FIG. 58 shows a graph showing the reaction profile for Example 25.

FIGS. 59A-1-59B-2 show HPLC chromatograms showing the HPLC assay results for Example 28.

FIGS. 60A-1-60B-2 show HPLC chromatograms showing the HPLC assay results for Example 29.

FIGS. 61A-61B show an HPLC chromatogram showing the HPLC assay results for Example 30.

FIG. 62 shows an LS-MS spectrogram showing the LS-MS assay results for Example 31.

FIGS. 63A-1-63C-2 show HPLC chromatograms showing the HPLC assay results for Example 32.

FIGS. 64A-64B show an HPLC chromatogram showing the HPLC assay results for Example 35.

FIGS. 65A-65B show an HPLC chromatogram showing the HPLC assay results for Example 37 FIG. 66 shows a graph showing the HPLC results for Example 43.

FIGS. 67A-67B show a graph showing the reaction profile for Example 46.

FIGS. 68A-68F show reaction profiles for Example 49.

FIGS. 69A-69C show graphs showing the HPLC results for Example 50.

FIGS. 70A-70D show reaction profile graphs for Example 51.

FIG. 71 shows a reaction profile graph for Example 52.

FIG. 72A shows a reaction profile graph for Example 54.

FIG. 72B shows an HPLC chromatogram showing the HPLC analysis for Example 54.

FIG. 73A shows a reaction profile graph for Example 55.

FIG. 73B shows an HPLC chromatogram showing the HPLC analysis for Example 55.

FIG. 74A shows a reaction profile graph for Example 56.

FIG. 74B shows an HPLC chromatogram showing the HPLC analysis for Example 56.

FIG. 75A shows a reaction profile graph for Example 57.

FIG. 75B shows an HPLC chromatogram showing the HPLC analysis for Example 57.

FIG. 76A shows a reaction profile graph for Example 58.

FIG. 76B shows an HPLC chromatogram showing the HPLC analysis for Example 58.

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, 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 are known in the art. Biocatalyst protein enzymes can be naturally occurring or recombinant proteins.

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

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 D, rebaudioside M2, 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

[Image Omitted]

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

[Image Omitted]

Rebaudioside D
β-Glc-β-Glc(2 → 1)

[Image Omitted]

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

Rebaudioside M

[Image Omitted]

(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.

In another embodiment, the starting composition is steviol glycoside(s).

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.

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

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

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 95% 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 one 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

In one embodiment of present invention, a microorganism is contacted with the starting composition to produce a composition comprising the target steviol glycoside. The microorganism can be any microorganism possessing biocatalysts suitable for converting the starting composition to the target steviol glycoside. These biocatalysts are encoded within the microorganism's genome.

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

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

The biocatalysts can be separated from the microorganism and used for conversion of starting composition to target steviol glycoside(s). The separation can be achieved by any means known to art, including but not limited to lysis of microbial cells, centrifugation, filtration.

The biocatalysts can be excreted from the microorganism (extracellular enzymes) and used for conversion of starting composition to target steviol glycoside(s).

In one embodiment, the biocatalysts are steviol biosynthesis enzymes and UDP-glycosyltransferases (UGTs), or their variants, having greater than 75% amino-acid sequence identity.

The steviol biosynthesis can be any steviol biosynthesis enzyme, or its variant, having greater than 75% amino-acid sequence identity.

In one embodiment the steviol biosynthesis enzymes include mevalonate (MVA) pathway enzymes, or their variants, having greater than 75% amino-acid sequence identity.

In another embodiment the steviol biosynthesis enzymes include non-mevalonate 2-C-methyl-D-erythritol-4-phosphate pathway (MEP/DOXP) enzymes, or their variants, having greater than 75% amino-acid sequence identity.

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., or their variants, having greater than 75% amino-acid sequence identity.

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 or UGT91D2 variant, having greater than 75% amino-acid sequence identity with 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 or UGTSL2 variant, having greater than 75% amino-acid sequence identity with 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 or UGT76G1 variant, having greater than 75% amino-acid sequence identity with 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 or UGT76G1 variant, having greater than 75% amino-acid sequence identity with 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 or their variant, having greater than 75% amino-acid sequence identity with UGT91D2 or UGTSL2.

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

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

In another embodiment, the UDP-glucosyltransferase capable of adding at least one glucose unit to produce target steviol glycoside, has greater than 75% amino-acid sequence identity with at least one enzyme 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 × ananassa

125534461
EAY81009.1
Oryza sativa 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 sativa Japonica Group

224055535
XP_002298527.1
Populus trichocarpa

224101569
XP_002334266.1
Populus trichocarpa

224120552
XP_002318358.1
Populus trichocarpa

224121288
XP_002330790.1
Populus trichocarpa

225444853
XP_002281094
Vitis vinifera

225454342
XP_002275850.1
Vitis vinifera

225454475
XP_002280923.1
Vitis vinifera

225461556
XP_002285222
Vitis vinifera

225469540
XP_002270294.1
Vitis vinifera

226495389
NP_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 × 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

GI number
Accession
Origin

460409128
XP.004249992.1
Solanum lycopersicum

460386018
XP.004238697.1
Solanum lycopersicum

460409134
XP.004249995.1
Solanum lycopersicum

460410132
XP.004250485.1
Solanum lycopersicum

460410130
XP.004250484.1
Solanum lycopersicum

460410128
XP.004250483.1
Solanum lycopersicum

460378310
XP.004234916.1
Solanum lycopersicum

209954733
BAG80557.1
Lycium barbarum

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 or UGT76G1 variant, having greater than 75% amino-acid sequence identity with 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.

Optionally, the method of the present invention further comprises hydrolysis of 1,6-β-glucosidic linkages in reb D2 and/or reb M2. In one embodiment, the method comprises hydrolysis of 1,6-β-glucosidic linkages in reb D2 and/or reb M2 by providing a β-glucosidase.

In one embodiment β-glucosidase is provided together with UDP-recycling biocatalyst and UGTs to minimize the content of reb D2 and/or reb M2 in final reaction mixture and maximize the yield of reb M In a particular embodiment to minimize the content of reb D2 and/or reb M2 in final reaction mixture and maximize the yield of reb M, β-glucosidase is provided together with UDP-recycling biocatalyst, UGT76G1 and UGTSL2, or their variants having greater than 75% amino-acid sequence identity with UGT76G1 or UGTSL2.

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 any 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:

[Image Omitted]

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]

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 provided in the form of a recombinant microorganism.

In one embodiment, the enzyme is immobilized. In another embodiment, the enzyme is provided in the form of 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 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 and is contained in E. coli.

In a still more particular embodiment, the enzyme is UGTSL2 and is contained in E. coli.

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:

[Image Omitted]

(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])

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 and is contained in E. coli.

In a still more particular embodiment, the enzyme is UGTSL2 and is contained in E. coli.

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 Min 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 other sweeteners, flavors and food ingredients.

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

Non-limiting examples of other food ingredients include flavors, acidulants, organic and amino acids, coloring agents, bulking agents, modified starches, gums, texturizers, preservatives, antioxidants, emulsifiers, stabilizers, thickeners and gelling agents.

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 mixtures 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 other 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, camosiflosides, cyclocarioside, pterocaryosides, polypodoside A, brazilin, hemandulcin, 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 others.

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 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, 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, sulfinyl, sulfamoyl, carboalkoxy, carboxamido, phosphonyl, phosphinyl, phosphoryl, phosphino, thioester, thioether, oximino, hydrazino, carbamyl, phospho, phosphonato, or any other viable functional group provided the carbohydrate derivative or substituted carbohydrate functions to improve the sweet taste of the sweetener composition.

Examples of carbohydrates which may be used in accordance with this invention include, but are not limited to, Psicose, turanose, allose, tagatose, trehalose, galactose, rhamnose, various cyclodextrins, cyclic oligosaccharides, various types of maltodextrins, dextran, sucrose, glucose, ribulose, fructose, threose, arabinose, xylose, lyxose, allose, altrose, mannose, idose, lactose, maltose, invert sugar, isotrehalose, neotrehalose, isomaltulose, erythrose, deoxyribose, gulose, idose, talose, erythrulose, xylulose, psicose, turanose, cellobiose, amylopectin, glucosamine, mannosamine, fucose, glucuronic acid, gluconic acid, glucono-lactone, 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 containing 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-arabinose, 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 sites 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 NO: 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 sites 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 NO: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 H₂SO₄and 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 shown in FIG. 51.

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 H₂SO₄and 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 H₂SO₄and 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 shown in FIG. 52.

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 Tuner™ (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 Nde1 and Sal1 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 Origami™2 (DE3) Competent Cells) by heat shock treatment. The obtained cells were grown on LB Agar medium in petri-dishes in the presence of Ampicillin. Suitable colonies were selected and allowed to grow in liquid LBGKP medium containing Ampicillin. Glycerol was added and 400 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 MgCl₂in 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*

Stevioside
Rebaudioside

Expression
to Rebaudio-
D to Rebaudio-

Example
Plasmid
strain
side A
side 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
ArcticExpress
15 U mL⁻¹
0.25 U mL⁻¹

11
pCOLDIII
ArcticExpress
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 substance 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 5 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 Nde1 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 Tuner™ (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 MgCl₂in 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.

Exam-

Transformation activity*

ple
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.

Conversion

Internal
RebA to

Microplate
Position
Gene Name
reference
RebD

C908201
A1
gi115454819_NP_
S115N01
Active

001051010.1
A1

C908201
G2
gi187373030_
S115N01
Active

ACD03249.1
G2

C908201
A7
gi460409128_XP_
S115N05
Active

004249992.1
A7

C912666
E1
gi222619587_
S115N06
Active

EEE55719.1
E1

C912666
C2
gi297795735_XP_
S115N06
Active

002865752.1
C2

The amino acid sequences are as follows:

SEQ ID NO: 3:

MDDAHSSQSPLHVVIFPWLAFGHLLPCLDLAERLAARGHRVSFVSTPRNL

ARLPPVRPELAELVDLVALPLPRVDGLPDGAEATSDVPFDKFELHRKAFD

GLAAPFSAFLDTACAGGKRPDWVLADLMHHWVALASQERGVPCAMILPCS

AAVVASSAPPTESSADQREAIVRSMGTAAPSFEAKRATEEFATEGASGVS

IMTRYSLTLQRSKLVAMRSCPELEPGAFTILTRFYGKPVVPFGLLPPRPD

GARGVSKNGKHDAIMQWLDAQPAKSVVYVALGSEAPMSADLLRELAHGLD

LAGTRFLWAMRKPAGVDADSVLPAGFLGRTGERGLVTTRWAPQVSILAHA

AVCAFLTHCGWGSVVEGLQFGHPLIMLPILGDQGPNARILEGRKLGVAVP

RNDEDGSFDRGGVAGAVRAVVVEEEGKTFFANARKLQEIVADREREERCI

DEFVQHLTSWNELKNNSDGQYP

SEQ ID NO: 4:

MAVKDEQQSPLHILLFPFLAPGHLIPIADMAALFASRGVRCTILTTPVNA

AIIRSAVDRANDAFRGSDCPAIDISVVPFPDVGLPPGVENGNALTSPADR

LKFFQAVAELREPFDRFLADNHPDAVVSDSFFHWSTDAAAEHGVPRLGFL

GSSMFAGSCNESTLHNNPLETAADDPDALVSLPGLPHRVELRRSQMMDPK

KRPDHWALLESVNAADQKSFGEVFNSFHELEPDYVEHYQTTLGRRTWLVG

PVALASKDMAGRGSTSARSPDADSCLRWLDTKQPGSVVYVSFGTLIRFSP

AELHELARGLDLSGKNFVWVLGRAGPDSSEWMPQGFADLITPRGDRGFII

RGWAPQMLILNHRALGGFVTHCGWNSTLESVSAGVPMVTWPRFADQFQNE

KLIVEVLKVGVSIGAKDYGSGIENHDVIRGEVIAESIGKLMGSSEESDAI

QRKAKDLGAEARSAVENGGSSYNDVGRLMDELMARRSSVKVGEDIIPTND

GL

SEQ ID NO: 5:

MSPKLHKELFFHSLYKKTRSNHTMATLKVLMFPFLAYGHISPYLNVAKKL

ADRGFLIYFCSTPINLKSTIEKIPEKYADSIHLIELHLPELPQLPPHYHT

TNGLPPNLNQVLQKALKMSKPNFSKILQNLKPDLVIYDILQRWAKHVANE

QNIPAVKLLTSGAAVFSYFFNVLKKPGVEFPFPGIYLRKIEQVRLSEMMS

KSDKEKELEDDDDDDDLLVDGNMQIMLMSTSRTIEAKYIDFCTALTNWKV

VPVGPPVQDLITNDVDDMELIDWLGTKDENSTVFVSFGSEYFLSKEDMEE

VAFALELSNVNFIWVARFPKGEERNLEDALPKGFLERIGERGRVLDKFAP

QPRILNHPSTGGFISHCGWNSAMESIDFGVPIIAMPMHLDQPMNARLIVE

LGVAVEIVRDDDGKIHRGEIAETLKGVITGKTGEKLRAKVRDISKNLKTI

RDEEMDAAAEELIQLCRNGN

SEQ ID NO: 6:

MHVVMLPWLAFGHILPFAEFAKRVARQGHRVTLFSTPRNTRRLIDVPPSL

AGRIRVVDIPLPRVEHLPEHAEATIDLPSNDLRPYLRRAYDEAFSRELSR

LLQETGPSRPDWVLADYAAYWAPAAASRHGVPCAFLSLFGAAALCFFGPA

ETLQGRGPYAKTEPAHLTAVPEYVPFPTTVAFRGNEARELFKPSLIPDES

GVSESYRFSQSIEGCQLVAVRSNQEFEPEWLELLGELYQKPVIPIGMFPP

PPPQDVAGHEETLRWLDRQEPNSVVYAAFGSEVKLTAEQLQRIALGLEAS

ELPFIWAFRAPPDAGDGDGLPGGFKERVNGRGVVCRGWVPQVKFLAHASV

GGFLTHAGWNSIAEGLANGVRLVLLPLMFEQGLNARQLAEKKVAVEVARD

EDDGSFAANDIVDALRRVMVGEEGDEFGVKVKELAKVFGDDEVNDRYVRD

FLKCLSEYKMQRQG

SEQ ID NO: 7:

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:

Internal
[Plasmid]

Microtiter plate
Position
reference
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

Internal
[Plasmid]

plate
Position
reference of test
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 4 u Hydro-
Phenomenex
00G-4375-E0
588582-12

RP 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 and shown in FIGS. 53a-e:

Steviol glycoside conversion

Internal
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.

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-
Posi-
GI

Internal
RebA to

plate
tion
number
Version
reference
RebD

C912666
G4
41469452
AAS07253.1
S115N08 G4
Active

The amino-acid sequence is as follows:

>gi|41469452|gb|AAS07253.1| putative UDP-glucoronosyl and UDP-glucosyl transferase [Oryza sativa japonica Group] EUGT11 enzyme from patent application WO/2013/022989A2

SEQ ID NO: 8:

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

Internal
[Plasmid]

plate
Position
reference of test
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 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 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: PGP-51T1

Microtiter

Internal
[Plasmid]

plate
Position
reference of test
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 (~1.5 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.

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

The HPLC assay results are shown in FIG. 54.

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 MgCl₂in 50 mM Sodium Phosphate buffer at pH 7.2. Samples were taken and analyzed by HPLC.

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

The results for the different enzymes are provided below and shown in FIGS. 55a-e.

Conversion
Reb D

GI Number
Version
after 45 hrs.
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

Example 23

Identification of Glycosides

The reaction mixtures representing GI No. 460409128, particularly the sample “12400 S115N05A7 T24h 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), thermostated 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 100 A,

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.

Steviol glycoside conversion in reaction mixture (% area)

Sample
Unknown@RT3.5
Reb D
Reb UNK
Reb A

S115N05A7
6.47
20.35
19.93
53.24

S129N04
6.05
23.73
21.22
49.00

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 along with Stevia rebaudiana Bertoni leaf extract “MLD1” produced by PureCircle Sdn Bhd (Malaysia) to determine the occurrence of S129N04 glycosides in nature.

The assays in FIGS. 57a-b show 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 H₂SO₄and 270 μL of methanol/water (6/4). The samples were immediately centrifuged and kept at 10° C. before analysis by HPLC (CAD detection). The reaction profile shown in FIG. 58 was obtained corresponding to a complete conversion of Rebaudioside E to Rebaudioside D.

Example 26

Directed Evolution of UGT76G1 for the Conversion of Rebaudioside D to Rebaudioside M

Starting from the amino acid sequence of UGT76G1, as is described in Genbank (AAR06912.1), different mutations at various amino acid positions were identified that could alter the activity of the enzyme for the transformation of Rebaudioside D (Reb D) to Rebaudioside M (Reb M). This list of mutations, designed by DNA2.0 ProteinGPS™ strategy, was subsequently used to synthesize 96 variant genes that contained 3, 4 or 5 of these mutations that were codon-optimized for expression in E. coli. The genes were subcloned in the pET30a+ plasmid and used for transformation of E. coli BL21 (DE3) chemically competent cells. The obtained cells were grown in Petri-dishes on solid LB medium in the presence of Kanamycin. Suitable colonies were selected and allowed to grow in liquid LB medium in tubes. Glycerol was added to the suspension as cryoprotectant and 400 μL aliquots were stored at −20° C. and at −80° C.

These storage aliquots of E. coli BL21(DE3) containing the pET30a+_UGT76Glvar plasmids were thawed and added to LBGKP medium (20 g/L Luria Broth Lennox; 50 mM PIPES buffer pH 7.00; 50 mM Phosphate buffer pH 7.00; 2.5 g/L glucose and 50 mg/L of Kanamycine). This culture was allowed to shake in a 96 microtiter plate at 135 rpm at 30° C. for 8 h.

3.95 mL of production medium containing 60 g/L of Overnight Express™ Instant TB medium (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
initial rate

to Reb M 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

UGTSL2 (GI_460410132/XP_004250485.1) amino acid

sequence (SEQ ID NO: 9):

MATNLRVLMFPWLAYGHISPFLNIAKQLADRGFLIYLCSTRINLESIIKK

IPEKYADSIHLIELQLPELPELPPHYHTTNGLPPHLNPTLHKALKMSKPN

FSRILQNLKPDLLIYDVLQPWAEHVANEQNIPAGKLLTSCAAVFSYFFSF

RKNPGVEFPFPAIHLPEVEKVKIREILAKEPEEGGRLDEGNKQMMLMCTS

RTIEAKYIDYCTELCNWKVVPVGPPFQDLITNDADNKELIDWLGTKHENS

TVFVSFGSEYFLSKEDMEEVAFALELSNVNFIWVARFPKGEERNLEDALP

KGFLERIGERGRVLDKFAPQPRILNHPSTGGFISHCGWNSAMESIDFGVP

IIAMPIHNDQPINAKLMVELGVAVEIVRDDDGKIHRGEIAETLKSVVTGE

TGEILRAKVREISKNLKSIRDEEMDAVAEELIQLCRNSNKSK

The pET30A+ vector containing the UGTSL2 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 cryoprotection 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 MgCl₂in 50 mM Sodium Phosphate buffer at pH 7.2. Samples were taken and analyzed by HPLC. HPLC Analysis. The HPLC assay was performed as described in EXAMPLE 20.

The results for the different enzymes and the corresponding chromatograms are provided below and shown in FIGS. 59a-b

Enzyme internal

Stevioside conv.¹
Rebaudioside E

reference
GI Number
Version
(reaction 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

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 MgCl₂in 50 mM Sodium Phosphate buffer at pH 7.2. Samples were taken and analyzed by HPLC. The HPLC assay was performed as described in EXAMPLE 20.

The results for the different enzymes and the corresponding chromatograms are provided below and shown in FIGS. 60a-b.

Enzyme internal

Rubusoside conv.¹
Rebaudioside E

reference
GI Number
Version
(reactiontime)
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

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 MgCl₂in 50 mM Sodium Phosphate buffer at pH 7.2. Samples were taken and analyzed by HPLC. The HPLC assay was performed as described in EXAMPLE 20.

The result after 23 h. of reaction and the corresponding chromatogram is provided below and shown in FIG. 61.

Enzyme internal

Rebaudioside A conv.¹
Rebaudioside D

reference
GI Number
Version
(reaction time)
formation¹

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

Note:

¹Based on initial concentration of Rebaudioside A

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), thermostated column compartment (G1316B), DAD detector (G1315C), connected with Agilent 6110A MSD, and interfaced with “LC/MSD Chemstation” software, was used.

Instrument Conditions

Column
Phenomenex Prodigy 3u C18 100 A,

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 shown in FIG. 62 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 X 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.

Example 32

In Vivo Preparation and Activity Determination of UGTLB

UGTLB (GI_209954733/BAG80557.1) amino acid

sequence (SEQ ID NO: 10):

_MGTEVTVHKNTLRVLMFPWLAYGHISPFLNVAKKLVDRGFLIYLCSTAI

NLKSTIKKIPEKYSDSIQLIELHLPELPELPPHYHTTNGLPPHLNHTLQ

KALKMSKPNFSKILQNLKPDLVIYDLLQQWAEGVANEQNIPAVKLLTSG

AAVLSYFFNLVKKPGVEFPFPAIYLRKNELEKMSELLAQSAKDKEPDGV

DPFADGNMQVMLMSTSRIIEAKYIDYFSGLSNWKVVPVGPPVQDPIADD

ADEMELIDWLGKKDENSTVFVSFGSEYFLSKEDREEIAFGLELSNVNFI

WVARFPKGEEQNLEDALPKGFLERIGDRGRVLDKFAPQPRILNHPSTGG

FISHCGWNSVMESVDFGVPIIAMPIHLDQPMNARLIVELGVAVEIVRDD

YGKIHREEIAEILKDVIAGKSGENLKAKMRDISKNLKSIRDEEMDTAAE

ELIQLCKNSPKLK

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 MgCl₂in 50 mM Sodium Phosphate buffer at pH 7.2. Samples were taken and analyzed by HPLC. The corresponding chromatograms are depicted in FIG. 63a.

Enzyme

Stevioside

internal

conv.¹
Rebaudioside E

reference
GI Number
Version
(reaction time)
formation¹

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

Note:

¹Based on initial concentration of Stevioside

Determination of Activity for Rubusoside to Rebaudioside E Conversion with UGTLB

Activity tests were performed at 3 mL scale with 600 μL of lysate for the transformation of Rubusoside using 0.5 mM of substrate, 2.5 mM of UDP-Glucose and 3 mM MgCl₂in 50 mM Sodium Phosphate buffer at pH 7.2. Samples were taken and analyzed by HPLC. The corresponding chromatograms are depicted in FIG. 63b.

Enzyme

Rubusoside

internal

conv.¹
Rebaudioside E

reference
GI Number
Version
(reaction time)
formation¹

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

Note:

¹Based on initial concentration of Rubusoside

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 MgCl₂in 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. 63c.

Enzyme

Rebaudioside
Rebaudioside

internal

A 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

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.

LC-MS, peak area ratio (%)

Unknown peak
Unknown peak
Unknown peak

Sample
UGT (reaction

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

ID
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

LC-MS, peak area ratio (%)

Unknown peak
Unknown peak
Unknown peak

UGT (reaction

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

Sample ID
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 NO: 11:

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 CelLytic™ 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 MgCl₂in 50 mM Sodium Phosphate buffer at pH 7.2. Samples were taken and analyzed by HPLC. The corresponding chromatogram is depicted in FIG. 64.

Enzyme
Rebaudioside D
Rebaudioside M

internal reference
conv.¹(reaction time)
selectivity¹

UGT76G1_Sc
85% (21 h.)
100%

Note:

¹Based on initial concentration of Rebaudioside D

Example 36

In Vivo Production of UGTSL in S. cerevisiae

SEQ ID NO: 12:

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 CelLytic™ Y Cell Lysis Reagent (Sigma). The lysates were used directly for activity testing (UGTSL_Sc).

Example 37

Determination of Activity of UGTSL_Sc for the Conversion of Rebaudioside A to Rebaudioside D UGTSL_Sc was prepared according to EXAMPLE 36. Activity tests were performed at 2 mL scale with 200 μL of lysate for the transformation of Rebaudioside A using 0.5 mM of substrate, 2.5 mM of UDP-Glucose and 3 mM MgCl₂in 50 mM Sodium Phosphate buffer at pH 7.2. Samples were taken and analyzed by HPLC. The corresponding chromatogram is depicted in FIG. 65.

Enzyme
Rebaudioside A
Rebaudioside D

internal reference
conv.¹(reaction time)
selectivity¹

UGTSL_Sc
46% (4 h)
42%

Note:

¹Based on initial concentration of Rebaudioside A

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

^aassignments made on the basis of COSY, HMQC and HMBC correlations;

^bChemical shift values are in δ (ppm);

^cCoupling 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

[Image Omitted]

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 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.

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-d₅and NMR data were acquired on a Bruker Avance 500 MHz instrument with a 2.5 mm inverse detection probe. The ¹H NMR spectrum was referenced to the residual solvent signal (δ_H8.74 and δ_C150.35 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 C₅₀H₇₉O₂₈1127.4758, 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), and 1D-TOCSY were performed to allow assignment of the compound.

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 δ_C69.9 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 δ_H1.29 to the carbonyl at δ_C177.7 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 δ_C38.9, 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 δ_C38.9 was a methylene group and the carbon at δ_C57.8 was a methine which were assigned as C-3 and C-5, respectively. This left the carbon at δ_C45.0, 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 (δ_H0.98 and 2j.36) and C-5 (δ_H1.04) were assigned using the HSQC-DEPT data. A COSY correlation between one of the H-3 protons (δ_H0.98) and a proton at δ_H1.43 allowed assignment of one of the H-2 protons which in turn showed a correlation with a proton at δ_H0.75 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 following table.

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 δ_H1.30 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 (δ_C40.3) and a methine carbon (δ_C54.5) which were assigned as C-10 and C-9, respectively. COSY correlations between H-5 (δ_H1.04) and protons at δ_H1.92 and 2.43 then allowed assignment of the H-6 protons which in turn showed correlations to protons at δ_H1.22 and 1.30 which were assigned to C-7. The ¹³C chemical shifts for C-6 (δ_C22.7) and C-7 (δ_C42.2) were then determined from the HSQC-DEPT data. COSY correlations between H-9 (δ_H0.88) and protons at δ_H1.65 and 1.69 allowed assignment of the H-11 protons which in turn showed COSY correlations to protons at δ_H1.99 and 2.25 which were assigned as the H-12 protons. The HSQC-DEPT data was then used to assign C-11 (δ_C21.1) and C-12 (δ_C37.5). HMBC correlations from the H-12 proton (δ_H2.25) to carbons at δ_C87.1 and 154.7 allowed assignment of C-13 and C-16, respectively. The olefinic protons observed at δ_H5.01 and 5.64 showed HMBC correlations to C-13 and were assigned to C-17 (δ_C105.2 via HSQC-DEPT). The olefinic protons H-17 and the methine proton H-9 showed HMBC correlations to a carbon at δ_C48.3 which was assigned as C-15. An additional HMBC correlation from H-9 to a methylene carbon at δ_C44.5 then allowed assignment of C-14. The ¹H chemical shifts at C-14 (δ_H1.80 and 2.65) and C-15 (δ_H1.31 and 2.04) were assigned using the HSQC-DEPT data.

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

[Image Omitted]

Analysis of the ¹H-¹³C HSQC-DEPT data confirmed the presence of five anomeric protons. Three of the anomeric protons were well resolved at δ_H6.02 (δ_C96.1), 5.57 (δ_C105.3), and 5.34 (δ_C105.3) in the ¹H NMR spectrum. The remaining two anomeric protons observed at δ_H5.04 (δ_C105.6) and 5.07 (δ_C98.7) which were obscured by solvent (HOD) resonance in the ¹H spectrum were identified by ¹H-¹³C HSQC-DEPT data. The anomeric proton observed at δ_H6.02 showed HMBC correlation to C-19 which indicated that it corresponds to the anomeric proton of Glc_I. Similarly, the anomeric proton observed at δ_H5.07 showed an HMBC correlation to C-13 allowing it to be assigned as the anomeric proton of Glc_II.

The Glc_Ianomeric proton (δ_H6.02) showed a COSY correlation to a proton at δ_H4.07 was assigned as Glc_IH-2 which in turn showed a COSY correlation to a proton at δ_H4.22 (Glc_IH-3) which showed a COSY correlation with a proton at δ_H4.12 (Glc_IH-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 Glc_Ianomeric proton with several different mixing times. In addition to confirming the assignments for Glc_IH-2 through H-4, the 1D-TOCSY data showed a proton at δ_H4.04 assigned as Glc_IH-5 and a proton at δ_H4.68 assigned as one of the Glc_IH-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 Glc_IH-1 to H-5 as well as the remaining methylene proton of H-6 (δ_H4.30). Assignment of the ¹³C chemical shifts for Glc_IC-2 (δ_C74.2), C-3 (δ_C79.1), C-4 (δ_C72.1), C-5 (δ_C78.5), and C-6 (δ_C69.9) was determined using the ¹H-¹³C HSQC-DEPT data to complete the assignment of Glc_I. Furthermore, the presence of a methylene ¹³C resonance at δ_C69.9 in the ¹H-¹³C HSQC-DEPT spectrum indicated a 1→6 sugar linkage of Glc_Iin the structure.

Out of four remaining unassigned glucose moieties, one was assigned as a substituent at C-6 of Glc_Ion the basis of ¹H-¹³C HSQC-DEPT, HMBC, and 1D-TOCSY correlations. The relatively downfield shift of a methylene ¹³C resonance of Glc_Iat δ_C69.9 in the HSQC-DEPT spectrum indicated a 1→6 sugar linkage of Glc_I. The anomeric proton observed at δ_H5.04 showed HMBC correlation to Glc_IC-6 and was assigned as the anomeric proton of Glc_V. Similarly, methylene protons of Glc_Ishowed HMBC correlations to anomeric carbon of Glc_Vconfirming the presence of a 1→6 sugar linkage between Glc_Iand Glc_V. The Glc_Vanomeric proton showed a COSY correlation to a proton at δ_H4.00 which was assigned as Glc_VH-2 which in turn showed a COSY correlation to a proton at δ_H4.22 (Glc_VH-3). Due to data overlap, the COSY spectrum did not allow assignment of Glc_VH-4 based on the COSY correlation of Glc_VH-3. However, in the HMBC spectrum, Glc_VH-3 showed a correlation to Glc_VC-5 (δ_C78.9). In HSQC-DEPT spectrum, Glc_VC-5 showed a correlation to δ_H3.89 (Glc_VH-5). The Glc_VH-5 showed COSY correlations to δ_H4.21, 4.37, and 4.48. In the HSQC-DEPT spectrum, δ_H4.21 showed a correlation to δ_C71.4 (Glc_VH-4), while δ_H4.37 and 4.48 showed a correlation to δ_C63.1 and were assigned to Glc_VH-6a and H-6b, respectively. Assignment of the ¹³C chemical shifts for Glc_VC-2 (δ_C75.7) and C-3 (δ_C79.1) was determined using the ¹H-¹³C HSQC-DEPT data to complete the assignment of Glc_V.

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

Glc_I-1
96.1
6.02
d (8.1)

Glc_I-2
74.2
4.07
m

Glc_I-3
79.1^#
4.22
m^#

Glc_I-4
72.1
4.12
m

Glc_I-5
78.5
4.04
m

Glc_I-6
69.9
4.30
m

4.68
d (10.7)

Glc_V-1
105.6
5.04*

Glc_V-2
75.7
4.00
m

Glc_V-3
79.1^#
4.22
m^#

Glc_V-4
71.4
4.21
m

Glc_V-5
78.9
3.89
m

Glc_V-6
63.1
4.37
m

4.48
m

*Anomeric proton was obscured by solvent (HDO) resonance. Therefore, the coupling constant value could not be determined.

^#1H and ¹³C values can be exchangeable between positions Glc_I-3, Glc_V-3 and Glc_IV-3.

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

[Image Omitted]

Assignment of Glc_IIwas carried out in a similar manner. The Glc_IIanomeric proton (δ_H5.07) showed a COSY correlation to a proton at δ_H4.37, assigned as Glc_IIH-2, which in turn showed a COSY correlation to a proton at δ_H4.18 (Glc_IIH-3). This latter proton showed an additional correlation with a proton at δ_H3.88 (Glc_IIH-4) which also showed a COSY correlation to a proton at δ_H3.79 (Glc_IIH-5). Glc_IIH-5 also showed a COSY correlation to Glc_IIH-6 protons (δ_H4.08 and 4.46). Assignment of the ¹³C chemical shifts for Glc_IIC-2 (δ_C81.3), C-3 (δ_C88.4), C-4 (δ_C71.1), C-5 (δ_C77.9), and C-6 (δ_C63.2) was determined using the HSQC-DEPT data. HMBC correlations from Glc_IIH-3 to C-2 and C-4 and also from Glc_IIH-4 to C-2 and C-5 confirmed the assignments made above. Additional HMBC correlations of Glc_IIH-4 to Glc_IIC-6 further support to complete the assignment of Glc_II.

Two of the remaining unassigned glucose moieties were assigned as substituents at C-2 and C-3 of Glc_IIon the basis of HMBC correlations. The anomeric proton observed at δ_H5.57 showed a HMBC correlation to Glc_IIC-2 and was assigned as the anomeric proton of Glc_II. The anomeric proton observed at δ_H5.34 showed a HMBC correlation to Glc_IIC-3 and was assigned as the anomeric proton of Glc_IV. The reciprocal HMBC correlations from Glc_IIH-2 to the anomeric carbon of Glc_IIand from Glc_IIH-3 to the anomeric carbon of Glc_IVwere also observed.

The anomeric proton of Glc_II(δ_H5.57) showed a COSY correlation with a proton at δ_H4.19 which was assigned as Glc_IIH-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 Glc_IIanomeric proton with several different mixing times. In addition to confirming the assignments for Glc_IIIH-2, the 1D-TOCSY data showed protons at δ_H4.24 (Glc_IIIH-3), δ_H4.27 (Glc_IIIH-4), and SH 3.94 (Glc_IIIH-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, Glc_IIIH-4 showed a correlation to Glc_IIIH-5, which in turn showed COSY correlations to δ_H4.41 and 4.50 of Glc_IIIH-6a and H-6b, respectively. The ¹³C chemical shifts for Glc_IIC-2 (δ_C76.8), C-3 (δ_C78.9), C-4 (δ_C72.4), C-5 (δ_C78.8), and C-6 (δ_C63.5) were then determined using the ¹H-¹³C HSQC-DEPT correlations to complete the assignment of Glc_III.

The anomeric proton of Glc_IV(δ_H5.34) showed a COSY correlation with a proton at δ_H4.06 which was assigned as Glc_IVH-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 Glc_IVanomeric proton with several different mixing times. In addition to confirming the assignments for Glc_IVH-2, the 1D-TOCSY data showed protons at δ_H4.22 (Glc_IVH-3), SH 4.18 (Glc_IVH-4), and SH 4.10 (Glc_IVH-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, Glc_IVH-4 showed a correlation to Glc_IVH-5, which in turn showed COSY correlations to δ_H4.32 and 4.58, Glc_IVH-6a and H-6b, respectively. The ¹³C chemical shifts for Glc_IVC-2 (δ_C75.8), C-3 (δ_C78.9), C-4 (δ_C72.0), C-5 (δ_C79.3), and C-6 (δ_C62.9) were then determined using the ¹H-¹³C HSQC-DEPT correlations to complete the assignment of Glc_IV.

A summary of the ¹H 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

Glc_II-1
98.7
5.07*

Glc_II-2
81.3
4.37
m

Glc_II-3
88.4
4.18
t (9.0)

Glc_II-4
71.1
3.88
t (8.6)

Glc_II-5
77.9
3.79
m

Glc_II-6
63.2
4.08
m

4.46
m

Glc_III-1
105.3
5.57
d (7.6)

Glc_III-2
76.8
4.19
m

Glc_III-3
78.9
4.24
m

Glc_III-4
72.4
4.27
m

Glc_III-5
78.8
3.94
m

Glc_III-6
63.5
4.41
m

4.50
m

Glc_IV-1
105.3
5.34
d (7.9)

Glc_IV-2
75.8
4.06
m

Glc_IV-3
78.9^#
4.22
m^#

Glc_IV-4
72.0
4.18
m

Glc_IV-5
79.3
4.10
m

Glc_IV-6
62.9
4.32
m

4.58
m

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

[Image Omitted]

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.

[Image Omitted]

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 μL. 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 QT of 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 D20 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 (δ_H4.79 ppm) and TSP (δ_C0.00 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. 21), ¹³C NMR (FIGS. 22 and 23), ¹H-¹H COSY (FIG. 24), HSQC-DEPT (FIG. 25), HMBC (FIGS. 26 and 27), and 1D-TOCSY 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 δ_C70.9 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 δ_H1.29 to the carbonyl at δ_C181.5 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 δ_C39.8, 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 δ_C39.8 was a methylene group and the carbon at δ_C59.2 was a methine which were assigned as C-3 and C-5, respectively. This left the carbon at δ_C43.7, 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 (δ_H1.16 and 2.28) and C-5 (δ_H1.24) were assigned using the HSQC-DEPT data. A COSY correlation between one of the H-3 protons (δ_H1.16) and a proton at δ_H1.49 allowed assignment of one of the H-2 protons which in turn showed a correlation with a proton at δ_H0.92 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 δ_H0.92 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 (δ_C42.4) and a methine (δ_C55.5) which were assigned as C-10 and C-9, respectively. COSY correlations between H-5 (δ_H1.24) and protons at δ_H1.73 and 1.94 then allowed assignment of the H-6 protons which in turn showed correlations to protons at δ_H1.49 and 1.56 which were assigned to C-7. The ¹³C chemical shifts for C-6 (δ_C24.4) and C-7 (δ_C44.2) were then determined from the HSQC-DEPT data. COSY correlations between H-9 (δ_H1.09) and protons at δ_H1.66 and 1.70 allowed assignment of the H-11 protons which in turn showed COSY correlations to protons at δ_H1.60 and 2.00 which were assigned as the H-12 protons. The HSQC-DEPT data was then used to assign C-11 (δ_C22.6) and C-12 (δ_C39.9). The olefinic protons observed at δ_H4.98 and 5.16 showed HMBC correlations to C-13 (δ_C90.9) and were assigned to C-17 (Sc 107.0 via HSQC-DEPT). The olefinic protons H-17 showed HMBC correlations to a carbon at δ_C49.4 which was assigned as C-15. An additional HMBC correlation from H-9 to a methylene carbon at δ_C46.9 then allowed assignment of C-14. The ¹H chemical shifts at C-14 (δ_H1.53 and 2.21) and C-15 (δ_H2.15 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:

[Image Omitted]

Analysis of the ¹H-¹³C HSQC-DEPT data confirmed the presence of six anomeric protons. Three of the anomeric protons were well resolved at δ_H5.65 (δ_C95.5), 4.92 (δ_C104.9), and 4.50 (δ_C105.7) in the ¹H NMR spectrum. The remaining three anomeric protons observed at δ_H4.85 (δ_C98.4), 4.84 (δ_C105.0), and 4.83 (δ_C105.3) were overlapped by the residual solvent resonance in the ¹H spectrum. The anomeric proton observed at δ_H5.65 showed a HMBC correlation to C-19 which indicated that it corresponds to the anomeric proton of Glc_I. Similarly, the anomeric proton observed at δ_H4.85 showed a HMBC correlation to C-13 allowing it to be assigned as the anomeric proton of Glc_II.

The Glc_Ianomeric proton (δ_H5.65) showed a COSY correlation to a proton at δ_H3.96 which was assigned as Glc_IH-2 which in turn showed a COSY correlation to a proton at δ_H3.89 (Glc_IH-3) which showed a COSY correlation with a proton at δ_H3.71 (Glc_IH-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 Glc_Ianomeric proton with several different mixing times. In addition to confirming the assignments for Glc_IH-2 through H-4, the 1D-TOCSY data showed a proton at δ_H3.73 assigned as Glc_IH-5 and a proton at δ_H4.15 assigned as one of the Glc_IH-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 Glc_IH-1 to H-5 as well as the remaining methylene proton of H-6 (δ_H4.00). Assignment of the ¹³C chemical shifts for Glc_IC-2 (δ_C80.5), C-3 (δ_C79.0), C-4 (δ_C71.5), C-5 (δ_C79.0), and C-6 (δ_C70.9) was determined using the ¹H-¹³C HSQC-DEPT data to complete the assignment of Glc_I. Furthermore, the presence of a methylene ¹³C resonance at δ_C70.9 in the ¹H-¹³C HSQC-DEPT spectrum indicated a 1→6 sugar linkage of Glc_Iin the structure.

Two of the unassigned glucose moieties were assigned as substituents at C-2 and C-6 of Glc_Ion the basis of HMBC correlations. The anomeric proton observed at δ_H4.83 showed an HMBC correlation to Glc_IC-2 and was assigned as the anomeric proton of Glc_V. The anomeric proton observed at δ_H4.50 showed a HMBC correlation to Glc_IC-6 and was assigned as the anomeric proton of Glc_VI. The reciprocal HMBC correlations from Glc_IH-2 to the anomeric carbon of Glc_Vand from Glc_IH-6 to the anomeric carbon of Glc_VIwere also observed.

The anomeric proton of Glc_V(δ_H4.83) showed a COSY correlation with a proton at δ_H3.32 which was assigned as Glc_VH-2. The Glc_VH-2 in turn showed a COSY correlation to a proton at δ_H3.51 (Glc_VH-3). This latter proton showed an additional correlation with a proton at δ_H3.38 (Glc_VH-4). H-4 also showed a COSY correlation to a proton at δ_H3.55 (Glc_VH-5) and Glc_VH-5 in turn showed a COSY correlation to Glc_VH-6 protons (δ_H3.76 and 3.97). Assignment of the ¹³C chemical shifts for Glc_VC-2 (δ_C78.5), C-3 (δ_C78.7), C-4 (δ_C72.9), C-5 (δ_C78.8), and C-6 (δ_C63.6) was determined using the HSQC-DEPT data. HMBC correlations from Glc_VH-3 to C-2 and C-4 and also from Glc_VH-4 to C-3 and C-6 confirmed the assignments made above to complete the assignment of Glc_V.

Another glucose moiety was assigned as a substituent at C-6 of Glc_Ion the basis of ¹H-¹³C HSQC-DEPT and HMBC correlations. The relatively downfield shift of a methylene ¹³C resonance of Glc_Iat δ_C70.9 in the HSQC-DEPT spectrum indicated a 1→6 sugar linkage of Glc_I. The anomeric proton observed at δ_H4.50 showed a HMBC correlation to Glc_IC-6 and was assigned as the anomeric proton of Glc_V1. Similarly, methylene protons of Glc_Ishowed HMBC correlations to the anomeric carbon of Glc_VIand this confirmed the presence of a 1→6 sugar linkage between Glc_Iand Glc_VI. The Glc_VIanomeric proton showed a COSY correlation to a proton at δ_H3.33 which was assigned as Glc_VIH-2 which in turn showed a COSY correlation to a proton at δ_H3.49 (Glc_VIH-3). Due to data overlap, the COSY spectrum did not allow assignment of Glc_VH-4 to H-6 based on the COSY correlations. Therefore, a series of 1D-TOCSY experiments were performed using selective irradiation of the Glc_VIanomeric proton with different mixing times. In addition to confirming the assignments for Glc_VIH-2 through H-3, the 1D-TOCSY data showed protons at δ_H3.45 (Glc_VIH-4) and δ_H3.48 (Glc_VIH-5) and protons at δ_H3.92 and 3.94 assigned for Glc_VIH-6 protons. Assignment of the ¹³C chemical shifts for Glc_V1 C-2 (δ_C78.1), C-3 (δ_C78.6), C-4 (δ_C72.3), C-5 (δ_C78.8), and C-6 (δ_C64.1) was determined using the ¹H-¹³C HSQC-DEPT data to complete the assignment of Glc_VI.

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

Glc_I-1
95.5
5.65
d (7.6)

Glc_I-2
80.5
3.96
m

Glc_I-3
79.0
3.89
m

Glc_I-4
71.5
3.71
m

Glc_I-5
79.0
3.73
m

Glc_I-6
70.9
4.00
m

4.15
d (11.7)

Glc_V-1
105.3*
4.83*
d (8.0)

Glc_V-2
78.5
3.32
m

Glc_V-3
78.7
3.51
m

Glc_V-4
72.9
3.38
m

Glc_V-5
78.8
3.55
m

Glc_V-6
63.6
3.76
m

3.97
m

Glc_VI-1
105.7
4.50
d (7.9)

Glc_VI-2
78.1
3.33
m

Glc_VI-3
78.6
3.49
m

Glc_VI-4
72.3
3.45
m

Glc_VI-5
78.8
3.48
m

Glc_VI-6
64.1
3.92
m

3.94
m

*¹H and ¹³C values can be exchangeable with Glc_IV-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

Glc_II-1
98.4
4.85
d (7.8)

Glc_II-2
81.7
3.75
m

Glc_II-3
88.0
3.98
m

Glc_II-4
71.3
3.54
m

Glc_II-5
80.5
3.96
m

Glc_II-6
63.6
3.45
m

3.77
m

Glc_III-1
104.9
4.92
d (7.9)

Glc_III-2
76.3
3.32
m

Glc_III-3
78.8
3.51
m

Glc_III-4
73.3
3.26
t (9.5)

Glc_III-5
78.8
3.44
m

Glc_III-6
64.4
3.75
m

3.94
m

Glc_IV-1
105.0
4.84
d (7.8)

Glc_IV-2
76.1
3.41
m

Glc_IV-3
78.8
3.46
m

Glc_IV-4
72.5
3.45
m

Glc_IV-5
81.7
3.75
m

Glc_IV-6
65.8
3.55
m

3.78
m

Assignment of Glc_IIwas carried out in a similar manner. The Glc_IIanomeric proton (δ_H4.85) showed a COSY correlation to a proton at δ_H3.75 which was assigned as Glc_IIH-2 which in turn showed a COSY correlation to a proton at δ_H3.98 (Glc_IIH-3). This latter proton showed an additional correlation with a proton at δ_H3.54 (Glc_IIH-4). H-4 also showed a COSY correlation to a proton at δ_H3.96 (Glc_IIH-5). Glc_IIH-5 also showed a COSY correlation to Glc_IIH-6 protons (δ_H3.77 and 3.45). Assignment of the ¹³C chemical shifts for Glc_IIC-2 (δ_C81.7), C-3 (δ_C88.0), C-4 (δ_C71.3), C-5 (δ_C80.5), and C-6 (δ_C63.6) was determined using the HSQC-DEPT data. HMBC correlations from Glc_IIH-3 to C-2 and C-4 and also from Glc_IIH-4 to C-3 and C-6 confirmed the assignments made above to complete the assignment of Glc_II.

Two of the remaining unassigned glucose moieties were assigned as substituents at C-2 and C-3 of Glc_IIon the basis of HMBC correlations. The anomeric proton observed at δ_H4.92 showed a HMBC correlation to Glc_IIC-2 and was assigned as the anomeric proton of Glc_III. The anomeric proton observed at δ_H4.84 showed a HMBC correlation to Glc_IIC-3 and was assigned as the anomeric proton of Glc_IV. The reciprocal HMBC correlations between Glc_IIH-2 and the anomeric carbon of Glc_IIIand between Glc_IIH-3 and the anomeric carbon of Glc_IVwere also observed.

The anomeric proton of Glc_III(δ_H4.92) showed a COSY correlation with a proton at δ_H3.32 which was assigned as Glc_IIIH-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 Glc_IIIanomeric proton with different mixing times. In addition to confirming the assignments for Glc_IIIH-2, the 1D-TOCSY data showed protons at δ_H3.51 (Glc_IIIH-3), δ_H3.26 (Glc_IIIH-4), and δ_H3.44 (Glc_IIIH-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, Glc_IIIH-4 showed a correlation to Glc_IIIH-5, which in turn showed COSY correlations to δ_H3.94 and 3.75 of Glc_IIIH-6a and H-6b, respectively. The ¹³C chemical shifts for Glc_IIC-2 (δ_C76.3), C-3 (δ_C78.8), C-4 (δ_C73.3), C-5 (δ_C78.8), and C-6 (δ_C64.4) were then determined using the ¹H-¹³C HSQC-DEPT correlations to complete the assignment of Glc_III.

The anomeric proton of Glc_IV(δ_H4.84) which showed a COSY correlation to a proton at δ_H3.41 was assigned as Glc_IVH-2 which in turn showed a COSY correlation to a proton at δ_H3.46 (Glc_IVH-3). This latter proton showed an additional correlation with a proton at δ_H3.45 (Glc_IVH-4) which also showed a COSY correlation to a proton at δ_H3.75 (Glc_IVH-5). Glc_IVH-5 also showed a COSY correlation to Glc_IVH-6 protons (δ_H3.55 and 3.78). Assignment of the ¹³C chemical shifts for Glc_IVC-2 (δ_C76.1), C-3 (δ_C78.8), C-4 (δ_C72.5), C-5 (δ_C81.7), and C-6 (δ_C65.8) was determined using the HSQC-DEPT data. HMBC correlations from Glc_IVH-3 to C-4 and C-5 and also from Glc_IVH-4 to C-3 and C-6 confirmed the assignments made above to complete the assignment of Glc_IV.

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

Glc_II-1
98.4
4.85
d (7.8)

Glc_II-2
81.7
3.75
m

Glc_II-3
88.0
3.98
m

Glc_II-4
71.3
3.54
m

Glc_II-5
80.5
3.96
m

Glc_II-6
63.6
3.45
m

3.77
m

Glc_III-1
104.9
4.92
d (7.9)

Glc_III-2
76.3
3.32
m

Glc_III-3
78.8
3.51
m

Glc_III-4
73.3
3.26
t (9.5)

Glc_III-5
78.8
3.44
m

Glc_III-6
64.4
3.75
m

3.94
m

Glc_IV-1
105.0
4.84
d (7.8)

Glc_IV-2
76.1
3.41
m

Glc_IV-3
78.8
3.46
m

Glc_IV-4
72.5
3.45
m

Glc_IV-5
81.7
3.75
m

Glc_IV-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:

[Image Omitted]

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.

[Image Omitted]

Example 41

Directed Evolution of UGT76G1 for the Conversion of Rebaudioside D to Rebaudioside X (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 ProteinGPS™ 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+_UGT76Glvar 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 Express™ 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 X. 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 initial

Clone
Mutations*
Reb D to Reb X after 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 NO: 13:

MANAERMITRVHSQRERLNETLVSERNEVLALLSRVEAKGKGILQQNQII

AEFEALPEQTRKKLEGGPFFDLLKSTQEAIVLPPWVALAVRPRPGVWEYL

RVNLHALVVEELQPAEFLHFKEELVDGVKNGNFTLELDFEPFNASIPRPT

LHKYIGNGVDFLNRHLSAKLFHDKESLLPLLKFLRLHSHQGKNLMLSEKI

QNLNTLQHTLRKAEEYLAELKSETLYEEFEAKFEEIGLERGWGDNAERVL

DMIRLLLDLLEAPDPCTLETFLGRVPMVFNVVILSPHGYFAQDNVLGYPD

TGGQVVYILDQVRALEIEMLQRIKQQGLNIKPRILILTRLLPDAVGTICG

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 Bl21(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 X 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 H₂SO₄and 115 μL of 60% methanol. 68% of Rebaudioside X and 26% of Rebaudioside M2 was obtained after 21 h of reaction time, as shown in FIG. 66.

Example 44

Directed Evolution of UGT76G1 for the Conversion of Rebaudioside D to Rebaudioside X (Round 3)

The most active clone from the second round of directed evolution of UGT76G1 (see EXAMPLE 41 round2_UGT76G1var66 containing mutations: S42A_F46I_I407V) was chosen as baseline clone for round 3. A list of 56 mutations was established containing different identified positive mutations from the second round and 30 new mutations obtained by DNA2.0 ProteinGPS™ strategy. This list of mutations was subsequently used to design 92 variant genes that contained each 3 or 4 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+_UGT76Glvar 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 Express™ 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 1, 2, 4, 6 and 22 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 X. In parallel the experiments were performed with baseline clone, Round2-Var66. The conversion after 22 h. and initial rate for this baseline clone was defined as 10000 and the normalized conversions and initial rates for the round 3 clones are depicted in the following table:

Normalized conversion
Normalized initial

Clone
Mutations*
Reb D to Reb X after 22 h.
rate (0-4 h)

Round2-Var66
UGT76G1
100%
100%

(S42A_F46I_Q266E_P272A_R334K_G348P_L379G_I407V)

Baseline clone

Round3-Var1
Round2-Var66 (I46F_L121I_E229A_K393R)
42%
96%

Round3-Var2
Round2-Var66 (F18V_A213N_E266S)
7%
36%

Round3-Var3
Round2-Var66 (F41L_I190L_A239V_K316R)
20%
64%

Round3-Var4
Round2-Var66 (N138K_S274G_Q425E_S456L)
92%
104%

Round3-Var5
Round2-Var66 (F22Y_E229S_V407I_N409R)
15%
66%

Round3-Var6
Round2-Var66 (F150A_G216A_T355S_S447A)
15%
50%

Round3-Var7
Round2-Var66 (V394I_N409R_Q425E_S447A)
72%
97%

Round3-Var8
Round2-Var66 (Y37H_F41L_N409R_Q425E)
6%
37%

Round3-Var9
Round2-Var66 (L121V_F182L_K303G_E331G)
75%
95%

Round3-Var10
Round2-Var66 (S274G_K303G_N409R_Q432E)
99%
106%

Round3-Var11
Round2-Var66 (F41L_K303G_F314S)
26%
67%

Round3-Var12
Round2-Var66 (F22Y_R141S_T284V)
3%
15%

Round3-Var13
Round2-Var66 (I190L_E229A_T284V)
31%
70%

Round3-Var14
Round2-Var66 (K303G_Q425E_S447A)
109%
114%

Round3-Var15
Round2-Var66 (K316R_L383V_V394I)
107%
117%

Round3-Var16
Round2-Var66 (I190L_K303G_S447A_S456L)
112%
110%

Round3-Var17
Round2-Var66 (N138G_V264C_A352G_S447A)
102%
107%

Round3-Var18
Round2-Var66 (S274K_V407I_Q425E)
91%
107%

Round3-Var19
Round2-Var66 (I190L_S274G_K393R_V394I)
120%
108%

Round3-Var20
Round2-Var66 (A213N_L277I_Q425E_E449D)
79%
101%

Round3-Var21
Round2-Var66 (I46L_K303G_K393R)
147%
117%

Round3-Var22
Round2-Var66 (S221T_S274G_S375Q)
19%
65%

Round3-Var23
Round2-Var66 (Y37H_L383V_S456L)
67%
99%

Round3-Var24
Round2-Var66 (N138G_I190L_I295T_N409R)
45%
84%

Round3-Var25
Round2-Var66 (A42S_S119A_K303G_V407I)
92%
99%

Round3-Var26
Round2-Var66 (F22Y_I46F_I190L_V394I)
76%
95%

Round3-Var27
Round2-Var66 (N138K_A213N_F314S)
83%
92%

Round3-Var28
Round2-Var66 (D301N_F314S_V394I_N409R)
76%
86%

Round3-Var29
Round2-Var66 (G216A_E266S_Q432E)
70%
88%

Round3-Var30
Round2-Var66 (N138K_A239V_P382R_K393R)
42%
76%

Round3-Var31
Round2-Var66 (I46L_S274G_K316R_S456L)
149%
109%

Round3-Var32
Round2-Var66 (F18V_I190L_S375Q_S456L)
1%
2%

Round3-Var33
Round2-Var66 (N138K_R141S_S274G)
18%
57%

Round3-Var34
Round2-Var66 (N138K_K393R_N409R_S447A)
59%
82%

Round3-Var35
Round2-Var66 (I295T_K303G_P382R_V394I)
31%
70%

Round3-Var36
Round2-Var66 (N138K_I218V_S456L)
54%
81%

Round3-Var37
Round2-Var66 (M145R_S274K_L383V)
1%
1%

Round3-Var38
Round2-Var66 (F182L_A352G_V394I)
86%
96%

Round3-Var39
Round2-Var66 (A42S_N138G_E229A_S456L)
21%
77%

Round3-Var40
Round2-Var66 (R141S_I190L_E331G_Q425E)
6%
35%

Round3-Var41
Round2-Var66 (E229S_K316R_T355S)
32%
81%

Round3-Var42
Round2-Var66 (I46F_N138K_F292L_N409R)
30%
83%

Round3-Var43
Round2-Var66 (A42S_F182L_L277I_T355S)
40%
89%

Round3-Var44
Round2-Var66 (S274G_T284V_Q425E)
85%
93%

Round3-Var45
Round2-Var66 (A272P_E331G_V394I_S447A)
88%
96%

Round3-Var46
Round2-Var66 (S274G_F314S_Q432E_S447A)
112%
104%

Round3-Var47
Round2-Var66 (L121I_K316R_S375Q_N409R)
24%
76%

Round3-Var48
Round2-Var66 (L121I_N138K_F150A_K303G)
40%
83%

Round3-Var49
Round2-Var66 (I46F_V264C_Q432E)
61%
98%

Round3-Var50
Round2-Var66 (F150A_A272P_D301N_K316R)
44%
88%

Round3-Var51
Round2-Var66 (I46L_R64V_A239V)
28%
71%

Round3-Var52
Round2-Var66 (L121I_I218V_F314S)
87%
94%

Round3-Var53
Round2-Var66 (I190L_G216A_E449D)
49%
90%

Round3-Var54
Round2-Var66 (S274G_I295M_F314S)
128%
106%

Round3-Var55
Round2-Var66 (F22Y_S274G_P382R_Q432E)
39%
48%

Round3-Var56
Round2-Var66 (N138K_I190L_K334R)
93%
97%

Round3-Var57
Round2-Var66 (N138G_I295M_K303G)
110%
100%

Round3-Var58
Round2-Var66 (L121V_G216A_Q425E_S456L)
28%
52%

Round3-Var59
Round2-Var66 (F182L_F314S_K393R)
92%
97%

Round3-Var60
Round2-Var66 (R64V_K316R_N409K)
16%
54%

Round3-Var61
Round2-Var66 (V264C_S274G_K393R)
102%
98%

Round3-Var62
Round2-Var66 (F41L_K393R_S456L)
12%
49%

Round3-Var63
Round2-Var66 (A42S_S274G_F292L_V394I)
75%
87%

Round3-Var64
Round2-Var66 (I190L_S221T_E266S_S447A)
34%
71%

Round3-Var65
Round2-Var66 (R64V_E229S_S274K)
12%
49%

Round3-Var66
Round2-Var66 (S221T_K334R_K393R_V394I)
72%
90%

Round3-Var67
Round2-Var66 (I190L_K393R_Q425E_Q432E)
101%
102%

Round3-Var68
Round2-Var66 (F18V_N138K_M145R)
1%
1%

Round3-Var69
Round2-Var66 (I218V_F292L_K316R_S447A)
40%
79%

Round3-Var70
Round2-Var66 (L121V_E229A_K316R_Q432E)
19%
63%

Round3-Var71
Round2-Var66 (Y37H_L121V_D301N)
35%
68%

Round3-Var72
Round2-Var66 (N138K_V394I_Q432E_S456L)
66%
89%

Round3-Var73
Round2-Var66 (T284V_I295M_A352G_L383V)
69%
89%

Round3-Var74
Round2-Var66 (S119A_F150A_V394I_Q425E)
66%
88%

Round3-Var75
Round2-Var66 (F18V_A239V_S447A)
8%
27%

Round3-Var76
Round2-Var66 (K303G_N409R_Q432E)
84%
97%

Round3-Var77
Round2-Var66 (Y37H_A272P_K334R_E449D)
75%
89%

Round3-Var78
Round2-Var66 (K303G_F314S_V394I_Q425E)
121%
104%

Round3-Var79
Round2-Var66 (R141S_I295T_F314S_Q432E)
9%
29%

Round3-Var80
Round2-Var66 (N138K_I190L_F314S_N409R)
90%
97%

Round3-Var81
Round2-Var66 (S119A_E331G_S456L)
87%
97%

Round3-Var82
Round2-Var66 (K303G_F314S_K393R_S456L)
100%
100%

Round3-Var83
Round2-Var66 (N138K_A352G_V407I_Q432E)
72%
95%

Round3-Var84
Round2-Var66 (S274G_L277I_I295T)
34%
81%

Round3-Var85
Round2-Var66 (R64V_L277I_F314S_S447A)
34%
61%

Round3-Var86
Round2-Var66 (S221T_N409K_Q432E)
39%
75%

Round3-Var87
Round2-Var66 (N409R_S447A_S456L)
52%
86%

Round3-Var88
Round2-Var66 (K393R_Q425E_Q432E)
102%
99%

Round3-Var89
Round2-Var66 (I46L_F292L_S375Q_N409K)
8%
35%

Round3-Var90
Round2-Var66 (M145R_K393R_N409R)
1%
1%

Round3-Var91
Round2-Var66 (S119A_M145R_T355S_P382R)
0%
1%

Round3-Var92
Round2-Var66 (I190L_E229S_V264C_F314S)
64%
82%

*Mutations are noted as follows: reference gene-original amino acid-position-new amino acid: For example the mutation of an isoleucine at position 190 to a leucine for variant 66 from the second round of directed evolution of UGT76G1 is noted as Round2-Var66 (I190L)

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: I46L, I295M, S119A, S274G, K334R, F314S, K303G, K316R, K393R, I190L, Q425E, Q432E, N138G, V394I, F182L, V407I, A272P, V264C, E449D, A352G.

Example 45

Directed Evolution of UGTSL2 for the Conversion of Rebaudioside A to Rebaudioside D (Round 1)

Starting from native enzyme UGTSL2 (GI_460410132) a list of 60 mutations was established containing different identified positive mutations from the first round and new mutations obtained by DNA2.0 ProteinGPS™ 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+_UGTSL2var 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 Express™ 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, 6 and 22 h. to determine the initial rate by HPLC (CAD detection) using the analytical method that was described above for the transformation of Rebaudioside A to Rebaudioside D. In parallel the experiments were performed with baseline clone, UGTSL2. The initial rate for this baseline clone was defined as 10000. As an indication of the specificity of the clones, Rebaudioside M2 content was determined at 100% UDP-Glucose conversion and Rebaudioside D2 content was determined at 50% UDP-Glucose conversion. Wherein UDP glucose conversion is defined as: ([Reb D]/[Reb A]₀)+([Reb D2]/[Reb A]₀)+2*([Reb M2]/[Reb A]₀).

The normalized initial rate, Rebaudioside M2 content at 100% UDP-glucose conversion and Rebaudioside D2 content at 50% UDP-glucose conversion are depicted in the following table

Reb D2

Normalized
Reb M2 content
content at 50%

initial rate
at 100% UDP-
UDP-Glc

Clone
Mutations*
(0-4 h)
Glc conversion
conversion

UGTSL2
baseline clone
100%
100%
12.5%

Round1-Var1
UGTSL2 (L276A_N278G_T329V)
220%
98%
8.5%

Round1-Var2
UGTSL2 (S19I_E259G_V270L)
0%
0%

Round1-Var3
UGTSL2 (I323V_S334T_V368E)
0%
0%

Round1-Var4
UGTSL2 (V125I_E259G_L393V)
0%
0%

Round1-Var5
UGTSL2 (Q27R_H247P_I333L)
185%
134%
15.0%

Round1-Var6
UGTSL2 (Q27R_N325S_G387E_T392A)
148%
116%
17.0%

Round1-Var7
UGTSL2 (F253Y_N325A_K365V_G371K)
0%
0%

Round1-Var8
UGTSL2 (T245R_N325A_G331A_S334T)
8%
17%

Round1-Var9
UGTSL2 (G331A_N339S_G371K)
2%
3%

Round1-Var10
UGTSL2 (R6H_F272L_I323V)
3%
6%

Round1-Var11
UGTSL2 (R6H_F21L_T329I)
0%
0%

Round1-Var12
UGTSL2 (F21L_N280P_I282L)
0%
0%

Round1-Var13
UGTSL2 (T245R_V254L_I333V)
0%
1%

Round1-Var14
UGTSL2 (L276A_I351L_M354L_I389L)
2%
2%

Round1-Var15
UGTSL2 (S19I_I240L_I351M)
4%
9%

Round1-Var16
UGTSL2 (I131V_I333V_S334T)
3%
8%

Round1-Var17
UGTSL2 (S200F_A285V_I351M_P361G)
0%
0%

Round1-Var18
UGTSL2 (R6H_L37F_A285L)
8%
21%

Round1-Var19
UGTSL2 (H247P_N249G_K289P)
8%
17%

Round1-Var20
UGTSL2 (R6H_S19I_N325A)
50%
59%

Round1-Var21
UGTSL2 (N280P_K289P_T329I_V368E)
0%
0%

Round1-Var22
UGTSL2 (I240L_N325S_V368E)
26%
43%

Round1-Var23
UGTSL2 (A205P_T245R_K365V)
0%
0%

Round1-Var24
UGTSL2 (L276A_A341V_T392A)
255%
115%
7.5%

Round1-Var25
UGTSL2 (L37F_I351L_K365V)
7%
17%

Round1-Var26
UGTSL2 (T199S_E259G_T329I)
80%
90%
12.0%

Round1-Var27
UGTSL2 (T245R_S258T_L405V)
7%
18%

Round1-Var28
UGTSL2 (K289S_I352V_P361G)
9%
15%

Round1-Var29
UGTSL2 (L37F_V254L_V270L_I323V)
0%
0%

Round1-Var30
UGTSL2 (I240L_S258T_G387E)
127%
107%
11.0%

Round1-Var31
UGTSL2 (V270I_I282L_T329V_N339S)
0%
0%

Round1-Var32
UGTSL2 (H247P_T329I_I351L)
0%
3%

Round1-Var33
UGTSL2 (N280P_A285L_I352V_G387E)
37%
62%

Round1-Var34
UGTSL2 (S19I_I323V_N325S_P361G)
0%
0%

Round1-Var35
UGTSL2 (L37F_Q65P_F272L)
14%
24%

Round1-Var36
UGTSL2 (H247P_N280R_A285V)
32%
54%

Round1-Var37
UGTSL2 (I240L_N339S_I352V_L405V)
0%
0%

Round1-Var38
UGTSL2 (V125I_N280P_G371K)
2%
5%

Round1-Var39
UGTSL2 (F253Y_I282L_A285V)
25%
45%

Round1-Var40
UGTSL2 (I282L_R312L_N325S)
4%
8%

Round1-Var41
UGTSL2 (T199S_S258T_N278G)
0%
9%

Round1-Var42
UGTSL2 (I114V_I351M_G387E)
0%
0%

Round1-Var43
UGTSL2 (S255C_S258T_V270L)
29%
59%

Round1-Var44
UGTSL2 (Q27R_R312L_T329V)
86%
92%
12.0%

Round1-Var45
UGTSL2 (V254L_N339S_I345L)
0%
0%
11.0%

Round1-Var46
UGTSL2 (I333V_A341V_M354L)
84%
86%

Round1-Var47
UGTSL2 (F253Y_F272L_T392A)
125%
116%
12.0%

Round1-Var48
UGTSL2 (F253Y_A285L_N339S)
50%
70%

Round1-Var49
UGTSL2 (K289S_I345L_G387E)
0%
2%

Round1-Var50
UGTSL2 (I131V_E259G_V270I)
0%
0%

Round1-Var51
UGTSL2 (F272L_N280R_T329V)
0%
4%

Round1-Var52
UGTSL2 (N278G_R312L_T329I_I333L)
100%
100%
13.0%

Round1-Var53
UGTSL2 (I114V_I131V_N325S)
10%
20%

Round1-Var54
UGTSL2 (A205P_K289P_I333V_G371K)
0%
0%

Round1-Var55
UGTSL2 (S19I_F21L_S200F)
0%
0%

Round1-Var56
UGTSL2 (I131V_H247P_N278G_A285L)
109%
120%
13.0%

Round1-Var57
UGTSL2 (R312L_A341V_M367V)
14%
25%

Round1-Var58
UGTSL2 (N280R_I333L_M354L)
0%
1%

Round1-Var59
UGTSL2 (S258T_E259G_A285V_I333V)
0%
0%

Round1-Var60
UGTSL2 (P361G_I389L_L405V)
0%
0%

Round1-Var61
UGTSL2 (S255C_N280R_I345L_V368E)
0%
0%

Round1-Var62
UGTSL2 (F21L_Q65P_N280R_K289S)
0%
0%

Round1-Var63
UGTSL2 (V270I_M367V_V368E)
20%
32%

Round1-Var64
UGTSL2 (T199S_V254L_A285L)
0%
0%

Round1-Var65
UGTSL2 (S255C_N280P_G331A)
73%
82%
11.5%

Round1-Var66
UGTSL2 (N249G_K365V_M367V_I389L)
0%
0%

Round1-Var67
UGTSL2 (S200F_I333L_I351L)
0%
0%

Round1-Var68
UGTSL2 (N249G_V270L_K289S)
13%
24%

Round1-Var69
UGTSL2 (I114V_V125I_N249G)
6%
9%

Round1-Var70
UGTSL2 (V125I_K289P_N325A)
0%
1%

Round1-Var71
UGTSL2 (N249G_N325A_I352V)
43%
76%
11.5%

Round1-Var72
UGTSL2 (V270I_A285V_M354L)
196%
158%
11.5%

Round1-Var73
UGTSL2 (Q65P_V254L_M367V)
0%
0%

Round1-Var74
UGTSL2 (V270I_K289P_S334T)
0%
0%

Round1-Var75
UGTSL2 (T199S_A205P_L393V)
0%
0%

Round1-Var76
UGTSL2 (V125I_I345L_M367V_T392A)
8%
19%

Round1-Var77
UGTSL2 (A205P_I323V_T392A)
0%
0%

Round1-Var78
UGTSL2 (F21L_L37F_I131V)
0%
0%

Round1-Var79
UGTSL2 (F272L_I282L_A341V_I351L)
0%
2%

Round1-Var80
UGTSL2 (N278G_I352V_I389L)
95%
113%
11.5%

Round1-Var81
UGTSL2 (I114V_G331A_A341V_L405V)
8%
20%

Round1-Var82
UGTSL2 (Q27R_Q65P_I351M)
0%
0%

Round1-Var83
UGTSL2 (R6H_T329V_M354L_L393V)
77%
100%
10.5%

Round1-Var84
UGTSL2 (S200F_G331A_L393V)
0%
0%

Round1-Var85
UGTSL2 (T199S_K289S_R312L_I351M)
0%
0%

Round1-Var86
UGTSL2 (Q65P_A205P_L405V)
0%
0%

Round1-Var87
UGTSL2 (V270L_I345L_K365V)
0%
0%

Round1-Var88
UGTSL2 (S200F_F253Y_S255C)
0%
0%

Round1-Var89
UGTSL2 (I114V_G371K_I389L)
0%
3%

Round1-Var90
UGTSL2 (L276A_I333L_S334T_L393V)
75%
94%
11.5%

Round1-Var91
UGTSL2 (I240L_S255C_P361G)
5%
13%

Round1-Var92
UGTSL2 (Q27R_T245R_L276A)
51%
81%
12.0%

*Mutations are noted as follows: reference gene-original amino acid-position-new amino acid: For example the mutation of an isoleucine at position 240 to a Leucine for UGTSL2 is noted as UGTSL2 (I240L)

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:

L276A, T392A, Q27R, N278G, T329V, A341V, I333L, G387E, H247P, M354L, A285V, V270I, N325S, I240L, F253Y, A285L, I352V.

The following mutations were determined as being beneficial for lower Rebaudioside M2 formation:

Q27R, N325S, G387E, I333L, H247P, T329I, R312L, T199S, E259G, S334T, I131V, A285L, I389L, L393V, V254L, N339S, I345L, T245R.

Example 46

Conversion of Rebaudioside A to Rebaudioside I Using UGT76G1

The reaction was conducted using UGT76G1-R1-F12 (also known as UGT76G1var94 (see EXAMPLE 26))

The total volume of the reaction was 40 mL with the following composition: 50 mM potassium phosphate buffer pH 7.5, 3 mM MgCl₂, 2.5 mM UDP-glucose, 0.5 mM Rebaudioside A and 4 mL of UGT76G1-R1-F12 lysate (2.5 U/mL). The reaction was run at 30° C. on an orbitary shaker at 135 rpm. For sampling 125 μL of the reaction mixture was quenched with 10 μL of 2N H₂SO₄and 115 μL of methanol/water (7/3). The samples were immediately centrifuged and kept at 10° C. before analysis by by LC-MS. An Agilent 1200 series HPLC system, equipped with binary pump (G1312B), autosampler (G1367D), thermostated 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
20 min

Injected volume
2 μL

Flow rate
0.8 mL/min

Mobile Phase Gradient Program

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

0
76
24

8.5
76
24

10.0
71
29

16.5
70
30

The reaction profile shown in FIG. 67a was obtained:

After 42 h. of reaction, 20 mL of the reaction mixture was quenched with 20 mL of ethanol and used for structure elucidation.

In similar manner the best clones of UGT76G1 directed evolution round 2 (UGT76G1-R2-B9 identified above as “Round2-Var66”, see EXAMPLE 41) and round 3 (UGT76G1-R3-G3 identified above as “Round3-Var21”, see EXAMPLE 44) and native UGT76G1 (see EXAMPLE 26) were tested for the conversion of Rebaudioside A to Rebaudioside I and the activities shown in FIG. 67b were determined.

Example 47

Isolation and Characterization of Reb I

Crude Reaction Sample. The sample, Lot Crude CB-2977-198, used for isolation, was prepared according to Example 46 with UGT76G1.

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% 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

0.0-8.5
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

Isolation by HPLC. The purification was performed using a Waters Atlantis dC18 (30×100 mm, 5 μm, p/n 186001375) column with isocratic mobile phase conditions of 80:20 water/MeCN. Flow rate was maintained at 45 mL/min and injection load was 180 mg. Detector wavelength was set at 210 nm.

The analyses of fractions were performed using a Waters Atlantis dC18 (4.6×150 mm, 5 μm, p/n 186001342) column; Mobile Phase A: water; Mobile Phase B: MeCN; Flow Rate: 1 mL/min; Isocratic mobile phase conditions: 75:25 A/B for 30 min.

MS and MS/MS. MS and MS/MS data were generated with a Waters QT of Micro mass spectrometer equipped with an electrospray ionization source. The sample was analyzed by negative ESI. The sample was diluted to a concentration of 0.25 mg/mL with H₂O:MeCN (1:1) and introduced via flow injection for MS data acquisition. The sample was diluted further to 0.01 mg/mL to yield good s/n to tune for MS/MS and acquired by direct infusion. The collision energy was set to 60 V in order to acquire MS/MS data with increased fragment ion peaks due to the nature of the molecule

NMR. The sample was prepared by dissolving ~1.0 mg in 180 μL of pyridine-d₅+TMS, and NMR data were acquired on a Bruker Avance 500 MHz instrument with either a 2.5 mm inverse probe or a 5 mm broad band probe. The 13C and HMBC NMR data were acquired at Rensselaer Polytechnic Institute using their Bruker Avance 600 MHz and 800 MHz instruments with 5 mm cryo-probe, respectively. The ¹H and ¹³C NMR spectra were referenced to the TMS resonance (δ_H0.00 ppm and δ_C0.0 ppm).

Isolation of Reb I was performed using a semi-synthetic steviol glycoside mixture, Lot number CB-2977-198. The material was analyzed by HPLC as described above. The Reb I peak was observed at a retention time (t_R) of approximately 17 min as shown in FIG. 28.

Results and Discussion

The reb I peak was isolated from the reaction crude as described above and shown in FIG. 29. The isolated fraction was pooled and lyophilized. Purity of the final product was 91% as confirmed by LC-CAD using the method described above (FIG. 30). Approximately 1 mg of Reb I was provided for spectroscopic and spectrometric analyses.

Mass Spectrometry. The ESI-TOF mass spectrum acquired by infusing a sample of reb I showed a [M-H]⁻ ion at m z 1127.4741 (FIG. 31). The mass of the [M-H]⁻ ion was in good agreement with the molecular formula C₅₀H₇₉O₂₈(calcd for C₅₀H₇₉O₂₈: 1127.4758, error: −1.5 ppm) expected for reb I (FIG. 32). The MS data confirmed that reb I has a nominal mass of 1128 Daltons with the molecular formula, C₅₀H₈₀O₂₈.

The MS/MS spectrum of reb I, selecting the [M-H]⁻ ion at m z 1127.4 for fragmentation, indicated loss of two sugar units at m z 803.5301, however did not show additional fragmentation with collision energy of 30 V (FIG. 33). When higher collision energy was applied (60 V) (FIG. 34), the parent ion was not observed but sequential loss of three sugar units at m z 641.4488, 479.3897, and 317.3023 were observed from m z 803.5301

NMR Spectroscopy. A series of NMR experiments including ¹H NMR (FIGS. 35-37), ¹³C NMR (FIGS. 38-39), ¹H-¹H COSY (FIG. 40), HSQC-DEPT (FIG. 41), HMBC (FIGS. 42-43), NOESY (FIGS. 44-45), and 1D TOCSY (FIGS. 46-50) were performed to allow assignment of reb I.

In the ¹H NMR spectrum of reb I acquired at 300 K (FIG. 35), one of the anomeric protons was completely obscured by the water resonance. Therefore, ¹H NMR spectrum of the sample was acquired at lower temperature (292 K), to shift out the water resonance, and at this temperature anomeric proton was sufficiently resolved (FIGS. 36-37). Thus, all other NMR data of reb I was acquired at 292 K.

The 1D and 2D NMR data indicated that the central core of the glycoside is a diterpene. An HMBC correlation from the methyl protons at δ_H1.22 to the carbonyl at δ_C176.9 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 δ_C38.5, 44.0, and 57.2 allowed assignment of C-3, C-4, and C-5. Analysis of the ¹H-¹³C HSQC-DEPT data indicated that the carbon at δ_C38.5 was a methylene group and the carbon at δ_C57.2 was a methine which were assigned as C-3 and C-5, respectively. This left the carbon at δ_C44.0, 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 (δ_H1.02 and 2.35) and C-5 (δ_H1.03) were assigned using the HSQC-DEPT data. A COSY correlation between one of the H-3 protons (δ_H1.02) and a proton at δ_H1.44 allowed assignment of one of the H-2 protons which in turn showed a correlation with a proton at δ_H0.74 which was assigned to H-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 150 MHz, pyridine-d₅), assignments of the

¹H and ¹³C NMR (500 and 150 MHz, pyridine-d₅),

assignments of the Rebaudioside I aglycone.

Position
¹³C
¹H

1
40.7
0.74
t (11.6)

1.75
m

2
19.4
1.44
m

2.20
m

3
38.5
1.02
m

2.35
m

4
44.0
—

5
57.2
1.03
m

6
22.2
1.90
m

2.33
m

7
41.7
1.29
m

1.31
m

8
42.3
—

9
54.1
0.88
d (6.3)

10
39.8
—

11
20.5
1.67
m

1.70
m

12
37.3
1.98
m

2.28
m

13
86.7
—

14
44.3
1.78
m

2.59
d (11.9)

15
47.6
2.04
brs

16
154.0
—

17
104.8
5.02
s

5.67
s

18
28.4
1.22
s

19
176.9
—

20
15.7
1.26
s

The other tertiary methyl singlet, observed at δ_H1.26, showed HMBC correlations to C-1 and C-5 and was assigned as H-20. The methyl protons showed additional HMBC correlations to a quaternary carbon (δ_C39.8) and a methine carbon (δ_C54.1) which were assigned as C-10 and C-9, respectively. COSY correlations between H-5 (δ_H1.03) and protons at δ_H1.90 and 2.33 then allowed assignment of the H-6 protons which in turn showed correlations to protons at δ_H1.29 and 1.31 which were assigned to H-7. The ¹³C chemical shifts for C-6 (δ_C22.2) and C-7 (δ_C41.7) were then determined from the HSQC-DEPT data. COSY correlations between H-9 (δ_H0.88) and protons at δ_H1.67 and 1.70 allowed assignment of the H-11 protons which in turn showed COSY correlations to protons at δ_H1.98 and 2.28 which were assigned as the H-12 protons. The HSQC-DEPT data was then used to assign C-11 (δ_C20.5) and C-12 (δ_C37.3). The olefinic protons observed at δ_H5.02 and 5.67 showed HMBC correlations to a quaternary carbon at δ_C86.7 (C-13) and thus were assigned to H-17 (δ_C104.8 via HSQC-DEPT). The methine proton H-9 showed HMBC correlations to carbons at δ_C42.3, 44.3 and 47.6 which were assigned as C-8, C-14 and C-15, respectively. The ¹H chemical shifts at C-14 (δ_H1.78 and 2.59) and C-15 (δ_H2.04) were assigned using the HSQC-DEPT data. Additional HMBC correlations from H-9 to C-11 and H-12 to C-9 further confirmed the assignments made above. HMBC correlations observed from H-14 to a quaternary carbon at δ_C154.0 allowed the assignment of C-16 to complete the assignment of the central core.

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 as well as H-5 and H-18. NOE correlations between H-9 and H-14 were not observed. The NOESY data thus indicate 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 the figure below. These data thus indicate that the relative stereochemistry in the central core was retained during the glycosylation step.

Analysis of the ¹H-¹³C HSQC-DEPT data for reb I confirmed the presence of five anomeric protons. All five anomeric protons were resolved in the spectra acquired at 292 K at δ_H6.14 (δ_C95.3), 5.57 (δ_C104.6), 5.38 (δ_C104.7), 5.29 (δ_C105.0), and 5.06 (δ_C98.0). Additionally, all five anomeric protons had large couplings (7.7 Hz-8.2 Hz) indicating that they had 0-configurations. The anomeric proton observed at δ_H6.14 showed an HMBC correlation to C-19 which indicated that it corresponds to the anomeric proton of Glc_I. Similarly, the anomeric proton observed at δ_H5.06 showed an HMBC correlation to C-13 allowing it to be assigned as the anomeric proton of Glc_II.

The Glc_Ianomeric proton (δ_H6.14) showed a COSY correlation to a proton at δ_H4.18 which was assigned as Glc_IH-2. Due to data overlap the COSY spectrum did not allow assignment of H-3 or H-4. Therefore, a series of 1D TOCSY experiments were performed using selective irradiation of the Glc_Ianomeric proton with several different mixing times (FIG. 46). In addition to confirming the assignment for Glc_IH-2, the TOCSY data showed protons at δ_H4.27, 4.25, and 3.93 which were assigned as H-3, H-4 and H-5, respectively. The proton observed at δ_H4.37 in the TOCSY spectrum was assigned to one of the Glc_IH-6 protons. The other H-6 methylene proton at δ_H4.27 was assigned based on COSY correlation from H-5 to δ_H4.27. The ¹³C chemical shifts for Glc_IC-2 (δ_C72.5), C-3 (Sc 89.4), C-4 (δ_C69.2), C-5 (δ_C78.2-78.8) and C-6 (δ_C61.7) were assigned using the HSQC-DEPT data. HMBC correlations from H-1 to C-3 and H-4 to C-6 further confirmed the assignments made above to complete the assignment of Glc_I.

Of the four remaining unassigned glucose moieties one was assigned as a substituent at C-3 of Glc_Ion the basis of HMBC correlations. The anomeric proton observed at δ_H5.29 showed an HMBC correlation to Glc_IC-3 and was assigned as the anomeric proton of Glc_V. The reciprocal HMBC correlation from Glc_IH-3 to the anomeric carbon of Glc_Vwas also observed.

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 150 MHz, pyridine-d₅)

assignments of Rebaudioside I C-19 glycoside.

Position
¹³C
¹H

Glc_I-1
95.3
6.14
d (8.2)

Glc_I-2
72.5
4.18
m

Glc_I-3
89.4
4.27
m

Glc_I-4
69.2
4.25
m

Glc_I-5
78.2-78.8^†
3.93
m

Glc_I-6
61.7
4.27
m, 4.37 m

Glc_V-1
105.0
5.29
d (7.9)

Glc_V-2
75.3 or 75.5
4.04
m

Glc_V-3
78.2-78.6^†
4.27
m

Glc_V-4
71.5 or 71.6
4.12
m

Glc_V-5
78.5 or 78.6^†
4.05
m

Glc_V-6
62.3 or 62.4
4.26
m, 4.56 m

^†Five carbon resonances in the range of 78.2-78.8 (78.16, 78.47, 78.50, 78.55, and 78.77), hence chemical shift could not be unequivocally assigned.

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

[Image Omitted]

The anomeric proton of Glc_V(δ_H5.29) showed a COSY correlation with a proton at δ_H4.04 which was assigned as Glc_VH-2. Glc_VC-2 (δ_C75.3 or 75.5) was then assigned using the HSQC-DEPT data. Due to overlap in the data the COSY spectrum did not allow assignment of the remaining protons. Therefore, a series of 1D TOCSY experiments were performed using selective irradiation of the Glc_Vanomeric proton with several different mixing times (FIG. 47). In addition to confirming the assignments for Glc_VH-2, the TOCSY data allowed assignment of Glc_VH-3 (δ_H4.27), H-4 (δ_H4.12), and H-5 (δ_H4.05). The proton observed at δ_H4.56 in the TOCSY spectrum was assigned to one of the Glc_VH-6 protons. The other H-6 methylene proton at δ_H4.26 was assigned based on COSY correlation from H-5 to SH 4.26. The ¹³C chemical shifts for Glc_VC-3 (δ_C78.2-78.6), C-4 (Sc 71.5 or 71.6), C-5 (δ_C78.5 or 78.6) and C-6 (δ_C62.3 or 62.4) were assigned using the HSQC-DEPT data to complete the assignment of Glc_V.

Assignment of Glc_IIwas carried out in a similar manner. The Glc_IIanomeric proton (δ_H5.06) showed a COSY correlation to a proton at δ_H4.34 which was assigned as Glc_IIH-2 and in turn showed a COSY correlation to a proton at δ_H4.20 (Glc_IIH-3) which showed an additional correlation with a proton at δ_H3.97 (Glc_IIH-4) which also showed a COSY correlation to a proton at δ_H3.80 (Glc_IIH-5). H-5 showed additional COSY correlations to protons at δ_H4.18 and 4.49 which were assigned to H-6. A series of 1D TOCSY experiments were also performed using selective irradiation of the Glc_IIanomeric proton with several different mixing times (FIG. 48). The TOCSY data confirmed the above proton assignments. Assignment of the ¹³C chemical shifts for Glc_IIC-2 (δ_C80.2), C-3 (δ_C87.5), C-4 (δ_C70.1), C-5 (δ_C77.6) and C-6 (δ_C62.5) was based on HSQC-DEPT data. HMBC correlations from Glc_IIH-3 to C-2 and C-4 and also from Glc_IIH-4 to C-3, C-5 and C-6 confirmed the assignments made above to complete the assignment of Glc_II.

The remaining two unassigned glucose moieties were assigned as substituents at C-2 and C-3 of Glc_IIon the basis of HMBC correlations. The anomeric proton observed at δ_H5.57 showed an HMBC correlation to Glc_IIC-2 and was assigned as the anomeric proton of Glc_III. The anomeric proton observed at δ_H5.38 showed an HMBC correlation to Glc_IIC-3 and was assigned as the anomeric proton of Glc_IV. The reciprocal HMBC correlations from Glc_IIH-2 to the anomeric carbon of Glc_IIIand from Glc_IIH-3 to the anomeric carbon of Glc_IVwere also observed.

The anomeric proton of Glc_III(δ_H5.57) showed a COSY correlation with a proton at δ_H4.21 which was assigned as Glc_IIIH-2. Glc_IIIC-2 (δ_C76.3) was then assigned using the HSQC-DEPT data. Due to data overlap the COSY spectrum did not allow assignment of the remaining protons. Therefore, a series of 1D TOCSY experiments were performed using selective irradiation of the Glc_IIIanomeric proton with several different mixing times (FIG. 49). In addition to confirming the assignments for Glc_IIIH-2, the TOCSY data allowed assignment of Glc_IIIH-3 (δ_H4.27), H-4 (δ_H4.25) and H-5 (δ_H3.94). The protons observed at δ_H4.41 and SH 4.53 in the TOCSY spectrum were assigned as the Glc_IIIH-6 protons. The ¹³C chemical shifts for C-3 (δ_C78.2-78.6), C-4 (δ_C72.1), C-5 (δ_C78.2-78.8) and C-6 (δ_C63.1) were assigned using the HSQC-DEPT data. HMBC correlations from H-5 to a carbon at δ_C63.1 further confirmed the assignment of Glc_IIIC-6 to complete the assignment of Glc_III.

The anomeric proton of Glc_IV(δ_H5.38) showed a COSY correlation with a proton at δ_H4.01 which was assigned as Glc_IVH-2. Glc_IVC-2 (δ_C75.3 or 75.5) was then assigned using the HSQC-DEPT data. Due to data overlap the COSY spectrum did not allow assignment of the remaining protons. Therefore a series of 1D TOCSY experiments were performed using selective irradiation of the Glc_IVanomeric proton with several different mixing times (FIG. 50). In addition to confirming the assignments for Glc_IVH-2, the 1D TOCSY data allowed assignment of H-3 (δ_H4.28), H-4 (δ_H4.11), H-5 (δ_H4.13) and H-6 (δ_H4.25 and 4.58). The proton at δ_H4.25 also showed COSY correlation with SH 4.58 further confirmed that these protons belong to H-6. The ¹³C chemical shifts for C-3 (δ_C78.2-78.6), C-4 (δ_C72.1), C-5 (δ_C78.2-78.6) and C-6 (δ_C62.3 or 62.4) were assigned using the HSQC-DEPT data. HMBC correlations from H-4 to C-6 and H-5 to C-1 further confirmed the assignment of Glc_IVC-6 to complete the assignment of Glc_IV.

A summary of the ¹H and ¹³C chemical shifts for the glycoside at C-13 are found are shown below:

¹H and ¹³C NMR (500 and 150 MHz, pyridine-d₅)

assignments of the Rebaudioside I C-13 glycoside.

Position
¹³C
¹H

Glc_II-1
98.0
5.06
d (7.9)

Glc_II-2
80.6
4.34
m

Glc_II-3
87.5
4.20
m

Glc_II-4
70.1
3.97
m

Glc_II-5
77.6
3.80
m

Glc_II-6
62.5
4.18
m, 4.49 m

Glc_III-1
104.6
5.57
d (7.7)

Glc_III-2
76.3
4.21
m

Glc_III-3
78.2-78.6^†
4.27
m

Glc_III-4
72.1
4.25
m

Glc_III-5
78.2-78.8^†
3.94
m

Glc_III-6
63.1
4.41
m, 4.53 m

Glc_IV-1
104.7
5.38
d (7.9)

Glc_IV-2
75.3 or 75.5
4.01
m

Glc_IV-3
78.2-78.6^†
4.28
m

Glc_IV-4
72.1
4.11
m

Glc_IV-5
78.2-78.6^†
4.13
m

Glc_IV-6
62.3 or 62.4
4.25
m, 4.58 m

^†Five carbon resonances in the range of 78.2-78.8 (78.16, 78.47, 78.50, 78.55, and 78.77), hence chemical shift could not be unequivocally assigned.

A summary of key HMBC and COSY correlations used to assign the C-13 glycoside region are provided below.

[Image Omitted]

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

[Image Omitted]

Example 48

Directed Evolution of UGTSL2 for the Conversion of Rebaudioside A to Rebaudioside D (Round 2)

Taking the native enzyme UGTSL2 (GI 460410132) as baseline, a list of 23 mutations was established containing different identified positive mutations for activity from the first round (EXAMPLE 45) and new mutations obtained by DNA2.0 ProteinGPS™ strategy. This list of mutations was subsequently used to design 46 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+_UGTSL2var 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 Express™ 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 lysates were recovered by centrifugation.

In order to measure the activity of the variants for the transformation of Rebaudioside A to Rebaudioside D, 100 μL of fresh lysate was added to a solution of Rebaudioside A (final concentration 0.5 mM), MgCl₂(final concentration 3 mM) and UDP-Glucose (final concentration 2.5 mM) in 50 mM phosphate buffer pH 7.2. The reaction was allowed to run at 30° C. and samples were taken after 2, 4, 6 and 22 h. to determine the initial rates after HPLC analysis (CAD detection) using the analytical method that was described above for the transformation of Rebaudioside A to Rebaudioside D.

In parallel for the most active clones, 100 μL of fresh lysate 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, 6 and 22 h. to determine the initial rates for Rebaudioside D conversion after HPLC analysis (CAD detection).

Apart from the new variants, both experiments were also performed with baseline clone, UGTSL2. The initial rates for the conversion of Rebaudioside A or Rebaudioside D for this baseline clone were defined as 100%.

Activity of each clone was defined as normalized activity compared to baseline clone UGTSL2 whereas specificity of each clone was expressed as the ratio between the initial rates for the conversion of Rebaudioside A and Rebaudioside D.

The normalized initial rate for the conversion of Rebaudioside A and the ratio between the initial rates for the conversion of Rebaudioside A and Rebaudioside D are depicted in the following table.

Ratio between initial

Normalized initial
rates for the conversion

rate for conversion of
of Rebaudioside A and

Clone
Mutations*
Rebaudioside A
Rebaudioside D

UGTSL2
Baseline clone
100%
1.67

Round2-var1
UGTSL2 (Q27R_V270I_A285V)
188%
1.21

Round2-var2
UGTSL2 (F253Y_S255C_M354L)
5%
Nd

Round2-var3
UGTSL2_S255C_I352V_L393V
28%
Nd

Round2-var4
UGTSL2_R6H_N278G_T329I
7%
Nd

Round2-var5
UGTSL2_H247P_V270I_A285L
75%
1.27

Round2-var6
UGTSL2_I240L_T392A_L393V
114%
1.85

Round2-var7
UGTSL2_A285L_R312L_T392A
135%
1.66

Round2-var8
UGTSL2_Q27R_G387E_T392A
164%
1.65

Round2-var9
UGTSL2_Q27R_N278G_A341V
178%
3.13

Round2-var10
UGTSL2_I240L_A285L_N325S
9%
Nd

Round2-var11
UGTSL2_S255C_S258T_N325S
26%
Nd

Round2-var12
UGTSL2_Q27R_N325S_I352V
6%
Nd

Round2-var13
UGTSL2_N325S_A341V_M354L
116%
1.89

Round2-var14
UGTSL2_S255C_A285V_T392A
98%
2.63

Round2-var15
UGTSL2_A285L_A341V_I352V
26%
Nd

Round2-var16
UGTSL2_F253Y_G387E_L393V
88%
1.69

Round2-var17
UGTSL2_V270I_T329I_L393V
88%
2.16

Round2-var18
UGTSL2_H247P_I333L_L393V
197%
1.75

Round2-var19
UGTSL2_L276A_R312L_N325S
53%
1.72

Round2-var20
UGTSL2_V270I_T329V_M354L
30%
Nd

Round2-var21
UGTSL2_A285V_I352V_G387E
30%
Nd

Round2-var22
UGTSL2_I240L_H247P_L276A
76%
2.00

Round2-var23
UGTSL2_A285V_R312L_T329I
4%
Nd

Round2-var24
UGTSL2_I240L_M354L_G387E
8%
Nd

Round2-var25
UGTSL2_N278G_R312L_I333L
50%
1.57

Round2-var26
UGTSL2_L276A_T329I_I352V
0%
Nd

Round2-var27
UGTSL2_L276A_T329V_G387E
73%
Nd

Round2-var28
UGTSL2_R6H_Q27R_L393V
9%
Nd

Round2-var29
UGTSL2_H247P_S258T_T329I
129%
1.21

Round2-var30
UGTSL2_N278G_N325S_T392A
206%
2.06

Round2-var31
UGTSL2_S255C_V270I_I333L
81%
2.87

Round2-var32
UGTSL2_R6H_H247P_A341V
119%
2.05

Round2-var33
UGTSL2_H247P_R312L_G387E
67%
Nd

Round2-var34
UGTSL2_R6H_I240L_T329V
0%
Nd

Round2-var35
UGTSL2_S258T_V270I_T392A
146%
1.71

Round2-var36
UGTSL2_F253Y_T329I_I333L
76%
1.69

Round2-var37
UGTSL2_S258T_A285V_A341V
187%
2.32

Round2-var38
UGTSL2_L276A_A285L_M354L
12%
Nd

Round2-var39
UGTSL2_Q27R_F253Y_T329V
35%
Nd

Round2-var40
UGTSL2_R6H_S258T_L276A
36%
Nd

Round2-var41
UGTSL2_S258T_N278G_M354L
88%
0.96

Round2-var42
UGTSL2_R6H_F253Y_A285L
72%
1.40

Round2-var43
UGTSL2_F253Y_R312L_I352V
8%
Nd

Round2-var44
UGTSL2_I240L_A285V_I333L
43%
1.06

Round2-var45
UGTSL2_S255C_N278G_T329V
45%
2.51

*Mutations are noted as follows: reference gene-original amino acid-position-new amino acid: For example the mutation of an isoleucine at position 240 to a Leucine for UGTSL2 is noted as UGTSL2 (I240L).

Nd means Not determined.

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:

N325S, G387E, A285V, I333L, V270I, Q27R, N278G, L393V, S258T, A341V, H247P and T392A.

The following mutations were determined as being beneficial for an improved ratio between initial rate for the conversion of Rebaudioside A and Rebaudioside D:

V270I, T392A, T329V, L276A, L393V, A341V and S255C.

Example 49

Use of β-Glucosidases for the Conversion of Rebaudioside M2 to Rebaudioside D

Different β-glucosidases were tested for the hydrolysis of Rebaudioside M2. The goal was to selectively hydrolyze the (1→6) glucoside bond in order to obtain Rebaudioside D. The desired general reaction scheme is as follows:

[Image Omitted]

First the selected β-glucosidases were tested on reference substrate 4-nitrophenyl-β-D-glucopyranoside to determine the activity. Based on the determined activities, the used quantities of enzyme were calculated as units for use in the hydrolysis of Rebaudioside M2.

The tested β-glucosidases are depicted in the following table:

Activity enzyme using 4-
Quantity of enzyme used

nitrophenyl-β-D-glucopyranoside
(mg/mL of reaction

β-glucosidase
Manufacturer*
(mmol · min⁻¹· mg⁻¹)
mixture)/(U/mL)

Isolase
NEC
0.29
1.50/0.44

Aromase
Amano
0.030
11.3/0.35

Naringinase
Amano
0.017
13.5/0.23

Cellulase Tr (Celluclast ® 1.5L)
Novozyme
0.026
20.4/0.53

Cellobiase As (Novozyme 188)
Novozyme
0.26
1.50/0.39

CWD (Viscozyme ®L)
Novozyme
0.0062
132/0.82

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

The assay conditions were as follows:

Reactions were performed at 30° C. at a total volume of 10 mL containing 15 mM of sodium acetate buffer (pH 4.5) and 1 mM Rebaudioside M2. The reaction was started by the addition of enzyme.

625 μL of reaction mixture was sampled after 0, 0.5, 1, 1.5, 2, 2.5, 3 and 3.3 hrs and quenched with a mixture of 575 μL of 80% methanol and 50 μL of 2N H₂SO₄. The samples were analyzed by HPLC analysis (CAD detection) using the analytical method that was described above.

The reaction profiles of these reactions with different β-glucosidases are shown in FIGS. 68a-f.

It can be concluded that Naringinase and CWD catalyzed the formation of Rebaudioside D2 and Rebaudioside A which indicates a (1→2) bond glycolysis and (1→6) bond glycolysis respectively. These enzymes can be considered as non-selective for the conversion of Rebaudioside M2.

Isolase, Cellulase Tr and Cellobiase As possess a clear-cut selectivity for the conversion of Rebaudioside M2 to Rebaudioside D (hydrolysis of (1→6) glucoside bond), whereas Aromase possessed low overall activity for the conversion of Rebaudioside M2.

Example 50

Stability of Rebaudiosides in the Presence of Isolase, Cellulase Tr and Cellobiase as

In order to assess the selectivity of Isolase, Cellulase Tr and Cellobiase As for Rebaudioside M2, Rebaudioside A, Rebaudioside D and Rebaudioside M were tested as substrates under the following conditions:

Reactions were performed over 24 h. at 30° C. at a total volume of 10 mL containing 15 mM of sodium acetate buffer (pH 4.5) and 1 mM of Rebaudioside A, Rebaudioside D or Rebaudioside M. The reaction was started by the addition of enzyme.

625 μL of reaction mixture was sampled after 0, 0.5, 1, 1.5, 2, 2.5, 3 and 3.3 hrs and quenched with a mixture of 575 μL of 80% methanol and 50 μL of 2N H₂SO₄. The samples were analyzed by HPLC.

The results shown in FIGS. 69a-c were obtained. It can be observed that no significant conversion of Rebaudioside A, Rebaudioside D and Rebaudioside M can be observed in the presence of Isolase, Cellulase Tr and Cellobiase As.

Example 51

Four-Enzyme Reaction for the Conversion of Rebaudioside A to Rebaudioside M

The influence of adding Isolase, Cellulase Tr or Cellobiase As to the one-pot reaction of Rebaudioside A to Rebaudioside M with UGTSL2, UGT76G1-1R-F12 and AtSUS was studied. The following reaction conditions were used:

Compound
Assay conc (mM)

Rebaudioside A
2

UDP
0.25

MgCl₂
3

Phosphate buffer pH 7.0
50

Sucrose
100

UGTSL2 lysate (2.1 U/mL)
25
μL/mL (0.053 U/mL)

UGT76G1-R1-F12 lysate (2.5 U/mL)
170
μL/mL (0.425 U/mL)

AtSUS (130 U/mL)
25
μL/mL (3.25 U/mL)

Isolase
0.3
mg/mL

Cellulase Tr
0.3
mg/mL

Cellobiase As
4.0
mg/mL

The results for the experiments without and with added β-glucosidase are shown in FIGS. 70a-d. It can be seen that addition of Cellobiase As is blocking the reaction and that addition of Cellulase Tr does not have an influence on the reaction profile. However, addition of Isolase to the reaction mixture has a positive effect on the quantity of Rebaudioside M that is formed in the reaction. An increase of almost 20% is observed when Isolase is added. The Rebaudioside M2 content is approximately 10% lower and Rebaudioside I content is approximately 15% lower when Isolase is added to the reaction mixture compared to the reaction without added β-glucosidase.

Further improvement Reb M yield and reduction of Reb M2 and Reb I content can be achieved through optimization of the reaction parameters and amount of Isolase.

Example 52

Use of β-Glucosidases for the Conversion of Rebaudioside I to Rebaudioside A

Three β-glucosidases were tested for the hydrolysis of Rebaudioside I to Rebaudioside A. The goal was to selectively hydrolyze the (1→6) glucoside bond in order to obtain Rebaudioside D. The desired general reaction scheme is as follows:

[Image Omitted]

The selected β-glucosidases were tested on reference substrate 4-nitrophenyl-β-D-glucopyranoside to determine the activity. Based on the determined activities, the used quantities of enzyme were calculated as units for use in the hydrolysis of Rebaudioside I. The tested β-glucosidases are depicted in the following table:

Activity enzyme using 4-

nitrophenyl-β-D-
Quantity of enzyme used

glucopyranoside
(mg/mL of reaction

β-glucosidase
Manufacturer*
(mmol · min⁻¹· mg⁻¹)
mixture)/(U/mL)

Isolase
NEC (011410)
0.29
1.50/0.44

Cellulase Tr (Celluclast ® 1.5L)
Novozymes*
0.026
22.3/0.58

Cellobiase As (Novozyme 188)
Novozymes*
0.26
1.60/0.42

*Isolase (011410; National Enzyme Company, USA); Cellulase from Trichoderma reesei ATCC 26921 (Sigma C2730); Cellobiase from Aspergillus niger (Sigma C6105)

The assay conditions were as follows. Reactions were performed at 30° C. at a total volume of 2 mL containing 15 mM of sodium acetate buffer (pH 4.5) and 1 mM Rebaudioside I. The reaction was started by the addition of enzyme.

125 μL of reaction mixture was sampled after 0, 1.5, 2.5 and 18 h. and quenched with a mixture of 115 μL of 80% methanol and 10 μL of 2N H₂SO₄. The samples were analyzed by HPLC analysis (CAD detection) using the analytical method that was described above. The reaction profiles of the different β-glucosidases with Rebaudioside I are depicted in the graph shown in FIG. 71.

It can be observed that all three tested β-glucosidases converted Rebaudioside I. The sole product was Rebaudioside A.

Example 53

Directed Evolution of UGTSL2 for the Conversion of Rebaudioside A to Rebaudioside D (Round 3)

Taking the native enzyme UGTSL2 (GI 460410132) as baseline, a list of 13 mutations that were identified during round 2 (EXAMPLE 48) and another list of 12 new mutations that were obtained by DNA2.0 ProteinGPS™ strategy were prepared. This list of mutations was subsequently used to design 46 variant genes that contained each 1 to 8 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+_UGTSL2var 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 Kanamycin). This culture was allowed to shake in a 96 microtiter plate at 30° C. for 8 h.

3.95 mL of production medium containing 60 g/L of Overnight Express™ Instant TB medium (Novagen®), 10 g/L of glycerol and 50 mg/L of 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 lysates were recovered by centrifugation.

In order to measure the activity of the variants for the transformation of Rebaudioside A to Rebaudioside D, 100 μL of fresh lysate was added to a solution of Rebaudioside A (final concentration 0.5 mM), MgCl₂(final concentration 3 mM) and UDP-Glucose (final concentration 2.5 mM) in 50 mM phosphate buffer pH 7.2. The reaction was allowed to run at 30° C. and samples were taken after 2, 4, 6 and 22 h. to determine the initial rates after HPLC analysis (CAD detection) using the analytical method that was described above for the transformation of Rebaudioside A to Rebaudioside D.

In parallel 100 μL of fresh lysate 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, 6 and 22 h. to determine the initial rates for Rebaudioside D conversion after HPLC analysis (CAD detection).

Apart from the new variants for this round, both experiments were also performed with baseline clone, UGTSL2. The initial rates for the conversion of Rebaudioside A or Rebaudioside D for this baseline clone were defined as 100%.

Activity of each clone was defined as normalized activity compared to baseline clone UGTSL2 whereas specificity of each clone was expressed as the ratio between the initial rates for the conversion of Rebaudioside A and Rebaudioside D.

The normalized initial rate for the conversion of Rebaudioside A and the ratio between the initial rates for the conversion of Rebaudioside A and Rebaudioside D are depicted in the following table.

Ratio between initial

Normalized initial
rates for the

rate for
conversion of

conversion of
Rebaudioside A and

Clone
Mutations*
Rebaudioside A
Rebaudioside D

UGTSL2
Baseline clone
100%
1.67

Round3-var1
UGTSL2_S255C_A285V_V349L_L393V
13%
1.86

Round3-var2
UGTSL2_N130G_S255C_N339G_T392A
264%
3.09

Round3-var3
UGTSL2_S255C_V270I_L276A_A285V
10%
4.50

Round3-var4
UGTSL2_S255C_A285I_T329V_H357Y_T392A
70%
4.87

Round3-var5
UGTSL2_S255C_A341V_T392A_I412M
359%
4.34

Round3-var6
UGTSL2_S255C_A285V_K301E_A341V_T392A_L393V
104%
4.34

Round3-var7
UGTSL2_S255C_L276A_K301E_T392A
79%
4.51

Round3-var8
UGTSL2_S255C_T392A_L393V_I412L
46%
2.12

Round3-var9
UGTSL2_F226V_S255C_V270I_T392A
226%
2.67

Round3-var10
UGTSL2_S255C_L276A_A285V_T329V_T392A_I412L
5%
8.57

Round3-var11
UGTSL2_S255C_H357Y_T392A_K408R
0%
Nd

Round3-var12
UGTSL2_S255C_V270I_A285V_A341V_T392A_I412L
403%
7.83

Round3-var13
UGTSL2_S255C_A285V_T329V_N339G_A341V_V349L_T392A
0%
Nd

Round3-var14
UGTSL2_N130G_A285V_A341V_T392A_K408R
475%
2.69

Round3-var15
UGTSL2_T329V
122%
2.62

Round3-var16
UGTSL2_P225L_F226V_S255C_A285V_T329V_T392A_L393V
14%
3.03

Round3-var17
UGTSL2_I203L_P225L_S255C_V349L_T392A
0%
Nd

Round3-var18
UGTSL2_V270I_A285I_K301E_T392A
390%
1.40

Round3-var19
UGTSL2_I203L_S255C_V270I_A285V_N339G_T392A_L393V
12%
1.81

Round3-var20
UGTSL2_N130G_S255C_L276A_A285I_T392A_L393V
262%
3.35

Round3-var21
UGTSL2_S255C_V270I_A285V_T329V_T392A_K408R_I412M
67%
3.33

Round3-var22
UGTSL2_I203L_F226V_S255C_L276A_A285V_T392A_I412M
0%
Nd

Round3-var23
UGTSL2_P225L_S255C_L276A_A285V_A341V_H357Y_T392A
1%
Nd

Round3-var24
UGTSL2_S258T_K408R
58%
3.12

Round3-var25
UGTSL2_F226V_H247P_S258T_A341V
85%
2.47

Round3-var26
UGTSL2_S258T_V270I_A341V_V349L
5%
1.74

Round3-var27
UGTSL2_S258T_L276A_A285V_K301E_A341V_L393V
297%
2.26

Round3-var28
UGTSL2_P225L_S258T_L276A_A341V
22%
1.08

Round3-var29
UGTSL2_S258T_L276A_N339G_A341V
18%
1.08

Round3-var30
UGTSL2_S258T_V270I_N278G_A285V_A341V_T392A
313%
2.29

Round3-var31
UGTSL2_F253Y_A341V_L393V
105%
3.88

Round3-var32
UGTSL2_N130G_S258T_N278G_A341V_H357Y
13%
1.66

Round3-var33
UGTSL2_H247P_S258T_N278G_A285V_A341V_L393V_K408R
286%
3.29

Round3-var34
UGTSL2_F253Y_S258T_V270I_L276A_A285I_A341V
362%
1.90

Round3-var35
UGTSL2_F253Y_S255C_S258T_A341V_T392A
24%
3.28

Round3-var36
UGTSL2_S255C_S258T_L276A_N278G_A285V_I333L_A341V
121%
3.36

Round3-var37
UGTSL2_F226V_S258T_I333L
5%
1.20

Round3-var38
UGTSL2_S255C_S258T_V270I_A285V_T329V
139%
2.59

Round3-var39
UGTSL2_S258T_L276A_A285V_H357Y_T392A
94%
1.98

Round3-var40
UGTSL2_S258T_N278G_K301E_T329V_A341V_I412L
179%
2.82

Round3-var41
UGTSL2_P225L_S258T_A285I_L393V_I412L
1%
0.59

Round3-var42
UGTSL2_I203L_N278G_A285V_I412M
3%
2.68

Round3-var43
UGTSL2_I203L_S258T_V270I_I333L_A341V_L393V
44%
6.27

Round3-var44
UGTSL2_S258T_A285V_T329V_N339G_A341V_V349L_T392A_L393V
0%
Nd

Round3-var45
UGTSL2_N130G_H247P_V270I_A285V_A341V_T392A
869%
2.69

Round3-var46
UGTSL2_S258T_A341V_T392A_I412M
132%
3.27

*Mutations are noted as follows: reference gene-original amino acid-position-new amino acid: For example the mutation of an isoleucine at position 240 to a Leucine for UGTSL2 is noted as UGTSL2 (I240L).

Nd means Not determined.

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:

N130G, H247P, F253Y, V270I, L276A, A2851, A285V, K301E, A341V, T392A, K408R, I412L.

The following mutations were determined as being beneficial for an improved ratio between initial rate for the conversion of Rebaudioside A and Rebaudioside D:

I203L, S255C, I333L, A341V, H357Y, L393V, K408R, 1412L.

Example 54

One-Pot, Four-Enzyme Conversion of Rebaudioside A to Rebaudioside M

10 mL of a reaction mixture containing 5.0 mM of Rebaudioside A, 0.25 mM of UDP, 2 mM of MgCl₂, 100 mM of sucrose, 50 mM of potassium phosphate buffer pH 7.5, 2.5 U of UGTSL2-R3-D2 (UGTSL2-Round3-var12, see EXAMPLE 53), 25 U of UGT76G1-R3-G3 (UGT76G1-Round3-var21, see EXAMPLE 44), 25 U of AtSUS and 5 U of Isolase® was filtered through a 0.2 μm filter in a sterile flask. The resulting reaction mixture was gently shaken at 30° C. for 65 h.

Samples were taken under sterile conditions at regular intervals by taking 125 μL of reaction mixture and quenching it with 10 μL of 2 N H₂SO₄and 765 μL of 50% methanol. After centrifugation, 200 μL of the supernatant was analyzed by HPLC.

The reaction profile shown in FIG. 72a was obtained. The HPLC analysis after 48 h of reaction is shown in FIG. 72b.

Example 55

One-Pot, Four-Enzyme Conversion of Rebaudioside A to Rebaudioside M

10 mL of a reaction mixture containing 10.0 mM of Rebaudioside A, 0.50 mM of UDP, 3 mM of MgCl₂, 100 mM of sucrose, 50 mM of potassium phosphate buffer pH 7.5, 5.0 U of UGTSL2-R3-D2 (UGTSL2-Round3-var12, see EXAMPLE 53), 50 U of UGT76G1-R3-G3 (UGT76G1-Round3-var21, see EXAMPLE 44), 50 U of AtSUS and 10 U of Isolase® was filtered through a 0.2 μm filter in a sterile flask. The resulting reaction mixture was gently shaken at 30° C. for 66 h.

Samples were taken under sterile conditions at regular intervals by taking 125 μL of reaction mixture and quenching it with 10 μL of 2N H₂SO₄and 765 μL of 50% methanol. After centrifugation, 200 μL of the supernatant was analyzed by HPLC.

The reaction profile shown in FIG. 73a was obtained. The HPLC analysis after 48 h of reaction is shown in FIG. 73b.

Example 56

One-Pot, Four-Enzyme Conversion of Rebaudioside A to Rebaudioside M

50 mL of a reaction mixture containing 10.0 mM of Rebaudioside A, 0.5 mM of UDP, 4 mM of MgCl₂, 100 mM of sucrose, 50 mM of potassium phosphate buffer pH 7.5, 25 U of UGTSL2-R3-D2 (UGTSL2-Round3-var12, see EXAMPLE 53), 250 U of UGT76G1-R3-G3 (UGT76G1-Round3-var21, see EXAMPLE 44), 250 U of AtSUS and 50 U of Isolase® was filtered through a 0.2 μm filter in a sterile flask. The resulting reaction mixture was gently shaken at 35° C. for 95 hrs.

Samples were taken under sterile conditions at regular intervals by taking 125 μL of reaction mixture and quenching it with 10 μL of 2 N H₂SO₄and 765 μL of 50% methanol. After centrifugation, 200 μL of the supernatant was analyzed by HPLC.

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

The reaction profile shown in FIG. 74a was obtained. The HPLC of the reaction mixture after 95 hrs is shown in FIG. 74b.

Example 57

One-Pot, Four-Enzyme Conversion of Rebaudioside A to Rebaudioside M (Addition of UGT76G1 and Isolase after 6.5 h)

A reaction mixture containing Rebaudioside A, UDP, MgCl₂, sucrose, potassium phosphate buffer pH 7.5, UGTSL2-R3-D2 (UGTSL2-Round3-var12, see EXAMPLE 53) and AtSUS was filtered through a 0.2 μm filter in a sterile flask. The resulting reaction mixture was gently shaken at 35° C. for 6.5 h. UGT76G1-R3-G3 (UGT76G1-Round3-var21, see EXAMPLE 44) and Isolase® were added and the reaction mixture was filtered through a 0.2 μm filter in a sterile flask and gently shaken for another 89 h at 35° C. The final volume of the reaction mixture was 50 mL and final concentrations of reagents and enzymes were as follows: 10.0 mM of Rebaudioside A, 0.5 mM of UDP, 4 mM of MgCl₂, 100 mM of sucrose, 50 mM of potassium phosphate buffer pH 7.5, 25 U of UGTSL2-R3-D2, 250 U of UGT76G1-R3-G3, 250 U of AtSUS and 50 U of Isolase®

Samples were taken under sterile conditions at regular intervals by taking 125 μL of reaction mixture and quenching it with 10 μL of 2 N H₂SO₄and 765 μL of 50% methanol. After centrifugation, 200 μL of the supernatant was analyzed by HPLC.

The reaction profile shown in FIG. 75a was obtained. The HPLC of the reaction mixture after 95 h is shown in FIG. 75b.

Example 58

One-Pot, Four-Enzyme Conversion of Rebaudioside A to Rebaudioside M (Addition of UGT76G1 and Isolase after 6.5 h)

A reaction mixture containing Rebaudioside A, UDP, MgCl₂, sucrose, potassium phosphate buffer pH 7.5, UGTSL2-R3-D2 (UGTSL2-Round3-var12, see EXAMPLE 53) and AtSUS was filtered through a 0.2 μm filter in a sterile flask. The resulting reaction mixture was gently shaken at 35° C. for 6.5 h. UGT76G1-R3-G3 (UGT76G1-Round3-var21, see EXAMPLE 44) and Isolase® were added and the reaction mixture was filtered through a 0.2 μm filter in a sterile flask and gently shaken for another 89 h at 35° C. The final volume of the reaction mixture was 50 mL and the final concentrations of reagents and enzymes were as follows: 10.0 mM of Rebaudioside A, 0.5 mM of UDP, 4 mM of MgCl₂, 100 mM of sucrose, 50 mM of potassium phosphate buffer pH 7.5, 25 U of UGTSL2-R3-D2, 250 U of UGT76G1-R3-G3, 250 U of AtSUS and 25 U of Isolase®.

Samples were taken under sterile conditions at regular intervals by taking 125 μL of reaction mixture and quenching it with 10 μL of 2 N H₂SO₄and 765 μL of 50% methanol. After centrifugation, 200 μL of the supernatant was analyzed by HPLC.

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

The reaction profile shown in FIG. 76a was obtained. The HPLC of the reaction mixture after 95 h is shown in FIG. 76b.

Example 59

Directed Evolution of UGTSL2 for the Conversion of Rebaudioside A to Rebaudioside D (Round 4)

The most active enzyme from the third round (see EXAMPLE 53) UGTSL2_round3-var45 was taken as starting point. The five best mutations for activity from round 3 were used to create a set of 10 variants containing each two of these 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+_UGTSL2var 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 Express™ 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 lysates were recovered by centrifugation. Lysates were diluted five-fold with water before activity testing.

In order to measure the activity of the variants for the transformation of Rebaudioside A to Rebaudioside D, 100 μL of fresh lysate was added to a solution of Rebaudioside A (final concentration 0.5 mM), MgCl₂(final concentration 3 mM) and UDP-Glucose (final concentration 2.5 mM) in 50 mM phosphate buffer pH 7.2. The reaction was allowed to run at 30° C. and samples were taken after 2, 4, 6 and 22 h. to determine the activities after HPLC analysis (CAD detection) using the analytical method that was described above for the transformation of Rebaudioside A to Rebaudioside D.

Selectivity of each clone was determined by measuring the amount of Rebaudioside M2 that was formed at 100% UDP-Glc conversion (defined as (2*[Reb M2]+[Reb D])/([Reb A]+[Reb D]+[Reb M2]).