC-DEPT and 'H-¹³C HMBC NMR data indicated that the central core of the glycoside is a diterpene. The presence of six anomeric protons observed in the Ή and ^lH-^l3C HSQC-DEPT spectra confirm six sugar units in the structure. The methylene ¹³C resonance at 5c 70.9 in the 'H-¹³C HSQC-DEPT spectrum indicated the presence of a 1→6 sugar linkage in the structure. The linkages of sugar units were assigned using 'H-^C HMBC and 1D-TOCSY correlations.

A HMBC correlation from the methyl protons at δπ 1.29 to the carbonyl at 5c 181.5 allowed assignment of one of the tertiary methyl groups (C-18) as well as C-19 and provided a starting point for the assignment of the rest of the aglycone. Additional HMBC correlations from the methyl protons (H-18) to carbons at 6c 39.8, 43.7, and 59.2 allowed assignment of C3, C4, and C5. Analysis of the ^!H-¹³C HSQC-DEPT data indicated that the carbon at 5c 39.8 was a methylene group and the carbon at 5c 59.2 was a methine which were assigned as C-3 and C-5, respectively. This left the carbon at 5c 43.7, which did not show a correlation in the HSQC-DEPT spectrum, to be assigned as the quaternary carbon, C-4. The Ή chemical shifts for C-3 (δ_Η 1.16 and 2.28) and C-5 (δ_Η 1.24) were assigned using the HSQC-DEPT data. A COSY correlation between one of the H-3 protons (δ_Η 1 .16) and a proton at δπ 1.49 allowed assignment of one of the H-2 protons which in turn showed a correlation with a proton at δπ 0.92 which was assigned to C-l . The remaining 'H and ¹³C chemical shifts for C-1 and C-2 were then assigned on the basis of additional COSY and HSQC-DEPT correlations and are summarized in the table below.

Ή NMR (500 MHz, D₂0) and ¹³C NMR (125 MHz, D₂0/TSP)

Assignments of the Reb M2 aglycone.

107.0 4.98 s

17

5.16 s

18 31.0 1.29 s

19 181.5 —

20 19.1 0.92 s

The other tertiary methyl singlet, observed at 8H 0.92 showed HMBC correlations to C-l and C-5 and was assigned as C-20. The methyl protons showed additional HMBC correlations to a quaternary carbon (5c 42.4) and a methine (5_C 55.5) which were assigned as C-10 and C-9, respectively. COSY correlations between H-5 (δ_Η 1.24) and protons at δ_Η 1.73 and 1.94 then allowed assignment of the H-6 protons which in turn showed correlations to protons at δ_Η 1.49 and 1.56 which were assigned to C-7. The ¹³C chemical shifts for C-6 (5_C 24.4) and C-7 (8_C 44.2) were then determined from the HSQC-DEPT data. COSY correlations between H-9 (δπ 1.09) and protons at δ_Η 1.66 and 1.70 allowed assignment of the H-l 1 protons which in turn showed COSY correlations to protons at 8H 1.60 and 2.00 which were assigned as the H-12 protons. The HSQC-DEPT data was then used to assign C-l 1 (δς 22.6) and C-12 (δο 39.9). The olefmic protons observed at 5H 4.98 and 5.16 showed HMBC correlations to C-13 (8c 90.9) and were assigned to C-17 (5c 107.0 via HSQC-DEPT). The olefinic protons H-17 showed HMBC correlations to a carbon at 5c 49.4 which was assigned as C-15. An additional HMBC correlation from H-9 to a methylene carbon at δο 46.9 then allowed assignment of C-14. The Ή chemical shifts at C-14 (δ_Η 1 .53 and 2.21) and C-15 (δ_Η 2.15 and 2.18) were assigned using the HSQC- DEPT data.

A summary of the key HMBC and COSY correlations used to assign the aglycone region are provided below:

Analysis of the 'H-¹³C HSQC-DEPT data confirmed the presence of six anomeric protons. Three of the anomeric protons were well resolved at 5H 5.65 (5c 95.5), 4.92 (5c 104.9), and 4.50 (5_C 105.7) in the *H NMR spectrum. The remaining three anomeric protons observed at δ_Η 4.85 (8_C 98.4), 4.84 (6_C 105.0), and 4.83 (8_C 105.3) were overlapped by the residual solvent resonance in the ^]H spectrum. The anomeric proton observed at δ_Η 5.65 showed a HMBC correlation to C-19 which indicated that it corresponds to the anomeric proton of Glci. Similarly, the anomeric proton observed at δ_Η 4.85 showed a HMBC correlation to C-13 allowing it to be assigned as the anomeric proton of Glen.

The Glci anomeric proton (δ_Η 5.65) showed a COSY correlation to a proton at δ_Η 3.96 which was assigned as Glci H-2 which in turn showed a COSY correlation to a proton at δ_Η 3.89 (Glci H-3) which showed a COSY correlation with a proton at δ_Η 3.71 (Glci H-4). Due to data overlap, the COSY spectrum did not allow assignment of the H-5 or H-6 protons. Therefore, a series of ID-TOCSY experiments were performed using selective irradiation of the Glci anomeric proton with several different mixing times. In addition to confirming the assignments for Glci H-2 through H-4, the ID-TOCSY data showed a proton at δπ 3.73 assigned as Glci H-5 and a proton at 8H 4.15 assigned as one of the Glci H-6 protons. The latter proton was also used for ID-TOCSY experiments. The selective irradiation of H-6 with several different mixing times also confirmed the assignment of Glci H-l to H-5 as well as the remaining methylene proton of H-6 (δ_Η 4.00). Assignment of the ^{l 3}C chemical shifts for Glci C-2 (8_C 80.5), C-3 (5_C 79.0), C-4 (5_C 71.5), C-5 (5c 79.0), and C-6 (5_C 70.9) was determined using the ^!H-¹³C HSQC-DEPT data to complete the assignment of Glci. Furthermore, the presence of a methylene C resonance at 5c 70.9 in the 'H-¹³C HSQC-DEPT spectrum indicated a 1→6 sugar linkage of Glci in the structure.

Two of the unassigned glucose moieties were assigned as substituents at C-2 and C-6 of Glci on the basis of HMBC correlations. The anomeric proton observed at δ_Η 4.83 showed an HMBC correlation to Glci C-2 and was assigned as the anomeric proton of Glcy. The anomeric proton observed at 5H 4.50 showed a HMBC correlation to Glci C-6 and was assigned as the anomeric proton of Glcyi. The reciprocal HMBC correlations from Glci H-2 to the anomeric carbon of Glcv and from Glci H-6 to the anomeric carbon of Glcyi were also observed.

The anomeric proton of Glcy (5H 4.83) showed a COSY correlation with a proton at δ_Η 3.32 which was assigned as Glc_v H-2. The Glcy H-2 in turn showed a COSY correlation to a proton at δ_Η 3.51 (Glc_v H-3). This latter proton showed an additional correlation with a proton at δ_Η 3.38 (Glc_v H-4). H-4 also showed a COSY correlation to a proton at δπ 3.55 (Glcy H-5) and Glcy H-5 in turn showed a COSY correlation to Glcy H- 6 protons (δ_Η 3.76 and 3.97). Assignment of the ¹³C chemical shifts for Glc_v C-2 (5c 78.5), C-3 (8c 78.7), C-4 (δ_€ 72.9), C-5 (δ₀ 78.8), and C-6 (6_C 63.6) was determined using the HSQC-DEPT data. HMBC correlations from Glc_v H-3 to C-2 and C-4 and also from Glcy H-4 to C-3 and C-6 confirmed the assignments made above to complete the assignment of Glcy.

Another glucose moiety was assigned as a substituent at C-6 of Glci on the basis of

'H-¹³C HSQC-DEPT and HMBC correlations. The relatively downfield shift of a methylene ¹³C resonance of Glci at 5c 70.9 in the HSQC-DEPT spectrum indicated a 1→6 sugar linkage of Glci. The anomeric proton observed at δπ 4.50 showed a HMBC correlation to Glci C-6 and was assigned as the anomeric proton of Glcyi. Similarly, methylene protons of Glci showed HMBC correlations to the anomeric carbon of Glcyi and this confirmed the presence of a 1→6 sugar linkage between Glci and Glcyi. The Glcyi anomeric proton showed a COSY correlation to a proton at δπ 3.33 which was assigned as

Glcyi H-2 which in turn showed a COSY correlation to a proton at δκ 3.49 (Glcyi H-3).

Due to data overlap, the COSY spectrum did not allow assignment of Glc_v H-4 to H-6 based on the COSY correlations. Therefore, a series of ID-TOCSY experiments were performed using selective irradiation of the Glcvi anomeric proton with different mixing times. In addition to confirming the assignments for Glcvi H-2 through H-3, the 1D- TOCSY data showed protons at δ_Η 3.45 (Glc_Vi H-4) and δ_Η 3.48 (Glcvi H-5) and protons at δ_Η 3.92 and 3.94 assigned for Glc_Vi H-6 protons. Assignment of the ¹³C chemical shifts for Glcvi C-2 (5_C 78.1), C-3 (5_C 78.6), C-4 (5_C 72.3), C-5 (5_C 78.8), and C-6 (5_C 64.1) was determined using the 'H-¹³C HSQC-DEPT data to complete the assignment of Glcvi.

A summary of the Ή and ^{l 3}C chemical shifts for the glycoside at C-19 are found in the table below:

H NMR (500 MHz, D₂0) and °C NMR (125 MHz, D₂0/TSP) Assignments of the Reb M2 glycoside.

Glcvi-4 72.3 3.45 m

Glcvi-5 78.8 3.48 m

Glcvi-6 64.1 3.92 m

3.94 m

Ή and ^{I j}C values can be exchangeable with Glciv-1 of the following table.

A summary of the key HMBC, COSY, and ID-TOCSY correlations used to the C-19 glycoside region are provided below:

Ή NMR (500 MHz, D₂0) and ¹³C NMR (125 MHz, D₂0/TSP) Assignments of the Reb M2 glycoside.

Gldv-5 81.7 3.75 m

Glciv-6 65.8 3.55 m

3.78 m

Assignment of Glcn was carried out in a similar manner. The Glcn anomeric proton (δ_Η 4.85) showed a COSY correlation to a proton at δ_Η 3.75 which was assigned as Glcn H-2 which in turn showed a COSY correlation to a proton at δ_Η 3.98 (Glc_n H-3). This latter proton showed an additional correlation with a proton at δπ 3.54 (Glcn H-4). H- 4 also showed a COSY correlation to a proton at δ_Η 3.96 (Glcn H-5). Glcn H-5 also showed a COSY correlation to Glcn H-6 protons (δ_Η 3.77 and 3.45). Assignment of the ^{l 3}C chemical shifts for Glc_n C-2 (5c 81.7), C-3 (5_C 88.0), C-4 (6_C 71.3), C-5 (5_C 80.5), and C-6 (δς 63.6) was determined using the HSQC-DEPT data. HMBC correlations from Glcn H-3 to C-2 and C-4 and also from Glc_n H-4 to C-3 and C-6 confirmed the assignments made above to complete the assignment of Glcn.

Two of the remaining unassigned glucose moieties were assigned as substituents at C-2 and C-3 of Glcn on the basis of HMBC correlations. The anomeric proton observed at δ_Η 4.92 showed a HMBC correlation to Glcn C-2 and was assigned as the anomeric proton of Glcm. The anomeric proton observed at δη 4.84 showed a HMBC correlation to Glcn C-3 and was assigned as the anomeric proton of Glciy. The reciprocal HMBC correlations between Glcn H-2 and the anomeric carbon of Glc_m and between Glcn H-3 and the anomeric carbon of Glcrv were also observed.

The anomeric proton of Glcm (δ_Η 4.92) showed a COSY correlation with a proton at δ_Η 3.32 which was assigned as Glcm H-2. Due to data overlap, the COSY spectrum did not allow assignment of H-3 to H-6 protons. Therefore, a series of ID-TOCSY experiments were performed using selective irradiation of the Glcm anomeric proton with different mixing times. In addition to confirming the assignments for Glc_m H-2, the 1D- TOCSY data showed protons at δ_Η 3.51 (Glc_m H-3), δ_Η 3.26 (Glc_m H-4), and δ_Η 3.44 (Glcm H-5). Once H-4 was assigned using ID-TOCSY data, COSY correlations from H-4 to H-5 and in turn to H-6 were used to assign H-6. In the COSY spectrum, Glcm H-4 showed a correlation to Glc_m H-5, which in turn showed COSY correlations to δ_Η 3.94 and 3.75 of Glcm H-6a and H-6b, respectively. The ¹³C chemical shifts for Glc_m C-2 (5c 76.3), C-3 (6c 78.8), C-4 (5_C 73.3), C-5 (5_C 78.8), and C-6 (5_C 64.4) were then determined using the *H-¹³C HSQC-DEPT correlations to complete the assignment of Glcm.

The anomeric proton of Glciv (6H 4.84) which showed a COSY correlation to a proton at δπ 3.41 was assigned as Glciv H-2 which in turn showed a COSY correlation to a proton at δκ 3.46 (Glcrv H-3). This latter proton showed an additional correlation with a proton at δ_Η 3.45 (Glcrv H-4) which also showed a COSY correlation to a proton at δ_Η 3.75 (Glciv H-5). Glciv H-5 also showed a COSY correlation to Glciv H-6 protons (6H 3.55 and 3.78). Assignment of the ¹³C chemical shifts for Glciv C-2 (5_C 76.1), C-3 (5_C 78.8), C-4 (5c 72.5), C-5 (5_C 81.7), and C-6 (8_C 65.8) was determined using the HSQC- DEPT data. HMBC correlations from Glc_]V H-3 to C-4 and C-5 and also from Glc_IV H-4 to C-3 and C-6 confirmed the assignments made above to complete the assignment of Glciv.

A summary of the ^!H and ¹³C chemical shifts for the glycoside at C-13 are found in the following table:

1H NMR (500 MHz, D₂0) and ¹³C NMR (125 MHz, D₂0/TSP)

Assignments of the Reb M2 glycoside.

Glcu-4 73.3 3.26 t (9.5)

Glcm-5 78.8 3.44 m

Glcin-6 64.4 3.75 m

3.94 m

Glc]v-1 105.0 4.84 d (7.8)

Glcrv-2 76.1 3.41 m

GIcrv-3 78.8 3.46 m

Glciv-4 72.5 3.45 m

Glcrv-5 81.7 3.75 m

Glciv-6 65.8 3.55 m

3.78 m

A summary of the key HMBC, COSY, and 1 D-TOCSY correlations used to assign the C-13 glycoside region are provided below:

^ = 'H-¹³C HMBC

= ¹H-¹H COSY

— = Ή^Η spin system based on ID TOCSY

NMR and MS analyses allowed a full assignment of its structure, shown below. The chemical name of the compound is 13-[(2-(3-p-D-glucopyranosyl-3-<3- β -D- glucopyranosyl- β -D-glucopyranosyl)oxy] <?«i-kaur-16-en-19-oic acid-[(2-C- β -D- glucopyranosyl-6-0- β -D-glucopyranosyl- β -D-glucopyranosyl) ester] (rebaudioside M2 or reb M2). The compound is an isomer of rebaudioside M.

EXAMPLE 41

Directed evolution of UGT76G1 for the conversion of Rebaudioside D to Rebaudioside M(Round 2)

The most active clone from the first round of directed evolution of UGT76G1 (see EXAMPLE 26 UGT76Glvar94 containing mutations: Q266EJP272AJR334K G348P L379G) was chosen as baseline clone for round 2. A list of 53 mutations was established containing different identified positive mutations from the first round and new mutations obtained by DNA2.0 ProteinGPStm strategy. This list of mutations was subsequently used to design 92 variant genes that contained each 3 different mutations. After codon-optimized for expression in E. coli the genes were synthesized, subcloned in the pET30a+ plasmid and used for transformation of E. coli BL21 (DE3) chemically competent cells. The obtained cells were grown in Petri-dishes on solid LB medium in the presence of Kanamycin. Suitable colonies were selected and allowed to grow in liquid LB medium in tubes. Glycerol was added to the suspension as cryoprotectant and 400 μΐ, aliquots were stored at -20°C and at -80°C.

These storage aliquots of E. coli BL21 (DE3) containing the pET30a+_UGT76Glvar plasmids were thawed and added to LBGKP medium (20 g/L Luria Broth Lennox; 50 niM PIPES buffer pi I 7.00; 50 mM Phosphate buffer pi I 7.00; 2.5 g/L glucose and 50 mg/L of Kanamycine). This culture was allowed to shake in a 96 microtiter plate at 30°C for 8 h.

3.95 mL of production medium containing 60 g/L of Overnight Express™ Instant TB medium (Novagen®), 10 g/L of glycerol and 50 mg/L of anamycin was inoculated with 50 i of above described culture. In a 48 deepwell plate the resulting culture was allowed to stir at 20°C. The cultures gave significant growth and a good OD (600 nm) was obtained. After 44 h, the cells were harvested by centrifugation and frozen.

Lysis was performed by addition of Bugbuster® Master mix (Novagen®) to the thawed cells and the lysate was recovered by centrifugation. Activity tests were performed with 100 \iL of fresh lysate that was added to a solution of Rebaudioside D (final concentration 0.5 mM), MgCl₂ (final concentration 3 raM) and UDP-Glucose (final concentration 2.5 mM) in 50 mM phosphate buffer pH 7.2.

The reaction was allowed to run at 30°C and samples were taken after 2, 4, 7 and 24 h. to determine conversion and initial rate by HPLC (CAD detection) using the analytical method that was described above for the transformation of Rebaudioside D to Rebaudioside M. In parallel the experiments were performed with baseline clone, Roundl- Var94. The conversion after 22 h. and initial rate for this baseline clone was defined as 100% and the normalized conversions and initial rates for the round 2 clones are depicted in the following table:

Clone Mutations* Normalized conversion Normalized initial

Reb D to Reb Mailer 22h. rate (0-4h)

Round 1-Var94 UGT76G1 100% 100%

(Q266E_P272A_R334K_G348P_L379G)

baseline clone

Round2-Varl Roundl-Var94 (A213N _P348G_I41 IV) 70% 86%

Round2-Var2 Roundl-Var94 (K303G_I423M_Q425E) 120% 134%

Round2-Var3 Roundl-Var94 (V20L_N138K_S147G) 14% 15%

Round2-Var4 Round 1-Var94 (I 16V_V133A L299I) 37% 43%

Round2-Var5 Roundl-Var94 (S241 V_S274G_Q432E) 75% 72%

Round2-Var6 Roundl-Var94 (I16V_L139V_I218V) 62% 68%

Round2-Var7 Roundl-Var94 (K334R_^N409K_Q432E) 104% 92% Clone Mutations* Normalized conversion Normalized initial

Reb D to Reb Matter 22h. rate (0-4h)

Round2-Var8 Roundl-Var94 (I 15L R141 TJ407V) 17% 26%

Round2-Var9 Roundl-Var94 (R141T_K303G_G379L) 31% 42%

Round2-Varl 0 Roundl-Var94 (I190L_ 303G_P348G) 131% 149%

Round2-Varl l Roundl -Var94 (E266Q_F314S_N409R) 106% 132%

Round2-Varl 2 Roundl-Var94 (V133A_I295V_K303G) 43% 49%

Round2-Varl 3 Round 1-Var94 (I 16V_S241 V__N409R) 80% 79%

Round2-Varl4 Roundl-Var94 (A239V_K334R_G379L) 58% 55%

Round2-Varl 5 Round 1-Var94 (I190L_ 393R_V396L) 118% 126%

Round2-Varl6 Roundl -Var94 (L101F_I295M_K393R) 84% 89%

Round2-Varl7 Roundl-Var94 (A239V_E266Q_Q425E) 96% 101%

Round2-Varl 8 Roundl-Var94 (V20L_I190L_I423M) 98% 98%

Round2-Varl 9 Roundl-Var94 (V20L_G379L_S456L) 84% 81 %

Round2-Var20 Roundl-Var94 (K334R P348G N409R) 73% 73%

Round2-Var21 Round 1 - Var94 (E231 A_S241 V_E449D) 53% 50%

Round2-Var22 Roundl-Var94 (K188R_L299I_V394I) 56% 59%

Round2-Var23 Roundl-Var94 (E231A_S274G_V394I) 1 10% 124%

Round2-Var24 Roundl-Var94 (S42A_I295V_Q432E) 71% 78%

Round2-Var25 Roundl-Var94 (A213N_A272P_ 334R) 95% 80%

Round2-Var26 Round 1-Var94 (L158Y_S274K_N409K) 80% 50%

Round2-Var27 Roundl-Var94 (K188R_I295M_Q425E) 132% 116%

Round2-Var28 Roundl -Var94 (I15L_I295M_V394I) 53% 36%

Round2-Var29 Roundl-Var94 (V133A_A239V_V394I) 47% 30%

Round2-Var30 Round 1 -Var94 (L 158 Y_F314SJC316R) 107% 72%

Round2-Var31 Round 1-Var94 (L158Y_A239V_A272P) 54% 30%

Round2-Var32 Roundl -Var94 (F46I_D301N_V396L) 109% 101%

Round2-Var33 Roundl-Var94 (L101F_I218V_Q432E) 78% 54%

Round2-Var34 Roundl-Var94 (I16V_F46I_I295M) 1 10% 95%

Round2-Var35 Round 1-Var94 (A213N_E266S_I407V) 98% 79%

Round2-Var36 Roundl-Var94 (A239V_S274 _I295M) 102% 89%

Round2-Var37 Roundl-Var94 (A239V_F314S_S450K) 105% 99%

Round2-Var38 Roundl-Var94 (L139V_K188R_D301N) 66% 51%

Round2-Var39 Roundl-Var94 (145 V_I218V_S274K) 87% 58%

Round2-Var40 Roundl -Var94 (S241 V_ K303G_V394I) 78% 57%

Round2-Var41 Roundl-Var94 (R141T_S274G_K334R) 41% 28%

Round2-Var42 Round 1 -Var94 (V217L_S274G_L299I) 47% 34%

Round2-Var43 Roundl-Var94 (S274G_D301N_P348G) 98% 91%

Round2-Var44 Roundl -Var94 (E231A_N409R_S450K) 87% 65%

Round2-Var45 Round 1-Var94 (R64H_E231 A_K316R) 88% 64%

Round2-Var46 Round 1-Var94 (V394I N409KJ41 IV) 110% 100%

Round2-Var47 Roundl-Var94 (I45V_I295M_K303G) 113% 88%

Round2-Var48 Roundl-Var94 (L101F_V396L_L398V) 46% 43%

Round2-Var49 Round 1-Var94 (N27S_L101F_S447A) 54% 37% Clone Mutations* Normalized conversion Normalized initial

Reb D to Reb Matter 22h. rate (0-4h)

Round2-Var50 Roundl-Var94 (S274G_F314S_L398V) 129% 156%

Round2-Var51 Roundl-Var94 (E266Q_L299I_K393R) 70% 51%

Round2-Var52 Roundl -Var94 (V217L_E266S_V394I) 62% 48%

Round2-Var53 Roundl-Var94 (N138K__A272P_N409R) 118% 102%

Round2-Var54 Roundl-Var94 (E266S_F314S_Q432E) 124% 146%

Round2-Var55 Roundl-Var94 (D301N_G379L_L398V) 56% 45%

Round2-Var56 Roundl-Var94 (F46I_E266S_K334R) 123% 142%

Round2-Var57 Roundl-Var94 (A272P_V394I_Q432E) 133% 142%

Round2-Var58 Roundl-Var94 (V394I_I407V_S456L) 118% 114%

Round2-Var59 Roundl-Var94 (I218V_E266Q_I423M) 106% 98%

Round2-Var60 Round 1-Var94 (A272P_G379L_I407V) 80% 63%

Round2-Var61 Roundl-Var94 (E231A_K303G_S456L) 1 13% 110%

Round2-Var62 Roundl-Var94 (1190L_E266Q_I407V) 150% 167%

Round2-Var63 Round 1-Var94 (N27SJL139VJ295V) 43% 25%

Round2-Var64 Roundl-Var94 (V217L_I423M__S447A) 67% 51%

Round2-Var65 Roundl-Var94 (L158Y_E266S_E449D) 68% 43%

Round2-Var66 Round 1-Var94 (S42A_F46I_I407V) 160% 203%

Round2-Var67 Round 1-Var94 (N138K_E231A_D301N) 118% 93%

Round2-Var68 Roundl-Var94 ( 188R_G379L_N409R) 52% 35%

Round2-Var69 Roundl-Var94 (I 15L_E231A_V396L) 38% 22%

Round2-Var70 Roundl-Var94 (E231A_Q425E_Q432E) 115% 119%

Round2-Var71 Roundl-Var94 (D301N_K316R_Q425E) 126% 121%

Round2-Var72 Roundl-Var94 (L139V_I295M_F314S) 76% 91%

Round2-Var73 Roundl-Var94 (S 147G_E266S_D301N) 30% 18%

Round2-Var74 Roundl -Var94 (R64H_S147G_S447A) 23% 12%

Round2-Var75 Roundl-Var94 (S42A_K303G_L398V) 95% 1 10%

Round2-Var76 Roundl-Var94 (I45V_D301N_E449D) 62% 60%

Round2-Var77 Roundl-Var94 (V133A_E266S_I411V) 37% 28%

Round2-Var78 Roundl-Var94 (I45V_N409R_Q425E) 63% 59%

Round2-Var79 Roundl-Var94 (R141T_A272P_F314S) 23% 10%

Round2-Var80 Round 1-Var94 (E266S_S274G_N409R) 81% 91%

Round2-Var81 Round 1-Var94 (N409K_Q425E_S450K) 81% 84%

Round2-Var82 Roundl-Var94 (N27S_R64H_K393R) 47% 37%

Round2-Var83 Round 1-Var94 (S42A_^A213N_V217L) 62% 46%

Round2-Var84 Roundl-Var94 (N27S_S274K_I407V) 49% 44%

Round2-Var85 Round 1-Var94 (I411V_Q425E_S456L) 75% 81 %

Round2-Var86 Roundl-Var94 (A239V_K316R_E449D) 83% 72%

Round2-Var87 Roundl-Var94 (S147G_A239V_P348G) 18% 7%

Round2-Var88 Round 1-Var94 (V20L_S274G_S450K) 71 % 68%

Round2-Var89 Roundl-Var94 (F314S V394I_S447A) 88% 123%

Round2-Var90 Roundl -Var94 (R64H_E266Q_I295M) 45% 47%

Round2-Var91 Roundl-Var94 (N138KJ295VJ407V) 50% 51 % Clone Mutations* Normalized conversion Normalized initial

Reb D to Reb Mafter 22h. rate (0-4h)

Round2-Var92 Roundl -Var94 (I15L_P348G_Q432E) 18% 13%

*Mutations are noted as follows: reference gene-original amino acid-position-new amino acid: For example the mutation of an alanine at position 33 to a glycine for variant 94 from the first round of directed evolution of UGT76G1 is noted as Roundl-Var94 (A33G)

Modeling of these results allowed to obtain a ranking of the effect of each mutation. The following mutations were determined as being beneficial for activity: S42A, F46I, I190L, S274G, I295M, K303G, F314S, K316R, K393R, V3941, I407V, N409K, N409R, Q425E, Q432E, S447A, S456L.

EXAMPLE 42

In vivo production of AtSUS

SEQ ID 13 AtSUS

>gi|79328294|ref|NP_001031915. l j sucrose synthase 1 [Arabidopsis thaliana]

MANAERMITRVHSQRERLNETLVSERNEVLALLSRVEAKGKGILQQNQIIAEFEAL

PEQTRKKLEGGPFFDLLKSTQEAIVLPPWVALAVRPRPGVWEYLRVNLHALVVEE

LQPAEFLHFKEELVDGVKNGNFTLELDFEPFNASIPRPTLHKYIGNGVDFLNRHLS

AKLFHDKESLLPLLKFLRLHSHQGKNLMLSEKIQNLNTLQHTLRKAEEYLAELKS

ETLYEEFEAKFEEIGLERGWGDNAERVLDMIRLLLDLLEAPDPCTLETFLGRVPMV

FNVVILSPHGYFAQDNVLGYPDTGGQVVYILDQVRALEIEMLQRIKQQGLNIKPRI

LILTRLLPDAVGTTCGERLERVYDSEYCDILRVPFRTEKGIVRKWISRFEVWPYLET

YTEDAAVELSKELNGKPDLIIGNYSDGNLVASLLAHKLGVTQCTIAHALEKTKYP

DSDIYWKKLDDKYHFSCQFTADIFAMNHTDFIITSTFQEIAGSKETVGQYESHTAF

TLPGLYRVVHGIDVFDPKFNIVSPGADMSIYFPYTEEKRRLTKFHSEIEELLYSDVE

NKEHLCVLKDKKKPILFTMARLDRVKNLSGLVEWYGKNTRLRELANLVVVGGD

RRKESKDNEEKAEMKKMYDLIEEYKLNGQFRWISSQMDRVRNGELYRYICDTKG

AFVQPALYEAFGLTVVEAMTCGLPTFATCKGGPAEIIVHGKSGFHIDPYHGDQAA

DTLADFFTKCKEDPSHWDEISKGGLQRIEEKYTWQIYSQRLLTLTGVYGFWKHVS

NLDRLEARRYLEMFYALKYRPLAQAVPLAQDD

The synthetic gene of AtSuS that was codon optimized for expression in E. coli and subcloned in the pET30a+ plasmid using the Ndel and Xhol restriction sites. The pET30A+ vector containing the AtSUS gene was used to transform electrocompetent E. coli B121(DE3) cells. The obtained cells were grown in petri-dishes in the presence of Kanamycin and suitable colonies were selected and allowed to grow in liquid LB medium (erlenmeyer flasks). Glycerol was added to the suspension as cryoprotectant and 400 aliquots were stored at -20°C and at -80°C.

The storage aliquots of E. coli BL21 (DE3) containing the pET30A+_AtSUS plasmids were 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 of Kanamycine). This culture was allowed to shake at 135 rpm at 30°C for 8 h.

The production medium contained 60 g/L of overnight express instant TB medium (Novagen), 10 g/L of glycerol and 50 mg/L of Kanamycine. The preculture was added to 800 mL of this medium and the solution was allowed to stir at 20°C while taking samples to measure the OD and pH. The culture gave significant growth and a good OD was obtained. After 40 h, the cells were harvested by centrifugation and frozen to obtain 30.1 g of cell wet weight.

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

EXAMPLE 43

Conversion of Rebaudioside A to Rebaudioside M with in situ prepared UDP- Glucose using UGTSL2, UGT76G1-R1-F12 and AtSUS

The reaction was performed at 1 mL scale using 100 mM of sucrose, 3 mM of MgCl₂, 0.25 mM of UDP and 0.5 mM of Rebaudioside A in potassium phosphate buffer (50 mM final concentration, pH 7.5). The reaction was started by adding 15 μΐ of UGTSL2 (see EXAMPLE 27) lysate (2 U/mL), 150 of UGT76Glvar94 (see EXAMPLE 26) (2.5 U/mL) and 15 of AtSUS (see EXAMPLE 42) (400 U/mL). The reaction was followed by HPLC after quenching 125 iL samples with 10 of 2 N H₂S0₄ and 1 15 μΐ, of 60% methanol. 68% of Rebaudioside M and 26% of Rebaudioside M2 was obtained after 21 h of reaction time. The results are presented in Figure 69.

Although various embodiments of the present invention have been disclosed in the foregoing description for purposes of illustration, it should be understood that a variety of changes, modifications and substitutions may be incorporated without departing from either the spirit of scope of the present invention.

Claims

CLAIMS We claim:

1. A method for producing highly purified target steviol glycosides, comprising the steps of: a. providing a starting composition comprising an organic compound with at least one carbon atom; b. providing a microorganism containing at least one enzyme selected from

steviol biosynthesis enzymes, UDP-glycosyltransferases, and optionally UDP- glucose recycling enzymes; c. contacting the microorganism with a medium containing the starting

composition to produce a medium comprising at least one target steviol glycoside.

2. A method for producing highly purified target steviol glycosides, comprising the steps of: a. providing a starting composition comprising an organic compound with at least one carbon atom; b. providing a biocatalyst comprising at least one enzyme selected from steviol biosynthesis enzymes, UDP-glycosyltransferases, and optionally UDP-glucose recycling enzymes; c. contacting the biocatalyst with a medium containing the starting composition to produce a medium comprising at least one target steviol glycoside.

3. The method of claim 1 or 2 further comprising the step of: d. separating the target steviol glycoside from the medium to provide a highly purified target steviol glycoside composition.

4. The method of claim 1 or 2, wherein the staring composition is selected from the group consisting polyols, carbohydrates, steviol, steviol glycosides and combinations thereof.

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

6. The method of claim 2, wherein the biocatalyst is selected from the group consisting of whole cell suspension, crude lysate or purified enzymes in free or immobilized form.

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

8. The method of claim 1 or 2, wherein the target steviol glycoside is produced within the cell or in outside medium and is separated using crystallization, separation by membranes, centrifugation, extraction, chromatographic separation or a combination of such methods.

9. The method of claim 1 or 2, wherein the target steviol glycoside content is greater than about 95% by weight on a dry basis.

10. The method of claim 9, wherein the target steviol glycoside is selected from stevioside, reb A, reb E, reb D, reb D2, reb M, and reb M2.

1 1. The method of claim 9, wherein the target steviol glycoside is reb M.

12. A highly purified target steviol glycoside composition prepared according to the method of claim 1 or 2, wherein the target steviol glycoside content is greater than about 95% by weight on a dry basis.

13. The highly purified target steviol glycoside composition of claim 12, wherein the target steviol glycoside is selected from reb D and reb M.

14. A highly purified target steviol glycoside composition prepared according to the method of claim 1 or 2, wherein the target steviol glycoside is polymorphic.

1 . The highly purified target steviol glycoside composition of claim 14, wherein the target steviol glycoside is reb D or reb M.

16. A consumable product comprising the highly purified target glycoside composition of claim 1 or 2, wherein the product is selected from the group consisting of a food, a beverage, a pharmaceutical composition, a tobacco product, a nutraceutical composition, an oral hygiene composition, and a cosmetic composition.

17. A consumable product comprising the highly purified target steviol glycoside composition of claim 1 or 2, wherein the product is selected from the group consisting of a food, a beverage, a pharmaceutical composition, a tobacco product, a nutraceutical composition, an oral hygiene composition, and a cosmetic composition, and wherein the target steviol glycoside is reb D.

18. A consumable product comprising the highly purified target steviol glycoside composition of claim 1 or 2, wherein the product is selected from the group consisting of a food, a beverage, a pharmaceutical composition, a tobacco product, a nutraceutical composition, an oral hygiene composition, and a cosmetic composition, and wherein the target steviol glycoside is reb M.

19. Isolated and purified reb D2 having the following structure:

Reb D2 having the following structure

21. A consumable product comprising reb D2.

22. The consumable product of claim 21 , wherein the composition is selected from the group consisting of beverages; natural juices; refreshing drinks; carbonated soft drinks; diet drinks; zero calorie drinks; reduced calorie drinks and foods; yogurt drinks; instant juices; instant coffee; powdered types of instant beverages; canned products; syrups; fermented soybean paste; soy sauce; vinegar; dressings; mayonnaise; ketchups; curry; soup; instant bouillon; powdered soy sauce; powdered vinegar; types of biscuits; rice biscuit; crackers; bread; chocolates; caramel; candy; chewing gum; jelly; pudding; preserved fruits and vegetables; fresh cream; jam; marmalade; flower paste; powdered milk; ice cream; sorbet; vegetables and fruits packed in bottles; canned and boiled beans; meat and foods boiled in sweetened sauce; agricultural vegetable food products; seafood; ham; sausage; fish ham; fish sausage; fish paste; deep fried fish products; dried seafood products; frozen food products; preserved seaweed; preserved meat; tobacco and medicinal products.

23. A beverage comprising reb D2.

24. A method for preparing reb D2 comprising: a. contacting a starting composition comprising reb A with an enzyme capable of transforming reb A to reb D2, UDP-glucose, and optionally UDP-glucose recycling enzymes to produce a composition comprising reb D2; and b. isolating a composition comprising reb D2.

25. The method of claim 24, further comprising purifying the composition comprising reb D2 to provide reb D2 having a purity greater than about 95% by weight on an anhydrous basis.

26. Isolated and purified reb M2 having the following structure:

Reb M2 having the following structure:

28. A consumable product comprising reb M2.

29. The consumable product of claim 28, wherein the composition is selected from the group consisting of beverages; natural juices; refreshing drinks; carbonated soft drinks; diet drinks; zero calorie drinks; reduced calorie drinks and foods; yogurt drinks; instant juices; instant coffee; powdered types of instant beverages; canned products; syrups; fermented soybean paste; soy sauce; vinegar; dressings; mayonnaise; ketchups; curry; soup; instant bouillon; powdered soy sauce; powdered vinegar; types of biscuits; rice biscuit; crackers; bread; chocolates; caramel; candy; chewing gum; jelly; pudding; preserved fruits and vegetables; fresh cream; jam; marmalade; flower paste; powdered milk; ice cream; sorbet; vegetables and fruits packed in bottles; canned and boiled beans; meat and foods boiled in sweetened sauce; agricultural vegetable food products; seafood; ham; sausage; fish ham; fish sausage; fish paste; deep fried fish products; dried seafood products; frozen food products; preserved seaweed; preserved meat; tobacco and medicinal products.

30. A beverage comprising reb M2.

31. A method for preparing reb M2 comprising: a. contacting a starting composition comprising reb D2 with an enzyme capable of transforming reb D2 to reb M2, UDP-glucose, and optionally UDP-glucose recycling enzymes to produce a composition comprising reb M2; and b. isolating a composition comprising reb M2.

32. The method of claim 31, further comprising purifying the composition comprising reb M2 to provide reb M2 having a purity greater than about 95% by weight on an anhydrous basis.

33. The consumable product of claim 21 , further comprising at least one additive selected from the group consisting of carbohydrates, polyols, amino acids and their corresponding salts, poly-amino acids and their corresponding salts, sugar acids and their corresponding salts, nucleotides, organic acids, inorganic acids, organic salts including organic acid salts and organic base salts, inorganic salts, bitter compounds, flavorants and flavoring ingredients, astringent compounds, proteins or protein hydrolysates, surfactants, emulsifiers, flavonoids, alcohols, polymers and combinations thereof.

34. The consumable product of claim 21, further comprising at least one functional ingredient selected from the group consisting of saponins, antioxidants, dietary fiber sources, fatty acids, vitamins, glucosamine, minerals, preservatives, hydration agents, probiotics, prebiotics, weight management agents, osteoporosis management agents, phytoestrogens, long chain primary aliphatic saturated alcohols, phytosterols and combinations thereof.

35. The consumable product of claim 21, further comprising a compound selected from the group consisting of reb A, reb B, reb D, NSF-02, Mogroside V, Luo Han Guo, allulose, allose, D-tagatose, erythritol and combinations thereof.

36. A tabletop sweetener composition comprising reb D2.

37. The consumable product of claim 28, further comprising at least one additive selected from the group consisting of carbohydrates, polyols, amino acids and their corresponding salts, poly-amino acids and their corresponding salts, sugar acids and their corresponding salts, nucleotides, organic acids, inorganic acids, organic salts including organic acid salts and organic base salts, inorganic salts, bitter compounds, flavorants and flavoring ingredients, astringent compounds, proteins or protein hydrolysates, surfactants, emulsifiers, flavonoids, alcohols, polymers and combinations thereof.

38. The consumable product of claim 28, further comprising at least one functional ingredient selected from the group consisting of saponins, antioxidants, dietary fiber sources, fatty acids, vitamins, glucosamine, minerals, preservatives, hydration agents, probiotics, prebiotics, weight management agents, osteoporosis management agents, phytoestrogens, long chain primary aliphatic saturated alcohols, phytosterols and combinations thereof.

39. The consumable product of claim 28, further comprising a compound selected from the group consisting of Reb A, Reb B, Reb D, NSF-02, Mogroside V, Luo Han Guo, allulose, allose, D-tagatose, erythritol and combinations thereof

40. A tabletop sweetener composition comprising reb M2.

41. A method for enhancing the sweetness of a beverage comprising a sweetener comprising:

a. ) providing a beverage comprising a sweetener; and

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

wherein reb D2 and/or reb M2 is present in a concentration at or below the sweetness recognition threshold.

42. Target steviol glycoside of claim 1 or 2, in combination with at least one naturally occurring high intensity sweetener selected from group including stevia, stevia extract, steviolmonoside, steviolbioside, rubusoside, dulcoside A, dulcoside B, rebaudioside B, rebaudioside G, stevioside, rebaudioside C, rebaudioside F, rebaudioside A, rebaudioside /, rebaudioside E, rebaudioside H, rebaudioside L, rebaudioside K, rebaudioside J rebaudioside M, rebaudioside M2, rebaudioside D, rebaudioside D2, rebaudioside N or rebaudioside O, or other glycoside of steviol found in Stevia rebaudiana, mogrosides, brazzein, neohesperidin dihydrochalcone, glycyrrhizic acid and its salts, thaumatin, perillartine, pernandulcin, mukuroziosides, baiyunoside, phlomisoside-I, dimethyl- hexahydrofluorene-dicarboxylic acid, abrusosides, periandrin, carnosiflosides, cyclocarioside, pterocaryosides, polypodoside A, brazilin, hernandulcin, phillodulcin, glycyphyllin, phlorizin, trilobatin, dihydroflavonol, dihydroquercetin-3 -acetate, neoastilibin, iram-cinnamaldehyde, monatin and its salts, selligueain A, hematoxylin, monellin, osladin, pterocaryoside A, pterocaryoside B, mabinlin, pentadin, miraculin, curculin, neoculin, chlorogenic acid, cynarin, Luo Han Guo sweetener, mogroside V, mogroside VI, grosmomomside, siamenoside or other glycoside of mogrol found in Siraitia grosvenorii and/or combination thereof.

43. The method of claim 1 or 2, wherein the target steviol glycoside content is greater than about 99% by weight on a dry basis.