# Patent

## Title
High-purity steviol glycosides

## 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.

## Background
RELATED APPLICATIONS
This application claims the benefit of priority from U.S. Provisional Application No. 61/827,922, filed on May 28, 2013, U.S. Provisional Application No. 61/843,544, filed on Jul. 8, 2013, U.S. Provisional Application No. 61/861,528, filed on Aug. 2, 2013, U.S. Provisional Application No. 61/881,166, filed on Sep. 23, 2013, U.S. Provisional Application No. 61/885,084, filed on Oct. 1, 2013, U.S. Provisional Application No. 61/904,751, filed on Nov. 15, 2013, U.S. Provisional Application No. 61/913,482, filed on Dec. 9, 2013, U.S. Provisional Application No. 61/921,635, filed on Dec. 30, 2013, U.S. Provisional Application No. 61/925,329, filed on Jan. 9, 2014, and U.S. Provisional Application No. 61/939,855, filed on Feb. 14, 2014.


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 C13 and C19. 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 M, 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-1-deoxyxylulose 5-phosphate reductoisomerase (DXR), 4-diphosphocytidyl-2-C-methyl-D-erythritol synthase (CMS), 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase (CMK), 4-diphosphocytidyl-2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (MCS), 1-hydroxy-2-methyl-2(E)-butenyl 4-diphosphate synthase (HDS), 1-hydroxy-2-methyl-2(E)-butenyl 4-diphosphate reductase (HDR), acetoacetyl-CoA thiolase, truncated HMG-CoA reductase, mevalonate kinase, phosphomevalonate kinase, mevalonate pyrophosphate decarboxylase, cytochrome P450 reductase 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, UGT76G1, 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 M. 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 M 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 biocatalysts 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 1H 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. 11 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 A peak 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 1H NMR spectrum of reb D2 (500 MHz, pyridine-d5).
FIG. 15 shows the 13C NMR spectrum of reb D2 (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 1H-1H COSY Spectrum of reb D2 (500 MHz, pyridine-d5).
FIG. 18 shows the HSQC-DEPT spectrum of reb D2 (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-d5).
FIG. 21 shows the 1H NMR spectrum of reb M2 (500 MHz, D2O).
FIG. 22 shows the 13C NMR spectrum of reb M2 (125 MHz, D2O/TSP).
FIG. 23 shows an expansion of the 13C NMR spectrum of reb M2 (125 MHz, D2O/TSP).
FIG. 24 shows the 1H-1H COSY spectrum of reb M2 (500 MHz, D2O).
FIG. 25 shows the HSQC-DEPT spectrum of reb M2 (500 MHz, D2O).
FIG. 26 shows the HMBC spectrum of reb M2 (500 MHz, D2O).
FIG. 27 shows an expansion of HMBC spectrum of reb M2 (500 MHz, D2O).
FIG. 28 shows another HMBC spectrum of reb M2.
FIG. 29 shows a 1H 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 a 1H-1H 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 1D-TOCSY spectrum of reb M2.
FIG. 37 shows a 1D-TOCSY spectrum of reb M2.
FIG. 38 shows a 1D-TOCSY spectrum of reb M2.
FIG. 39 shows a 1D-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 I, rebaudioside E, rebaudioside H, rebaudioside L, rebaudioside K, rebaudioside J, rebaudioside M, rebaudioside M2, rebaudioside D, rebaudioside D2, rebaudioside N, rebaudioside O, synthetic steviol glycosides, e.g. enzymatically glucosylated steviol glycosides and combinations thereof.










Chemical structures of steviol and its glycosides








 









Compound
R1
R2





Steviol
H
H


Steviolmonoside
H
β-Glc


Steviol monoglucosyl ester
β-Glc
H


Rubusoside
β-Glc
β-Glc


Steviolbioside
H
β-Glc-β-Glc (2→1)


Stevioside
β-Glc
β-Glc-β-Glc (2→1)


 


Rebaudioside A
β-Glc






 


Rebaudioside D
β-Glc-β-Glc (2→1)






 


Rebaudioside E
β-Glc-β-Glc (2→1)
β-Glc-β-Glc (2→1)


 


Rebaudioside M













(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 (CH2O)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 I, rebaudioside E, rebaudioside H, rebaudioside L, rebaudioside K, rebaudioside J, rebaudioside M, rebaudioside M2, rebaudioside D, rebaudioside D2, rebaudioside N or rebaudioside O, or other 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.
The target steviol glycoside can be in any polymorphic or amorphous form, including hydrates, solvates, anhydrous or combinations thereof.
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 I content by weight on a dry basis. In another particular embodiment, when the target steviol glycoside is reb I, the process described herein provides a composition comprising greater than about 95% reb I 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 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-1-deoxyxylulose 5-phosphate reductoisomerase (DXR), 4-diphosphocytidyl-2-C-methyl-D-erythritol synthase (CMS), 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase (CMK), 4-diphosphocytidyl-2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (MCS), 1-hydroxy-2-methyl-2(E)-butenyl 4-diphosphate synthase (HDS), 1-hydroxy-2-methyl-2(E)-butenyl 4-diphosphate reductase (HDR), acetoacetyl-CoA thiolase, truncated HMG-CoA reductase, mevalonate kinase, phosphomevalonate kinase, mevalonate pyrophosphate decarboxylase, cytochrome P450 reductase 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, 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 GenInfo identifier numbers, preferably from the group presented in Table 1, and more preferably the group presented in Table 2.

















397567
30680413
115480946
147798902
218193594
225443294


454245
32816174
116310259
147811764
218193942 
225444853


1359905
32816178 
116310985
147827151
219885307 
225449296


1685003
34393978
116788066
147836230
222615927
225449700


1685005
37993665 
116788606
147839909
222619587
225454338


2191136
37993671 
116789315
147846163
222623142
225454340


2501497
37993675
119394507
147855977
222625633
225454342


2911049
39104603
119640480
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


15231757 
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
226531147


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
224114583
242043290


18086351
82791223 
126635845
187373042
224116284
242044836


18418378
83778990 
126635847
190692175
224120552
242051252


18418380 
89953335 
126635863
194701936
224121288
242056217


18418382 
110741436
126635867
195620060
224121296
242056219


19743740 
110743955
126635883
209954691
224121300
242056663


19911201 
115438196
126635887
209954719
224130358
242059339


20149064 
115438785
133874210
209954725
224140703
242059341


20260654
115441237
133874212
209954733
224143404
242060922


21435782
115454819
145358033
210063105
224143406
242067411


21553613
115456047
147772508
210063107
224144306
242067413


21593514
115457492
147776893
212275846
224285244
242076258


22759895
115459312
147776894
216296854
225431707
242076396


23955910
115464719
147776895
217074506
225435532
242084750


26452040
115471069
147786916
218185693
225436321
242091005


28393204
115471071
147798900
218187075
225440041
242095206


30679796
115474009
147798901
218189427
225441116
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
326511261
356527181
357445729 
359493636


255555361
297796257
326511866
356533209
357445731
359493815


255555363
297796261
326512412
356533852
357445733
359495856


255555365
297797587
326517673
356534718
357446799
359495858


255555369
297798502
326518800
356535480
357446805
359495869


255555373
297799226
326521124
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
297811403
326530093
356554358
357452791 
378405177


255564531
297820040
326534036
356554360
357452797 
378829085


255572878
297821483
326534312
356558606
357452799 
387135070


255577901
297825217
332071132
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
357114993
357497581
387135184


260279126
302142947
356501328
357115447
357497671 
387135186


260279128
302142948
356502523
357115451
357500579 
387135188


261343326
302142950
356503180
357115453
357504663 
387135190


283132367
302142951
356503184
357116080
357504691 
387135192


283362112
302765302
356503295
357116928
357504699 
387135194


289188052
302796334
356504436
357117461
357504707 
387135282


295841350
302811470
356504523
357117463
357505859 
387135284


296088529 
302821107
356504765
357117829
357510851 
387135294


296090415 
302821679
356511113
357117839
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


297611791 
326490273
356522588
357138503
359484299
387135314


297722841 
326491131
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
470104211 
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
470115448 
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
462402118
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
462411467
470143607 
482562795


414590349
449519559
460409134
462414311
470143939 
482562850


414590661
449522783
460409136
462414416
470145404
482565074


414591157
449524530
460409459
462414476
473923244
482566269


414879558
449524591
460409461
462415526
474114354
482566296


414879559
449528823
460409463
462415603
474143634
482566307


414879560
449528825
460409465
462415731
474202268 
482568689


414888074
449534021
460409467
462416307
474299266 
482570049


431812559
460365546
460410124
462416920
474363119 
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
475511330



449440431 
460374714
460411200
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
ABK24743.1
Picea sitchensis


122209731
Q2V6J9.1
Fragaria x ananassa


125534461
EAY81009.1
Oryza saliva Indica Group


125559566
EAZ05102.1
Oryza sativa Indica Group


125588307
EAZ28971.1
Oryza sativa Japonica Group


148907340
ABR16806.1
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_172511.3
Arabidopsis thaliana


187373030
ACD03249.1
Avena strigosa


194701936
ACF85052.1
Zea mays


19743740
AAL92461.1
Solanum lycopersicum


212275846
NP_001131009.1
Zea mays


222619587
EEE55719.1
Oryza saliva 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_001148083.1
Zea mays


226502400
NP_001147674.1
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


297611791
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


302821107
XP_002992218.1
Selaginella moellendorffii


30680413
NP_179446.2
Arabidopsis thaliana


319759266
ADV71369.1
Pueraria montana var. lobata


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 truncatula


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
Solanum lycopersicum


460409128
XP_004249992.1
Solanum lycopersicum


460409461
XP_004250157.1
Solanum lycopersicum


460409465
XP_004250159.1
Solanum lycopersicum


462396388
EMJ02187.1
Prunus persica


462402118
EMJ07675.1
Prunus persica


462409359
EMJ14693.1
Prunus persica


462416923
EMJ21660.1
Prunus persica


46806235
BAD17459.1
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








Internal 


GI number
Accession
Origin
reference







460409128 
XP.004249992.1
Solanum lycopersicum
UGTSL


460386018 
XP.004238697.1
Solanum lycopersicum
—


460409134
XP.004249995.1
Solanum lycopersicum
—


460410132
XP.004250485.1
Solanum lycopersicum
UGTSL2


460410130 
XP.004250484.1
Solanum lycopersicum
—


460410128 
XP.004250483.1
Solanum lycopersicum
—


460378310 
XP.004234916.1
Solanum lycopersicum
—


209954733
BAG80557.1
Lycium barbarum
UGTLB


209954725
BAG80553.1
Lycium barbarum
—









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-glucosyltransferase.
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:




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% 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. 115454819 version NP_001051010.1, GI No. 187373030, version ACD03249.1. GI No. 222619587 version EEE55719.1, GI No. 297795735 version XP_002865752.1 or EUGT11.
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.
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.
Suitable microorganisms include, but are not limited to, E. coli, Saccharomyces sp., Aspergillus sp., Pichia sp., Bacillus sp., Yarrowia 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) version XP_004249992.1, GI No. 115454819 version NP_001051010.1, GI No. 187373030, version ACD03249.1. GI No. 222619587 version EEE55719.1, GI No. 297795735 version XP_002865752.1 or EUGT11 and UGTs having substantial (>85%) sequence identity to these.
In a still more particular embodiment, the enzyme is UGTSL2 or its improved variant produced by directed evolution and having higher activity.
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.
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 C18 column with a mobile phase containing A (25% MeCN in water) and B (30% MeCN in water) with the following gradient:












Time (min)
% A
% B

















0.0-5.0 
100
0


20
20
80


25
20
80


30
100
0









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 M and has the following structure:




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 XP_004250485.1), GI No. 460409128 (UGTSL) version XP_004249992.1, GI No. 115454819 version NP_001051010.1, GI No. 187373030, version ACD03249.1. GI No. 222619587 version EEE55719.1, GI No. 297795735 version XP_002865752.1 or EUGT11.
The enzyme 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.
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.
Suitable microorganisms include, but are not limited to, E. coli, Saccharomyces sp., Aspergillus sp., Pichia sp., Bacillus sp., Yarrowia sp.
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 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) version XP_004249992.1, GI No. 115454819 version NP_001051010.1, GI No. 187373030, version ACD03249.1. GI No. 222619587 version EEE55719.1, GI No. 297795735 version XP_002865752.1 or EUGT11.
In a still more particular embodiment, the enzyme is UGTSL2 or its improved variant produced by directed evolution and having higher activity.
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 C18 column with a mobile phase containing A (25% MeCN in water) and B (30% MeCN in water) with the following gradient:












Time (min)
% A
% B

















0.0-5.0 
100
0


20
20
80


25
20
80


30
100
0









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.
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 C56H90O33, 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 [α]D25 −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, trans-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 (CH2O)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, sulfonyl, 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 11.0% by weight, about 11.5% 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
NcoI and NdeI 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):







CCATGGCCCATATGGAAAACAAAACCGAAACCACCGTTCGTCGTCGTCGC


 


CGTATTATTCTGTTTCCGGTTCCGTTTCAGGGTCATATTAATCCGATTCT


 


GCAGCTGGCAAATGTGCTGTATAGCAAAGGTTTTAGCATTACCATTTTTC


 


ATACCAATTTTAACAAACCGAAAACCAGCAATTATCCGCATTTTACCTTT


 


CGCTTTATTCTGGATAATGATCCGCAGGATGAACGCATTAGCAATCTGCC


 


GACACATGGTCCGCTGGCAGGTATGCGTATTCCGATTATTAACGAACATG


 


GTGCAGATGAACTGCGTCGTGAACTGGAACTGCTGATGCTGGCAAGCGAA


 


GAAGATGAAGAAGTTAGCTGTCTGATTACCGATGCACTGTGGTATTTTGC


 


ACAGAGCGTTGCAGATAGCCTGAATCTGCGTCGTCTGGTTCTGATGACCA


 


GCAGCCTGTTTAACTTTCATGCACATGTTAGCCTGCCGCAGTTTGATGAA


 


CTGGGTTATCTGGATCCGGATGATAAAACCCGTCTGGAAGAACAGGCAAG


 


CGGTTTTCCGATGCTGAAAGTGAAAGATATCAAAAGCGCCTATAGCAATT


 


GGCAGATTCTGAAAGAAATTCTGGGCAAAATGATTAAACAGACCAAAGCA


 


AGCAGCGGTGTTATTTGGAATAGCTTTAAAGAACTGGAAGAAAGCGAACT


 


GGAAACCGTGATTCGTGAAATTCCGGCACCGAGCTTTCTGATTCCGCTGC


 


CGAAACATCTGACCGCAAGCAGCAGCAGCCTGCTGGATCATGATCGTACC


 


GTTTTTCAGTGGCTGGATCAGCAGCCTCCGAGCAGCGTTCTGTATGTTAG


 


CTTTGGTAGCACCAGCGAAGTTGATGAAAAAGATTTTCTGGAAATTGCCC


 


GTGGTCTGGTTGATAGCAAACAGAGCTTTCTGTGGGTTGTTCGTCCGGGT


 


TTTGTTAAAGGTAGCACCTGGGTTGAACCGCTGCCGGATGGTTTTCTGGG


 


TGAACGTGGTCGTATTGTTAAATGGGTTCCGCAGCAAGAAGTTCTGGCAC


 


ACGGCGCAATTGGTGCATTTTGGACCCATAGCGGTTGGAATAGCACCCTG


 


GAAAGCGTTTGTGAAGGTGTTCCGATGATTTTTAGCGATTTTGGTCTGGA


 


TCAGCCGCTGAATGCACGTTATATGAGTGATGTTCTGAAAGTGGGTGTGT


 


ATCTGGAAAATGGTTGGGAACGTGGTGAAATTGCAAATGCAATTCGTCGT


 


GTTATGGTGGATGAAGAAGGTGAATATATTCGTCAGAATGCCCGTGTTCT


 


GAAACAGAAAGCAGATGTTAGCCTGATGAAAGGTGGTAGCAGCTATGAAA


 


GCCTGGAAAGTCTGGTTAGCTATATTAGCAGCCTGTAATAACTCGAG






After synthesis of the gene and subcloning into pET30A+ vector using NdeI and XhoI cloning sites, the UGT76G1_pET30a+ plasmid was introduced in E. coli Bl21(DE3) and E. coli EC100 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 μL aliquots were stored at −20° C. and at −80° C.
The storage aliquots of E. coli BL21(DE3) containing the pET30A+_UGT76G1 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 UGT76G1_pET30a+ plasmid from E. coli EC100 was mixed with 80 μL of S30 premix plus and 72 μL of S30 T7 extract was added. Nuclease-free water was added in order to obtain a total volume of 200 μL 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
NcoI and NdeI 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):







CCATGGCACATATGGCAACCAGCGATAGCATTGTTGATGATCGTAAACAG


 


CTGCATGTTGCAACCTTTCCGTGGCTGGCATTTGGTCATATTCTGCCGTA


 


TCTGCAGCTGAGCAAACTGATTGCAGAAAAAGGTCATAAAGTGAGCTTTC


 


TGAGCACCACCCGTAATATTCAGCGTCTGAGCAGCCATATTAGTCCGCTG


 


ATTAATGTTGTTCAGCTGACCCTGCCTCGTGTTCAAGAACTGCCGGAAGA


 


TGCCGAAGCAACCACCGATGTTCATCCGGAAGATATTCCGTATCTGAAAA


 


AAGCAAGTGATGGTCTGCAGCCGGAAGTTACCCGTTTTCTGGAACAGCAT


 


AGTCCGGATTGGATCATCTATGATTATACCCATTATTGGCTGCCGAGCAT


 


TGCAGCAAGCCTGGGTATTAGCCGTGCACATTTTAGCGTTACCACCCCGT


 


GGGCAATTGCATATATGGGTCCGAGCGCAGATGCAATGATTAATGGTAGT


 


GATGGTCGTACCACCGTTGAAGATCTGACCACCCCTCCGAAATGGTTTCC


 


GTTTCCGACCAAAGTTTGTTGGCGTAAACATGATCTGGCACGTCTGGTTC


 


CGTATAAAGCACCGGGTATTAGTGATGGTTATCGTATGGGTCTGGTTCTG


 


AAAGGTAGCGATTGTCTGCTGAGCAAATGCTATCATGAATTTGGCACCCA


 


GTGGCTGCCGCTGCTGGAAACCCTGCATCAGGTTCCGGTTGTTCCGGTGG


 


GTCTGCTGCCTCCGGAAGTTCCGGGTGATGAAAAAGATGAAACCTGGGTT


 


AGCATCAAAAAATGGCTGGATGGTAAACAGAAAGGTAGCGTGGTTTATGT


 


TGCACTGGGTAGCGAAGTTCTGGTTAGCCAGACCGAAGTTGTTGAACTGG


 


CACTGGGTCTGGAACTGAGCGGTCTGCCGTTTGTTTGGGCATATCGTAAA


 


CCGAAAGGTCCGGCAAAAAGCGATAGCGTTGAACTGCCGGATGGTTTTGT


 


TGAACGTACCCGTGATCGTGGTCTGGTTTGGACCAGCTGGGCACCTCAGC


 


TGCGTATTCTGAGCCATGAAAGCGTTTGTGGTTTTCTGACCCATTGTGGT


 


AGCGGTAGCATTGTGGAAGGTCTGATGTTTGGTCATCCGCTGATTATGCT


 


GCCGATTTTTGGTGATCAGCCGCTGAATGCACGTCTGCTGGAAGATAAAC


 


AGGTTGGTATTGAAATTCCGCGTAATGAAGAAGATGGTTGCCTGACCAAA


 


GAAAGCGTTGCACGTAGCCTGCGTAGCGTTGTTGTTGAAAAAGAAGGCGA


 


AATCTATAAAGCCAATGCACGTGAACTGAGCAAAATCTATAATGATACCA


 


AAGTGGAAAAAGAATATGTGAGCCAGTTCGTGGATTATCTGGAAAAAAAC


 


ACCCGTGCAGTTGCCATTGATCACGAAAGCTAATGACTCGAG






After synthesis of the gene and subcloning into pET30A+ vector using NcoI and XhoI cloning sites, the UGT91D2_pET30a+ plasmid was introduced into E. coli EC100 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 μL 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 μL of S30 premix plus and 72 μL of S30 T7 extract was added. Nuclease-free water was added in order to obtain a total volume of 200 μL and the resulting solution was incubated for 2 h at 30° C. 5 μL was used for SDS-page analysis while the remaining 45 μL 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 μL of UGT76G1 thawed lysate. The reactions were run at 30° C. on an orbitary shaker at 135 rpm. For each sample, 460 μL of the reaction mixture was quenched with 40 μL of 2N H2SO4 and 420 μL of methanol/water (6/4). The samples were immediately centrifuged and kept at 10° C. before analysis by HPLC (CAD). HPLC indicated almost complete conversion of stevioside to rebaudioside A as seen in FIG. 40.
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 μL of in-vitro produced UGT91D2. The reactions were run at 30° C. on an orbitary shaker at 135 rpm. For each sample, 450 μL of reaction mixture was quenched with 45 μL of 2N H2SO4 and 405 μL of 60% MeOH. After centrifugation, the supernatant was analyzed by HPLC (CAD). HPLC indicated 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 μL of in-vitro produced UGT76G1. The reactions were run at 30° C. on an orbitary shaker at 135 rpm. For each sample, 400 μL of reaction mixture was quenched with 40 μL of 2N H2SO4 and 360 μL of 60% MeOH. After centrifugation, the supernatant was analyzed by HPLC (CAD). HPLC indicated 80% conversion of rebaudioside D to rebaudioside M after 120 h as seen in FIG. 41.
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+_UGT76G1 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 μL 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+_UGT76G1 plasmid was transformed into Tuner (DE3) expression strain (Novagen TunerTM (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 μL 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 μL 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 NdeI and SalI cloning sites, the pMAL_UGT76G1 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 μL 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_UGT76G1 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 μL 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 Nde1 and Xho1 cloning sites, the pCOLDIII_UGT76G1 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 μL 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_UGT76G1 plasmid was transformed into Origami2 (DE3) expression strain (Novagen OrigamiTM2 (DE3) Competent Cells) by heat shock treatment. The obtained cells were grown on LB Agar medium in 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 μL 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 μL 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.
















Transformation activity*













Expression
Stevioside to
Rebaudioside D


Example
Plasmid
strain
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−1
0.25 U mL−1


11
pCOLDIII
ArticExpress
15 U mL−1
0.11 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 μmol 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 μL/min. The column used was Phenomenex Prodigy 5μ ODS (3) 100 A; 250×2 mm. The column temperature was maintained at 40° C. The injection volume was 20-40 μl.
Example 15
Preparation of UGT91D2 Using pMAL Plasmid and BL21 Expression Strain
After subcloning the synthetic UGT91D2 gene into the pMAL plasmid using NdeI and Sal1 cloning sites, the pMAL_UGT91D2 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 μl, 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 12.32 g.
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 μL 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 TunerTM (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 μL 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 μL of a 100 mM IPTG solution was added (IPTG concentration in medium is 100 μM) 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 μL 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 16 h. 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 11.4 mL of “Bugbuster Master Mix” (Novagen, reference 71456) and 4.8 mL of water. The lysate was recovered by centrifugation and used directly for activity testing.
Example 19
Determination of Activity of In-Vivo Preparations of UGT91D2
Activity tests were performed at 5 mL scale with 1000 μL 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.
















Transformation activity*


Example
Plasmid
Expression strain
Rubusoside to Stevioside


















15
pMAL
BL21
 9 mU mL−1


16
pMAL
ArcticExpress
60 mU mL−1


17
pET30a+
Tuner (DE3)
28 mU mL−1


18
pET30a+
ArcticExpress (DE3)
21 mU mL−1





* Note:


The activities are mentioned per mL of lysate. 1 U will transform 1 μmol 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 public databases, synthesized by DNA2.0 and subsequently subcloned in pET30a+ vector.


















Con-






version


Micro-


Internal 
RebA to 


plate
Position
Gene Name
reference
RebD



















C908201
A1
gi115454819_NP_001051010.1
S115N01 
Active





A1



C908201
G2
gi187373030_ACD03249.1
S115N01 
Active





G2



C908201
A7
gi460409128_XP_004249992.1
S115N05 
Active





A7



C912666
E1
gi222619587_EEE55719.1
S115N06 
Active





E1



C912666
C2
gi297795735_XP_002865752.1
S115N06 
Active





C2









The aminoacid sequences are as follows:







SEQ ID 3


>gi|115454819|ref|NP_001051010.1|Os03g0702500


[Oryza sativa Japonica Group]


MDDAHSSQSPLHVVIFPWLAFGHLLPCLDLAERLAARGHRVSFVSTPRNL


 


ARLPPVRPELAELVDLVALPLPRVDGLPDGAEATSDVPFDKFELHRKAFD


 


GLAAPFSAFLDTACAGGKRPDWVLADLMHHWVALASQERGVPCAMILPCS


 


AAVVASSAPPTESSADQREAIVRSMGTAAPSFEAKRATEEFATEGASGVS


 


IMTRYSLTLQRSKLVAMRSCPELEPGAFTILTRFYGKPVVPFGLLPPRPD


 


GARGVSKNGKHDAIMQWLDAQPAKSVVYVALGSEAPMSADLLRELAHGLD


 


LAGTRFLWAMRKPAGVDADSVLPAGFLGRTGERGLVTTRWAPQVSILAHA


 


AVCAFLTHCGWGSVVEGLQFGHPLIMLPILGDQGPNARILEGRKLGVAVP


 


RNDEDGSFDRGGVAGAVRAVVVEEEGKTFFANARKLQEIVADREREERCI


 


DEFVQHLTSWNELKNNSDGQYP


 


SEQ ID 4


>gi|187373030|gb|ACD03249.1|UDP-glycosyltrans-


ferase [Avena strigosa]


MAVKDEQQSPLHILLFPFLAPGHLIPIADMAALFASRGVRCTILTTPVNA


 


AIIRSAVDRANDAFRGSDCPAIDISVVPFPDVGLPPGVENGNALTSPADR


 


LKFFQAVAELREPFDRFLADNHPDAVVSDSFFHWSTDAAAEHGVPRLGFL


 


GSSMFAGSCNESTLHNNPLETAADDPDALVSLPGLPHRVELRRSQMMDPK


 


KRPDHWALLESVNAADQKSFGEVFNSFHELEPDYVEHYQTTLGRRTWLVG


 


PVALASKDMAGRGSTSARSPDADSCLRWLDTKQPGSVVYVSFGTLIRFSP


 


AELHELARGLDLSGKNFVWVLGRAGPDSSEWMPQGFADLITPRGDRGFII


 


RGWAPQMLILNHRALGGFVTHCGWNSTLESVSAGVPMVTWPRFADQFQNE


 


KLIVEVLKVGVSIGAKDYGSGIENHDVIRGEVIAESIGKLMGSSEESDAI


 


QRKAKDLGAEARSAVENGGSSYNDVGRLMDELMARRSSVKVGEDIIPTND


 


GL


 


SEQ ID 5


>gi|460409128|ref|XP_004249992.1| PREDICTED:


cyanidin-3-O-glucoside 2-O-glucuronosyltrans-


ferase-like [Solanum lycopersicum]


MSPKLHKELFFHSLYKKTRSNHTMATLKVLMFPFLAYGHISPYLNVAKKL


 


ADRGFLIYFCSTPINLKSTIEKIPEKYADSIHLIELHLPELPQLPPHYHT


 


TNGLPPNLNQVLQKALKMSKPNFSKILQNLKPDLVIYDILQRWAKHVANE


 


QNIPAVKLLTSGAAVFSYFFNVLKKPGVEFPFPGIYLRKIEQVRLSEMMS


 


KSDKEKELEDDDDDDDLLVDGNMQIMLMSTSRTIEAKYIDFCTALTNWKV


 


VPVGPPVQDLITNDVDDMELIDWLGTKDENSTVFVSFGSEYFLSKEDMEE


 


VAFALELSNVNFIWVARFPKGEERNLEDALPKGFLERIGERGRVLDKFAP


 


QPRILNHPSTGGFISHCGWNSAMESIDFGVPIIAMPMHLDQPMNARLIVE


 


LGVAVEIVRDDDGKIHRGEIAETLKGVITGKTGEKLRAKVRDISKNLKTI


 


RDEEMDAAAEELIQLCRNGN


 


SEQ ID 6


>gi|222619587|gb|EEE55719.1| hypothetical protein


OsJ_04191 [Oryza sativa Japonica Group]


MHVVMLPWLAFGHILPFAEFAKRVARQGHRVTLFSTPRNTRRLIDVPPSL


 


AGRIRVVDIPLPRVEHLPEHAEATIDLPSNDLRPYLRRAYDEAFSRELSR


 


LLQETGPSRPDWVLADYAAYWAPAAASRHGVPCAFLSLFGAAALCFFGPA


 


ETLQGRGPYAKTEPAHLTAVPEYVPFPTTVAFRGNEARELFKPSLIPDES


 


GVSESYRFSQSIEGCQLVAVRSNQEFEPEWLELLGELYQKPVIPIGMFPP


 


PPPQDVAGHEETLRWLDRQEPNSVVYAAFGSEVKLTAEQLQRIALGLEAS


 


ELPFIWAFRAPPDAGDGDGLPGGFKERVNGRGVVCRGWVPQVKFLAHASV


 


GGFLTHAGWNSIAEGLANGVRLVLLPLMFEQGLNARQLAEKKVAVEVARD


 


EDDGSFAANDIVDALRRVMVGEEGDEFGVKVKELAKVFGDDEVNDRYVRD


 


FLKCLSEYKMQRQG


 


SEQ ID 7


>gi|297795735|ref|XP_002865752.1| UDP-gluco-


ronosyl/UDP-glucosyl transferase family protein


[Arabidopsis lyrata subsp. lyrata]


MDDKKEEVMHIAMFPWLAMGHLLPFLRLSKLLAQKGHKISFISTPRNILR


 


LPKLPSNLSSSITFVSFPLPSISGLPPSSESSMDVPYNKQQSLKAAFDLL


 


QPPLTEFLRLSSPDWIIYDYASHWLPSIAKELGISKAFFSLFNAATLCFM


 


GPSSSLIEESRSTPEDFTVVPPWVPFKSTIVFRYHEVSRYVEKTDEDVTG


 


VSDSVRFGYTIDGSDAVFVRSCPEFEPEWFSLLQDLYRKPVFPIGFLPPV


 


IEDDDDDTTWVRIKEWLDKQRVNSVVYVSLGTEASLRREELTELALGLEK


 


SETPFFWVLRNEPQIPDGFEERVKGRGMVHVGWVPQVKILSHESVGGFLT


 


HCGWNSVVEGIGFGKVPIFLPVLNEQGLNTRLLQGKGLGVEVLRDERDGS


 


FGSDSVADSVRLVMIDDAGEEIREKVKLMKGLFGNMDENIRYVDELVGFM


 


RNDESSQLKEEEEEDDCSDDQSSEVSSETDEKELNLDLKEEKRRISVYKS


 


LSSEFDDYVANEKMG






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 μL 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 μL of suspension. Determined quantities of plasmids were:
















Microtiter plate
Position
Internal reference
[Plasmid] ng/μL









C908201
A1
S115N01 A1
32.8



C908201
G2
S115N01 G2
41.0



C908201
A7
S115N05 A7
56.6



C912666
E1
S115N06 E1
64.0



C912666
C2
S115N06 C2
31.4










Transformation of Competent Cells with Plasmids.
Aliquots of chemically competent EC100 cells were taken from freezer at −80° C. and stored on ice. The cells were allowed to thaw on ice for 10 minutes. 10 μL 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 μL 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 μL 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 μL 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 quantities were determined as follows:













Microtiter plate
Position
Internal reference of test
[Plasmid] ng/μL


















C908201
A1
S115N01 A1
115.7


C908201
G2
S115N01 G2
120.4


C908201
A7
S115N05 A7
293.8


C912666
E1
S115N06 E1
126.1


C912666
C2
S115N06 C2
98.8









In-Vitro Expression of Enzymes.
18 μL of plasmid solution (containing approximately 1.5 μg of plasmid) was used for in-vitro expression according to the Promega S30 T7 High-Yield Protein Expression System (ref: L1110) kit. The expression medium was produced as follows:














S30 Premix Plus
T7 S30 Extract
Total







Trials
30 μL
27 μL
57 μL


reference
20 μL
18 μL
38 μL









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 μL 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 μL of a reaction mixture containing 0.5 mM Rebaudioside A, 3 mM MgCl2, 50 mM phosphate buffer (pH7.2) and 2.5 mM UDP-glucose was added to a 1.5 mL sterile microtube. 52 μL of the enzyme expression medium was added and the resulting mixture was allowed to react at 30° C. for 24 hours. 125 μL samples were taken after 2 hours, 16 hours and 24 hours and added to a 115 μL of 60% methanol and 10 μL of 2 N H2SO4. The quenched sample was centrifuged at 18,000 g for 2 minutes at RT. 200 μL was transferred to an HPLC vial and analyzed.
HPLC Analysis
The HPLC assay was performed as follows:
Apparatus













Equipment
Supplier
Reference
Lot#







Elite
Hitachi
L-2130
NA


Photodiode Array
Hitachi
L-2455
NA


Corona CAD detector
ESA
70-6186A
CO-2044


Injector 100 μL
Hitachi

NA


Column Synergy 4u Hydro-RP 
Phenomenex
00G-4375-E0
588582-12


80A (250 × 4.60 mm)









Instrument Conditions














Column Temperature
55° C.


Detection
UV 205 nm; bw 400 nm CAD detection


Analysis duration
15 min


Injected volume
10 μL


Flow rate
1 mL/min









Mobile Phase Gradient Program
















Time (min)
% Water containing 0.04% acetic acid
% methanol




















0
40
60



8
25
75



10
25
75



11
40
60



15
40
60










The HPLC assay results are provided below:











Internal
Steviol glycoside conversion in reaction mixture (% area)










reference 
Reb D
Reb UNK
Reb A













S115N01 A1
2.1
ND
96.7


S115N01 G2
0.6
ND
99.4


S115N05 A7
22.4
23.3
46.7


S115N06 E1
0.14
7.0
92.8


S115N06 C2
0.28
3.9
95.8









The enzyme S115N05 A7 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 FIGS. 42-46.
The below table accompanies FIG. 42.












Sample: 12400 S115N01A1 T24h 13062 lABA


gi|115454819|ref|NP_001051010.1| Os03g0702500 


[Oryza saliva Japonica Group]


Filename: 130621_12400_011.dat





CAD Ch 1 Results









Compound
Retention time
Integration (area)





Rebaudioside D
5.797
 13,532,277


Unknown@RT7.890
7.890
 7,094,778


Rebaudioside A
9.157
613,483,011


Total

634,110,066









The below table accompanies FIG. 43.















Sample: 12400 S115NOIG2 T24h 130621ABA



>gi|187373030|gb|ACD03249.1| UDP-



glycosyltransferase [Avena 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










The below table accompanies FIG. 44.












Sample: 12400 S115N05A7 T24h 130627ABA


>gi|460409128|ref|XP_004249992.1| PREDICTED: 


cyanidin-3-O-glucoside 2-O-glucuronosyltransferase-


like [Solanum lycopersicum]


Filename: 130627_12400_027.dat





CAD Ch 1 Results









Compound
Retention time
Integration (area)





Unknown@RT4.508
4.508
 64,361,822


Rebaudioside D
5.761
191,273,935


Rebaudioside UNK
6.685
198,934,644


Rebausioside A
9.106
398,115,681


Total

852,686,082









The below table accompanies FIG. 45.












Sample: 12400 S115N06E1 T24h 130627ABA


>gi|222619587|gb|EEE55719.1| hypothetical


protein OsJ_04191 (Oryza saliva 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
9.113
606,312,523


Total

653,304,599









The below table accompanies FIG. 46.












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
EUGT11 gene as was described in the Patent application WO/2013/022989A2 was synthesized by DNA2.0 and subsequently subcloned in pET30a+ vector.




















Conversion


Micro-

GI 

Internal 
RebA to 


plate
Position
number
Version
reference
RebD







C912666
G4
41469452
AAS07253.1
S115N08 G4
Active









The amino-acid sequence is as follows:







SEQ ID 8


>gi|41469452|gb|AAS07253.1| putative UDP-gluco-


ronosyl and UDP-glucosyl transferase [Oryza sativa


Japonica Group] EUGT11 enzyme from patent


application WO/2013/022989A2


MHVVICPLLAFGHLLPCLDLAQRLACGHRVSFVSTPRNISRLPPVRPSLA


 


PLVSFVALPLPRVEGLPNGAESTHNVPHDRPDMVELHLRAFDGLAAPFSE


 


FLGTACADWVMPTSSAPRQTLSSNIHRNSSRPGTPAPSGRLLCPITPHSN


 


TLERAAEKLVRSSRQNARARSLLAFTSPPLPYRDVFRSLLGLQMGRKQLN


 


IAHETNGRRTGTLPLNLCRWMWKQRRCGKLRPSDVEFNTSRSNEAISPIG


 


ASLVNLQSIQSPNPRAVLPIASSGVRAVFIGRARTSTPTPPHAKPARSAA


 


PRAHRPPSSVMDSGYSSSYAAAAGMHVVICPWLAFGHLLPCLDLAQRLAS


 


RGHRVSFVSTPRNISRLPPVRPALAPLVAFVALPLPRVEGLPDGAESTND


 


VPHDRPDMVELHRRAFDGLAAPFSEFLGTACADWVIVDVFHHWAAAAALE


 


HKVPCAMMLLGSAHMIASIADRRLERAETESPAAAGQGRPAAAPTFEVAR


 


MKLIRTKGSSGMSLAERFSLTLSRSSLVVGRSCVEFEPETVPLLSTLRGK


 


PITFLGLMPPLHEGRREDGEDATVRWLDAQPAKSVVYVALGSEVPLGVEK


 


VHELALGLELAGTRFLWALRKPTGVSDADLLPAGFEERTRGRGVVATRWV


 


PQMSILAHAAVGAFLTHCGWNSTIEGLMFGHPLIMLPIFGDQGPNARLIE


 


AKNAGLQVARNDGDGSFDREGVAAAIRAVAVEEESSKVFQAKAKKLQEIV


 


ADMACHERYIDGFIQQLRSYKD






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 μL 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 μL of suspension. Plasmid quantity was determined as follows:













Microtiter plate
Position
Internal reference of test
[Plasmid] ng/μL







C912666
G4
S115N08 G4
19.2









Transformation of Competent Cells with Plasmid.
An aliquot of chemically competent EC100 cells was taken from freezer at −80° C. and stored on ice. The cells were allowed to thaw on ice for 10 minutes. 10 μL 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 μL 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 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 μL of a 60% sterile glycerol solution, 600 μL 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:













Microtiter plate
Position
Internal reference of test
[Plasmid] ng/μL







C912666
G4
S115N08 G4
38.4









In-Vitro Expression of EUGT11.
18 μL 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: L1110) kit. The expression medium was produced as follows:















S30 Premix Plus
T7 S30 Extract
DNA template
Total







Trials
30 μL
27 μL
18 μL (~1.8 μg)
75 μL


reference 
20 μL
18 μL
12 μL (~1.0 μg)
50 μL









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 μL 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 μL of a reaction mixture containing 0.5 mM Rebaudioside A, 3 mM MgCl2, 50 mM phosphate buffer (pH7.2) and 2.5 mM UDP-glucose was added to a 1.5 mL sterile microtube. 52 μL of the enzyme expression medium was added and the resulting mixture was allowed to react at 30° C. for 24 hours. 125 μL samples were taken after 2 hours, 16 hours and 24 hours and added to a 115 μL of 60% methanol and 10 μL of 2 N H2SO4. The quenched sample was centrifuged at 18,000 g for 2 minutes at RT. 200 μL was transferred to HPLC vial and analyzed as seen in FIG. 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 a 


and UDP-glucosyl transferase [Oryza sativ Japonica Group] 


(EUGT11 enzyme from patent application WO/2013/022989A2)


Filename: 130702_12400_026.dat





CAD Ch 1 Results









Compound
Retention time
Integration (area)





Rebaudioside 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 μL 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 8 hrs.
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 40 hrs, the cells were harvested by centrifugation and frozen. The following yields of cell wet weights (CWW) are mentioned below.












GI number
Version
CWW







115454819
NP_001051010.1
9.2 g


187373030
ACD03249.1
7.4 g


460409128
XP_004249992.1
6.8 g


222619587
EEE55719.1
7.5 g


297795735
XP_002865752.1
8.8 g









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 μL 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 FIGS. 48-52.
HPLC Analysis.
The HPLC assay was performed as described in EXAMPLE 20.
The results for the different enzymes are provided below.















Conversion after 



GI Number
Version
45 hrs. 
Reb D selectivity


















115454819
NP_001051010.1
1.1%
 100%


187373030
ACD03249.1
0.8%
 100%


460409128
XP_004249992.1
62.1%
43.6%


222619587
EEE55719.1
2.9%
Reb D Not detected


297795735
XP_002865752.1
0.0%
Reb D Not detected









The below table accompanies FIG. 48.












SAMPLE: 12400 S129N01 T45h 130712ABA


>gi115454819 / NP_001051010.1


Filename: 130712_12400_003.dat





CAD Ch 1 Results









Compound
Retention time
Integration (area)





Rebaudioside D
5.775
3,264,475


Unknown@RT6.986
6.986
4,110,607


Unknown@RT7.330
7.330
564,033,104


Unknown@RT7.700
7.700
328,710,539


Unknown@RT8.158
8.158
6,344,796


Rebaudioside A
9.135
673,271,863


Unknown@RT9.653
9.653
616,489,141


Total

2,196,224,525









The below table accompanies FIG. 49.












Sample: 12400 S129N02 T45h 130712ABA


>gi187373030 / ACD03249.1


Filename: 130712_12400_004.dat





CAD Ch 1 Results









Compound
Retention time
Integration (area)





Rebaudioside D
5.772
1,997,401


Unknown@RT6.977
6.977
3,341,419


Unknown@RT7.252
7.252
10,576,676


Unknown@RT7.687
7.687
298,862,034


Rebaudioside A
9.117
675,210,845


Unknown@RT9.639
9.639
874,680,345


Total

1,864,668,720









The below table accompanies FIG. 50.












Sample: 12400 S129N04 T45h 130712ABA


>gi460409128 / XP_004249992.1


Filename: 130712_12400_006.dat





CAD Ch 1 Results









Compound
Retention time
Integration (area)





Unknown@RT4.526
4.526
89,809,997


Rebaudioside D
5.777
217,830,913


Rebaudioside UNK
6.711
192,129,243


Unknown@RT6.986
6.986
10,241,417


Unknown@RT7.331
7.331
41,195,765


Unknown@RT7.701
7.701
310,640,254


Unknown@RT8.162
8.162
7,950,609


Rebaudioside A
9.137
304,611,760


Unknown@RT9.656
9.656
863,496,704


Total

2,037,906,662









The below table accompanies FIG. 51.












Sample: 12400 S129N05 T45h 130712ABA


>gi222619587 / EEE55719.1


Filename: 130712_12400_007.dat





CAD Ch 1 Results









Compound
Retention time
Integration (area)





Rebaudioside UNK
6.708
  20,047,847


Unknown@RT6.999
6.999
  598,924,958


Unknown@RT7.699
7.699
  303,182,042


Rebaudioside A
9.133
  672,777,773


Unknown@RT9.655
9.655
  606,371,969


Total

2,201,304,589









The below table accompanies FIG. 52.












Sample: 12400 S129N06 T45h 130712ABA


>gi297795735 / XP_002865752.1


Filename: 130712_12400_008.dat





CAD Ch 1 Results









Compound
Retention time
Integration (area)





Unknown@RT6.998
6.998
  920,620,332


Unknown@RT7.696
7.696
  314,421,575


Rebaudioside A
9.128
  688,195,594


Unknown@RT9.645
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 S115N05A7 T24 h 130627ABA” of EXAMPLE 20 (hereinafter S115N05A7), 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 6110A MSD, and interfaced with “LC/MSD Chemstation” software, was used.
Instrument Conditions
















Column
Phenomenex Kinetex 2.6u C18 100A, 




4.6 mm × 150 mm, 2.6 μm



Column Temperature
55° C.



Detection
DAD at 210 nm bw 360 nm




MSD (Scan and SIM mode)




Mode: ES-API, Negative Polarity




Drying gas flow: 13.0 L/min




Nebulizer pressure: 30 psig




Drying gas temperature: 270° C.



Analysis duration 
25 min



Injected volume
2 μL



Flow rate
1 mL/min










Mobile Phase Gradient Program













Time (min)
A (%): Formic acid 0.1%
B (%): Acetonitrile

















0
75
25


8.5
75
25


10.0
71
29


16.5
70
30









The compound observed on LCMS system at 3.5 min, corresponds to compound “Unknown@4.508” in sample “S115N05A7” (EXAMPLE 20), and compound “Unknown@RT4.526” in sample “S129N04” (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 7.6 min, corresponds with compound “reb UNK” in sample “S115N05A7” (EXAMPLE 20), and compound “reb UNK” in sample “S129N04” (EXAMPLE 22), The LCMS data suggests that “reb UNK” has five glucosidic residues (C50H80O28) 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 FIGS. 53-54.












Steviol glycoside conversion in reaction mixture (% area)











Sample
Unknown@RT 3.5
Reb D
Reb UNK
Reb A





S115N05A7
6.47
20.35
19.93
53.24


S129N04
6.05
23.73
21.22
49.00









The below table accompanies FIG. 53.










Sample: 12400 S115N05A7 T24h 130627ABA


>gi|460409128/XP_004249992.1


MSD SIM Results










Compound
Retention time
MW
Integration (area)













Unknown@RT3.567
3.567
1,291
79,060


Rebaudioside D
5.654
1,129
248,604


Rebaudioside UNK
7.659
1,129
243,469


Rebausioside A
13.793
967
650,372


Total


1,221,505









The below table accompanies FIG. 54.










Sample: 12400 S129N04 T45h 130712ABA >


gi460409128/XP_004249992.1


MSD SIM Results










Compound
Retention time
MW
Integration (area)













Unknown@RT3.550
3.550
1,291
151,414


Rebaudioside D
5.633
1,129
593,709


Rebaudioside UNK
7.638
1,129
530,836


Rebaudioside A
13.782
967
1,225,970


Total


2,501,929









Example 24
Identification of Glycosides
The reaction mixture representing GI No. 460409128, particularly the sample “12400 S129N04 T45h 130712ABA” of EXAMPLE 22 (hereinafter S129N04) were additionally assayed by LC-MS, as seen in FIGS. 55-56, along with Stevia rebaudiana Bertoni leaf extract “MLD1” produced by PureCircle Sdn Bhd (Malaysia) to determine the occurrence of S129N04 glycosides in nature.
The below table accompanies FIG. 55.










Samples:


1) 12400 S129N04 T45h 130712ABA > 


gi460409128/XP_004249992.1


2) MLD1 Stevia rebaudiana Bertoni extract


MSD SIM 1,129 Results










Compound
MW







Rebaudioside D
1,129



Rebaudioside UNK
1,129










The below table accompanies FIG. 56.










Samples:


1) 12400 S129N04 T45h 130712ABA >


gi460409128/XP_004249992.1


2) MLD1 Stevia rebaudiana Bertoni extract


MSD SIM 1,291 Results










Compound
MW







Unknown@RT3.550
1,291



Rebaudioside M
1,291










The assay shows that the compound observed on LCMS system at 3.5 min, in EXAMPLE 23 (C56H90O33; later confirmed as reb M2), and the compound observed on LCMS system at 7.6 min, in EXAMPLE 23 (C50H80O28, 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 mM potassium phosphate buffer pH 7.5, 3 mM MgCl2, 2.5 mM UDP-glucose, 0.5 mM Rebaudioside E and 500 μL 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 μL of the reaction mixture was quenched with 30 μl of 2N H2SO4 and 270 μL 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 FIG. 57.
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 ProteinGPSTM 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 μL aliquots were stored at −20° C. and at −80° C.
These storage aliquots of E. coli BL21(DE3) containing the pET30a+_UGT76G1var 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 ExpressTM Instant TB medium (Novagen®), 10 g/L of glycerol and 50 mg/L of Kanamycin was inoculated with 50 μL 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 μL of fresh lysate that was added to a solution of Rebaudioside D (final concentration 0.5 mM), MgCl2 (final concentration 3 mM) and UDP-Glucose (final concentration 2.5 mM) in 50 mM phosphate buffer pH 7.2.
The reaction was 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.















conversion Reb D 





to Reb M
initial rate




after 24 h
(Reb M


Clone
Mutations*
(%)
area/min)


















UGT76G1var1
E224A_F314S_R334K
51.8
5.5E+07


UGT76G1var2
S274G_T284I_L379G
49.3
4.7E+07


UGT76G1var3
I295T_S357C_V366I
9.6
1.6E+06


UGT76G1var4
E224D_E231A_F265I
14.7
8.6E+06


UGT76G1var5
F22Y_I373L_P382M
3.5
2.3E+06


UGT76G1var6
Q266S_S357N_I373L
0.5
1.8E+06


UGT76G1var7
F22L_I43V_A239V
0.2
−6.0E+04  


UGT76G1var8
E224A_Q266S_Q342E
0.5
2.3E+04


UGT76G1var9
E231A_D301N_G348P
52.0
4.9E+07


UGT76G1var10
A33G_L246F_Q342E
0.3
−7.7E+02  


UGT76G1var11
F22L_A33G_V310I
0.4
3.8E+04


UGT76G1var12
L243P_K303G_A352G
0.5
8.7E+04


UGT76G1var13
L243A_S357C_A385T
0.2
−3.3E+04  


UGT76G1var14
A239I_F265I_V396F
5.3
1.5E+06


UGT76G1var15
F41L_L246F_Q425E
5.6
1.5E+06


UGT76G1var16
F265I_P272A_I335V
18.6
5.8E+06


UGT76G1var17
F265L_Q266E_Q342K
0.7
7.2E+05


UGT76G1var18
L243P_S274G_N409R
1.9
5.0E+05


UGT76G1var19
E224D_E229A_Q432E
10.5
5.5E+06


UGT76G1var20
S375M_K393G_Y397E
1.8
1.9E+06


UGT76G1var21
A239V_V300A_K303G
41.9
3.3E+07


UGT76G1var22
E231A_V310I_R334K
34.4
2.4E+07


UGT76G1var23
T263S_G348P_A352G
47.8
4.1E+07


UGT76G1var24
A239I_P272A_Q425E
31.0
2.1E+07


UGT76G1var25
T284L_Q342K_Y397Q
0.9
6.3E+04


UGT76G1var26
S241I_F265L_F377C
1.8
7.5E+05


UGT76G1var27
A239I_L379A_V394I
29.0
1.5E+07


UGT76G1var28
L243A_S274G_P382M
6.1
2.4E+06


UGT76G1var29
F22Y_V279I_N409R
41.0
2.9E+07


UGT76G1var30
I43V_E224A_S241I
13.6
5.6E+06


UGT76G1var31
E224D_L243P_V300A
0.4
2.4E+05


UGT76G1var32
A239V_L243A_S375M
0.0
−4.4E+04  


UGT76G1var33
A33G_R334H_Y397Q
1.0
7.5E+06


UGT76G1var34
I43V_T284I_I295T
3.4
1.5E+06


UGT76G1var35
T284L_F314S_S357N
0.5
1.8E+05


UGT76G1var36
F265L_L379A_V396F
20.0
8.8E+06


UGT76G1var37
E229A_L379G_I407V
39.1
2.8E+07


UGT76G1var38
F41L_I295M_F377C
8.2
3.7E+06


UGT76G1var39
F22Y_F41L_V366I
7.2
3.3E+06


UGT76G1var40
T263S_Q266E_S375R
47.6
3.3E+07


UGT76G1var41
L246F_A385T_K393G
0.8
1.4E+06


UGT76G1var42
T263S_Q266S_R334H
34.6
2.2E+07


UGT76G1var43
S241I_P272A_V279I
19.9
9.4E+06


UGT76G1var44
I335V_S375R_I407V
35.3
2.3E+07


UGT76G1var45
V279I_D301N_S389E
38.6
2.3E+07


UGT76G1var46
F22L_Q266E_I295M
0.6
9.8E+05


UGT76G1var47
E229A_T284I_S389E
4.8
2.7E+06


UGT76G1var48
V394I_Y397E_Q432E
47.6
3.8E+07


UGT76G1var49
F41L_Q266E_T284I_Y397Q
2.6
1.1E+06


UGT76G1var50
F22Y_V310I_S375M_F377C
1.9
−7.9E+05  


UGT76G1var51
K303G_S357C_S389E_V396F
18.7
9.5E+06


UGT76G1var52
D301N_I373L_F377C_I407V
12.9
4.6E+06


UGT76G1var53
R334K_A352G_P382M_S389E
9.3
4.1E+06


UGT76G1var54
E229A_T284L_R334K_Q342E
0.7
4.3E+05


UGT76G1var55
I295M_Q342E_V366I_N409R
1.0
2.2E+05


UGT76G1var56
L246F_A352G_S357N_Q432E
0.4
4.1E+04


UGT76G1var57
S241I_T263S_L379G_A385T
0.8
1.5E+05


UGT76G1var58
S357C_S375M_N409R_Q425E
7.5
2.2E+06


UGT76G1var59
I335V_K393G_V394I_Y397Q
33.0
2.7E+07


UGT76G1var60
E231A_L243A_V279I_S357N
0.5
9.5E+04


UGT76G1var61
I43V_F265I_Q266S_L379A
6.4
2.0E+06


UGT76G1var62
L243P_P272A_V394I_V396F
0.1
3.4E+04


UGT76G1var63
F314S_R334H_Q342K_L379G
3.4
1.2E+06


UGT76G1var64
F22L_A239I_R334H_I407V
0.3
3.1E+04


UGT76G1var65
A33G_A239V_P382M_Q425E
1.2
3.3E+05


UGT76G1var66
F265L_V310I_V366I_A385T
0.8
3.7E+05


UGT76G1var67
E224D_F314S_S375R_Y397E
−2.1
−5.6E+05  


UGT76G1var68
Q342K_G348P_I373L_Y397E
−1.4
−1.1E+05  


UGT76G1var69
S274G_I295T_I335V_L379A
24.7
8.3E+06


UGT76G1var70
E224A_I295T_V300A_G348P
24.0
8.4E+06


UGT76G1var71
I295M_V300A_K393G_Q432E
42.9
2.1E+07


UGT76G1var72
T284L_D301N_K303G_S375R
19.2
9.1E+06


UGT76G1var73
F22Y_D301N_R334H_Q342E_V396F
0.8
8.7E+05


UGT76G1var74
I295T_I373L_S375R_Y397Q_Q432E
0.6
9.6E+04


UGT76G1var75
F41L_A239I_Q266S_S375M_P382M
0.8
−1.3E+05  


UGT76G1var76
F22Y_A239I_L246F_I295M_R334K
2.6
7.2E+05


UGT76G1var77
A239V_F265I_I295T_D301N_K393G
1.9
4.4E+05


UGT76G1var78
V279I_V300A_V310I_I335V_S357C
3.2
8.2E+05


UGT76G1var79
E224D_T284I_V366I_I373L_K393G
8.5
3.8E+06


UGT76G1var80
L243P_L379A_S389E_Q425E_Q432E
1.0
2.1E+05


UGT76G1var81
A33G_T263S_S274G_V279I_Y397E
15.0
6.5E+06


UGT76G1var82
E224D_L243A_F265L_R334H_A352G
1.1
2.5E+05


UGT76G1var83
I43V_Q342E_S357N_S375R_L379G
0.5
4.3E+04


UGT76G1var84
F22L_Q266S_F314S_A352G_S357C
1.2
2.3E+05


UGT76G1var85
T284L_G348P_F377C_P382M_N409R
1.8
4.0E+05


UGT76G1var86
E224A_T284L_V396F_Y397E_I407V
1.6
3.8E+05


UGT76G1var87
S241I_L243A_V300A_F314S_N409R
35.7
2.1E+07


UGT76G1var88
A239V_T284I_V310I_Q342K_L379A
1.6
3.8E+05


UGT76G1var89
F41L_E229A_E231A_F265L_P272A
1.2
2.1E+05


UGT76G1var90
E231A_S241I_S274G_Y397Q_Q425E
34.5
1.9E+07


UGT76G1var91
E224A_L246F_T263S_F265I_Q342K
1.2
2.3E+05


UGT76G1var92
K303G_S357N_V366I_V394I_I407V
1.6
3.6E+05


UGT76G1var93
I43V_Q266E_S375M_S389E_V394I
1.8
4.5E+05


UGT76G1var94
Q266E_P272A_R334K_G348P_L379G
72.0
7.9E+07


UGT76G1var95
A33G_I295M_K303G_I335V_A385T
−1.3
−1.7E+05  


UGT76G1var96
F22L_E229A_L243P_F377C_A385T
1.2
2.7E+05





*Mutations are noted as follows: original amino acid-position-new amino acid: For example the mutation of an alanine at position 33 to a glycine is noted as A33G.






Example 27
In-Vivo Production of UGTSL2







SEQ ID 9


UGTSL2 (GI_460410132 / XP_004250485.1) amino acid


sequence:


MATNLRVLMFPWLAYGHISPFLNIAKQLADRGFLIYLCSTRINLESIIKK


 


IPEKYADSIHLIELQLPELPELPPHYHTTNGLPPHLNPTLHKALKMSKPN


 


FSRILQNLKPDLLIYDVLQPWAEHVANEQNIPAGKLLTSCAAVFSYFFSF


 


RKNPGVEFPFPAIHLPEVEKVKIREILAKEPEEGGRLDEGNKQMMLMCTS


 


RTIEAKYIDYCTELCNWKVVPVGPPFQDLITNDADNKELIDWLGTKHENS


 


TVFVSFGSEYFLSKEDMEEVAFALELSNVNFIWVARFPKGEERNLEDALP


 


KGFLERIGERGRVLDKFAPQPRILNHPSTGGFISHCGWNSAMESIDFGVP


 


IIAMPIHNDQPINAKLMVELGVAVEIVRDDDGKIHRGEIAETLKSVVTGE


 


TGEILRAKVREISKNLKSIRDEEMDAVAEELIQLCRNSNKSK






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 μL 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 μL 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 FIGS. 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.

















Stevioside 



Enzyme


conv.1
Rebaudioside


internal


(reaction 
E


reference
GI Number
Version
time)
formation1



















UGTSL
460409128
XP_004249992.1
74% (22 h.)
46%


UGTSL2
460410132
XP_004250485.1
77% (2 h.) 
50%





Note:


1Based on initial concentration of Stevioside






The below table accompanies FIG. 58.










SAMPLE: 12400 S151N33 T22h 130926ABA


Gene references: UGTSL (XP_004249992.1)


Filename: 130926_12400_042.dat


CAD Ch 1 Results









Compound
Retention time
Integration (area)












Unknown@RT4.27
4.270
45,634,692


Rebaudioside E
5.398
215,079,800


Unknown@RT6.79
6.790
11,0326,212


Unknown@RT7.32
7.320
33,855,010


Unknown@RT7.69
7.689
271,186,269


Unknown@RT8.18
8.178
6,003,490


Unknown@RT8.78
8.779
20,739,231


Stevioside
9.201
114,734,548


Unknown@RT9.65
9.648
779,225,521


Total

1,596,784,773









The below table accompanies FIG. 59.










SAMPLE: 12400 S151N26 T2h 130927ABA


Gene references: UGTSL2 (XP_004250485.1)


Filename: 130927_12400_093.dat


CAD Ch 1 Results









Compound
Retention time
Integration (area)












Unknown@RT3.84
3.841
16,182,482


Unknown@RT4.25
4.255
20,078,830


Unknown@RT4.91
4.910
27,630,795


Rebaudioside E
5.389
203,768,956


Unknown@RT5.75
5.751
8,018,638


Unknown@RT6.82
6.817
200,959,602


Unknown@RT7.31
7.310
370188401


Unknown@RT7.68
7.680
294,963,186


Stevioside
9.186
101,729,292


Unknown@RT9.63
9.635
727,903,255


Total

1,971,423,437









Example 29
Determination of Activity for Rubusoside 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 μL 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 FIGS. 60-61. The HPLC assay was performed as described in EXAMPLE 20.
The results for the different enzymes and the corresponding chromatograms are provided below.

















Rubusoside 



Enzyme


conv.1
Rebaudioside 


internal


(reaction 
E


reference
GI Number
Version
time)
formation1



















UGTSL
460409128
XP_004249992.1
70% (45 h.)
27%


UGTSL2
460410132
XP_004250485.1 
80% (2 h.) 
55%





Note:


1Based on initial concentration of Rubusoside






The below table accompanies FIG. 60.










SAMPLE: 12400 S151N22 T45h 130927ABA


Gene references: UGTSL (XP_004249992.1)


Filename: 130927_12400_092.dat


CAD Ch 1 Results









Compound
Retention time
Integration (area)












Rebaudioside E
5.499
135,984,743


Unknown@RT7.03
7.027
54,448,761


Unknown@RT7.30
7.302
41,308,528


Unknown@RT7.68
7.682
283,852,603


Unknown@RT8.14
8.145
5,484,731


Unknown@RT8.74
8.742
290,946,055


Stevioside
9.178
8,774,098


Unknown@RT9.64
9.637
761,299,117


Unknown@RT10.54
10.542
18,276,224


Rubusoside
11.233
155,492,389


Total

1,755,867,249









The below table accompanies FIG. 61.










SAMPLE: 12400 S151N15 T2h 130927ABA


Gene references: UGTSL2 (XP_004250485.1)


Filename: 130927_12400_080.dat


CAD Ch 1 Results









Compound
Retention time
Integration (area)












Unknown@RT5.14
5.138
5,555,472


Rebaudioside E
5.505
278,529,547


Unknown@RT6.64
6.643
23,812,633


Unknown@RT7.01
7.012
84,543,823


Unknown@RT7.31
7.315
283,724,517


Unknown@RT7.69
7.687
264,400,008


Unknown@RT8.78
8.767
188,634,123


Stevioside
9.193
9,365,107


Unknown@RT9.64
9.643
700,878,865


Rubusoside
11.238
102,484,386


Totals

1,941,928,481









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 μL 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 FIG. 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.

















Rebaudioside 



Enzyme


A conv.1
Rebaudioside 


internal


(reaction 
D


reference
GI Number
Version
time)
formation1







UGTSL2
460410132
XP_004250485.1
78% (23 h.)
75%





Note:


1Based on initial concentration of Rebaudioside A






The below table accompanies FIG. 62.










SAMPLE: 12400 S154N14 T23h 131003ABA


Gene references: UGTSL2 (XP_004250485.1)


Filename: 131003_12400_086.dat


CAD Ch 1 Results









Compound
Retention time
Integration (area)












Unknown@RT4.53
4.530
55,894,278


Rebaudioside D
5.788
461,768,318


Unknown@RT6.71
6.713
7,942,480


Unknown@RT6.99
6.993
11,192,896


Unknown@RT7.33
7.327
120,255,606


Unknown@RT7.70
7.700
38,994,186


Rebaudioside A
9.140
137,037,966


Unknown@RT9.65
9.652
314,468,535


Total

1,147,554,265









Example 31
Identification of Glycosides
The reaction mixtures prepared according to EXAMPLE 30 and incubated for 45 hrs was analyzed by LC-MS, along with Stevia 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 6110A MSD, and interfaced with “LC/MSD Chemstation” software, was used, and the chromatogram is shown in FIG. 63.
Instrument Conditions










Instrument conditions


















Column
Phenomenex Prodigy 3u C18 100A, 




4.6 mm × 250 mm, 3 μm



Column Temperature
55° C.



Detection
DAD at 210 nm bw 360 nm




MSD (Scan and SIM mode)




Mode: ES-API, Negative Polarity




Drying gas flow: 13.0 L/min




Nebulizer pressure: 30 psig




Drying gas temperature: 270° C.



Analysis duration
75 min



Injected volume
10 μL



Flow rate
0.5 mL/min










Mobile Phase Gradient Program













Time (min)
A (%): Formic acid 0.1%
B (%): Acetonitrile







 0
75
25


30
75
25


33
68
32


75
68
32









The assay shows that the compound observed on LC-MS system at 11.77 min is the same as the compound at 3.5 min, in EXAMPLE 23 (C56H90O33; later confirmed as reb M2), and the compound observed at 26.64 min is the same as the compound at 7.6 min, in EXAMPLE 23 (C50H80O28, reb UNK; later confirmed as reb D2). Other isomers of reb M were observed at 13.96 min and also another isomer form of reb D was observed at 25.06 min. All observed compounds occurred in the extract of Stevia rebaudiana Bertoni plant.
The below table accompanies FIG. 63.










SAMPLE: UGTSL2 T45h


Gene references: UGTSL2 (XP_004250485.1)


MSD SIM Results











Compound
Retention time
MW















Unknown@RT11.77
11.775
1,291



Unknown@RT13.96
13.965
1,291



Rebaudioside D
19.598
1,129



Unknown@RT25.06
25.061
1,129



Unknown@RT26.64
26.637
1,129



Rebausioside A
52.258
967










Example 32
In Vivo Preparation and Activity Determination of UGTLB







SEQ ID 10


UGTLB (GI_209954733 / BAG80557.1) amino acid


sequence


MGTEVTVHKNTLRVLMFPWLAYGHISPFLNVAKKLVDRGFLIYLCSTAIN


 


LKSTIKKIPEKYSDSIQLIELHLPELPELPPHYHTTNGLPPHLNHTLQKA


 


LKMSKPNFSKILQNLKPDLVIYDLLQQWAEGVANEQNIPAVKLLTSGAAV


 


LSYFFNLVKKPGVEFPFPAIYLRKNELEKMSELLAQSAKDKEPDGVDPFA


 


DGNMQVMLMSTSRIIEAKYIDYFSGLSNWKVVPVGPPVQDPIADDADEME


 


LIDWLGKKDENSTVFVSFGSEYFLSKEDREEIAFGLELSNVNFIWVARFP


 


KGEEQNLEDALPKGFLERIGDRGRVLDKFAPQPRILNHPSTGGFISHCGW


 


NSVMESVDFGVPIIAMPIHLDQPMNARLIVELGVAVEIVRDDYGKIHREE


 


IAEILKDVIAGKSGENLKAKMRDISKNLKSIRDEEMDTAAEELIQLCKNS


 


PKLK






The pET30A+ vector containing the UGTLB gene 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 cryoprotecteur and 400 μL 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 μL 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 FIGS. 64-66. The corresponding chromatograms are depicted in FIGS. 64-66.

















Stevioside 



Enzyme 


conv.1
Rebaudioside 


internal


(reaction 
E


reference
GI Number
Version
time)
formation1







UGTLB
209954733
BAG80557.1
89% (22 h.)
3%





Note:


1Based on initial concentration of Stevioside






The below table accompanies FIG. 64.










SAMPLE: 12400 S151N28 T22h 130926ABA


Gene references: UGTLB (BAG80557.1)


Filename: 130926_12400_037.dat


CAD Ch 1 Results











Compound
Retention time
Integration (area)















Unknown@4.27
4.270
101,580,340



Unknown@4.88
4.884
2,979,482



Rebaudioside E
5.397
13,747,837



Unknown@6.80
6.796
378,936,196



Unknown@7.32
7.319
54,838,779



Unknown@7.69
7.693
291,189,747



Unknown@8.78
8.784
21,079,018



Stevioside
9.200
50,143,248



Unknown@9.65
9.650
888,211,556



Unknown@10.70
10.697
5,878,160



Totals

1,808,584,363










Determination of Activity for Rubusoside to Rebaudioside E Conversion with UGTLB

Activity tests were performed at 3 mL scale with 600 μL 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 chromatogram is depicted in FIG. 65.

















Rubusoside 



Enzyme 


conv.1
Rebaudioside 


internal


(reaction 
E


reference
GI Number
Version
time)
formation1







UGTLB
209954733
BAG80557.1
65% (5 h.)
4%





Note:


1Based on initial concentration of Rubusoside






The below table accompanies FIG. 65.










SAMPLE: 12400 S151N17 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



Unknown@7.01
7.010
9764063



Unknown@7.29
7.295
12510947



Unknown@7.68
7.677
283386906



Unknown@8.73
8.728
402240506



Unknown@9.63
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 μL 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 in FIG. 66.

















Rebaudioside 



Enzyme 


A
Rebaudioside


internal


conv.1 
D


reference
GI Number
Version
(reaction time)
formation1







UGTLB
209954733
BAG80557.1
72% (22 h.)
10%





Note:


1Based on initial concentration of Rebaudioside A






The below table accompanies FIG. 66.










SAMPLE: 12400 S154N6 T22h 130926ABA


Gene references: UGTLB (BAG80557.1)


Filename: 130926_12400_015.dat


CAD Ch 1 Results











Compound
Retention time
Integration (area)















Unknown@4.42
4.522
137,916,950



Unknown@4.90
4.903
2,015,271



Rebaudioside D
5.762
59,876,764



Unknown@6.69
6.689
364,185,331



Unknown@6.97
6.973
26,368,965



Unknown@7.32
7.318
110,284,197



Unknown@7.69
7.689
294,579,799



Unknown@8.29
8.293
7,867,452



Unknown@8.78
8.779
15,928,550



Rebausioside A
9.118
165,602,247



Unknown@9.64
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 products











LC-MS, peak area ratio (%)





















Unknown peak 
Unknown peak
Unknown peak


Sample
UGT




#1 (MW804)
#2 (MW804)
#3 (MW804)


ID
(reaction time)
Rub
Stev
Reb E
Reb D
RT 30.70 min
RT 49.50 min
RT 50.40 min


















S151N15
UGTSL2 (2 hrs)
3.54
2.12
52.88
6.73
12.02
9.94
12.77


S151N17
UGTLB (5 hrs)
13.49
ND
9.21
1.29
4.07
66.67
5.27


S151N22
UGTSL (45 hrs)
7.82
2.37
35.88
3.45
20.38
27.75
2.35



















Stevioside conversion products











LC-MS, peak area ratio (%)



















Unknown peak 
Unknown peak
Unknown peak


Sample 
UGT 



#1 (MW966)
#2 (MW966)
#3 (MW966)


ID
(reaction time)
Stev
Reb E
Reb D
RT = 22.60 min
RT = 26.50 min
RT = 29.50 min

















S151N26
UGTSL2 (2 hrs)
20.01
42.56
1.70
4.48
5.56
25.70


S151N28
UGTLB (2 hrs) 
43.11
3.12
ND
ND
53.78
ND


S151N33
UGTSL (22 hrs)
25.24
49.68
0.54
3.97
20.56
ND









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 11


UGT76G1 [Stevia rebaudiana] (gi_37993653 /gb_


AAR06912.1)


MENKTETTVRRRRRIILFPVPFQGHINPILQLANVLYSKGFSITIFHTNF


 


NKPKTSNYPHFTFRFILDNDPQDERISNLPTHGPLAGMRIPIINEHGADE


 


LRRELELLMLASEEDEEVSCLITDALWYFAQSVADSLNLRRLVLMTSSLF


 


NFHAHVSLPQFDELGYLDPDDKTRLEEQASGFPMLKVKDIKSAYSNWQIL


 


KEILGKMIKQTKASSGVIWNSFKELEESELETVIREIPAPSFLIPLPKHL


 


TASSSSLLDHDRTVFQWLDQQPPSSVLYVSFGSTSEVDEKDFLEIARGLV


 


DSKQSFLWVVRPGFVKGSTWVEPLPDGFLGERGRIVKWVPQQEVLAHGAI


 


GAFWTHSGWNSTLESVCEGVPMIFSDFGLDQPLNARYMSDVLKVGVYLEN


 


GWERGEIANAIRRVMVDEEGEYIRQNARVLKQKADVSLMKGGSSYESLES


 


LVSYISSL






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_UGT76G1_Sc vector was used to transform chemically competent S. cerevisiae INVSc1 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 19 h30 at 30° C.
Cells were obtained by centrifugation and lysis with five volumes of CelLyticTM Y Cell Lysis Reagent (Sigma). The lysates were used directly for activity testing (UGT76G1_Sc).
Example 35
Determination of Activity of UGT76G1_Sc for the Conversion of Rebaudioside D to Rebaudioside M
UGT76G1_Sc was prepared according to EXAMPLE 34. Activity tests were performed at 2 mL scale with 200 μL 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 FIG. 67. The corresponding chromatogram is depicted in FIG. 67.















Enzyme 
Rebaudioside D 




internal
conv.1
Rebaudioside



reference
(reaction time)
M selectivity1









UGT76G1_Sc
85% (21 h.)
100%







Note:



1Based on initial concentration of Rebaudioside D






The below table accompanies FIG. 67.










SAMPLE: 12400 S169N10 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



Unknown@6.23
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@11.31
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


MSPKLHKELFFHSLYKKTRSNHTMATLKVLMFPFLAYGHISPYLNVAKKL


 


ADRGFLIYFCSTPINLKSTIEKIPEKYADSIHLIELHLPELPQLPPHYHT


 


TNGLPPNLNQVLQKALKMSKPNFSKILQNLKPDLVIYDILQRWAKHVANE


 


QNIPAVKLLTSGAAVFSYFFNVLKKPGVEFPFPGIYLRKIEQVRLSEMMS


 


KSDKEKELEDDDDDDDLLVDGNMQIMLMSTSRTIEAKYIDFCTALTNWKV


 


VPVGPPVQDLITNDVDDMELIDWLGTKDENSTVFVSFGSEYFLSKEDMEE


 


VAFALELSNVNFIWVARFPKGEERNLEDALPKGFLERIGERGRVLDKFAP


 


QPRILNHPSTGGFISHCGWNSAMESIDFGVPIIAMPMHLDQPMNARLIVE


 


LGVAVEIVRDDDGKIHRGEIAETLKGVITGKTGEKLRAKVRDISKNLKTI


 


RDEEMDAAAEELIQLCRNGN






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_UGTSL_Sc vector was used to transform chemically competent S. cerevisiae INVSc1 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 19 h30 at 30° C.
Cells were obtained by centrifugation and lysis with five volumes of CelLyticTM 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 μL 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 FIG. 68. The corresponding chromatogram is depicted in FIG. 68.















Enzyme 
Rebaudioside A 




internal 
conv.1 
Rebaudioside 



reference
(reaction time)
D selectivity1









UGTSL_Sc
46% (4 h)
42%







Note:



1Based on initial concentration of Rebaudioside A






The below table accompanies FIG. 68.










SAMPLE: 12400 S169N02 T4h 131119CJA


Gene references: UGTSL_Sc


Filename: 131122_12400_203.dat


CAD Ch 1 Results











Compound
Retention time
Integration (area)















Unknown@4.50
4.500
75,046,986



Rebaudioside D
5.731
223,409,643



Unknown@6.66
6.658
228,651,278



Rebaudioside A
9.084
404,642,305



Unknown@10.08
10.079
43,992,253



Unknown@11.21
11.211
29,776,761



Unknown@11.90
11.905
2,185,316



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 M 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 M is shown below:










1H and 13C NMR spectral data for Rebaudioside M in C5D5N a-c.











Position
13C NMR
1H NMR















1
40.3
0.75 t (13.2)





1.76 m



2
19.6
1.35 m





2.24 m



3
38.4
1.01 m





2.30 d (13.3)



4
44.3
—



5
57.4
1.06 d (12.8)



6
23.5
2.23 m





2.41 q (13.2)



7
42.6
1.41 m





1.80 m



8
41.2
—



9
54.3
0.91 d (7.7)



10
39.7
—



11
20.2
1.65 m





1.75 m



12
38.5
1.86 m





2.73 m



13
87.6
—



14
43.3
2.02 m





2.74 m



15
46.5
1.88 d (16.4)





2.03 m



16
153.3
—



17
104.9
4.90 s





5.69 s



18
28.2
1.32 s



19
176.9
—



20
16.8
1.38 s



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



6′
61.8
4.20 m





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



6′′
62.6
4.19 m





4.32 m



1′′′
104.8
5.48 d (7.7)



2′′′
75.8
4.15 m



3′′′
78.6
4.13 m



4′′′
73.2
3.98 m



5′′′
77.6
3.74 ddd (2.8, 6.4, 9.9)



6′′′
64.0
4.27 m





4.51 m



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



6′′′′
62.1
4.20 m





4.32 m



1′′′′′
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)



6′′′′′
64.0
4.32 m





4.64 d (10.3)



1′′′′′′
104.1
5.31 d (8.0)



2′′′′′′
75.5
3.95 m



3′′′′′′
78.0
4.37 t (9.1)



4′′′′′′
71.1
4.10 m



5′′′′′′
78.1
3.85 ddd (1.7, 6.1, 9.9)



6′′′′′′
62.1
4.10 m





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 M 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, 4.6×250 mm, 4 μm (p/n 00G-4375-E0); Column Temp: 55° C.; Mobile Phase A: 0.0284% ammonium acetate (NH4OAc) and 0.0116% acetic acid (HOAc) in water; Mobile Phase B: Acetonitrile (MeCN); Flow Rate: 1.0 mL/min; Injection volume: 10 μL. Detection was by UV (210 nm) and CAD.
Gradient:












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









Analyses of semi-preparative purification fractions were performed with the following method: Waters Atlantis dC18, 4.6×100 mm, 5 μm (p/n 186001340); Mobile Phase A: 25% MeCN in water; Mobile Phase B: 30% MeCN in water; Flow Rate: 1.0 mL/min; Injection volume: 10 μL. Detection was by CAD.
Gradient:












Time (min)
% A
% B

















0.0-5.0
100
0


20
20
80


25
20
80


30
100
0









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, 4.6×250 mm, 4 μm (p/n 00G-4375-E0); Column Temp: 55° C.; Mobile Phase A: 0.0284% NH4OAc and 0.0116% HOAc in water; Mobile Phase B: Acetonitrile; Flow Rate: 1.0 mL/min; Injection volume: 10 μL. Detection was 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, 30×100 mm, 5 (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









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 C18, 50×250 mm, 7 μm (p/n WAT248000); Isocratic mobile phase: 50% methanol (MeOH) in water with 0.05% HOAc; Flow Rate: 85 mL/min; Injection load: 6 g crude extract dissolved in 50 mL 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, 50×250 mm, 7 μm (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, 50×250 mm, 7 μm (p/n WAT248000); Isocratic mobile phase: 20% MeCN in water; Flow Rate: 100 mL/min; Injection load: 0.5 g secondary 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 H2O: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.
NMR.
The sample was prepared by dissolving 1-2 mg in 150 μL of pyridine-d5 and NMR data were acquired on a Bruker Avance 500 MHz instrument with a 2.5 mm inverse detection probe. The NMR spectrum was referenced to the residual solvent signal (δH 8.74 and δC 150.35 for pyridine-d5).
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 FIG. 11. 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 1127.6. 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 FIG. 12. From this semi-preparative collection, the compound was isolated with a purity>99% (CAD, AUC). The fraction analysis is provided in FIG. 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 1127.4709. The mass of the [M-H]− ion was in good agreement with the molecular formula C50H80O28 (calcd for C50H79O28: 1127.4758, error: −4.3 ppm). The MS data confirmed a nominal mass of 1128 Daltons with the molecular formula, C50H80O28.
The MS/MS spectrum (selecting the [M-H]− ion at m/z 1127.5 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 1H NMR (FIG. 14), 13C NMR (FIGS. 15 and 16), 1H-1H COSY (FIG. 17), HSQC-DEPT (FIG. 18), HMBC (FIGS. 19 and 20), NOESY (FIG. 21) and 1D-TOCSY (FIG. 22-26) were performed to allow assignment of the compound. In the 1H NMR acquired after  ̃46 hrs of sample preparation (FIGS. 27-28), the anomeric resonance at δH 5.04 is resolved which was obscured by the solvent (HOD) in the original spectrum (FIG. 14)
The 1H, 1H-1H COSY, 1H-13C HSQC-DEPT and 1H-13C HMBC NMR data indicated that the central core of the glycoside is a diterpene. The presence of five anomeric protons observed in the 1H and 1H-13C HSQC-DEPT spectra confirm five sugar units in the structure. The methylene 13C resonance at δC 69.9 in the 1H-13C HSQC-DEPT spectrum indicated the presence of a sugar linkage in the structure. The linkages of sugar units were assigned using 1H-13C HMBC and 1D-TOCSY correlations.
A HMBC correlation from the methyl protons at δH 1.29 to the carbonyl at δC 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 δC 38.9, 45.0, and 57.8 allowed assignment of C-3, C-4, and C-5. Analysis of the 1H-13C HSQC-DEPT data indicated that the carbon at δC 38.9 was a methylene group and the carbon at δC 57.8 was a methine which were assigned as C-3 and C-5, respectively. This left the carbon at δC 45.0, which did not show a correlation in the HSQC-DEPT spectrum, to be assigned as the quaternary carbon, C-4. The 1H chemical shifts for C-3 (δH 0.98 and 2j.36) and C-5 (δH 1.04) were assigned using the HSQC-DEPT data. A COSY correlation between one of the H-3 protons (δH 0.98) and a proton at δH 1.43 allowed assignment of one of the H-2 protons which in turn showed a correlation with a proton at δH 0.75 which was assigned to C-1. The remaining 1H 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.










1H and 13C NMR (500 and 125 MHz, pyridine-d5), Assignments of Reb D2.











Reb D2











Position
13C
1H















1
41.3
0.75 t (11.0)





1.76 m



2
19.9
1.43 m





2.20 m



3
38.9
0.98 m





2.36 d (12.1)



4
45.0
—



5
57.8
1.04 d (12.5)



6
22.7
1.92 m





2.43 m



7
42.2
1.22 m





1.30 m



8
43.1
—



9
54.5
0.88 brs



10
40.3
—



11
21.1
1.65 m





1.69 m



12
37.5
1.99 m





2.25 m



13
87.1
—



14
44.5
1.80 d (11.7)





2.65 d (11.7)



15
48.3
1.31 m





2.04 brs



16
154.7
—



17
105.2
5.01 s





5.64 s



18
28.8
1.29 s



19
177.7
—



20
16.0
1.30 s










The other tertiary methyl singlet, observed at δH 1.30 showed HMBC correlations to C-1 and C-5 and was assigned as C-20. The methyl protons showed additional HMBC correlations to a quaternary carbon (δC 40.3) and a methine carbon (δC 54.5) which were assigned as C-10 and C-9, respectively. COSY correlations between H-5 (δH 1.04) and protons at δH 1.92 and 2.43 then allowed assignment of the H-6 protons which in turn showed correlations to protons at δH 1.22 and 1.30 which were assigned to C-7. The 13C chemical shifts for C-6 (δC 22.7) and C-7 (δC 42.2) were then determined from the HSQC-DEPT data. COSY correlations between H-9 (δH 0.88) and protons at δH 1.65 and 1.69 allowed assignment of the H-11 protons which in turn showed COSY correlations to protons at δH 1.99 and 2.25 which were assigned as the H-12 protons. The HSQC-DEPT data was then used to assign C-11 (δC 21.1) and C-12 (δC 37.5). HMBC correlations from the H-12 proton (δH 2.25) to carbons at δC 87.1 and 154.7 allowed assignment of C-13 and C-16, respectively. The olefinic protons observed at δH 5.01 and 5.64 showed HMBC correlations to C-13 and were assigned to C-17 (δC 105.2 via HSQC-DEPT). The olefinic protons H-17 and the methine proton H-9 showed HMBC correlations to a carbon at δC 48.3 which was assigned as C-15. An additional HMBC correlation from H-9 to a methylene carbon at δC 44.5 then allowed assignment of C-14. The 1H chemical shifts at C-14 (δH 1.80 and 2.65) and C-15 δH 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-18 but NOE correlations were not observed between H-9 and H-14 indicating that H-5, H-9 and H-18 were on the opposite face of the rings compared to H-14 and H-20 as presented in FIG. 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 1H-13C HSQC-DEPT data confirmed the presence of five anomeric protons. Three of the anomeric protons were well resolved at δH 6.02 (δC 96.1), 5.57 (δC 105.3), and 5.34 (δC 105.3) in the 1H NMR spectrum. The remaining two anomeric protons observed at δH 5.04 (δC 105.6) and 5.07 (δC 98.7) which were obscured by solvent (HOD) resonance in the 1H spectrum were identified by 1H-13C HSQC-DEPT data. The anomeric proton observed at δH 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 δH 5.07 showed an HMBC correlation to C-13 allowing it to be assigned as the anomeric proton of GlcII.
The GlcI anomeric proton (δH 6.02) showed a COSY correlation to a proton at δH 4.07 was assigned as GlcI H-2 which in turn showed a COSY correlation to a proton at δH 4.22 (GlcI H-3) which showed a COSY correlation with a proton at δH 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 1D-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 1D-TOCSY data showed a proton at δH 4.04 assigned as GlcI H-5 and a proton at δH 4.68 assigned as one of the GlcI H-6 protons. The latter proton was also used for 1D-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 (δH 4.30). Assignment of the 13C chemical shifts for GlcI C-2 (δC 74.2), C-3 (δC 79.1), C-4 (δC 72.1), C-5 (δC 78.5), and C-6 (δC 69.9) was determined using the 1H-13C HSQC-DEPT data to complete the assignment of GlcI. Furthermore, the presence of a methylene 13C resonance at δC 69.9 in the 1H-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 GlcI on the basis of 1H-13C HSQC-DEPT, HMBC, and 1D-TOCSY correlations. The relatively downfield shift of a methylene 13C resonance of GlcI at δC 69.9 in the HSQC-DEPT spectrum indicated a 1→6 sugar linkage of GlcI. The anomeric proton observed at δH 5.04 showed HMBC correlation to GlcI C-6 and was assigned as the anomeric proton of GlcV. Similarly, methylene protons of GlcI showed HMBC correlations to anomeric carbon of GlcV confirming the presence of a 1→6 sugar linkage between GlcI and GlcV. The GlcV anomeric proton showed a COSY correlation to a proton at δH 4.00 which was assigned as GlcV H-2 which in turn showed a COSY correlation to a proton at δH 4.22 (GlcV H-3). Due to data overlap, the COSY spectrum did not allow assignment of GlcV H-4 based on the COSY correlation of GlcV H-3. However, in the HMBC spectrum, GlcV H-3 showed a correlation to GlcV C-5 (δC 78.9). In HSQC-DEPT spectrum, GlcV C-5 showed a correlation to δH 3.89 (GlcV H-5). The GlcV H-5 showed COSY correlations to δH 4.21, 4.37, and 4.48. In the HSQC-DEPT spectrum, δH 4.21 showed a correlation to δC 71.4 (GlcV H-4), while δH 4.37 and 4.48 showed a correlation to δC 63.1 and were assigned to GlcV H-6a and H-6b, respectively. Assignment of the 13C chemical shifts for GlcV C-2 (δC 75.7) and C-3 (δC 79.1) was determined using the 1H-13C HSQC-DEPT data to complete the assignment of GlcV.
A summary of the 1H 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-d5), Assignments of the reb D2 C-19 glycoside.









Reb D2











Position
13C
1H














GlcI-1
96.1
6.02 d (8.1)



GlcI-2
74.2
4.07 m



GlcI-3
79.1#
4.22 m#



GlcI-4
72.1
4.12 m



GlcI-5
78.5
4.04 m



GlcI-6
69.9
4.30 m





4.68 d (10.7)



GlcV-1
105.6
5.04 (8.1)



GlcV-2
75.7
4.00 m



GlcV-3
79.1#
4.22 m#



GlcV-4
71.4
4.21 m



GlcV-5
78.9
3.89 m



GlcV-6
63.1
4.37 m





4.48 m





#1H and 13C values can be exchangeable between positions Glc1-3, GlcV-3 and GlcIV-3.






A summary of the key HMBC, COSY, and 1D-TOCSY correlations used to assign the C-19 glycoside region are provided below.




Assignment of GlcII was carried out in a similar manner. The GlcII anomeric proton (δH 5.07) showed a COSY correlation to a proton at δH 4.37, assigned as GlcII H-2, which in turn showed a COSY correlation to a proton at δH 4.18 (GlcII H-3). This latter proton showed an additional correlation with a proton at δH 3.88 (GlcII H-4) which also showed a COSY correlation to a proton at δH 3.79 (GlcII H-5). GlcII H-5 also showed a COSY correlation to GlcII H-6 protons (δH 4.08 and 4.46). Assignment of the 13C chemical shifts for GlcII C-2 (δC 81.3), C-3 (δC 88.4), C-4 (δC 71.1), C-5 (δC 77.9), and C-6 (δC 63.2) was determined using the HSQC-DEPT data. HMBC correlations from GlcII H-3 to C-2 and C-4 and also from GlcII H-4 to C-2 and C-5 confirmed the assignments made above. Additional HMBC correlations of GlcII H-4 to GlcII C-6 further support to complete the assignment of GlcII.
Two of the remaining unassigned glucose moieties were assigned as substituents at C-2 and C-3 of GlcII on the basis of HMBC correlations. The anomeric proton observed at δH 5.57 showed a HMBC correlation to GlcII C-2 and was assigned as the anomeric proton of GlcIII. The anomeric proton observed at δH 5.34 showed a HMBC correlation to GlcII C-3 and was assigned as the anomeric proton of GlcIV. The reciprocal HMBC correlations from GlcII H-2 to the anomeric carbon of GlcIII and from GlcII H-3 to the anomeric carbon of GlcIV were also observed.
The anomeric proton of GlcIII (δH 5.57) showed a COSY correlation with a proton at δH 4.19 which was assigned as GlcIII H-2. Due to data overlap, the COSY spectrum did not allow assignment of H-3 to H-6 protons. Therefore, a series of 1D-TOCSY experiments were performed using selective irradiation of the GlcIII anomeric proton with several different mixing times. In addition to confirming the assignments for GlcIII H-2, the 1D-TOCSY data showed protons at δH 4.24 (GlcIII H-3), δH 4.27 (GlcIII H-4), and δH 3.94 (GlcIII 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, GlcIII H-4 showed a correlation to GlcIII H-5, which in turn showed COSY correlations to δH 4.41 and 4.50 of GlcIII H-6a and H-6b, respectively. The 13C chemical shifts for GlcIII C-2 (δC 76.8), C-3 (δC 78.9), C-4 (δC 72.4), C-5 (δC 78.8), and C-6 (δC 63.5) were then determined using the 1H-13C HSQC-DEPT correlations to complete the assignment of GlcIII.
The anomeric proton of GlcIV (δH 5.34) showed a COSY correlation with a proton at δH 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 1D-TOCSY experiments were performed using selective irradiation of the GlcIV anomeric proton with several different mixing times. In addition to confirming the assignments for GlcIV H-2, the 1D-TOCSY data showed protons at δH 4.22 (GlcIV H-3), δH 4.18 (GlcIV H-4), and δH 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 δH 4.32 and 4.58, GlcIV H-6a and H-6b, respectively. The 13C chemical shifts for GlcIV C-2 (δC 75.8), C-3 (δC 78.9), C-4 (δC 72.0), C-5 (δC 79.3), and C-6 (δC 62.9) were then determined using the 1H-13C HSQC-DEPT correlations to complete the assignment of GlcIV.
The large coupling constants observed for the anomeric protons of the glucose moieties at δH 6.02 (d, J=8.1 Hz), 5.57 (d, J=7.6 Hz), 5.34 (d, J=7.9 Hz) and δH 5.04 (d, J=8.1 Hz), suggested their β-orientation (FIGS. 14, 27, and 28). While the remaining anomeric proton at δH 5.07 was obscured by the solvent resonance (HDO) it's coupling constant (J= ̃8 Hz) evident from 1D TOCSY data (FIG. 24) also indicated β-orientation.
A summary of the 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.









Reb D2











Position
13C
1H














GlcII-1
98.7
5.07 (~8)*



GlcII-2
81.3
4.37 m



GlcII-3
88.4
4.18 m



GlcII-4
71.1
3.88 m



GlcII-5
77.9
3.79 m



GlcII-6
63.2
4.08 m





4.47 m



GlcIII-1
105.3
5.57 d (7.6)



GlcIII-2
76.8
4.19 m



GlcIII-3
78.9
4.24 m



GlcIII-4
72.4
4.27 m



GlcIII-5
78.8
3.94 m



GlcIII-6
63.5
4.41 m





4.50 m



GlcIV-1
105.3
5.34 d (7.9)



GlcIV-2
75.8
4.06 m



GlcIV-3
78.9#
4.22 m#



GlcIV-4
72.0
4.18 m



GlcIV-5
79.3
4.10 m



GlcIV-6
62.9
4.32 m





4.58 m





*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, GlcV-3 and GlcIV-3.






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




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




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, 4.6×250 mm, 4 μm (p/n 00G-4375-E0); Column Temp: 55° C.; Mobile Phase A: 0.0284% NH4OAc and 0.0116% HOAc in water; Mobile Phase B: Acetonitrile (MeCN); Flow Rate: 1.0 mL/min; Injection volume: 10 μL. Detection was by UV (210 nm) and CAD.
Gradient:












Time (min)
% A
% B

















0.0-5.0
100
0


20
20
80


25
20
80


30
100
0









Analyses of semi-preparative purification fractions were performed with the following method: Waters Atlantis dC18, 4.6×100 mm, 5 μm (p/n 186001340); Mobile Phase A: 25% MeCN in water; Mobile Phase B: 30% MeCN in water; Flow Rate: 1.0 mL/min; Injection volume: 10 Detection was by 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









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, 4.6×250 mm, 4 μm (p/n 00G-4375-E0); Column Temp: 55° C.; Mobile Phase A: 0.0284% NH4OAc and 0.0116% HOAc in water; Mobile Phase B: MeCN; Flow Rate: 1.0 mL/min; Injection volume: 10 μL. Detection was 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, 30×100 mm, 5 μm (p/n 186001375); Mobile Phase A: 25% MeCN in water; Mobile Phase B: 30% MeCN in water; Flow Rate: 45 mL/min; Injection load: 160 mg dissolved in 20 mL of water. Detection was by UV (205 nm).
Gradient:












Time (min)
% A
% B

















0.0-5.0
100
0


20
20
80


25
20
80


30
100
0









The secondary purification used the same column and conditions, but isocratic mobile phase: 20% MeCN in water.
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 H2O: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 μL of D2O and NMR data were acquired on a Bruker Avance 500 MHz instrument with a 2.5 mm inverse detection probe. The 1H NMR and 13C NMR spectra were referenced to the residual solvent signal HDO (δH 4.79 ppm) and TSP (δC 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 (FIG. 11). 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 FIG. 12. From this semi-preparative collection, the peak was isolated with a purity>99% (CAD, AUC). The fraction analysis is provided in FIG. 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 m/z 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 1H NMR (FIG. 29), 13C NMR (FIGS. 30 and 31), 1H-1H COSY (FIG. 32), HSQC-DEPT (FIG. 33), HMBC (FIGS. 34 and 35), and 1D-TOCSY (FIG. 36-39) were performed to allow assignment of reb M2.
The 1H, 1H-1H COSY, 1H-13C HSQC-DEPT and 1H-13C HMBC NMR data indicated that the central core of the glycoside is a diterpene. The presence of six anomeric protons observed in the 1H and 1H-13C HSQC-DEPT spectra confirm six sugar units in the structure. The methylene 13C resonance at δC 70.9 in the 1H-13C HSQC-DEPT spectrum indicated the presence of a 1→6 sugar linkage in the structure. The linkages of sugar units were assigned using 1H-13C HMBC and 1D-TOCSY correlations.
A HMBC correlation from the methyl protons at δH 1.29 to the carbonyl at δC 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 δC 39.8, 43.7, and 59.2 allowed assignment of C3, C4, and C5. Analysis of the 1H-13C HSQC-DEPT data indicated that the carbon at δC 39.8 was a methylene group and the carbon at δC 59.2 was a methine which were assigned as C-3 and C-5, respectively. This left the carbon at δC 43.7, which did not show a correlation in the HSQC-DEPT spectrum, to be assigned as the quaternary carbon, C-4. The 1H chemical shifts for C-3 (δH 1.16 and 2.28) and C-5 (δH 1.24) were assigned using the HSQC-DEPT data. A COSY correlation between one of the H-3 protons (δH 1.16) and a proton at δH 1.49 allowed assignment of one of the H-2 protons which in turn showed a correlation with a proton at δH 0.92 which was assigned to C-1. The remaining 1H 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.










1H NMR (500 MHz, D2O) and 13C NMR (125 MHz,


D2O/TSP) Assignments of the Reb M2 aglycone.











Position
13C
1H















1
41.9
0.92 m





1.93 m



2
21.8
1.49 m





1.86 m



3
39.8
1.16 m





2.28 d (13.4)



4
43.7
—



5
59.2
1.24 d (12.1)



6
24.4
1.73 m





1.94 m



7
44.2
1.49 m





1.56 m



8
46.9
—



9
55.5
1.09 d (7.7)



10
42.4
—



11
22.6
1.66 m





1.70 m



12
39.9
1.60 m





2.00 m



13
90.9
—



14
46.9
1.53 d (12.6)





2.21 d (13.6)



15
49.4
2.15 d (17.2)





2.18 d (18.1)



16
164.0
—



17
107.0
4.98 s





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 δH 0.92 showed HMBC correlations to C-1 and C-5 and was assigned as C-20. The methyl protons showed additional HMBC correlations to a quaternary carbon (δC 42.4) and a methine (δC 55.5) which were assigned as C-10 and C-9, respectively. COSY correlations between H-5 (δH 1.24) and protons at δH 1.73 and 1.94 then allowed assignment of the H-6 protons which in turn showed correlations to protons at δH 1.49 and 1.56 which were assigned to C-7. The 13C chemical shifts for C-6 (δC 24.4) and C-7 (δC 44.2) were then determined from the HSQC-DEPT data. COSY correlations between H-9 (δH 1.09) and protons at δH 1.66 and 1.70 allowed assignment of the H-11 protons which in turn showed COSY correlations to protons at δH 1.60 and 2.00 which were assigned as the H-12 protons. The HSQC-DEPT data was then used to assign C-11 (δC 22.6) and C-12 (δC 39.9). The olefinic protons observed at δH 4.98 and 5.16 showed HMBC correlations to C-13 (δC 90.9) and were assigned to C-17 (δC 107.0 via HSQC-DEPT). The olefinic protons H-17 showed HMBC correlations to a carbon at δC 49.4 which was assigned as C-15. An additional HMBC correlation from H-9 to a methylene carbon at δC 46.9 then allowed assignment of C-14. The 1H chemical shifts at C-14 (δH 1.53 and 2.21) and C-15 (δH 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 1H-13C HSQC-DEPT data confirmed the presence of six anomeric protons. Three of the anomeric protons were well resolved at δH 5.65 (δC 95.5), 4.92 (δC 104.9), and 4.50 (δC 105.7) in the 1H NMR spectrum. The remaining three anomeric protons observed at δH 4.85 (δC 98.4), 4.84 (δC 105.0), and 4.83 (δC 105.3) were overlapped by the residual solvent resonance in the 1H spectrum. The anomeric proton observed at δH 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 δH 4.85 showed a HMBC correlation to C-13 allowing it to be assigned as the anomeric proton of GlcII.
The GlcI anomeric proton (δH 5.65) showed a COSY correlation to a proton at δH 3.96 which was assigned as GlcI H-2 which in turn showed a COSY correlation to a proton at δH 3.89 (GlcI H-3) which showed a COSY correlation with a proton at δH 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 1D-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 1D-TOCSY data showed a proton at δH 3.73 assigned as GlcI H-5 and a proton at δH 4.15 assigned as one of the GlcI H-6 protons. The latter proton was also used for 1D-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 (δH 4.00). Assignment of the 13C chemical shifts for GlcI C-2 (δC 80.5), C-3 (δC 79.0), C-4 (δC 71.5), C-5 (δC 79.0), and C-6 (δC 70.9) was determined using the 1H-13C HSQC-DEPT data to complete the assignment of GlcI. Furthermore, the presence of a methylene 13C resonance at δC 70.9 in the 1H-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 δH 4.83 showed an HMBC correlation to GlcI C-2 and was assigned as the anomeric proton of GlcV. The anomeric proton observed at δH 4.50 showed a HMBC correlation to GlcI C-6 and was assigned as the anomeric proton of GlcVI. The reciprocal HMBC correlations from GlcI H-2 to the anomeric carbon of GlcV and from GlcI H-6 to the anomeric carbon of GlcVI were also observed.
The anomeric proton of GlcV (δH 4.83) showed a COSY correlation with a proton at δH 3.32 which was assigned as GlcV H-2. The GlcV H-2 in turn showed a COSY correlation to a proton at δH 3.51 (GlcV H-3). This latter proton showed an additional correlation with a proton at δH 3.38 (GlcV H-4). H-4 also showed a COSY correlation to a proton at δH 3.55 (GlcV H-5) and GlcV H-5 in turn showed a COSY correlation to GlcV H-6 protons (δH 3.76 and 3.97). Assignment of the 13C chemical shifts for GlcV C-2 (δC 78.5), C-3 (δC 78.7), C-4 (δC 72.9), C-5 (δC 78.8), and C-6 (δC 63.6) was determined using the HSQC-DEPT data. HMBC correlations from GlcV H-3 to C-2 and C-4 and also from GlcV H-4 to C-3 and C-6 confirmed the assignments made above to complete the assignment of GlcV.
Another glucose moiety was assigned as a substituent at C-6 of GlcI on the basis of 1H-13C HSQC-DEPT and HMBC correlations. The relatively downfield shift of a methylene 13C resonance of GlcI at δC 70.9 in the HSQC-DEPT spectrum indicated a 1-46 sugar linkage of GlcI. The anomeric proton observed at δH 4.50 showed a HMBC correlation to GlcI C-6 and was assigned as the anomeric proton of GlcVI. Similarly, methylene protons of GlcI showed HMBC correlations to the anomeric carbon of GlcVI and this confirmed the presence of a 1→6 sugar linkage between GlcI and GlcVI. The GlcVI anomeric proton showed a COSY correlation to a proton at δH 3.33 which was assigned as GlcVI H-2 which in turn showed a COSY correlation to a proton at δH 3.49 (GlcVI H-3). Due to data overlap, the COSY spectrum did not allow assignment of GlcV H-4 to H-6 based on the COSY correlations. Therefore, a series of 1D-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 δH 3.45 (GlcVI H-4) and δH 3.48 (GlcVI H-5) and protons at δH 3.92 and 3.94 assigned for GlcVI H-6 protons. Assignment of the 13C chemical shifts for GlcVI C-2 (δC 78.1), C-3 (δC 78.6), C-4 (δC 72.3), C-5 (δC 78.8), and C-6 (δC 64.1) was determined using the 1H-13C HSQC-DEPT data to complete the assignment of GlcVI.
A summary of the 1H and 13C chemical shifts for the glycoside at C-19 are found in the table below:










H NMR (500 MHz, D2O) and 13C NMR (125 MHz,


D2O/TSP) Assignments of the Reb M2 glycoside.











Position
13C
1H














GlcI-1
95.5
5.65 d (7.6)



GlcI-2
80.5
3.96 m



GlcI-3
79.0
3.89 m



GlcI-4
71.5
3.71 m



GlcI-5
79.0
3.73 m



GlcI-6
70.9
4.00 m





4.15 d (11.7)



GlcV-1
105.3*
4.83* d (8.0)



GlcV-2
78.5
3.32 m



GlcV-3
78.7
3.51 m



GlcV-4
72.9
3.38 m



GlcV-5
78.8
3.55 m



GlcV-6
63.6
3.76 m





3.97 m



GlcVI-1
105.7
4.50 d (7.9)



GlcVI-2
78.1
3.33 m



GlcVI-3
78.6
3.49 m



GlcVI-4
72.3
3.45 m



GlcVI-5
78.8
3.48 m



GlcVI-6
64.1
3.92 m





3.94 m





*1H and 13C values can be exchangeable with GlcIV-1 of the following table.






A summary of the key HMBC, COSY, and 1D-TOCSY correlations used to assign the C-19 glycoside region are provided below:










1H NMR (500 MHz, D2O) and 13C NMR (125 MHz,


D2O/TSP) Assignments of the Reb M2 glycoside.











Position
13C#
1H














GlcII-1
98.4
4.85 d (7.8)



GlcII-2
81.7
3.75 m



GlcII-3
88.0
3.98 m



GlcII-4
71.3
3.54 m



GlcII-5
80.5
3.96 m



GlcII-6
63.6
3.45 m





3.77 m



GlcIII-1
104.9
4.92 d (7.9)



GlcIII-2
76.3
3.32 m



GlcIII-3
78.8
3.51 m



GlcIII-4
73.3
3.26 t (9.5)



GlcIII-5
78.8
3.44 m



GlcIII-6
64.4
3.75 m





3.94 m



GlcIV-1
105.0
4.84 d (7.8)



GlcIV-2
76.1
3.41 m



GlcIV-3
78.8
3.46 m



GlcIV-4
72.5
3.45 m



GlcIV-5
81.7
3.75 m



GlcIV-6
65.8
3.55 m





3.78 m









Assignment of GlcII was carried out in a similar manner. The GlcII anomeric proton (δH 4.85) showed a COSY correlation to a proton at δH 3.75 which was assigned as GlcII H-2 which in turn showed a COSY correlation to a proton at δH 3.98 (GlcII H-3). This latter proton showed an additional correlation with a proton at δH 3.54 (GlcII H-4). H-4 also showed a COSY correlation to a proton at δH 3.96 (GlcII H-5). GlcII H-5 also showed a COSY correlation to GlcII H-6 protons (δH 3.77 and 3.45). Assignment of the 13C chemical shifts for GlcII C-2 (δC 81.7), C-3 (δC 88.0), C-4 (δC 71.3), C-5 (δC 80.5), and C-6 (δC 63.6) was determined using the HSQC-DEPT data. HMBC correlations from GlcII H-3 to C-2 and C-4 and also from GlcII H-4 to C-3 and C-6 confirmed the assignments made above to complete the assignment of GlcII.
Two of the remaining unassigned glucose moieties were assigned as substituents at C-2 and C-3 of GlcII on the basis of HMBC correlations. The anomeric proton observed at δH 4.92 showed a HMBC correlation to GlcII C-2 and was assigned as the anomeric proton of GlcIII. The anomeric proton observed at δH 4.84 showed a HMBC correlation to GlcII C-3 and was assigned as the anomeric proton of GlcIV. The reciprocal HMBC correlations between GlcII H-2 and the anomeric carbon of GlcIII and between Glen H-3 and the anomeric carbon of GlcIV were also observed.
The anomeric proton of Glciii (δH 4.92) showed a COSY correlation with a proton at δH 3.32 which was assigned as GlcIII H-2. Due to data overlap, the COSY spectrum did not allow assignment of H-3 to H-6 protons. Therefore, a series of 1D-TOCSY experiments were performed using selective irradiation of the GlcIII anomeric proton with different mixing times. In addition to confirming the assignments for GlcIII H-2, the 1D-TOCSY data showed protons at δH 3.51 (GlcIII H-3), δH 3.26 (Glciii H-4), and δH 3.44 (Glciii 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, Glciii H-4 showed a correlation to GlcIII H-5, which in turn showed COSY correlations to δH 3.94 and 3.75 of Glciii H-6a and H-6b, respectively. The 13C chemical shifts for GlcIII C-2 (δC 76.3), C-3 (δC 78.8), C-4 (δC 73.3), C-5 (δC 78.8), and C-6 (δC 64.4) were then determined using the 1H-13C HSQC-DEPT correlations to complete the assignment of GlcIII.
The anomeric proton of GlcIV (δH 4.84) which showed a COSY correlation to a proton at δH 3.41 was assigned as GlcIV H-2 which in turn showed a COSY correlation to a proton at δH 3.46 (GlcIV H-3). This latter proton showed an additional correlation with a proton at δH 3.45 (GlcIV H-4) which also showed a COSY correlation to a proton at δH 3.75 (GlcIV H-5). GlcIV H-5 also showed a COSY correlation to GlcIV H-6 protons OH 3.55 and 3.78). Assignment of the 13C chemical shifts for GlcIV C-2 (δC 76.1), C-3 (δC 78.8), C-4 (δC 72.5), C-5 (δC 81.7), and C-6 (δC 65.8) was determined using the HSQC-DEPT data. HMBC correlations from GlcIV H-3 to C-4 and C-5 and also from GlcIV H-4 to C-3 and C-6 confirmed the assignments made above to complete the assignment of GlcIV.
A summary of the 1H and 13C chemical shifts for the glycoside at C-13 are found in the following table:










1H NMR (500 MHz, D2O) and 13C NMR (125 MHz,


D2O/TSP) Assignments of the Reb M2 glycoside.











Position
13C#
1H














GlcII-1
98.4
4.85 d (7.8)



GlcII-2
81.7
3.75 m



GlcII-3
88.0
3.98 m



GlcII-4
71.3
3.54 m



GlcII-5
80.5
3.96 m



GlcII-6
63.6
3.45 m





3.77 m



GlcIII-1
104.9
4.92 d (7.9)



GlcIII-2
76.3
3.32 m



GlcIII-3
78.8
3.51 m



GlcIII-4
73.3
3.26 t (9.5)



GlcIII-5
78.8
3.44 m



GlcIII-6
64.4
3.75 m





3.94 m



GlcIV-1
105.0
4.84 d (7.8)



GlcIV-2
76.1
3.41 m



GlcIV-3
78.8
3.46 m



GlcIV-4
72.5
3.45 m



GlcIV-5
81.7
3.75 m



GlcIV-6
65.8
3.55 m





3.78 m









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




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




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 UGT76G1var94 containing mutations: Q266E_P272A_R334K_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 μL aliquots were stored at −20° C. and at −80° C.
These storage aliquots of E. coli BL21(DE3) containing the pET30a+_UGT76G1var 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 30° C. for 8 h.
3.95 mL of production medium containing 60 g/L of Overnight ExpressTM Instant TB medium (Novagen®), 10 g/L of glycerol and 50 mg/L of Kanamycin was inoculated with 50 μL 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 μL of fresh lysate that was added to a solution of Rebaudioside D (final concentration 0.5 mM), MgCl2 (final concentration 3 mM) and UDP-Glucose (final concentration 2.5 mM) in 50 mM phosphate buffer pH 7.2.
The reaction was 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, Round1-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:















Normalized





conversion
Normalized




Reb D to Reb
initial


Clone
Mutations*
Mafter 22 h.
rate (0-4 h)







Round1-Var94
UGT76G1
100% 
100% 



(Q266E_P272A_R334K_G348P_L379G)





baseline clone




Round2-Var1
Round1-Var94 (A213N_P348G_I411V)
70%
86%


Round2-Var2
Round1-Var94 (K303G_I423M_Q425E)
120% 
134% 


Round2-Var3
Round1-Var94 (V20L_N138K_S147G)
14%
15%


Round2-Var4
Round1-Var94 (I16V_V133A_L299I)
37%
43%


Round2-Var5
Round1-Var94 (S241V_S274G_Q432E)
75%
72%


Round2-Var6
Round1-Var94 (I16V_L139V_I218V)
62%
68%


Round2-Var7
Round1-Var94 (K334R_N409K_Q432E)
104% 
92%


Round2-Var8
Round1-Var94 (I15L_R141T_I407V)
17%
26%


Round2-Var9
Round1-Var94 (R141T_K303G_G379L)
31%
42%


Round2-Var10
Round1-Var94 (I190L_K303G_P348G)
131% 
149% 


Round2-Var11
Round1-Var94 (E266Q_F314S_N409R)
106% 
132% 


Round2-Var12
Round1-Var94 (V133A_I295V_K303G)
43%
49%


Round2-Var13
Round1-Var94 (I16V_S241V_N409R)
80%
79%


Round2-Var14
Round1-Var94 (A239V_K334R_G379L)
58%
55%


Round2-Var15
Round1-Var94 (I190L_K393R_V396L)
118% 
126% 


Round2-Var16
Round1-Var94 (L101F_I295M_K393R)
84%
89%


Round2-Var17
Round1-Var94 (A239V_E266Q_Q425E)
96%
101% 


Round2-Var18
Round1-Var94 (V20L_I190L_I423M)
98%
98%


Round2-Var19
Round1-Var94 (V20L_G379L_S456L)
84%
81%


Round2-Var20
Round1-Var94 (K334R_P348G_N409R)
73%
73%


Round2-Var21
Round1-Var94 (E231A_S241V_E449D)
53%
50%


Round2-Var22
Round1-Var94 (K188R_L299I_V394I)
56%
59%


Round2-Var23
Round1-Var94 (E231A_S274G_V394I)
110% 
124% 


Round2-Var24
Round1-Var94 (S42A_I295V_Q432E)
71%
78%


Round2-Var25
Round1-Var94 (A213N_A272P_K334R)
95%
80%


Round2-Var26
Round1-Var94 (L158Y_S274K_N409K)
80%
50%


Round2-Var27
Round1-Var94 (K188R_I295M_Q425E)
132% 
116% 


Round2-Var28
Round1-Var94 (I15L_I295M_V394I)
53%
36%


Round2-Var29
Round1-Var94 (V133A_A239V_V394I)
47%
30%


Round2-Var30
Round1-Var94 (L158Y_F314S_K316R)
107% 
72%


Round2-Var31
Round1-Var94 (L158Y_A239V_A272P)
54%
30%


Round2-Var32
Round1-Var94 (F46I_D301N_V396L)
109% 
101% 


Round2-Var33
Round1-Var94 (L101F_I218V_Q432E)
78%
54%


Round2-Var34
Round1-Var94 (I16V_F46I_I295M)
110% 
95%


Round2-Var35
Round1-Var94 (A213N_E266S_I407V)
98%
79%


Round2-Var36
Round1-Var94 (A239V_S274K_I295M)
102% 
89%


Round2-Var37
Round1-Var94 (A239V_F314S_S450K)
105% 
99%


Round2-Var38
Round1-Var94 (L139V_K188R_D301N)
66%
51%


Round2-Var39
Round1-Var94 (I45V_I218V_S274K)
87%
58%


Round2-Var40
Round1-Var94 (S241V_K303G_V394I)
78%
57%


Round2-Var41
Round1-Var94 (R141T_S274G_K334R)
41%
28%


Round2-Var42
Round1-Var94 (V217L_S274G_L299I)
47%
34%


Round2-Var43
Round1-Var94 (S274G_D301N_P348G)
98%
91%


Round2-Var44
Round1-Var94 (E231A_N409R_S450K)
87%
65%


Round2-Var45
Round1-Var94 (R64H_E231A_K316R)
88%
64%


Round2-Var46
Round1-Var94 (V394I_N409K_I411V)
110% 
100% 


Round2-Var47
Round1-Var94 (I45V_I295M_K303G)
113% 
88%


Round2-Var48
Round1-Var94 (L101F_V396L_L398V)
46%
43%


Round2-Var49
Round1-Var94 (N27S_L101F_S447A)
54%
37%


Round2-Var50
Round1-Var94 (S274G_F314S_L398V)
129% 
156% 


Round2-Var51
Round1-Var94 (E266Q_L299I_K393R)
70%
51%


Round2-Var52
Round1-Var94 (V217L_E266S_V394I)
62%
48%


Round2-Var53
Round1-Var94 (N138K_A272P_N409R)
118% 
102% 


Round2-Var54
Round1-Var94 (E266S_F314S_Q432E)
124% 
146% 


Round2-Var55
Round1-Var94 (D301N_G379L_L398V)
56%
45%


Round2-Var56
Round1-Var94 (F46I_E266S_K334R)
123% 
142% 


Round2-Var57
Round1-Var94 (A272P_V394I_Q432E)
133% 
142% 


Round2-Var58
Round1-Var94 (V394I_I407V_S456L)
118% 
114% 


Round2-Var59
Round1-Var94 (I218V_E266Q_I423M)
106% 
98%


Round2-Var60
Round1-Var94 (A272P_G379L_I407V)
80%
63%


Round2-Var61
Round1-Var94 (E231A_K303G_S456L)
113% 
110% 


Round2-Var62
Round1-Var94 (I190L_E266Q_I407V)
150% 
167% 


Round2-Var63
Round1-Var94 (N27S_L139V_I295V)
43%
25%


Round2-Var64
Round1-Var94 (V217L_I423M_S447A)
67%
51%


Round2-Var65
Round1-Var94 (L158Y_E266S_E449D)
68%
43%


Round2-Var66
Round1-Var94 (S42A_F46I_I407V)
160% 
203% 


Round2-Var67
Round1-Var94 (N138K_E231A_D301N)
118% 
93%


Round2-Var68
Round1-Var94 (K188R_G379L_N409R)
52%
35%


Round2-Var69
Round1-Var94 (I15L_E231A_V396L)
38%
22%


Round2-Var70
Round1-Var94 (E231A_Q425E_Q432E)
115% 
119% 


Round2-Var71
Round1-Var94 (D301N_K316R_Q425E)
126% 
121% 


Round2-Var72
Round1-Var94 (L139V_I295M_F314S)
76%
91%


Round2-Var73
Round1-Var94 (S147G_E266S_D301N)
30%
18%


Round2-Var74
Round1-Var94 (R64H_S147G_S447A)
23%
12%


Round2-Var75
Round1-Var94 (S42A_K303G_L398V)
95%
110% 


Round2-Var76
Round1-Var94 (I45V_D301N_E449D)
62%
60%


Round2-Var77
Round1-Var94 (V133A_E266S_I411V)
37%
28%


Round2-Var78
Round1-Var94 (I45V_N409R_Q425E)
63%
59%


Round2-Var79
Round1-Var94 (R141T_A272P_F314S)
23%
10%


Round2-Var80
Round1-Var94 (E266S_S274G_N409R)
81%
91%


Round2-Var81
Round1-Var94 (N409K_Q425E_S450K)
81%
84%


Round2-Var82
Round1-Var94 (N27S_R64H_K393R)
47%
37%


Round2-Var83
Round1-Var94 (S42A_A213N_V217L)
62%
46%


Round2-Var84
Round1-Var94 (N27S_S274K_I407V)
49%
44%


Round2-Var85
Round1-Var94 (I411V_Q425E_S456L)
75%
81%


Round2-Var86
Round1-Var94 (A239V_K316R_E449D)
83%
72%


Round2-Var87
Round1-Var94 (S147G_A239V_P348G)
18%
 7%


Round2-Var88
Round1-Var94 (V20L_S274G_S450K)
71%
68%


Round2-Var89
Round1-Var94 (F314S_V394I_S447A)
88%
123% 


Round2-Var90
Round1-Var94 (R64H_E266Q_I295M)
45%
47%


Round2-Var91
Round1-Var94 (N138K_I295V_I407V)
50%
51%


Round2-Var92
Round1-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 Round1-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, V394I, I407V, N409K, N409R, Q425E, Q432E, S447A, S456L.
Example 42
In Vivo Production of AtSUS







SEQ ID 13


AtSUS


>gi|79328294|ref|NP_001031915.1| sucrose synthase


1 [Arabidopsis thaliana]


MANAERMITRVHSQRERLNETLVSERNEVLALLSRVEAKGKGILQQNQII


 


AEFEALPEQTRKKLEGGPFFDLLKSTQEAIVLPPWVALAVRPRPGVWEYL


 


RVNLHALVVEELQPAEFLHFKEELVDGVKNGNFTLELDFEPFNASIPRPT


 


LHKYIGNGVDFLNRHLSAKLFHDKESLLPLLKFLRLHSHQGKNLMLSEKI


 


QNLNTLQHTLRKAEEYLAELKSETLYEEFEAKFEEIGLERGWGDNAERVL


 


DMIRLLLDLLEAPDPCTLETFLGRVPMVFNVVILSPHGYFAQDNVLGYPD


 


TGGQVVYILDQVRALEIEMLQRIKQQGLNIKPRILILTRLLPDAVGTTCG


 


ERLERVYDSEYCDILRVPFRTEKGIVRKWISRFEVWPYLETYTEDAAVEL


 


SKELNGKPDLIIGNYSDGNLVASLLAHKLGVTQCTIAHALEKTKYPDSDI


 


YWKKLDDKYHFSCQFTADIFAMNHTDFIITSTFQEIAGSKETVGQYESHT


 


AFTLPGLYRVVHGIDVFDPKFNIVSPGADMSIYFPYTEEKRRLTKFHSEI


 


EELLYSDVENKEHLCVLKDKKKPILFTMARLDRVKNLSGLVEWYGKNTRL


 


RELANLVVVGGDRRKESKDNEEKAEMKKMYDLIEEYKLNGQFRWISSQMD


 


RVRNGELYRYICDTKGAFVQPALYEAFGLTVVEAMTCGLPTFATCKGGPA


 


EIIVHGKSGFHIDPYHGDQAADTLADFFTKCKEDPSHWDEISKGGLQRIE


 


EKYTWQIYSQRLLTLTGVYGFWKHVSNLDRLEARRYLEMFYALKYRPLAQ


 


AVPLAQDD






The synthetic gene of AtSuS that was codon optimized for expression in E. coli and subcloned in the pET30a+ plasmid using the NdeI and XhoI 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 μL 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, 3×40 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 μL of UGTSL2 (see EXAMPLE 27) lysate (2 U/mL), 150 μL of UGT76G1var94 (see EXAMPLE 26) (2.5 U/mL) and 15 μL of AtSUS (see EXAMPLE 42) (400 U/mL). The reaction was followed by HPLC after quenching 125 μL samples with 10 μL of 2 N H2SO4 and 115 μL 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 FIG. 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

1. A method for preparing reb D2 comprising:
a. contacting a starting composition comprising reb A with UDP-glucosyltransferase, to produce a composition comprising reb D2; and
b. isolating the composition comprising reb D2.

2. The method of claim 1, 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.

3. A method for preparing reb M2 comprising:
a. contacting a starting composition comprising reb D2 with UDP-glucosyltransferase to produce a composition comprising reb M2; and
b. isolating the composition comprising reb M2.

4. The method of claim 3, 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.