Xylosylated steviol glycosides and enzymatic methods for production

Abstract

Methods and compositions are disclosed for producing xylosylated steviol glycosides that contain a xylose residue or an oligosaccharide moiety that contains a xylose residue attached to the 19 carbon of the steviol base. A glycosyltransferase having UDP-xylose:19-steviol xylosyltransferase activity (e.g., one of SEQ ID NO:1 or a homolog or variant thereof) forms a xylosylated steviol glycoside using a steviol glycoside acceptor that has a glucose residue attached to the 19 carbon of the steviol base. Glycosyltransferase mutants with increased activity are also described.

Description

[Technical field]

[0001] (CROSS REFERENCE TO RELATED APPLICATIONS) This application claims the benefit of U.S. Provisional Patent Application No. 63 / 155,229, filed March 1, 2021, which is incorporated by reference in its entirety.

[0002] (Reference to sequence listing submitted via EFS-WEB) The contents of the ASCII text file of the Sequence Listing entitled "PT_923_WO_ST25.txt", created on March 1, 2022, and submitted electronically to EFS-Web with this application, having a size of 43.7 kb, are hereby incorporated by reference in their entirety.

[0003] FIELD OF THEINVENTION The present disclosure relates to xylosylated steviol glycosides and methods for their enzymatic production. [Background technology]

[0004] Sugars such as sucrose, fructose and glucose are utilized to provide a pleasant taste to beverages, foods, medicines and oral hygiene / beauty products. Sucrose, in particular, imparts a taste that is favored by consumers. Sucrose provides excellent sweetness characteristics, but is high in calories. Non-caloric or lower caloric sweeteners have been introduced to meet consumer demand, and these types of sweeteners with desirable taste characteristics are in demand.

[0005] Stevia is a group of about 240 species of herbs and shrubs in the genus Helianthus (family Asteraceae), native to subtropical and tropical regions of western North and South America. Stevia rebaudiana, commonly known as sweetleaf, sweet leaf, sugarleaf, or simply stevia, is widely cultivated for its sweet leaves. Stevia-based sweeteners can be obtained by extracting one or more sweet compounds from the leaves. Many of these compounds are steviol glycosides, which are glycosides of the diterpene compound steviol. These diterpene glycosides are about 150 to 450 times sweeter than sugar.

[0006] Examples of steviol glycosides are described in WO 2013 / 096420 (see, e.g., the listing in Figure 1) and Ohta et.al., (2010) "Characterization of Novel Steviol Glycosides from Leaves of Stevia rebaudiana Morita," J. Appl. Glycosi., 57:199-209 (see, e.g., Table 4 on p. 204). Structurally, diterpene glycosides are characterized by a single base, steviol, and differ by the carbohydrate residues present at the C13 and C19 positions, as presented in Figures 2a-k. See also PCT Patent Publication No. WO20013 / 096420.

[0007] The most abundant steviol glucosides in S. rebaudiana are 1,2-stevioside, rebaudioside A (Reb A) and rebaudioside C (Reb C), which constitute 5-10%, 2-4% and 1-2% of the leaf dry weight, respectively (Chatsudthipong, V., and Muanprasat, C. (2009) Stevioside and related compounds: therapeutic benefits beyond sweetness. Pharmacol Ther.; 121: 41-54). Reb A is 250-300 times sweeter than sucrose but has a bitter, lingering aftertaste (Prakash, I., et al. (2014) Development of next generation stevia sweetener: rebaudioside M. Foods 3: 162-75). Reb C is only 30 times sweeter than glucose (distinguished by a rhamnose moiety attached to the glucose moiety at the C13 position (DuBois, GE, and Stephenson, RA (1985) Diterpenoid sweeteners. Synthesis and sensory evaluation of stevioside analogues with improved organoleptic properties. J. Med. Chem. 28:93-8). Rebaudioside D (Reb D) and Rebaudioside M (Reb M) have a sweetness up to 350 times that of sucrose and are less bitter (Prakash). Reb M is characterized by high sweetness intensity, fast sweetness onset, clean taste, and greatly reduced licorice, bitter, sour, and astringent aftertastes compared to Reb A and other steviol glucosides. Reb D and Reb M is present only in trace amounts in the leaves of S. rebaudiana (approximately 0.4–1.5% (w / w) total dry weight in the traditional Chinese plant). Summary of the Invention

[0008] The present disclosure provides enzymatic methods for producing xylosylated steviol glycosides. In aspects, xylosylation can be performed in a reaction composition or can be performed in an engineered cell. The present disclosure also provides engineered cells capable of producing xylosylated steviol glycosides, as well as compositions comprising xylosylated steviol glycosides. Aspects of the present disclosure are based on experimental findings related to the identification of glycosyltransferase polypeptides having UDP-xylose:19-steviol xylosyltransferase activity (including those of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3) that can utilize activated xylose to provide steviol glycosides having a xylose residue or an oligosaccharide moiety that includes a xylose residue attached to the 19C position of the steviol base.

[0009] In some aspects, the disclosure provides methods for forming xylosylated steviol glycosides, the methods comprising using a glycosyltransferase having UDP-xylose:19-steviol xylosyltransferase activity in vitro or in an engineered cell to form a compound of formula II from a compound of formula I, where formula I is as follows:

[0010] [ka] In the formula, R 1 contains a glucose residue, and R 2 contains one or more sugar residues or is hydrogen. The glycosyltransferase transfers xylose from activated xylose to the compound of formula I to form the compound of formula II,

[0011] [ka] In the formula, R 3 contains one or more xylose residues added by a glycosyltransferase, and R 4 is R 2or contains one or more additional sugar residues.

[0012] Glycosyltransferase polypeptides having UDP-xylose:19-steviol xylosyltransferase activity include those set forth in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, variants thereof, and homologs thereof.

[0013] In the xylosylation product of formula II, R 3 contains a xylose residue, and R 4 is R in the compound of formula I 2 or R 4 is R 2 In some embodiments, R may contain one or more additional sugar residues compared to 4 is an oligosaccharide moiety that includes a xylose residue, such as β-Glc-β-Xyl, which may have a Glc2C→Xyl1C glycosidic bond. 4 can include one or more other sugar residues, such as glucose or rhamnose. In yet other embodiments, the compound of formula I that is xylosylated at position 19 is selected from stevioside, rebaudioside A (RebA), rebaudioside C (RebC), rebaudioside D (RebD), rebaudioside F, rebaudioside G, and dulcoside-A, with stevioside, RebA, and RebC being preferred substrates.

[0014] In some approaches, the methods disclosed herein provide a compound of formula II.

[0015] [ka] The compound was chemically annotated as SG-[13-β-Glc[(3→1)β-Glc](2→1)β-Glc(1-2)]-[19-β-Glc(2→1)βXyl and named rebaudioside DG (RebDG).

[0016] In an aspect, the disclosure provides a method of forming a xylosylated steviol glycoside, the method employing a reaction composition comprising (i) a steviol glycoside compound of formula I, (ii) activated xylose, and (iii) a glycosyltransferase having UDP-xylose:19-steviol xylosyltransferase activity. A product composition is formed from the reaction composition, where a xylosylated steviol glycoside compound of formula II, such as RebDG, is formed by the glycosyltransferase transferring xylose from the activated xylose to the compound of formula I.

[0017] In some embodiments, in the reaction composition, the compound of formula I may be present in an amount of more than 50% (mol) up to 100% (mol) of the steviol glycoside acceptor therein. The reaction composition may include components such as non-activated sugars and salts, a desired range of pH, activated xylose, a steviol glycoside acceptor, and a desired amount of a polypeptide relative to the activated xylose relative to the steviol glycoside acceptor to provide optimized reaction conditions for the transfer of xylose to the steviol glycoside acceptor. In some embodiments, the activated xylose is used in molar excess relative to the steviol glycoside acceptor. In the method, more than 2%, 5%, 7%, 10%, 20%, 30%, 40%, 50%, 60%, 65%, 75%, or more than 80% of the steviol glycoside compound of formula I may be converted to a xylosylated steviol glycoside compound of formula II, such as RebDG.

[0018] In other aspects, the disclosure provides methods for forming xylosylated steviol glycosides using engineered cells capable of making a steviol glycoside compound of formula I, wherein the cells express a glycosyltransferase having UDP-xylose:19-steviol xylosyltransferase activity, such as one of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3, or a polypeptide having at least 50% identity to the glycosyltransferase polypeptide of any of SEQ ID NOs:1-3.

[0019] Activated xylose can be provided in the engineered cells, for example, by feeding the cells with activated xylose from an external source, or the engineered cells can make activated xylose. The polypeptide transfers xylose from the activated xylose to the compound of formula I to form a xylosylated steviol glycoside compound of formula II. In some embodiments, the cells can be provided with a steviol glycoside (precursor) compound used to make the steviol glycoside compound of formula I used as an acceptor molecule. In some embodiments, the cells can be provided with a steviol glycoside compound of formula I, such as RebA, which can then be used directly as an acceptor for xylosylation to form a compound of formula II, such as RebDG.

[0020] In another aspect, the disclosure provides engineered cells having a pathway for forming a steviol glycoside compound of formula I. The cells are engineered to provide a steviol glycoside compound of formula I, such as stevioside, RebA, or RebC, in an amount that is greater than the amount of a steviol glycoside that is not of formula I that can be formed in the cell. The cells also express a glycosyltransferase having UDP-xylose:19-steviol xylosyltransferase activity, where the polypeptide is capable of transferring xylose from activated xylose to the compound of formula I to form a xylosylated steviol glycoside compound of formula II.

[0021] In other aspects, the disclosure provides ingestible or aqueous compositions comprising a mixture of steviol glycosides including a compound of formula II, where the compound of formula II, such as RebDG, is present in an amount greater than any other single steviol glycoside in the composition. In further aspects, two or more compounds of formula II are present in such ingestible or aqueous compositions, where the compound of formula II is present in a total amount greater than the total amount of other steviol glycosides in the composition. In another aspect, an ingestible or aqueous composition comprising a mixture of steviol glycosides including a compound of formula II, optionally including rebaudioside M, where rebaudioside M, if present, is present in an amount greater than rebaudioside M, where the compound of formula II optionally constitutes 1% (mol) or more, e.g., 2%, 3%, or 5% (mol) or more, of all steviol glycosides in the ingestible or aqueous composition.

[0022] A composition comprising a mixture of steviol glycosides can be obtained from a reaction composition or engineered cell that contains a glycosyltransferase having UDP-xylose:19-steviol xylosyltransferase activity.

[0023] In yet another aspect, the disclosure provides engineered glycosyltransferase mutants having surprisingly high levels of UDP-xylose:19-steviol xylosyltransferase activity. The glycosyltransferase mutants contain a mutant amino acid that changes a serine (wild type) to an isoleucine (mutant) residue at position 152 relative to SEQ ID NO:1. The amino acid change results in a significant increase in xylosylation of steviol glycoside molecules at position 19 in the presence of activated xylose.

[0024] Thus, in another aspect, the disclosure provides a glycosyltransferase variant having UDP-xylose:19-steviol xylosyltransferase activity, comprising a polypeptide having 50% or more, 90% or more, 95% or more, or 98% or more identity to SEQ ID NO:1 and the following amino acid: I152. In some aspects, the glycosyltransferase variant is SEQ ID NO:2, which has a sequence with a single serine to isoleucine change at position 152 compared to SEQ ID NO:1. [Brief description of the drawings]

[0025] [Figure 1] 1 shows the chemical structure of steviol base and a table of various steviol glycosides with chemical definitions of the R1 and R2 groups. [Diagram 2] FIG. 1 shows the mevalonate (MVA) pathway for the conversion of acetyl-CoA to isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) using eukaryotic and / or archaeal enzymes. [Diagram 3] 1 shows a non-mevalonate (MEP) pathway for the enzymatic conversion of glyceraldehyde-3-phosphate (G3P) and pyruvate (PYR) to IPP and DMAPP. [Figure 4] 1 shows the enzymatic pathway for the conversion of IPP and farnesyl pyrophosphate (FPP) to steviol. [Diagram 5] 1 is a graph showing average % SG acceptor conversion with UDP-xylose using the enzyme of SEQ ID NO:1 and the UGT76G1 enzyme. [Figure 6] This is the amino acid sequence of SEQ ID NO:1 (AC133334). [Figure 7] 1 is an amino acid sequence alignment of SEQ ID NO:1 with other glycosyltransferases or variants thereof. [Figure 8A] 1 is a graph showing the transfer of glucose and xylose from UDP-glucose and UDP-xylose to RebA over time in the presence of the polypeptide of SEQ ID NO:1 and 1 mM activated sugar. [Figure 8B] FIG. 1 is a graph showing the transfer of glucose, xylose, and rhamnose from UDP-glucose, UDP-xylose, and UDP-rhamnose to RebA over time in the presence of the polypeptide of SEQ ID NO:1 and 2 mM activated sugar. [Figure 9A] FIG. 1 is a graph showing the transfer of UDP-glucose, UDP-xylose, and UDP-rhamnose to -19C(O))-β-Glc residues of Reb F, Reb G, and Dulcoside A over time in the presence of the polypeptide of SEQ ID NO:1. [Figure 9B] FIG. 1 is a graph showing the transfer of UDP-glucose, UDP-xylose, and UDP-rhamnose to -19C(O))-β-Glc residues of Reb F, Reb G, and Dulcoside A over time in the presence of the polypeptide of SEQ ID NO:1. [Figure 9C] FIG. 1 is a graph showing the transfer of UDP-glucose, UDP-xylose, and UDP-rhamnose to -19C(O))-β-Glc residues of Reb F, Reb G, and Dulcoside A over time in the presence of the polypeptide of SEQ ID NO:1. [Figure 10] 1 is an amino acid sequence alignment of SEQ ID NO:1 with SEQ ID NOs:2 to 4. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0026] The aspects of the disclosure described herein are not intended to be exhaustive or to limit the scope of the claims to the precise forms disclosed in the following detailed description. Rather, the purpose of the aspects selected and described is to facilitate a deeper appreciation and understanding by those skilled in the art of the principles and practices of the disclosure.

[0027] Methods of the disclosure provide for forming xylosylated steviol glycosides from steviol glycoside acceptor molecules using a glycosyltransferase having UDP-xylose:19-steviol xylosyltransferase activity (which in some embodiments is a polypeptide having at least 50% identity to any of SEQ ID NOs:1-3) and an activated xylose molecule. It has been found that in the presence of the glycosyltransferase polypeptide of SEQ ID NO:1 and activated xylose, steviol glycoside acceptor molecules are preferentially xylosylated at the 19 carbon to form xylosylated steviol glycosides that have a xylose residue attached to the 19 carbon or that have one or more xylose residues present in an oligosaccharide attached to the 19 carbon.

[0028] The methods of the disclosure can be carried out using a reaction composition (e.g., an in vitro method not requiring engineered cells) that includes a steviol glycoside acceptor, a glycosyltransferase having UDP-xylose:19-steviol xylosyltransferase activity, such as a glycosyltransferase polypeptide having at least 50% identity to any of SEQ ID NOs:1-3, and an activated xylose, such as UDP-xylose.

[0029] The methods of the disclosure can also be practiced using engineered cells expressing a glycosyltransferase having UDP-xylose:19-steviol xylosyltransferase activity, such as a glycosyltransferase polypeptide having at least 50% identity to any of SEQ ID NOs: 1-3, and an activated xylose, such as UDP-xylose. The engineered cells can also have a pathway to make steviol glycoside compounds that are acceptor molecules for xylosylation at the 19 carbon. In some embodiments, the engineered cells can also have a pathway to make activated xylose that can be utilized by the polypeptide to xylosylate steviol glycoside compounds. For example, the engineered cells can also include one or more of UDP-glucose dehydrogenase and / or UDP-glucuronic acid decarboxylase for the production of UDP-xylose. Alternatively, the engineered cells can be fed activated xylose that can be taken up by the cells and used by the glycosyltransferase polypeptide. Alternatively, engineered cells can be supplied with a steviol glycoside molecule that can serve as an acceptor molecule for xylosylation at the C19 position or that can serve as a precursor for the formation of a steviol glycoside acceptor molecule.

[0030] As used herein, the term "steviol glycoside" refers to a glycoside of steviol, i.e., one or more sugar residues attached to a steviol base. Structurally, steviol glycosides have a central molecular moiety that is a single steviol base, and glucopyranosyl residues attached to the C13 and / or C19 atoms of the steviol base, according to the atomic numbering for the base shown below. That is, one or more glycopyranosyl residues are represented by the group R in formula I. 1 and / or R 2 may be present in

[0031] [ka]

[0032] Glycopyranosyl residues that may be present in steviol glycosides include those based on glucose, rhamnose, arabinose, and xylose. Other sugar residues such as fructose and deoxyglucose may be present in steviol glycosides. 1 and / or R 2 When one or both of R has a single glycopyranosyl group, it can be referred to as a monosaccharide portion or group of the steviol glycoside. 1 and / or R 2 If one or both of the glycosyl groups have more than one glycopyranosyl group, they can be referred to as oligosaccharide moieties / groups of steviol glycosides. An oligosaccharide moiety can consist of the same type of glycopyranosyl residues within the moiety (homo-oligosaccharide moiety) or can consist of different types of glycopyranosyl residues within the moiety (hetero-oligosaccharide moiety).

[0033] The oligosaccharide moiety can also be described in terms of the chemical bond between the glycopyranosyl residues in the oligosaccharide. For example, in the oligosaccharide moiety, the bond can be 1→2, 1→3, 1→4, or 1→6, alpha (α) or beta (β) glycosidic bond, based on the numbering on the glycopyranosyl ring and the stereochemistry of the glycosidic bond. In an alpha (α) glycosidic bond, the bond from the anomeric carbon to the oxygen atom of the glycosidic bond is oriented downward from the glycopyranosyl ring, while in a beta (β) glycosidic bond, the bond is oriented upward from the glycopyranosyl ring. The oligosaccharide moiety of steviol glycosides can have a linear or branched structure, with the branched structure having at least one sugar residue bound to two or more other sugar residues in the oligosaccharide moiety. The glycopyranosyl residues can optionally be described in terms of ordered attachment to the 19C and 13C of the steviol base, such as primary, secondary, tertiary residues, with primary glycopyranosyl residues directly attached to the 19C and / or 13C atoms, secondary glycopyranosyl residues directly attached to primary glycopyranosyl residues, tertiary glycopyranosyl residues directly attached to secondary glycopyranosyl residues, etc. Various known steviol glycosides can be represented by the steviol base structure of Formula I, as well as R 1 and R 2 groups attached to the 19C and 13C positions of the steviol base, respectively. 1 and R 2 In relation to the groups, the additives are shown in Figure 1. See, for example, the Compendium of Food Additive Specifications (Joint FAO / WHO Expert Committee on Food Additives; 84th Meeting 2017, FAO JECFA Monographs).

[0034] The disclosed methods use glycosyltransferases having UDP-xylose:19-steviol xylosyltransferase activity. "Glycosyltransferase" generally refers to an enzyme capable of transferring a sugar moiety to an acceptor molecule, with "steviol glycosyltransferase" or "steviol glycoside glycosyltransferase" being capable of transferring a sugar moiety to either a steviol base acceptor or a steviol glycoside acceptor, respectively. Glycosyltransferases may also be described in terms of their activity of adding a specific sugar moiety or moieties to a specific position or positions on a steviol or steviol glycoside molecule. In accordance with the disclosed methods, compositions, and procedures, glycosyltransferases having UDP-xylose:19-steviol xylosyltransferase activity are used, such as the polypeptide of SEQ ID NO:1. UDP-xylose:19-steviol xylosyltransferase activity refers to an enzymatic activity that transfers a xylose moiety from UDP-xylose to a 19C atom or a sugar residue directly or indirectly attached to (and extending from) a 19C atom to a steviol acceptor molecule. The glycosyltransferase may not exclusively have UDP-xylose:19-steviol xylosyltransferase activity, but may (or may not) also have different glycosyltransferase activities, such as UDP-xylose:13-steviol xylosyltransferase activity, and / or UDP-glucose:19-steviol glucosyltransferase activity, and / or UDP-glucose:19-steviol glucosyltransferase activity, and / or UDP-rhamnose:19-steviol rhamnosyltransferase activity, and / or UDP-rhamnose:19-steviol rhamnosyltransferase activity. The glycosyltransferases of the present disclosure can preferentially add xylose to the 19-position of steviol / steviol glycosides (rather than the 13-position).The ability of a glycosyltransferase to preferentially add xylose at position 19 can be determined by performing an assay using a composition comprising a glycosyltransferase and UDP-xylose and analyzing the reaction products, where greater than 50 mol % (or >60%, >70%, >80%, >90%, >95%, >97%, >98%, or >99%) of the products are steviol glycosides xylosylated at position 19(c).

[0035] In an embodiment, a glycosyltransferase polypeptide having at least 50% identity to any of SEQ ID NOs: 1-3 is used to transfer one or more xylose residues to the 19-carbon of a steviol glycoside acceptor molecule. In this method, the steviol glycoside acceptor molecule has a single glucopyranose moiety or an oligosaccharide moiety that includes one or more glucopyranose residues attached to the 19-carbon of a steviol glycoside. The steviol glycoside acceptor can be a compound of Formula I.

[0036] [ka] In the formula, R 1 contains a glucose residue, and R 2 contains one or more sugar residues or is hydrogen. Exemplary steviol glycoside acceptors for xylosylation are selected from stevioside, rebaudioside A (RebA), rebaudioside C (RebC), rebaudioside D (RebD), rebaudioside F, rebaudioside G, and dulcoside-A, with stevioside, RebA, and RebC being preferred substrates.

[0037] In some cases, the glycosyltransferase polypeptide of SEQ ID NO: 1, 2, or 3 utilizes activated xylose to attach xylose to a glucopyranose residue at carbon 19, resulting in an oligosaccharide comprising a glucopyranose residue glycosidically linked to a xylose residue. The enzymatic reaction can result in a compound of formula II:

[0038] [ka] In the formula, R 3 contains a xylose residue, and R 4 is R in the compound of formula I 2 or R 4 is R 2 In some cases, the glycosyltransferase adds one or more xylose, glucose, or rhamnose moieties to the 13 carbon (e.g., to a sugar residue attached to the 13 carbon). 3 is an oligosaccharide moiety that contains a xylose residue, such as β-Glc-β-Xyl, which may have a Glc2C→Xyl1C glycosidic bond. For example, xylosylated steviol glycosides contain an oligosaccharide moiety (R 3 ), where the oligosaccharide moiety comprises β-Glc-β-Xyl, which may have a Glc2C→Xyl1C glycosidic linkage. An exemplary product of formula II is SG-[13-β-Glc[(3→1)β-Glc](2→1)β-Glc(1-2)]-[19-β-Glc(2→1)β-Xyl, referred to as rebaudioside DG.

[0039] Glycosyltransferases constitute a family of enzymes that catalyze the transfer of sugar (glycosyl) moieties to acceptor molecules. Common acceptor molecules include sugars and non-sugars, including polysaccharides, glycoproteins, glycolipids, and terpenes. Activated monosaccharides, typically in the form of nucleoside diphosphate sugars, are used as substrates for the transfer of sugar moieties to acceptor molecules. Monosaccharide units can be transferred to hydroxyl or carboxyl moieties on steviol or steviol glycoside molecules, or to hydroxyl groups on glucose groups attached to the steviol base. Glycosyltransferases can either invert the anomeric configuration of the sugar, such as forming β-glucosides from UDP-glucose, or preserve the anomeric configuration, such as forming α-glucosides from UDP-glucose.

[0040] Uridine diphosphate (UDP) glycosyltransferases (UGTs) are members of family 1 glycosyltransferases (GTs) that are involved in the glycosylation of natural products, particularly the transfer of various sugars to plant natural products. There are also many UGT gene subfamilies involved in the glycosylation of different types of plant products. See, for example, Wang, X. (2009) FEBS Letters 583:3303-3309. GTs have been classified into families and subfamilies based on sequence homology. See, Li, et al., 2001, J. Biol. Chem. 276:4338-4343. A superfamily of over 100 genes encoding UGTs, each containing a 42 amino acid consensus sequence, has been identified in the model plant Arabidopsis thaliana, and genes encoding UGTs have also been identified in several other higher plant species. Each GT family is composed of proteins related by sequence and, consequently, by the type of protein fold. Thus, predictability exists within the family based on conservation of catalytic mechanism. GT polypeptides have also been classified into families by amino acid similarity (see, for example, Coutinho et al., 2003). An actively curated list of GT families and members can be found in the Carbohydrate Active Enzyme database at afmb.cnrs-mrs.fr / CAZY.

[0041] The crystal structures of several plant UGTs reveal the presence of a GT-B fold, one of two common folds in the GT superfamily of enzymes, and that these UGTs have two N- and C-terminal domains with similar Rossmann-like folds. Some UGT crystal structures include those of M. truncatula UGT71G1, UGT85H2, and UGT78G1, grapevine (Vitis vinifera) VvGT1 26, and Arabidopsis (A. thaliana) UGT72B1. For example, Shao, H., et al. (2005) Plant Cell, 17:3141-3154), Modolo, LV, et al. (2009), pp. J. Mol. Biol., 392: 1292-1302, L. Li, L. et al. al.(2007) J.Mol.Biol.,370:951-963, Offen,W.,et al.(2006)EMBO J.,25:1396-1405, Brazier,M.et al.(2007),Proc.Natl.Acad.Sci.104:20238-20243, and Bourne,Y.,and See Henrissat, B. (2001) Curr. Opin. Structure. Biol., 11:593-600. See also Lin, M. et al. (Biochemical Engineering Journal, 59, 2020), who describe a modeled structure of SEQ ID NO:1 using a computational strategy combining protein structure prediction, sequence optimization, and molecular dynamics simulations to improve the catalytic efficiency of UDP-dependent glycosyltransferase SEQ ID NO:1 for the synthesis of rebaudioside D from rebaudioside A.

[0042] The enzyme of SEQ ID NO:1 belongs to the GT-B fold subfamily UGT. SEQ ID NO:1 corresponds to a 462 amino acid uridine 5'-diphospho (UDP) glycosyltransferase from rice (Oryza sativa Japonica Group) (see, for example, U.S. Pat. No. 9,631,215 (#152) and Accession No. AC133334). Based on alignment with other EUGTs, amino acid residues of conserved domains including the active site, substrate binding pocket and TDP binding site can be identified in SEQ ID NO:1.

[0043] The methods of the disclosure use a polypeptide having at least 50% identity to SEQ ID NO: 1. Preferably, the glycosyltransferase has at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identity to SEQ ID NO: 1. In some embodiments, the glycosyltransferase has 100% identity to SEQ ID NO: 1.

[0044] An in silico modeling system was used to predict interactions between the polypeptide of SEQ ID NO: 1 and ligands, which in turn were based on the solved structures of homologues of SEQ ID NO: 1. This in silico modeling provided data for SEQ ID NO: 1-RebA-UDP-xylose and SEQ ID NO: 1-RebA-UDP-glucose that revealed the amino acid residues with SEQ ID NO: 1 that contact the substrate. The data revealed the following amino acids involved in the RebA-UDP-xylose (ligand) interaction: W22, L23, A24, F25, G26, H27, L28, L29, P30, L57, P59, D90, V91, P92, H93, D94, R95, P96, D97, M98, V99, D128, F130, L150, G151, S152, M155, R190, M191, K192, R195, T196, K197, S199, S200, G201, M202, S 203, L204, A205, R221, P249, P250, L251, Y277, A279, L280, G281, S282, E283, V284, P285, A308, L309, R310, R338, W339, V340, P341, Q342, M343, L346, F354, H357, C358, G359, W360, N361, S362, T363, E365, I378, F379, G380, D381, Q382, and N385 (Amino Acid Table Listing "A"). The data revealed that the same amino acids are involved in RebA-UDP-glucose (ligand) interactions with the following exceptions: P59 and D128 are involved in UDP-xylose (ligand) interactions but not UDP-glucose interactions, L149, S203, R310, and I378 are involved in UDP-glucose (ligand) interactions but not UDP-xylose (ligand) interactions, however, S203, R310, and I378 are still involved in RebA interactions.

[0045] Comparative sequence analysis revealed that the following amino acids are highly conserved between SEQ ID NO:1 and other UGTs: M1, H16, V18, P21, W22, L23, A24, F25, G26, H27, P30, L34, L38, A39, G42, H43, S46, S49, T50, P51, N53, R56, L57, P58, V 71, P76, L81, P82, A85, E86, T88, D90, A105, D107, L109, L117, D123, D128, W133, A138, A153 , P180, E187, S200, R207, R221, S222, E225, E227, P241, G246, P249, W266, L267, Q270, S274 , V275, Y277, V278, A279, G281, S282, E283, E293, L294, A295, G297, L298, E299, F305, W307 , R310, L321, P322, G324, F325, R328, G333, V335, W339, P341, Q342, I345, L346, H348, V351 , G352, F354, L355, T356, H357, G359, S362, E365, L373, L376, P377, D381, Q382, G383, N385, A386, R387, G395, V398, R400, D404, G405, F407, V412, A413, V419, and A433 (amino acid list "B").

[0046] Comparative analysis also revealed the following amino acids to be highly conserved in UGTs but not in SEQ ID NO:1: M15, A35, Q36, S87, H102, R103, V143, F244, M248, A301, R304, A350, A415, S425, and Q430 (amino acid list "C").

[0047] In some aspects, the glycosyltransferase has less than 99.8% identity but greater than 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or greater identity to SEQ ID NO:1, and the glycosyltransferase has one or more mutated amino acids that are not at a position in amino acid list "A", that are not at a position in amino acid list "B", or that are not at both positions "A" and "B". In some aspects, the glycosyltransferase based on SEQ ID NO:1 has one or more mutated amino acids at a position in amino acid list "C".

[0048] In some aspects, glycosyltransferases having UDP-xylose:19-steviol xylosyltransferase activity include one or more of the following amino acid motifs: SEQ ID NO:1: WLAFGHLLP (SEQ ID NO:5), LPP; NDVPHDRPDMV (SEQ ID NO:6), DVF, LLGSAHM (SEQ ID NO:7); RMKLIRTKGSSGMSLA (SEQ ID NO:8); PPL; YVALGSEVP (SEQ ID NO:9); ALR; RWVPQMSIL (SEQ ID NO:10); FLTHCGWNSTIE (SEQ ID NO:11); IFGDQGPN (SEQ ID NO:12). These motifs correspond to the following amino acid positions in SEQ ID NO:1: 22-30, 57-59, 90-99, 128-130, 149-155, 190-205, 249-251, 277-285, 308-310, 338-346, 354-365, and 378-385.

[0049] Hughes and Hughes (DNA Sequence, 5:41-49, 1994) showed that the consensus motif for all glycosyltransferases could be refined and extended toward the N-terminus of the protein to a 44 amino acid consensus typical of secondary metabolism plant UGTs. This plant secondary product glycosyltransferase (PSPG) motif was later found in all cloned secondary metabolism UGTs from other plant species.

[0050] In some embodiments, glycosyltransferases having UDP-xylose:19-steviol xylosyltransferase have one or more mutated amino acids at positions involved in RebA-UDP-glucose (ligand) interaction but not involved in RebA-UDP-xylose (ligand) interaction. As described herein, data has revealed that the same amino acids are involved in RebA-UDP-glucose (ligand) interaction with the following exceptions: P59 and D128 are involved in UDP-xylose (ligand) interaction but not UDP-glucose interaction, L149, S203, R310, and I378 are involved in UDP-glucose (ligand) interaction but not UDP-xylose (ligand) interaction, but S203, R310, and I378 are still involved in RebA interaction. Thus, compared to SEQ ID NO: 1 or 2, the glycosyltransferase can have a variant amino acid at one or more of the following positions: L149, S203, R310, and I378W339.

[0051] The one or more amino acid variants can be non-conservative substitutions (more preferred) or conservative substitutions (less preferred). Such variants can result in a reduced interaction of the glycosyltransferase with UDP-glucose, which in turn favors interaction with UDP-xylose, driving greater xylosylation of the steviol glycoside acceptor molecule. In other aspects, with respect to SEQ ID NO: 1 or 2, the glycosyltransferase can have variant amino acids at one or more of the following positions: M15, A35, Q36, S87, H102, R103, V143, F244, M248, A301, R304, A350, A415, S425, and Q430 (amino acid list "C").

[0052] In some embodiments, the glycosyltransferase having UDP-xylose:19-steviol xylosyltransferase activity comprises the following amino acid mutations relative to SEQ ID NO:1: I152. In embodiments using a polypeptide having less than 100% identity to any one of SEQ ID NOs:1-3, the difference in identity can be due to one or more amino acid substitutions in one or more regions of the polypeptide, for example, regions outside of the regions understood to be important for the active site, substrate binding pocket, and TDP binding site based on alignment with one or more other UGTs of the same family as SEQ ID NO:1, and / or regions outside of the conserved regions. Alternatively, when one or more amino acid substitutions, deletions, or additions are made to any of SEQ ID NOs:1-3, they are preferably made at positions that have a lower degree of identity between any of SEQ ID NOs:1-3 and other UGTs of the same family. In some embodiments, the glycosyltransferase has one or more amino acid substitutions, deletions, or additions that change the sequence from that of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3 while retaining certain sequence features. That is, when SEQ ID NO:1 is modified, it is modified at one or more amino acid positions outside of the conserved regions and / or regions important for the enzymatic function of the protein.

[0053] Figure 7 shows an alignment of SEQ ID NO:1 with mutant or other glycosyltransferases having homology to SEQ ID NO:1. As shown in the alignment, the row of shaded amino acids at a particular position represents the amino acid in the SEQ ID NO:1 homolog that has identity or similarity to the corresponding amino acid in the SEQ ID NO:1 template. Similarly, Figure 10 shows an alignment of SEQ ID NO:1 with SEQ ID NOs:2-4, with the shading representing the characteristics of different groups of amino acids.

[0054] In preferred aspects, any variant of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3 either does not have a mutated amino acid at that position, or, if a variant is present, it is a conservative amino acid substitution. For example, using any alignment of two or more glycosyltransferase sequences (one of which is SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3), variant glycosyltransferases can be generated by one or more amino acid substitutions made in the glycosyltransferase template (e.g., SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3) at any amino acid position that aligns with SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3, such as positions 3-15, 18-20, etc., relative to SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3 (as reflected by unshaded amino acids). Such substitutions can preferably be conservative substitutions, although non-conservative substitutions can be used if function is not affected. Shaded amino acids in a template such as SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3, such as 1, 2, 16, 17, 21-24, etc., preferably do not undergo any substitution if variants are used or prepared, or if at most conservative amino acid substitutions are used.

[0055] A "conservative amino acid substitution" or "conservative variation" of a particular sequence refers to the replacement of an amino acid or set of amino acids with a functionally identical amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art and include the replacement of one amino acid with another amino acid having the same type of functional group or side chain polarity, size, shape or charge (e.g., an aliphatic residue, an aromatic residue, a positively charged residue, a negatively charged residue, a polar residue, a non-polar residue, a positive polar residue, a negative polar residue, an uncharged polar residue, a non-polar hydrophobic residue, an ionic acidic residue, an ionic basic residue, or a sulfur-containing residue). Each of the following six groups contains amino acids that can be conservative substitutions for one another: 1) alanine (A), serine (S), threonine (T); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K); histidine (H); 5) isoleucine (I), leucine (L), methionine (M), valine (V); and 6) phenylalanine (F), tyrosine (Y), tryptophan (W).

[0056] Areas of polypeptide sequence identity between SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3 and homologous polypeptides can be understood using the sequence alignment tools described herein.

[0057] Glycosyltransferase polypeptides of the disclosure can also have deletions to one or more regions of a native glycosyltransferase polypeptide, where the deletions do not affect the glycosyltransferase activity of the polypeptide. Deletions can be based on known information about the structure and function of native glycosyltransferases, including information about regions that are conserved and / or important to the enzymatic function of the protein, such as, for example, sequences (a)-(i) described herein.

[0058] The determination of "corresponding" amino acids from two or more glycosyltransferases can be understood by aligning all or part of their amino acid sequences. Sequence alignment and sequence identity generation includes global alignment and local alignment, which typically use computational approaches. To provide a global alignment, a global optimization is used that forces sequence alignment over the entire length of all query sequences. By comparison, local alignment identifies shorter regions of similarity within a long sequence.

[0059] As used herein, "equivalent positions" refers to positions that are common to two sequences (e.g., SEQ ID NO:1 and a different UGT sequence having a desired substitution) based on a best alignment of the amino acid sequences of one glycosyltransferase or as an alignment of the three-dimensional structures. Thus, either sequence alignment or structural alignment, or both, can be used to determine equivalence.

[0060] The amino acid positions described herein are related to the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2, but the corresponding positions in other glycosyltransferases that do not have the same sequence length but can still be aligned using the MUSCLE algorithm with an opening gap of 10 positions and an extension gap of 0.2 as implemented in the Schrodinger platform v 2020-4 are used in the methods of the present disclosure. As shown in FIG. 10, SEQ ID NO:1 or SEQ ID NO:2 have good alignment with SEQ ID NO:3 and SEQ ID NO:4, and therefore the identification of the corresponding positions in either SEQ ID NO:3 and SEQ ID NO:4 to SEQ ID NO:1 and SEQ ID NO:3 can be easily understood. For example, in SEQ ID NO:3, the amino acid positions are shifted from SEQ ID NO:1 and SEQ ID NO:2 by -10 for the first 44 amino acids (i.e., position 11 in SEQ ID NO:1 is position 1 in SEQ ID NO:3), and then shifted by -9 for the next 49 amino acids in SEQ ID NO:3.

[0061] In some implementations, the BLAST algorithm is used to compare and determine sequence similarity or identity. In addition, the presence or significance of gaps in the sequence can be determined, which can be assigned a weight or score. These algorithms can also be used to determine nucleotide sequence similarity or identity. Parameters for determining relatedness are calculated based on methods known in the art for calculating statistical similarity and the significance of the determined match. Related gene products are expected to have high similarity (e.g., greater than 50% sequence identity). Exemplary parameters for determining relatedness of two or more sequences using the BLAST algorithm can be as follows:

[0062] In some implementations, the alignment is performed using BLAST (National Center for Biological Information (NCBI)) Basic Local Alignment Search Tool version 2.2.29 software with default parameters. A sequence having an identity score of XX% (e.g., 80%) to a reference sequence using the BLAST version 2.2.29 algorithm with default parameters is considered to be at least XX% identical to the reference sequence, or equivalently, to have XX% sequence identity.

[0063] When glycosyltransferase mutants are used, a global alignment can be used to align sequences with significant identity to SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3 to determine which corresponding amino acid positions in the target sequence (e.g., a glycosyltransferase ortholog) can be substituted with one or more amino acids.

[0064] In an embodiment of the present disclosure, the method of forming xylosylated steviol glycosides is carried out in a reaction composition. The reaction composition can be prepared to include the desired type and amount of the following reagents: (i) a steviol glycoside compound of formula I (steviol glycoside acceptor), (ii) activated xylose, and (iii) a glycosyltransferase having UDP-xylose:19-steviol xylosyltransferase activity, e.g., one having at least 50% identity to SEQ ID NO:1. The reaction composition can include components (i), (ii), and (iii), which means that other components different from (i), (ii), and (iii) can be optionally included in the reaction composition. The reaction composition can include other components, such as non-activated sugars, cofactors, and the liquid or liquid mixture in which the reaction is carried out.

[0065] The reaction composition includes a steviol glycoside acceptor of formula I,

[0066] [ka] In the formula, R 1 contains a glucose residue, R 2 comprises one or more sugar residues or is hydrogen. 1 is a single glucose residue, such as β-Glc. 1 Examples of steviol glycoside acceptors with a single glucose residue at the carboxyl end include steviol monoglucosyl esters (R 1 = β-Glu, R 2 =-Η), rubusodide (R 1 = β-Glc, R 2 = β-Glc, stevioside (R 1 = β-Glc, R 2 =-β-Glc(1-2)β-Glc), rebaudioside A (RebA;R 1 = β-Glc, R 2 =-β-Glc[β-Glc(1-3)]β-Glc(1-2)]), rebaudioside C (RebC;R 1 = β-Glc, R 2=-α-Rha(1-2)[β-Glc(1-3)]β-Glc), rebaudioside F (RebF; R 1 = β-Glc, R 2 =β-Glc(1-2)[β-Xyl(1-3)]β-Glc), rebaudioside G (RebG; R 1 = β-Glu, R 2 =-β-Glu(1-3)β-Glu), and dulcoside A (R 1 = β-Glu, R 2 =α-Rha(1-2)β-Glu-). In some embodiments, the steviol glycoside acceptor of Formula I is provided to the reaction composition in a pure form (e.g., greater than 99.9% (by weight) of the total steviol glycosides) or in a substantially pure form (e.g., greater than 99% (by weight) of the total steviol glycosides).

[0067] In some embodiments, the steviol glycoside acceptor of formula I can be provided to the reaction composition in a mixture with one or more other steviol glycosides. For example, the mixture of steviol glycosides can be obtained from a process of extracting steviol glycosides from a plant or plant cell. Alternatively, the mixture of steviol can be obtained from an engineered organism capable of producing one or more steviol glycosides. For example, a cell culture or fermentation medium can be used to obtain a mixture of steviol glycosides (see, for example, U.S. Pat. No. 9,631,215, WO 2016 / 196321 (CAR0210 / WO), WO 2016 / 196345 (CAR0211 / WO), WO 2016 / 196368 (CAR0212 / WO)).

[0068] In some embodiments, in the reaction composition, the steviol glycoside acceptor (a compound of formula I) can be, for example, any one or mixture of steviol monoglucosyl esters, stevioside, rebaudioside A, rebaudioside C, rebaudioside D, rebaudioside F, rebaudioside G, and dulcoside A. In addition to the steviol glycoside acceptor of formula I, the reaction composition can include one or more other steviol glycosides that, unlike those of formula I, are not acceptor molecules for xylosylation. A reaction composition that includes a mixture of acceptor and non-acceptor steviol glycosides can be obtained from a leaf (e.g., steviol leaf) extract.

[0069] In other embodiments, the reaction composition can be enriched in one or more steviol glycoside acceptors of formula I. For example, in the reaction composition, the steviol glycoside acceptor of formula I constitutes more than 50% (mol), 75% (mol) or more, 85% (mol) or more, 90% (mol) or more, 92.5% (mol) or more, 95% (mol) or more, 97% (mol) or more, 98% (mol) or more, 99% (mol) or more, 99.5% (mol) or more, or 99.9% (mol) or more, or essentially all, of the steviol glycosides in the reaction composition. In some embodiments, the steviol glycoside component of the reaction mixture consists essentially of one or more compounds of formula I, which means that other steviol glycosides not of formula I may be present in very small amounts (less than 1% (by weight) of the total steviol glycosides) in the steviol glycoside component of the reaction mixture. In yet other embodiments, the steviol glycoside component of the reaction mixture consists of one or more compounds of Formula I, meaning that other steviol glycosides not of Formula I are not present in detectable amounts in the steviol glycoside component of the reaction mixture.

[0070] In some embodiments, the reactant composition comprises greater than 50% (mol), 75% (mol) or more, 85% (mol) or more, 90% (mol) or more, 92.5% (mol) or more, 95% (mol) or more, 97% (mol) or more, 98% (mol) or more, 99% (mol) or more, 99.5% (mol) or more, or 99.9% (mol) or more, or essentially all, of the steviol glycosides therein. In other embodiments, the steviol glycoside component of the reactant composition consists of rebaudioside A.

[0071] In embodiments, the steviol glycoside (SG) acceptor can be any one or a mixture of steviol glycoside acceptors and is present in the reaction composition in an amount such that the SG acceptor is soluble in the reaction mixture. For example, the (SG) acceptor can be present in a range of about 0.01 to about 1.0 moles (e.g., about 0.01 μmol to about 1 μmol of SG per 100 μL of reaction volume), about 0.025 to about 0.5 moles, or about 0.05 to about 0.2 moles.

[0072] Compositions also include glycosyltransferase polypeptides having UDP-xylose:19-steviol xylosyltransferase activity. In some embodiments, the glycosyltransferase comprises the following amino acids of Amino Acid List "A" and / or Amino Acid List "B" for SEQ ID NO:1 or 2. Glycosyltransferases of the disclosure can be polypeptides having at least 50% identity to SEQ ID NO:1 or 2, preferably at least 60%, 75%, 85%, 90%, 95%, 98%, or 99% or more identity to SEQ ID NO:1 or SEQ ID NO:2. U.S. Patent No. 9,631,215 describes the purification of 6HIS-tagged or GST-tagged SEQ ID NO:1 from recombinant E. coli strains expressing the fusion protein.

[0073] In aspects, the glycosyltransferase polypeptide is present in the reaction composition in an amount ranging from about 0.25% (by weight) to about 10% (by weight) (e.g., from about 0.25 μg to about 10 μg of protein per 100 μL reaction volume), from about 0.5% (by weight) to about 5% (by weight), or from about 1% (by weight) to about 3% (by weight).

[0074] If desired, the amounts of steviol glycoside (SG) acceptor and glycosyltransferase polypeptide present in a reaction composition can be described relative to one another. For example, a reaction composition can have a ratio of glycosyltransferase polypeptide (by weight) to steviol glycoside (SG) acceptor (by mole) in the range of 25 μg:1 μmol to 5 μg:1 μmol, in the range of 15 μg:1 μmol to 7.5 μg:1 μmol, or about 10 μg:1 μmol.

[0075] The reaction composition is also preferably in the form of UDP-xylose (uridine [5'] diphospho-α-D-xylopyranoside), which is commercially available (e.g., Biosynth Carbosynth, UK; Sigma-Aldrich). In embodiments, UDP-xylose is present in the reaction composition in an amount ranging from about 0.02 to about 2.0 moles (e.g., about 0.02 μmol to about 2 μmol UDP-xylose per 100 μL reaction volume), about 0.05 to about 1.0 moles, or about 0.1 to about 0.4 moles. UDP-xylose has the following chemical structure:

[0076] [ka]

[0077] Optionally, the amounts of UDP-xylose and steviol glycoside (SG) acceptor, or UDP-xylose and glycosyltransferase polypeptide present in a reaction composition may be described relative to one another. For example, in some embodiments, the reaction composition comprises a molar excess of UDP-xylose (UDP-X) relative to the steviol glycoside compound of formula I (SGI), or a molar ratio of UDP-X:SGI ranging from 1.1:1 to 100:1, 1.1:1 to 10:1, 1.2:1 to 5:1, or 1.5:1 to 3:1. In some embodiments, the reaction composition comprises a glycosyltransferase polypeptide (by weight) to UDP-X (mol) ratio ranging from 50 μg:1 μmol to 10 μg:1 μmol, ranging from 25 μg:1 μmol to 15 μg:1 μmol, or about 20 μg:1 μmol.

[0078] The reaction composition may also include one or more non-activated sugar components to facilitate the reaction. For example, the composition may include a non-activated sugar, such as sucrose, to facilitate the stabilization of the enzymatic reaction. Other non-activated sugars include maltose, trehalose, glucose, and starch hydrolysates, such as glucose syrup and maltodextrin. The non-activated sugar may be used in an amount ranging from about 10 mM to about 0.5 M, or from about 50 mM to about 0.2 M. Optionally, the amount of non-activated sugar and steviol glycoside (SG) acceptor may be described relative to one another. For example, in some embodiments, the reaction composition includes a molar excess of non-activated sugar to steviol glycoside compound of formula I (SGI) in the range of 0.1:1 to 1:1, 1:1 to 5:1, 2:1 to 5:1, 5:1 to 10:1, or 10:1 to 20:1;

[0079] The compositions can also include salts that provide divalent cations that can be used as cofactors for the glycosyltransferase polypeptides. Exemplary divalent metal salts include magnesium and / or manganese salts, which can be used in amounts ranging from about 0.5 mM to about 5 mM or from about 1 mM to about 4 mM in the reaction mixture.

[0080] The reaction can be carried out at a neutral or slightly basic pH, such as a pH in the range of 4.0-8.0, 6.8-7.8, or 7.1-7.5, using buffers such as those containing citrate, phosphate, or tris(hydroxymethyl)aminomethane (Tris).

[0081] In some embodiments, the reaction composition consists essentially of (i) a steviol glycoside compound of formula I (steviol glycoside acceptor), (ii) activated xylose, (iii) a polypeptide having at least 50% identity to SEQ ID NO:1, (iv) a non-activated sugar, (v) a salt of a divalent cation, and (vii) a buffering agent.

[0082] The reaction can be carried out at a desired temperature and time to promote xylosylation of the steviol glycoside acceptor molecule. For example, the reaction can be carried out for a period of at least 1 hour and up to about 10 days, e.g., for a period of time ranging from 1 hour to 24 hours, from 1 hour to 12 hours, from about 12 hours to about 7 days, or from about 1 day to about 5 days. The reaction can be carried out at a temperature ranging from about 5 to 95°C, 25 to 80°C, 25 to 40°C, 30 to 40°C, 40 to 50°C, 50 to 60°C, 60 to 70°C, 70 to 80°C, or 25 to 35°C, or 28 to 32°C.

[0083] As a result of the reaction, a "product composition" is formed that contains one or more xylosylated steviol glycoside residual reaction components, including the polypeptide and any excipient components or components resulting from the reaction. In some cases, the product composition may contain some amount of unreacted steviol glycoside acceptor reactant.

[0084] After the reaction, the product composition can be subjected to purification to separate the components of the reaction mixture. The xylosylated steviol glycoside products can be purified by methods such as crystallization as described herein or by using a reverse phase chromatography column. The hydrophilic components of the reaction mixture can be removed with water, and the xylosylated steviol glycoside compounds can be removed by eluting with a solvent such as methanol. Additionally, the xylosylated steviol glycoside compounds can be further resolved using preparative HPLC (see, for example, WO 2009 / 140394).

[0085] The reaction composition can provide a xylosylated steviol glycoside compound of formula II,

[0086] [ka]

[0087] [ka] In the formula, R 3 contains a xylose residue, R 4 comprises one or more sugar residues or is hydrogen. 3 is an oligosaccharide moiety that contains a xylose residue, such as β-Glc-β-Xyl, and has a Glc2C→Xyl1C glycosidic bond. In some embodiments, the xylosylated steviol glycoside is chosen from one or more of the following compounds:

[0088] [ka]

[0089] [ka]

[0090] [ka]

[0091] [ka] The chemical structure of compound E (RebDG) is shown below.

[0092] [ka]

[0093] The reaction can also provide high yields of xylosylated steviol glycosides, particularly where greater than 50%, greater than 60%, greater than 65%, greater than 75%, or greater than 80% of the steviol glycoside compounds of formula I are converted to the xylosylated steviol glycoside compounds of formula II in the reaction composition.

[0094] The reaction can also provide a product composition that includes one or more compounds of formula II described herein. The types and relative amounts of xylosylated steviol glycoside products can depend on the types and relative amounts of steviol glycoside acceptors used in the reaction composition.

[0095] In some embodiments, the product composition comprises a mixture of steviol glycoside components comprising one, two, three or more xylosylated steviol glycosides of compounds A-F. In some embodiments, the product composition comprises a mixture of steviol glycoside components comprising at least xylosylated steviol glycosides of compounds E and / or F. In some embodiments, compounds E and / or F are the major steviol glycoside components in the product composition, meaning that the compounds are present in a greater amount than any other steviol glycoside in the product composition. In other embodiments, more than one of compounds E and F are present as the major steviol glycoside components in the product composition, meaning that the compounds are present in a greater amount than any other steviol glycoside in the product composition. In some embodiments, compounds E and / or F are present in the product composition in an amount greater than 50%, greater than 60%, greater than 65%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, or greater than 95% of the total amount of steviol glycosides in the composition.

[0096] After the reaction, the product composition can be subjected to purification to separate the components of the reaction mixture. The glycosylated steviol glycoside products can be purified by methods such as crystallization or by using a reverse phase chromatography column. The hydrophilic components of the reaction mixture can be removed with water, and the glycosylated steviol glycoside compounds can be removed by eluting with a solvent such as methanol. Additionally, the glycosylated steviol glycoside compounds can be further resolved using preparative HPLC (see, for example, WO 2009 / 140394).

[0097] In some embodiments, engineered cells are used to prepare xylosylated steviol glycoside compounds of formula II. The engineered cells can have a pathway capable of making a steviol glycoside acceptor compound of formula I and can express a glycosyltransferase polypeptide having UDP-xylose:19-steviol xylosyltransferase activity, such as a polypeptide having at least 50% identity to SEQ ID NO: 1. The cells can also express a pathway for forming activated xylose, such as UDP-xylose.

[0098] In aspects, the engineered cells contain a pathway to steviol, a precursor of the compound of formula I, that can be used as a substrate to form a compound of formula II using a glycosyltransferase polypeptide having UDP-xylose:19-steviol xylosyltransferase activity, such as a polypeptide having at least 50% identity to SEQ ID NO:1, and activated xylose. The cells can be engineered to provide a greater amount of steviol glycoside compounds of formula I than the amount of steviol glycosides that are not of formula I that can be formed in the cells. The cells can also express a glycosyltransferase polypeptide having UDP-xylose:19-steviol xylosyltransferase activity, such as a polypeptide having at least 50% identity to SEQ ID NO:1, that can transfer xylose from activated xylose to a compound of formula I to form a xylosylated steviol glycoside compound of formula II. Exemplary engineered cells include those that are engineered yeast, bacteria, and fungi.

[0099] The terpenoid compounds isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) can serve as chemical precursors of steviol glycosides in engineered cells. Some organisms, including plants, insects, and some microbial species, have the mevalonate (MVA) pathway, which converts acetyl-CoA to IPP and DMAPP through a series of chemical intermediates. Some organisms produce IPP and DMAPP through the non-mevalonate pathway (also known as the methyl D-erythritol 4-phosphate or MEP pathway), which starts with glyceraldehyde-3-phosphate (G3P) and pyruvate (PYR).

[0100] Figure 2 shows a representative mevalonate pathway, in which the mevalonate pathway genes include (a1) acetoacetyl-CoA thiolase (EC 2.3.1.9); (b1) 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) synthase (EC 4.1.3.5); (c1) HMG-CoA reductase (EC 1.1.1.34); (d1) mevalonate kinase (EC 2.7.1.36); (e1) phosphomevalonate kinase (EC 2.7.4.2); and (f1) mevalonate diphosphate decarboxylase (EC 4.1.1.33). Enzymes in the mevalonate pathway convert acetyl-CoA to IPP as follows: acetyl-CoA → acetoacetyl-CoA → 3-hydroxy-3-methylglutaryl-CoA → mevalonate → mevalonate-5-phosphate → mevalonate-5-pyrophosphate → IPP. Alternatively, the pathway can include enzymes from Archaea that use mevalonate-3-kinase to convert mevalonate to mevalonate-3-phosphate, then mevalonate-3-phosphate-5-kinase to convert mevalonate-3-phosphate to mevalonate-3,5-diphosphate, mevalonate-5-phosphate decarboxylase to convert mevalonate-3,5-diphosphate to isopentyl phosphate, and then isopentyl phosphate kinase to convert isopentyl phosphate to IPP.

[0101] Some host cells may not contain any of the enzymes required for the mevalonate pathway, some cells may contain some but not all of the mevalonate pathway genes, and some host cells may naturally contain all of the mevalonate pathway genes. In some cases, host cells that contain none or some can be engineered to contain missing mevalonate pathway genes. The yeast Saccharomyces cerevisiae naturally expresses the mevalonate pathway genes but can be engineered to increase expression of these pathway genes.

[0102] In some embodiments, the prokaryotic cell is engineered with a mevalonate pathway. Martin, VJ et al. describe the engineering of the mevalonate pathway in Escherichia coli for the production of terpenoids (Nature Biotechnology 21:796-802, 2003). Wang, J. et al. describe the engineering of a highly efficient Escherichia coli strain for mevalonate fermentation by chromosomal integration (Appl. Environ. Microbiol. 82:7176-7184, 2016).

[0103] In some embodiments, eukaryotic cells are engineered with a mevalonate pathway or engineered to provide higher amounts of IPP via modification of mevalonate pathway genes. For example, Gold, ND et al. describe a combinatorial approach to study cytochrome P450 enzymes for de novo production of steviol glucoside in baker's yeast. ACS Synth. Biol. 7:2918-2929 (2018).

[0104] Alternatively, the non-mevalonate (MEP) pathway can be used to provide IPP and DMAPP as precursors for steviol production. Theoretically, the MEP pathway is energetically more efficient since less carbon is generally lost as CO2 compared to the MVA pathway (MEP pathway: 1 CO2 / IPP; MVA pathway: 4 CO2 / IPP; sugars as carbon source).

[0105] In particular, in the non-mevalonate (MEP) pathway, the compounds isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) are produced through a series of intermediates arising from glyceraldehyde-3-phosphate (G3P) and pyruvate (PYR), and several enzymes are involved in this conversion. Figure 3 shows a representative non-mevalonate pathway. Enzymes involved in the biosynthetic pathway from G3P and PYR to IPP and DMAPP include (a2) 1-deoxy-D-xylulose-5-phosphate synthase (1-D-xylulose-5-phosphate synthase). synthase (DXS), (b2) 1-deoxy-D-xylulose-5-phosphate reductoisomerase (ispC), (c2) 4-diphosphocytidyl-2C-methyl-D-erythritol synthase (IspD), (d2) 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase (IspE), (e2) 2C methyl-D-erythritol-2,4-cyclodiphosphate synthase (IspF), (f2) 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate synthase (IspG), (g2) 4-hydroxy-3-methyl-2-(E)-butenyl-4-diphosphate reductase (IspH), and (h2) isopentenyl-diphosphate isomerase (IDI).

[0106] U.S. Patent No. 9,284,570 describes a method for producing steviol or steviol glycosides in E. coli using the upper methylerythritol pathway (MEP) to produce isopentyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP).

[0107] The yeast Saccharomyces cerevisiae naturally expresses genes for the MEP pathway, but can be engineered to provide MEP pathway genes if desired.

[0108] In some embodiments, engineered cells can include one or more modifications to increase the flux of acetyl-CoA to IPP and / or DMAPP, thereby providing an increased pool of IPP and / or DMAPP for use in the pathway to steviol. Modifications can include, for example, increasing expression or activity of one or more mevalonate pathway enzymes (a1)-(f1), such as by placing a nucleic acid encoding an enzyme that is homologous or heterologous to the cell under the control of a promoter that results in increased expression, by using multiple copies of a nucleic acid, and / or by using a heterologous enzyme, a mutant enzyme (e.g., one that contains one or more amino acid substitutions), or a mutant heterologous enzyme that results in a higher level of enzyme activity compared to the native enzyme.

[0109] Methods of the disclosure for producing xylosylated steviol glycosides using engineered cells can include cells with one or more genetic modifications that increase the flux of G3P and PYR to IPP and / or DMAPP, thereby providing an increased pool of IPP and / or DMAPP for use in the pathway to steviol. The modifications can include, for example, increasing the expression or activity of one or more of the enzymes (a2)-(h2), such as by placing a nucleic acid encoding an enzyme heterologous to the host cell under the control of a promoter that results in increased expression, using multiple copies of the nucleic acid, and / or using a heterologous enzyme, a mutant enzyme (e.g., one containing one or more amino acid substitutions), or a mutant heterologous enzyme that results in an increased level of enzyme activity.

[0110] The disclosed methods for producing xylosylated steviol glycosides can use engineered cells that may also contain pathways to convert IPP and / or DMAPP and farnesyl pyrophosphate (FPP) to steviol. For example, with reference to Figure 4, in some embodiments, engineered cells can contain exogenous nucleic acids expressing the following enzymes: (a3) ​​geranyl geranyldiphosphate synthase (GGPPS), (b3) copalyl diphosphate synthase (CPS), (c3) kaurene synthase (KS), (d3) kaurene oxidase (KO), and (e3) kaurenoic acid 13-hydroxylase (KAH). Enzymes in the mevalonate pathway convert IPP and / or DMAPP to steviol as follows: IPP / DMAPP→geranylgeranyl diphosphate→copalyl diphosphate→kaurene→kaurenoic acid→steviol. The exogenous nucleic acid encoding the enzymes (a3)-(e3) that are heterologous to the yeast cell can be placed under the control of a promoter that provides for increased expression, using multiple copies of the nucleic acid and / or using mutant enzymes (e.g., those containing one or more amino acid substitutions) or mutant heterologous enzymes that provide for a high level of enzyme activity.

[0111] No. 9,284,570 describes a method for producing steviol or steviol glycosides in E. coli using an upstream methylerythritol pathway (MEP) that produces isopentyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP), and a downstream pathway that produces steviol or steviol glycosides from IPP and DMAPP, expressing copalyl diphosphate synthase (CPS), kaurene synthase (KS), geranylgeranyl diphosphate synthase (GGPPS), kaurenoic acid 13-hydroxylase (KAH) and kaurene oxidase (KO), and optionally one or more stevia UDP glycosyltransferase enzymes.

[0112] The engineered cells also express a glycosyltransferase polypeptide having UDP-xylose:19-steviol xylosyltransferase activity, e.g., a polypeptide having at least 50% identity to SEQ ID NO:1, preferably at least 60%, 75%, 85%, 90%, 95%, 98%, or 99% or more identity to SEQ ID NO:1.

[0113] Regulation of heterologous gene expression can be controlled using desired promoters, terminators, and gene copy number.

[0114] In some embodiments, the engineered cells are engineered to make more Reb A, Reb C, or stevioside than other steviol glycosides.

[0115] The engineered cells can contain one or more uridine diphosphate (UDP) glycosyltransferases (UGTs) that mediate the transfer of glycosyl residues from activated nucleotide sugars to the steviol acceptor molecule, distinct from SEQ ID NO:1 or a homolog or variant of SEQ ID NO:1.

[0116] Exemplary UDP-glucosyltransferases other than those having SEQ ID NO:1 can be any UDP-glucosyltransferase capable of adding at least one glucose unit to a steviol and / or steviol glycoside substrate to provide a target steviol glycoside. In one aspect, the engineered cell can comprise one or more UDP-glucosyltransferases selected from the group of UGT74G1, UGT85C2, UGT76G1, UGT91d2, and UGTs having substantial (>85%) identity to these polypeptides. The engineered cell can comprise one or more exogenous nucleic acid molecules encoding these UGTs.

[0117] The engineered cells can also include one or more UDP-glucose salvage enzymes and / or one or more UGTs under heterologous genetic control. An exemplary UDP-glucosyltransferase that can add at least one glucose unit to rubusoside to form stevioside is UGT91d2. An exemplary UDP-glucosyltransferase that can add at least one glucose unit to stevioside to form rebaudioside A is UGT76G1. An exemplary UDP-glucosyltransferase that can add at least one glucose unit to rebaudioside A to form rebaudioside D is UGT91d2. An exemplary UDP-glucosyltransferase that can add at least one glucose unit to rebaudioside D to form stevioside M is UGT76G1.

[0118] Exemplary publications describing engineered microorganisms for the production of steviol glycosides and steviol glycoside pathway enzymes include, for example, U.S. Patent Application Publication No. 2014 / 0357588, WO 2014 / 193934, WO 2014 / 193888, and WO 2014 / 222227.

[0119] In one aspect, genetically engineered cells useful for producing steviol glycosides express some or all of the following enzymes: geranylgeranyl diphosphate synthase (GGPPS), ent-copalyl diphosphate synthase (CDPS), kaurene oxidase (KO), kaurene synthase (KS); cysteine ​​synthase (KAH), cytochrome P450 reductase (CPR), UGT74G1, UGT76G1, UGT91d2, and the polypeptide of SEQ ID NO: 1. WO 2014 / 122227 describes engineered yeast strains expressing these enzymes. The UGT74G1 enzyme functions as a uridine 5'-diphosphoglucosyl:steviol 19-COOH transferase and a uridine 5'-diphosphoglucosyl:steviol-13-O-glucoside 19-COOH transferase. The UGT76G1 enzyme is a steviauridine diphosphate-dependent glycosyltransferase that catalyzes several glycosylation reactions on the steviol backbone. The UGT76G1 enzyme can catalyze the glycosylation of steviol and steviol glycosides at the 19-0 or 13-0 positions.

[0120] The UGT91d2 enzyme can function as a uridine 5'-diphosphoglucosyl:steviol-13-O-glucoside transferase (also referred to as steviol-13-monoglucoside 1,2-glycosylase) that transfers a glucose moiety to the C-2' of the 13-O-glucose of the acceptor molecule steviol-13-O-glucoside to produce stevioside, or as a uridine 5'-diphosphoglucosyl:rubusoside transferase that transfers a glucose moiety to the C-2' of the 13-O-glucose of the acceptor molecule rubusoside to produce 19-O-1,2-diglycosylated rubusoside. The enzyme of SEQ ID NO:1 can also transfer a glucose moiety to the C-2' of the 19-O-glucose of the acceptor molecule rubusoside to produce 19-O-1,2-diglycosylated rubusoside.

[0121] Activated xylose can be provided in the engineered cells, for example, by feeding the cells with activated xylose from an external source, or the engineered cells can make activated xylose. The polypeptide transfers xylose from the activated xylose to the compound of formula I to form the xylosylated steviol glycoside compound of formula II.

[0122] An "engineered cell" refers to a host cell that has at least one exogenous DNA sequence introduced into the cell, either integrated into the genome of the cell or present on an extrachromosomal construct such as a plasmid or episome. The term "exogenous" refers to a molecule, such as a nucleic acid, or an activity, such as an enzymatic activity, that is introduced into a host cell. Exogenous nucleic acids can be introduced into a host cell by well-known techniques and can be maintained outside the host chromosomal material (e.g., maintained on a non-integrated vector) or can be integrated into the chromosome of the cell, such as by a recombination event. Generally, the genome of an engineered cell is augmented through the stable introduction of one or more recombinant genes. Exogenous nucleic acids can encode enzymes or portions thereof that are either homologous or heterologous to the cell. Exogenous nucleic acids can be in the form of "recombinant genes or DNA constructs," which refers to nucleic acids that have been manipulated in one or more ways by molecular techniques such that they are in a form that does not occur in nature.

[0123] The term "heterologous" (e.g., "non-naturally occurring") refers to a molecule or activity that originates from a source different from the referenced molecule or organism. Thus, a gene or protein that is heterologous to a referenced organism is a gene or protein that is not found in that organism. In the context of this disclosure, a "heterologous glycosyltransferase" refers to a glycosyltransferase polypeptide that is different from any glycosyltransferase polypeptide that may be native to the host organism. For example, a particular glycosyltransferase gene found in a first species and exogenously introduced into a host cell organism different from the first species is "heterologous" to the host cell.

[0124] In some embodiments, the engineered cells that produce xylosylated steviol glycosides are prokaryotic cells. Exemplary bacteria that can be used as hosts for exogenous DNA constructs encoding steviol glycoside pathway enzymes include bacteria of the genera Escherichia, Streptococcus, Lactobacillus, Pseudomonas, Lactococcus, Streptomyces, Bacillus, Clostridium, and the like. , Ralstonia genus, Mycobacterium genus, Agrobacterium genus, Geobacter genus, Zymomonas genus, Acetobacter genus, Citrobacter genus, Synechocystis genus, Rhizobium genus, Corynebacterium genus, Xanthomonas genus, homonas, Alcaligenes, Aeromonas, Azotobacter, Comamonas, Rhodococcus, Gluconobacter, Acidithiobacillus, Microlunatus, Geobacillus, Arthrobacter, These include, but are not limited to, species of the genera Flavobacterium, Serratia, Saccharopolyspora, Thermus, Stenotrophomonas, Chromobacterium, Sinorhizobium, Saccharopolyspora, and Pantoea.An exemplary bacterial species is Escherichia coli.

[0125] In some aspects, the engineered cells that produce xylosylated steviol glycosides are eukaryotic cells.

[0126] For example, various yeast hosts have been engineered to provide pathways to one or more xylosylated steviol glycosides. Such cells can be transformed with one or more DNA constructs encoding enzymes for the synthesis of xylosylated steviol glycosides. Exemplary yeasts that can be used to host exogenous DNA constructs encoding steviol glycoside pathway enzymes include those of the genera Agaricus, Aspergillus, Brettanomyces, Candida, Fusarium, Gibberella, Eluyveromyces, Haemophilus, and the like. The genus Hansenula, the genus Humicola, the genus Issatchenkia, the genus Kloeckera (Hanseniaspora), the genus Kluyveromyces, the genus Laetiporus, the genus Lentinus, the genus Lipomyces, Pachysolen, Examples of yeast fungi include, but are not limited to, species of the genera Phaffia, Phanerochaete, Physcomitrella, Pichia (Hansenula), Rhodotorula, Saccharomycete, Saccharomyces, Schizosaccharomyces, Sphaceloma, Torulopsis, Torulaspora, Trichosporon, Xanthophyllomyces, Yamadazyma, Yarrowia, and Zygosaccharomyces.Exemplary species include Arxula adeninivorans, Ashbya gossypii, Candida albicans, Candida boidinii, Candida glabrata, Candida krusei, Cyberlindnera jadinii, Eluyveromyces lactis, Hansenula polymorpha, Issatchenkia orientalis, Pichia pastoris, Saccharomyces cerevisiae, and the like. cerevisiae, Schizosaccharomyces pombe, Xanthophyllomyces dendrorhous, and Yarrowia lipolytica. Additionally, the host cell can also contain genetic modifications other than the steviol glycoside pathway genetic modifications that can provide improved performance during fermentation.

[0127] The genetically engineered yeast may use auxotrophic markers suitable for selecting transformants having nucleic acids encoding steviol glycoside pathway enzymes. The host yeast may include modifications (such as deletions) in one or more genes that control auxotrophy, such as LYS2, LEU2, HIS3, URA3, URA5, and TRP1. Using a host cell with the desired genetic background for the introduction of one or more exogenous genes, one or more genetic constructs are introduced into the cell and integrated into the genome or stably maintained to allow expression. Methods for introducing genetic constructs into host cells include transformation, transduction, transfection, co-transfection, and electroporation. In particular, yeast transformation may be performed using the lithium acetate method, the protoplast method, and the like. The introduced genetic construct may be integrated into the chromosome in the form of a plasmid, inserted into the host's gene, or integrated by homologous recombination with the host's gene. The transformed yeast into which the genetic construct has been introduced may be selected using a selection marker (e.g., an auxotrophic marker as described above). Further confirmation can be performed by measuring the activity of the expressed protein or the production of a biological product, such as steviol glycosides.

[0128] Transformation of exogenous nucleic acid sequences, including steviol pathway genes, can be confirmed using methods well known in the art. Such methods include, for example, nucleic acid analysis, such as Northern blot or polymerase chain reaction (PCR) amplification of mRNA, or immunoblotting for expression of gene products, or other suitable analytical methods for testing expression of the introduced nucleic acid sequences or their corresponding gene products. It will be understood by those of skill in the art that the exogenous nucleic acids will be expressed in sufficient amounts to produce the desired product, and it will be further understood that expression levels can be optimized to obtain sufficient expression using methods well known in the art and disclosed herein.

[0129] The disclosed method for producing steviol glycosides by cell culture can use engineered cells with a pathway that converts steviol to xylosylated steviol glycosides of formula II. When more than one steviol glycoside pathway enzyme is present in the engineered yeast, the yeast can be capable of producing different steviol glycosides, at least one of which is a steviol xylosylated steviol glycoside of formula II. For example, the yeast can be capable of producing two, three, four, five, six, seven, eight, nine, ten, or more than ten different steviol glycoside species, one or more of which is a xylosylated steviol glycoside.

[0130] The term "medium" refers to a liquid composition in which engineered cells may be maintained, grown, fermented, or a combination thereof. "Medium" may also be referred to as "broth" or "cell culture", and terms such as "growth", "division", "respiration", and "fermentation" may be used to more specifically define the type of cellular activity occurring in the medium. "Cell culture" refers to the process of growing cells under controlled conditions, and includes the growth of prokaryotic and eukaryotic cells in one or more of a defined medium, a defined time period, and a defined temperature. Cell culture is included.

[0131] The medium can be defined in terms of the components present in the medium, such as (a) a carbon source, including carbohydrates such as glucose and starch products such as maltodextrin, (b) a nitrogen source, such as yeast nitrogen base, ammonium hydroxide, urea, ammonium sulfate, or any combination thereof, (c) salts, such as potassium phosphate (monobasic, dibasic), magnesium sulfate, sodium chloride, and calcium chloride, (d) vitamins, such as biotin, calcium pantothenate, folic acid, (myo)-inositol, nicotinic acid, p-aminobenzoic acid, pyridoxine HCl, riboflavin, thiamine HCL, and citric acid, and (e) trace metals, such as boric acid, copper sulfate, cobalt chloride, calcium chloride, potassium iodide, ferric chloride, magnesium sulfate, manganese chloride, sodium molybdate, zinc sulfate, and the like, and their amounts. The components in the medium can be defined on a dry weight basis. Additionally, the medium is a water-based or "aqueous" composition. A medium may also be defined in terms of its pH and the biocompatible acids, bases, and buffers used to control the pH in the medium.

[0132] A composition ("feed composition") can be added to the medium containing the engineered cells to increase the volume of the medium and to increase the amount of biomass as the engineered cells grow in the medium.

[0133] In some embodiments, the cell culture can be performed in a medium containing a steviol-containing compound. In particular, the medium can contain one or more steviol glycoside compounds of formula I. Such compounds can be used directly by a glucosyltransferase polypeptide, e.g., one having at least 50% identity to SEQ ID NO:1, which can transfer xylose from activated xylose to the compound of formula I to form a xylosylated steviol glycoside compound of formula II. Other sugars, such as rhamnose, galactose, arabinose, and / or glucose, can also be transferred to the compound of formula I. In these aspects, the engineered cells do not need to have a pathway to a steviol glycoside precursor (e.g., the MVA, MEP, or SG pathway as described herein).

[0134] Exemplary engineered cells include those that are engineered yeast, bacteria, and fungi.

[0135] "Total steviol glycosides" refers to all steviol glycosides present in the medium after a period of cell culture, including the amount of steviol glycosides, including xylosylated steviol glycosides of the present disclosure, in a liquid medium and can be obtained from a genetically engineered yeast. The steviol glycoside content can be expressed in terms of the amount of total steviol glycosides in the medium, or the amount of one or more, but not all, steviol glycosides in the medium. The amount of steviol glycosides, including xylosylated steviol glycosides, in the composition can be expressed in terms of weight percent or mole percent, for example, a weight percentage of the total amount of steviol glycosides, or a ratio or ratio range, relative to each other or to the total amount of steviol glycosides.

[0136] For recovery of xylosylated steviol glycosides, the medium can then be centrifuged or filtered to remove the engineered cells. The medium can optionally be treated to remove low molecular weight components (glucose, basic nutrients, and salts), for example, by membrane dialysis. Depending on the desired application, a composition comprising one or more steviol glycoside compounds can be used.

[0137] Further purification can be performed if it is desired to provide a composition comprising xylosylated steviol glycosides in a concentrated or purified form, or if certain steviol glycosides are to be separated from one another. Such concentration or purification of the steviol glycoside components can be performed on the medium in which fermentation is occurring, or the medium can then be dried prior to purification. For example, the medium can be dried using lyophilization to form a dry composition comprising steviol glycosides (e.g., a powder or flakes) that can be subsequently processed.

[0138] As used herein, the term "total steviol glycosides" (TSG) is calculated as the sum of the content of all steviol glycosides in a composition on a dry (anhydrous) basis.

[0139] In some modes of implementation, a dried fermentation broth enriched in steviol glycosides is used as the starting material for purification. For example, a solvent or combination of solvents may be added to the dried fermentation broth to dissolve or suspend the material containing steviol glycosides. An exemplary combination for dissolving steviol glycosides is a mixture of water and alcohol (e.g., 50:50 ethanol:water). To facilitate dissolution or suspension, the dried broth material may be heated to a temperature higher than room temperature, such as in the range of 40°C to 60°C. Mechanical disruption of the dried broth material may be performed, such as by sonication. The dissolved or suspended broth material may be filtered using a micron or submicron filter prior to further purification, such as by preparative chromatography.

[0140] The dried fermentation broth, enriched in steviol glycoside compounds, can be subjected to purification or refinement using methods such as crystallization or by using reverse phase liquid chromatography. Techniques known in the art for the concentration and purification of steviol glycoside compounds include extraction using different solvents, adsorption and ion exchange chromatography, supercritical fluid extraction, crystallization, and ultra- and nanomembrane filtration (see, e.g., Kumari, N. et al. (2017) Indian J Pharm Sci; 79: 617-624; Zhang, SQ, et al. (2000) Food Res Int; 33: 617-20; Pol, J., et al. (2007) Anal Bioanal Chem; 388: 1847-57; Puri, M., et al. (2012) Food Chem; 132: 1113-20; Teo, CC, et al, Tan, SN, et al. (2010) J Chromatogr A; 121: 2484-91). U.S. Patent No. 5,962,678 discloses the recrystallization of rebaudioside A using anhydrous methanol solution to obtain 80% pure rebaudioside A. U.S. Patent Publication No. 2006 / 0083838 discloses the purification of rebaudioside A by recrystallization using a solvent containing ethanol and 4-15% water. Japanese Patent Application No. 55-23756 discloses a method for purifying rebaudioside A and stevioside by crystallization from aqueous ethanol (>70%) to obtain 80% pure rebaudioside A. U.S. Patent Publication No. 2007 / 0082103 discloses a method for purifying rebaudioside A by recrystallization from aqueous ethanol, claiming that a two-step recrystallization from crude rebaudioside (60%) results in the formation of rebaudioside with a purity of over 98% in 97% yield. WO 2007 / 149672 and WO 2011 / 082288 disclose a one-step crystallization method using an organic solvent.

[0141] When chromatography is used, a suitable resin can be used to retain the steviol glycoside compounds on the column while removing the hydrophilic compounds which are washed through the column with a liquid such as water. Elution of the steviol glycosides from the column can be accomplished with a suitable solvent or combination of solvents such as acetonitrile or methanol.

[0142] For example, steviol glycoside compounds can be purified using preparative liquid chromatography, such as high pressure liquid chromatography (HPLC) or ultra-high pressure liquid chromatography (UHPLC). A steviol glycoside composition having xylosylated steviol glycosides can be dissolved in a mobile phase, such as a mixture of water and alcohol (e.g., methanol) in a desired ratio (e.g., 60% water, 40% methanol, v / v). The composition can also be heated to facilitate dissolution of the steviol glycoside material, such as heating at about 50° C. The solution can also be filtered prior to injection into the column, for example, using a 0.2 μm filter. Phenomenex Kinetex XB-C18 5 μm, core-shell silica solid support, with a stationary phase of C18 with isobutyl side chains and TMS end caps. The flow rate through the column can be based on the column characteristics (e.g., about 20 mL / min), with a maximum pressure of 400 bar. The various xylosylated steviol glycosides can be identified by their elution time from the column. One of skill in the art will appreciate that the elution time of xylosylated steviol glycosides may change with changes in solvent and / or equipment.

[0143] Elution of the xylosylated steviol glycosides from the reverse phase column can result in a composition that can be useful for any one of a variety of purposes. For example, the purified xylosylated steviol glycoside composition can be used as a sweetener composition for oral ingestion or use, etc. The composition can be defined in terms of the steviol glycosides in the composition.

[0144] As used herein, a sweetener composition (also referred to as a sweetening composition) refers to a composition comprising one or more xylosylated steviol glycosides of formula II. In a preferred embodiment, the sweetener composition comprises compound E (SG-[13-β-Glu[(3→1)β-Glu](2→1)β-Glu(1-2)]-[19-β-Glu(2→1)β-Xyl]). For example, a sweetener composition can comprise one or more steviol glycosides of formula II (SG of formula II) together with another steviol glycoside other than formula II (SG of non-formula II). In some embodiments, when multiple steviol glycosides are present in a sweetener composition, SVs that are not of formula II may be present in the composition in small amounts (e.g., less than about 25%, less than about 20%, less than about 15%, or less than about 10%) of the total amount of steviol glycosides in the composition. One or more SGs of formula II may be present in the composition in a major amount, such as greater than about 75%, greater than about 80%, greater than about 85%, greater than about 90%, or greater than about 95% of the total amount of steviol glycosides in the composition. In some embodiments, compound E (RebDG; SG-[13-β-Glu[(3→1)β-Glu](2→1)β-Glu(1-2)]-[19-β-Glu(2→1)β-Xyl]) is the major SG of the SGs in the sweetener composition, meaning that it is present in an amount greater than any other SG in the composition. In some embodiments, compound E is present in an amount greater than the combined amount of all other SGs in the composition, i.e., compound E is present in an amount greater than 50% (by weight) of the total SGs in the sweetener composition, and in some embodiments, greater than 60%, greater than 70%, greater than 80%, greater than 90%, greater than 95%, greater than 98%, or greater than 99% (by weight) of the total amount of steviol glycosides in the composition.

[0145] The sweetener composition may optionally contain another sweetener, an additive, a liquid carrier, or a combination thereof. The sweetener composition is used to sweeten other compositions (sweetenable compositions), such as foods, beverages, pharmaceuticals, oral hygiene compositions, dietary supplements, etc.

[0146] As used herein, "sweetable composition" refers to a material that contacts the oral cavity of a human or animal, including materials that are placed in the oral cavity but are subsequently spat out (such as mouthwash rinses) and materials that are suitable for consumption by a human or animal when drunk, eaten, swallowed, or digested and used within normally acceptable limits. A sweetenable composition is a precursor composition to a sweetened composition, which is converted to a sweetened composition by combining the sweetenable composition with at least one sweetening composition comprising one or more xylosylated steviol glycosides of formula II. For example, a beverage that does not contain a sweetener component is one type of sweetenable composition. A sweetener composition comprising one or more xylosylated steviol glycosides of formula II can be added to a non-sweetened beverage, thereby providing a sweetened beverage. A sweetened beverage is one type of sweetened composition.

[0147] In some preparations, one or more xylosylated steviol glycosides of formula II provide the sole sweetening component in the sweetened composition.

[0148] In some embodiments, a sweetening composition comprises one or more xylosylated steviol glycosides of formula II in an amount effective to provide a sweetness intensity equivalent to a particular amount of sucrose. The amount of sucrose in a reference solution may be described in degrees Brix (°Bx). One degree Brix is ​​1 gram of sucrose in 100 grams of solution, expressing the strength of the solution as a weight percentage (% w / w). For example, a sweetener composition contains one or more xylosylated steviol glycosides of formula II in an amount effective to provide a sweetness equivalent to about 0.50 to 14 degrees Brix of sugar, e.g., about 5 to about 11 degrees Brix, about 4 to about 7 degrees Brix, or about 5 degrees Brix, when present in a sweetened composition.

[0149] The amount of the xylosylated steviol glycoside of formula II in the sweetener composition may vary. One or more xylosylated steviol glycosides of formula II can be present in the sweetener composition in any amount that imparts the desired sweetness when the sweetener composition is incorporated into a sweetened composition. For example, one or more xylosylated steviol glycosides of Formula II, when present in a sweetener composition, are present in the sweetener composition in an amount effective to provide a total steviol glycoside concentration of from about 1 ppm to about 10,000 ppm, and in another embodiment, one or more xylosylated steviol glycosides of Formula II are present in the sweetener composition in an amount effective to provide a steviol glycoside concentration in the range of from about 10 ppm to about 2,500 ppm, more specifically from about 10 ppm to about 2,000 ppm, from about 10 ppm to about 1500 ppm, from about 10 ppm to about 1250 ppm, from about 10 ppm to about 1000 ppm, from about 10 ppm to about 800 ppm, from about 50 ppm to about 800 ppm, from about 50 ppm to about 600 ppm, or from about 200 ppm to about 500 ppm. Unless otherwise specified, ppm is by weight.

[0150] Optionally, the sweetener composition may also contain one or more additional non-steviol glycoside sweetener compounds, such as natural sweeteners, such as sucrose, fructose, glucose, erythritol, or one or more synthetic sweeteners, such as sucralose, acesulfame potassium, acesulfame acid and its salts, aspartame, saccharin and its salts.

[0151] In addition to one or more xylosylated steviol glycosides of formula II, the sweetener composition may optionally include liquid carriers, binder matrices, and / or additional additives. In some embodiments, the sweetener composition contains additives, including, but not limited to, carbohydrates, polyols, amino acids and their corresponding salts, polyamino acids and their corresponding salts, sugar acids and their corresponding salts, nucleotides, organic acids, inorganic acids, organic salts including organic acid salts and organic base salts, inorganic salts, bitter compounds, flavorings and fragrances, astringent compounds, proteins or protein hydrolysates, surfactants, emulsifiers, bulking agents, gums, antioxidants, colorants, flavonoids, alcohols, polymers, and combinations thereof. In some embodiments, the additives act to improve the temporal and flavor profile to provide the sweetener composition with a desirable taste, such as a taste similar to sucrose.

[0152] The sweetener composition may contain one or more functional ingredients that provide a real or perceived health benefit to the composition, including, but not limited to, saponins, antioxidants, dietary fiber sources, fatty acids, vitamins glucosamine, minerals, preservatives, hydrating agents, probiotics, prebiotics, weight control agents, osteoporosis control agents, phytoestrogens, long chain primary aliphatic saturated alcohols, phytosterols, and combinations thereof.

[0153] Generally, the amount of functional ingredient in a sweetener or sweetened composition will vary widely depending on the particular sweetener or sweetened composition and the desired functional ingredient, and one of ordinary skill in the art will be able to readily ascertain the appropriate amount of functional ingredient for each sweetener or sweetened composition.

[0154] One or more xylosylated steviol glycosides of formula II, or sweetener compositions comprising these steviol glycosides, can be incorporated into any known foodstuff (referred to herein as a "sweetened composition") or other composition intended for ingestion and / or ingestion by the oral cavity of a human or animal, such as pharmaceutical compositions, edible gel mixes and compositions, dental and oral hygiene compositions, foods (confections, condiments, chewing gum, cereal compositions, baked goods, semi-prepared foods, food additives, dairy products, and tabletop sweetener compositions), beverages, and other beverage products (e.g., beverage mixes, beverage concentrates, etc.).

[0155] In one embodiment, the sweetened composition is a beverage product comprising one or more xylosylated steviol glycosides of formula II. As used herein, a "beverage product" is an instant beverage, a beverage concentrate, a beverage syrup, a frozen beverage, or a powdered beverage. Suitable ready-to-drink beverages include carbonated and non-carbonated beverages. Carbonated beverages include, but are not limited to, high-effervescent soft drinks, cola, lemon-lime flavored sparkling soft drinks, orange flavored sparkling soft drinks, grape flavored sparkling soft drinks, strawberry flavored sparkling soft drinks, pineapple flavored sparkling soft drinks, ginger ale, soft drinks, and root beer. Non-carbonated beverages include, but are not limited to, fruit juices, fruit-flavored juices, juice drinks, nectars, vegetable juices, vegetable-flavored juices, sports drinks, energy drinks, enhanced water drinks, vitamin-fortified water drinks, mostly water drinks (e.g., water with added natural or synthetic flavorings), coconut water, tea-type beverages (e.g., black tea, green tea, black tea, oolong tea), coffee, cocoa beverages, beverages containing dairy ingredients (e.g., dairy drinks, dairy coffee, caffe latte, milk tea, fruit milk beverages), beverages containing grain extracts, smoothies, and combinations thereof.

[0156] Examples of frozen beverages include, but are not limited to, icees, frozen cocktails, daiquiris, pina coladas, margaritas, milkshakes, frozen coffees, frozen lemonades, granitas, and slushies.

[0157] Beverage concentrates and beverage syrups can be prepared with an initial volume of a liquid matrix (e.g., water) and the desired beverage ingredients. A final strength beverage is then prepared by adding an additional volume of water. Powdered beverages are prepared by dry mixing all of the beverage ingredients in the absence of a liquid matrix. Full strength beverages are then prepared by adding the entire volume of water.

[0158] In one embodiment, the beverage contains a sweetener composition comprising steviol glycosides, comprising one or more xylosylated steviol glycosides of formula II. Any sweetener composition comprising one or more xylosylated steviol glycosides of formula II detailed herein can be used in the beverage. In another embodiment, a method of preparing a beverage comprises combining a liquid matrix with steviol glycosides, comprising one or more xylosylated steviol glycosides of formula II. The method may further comprise the addition of one or more sweeteners, additives, and / or functional materials. In yet another embodiment, a method of preparing a beverage comprises combining a liquid matrix with a sweetener composition comprising steviol glycosides, comprising one or more xylosylated steviol glycosides of formula II.

[0159] In another embodiment, a beverage contains a sweetener composition containing one or more xylosylated steviol glycosides of formula II, wherein the steviol glycosides are present in the beverage in an amount ranging from about 1 ppm to about 10,000 ppm, such as, for example, from about 25 ppm to about 800 ppm. In another aspect, the steviol glycosides are present in the beverage in an amount ranging from about 100 ppm to about 600 ppm. In yet another aspect, the steviol glycosides are present in the beverage in an amount ranging from about 100 ppm to about 200 ppm, about 100 ppm to about 300 ppm, about 100 ppm to about 400 ppm, or about 100 ppm to about 500 ppm. In yet another aspect, the steviol glycosides are present in the beverage in an amount ranging from about 300 ppm to about 700 ppm, such as, for example, from about 400 ppm to about 600 ppm. In certain embodiments, steviol glycosides are present in the beverage in an amount of about 500 ppm.

[0160] In another embodiment, a granular form of one or more xylosylated steviol glycosides of formula II is provided. As used herein, the terms "granules," "granular form," and "granular form" are synonymous and refer to free-flowing, substantially dust-free, mechanically strong agglomerates of the steviol glycoside sweetener composition. Granulation methods are known to those skilled in the art and are described in more detail in PCT Publication No. WO01 / 60842.

[0161] Preparation of UGT enzymes The amino acid sequence of SEQ ID NO:1 was fused to the C-terminus of the GST protein and the sequence was codon optimized for E. coli expression. (DNA2.0, Menlo Park, CA). The resulting sequence was cloned into a proprietary expression vector (DNA2.0) containing an IPTG-inducible T5 promoter and a strong RBS. The plasmids were transformed into E. coli. The appropriate plasmids from Table 1 were transformed into BL21 cells using standard methods. Overnight cultures were grown in 250 mL flasks containing 50 mL of LB + 50 mg / mL kanamycin at 30°C and 250 RPM. The next morning the seed culture had an OD600=2.4+ / -0.5. The following morning, 3 liter Fernbach flasks containing either LB or Terrific Broth + 50 mg / ml kanamycin were inoculated with 16 ml of the overnight culture, aiming for an initial OD600=0.04. Cultures were grown at 250 RPM for 5-7 hours at the temperatures shown in Table 1 and then induced with 0.8 mM IPTG. After induction, cultures were incubated overnight at 250 RPM at the temperatures shown in Table 1. Biomass was pelleted in 1 liter centrifuge bottles at 8000 RPM for 15 minutes at 4°C. Cells were resuspended in lysis buffer and lysed by sonication. Cell suspensions were sonicated for 2 minutes with 20 second pulses followed by a 20 second rest on ice. Cells were transferred to a 30 mL Oak Ridge tube and centrifuged at 15,000 RPM for 15 minutes. Supernatant was decanted into a new conical tube.

[0162] Example 1: 19C-xylosylation of stevioside, Reb A, Reb C, and Reb D using the enzyme of SEQ ID NO:1 and UDP-xylose Six steviol glycoside (SG) "acceptors" Reb A, B, C, D, M and stevioside were tested using two glucosyltransferase enzymes (SEQ ID NO:1 and UGT76G1) known to play important roles in SG biosynthesis in Stevia leaves and activated xylose (UDP-xylose).

[0163] UDP-xylose (98.9% purity) and UDP-rhamnose disodium salt (95% purity) were purchased from BOC Sciences. Activated sugar donors were prepared at 10 mM concentration in 0.05 M Tris buffer and stored according to the manufacturer's recommendations before use.

[0164] The enzyme of SEQ ID NO:1 and UGT76G1 enzyme were prepared in E. coli host cells as tagged proteins with expression induced by chemical inducers. After induction, cells were incubated overnight and harvested. Cells were then lysed (either by sonication, mechanical disruption or chemical lysis) and proteins were purified by chromatography and stored frozen at -20°C until use. In preliminary tests, enzyme activity was confirmed using UDP-glucose. Frozen enzyme solutions were thawed immediately before use and returned to frozen storage after use. Previous studies have shown that repeated freeze-thawing has little effect on enzyme activity.

[0165] SG materials were obtained from various sources with purity of 90% or higher. All SG materials were prepared at 20 mM with 90% DMSO and stored at ambient temperature. The final DMSO concentration in the reaction mixture was 4.5%. Previous studies have shown that this level of DMSO does not have any effect on biotransformation.

[0166] Sucrose, which has previously been shown to have a stabilizing effect on biotransformation reactions, was prepared at 0.1 M in 0.05 M Tris buffer.

[0167] Tris (tris(hydroxymethyl)aminomethane) buffer was prepared at 0.05 M, pH 7.3, and contained 3 mM each of MgCl2 and MnCl2 as cofactors.

[0168] Biotransformation reactions were carried out in sealed vials containing a total of 100 uL of Tris buffer, 0.1 μmol SG acceptor, 0.2 μmol activated sugar donor, 1 μmol sucrose and 2 ug of enzyme protein. To each vial, buffer, sucrose, UDP-sugar donor, and SG acceptor were added in that order, followed by enzyme addition last. For each SG donor, a control was included containing the same amount of sucrose and enzyme without the UDP-sugar donor. After hand mixing, the vials were placed in a temperature-controlled shaker set at 30° C. and 100 rpm and rotated for 96 hours (4 days).

[0169] After 4 days of reaction, 0.9 mL of 80% acetonitrile was added to each vial, and the obtained samples were analyzed by LC / MS. Xylose modification of -19C(O)-β-Glc residues was confirmed.

[0170] As shown in Table 1 and FIG. 5, the polypeptide of SEQ ID NO: 1 was more effective in transferring xylose to SG acceptors than UGT76G1. This result was rather unexpected, since it was assumed by others that UGT76G1 has a broader activity toward acceptors and donors than SEQ ID NO: 1. When the polypeptide of SEQ ID NO: 1 was used, stevioside, Reb A and Reb C all showed very high conversion, and one of the RebD copies showed about 10% conversion. When UGT76G1 was used, stevioside showed a higher conversion rate than Reb A and Reb C.

[0171] [Table 1]

[0172] Example 2: Time course analysis of glycosylation of Reb A with SEQ ID NO:1 and activated sugars The ability of SEQ ID NO:1 to glycosylate Reb A was examined with different activated sugars used at different concentrations and the reaction products formed were measured over the course of the reaction.

[0173] The purified protein of SEQ ID NO:1 was used along with UDP-glucose, UDP-xylose, and UDP-rhamnose disodium salt, and the reagents and reaction conditions were as described in Example 1.

[0174] [Table 2]

[0175] [Table 3]

[0176] The polypeptide of SEQ ID NO:1 was able to efficiently transfer glucose and xylose from UDP-glucose and USP-xylose, respectively, to RebA. In the presence of purified protein of SEQ ID NO:1 and 1 mM activated sugar, glucosylation and xylosylation of RebA reached a plateau after 20 min, with RebA having a slightly higher level of glucosylation than xylosylation (see Table 2 and Figure 8A). In the presence of purified protein of SEQ ID NO:1 and 2 mM activated sugar, glucosylation and xylosylation of RebA reached a plateau after 20 min, with similar levels, whereas rhamnosylation with UDP-rhamnose was significantly lower (see Table 3 and Figure 8B).

[0177] Example 3: Glycosylation of Reb F, Reb G, and Dulcoside A using SEQ ID NO:1 and activated sugars The ability of SEQ ID NO:1 to glycosylate the -19C(O)-β-Glc residues of RebF, RebG, and Dulcoside A was examined with different activated sugars used at different concentrations, and the reaction products formed were measured over the course of the reaction.

[0178] UDP-glucose, UDP-xylose, and UDP-rhamnose disodium salt were used, and the reagents and reaction conditions were as described in Example 1.

[0179] [Table 4]

[0180] [Table 5]

[0181] [Table 6]

[0182] SEQ ID NO:1 could effectively transfer glucose and xylose from UDP-glucose and UDP-xylose, respectively, to the -19C(O)-βGlc residues of Reb F, Reb G, and Dulcoside A, with glucosylation and xylosylation reaching a plateau after 20 min. Rhamnosylation using UDP-rhamnose was significantly lower (see Tables 4-6 and Figures 9A-C).

[0183] Example 4: Glycosylation of Reb A using homologues and variants of SEQ ID NO:1 In vitro enzyme production Candidate xylosyltransferase enzymes are produced in vitro using the PURExpress In Vitro Protein Synthesis kit from New England Biolabs (NEB#E6800) according to the manufacturer's instructions, including the addition of 250 ng of plasmid DNA and 20 units of the RNase inhibitor, Murine (NEB#M0314).

[0184] Glycosyltransferase assays for enzymes produced by in vitro protein synthesis The steviol glycoside (SG) "acceptor" Reb A was tested with four glucosyltransferase enzymes (SEQ ID NOs: 1-4) that are homologous to glucosyltransferases in SG biosynthesis in Stevia leaves. The identities of SEQ ID NOs: 2-4 to SEQ ID NO: 1 are listed in Table 7.

[0185] [Table 7]

[0186] UDP-xylose (98.9% purity) was purchased from BOC Sciences. Activated sugar donors were prepared at 10 mM concentration in 0.05 M Tris buffer and stored according to the manufacturer's recommendations before use. SG materials were obtained from various sources at 90% purity or higher. All SG materials were prepared at 20 mM with 90% DMSO and stored at ambient temperature. The final DMSO concentration in the reaction mixture was 4.5%. Previous studies have shown that this level of DMSO does not have any effect on bioconversion.

[0187] Tris (tris(hydroxymethyl)aminomethane) buffer was prepared at 0.05 M, pH 7.3, and contained 3 mM each of MgCl2 and MnCl2 as cofactors.

[0188] The enzymes were produced via in vitro transcription-translation (IVTT) and activity was confirmed using UDP-glucose in preliminary tests. Bioconversion reactions were carried out in sealed vials containing Tris buffer, 0.1 μmol SG acceptor, 0.2 μmol activated sugar donor, 1 μmol sucrose and 2 μg enzyme protein totaling 100 μL. To each vial, buffer, UDP-sugar donor, SG acceptor were added in that order, followed by enzyme addition last. For each SG donor, a control was included containing the same amount of enzyme without UDP-sugar donor. After hand mixing, the vials were placed in a temperature-controlled shaker set at 30° C. and 100 rpm and rotated for 96 hours (4 days).

[0189] After 4 days of reaction, 0.9 mL of 80% acetonitrile was added to each vial, and the obtained samples were analyzed by LC / MS. Xylose modification of -19C(O)-β-Glc residues was confirmed.

[0190] The activities of SEQ ID NOs:2-4 are expressed relative to the activity of SEQ ID NO:1. As shown in Table 8, SEQ ID NO:1 was more effective than either SEQ ID NO:3 or SEQ ID NO:4 in transferring xylose to the SG acceptor. The substantial overall increase in activity of SEQ ID NO:2 compared to SEQ ID NO:1 was rather unexpected. In the presence of SEQ ID NOs:1 and 2, very high conversion of Reb A to xylosylated products occurred in the presence of both UDP-glucose and UDP-xylose. [SEQ ID NO:3 was run more than once in this experiment.]

[0191] [Table 8]

Claims

1. 1. A method for forming glycosylated steviol glycosides, the method comprising: forming a compound of formula II from a compound of formula I in vitro or in an engineered cell using a glycosyltransferase having UDP-xylose:19-steviol xylosyltransferase activity, and (ii) activated xylose; Formula I is as follows: 【Chemistry 1】 In the formula, R １ contains a glucose residue, R ２ contains one or more sugar residues or is hydrogen; the glycosyltransferase transfers xylose from activated xylose to the compound of formula I; Formula II is as follows: 【Chemistry 2】 In the formula, R ３ comprises one or more xylose residues added by said glycosyltransferase, and R ４ But, R ２ or containing one or more additional sugar residues. (i) R of formula II ３ is an oligosaccharide moiety containing one or more xylose residues; and / or (ii) the oligosaccharide moiety is -β-Glu-β-Xyl or -β-Glu-β-Xyl-R 5 , where R 5 comprises one or more sugar residues; and / or (iii) The method of claim 1, wherein R 3 of formula II consists of -Glu-β-Xyl.

3. 2. The method of claim 1, wherein the compound of formula I is selected from the group consisting of stevioside, rebaudioside A, rebaudioside C, rebaudioside D, rebaudioside F, rebaudioside G, and dulcoside A.

4. 4. The method of claim 3, wherein the compound of formula I is selected from the group consisting of stevioside, rebaudioside A, and rebaudioside C.

5. 4. The method of claim 3, wherein the compound of formula I is rebaudioside A.

6. The method according to any one of claims 1 to 5, wherein the activated xylose is UDP-xylose.

7. 7. The method of any one of claims 1 to 6, wherein the glycosyltransferase has 85% or more, 90% or more, 92.5% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity to SEQ ID NO:

1.

8. The glycosyltransferase has the following amino acid sequence relative to SEQ ID NO:1: (A) W22, L23, A24, F25, G26, H27, L28, L29, P30, L57, P59, D90, V91, P92, H93, D94, R95, P96, D97, M98, V99, D128, F 130, L149, L150, G151, S152, M155, R190, M191, K192, R195, T196, K197, S199, S200, G201, M202, S203, L204, A205, R221, P249, P250, L251, Y277, A279, L280, G281, S282, E283, V284, P285, A308, L309, R310, R338, W339, V340, P341, Q342, M343, L346, F354, H357, C358, G359, W360, N361, S362, T363, E365, I378, F379, G380, D381, Q382, and N385, (B) M1, H16, V18, P21, W22, L23, A24, F25, G26, H27, P30, L34, L38, A39, G42, H43, S 46, S49, T50, P51, N53, R56, L57, P58, V71, P76, L81, P82, A85, E86, T88, D90, A105 , D107, L109, L117, D123, D128, W133, A138, A153, P180, E187, S200, R207, R221, S 222, E225, E227, P241, G246, P249, W266, L267, Q270, S274, V275, Y277, V278, A279 , G281, S282, E283, E293, L294, A295, G297, L298, E299, F305, W307, R310, L321, P 322, G324, F325, R328, G333, V335, W339, P341, Q342, I345, L346, H348, V351, G352 , F354, L355, T356, H357, G359, S362, E365, L373, L376, P377, D381, Q382, G383, N385, A386, R387, G395, V398, R400, D404, G405, F407, V412, A413, V419, and A433, or The method according to any one of claims 1 to 7, comprising both (A) and (B).

9. The method of any one of claims 1 to 8, wherein the glycosyltransferase comprises one or more of the following amino acid motifs relative to SEQ ID NO:1: WLAFGHLLP (SEQ ID NO:5), LPP; NDVPHDRPDMV (SEQ ID NO:6), DVF, LLGSAHM (SEQ ID NO:7); RMKLIRTKGSSGMSLA (SEQ ID NO:8); PPL; YVALGSEVP (SEQ ID NO:9); ALR; RWVPQMSIL (SEQ ID NO:10); FLTHCGWNSTIE (SEQ ID NO:11); IFGDQGPN (SEQ ID NO:12).

10. 10. The method of any one of claims 1 to 9, wherein the method is carried out in vitro using a steviol glycoside reaction composition comprising: (i) a steviol glycoside compound of formula I; (ii) an activated xylose sugar; and (iii) a glycosyltransferase having UDP-xylose:19-steviol xylosyltransferase activity. (i) the compound of formula I constitutes more than 50% (mol), 75% (mol) or more, 85% (mol) or more, 90% (mol) or more, 92.5% (mol) or more, 95% (mol) or more, 97% (mol) or more, 98% (mol) or more, 99% (mol) or more, 99.5% (mol) or more, or 99.9% (mol) or more, or essentially all, of the steviol glycosides in the reaction composition; and / or 11. The method of claim 10, wherein (ii) in the reaction composition, greater than 10%, greater than 20%, greater than 30%, greater than 40%, greater than 50%, greater than 60%, greater than 65%, greater than 75%, or greater than 80% of the steviol glycoside compounds of formula I are converted to xylosylated steviol glycoside compounds of formula II.

12. The compound of formula II is 【Chemistry 3】 The method of any one of claims 1 to 11, wherein the compound is rebaudioside DG.

13. The reaction composition comprises: (a) a magnesium and / or manganese salt; (b) a pH in the range of 3 to 9, 4 to 8, 6.8 to 7.8, or 7.1 to 7.5; and (c) a molar excess of UDP-xylose (UDP-X) relative to the steviol glycoside compound of formula I (SGI), or a molar ratio of UDP-X:SGI in the range of 1:1 to 1:100, 1:5 to 1:10, 1:20 to 1:50, 1:50 to 1:100, or 1.1:1 to 10:1, 1.2:1 to 5:1, or 1.5:1 to 3:

1.

13. The method of any one of claims 10 to 12, comprising one or more of the following: (a) a molar excess of UDP-xylose (UDP-X), or a UDP-X:SGI molar ratio, including at least one of: (d) a polypeptide (by weight) to UDP-X (mol) ratio in the range of 50 μg:1 μmol to 10 μg:1 μmol, in the range of 25 μg:1 μmol to 15 μg:1 μmol, or about 20 μg:1 μmol; (e) a polypeptide (by weight) to SGI (mol) ratio in the range of 25 μg:1 μmol to 5 μg:1 μmol, in the range of 15 μg:1 μmol to 7.5 μg:1 μmol, or about 10 μg:1 μmol.

14. 14. The method of any one of claims 10 to 13, wherein the reaction comprises one or more of the following conditions: (a) a time in the range of 1 hour to 10 days, in the range of 1 hour to 24 hours, in the range of 1 hour to 12 hours, in the range of 12 hours to 7 days, or in the range of 1 day to 5 days; (b) at a temperature in the range of 5 to 95°C, 25 to 80°C, 25 to 40°C, 30 to 40°C, 40 to 50°C, 50 to 60°C, 60 to 70°C, 70 to 80°C, or 25 to 35°C, or 28 to 32°C.

15. forming is carried out in an engineered cell capable of producing the steviol glycoside compound of formula I; 10. The method of any one of claims 1 to 9, comprising: providing (exogenously) the activated xylose to the cells or allowing the engineered cells to make the activated xylose, which is transferred from the activation to the compound of formula I to form a xylosylated steviol glycoside compound of formula II.

16. 1. An engineered cell comprising: A pathway for forming a steviol glycoside compound of formula I, comprising: 【Chemistry 4】 In the formula, R １ contains a glucose residue, R ２ contains one or more sugar residues or is hydrogen; a pathway that provides an amount of a steviol glycoside compound of Formula I that is greater than the amount of a steviol glycoside not of Formula I that can be formed in the cell; and a glycosyltransferase having UDP-xylose:19-steviol xylosyltransferase activity, said glycosyltransferase capable of transferring one or more xylose residues to said compound of formula I to form a glycosylated steviol glycoside compound of formula II; 【Chemistry 5】 In the formula II, R ３ contains one or more xylose residues, R ４ But, R ２ and a glycosyltransferase which is the same as or contains one or more additional sugar residues.

17. 17. The engineered cell of claim 16, wherein the engineered cell comprises a pathway for the production of UDP-xylose.

18. 18. The engineered cell of claim 17, wherein in the engineered cell, the pathway for the production of UDP-xylose comprises UDP-glucose dehydrogenase, UDP-glucuronic acid decarboxylase, UDP-xylose synthase, or a combination thereof.

19. 19. The engineered cell of any one of claims 16 to 18, wherein the engineered cell comprises one or more UDP-glucose recycling enzymes and / or one or more UGTs under heterologous genetic control.

20. The engineered cells may be selected from the group consisting of Candida, Kloeckera (Hanseniaspora), Issatchenkia, Kluyveromyces, Lipomyces, Pichia (Hansenula), Rhodotorula, Saccharomycete, Saccharomyces, Schizosaccharomyces, S ... or an engineered yeast selected from the group consisting of Saccharomyces, Torulopsis, Torulaspora, Yarrowia, and Zygosaccharomyces species, preferably selected from the group consisting of Pichia (Hansenula), Saccharomycete, Saccharomyces, and Yarrowia species; or 20. The engineered cell of any one of claims 16 to 19, wherein the engineered cell is an engineered bacterial cell selected from the group consisting of species of the genera Escherichia and Bacillus.

21. 21. The engineered cell of any one of claims 16-20, wherein the engineered cell expresses one or more exogenous nucleic acids encoding one or more of the following proteins heterologous to the cell: a GGPPS polypeptide, an ent-copalyl diphosphate synthase (CDPS) polypeptide, a kaurene oxidase (KO) polypeptide, a kaurene synthase (KS) polypeptide, a steviol synthase (KAH) polypeptide, a cytochrome P450 reductase (CPR) polypeptide, a UGT74G1 polypeptide, a UGT76G1 polypeptide.

22. 1. A method for forming xylosylated steviol glycosides, the method comprising: forming a xylosylated steviol glycoside product from steviol glycoside reactants in a reaction composition comprising one or more of: (i) stevioside, rebaudioside A, rebaudioside C, rebaudioside F, rebaudioside G, or dulcoside A; (ii) UDP-xylose; (iii) a polypeptide of SEQ ID NO:1 or SEQ ID NO:2; (iv) a non-activated sugar; and (v) a magnesium and / or manganese salt; the reaction composition has a pH in the range of 4.8 to 7.8; one or more of stevioside, rebaudioside A, rebaudioside C, rebaudioside F, rebaudioside G, or dulcoside A is present at 80% or more (mol) of the total of all steviol glycosides in the reacted composition; forming a ratio of said polypeptide (by weight) to said one or more of stevioside, rebaudioside A, rebaudioside C, rebaudioside F, rebaudioside G, or dulcoside A (by mole) in the range of 15 μg:1 μmol to 7.5 μg:1 μmol; and reacting the reaction composition at a temperature in the range of 25 to 95° C. for at least 12 hours to provide a product composition comprising one or more of compounds D, E, G, H, I, or J. 【Chemistry 6】 【Chemistry 7】 【Chemistry 8】

23. 23. A composition comprising the xylosylated steviol glycoside compound formed according to claim 22.

24. 1. An ingestible or aqueous composition comprising a mixture of steviol glycosides, including a compound of formula II, 【Chemistry 9】 In the formula, R ３ contains a xylose residue, R ４ comprises one or more sugar residues or is hydrogen, and the compound of formula II is present in an amount greater than any other single steviol glycoside in the composition.

25. A glycosyltransferase having UDP-xylose:19-steviol xylosyltransferase activity, comprising a polypeptide having at least 90%, at least 95%, or at least 98% identity to SEQ ID NO:1 and the following amino acid sequence relative to SEQ ID NO:1: I152.

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