Xylosylated steviol glycoside and enzymatic production method

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 of related applications) This application claims the interests of U.S. Provisional Patent Application No. 63 / 155,229, filed on March 1, 2021, which is incorporated herein by reference in its entirety.

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

[0003] (Field of Invention) This disclosure relates to xylosylated steviol glycosides and methods for their enzymatic production. [Background technology]

[0004] Sugars such as sucrose, fructose, and glucose are used to provide a desirable taste in beverages, foods, pharmaceuticals, and oral hygiene / cosmetic products. Specifically, sucrose imparts a taste that is preferred by consumers. While sucrose offers excellent sweetness characteristics, it is high in calories. Zero-calorie or lower-calorie sweeteners have been introduced to meet consumer demand, and there is a need for these types of sweeteners with desirable taste characteristics.

[0005] Stevia is a genus of herbs and shrubs belonging to the Asteraceae family, comprising approximately 240 species, and is native to the subtropical and tropical regions of western North America and South America. Stevia rebaudiana, commonly known as sweetleaf, 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 steviol, a diterpene compound. These diterpene glycosides are about 150 to 450 times sweeter than sugar.

[0006] Examples of steviol glycosides are described in International Publication No. 2013 / 096420 (see, for example, the list in Figure 1), and in Ohta et al., (2010) "Characterization of Novel Steviol Glycosides from Leaves of Stevia rebaudiana Morita," J.Appl.Glycosi., 57:199-209 (see, for example, Table 4 on p. 204). Structurally, diterpene glycosides are characterized by a single base, steviol, and differ in the presence of carbohydrate residues at positions C13 and C19, as shown in Figures 2a to 2k. 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 dry weight of the leaves, 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 position C13 [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 level up to 350 times that of sucrose and less bitterness (Prakash). Compared to Reb A and other steviol glucosides, Reb M is characterized by a higher sweetness intensity, faster onset of sweetness, a cleaner taste, and a significantly reduced aftertaste of sweetness (licorice), bitterness, sourness, and astringency. M is present only in trace amounts in the leaves of S. rebaudiana (approximately 0.4-1.5% (w / w) of total dry weight in traditional Chinese plants). [Overview of the Initiative]

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

[0009] In some embodiments, the present disclosure provides methods for forming xylosylated steviol glycosides. The methods comprise 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. Formula I is as follows,

[0010]

Chemical Formula

[0011]

Chemical Formula

[0012] Examples of glycosyltransferase polypeptides having UDP-xylose:19-steviol xylosyltransferase activity include those of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, variants thereof, and homologs thereof.

[0013] In the xylosylated product of Formula II, R 3 comprises a xylose residue, and R 4 may be the same chemistry as R in the compound of Formula I 2 , or R 4 may comprise one or more additional sugar residues compared to R 2 . In an embodiment, R 4 is an oligosaccharide moiety comprising a xylose residue such as β-Glc-β-Xyl, which may have a Glc2C→Xyl1C glycosidic bond. In an embodiment, R 4 may comprise one or more other sugar residues such as glucose or rhamnose. In still other embodiments, the compound of Formula I that is xylosylated at the 19-position is selected from stevioside, rebaudioside A (RebA), rebaudioside C (RebC), rebaudioside D (RebD), rebaudioside F, rebaudioside G, and dulcoside-A, wherein stevioside, RebA, and RebC are preferred substrates.

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

[0015]

Chemical Formula

[0016] In some embodiments, the present disclosure provides a method for forming xylosylated steviol glycosides, the method using a reaction composition. The reaction composition comprises (i) a steviol glycoside compound of formula I, (ii) activated xylose, and (iii) a glycosyltransferase having UDP-xylose:19-steviol xylosyltransferase activity. The 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 activated xylose to the compound of formula I.

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

[0018] In other embodiments, the Disclosure provides a method for forming xylosylated steviol glycosides using engineered cells, the engineered cells being able to produce steviol glycoside compounds of formula I, the cells expressing a glycosyltransferase having UDP-xylose:19-steviol xylosyltransferase activity, such as one of SEQ ID NOs: 1, SEQ ID NOs: 2, or SEQ ID NOs: 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 into the engineered cells, for example, by supplying activated xylose to the cells from an external source, or the engineered cells can produce activated xylose. Polypeptides transfer xylose from activated xylose to the compound of formula I to form the xylosylated steviol glycoside compound of formula II. In some embodiments, a steviol glycoside (precursor) compound used to produce the steviol glycoside compound of formula I, which is used as an acceptor molecule, can be supplied to the cells. In some embodiments, a steviol glycoside compound of formula I, such as RebA, can be supplied to the cells and then used directly as an acceptor for xylosylation to form the compound of formula II, such as RebDG.

[0020] In other embodiments, the disclosure provides engineered cells having a pathway for forming steviol glycoside compounds of formula I. The cells are engineered to provide steviol glycoside compounds of formula I, such as stevioside, RebA, or RebC, in amounts greater than the amount of non-formula I steviol glycosides that can be formed intracellularly. The cells also express a glycosyltransferase having UDP-xylose:19-steviol xylosyltransferase activity, and the polypeptide can transfer xylose from activated xylose to a compound of formula I to form a xylosylated steviol glycoside compound of formula II.

[0021] In other embodiments, the Disclosure provides an ingestible composition or aqueous composition comprising a mixture of steviol glycosides comprising a compound of formula II, wherein the compound of formula II, such as RebDG, is present in a greater amount than any other single steviol glycoside in the composition. In further embodiments, two or more compounds of formula II are present in such an ingestible composition or aqueous composition, wherein the compounds of formula II are present in a greater total amount than the total amount of other steviol glycosides in the composition. In another embodiment, an ingestible composition or aqueous composition comprising a mixture of steviol glycosides comprising rebaudioside M, and if rebaudioside M is present, the compound of formula II is present in a greater amount than rebaudioside M, wherein the compound of formula II optionally constitutes 1% (mol) or more, for example, 2%, 3%, or 5% (mol) or more of all steviol glycosides in the ingestible composition or aqueous composition.

[0022] Compositions containing a mixture of steviol glycosides can be obtained from reaction compositions or manipulated cells containing a glycosyltransferase having UDP-xylose:19-steviol xylosyltransferase activity.

[0023] In yet another aspect, the disclosure provides an engineered glycosyltransferase variant having remarkably high levels of UDP-xylose:19-steviol xylosyltransferase activity. The glycosyltransferase variant contains a mutant amino acid that changes 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 the steviol glycoside molecule at position 19 in the presence of activated xylose.

[0024] Accordingly, in another embodiment, the present 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 with SEQ ID NO: 1 and the following amino acid: I152. In some embodiments, the glycosyltransferase variant is SEQ ID NO: 2, which has a sequence with a change from a single serine to an isoleucine at position 152 compared to SEQ ID NO: 1. [Brief explanation of the drawing]

[0025] [Figure 1] The table shows the chemical structures of steviol bases and various steviol glycosides with chemical definitions for the R1 and R2 groups. [Figure 2] This describes the mevalonate (MVA) pathway for the conversion of acetyl-CoA to isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) using eukaryotic and / or archaeal enzymes. [Figure 3] This diagram shows the non-mevalonate (MEP) pathway for the enzymatic conversion of glyceraldehyde-3-phosphate (G3P) and pyruvate (PYR) to IPP and DMAPP. [Figure 4] This diagram shows the enzymatic pathways for the conversion of IPP and farnesyl pyrophosphate (FPP) to steviol. [Figure 5] This graph shows the average %SG acceptor conversion by UDP-xylose using the enzyme SEQ ID NO: 1 and the UGT76G1 enzyme. [Figure 6] This is the amino acid sequence of sequence number 1 (AC133334). [Figure 7] This is an amino acid sequence alignment of SEQ ID NO: 1 with other glycosyltransferases or their variants. [Figure 8A] This graph shows the time-dependent transfer of glucose and xylose from UDP-glucose and UDP-xylose to RebA in the presence of the polypeptide of Sequence ID No. 1 and 1 mM activated sugar. [Figure 8B] This graph shows the time-dependent transfer of glucose, xylose, and rhamnose from UDP-glucose, UDP-xylose, and UDP-rhamnose to RebA in the presence of the polypeptide of Sequence ID No. 1 and 2 mM activated sugar. [Figure 9A] This graph shows the time-dependent transfer of UDP-glucose, UDP-xylose, and UDP-rhamnose to the -19C(O)-β-Glc residue of Reb F, Reb G, and dulcoside A in the presence of the polypeptide of Sequence ID No. 1. [Figure 9B] This graph shows the time-dependent transfer of UDP-glucose, UDP-xylose, and UDP-rhamnose to the -19C(O)-β-Glc residue of Reb F, Reb G, and dulcoside A in the presence of the polypeptide of Sequence ID No. 1. [Figure 9C] This graph shows the time-dependent transfer of UDP-glucose, UDP-xylose, and UDP-rhamnose to the -19C(O)-β-Glc residue of Reb F, Reb G, and dulcoside A in the presence of the polypeptide of Sequence ID No. 1. [Figure 10] This is the amino acid sequence alignment between SEQ ID NO: 1 and SEQ ID NOs: 2-4. [Modes for carrying out the invention]

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

[0027] The present disclosure provides a method for forming a xylosylated steviol glycoside from a steviol glycoside acceptor molecule using a glycosyltransferase having UDP-xylose:19-steviol xylosyltransferase activity (in some embodiments, a polypeptide having at least 50% identity with 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, the steviol glycoside acceptor molecule is preferentially xylosylated at the 19th carbon to form a xylosylated steviol glycoside having a xylose residue bonded to the 19th carbon or having one or more xylose residues present in an oligosaccharide bonded to the 19th carbon.

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

[0029] The methods of the present disclosure can also be carried out using engineered cells expressing a glycosyltransferase having UDP-xylose:19-steviol xylosyltransferase activity, such as a glycosyltransferase polypeptide having at least 50% identity with any of SEQ ID NOs: 1-3, and activated xylose, such as UDP-xylose. The engineered cells may also have a pathway for producing steviol glycoside compounds, which are acceptor molecules for xylosylation at the 19th carbon. In some embodiments, the engineered cells may also have a pathway for producing activated xylose, which can be utilized by a polypeptide for xylosylation of steviol glycoside compounds. For example, the engineered cells may also contain one or more UDP-glucose dehydrogenases and / or UDP-glucuronide decarboxylases for the production of UDP-xylose. Alternatively, the engineered cells may be supplied with activated xylose, which can be taken up by the cells and used by the glycosyltransferase polypeptide. Alternatively, the manipulated cells can be supplied with a steviol glycoside molecule that can act as an acceptor molecule for xylosylation at position 19, or 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 bonded to a steviol base. Structurally, a steviol glycoside has a central molecular moiety which is a single steviol base, and glucopyranosyl residues bonded to the C13 and / or C19 atoms of the steviol base, according to the atomic number assignment on the base shown below. That is, one or more glycopyranosyl residues bond to the group R in formula I. 1 and / or R 2 It may exist in [location].

[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 also be present in steviol glycosides. 1 and / or R 2 If one or both of them have a single glycopyranosyl group, they can be referred to as the monosaccharide portion or monosaccharide group of a steviol glycoside. 1 and / or R 2 If one or both of these have two or more glycopyranosyl groups, they can be referred to as the oligosaccharide moiety / group of a steviol glycoside. The oligosaccharide moiety may consist of glycopyranosyl residues of the same type within it (homooligosaccharide moiety), or it may consist of glycopyranosyl residues of different types within it (heterooligosaccharide moiety).

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

[0034] The methods of this disclosure utilize a glycosyltransferase having UDP-xylose:19-steviol xylosyltransferase activity. A “glycosyltransferase” generally refers to an enzyme capable of transferring a sugar moiety to an acceptor molecule, where “steviol glycosyltransferase” or “steviol glycoside glycosyltransferase” can transfer a sugar moiety to either a steviol base acceptor or a steviol glycoside acceptor, respectively. The activity of a glycosyltransferase in attaching a specific sugar moiety(s) to a specific position(s) on a steviol or steviol glycoside molecule may also be described. According to the methods, compositions, and operations of this disclosure, a glycosyltransferase having UDP-xylose:19-steviol xylosyltransferase activity, such as the polypeptide of SEQ ID NO: 1, is used. UDP-xylose:19-steviol xylosyltransferase activity refers to the enzymatic activity that transfers the xylose moiety from UDP-xylose to the 19C atom, or a sugar residue directly or indirectly bound to (and extending from) the 19C atom, to a steviol acceptor molecule. Glycosyltransferases do not exclusively possess UDP-xylose:19-steviol xylosyltransferase activity, and may further possess (or not possess) other 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 this disclosure can preferentially add xylose to the 19th position of the steviol / steviol glycoside (rather than the 13th position).The ability of glycosyltransferase to preferentially add xylose at position 19 can be determined by performing an assay using a composition containing glycosyltransferase and UDP-xylose and analyzing the reaction product, wherein more than 50 mol% (or >60%, >70%, >80%, >90%, >95%, >97%, >98%, or >99%) of the product is a steviol glycoside xylosylated at position 19(c).

[0035] In one embodiment, a glycosyltransferase polypeptide having at least 50% identity with any of Sequence IDs 1 to 3 is used to transfer one or more xylose residues to the carbon at position 19 of a steviol glycoside acceptor molecule. In this method, the steviol glycoside acceptor molecule has a single glucopyranose moiety or an oligosaccharide moiety containing one or more glucopyranose residues bonded to the carbon at position 19 of a steviol glycoside. The steviol glycoside acceptor may be a compound of formula I.

[0036] [ka] In the formula, R 1 It contains a glucose residue, R 2 is a compound containing one or more sugar residues or 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 bond xylose to a glucopyranose residue at position 19, yielding an oligosaccharide containing a glucopyranose residue glycosidically bonded to the xylose residue. The enzymatic reaction can yield the compound of formula II,

[0038] [ka] In the formula, R 3 It contains xylose residues, R 4 R is in the compound of formula I. 2 It has the same chemistry as R 4 is R 2 It contains one or more additional sugar residues compared to [the other component]. In some cases, the glycosyltransferase adds one or more xylose, glucose, or rhamnose moieties to the carbon at position 13 (for example, to a sugar residue bonded to the carbon at position 13). In some embodiments, R 3 This is an oligosaccharide moiety containing xylose residues such as β-Glc-β-Xyl (which may have a Glc2C→Xyl1C glycosidic bond). For example, xylosylated steviol glycoside has an oligosaccharide moiety (R) bonded to the carbon at position 19 of the steviol base. 3 It can contain β-Glc-β-Xyl in the oligosaccharide portion, and β-Glc-β-Xyl may have a Glc2C→Xyl1C glycosidic bond. An exemplary product of formula II is called rebaudioside DG, which is SG-[13-β-Glc[(3→1)β-Glc](2→1)β-Glc(1-2)]-[19-β-Glc(2→1)β-Xyl].

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

[0040] Uridine diphosphate (UDP) glycosyltransferase (UGT) is a member of Family I glycosyltransferase (GT), which is responsible for the glycosylation of natural products, particularly the transfer of various sugars to plant natural products. Many UGT gene subfamilies also exist that are involved in the glycosylation of different types of plant products. See, for example, Wang, X. (2009) FEBS Letters 583:3303-3309. GTs are 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 UGT-encoding genes have also been identified in several other higher plant species. Each GT family consists of proteins associated by sequence, and consequently by type of protein folding. Therefore, predictability exists within the family based on the conservation of catalytic mechanisms. GT polypeptides are also classified into families by amino acid similarity (see, e.g., Coutinho et al., 2003). A list of actively curated 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 the GT-B fold, one of the two common folds of the enzyme's GT superfamily, and also reveal that these UGTs possess two N-terminal and C-terminal domains with similar Rossmann-like folds. Some of the UGT crystal structures include those of *Medicago truncatula* UGT71G1, UGT85H2, UGT78G1, *Vitis vinifera* VvGT1 26, and *Arabidopsis 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), which describes a modeled structure of Sequence ID No. 1 using a computational strategy combining protein structure prediction, sequence optimization, and molecular dynamics simulation to improve the catalytic efficiency of Sequence ID No. 1 for synthesizing 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 derived from rice (Oryza sativa Japonica Group) (see, for example, U.S. Patent No. 9,631,215 (#152) and access number AC133334). Based on alignment with other EUGTs, the amino acid residues of the conserved domain, including the active site, substrate binding pocket, and TDP binding site, can be identified in SEQ ID NO: 1.

[0043] The method of this disclosure uses a polypeptide having at least 50% identity with 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 with SEQ ID NO: 1. In some embodiments, the glycosyltransferase has 100% identity with SEQ ID NO: 1.

[0044] Using an in silico modeling system, the interaction between the polypeptide of SEQ ID NO: 1 and its ligand was predicted, which was then based on the elucidated structure of the homolog of SEQ ID NO: 1. This in silico modeling yielded data revealing the amino acid residues containing SEQ ID NO: 1 that come into contact with the substrate for SEQ ID NO: 1-RebA-UDP-xylose and SEQ ID NO: 1-RebA-UDP-glucose. The data identified 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 list "A"). The data revealed that, with the following exceptions, the same amino acids are involved in RebA-UDP-glucose (ligand) interactions: P59 and D128 are involved in UDP-xylose (ligand) interactions but not in UDP-glucose interactions; L149, S203, R310, and I378 are involved in UDP-glucose (ligand) interactions but not in 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 that are highly conserved in UGT 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 embodiments, the glycosyltransferase has less than 99.8% identity with SEQ ID NO: 1, but greater than 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identity, and the glycosyltransferase has one or more mutant amino acids that are not in the position of amino acid list "A", not in the position of amino acid list "B", or not in both the position of "A" and "B". In some embodiments, the glycosyltransferase based on SEQ ID NO: 1 has one or more mutant amino acids at the position of amino acid list "C".

[0048] In some embodiments, the glycosyltransferase having UDP-xylose:19-steviol xylosyltransferase activity includes one or more 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) demonstrated that the consensus motif for all glycosyltransferases can be purified and extended toward the N-terminus of the protein to a 44-amino acid consensus typical of secondary metabolite plant UGTs. This plant secondary product glycosyltransferase (PSPG) motif was later found in all cloned secondary metabolite UGTs from other plant species.

[0050] In some embodiments, glycosyltransferases having UDP-xylose:19-steviol xylosyltransferase have one or more mutant amino acids at positions that are involved in RebA-UDP-glucose (ligand) interactions but not in RebA-UDP-xylose (ligand) interactions. As described herein, data have 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 in UDP-glucose interactions; L149, S203, R310, and I378 are involved in UDP-glucose (ligand) interactions but not in UDP-xylose (ligand) interactions; however, S203, R310, and I378 are still involved in RebA interactions. Therefore, compared to SEQ ID NO: 1 or 2, glycosyltransferase may have one or more mutant amino acids at the following positions: L149, S203, R310, and I378W339.

[0051] One or more amino acid variants may be non-conservative substitutions (more preferred) or conservative substitutions (less preferred). Such mutations can result in a reduced interaction of the glycosyltransferase with UDP-glucose, which then favorably interacts with UDP-xylose, driving greater xylosylation of the steviol glycoside acceptor molecule. In other embodiments, with respect to SEQ ID NO: 1 or 2, the glycosyltransferase may have a mutant amino acid 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, a glycosyltransferase having UDP-xylose:19-steviol xylosyltransferase activity includes the following amino acid mutations compared to SEQ ID NO: 1:152. In embodiments using polypeptides having less than 100% identity to any one of SEQ ID NOs: 1-3, the difference in identity may be due to one or more amino acid substitutions in one or more regions of the polypeptide, e.g., regions outside the regions understood to be important for the active site, substrate binding pocket, and TDP binding site, and / or regions outside the conserved region, based on alignment with one or more other UGTs of the same family as SEQ ID NO: 1. Alternatively, if 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 with other UGTs of the same family as any of SEQ ID NOs: 1-3. 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. In other words, if Sequence ID No. 1 is modified, it is modified at one or more amino acid positions outside the conserved region and / or the region important for the enzymatic function of the protein.

[0053] Figure 7 shows the alignment of Sequence ID No. 1 with homologous variants or other glycosyltransferases to Sequence ID No. 1. As shown in the alignment, the shaded amino acid rows at specific positions represent amino acids in the Sequence ID No. 1 homolog that are identical or similar to the corresponding amino acids in the Sequence ID No. 1 template. Similarly, Figure 10 shows the alignment of Sequence ID No. 1 with Sequence ID Nos. 2-4, where shading represents characteristics of different groups of amino acids.

[0054] In a preferred embodiment, any variant of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3 either has no mutant amino acid at that position, or, if a variant exists, the mutant amino acid substitution is a conserved 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), a mutant glycosyltransferase can be produced by one or more amino acid substitutions made at any amino acid position in the glycosyltransferase template (e.g., SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3) that aligns with SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, such as positions 3-15, 18-20, etc. (as reflected by unshadowed amino acids). Such substitutions may preferably be conserved substitutions, but non-conservative substitutions may be used if the function is not affected. Shadowed amino acids in templates such as SEQ ID NO: 1, 2, 16, 17, 21-24, etc., in SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, preferably do not undergo any substitutions when a variant is used or prepared, or when at most, conserved amino acid substitutions are used.

[0055] A "conservative amino acid substitution" or "conservative mutation" of a particular sequence refers to the substitution of one amino acid or a series of amino acids with a functionally identical amino acid. Tables of conservative substitutions that provide functionally similar amino acids are well known in the art and include substitutions 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., aliphatic residues, aromatic residues, positively charged residues, negatively charged residues, polar residues, nonpolar residues, positively polar residues, negatively polar residues, uncharged residues, nonpolar hydrophobic residues, ionic acidic residues, ionic basic residues, or sulfur-containing residues). The following six groups each contain amino acids that can be conservedly substituted 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] The polypeptide sequence identity region between homologous polypeptides and sequence numbers 1, 2, or 3 can be identified using the sequence alignment tools described herein.

[0057] The glycosyltransferase polypeptides of this disclosure may also have deletions in one or more regions of a natural glycosyltransferase polypeptide, wherein the deletions do not affect the glycosyltransferase activity of the polypeptide. The deletions may be based on known information regarding the structure and function of natural glycosyltransferases, including, for example, information regarding regions that are conserved and / or important to the enzymatic function of the protein, such as sequences (a) to (i) described herein.

[0058] The determination of "corresponding" amino acids from two or more glycosyltransferases can be understood by the alignment of all or part of their amino acid sequences. Sequence alignment and sequence identity generation include global and local alignments, which typically utilize computational approaches. To provide a global alignment, global optimization is used, forcing a full-length sequence alignment of all query sequences. Local alignments, through comparison, identify shorter regions of similarity within longer sequences.

[0059] As used herein, “equivalent position” means a position common to two sequences (e.g., SEQ ID NO: 1 and different UGT sequences having the desired substitution) based on the best alignment of the amino acid sequences of one glycosyltransferase, or as a three-dimensional structural alignment. Therefore, equivalence can be determined using either sequence alignment or structural alignment, or both.

[0060] The amino acid positions described herein are related to the amino acid sequences of SEQ ID NO: 1 or SEQ ID NO: 2, but do not have the same sequence length. However, the corresponding positions in other glycosyltransferases used in the methods of this disclosure are those that can still be aligned using the MUSCLE algorithm, which has 10 positional opening gaps and 0.2 extension gaps, as performed on the Schrodinger platform v 2020-4. As shown in Figure 10, SEQ ID NO: 1 or SEQ ID NO: 2 aligns well with SEQ ID NOs: 3 and 4, and therefore the identification of the corresponding positions in either SEQ ID NOs: 3 and 4 for SEQ ID NOs: 1 and 3 is easily understood. For example, in SEQ ID NO: 3, the amino acid positions are shifted by -10 for the first 44 amino acids from SEQ ID NOs: 1 and 2 (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 several implementations, the BLAST algorithm is used to compare and determine sequence similarity or identity. Furthermore, it can determine the presence or significance of gaps in sequences to which weights or scores can be assigned. These algorithms can also be used to determine nucleotide sequence similarity or identity. Parameters for determining relevance are calculated based on methods known in the art for calculating statistical similarity and the significance of determined agreements. Related gene products are expected to have high similarity (e.g., sequence identity greater than 50%). Exemplary parameters for determining the relevance of two or more sequences using the BLAST algorithm may be as follows:

[0062] In some implementations, 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 that achieves an identity score of XX% (e.g., 80%) relative 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 XX% identical.

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

[0064] In aspects of this disclosure, the method for forming xylosylated steviol glycosides is carried out in a reaction composition. The reaction composition can be prepared to contain the following reagents of a desired type and amount: (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 with SEQ ID NO: 1. The reaction composition may contain components (i), (ii), and (iii), meaning that other components different from (i), (ii), and (iii) may be optionally included in the reaction composition. The reaction composition may also contain other components such as inactivated sugars, cofactors, and the liquid or liquid mixture in which the reaction is carried out.

[0065] The reaction composition comprises the steviol glycoside acceptor of formula I,

[0066] [ka] In the formula, R 1 It contains a glucose residue, R 2 R contains one or more sugar residues or is hydrogen. In some cases, R 1 This is a single glucose residue such as β-Glc. 1 An example of a steviol glycoside acceptor having a single glucose residue is steviol monoglucosyl ester (R 1 =β-Glu, R 2 =-H), rubsozide (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 Examples include α-Rha(1-2)β-Glu-). In some embodiments, the steviol glycoside acceptor of formula I is provided in the reaction composition in a pure form (e.g., more than 99.9% (by weight) of total steviol glycosides) or a substantially pure form (e.g., more than 99% (by weight) of total steviol glycosides).

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

[0068] In some embodiments, the steviol glycoside acceptor (compound of formula I) in the reaction composition may be one or a mixture of, for example, steviol monoglucosyl ester, 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 may contain one or more other steviol glycosides that, unlike those of formula I, are not acceptor molecules for xylosylation. A reaction composition containing a mixture of acceptor and non-acceptor steviol glycosides can be obtained from a leaf extract (e.g., steviol leaf).

[0069] In other embodiments, the reaction composition can be enriched with one or more steviol glycoside acceptors of formula I. For example, in the reaction composition, the steviol glycoside acceptors of formula I constitute 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 essentially consists of one or more compounds of formula I, meaning that other steviol glycosides that are not of formula I may be present in very small amounts (less than 1%(weight) of the total steviol glycosides) in the steviol glycoside component of the reaction mixture. In yet another embodiment, the steviol glycoside component of the reaction mixture consists of one or more compounds of formula I, meaning that other steviol glycosides that are not of formula I are not present in any detectable amount in the steviol glycoside component of the reaction mixture.

[0070] In some embodiments, the reaction composition consists of 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 glycoside therein. In other embodiments, the steviol glycoside component of the reaction composition consists of rebaudioside A.

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

[0072] The composition also comprises a glycosyltransferase polypeptide 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. The glycosyltransferases of this disclosure may 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 2. U.S. Patent No. 9,631,215 describes the purification of 6HIS-tagged or GST-tagged SEQ ID NO: 1 from recombinant Escherichia coli (E. coli) strains expressing this fusion protein.

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

[0074] If necessary, the amounts of steviol glycoside (SG) acceptor and glycosyltransferase polypeptide present in the reaction composition may be described in relation to each other. For example, the reaction composition may have a glycosyltransferase polypeptide (weight) to steviol glycoside (SG) acceptor (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.

[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 some 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 of UDP-xylose per 100 μL reaction volume), about 0.05 to about 1.0 mole, or about 0.1 to about 0.4 moles. UDP-xylose has the following chemical structure:

[0076] [ka]

[0077] If necessary, the amounts of UDP-xylose and steviol glycoside (SG) acceptor, or UDP-xylose and glycosyltransferase polypeptide present in the reaction composition may be described in relation to each other. For example, in some embodiments, the reaction composition includes UDP-xylose (UDP-X) in a molar excess to the steviol glycoside compound of formula I (SGI), or UDP-X:SGI molar ratios in the range of 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 includes glycosyltransferase polypeptide (weight) to UDP-X (mol) ratios in the range of 50 μg:1 μmol to 10 μg:1 μmol, 25 μg:1 μmol to 15 μg:1 μmol, or about 20 μg:1 μmol.

[0078] The reaction composition may also contain one or more inactivated sugar components to facilitate the reaction. For example, the composition may contain an inactivated sugar such as sucrose to facilitate the stabilization of the enzymatic reaction. Other inactivated sugars include maltose, trehalose, glucose, and starch hydrolysates such as glucose syrup and maltodextrin. The inactivated sugar may be used in amounts ranging from about 10 mM to about 0.5 M, or from about 50 mM to about 0.2 M. Optionally, the amounts of the inactivated sugar and the steviol glycoside (SG) acceptor may be described in relation to each other. For example, in some embodiments, the reaction composition contains a molar excess of the inactivated sugar pair steviol glycoside compound of formula I (SGI) in the ranges 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 composition may also include salts that provide divalent cations that can be used as cofactors for glycosyltransferase polypeptides. Examples of divalent metal salts include magnesium and / or manganese salts, which can be used in the reaction mixture in amounts ranging from about 0.5 mM to about 5 mM or about 1 mM to about 4 mM.

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

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

[0082] The reaction can be carried out at a desired temperature and time to promote the xylosylation of the steviol glycoside acceptor molecule. For example, the reaction can be carried out for at least 1 hour and up to about 10 days, for example, in the range of 1 to 24 hours, 1 to 12 hours, about 12 hours to about 7 days, or about 1 to about 5 days. The reaction can be carried out at temperatures in the range of 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, which includes one or more xylosylated steviol glycoside residual reaction components, including polypeptides 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 reaction product.

[0084] After the reaction, the product composition can be purified to separate the components of the reaction mixture. The xylosylated steviol glycoside product can be purified by methods such as crystallization as described herein, or by using a reversed-phase chromatography column. The hydrophilic components of the reaction mixture can be removed with water, and the xylosylated steviol glycoside compound can be removed by elution with a solvent such as methanol. Furthermore, the xylosylated steviol glycoside compound can be further separated using preparative HPLC (see, for example, International Publication No. 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 It contains xylose residues, R 4 R contains one or more sugar residues or is hydrogen. In this embodiment, R 3 This is an oligosaccharide moiety containing xylose residues such as β-Glc-β-Xyl and having a Glc2C→Xyl1C glycosidic bond. In some embodiments, the xylosylated steviol glycoside is selected 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 xylosylated steviol glycosides in high yield. In particular, more than 50%, more than 60%, more than 65%, more than 75%, or more than 80% of the steviol glycoside compound of formula I in the reaction composition is converted to the xylosylated steviol glycoside compound of formula II.

[0094] The reaction can also provide a product composition comprising one or more compounds of Formula II described herein. The type and relative amount of the xylosylated steviol glycoside product may depend on the type and relative amount of the steviol glycoside acceptor used in the reaction composition.

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

[0096] After the reaction, the product composition can be purified to separate the components of the reaction mixture. The glycosylated steviol glycoside product can be purified by methods such as crystallization or by using a reversed-phase chromatography column. The hydrophilic components of the reaction mixture can be removed with water, and the glycosylated steviol glycoside compound can be removed by elution with a solvent such as methanol. Furthermore, the glycosylated steviol glycoside compound can be further separated using preparative HPLC (see, for example, International Publication No. 2009 / 140394).

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

[0098] In some embodiments, the engineered cells include a pathway to steviol, a precursor of the compound of formula I, which can be used as a substrate for forming the compound of formula II using a glycosyltransferase polypeptide having UDP-xylose:19-steviol xylosyltransferase activity, such as a polypeptide having at least 50% identity with SEQ ID NO: 1, and activated xylose. The cells may be engineered to provide a greater amount of the steviol glycoside compound of formula I than the amount of non-SEQ ID NO 1 steviol glycoside that can be formed intracellularly. The cells may also express a glycosyltransferase polypeptide having UDP-xylose:19-steviol xylosyltransferase activity, for example a polypeptide having at least 50% identity with SEQ ID NO: 1, and the polypeptide can transfer xylose from activated xylose to the compound of formula I to form the xylosylated steviol glycoside compound of formula II. Exemplary engineered cells include engineered yeast, bacteria, and fungi.

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

[0100] Figure 2 shows a typical mevalonate pathway. In this pathway, 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). The 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 may include archaeal enzymes that use mevalonate-3-kinase to convert 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 lack any of the enzymes required for the mevalonate pathway, some cells may contain some, but not all, 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 those lacking mevalonate pathway genes. The yeast Saccharomyces cerevisiae naturally expresses mevalonate pathway genes, but it can be engineered to increase the expression of these pathway genes.

[0102] In some embodiments, prokaryotic cells can have their mevalonate pathway manipulated. Martin, VJ et al. described the manipulation of the mevalonate pathway in E. coli for terpenoid production (Nature Biotechnology 21:796-802, 2003). Wang, J. et al. described the manipulation of a highly efficient E. coli strain for mevalonate fermentation by chromosomal integration (Appl. Environ. Microbiol. 82:7176-7184, 2016).

[0103] In some embodiments, eukaryotic cells are manipulated via the mevalonate pathway or modified via mevalonate pathway genes to provide greater amounts of IPP. For example, Gold, ND et al. describe a combinatorial approach to studying 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 generally more energetically efficient than the MVA pathway because it loses less carbon as CO2 (MEP pathway: 1 CO2 / IPP; MVA pathway: 4 CO2 / IPP; sugar as carbon source).

[0105] In particular, in the non-mevalonate (MEP) pathway, the compounds isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) are produced via a series of intermediates derived from glyceraldehyde-3-phosphate (G3P) and pyruvate (PYR), and several enzymes are involved in this conversion. Figure 3 shows a typical non-mevalonate pathway. Enzymes involved in the biosynthesis pathway from G3P and PYR to IPP and DMAPP include (a2) 1-deoxy-D-xylulose-5-phosphate synthase Examples include (b) 1-deoxy-D-xylulose-5-phosphate reductosomerase (ispC), (c) 4-diphosphocytidyl-2C-methyl-D-erythritol synthase (IspD), (d) 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase (IspE), (e) 2C-methyl-D-erythritol-2,4-cyclodiphosphate synthase (IspF), (f) 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate synthase (IspG), (g) 4-hydroxy-3-methyl-2-(E)-butenyl-4-diphosphate reductase (IspH), and (h) isopentenyl-diphosphate isomerase (IDI).

[0106] U.S. Patent No. 9,284,570 describes a method for producing steviol or steviol glycoside in Escherichia coli using the upstream methylerythritol pathway (MEP), which produces isopentyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP).

[0107] The yeast Saccharomyces cerevisiae naturally expresses MEP pathway genes, but can be engineered to provide MEP pathway genes as needed.

[0108] In some embodiments, the manipulated cells may include one or more modifications to increase the flux from acetyl-CoA to IPP and / or DMAPP, thereby providing an increased pool of IPP and / or DMAPP for use in the steviol pathway. Modifications include, for example, increasing the expression or activity of one or more mevalonate pathway enzymes (a1) to (f1) by placing nucleic acids encoding enzymes that are homogeneous or heterogeneous to the cell under the control of a promoter that results in increased expression, using multiple copies of the nucleic acid, and / or using heterogeneous enzymes, mutant enzymes (e.g., those containing one or more amino acid substitutions), or mutant heterogeneous enzymes that result in a higher level of enzymatic activity compared to the native enzyme.

[0109] The methods of this disclosure for producing xylosylated steviol glycosides using engineered cells may include cells having one or more genetic modifications that increase the flux from G3P and PYR to IPP and / or DMAPP, thereby providing an increased pool of IPP and / or DMAPP for use in the steviol pathway. Modifications include, for example, increasing the expression or activity of one or more enzymes (a2) to (h2) by placing a nucleic acid encoding an enzyme that is 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 or more amino acid substitutions), or a mutant heterologous enzyme that results in a high level of enzymatic activity.

[0110] The methods of the present disclosure for producing xylosylated steviol glycosides may utilize engineered cells, which may also include pathways for converting IPP and / or DMAPP and farnesyl pyrophosphate (FPP) to steviol. For example, referring to Figure 4, in some embodiments the engineered cells may comprise exogenous nucleic acids expressing the following enzymes: (a3) ​​geranyl geranyl diphosphate synthase (GGPPS), (b3) copalyl diphosphate synthase (CPS), (c3) kaurene synthase (KS), (d3) kaurene oxidase (KO), and (e3) kaurenoic acid 13-hydroxylase (KAH). The enzymes of the mevalonate pathway convert IPP and / or DMAPP to steviol as follows: IPP / DMAPP → geranylgeranyl diphosphate → copalyl diphosphate → kauren → kaurenic acid → steviol. Exogenous nucleic acids encoding enzymes (a3)~(e3) that are heterologous to yeast cells can be placed under the control of promoters that provide increased expression using multiple copies of the nucleic acid and / or mutant enzymes (e.g., those containing one or more amino acid substitutions) or mutant heterologous enzymes that provide high levels of enzymatic activity.

[0111] U.S. Patent No. 9,284,570 describes a method for producing steviol or steviol glycosides in Escherichia 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), kauren synthase (KS), geranylgeranyl diphosphate synthase (GGPPS), kaurenic acid 13-hydroxylase (KAH), kauren oxidase (KO), and optionally one or more stevia UDP glycosyltransferase enzymes.

[0112] The manipulated cells also express a glycosyltransferase polypeptide having UDP-xylose:19-steviol xylosyltransferase activity, for example, 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] The regulation of heterologous gene expression can be controlled using desired promoters, terminators, and gene copy numbers.

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

[0115] Unlike SEQ ID NO: 1 or its homologs or mutants, the manipulated cells may contain one or more uridine diphosphate (UDP) glycosyltransferases (UGTs) that mediate the transfer of glycosyl residues from activated nucleotide sugars to steviol acceptor molecules.

[0116] Exemplary UDP-glucosyltransferases other than those having Sequence ID No. 1 may 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 embodiment, the engineered cell may contain one or more UDP-glucosyltransferases selected from the group of UGTs having substantial (>85%) identity to UGT74G1, UGT85C2, UGT76G1, UGT91d2, and these polypeptides. The engineered cell may contain one or more exogenous nucleic acid molecules encoding these UGTs.

[0117] The manipulated cells may also contain one or more UDP-glucose recyclases and / or one or more UGTs under heterologous gene regulation. An exemplary UDP-glucosyltransferase capable of adding at least one glucose unit to rubusoside to form stevioside is UGT91d2. An exemplary UDP-glucosyltransferase capable of adding at least one glucose unit to stevioside to form rebaudioside A is UGT76G1. An exemplary UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside A to form rebaudioside D is UGT91d2. An exemplary UDP-glucosyltransferase capable of adding at least one glucose unit to rebaudioside D to form stevioside M is UGT76G1.

[0118] Exemplary publications describing engineered microorganisms for steviol glycoside production and steviol glycoside pathway enzymes include, for example, U.S. Patent Application Publication 2014 / 0357588, International Publication 2014 / 193934, International Publication 2014 / 193888, and International Publication 2014 / 222227.

[0119] In one embodiment, genetically engineered cells useful for the production of steviol glycosides express some or all of the following enzymes: geranylgeranyl diphosphate synthase (GGPPS), ent-coparyl diphosphate synthase (CDPS), kauren oxidase (KO), kauren synthase (KS); cysteine ​​synthase (KAH), cytochrome P450 reductase (CPR), UGT74G1, UGT76G1, UGT91d2, and the polypeptide of Sequence ID No. 1. International Publication No. 2014 / 122227 describes engineered yeast strains expressing these enzymes. The UGT74G1 enzyme functions as uridine 5'-diphosphoglucosyl;steviol 19-COOH transferase and 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 skeleton. 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-glucosidetransferase (also called steviol-13-monoglucoside 1,2-glycosylase) to produce stevioside, by transferring the glucose moiety to the C-2' position of the 13-O-glucose of the receptor molecule steviol-13-O-glucoside, or as a uridine 5'-diphosphoglucosyl:rubusosidetransferase to transfer the glucose moiety to the C-2' position of the 13-O-glucose of the receptor molecule rubusoside. The enzyme of Sequence ID No. 1 can also produce 19-O-1,2-diglycosylated rubusoside by transferring the glucose moiety to the C-2' position of the 19-O-glucose of the receptor molecule rubusoside.

[0121] Activated xylose can be provided into the manipulated cells, for example, by supplying activated xylose to the cells from an external source, or the manipulated cells can produce activated xylose. Polypeptides transfer xylose from activated xylose to the compound of formula I to form the xylosylated steviol glycoside compound of formula II.

[0122] A “manipulated cell” refers to a host cell that has at least one exogenous DNA sequence introduced into the cell, either by integration into the cell’s genome or by presence on an extrachromosomal construct such as a plasmid or episome. The term “exogenous” refers to a molecule, such as nucleic acid, or an activity, such as enzyme activity, introduced into the host cell. Exogenous nucleic acids can be introduced into a host cell by known techniques and maintained outside the host chromosomal material (e.g., maintained on a non-integration vector), or integrated into the cell’s chromosome by a recombination event. Generally, the genome of a manipulated cell is enhanced through the stable introduction of one or more recombinant genes. Exogenous nucleic acids can encode an enzyme or part thereof that is either homogeneous or heterogeneous to the cell. Exogenous nucleic acids can be in the form of a “recombinant gene or DNA construct,” which means a nucleic acid that has been manipulated in one or more ways by molecular techniques to be in a form that does not exist in nature.

[0123] The term “heterogeneous” (e.g., “non-natural”) refers to a molecule or activity that originates from a source different from the molecule or organism being referenced. Therefore, a gene or protein that is heterogeneous to the organism being referenced is a gene or protein that is not found in that organism. In the context of this disclosure, “heterogeneous 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 of a different species is “heterogeneous” 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 the genera Escherichia, Streptococcus, Lactobacillus, Pseudomonas, Lactococcus, Streptomyces, Bacillus, and Clostridium. Ralstonia, Mycobacterium, Agrobacterium, Geobacter, Zymonas, Acetobacter, Citrobacter, Synechocystis, Rhizobium, Corynebacterium, Xanthomania Homonas, Alcaligenes, Aeromonas, Azotobacter, Comamonas, Rhodococcus, Gluconobacter, Acidithiobacillus, Microlunatus, Geobacillus, Arthrobacter, This includes, but is not limited to, species of the genera Flavobacterium, Serratia, Saccharopolyspora, Thermus, Stenotrophomonas, Chromobacterium, Sinorhizobium, Saccharopolyspora, and Pantoea.A typical example of a bacterial species is Escherichia coli.

[0125] In some embodiments, 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 xylosylated steviol glycoside synthesis. Exemplary yeasts that can be used as hosts for exogenous DNA constructs encoding steviol glycoside pathway enzymes include the genera Agaricus, Aspergillus, Brettanomyces, Candida, Fusarium, Gibberella, Eluyveromyces, and Ha Hansenula, Humicola, Issatchenkia, Kloeckera (Hanseniaspora), Kluyveromyces, Laetiporus, Lentinus, Lipomyces, Pachysolen, This includes, but is 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, and Saccharomyces cerevisiae. These include *Cerevisiae*, *Schizosaccharomyces pombe*, *Xanthophyllomyces dendrorhous*, and *Yarrowia lipolytica*. Furthermore, the host cells may also include genetic modifications other than those of the steviol glycoside pathway, which may provide improved performance during fermentation.

[0127] Genetically modified yeast can be used with suitable nutritional requirement markers to select transformants having nucleic acids encoding steviol glycoside pathway enzymes. The host yeast may contain modifications (such as deletions) in one or more genes that control nutritional requirements, e.g., LYS2, LEU2, HIS3, URA3, URA5, and TRP1. Using host cells with the desired genetic background for the introduction of one or more exogenous genes, one or more gene constructs are introduced into the cells, integrated into the genome, or stably maintained to enable expression. Methods for introducing gene constructs into host cells include transformation, transduction, transfection, co-transfection, and electroporation. In particular, yeast transformation can be performed using lithium acetate, protoplast, etc. The introduced gene construct may be integrated into the chromosome in plasmid form, inserted into host genes, or integrated by homologous recombination with host genes. Transformed yeast into which the gene construct has been introduced can be selected using selection markers (e.g., the nutritional requirement markers mentioned above). Further confirmation can be performed by measuring the activity of the expressed protein or the production of biological products such as steviol glycosides.

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

[0129] The method of producing steviol glycosides by cell culture according to this disclosure can use engineered cells having a pathway that converts steviol to xylosylated steviol glycosides of formula II. If two or more steviol glycoside pathway enzymes are present in genetically engineered yeast, the yeast may be able to produce different steviol glycosides, at least one of the steviol xylosylated steviol glycosides of formula II. For example, yeast may be able to produce two, three, four, five, six, seven, eight, nine, ten, or more than ten different steviol glycoside species, one or more of which are xylosylated steviol glycosides.

[0130] The term “culture medium” refers to a liquid composition in which manipulated cells can be maintained, grown, fermented, or a combination thereof. “Culture 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 culture 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 limited medium, a limited period, and a limited temperature.

[0131] A culture medium can be defined in terms of the components present in the medium and their amounts, such as (a) a carbon source containing 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, and zinc sulfate. Components in the medium can be defined on a dry weight basis. Furthermore, the medium is water-based or an "aqueous" composition. The culture medium may also be defined in terms of its pH, as well as the biocompatible acids, bases, and buffers used to control the pH in the medium.

[0132] The composition ("feed composition") can be added to the culture medium containing the manipulated cells to increase the volume of the medium, and as the manipulated cells grow in the medium, the amount of biomass can be increased.

[0133] In some embodiments, cell culture can be carried out 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, for example, one having at least 50% identity to SEQ ID NO: 1, and the polypeptide 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 embodiments, the manipulated cells do not need to have a pathway to the steviol glycoside precursor (e.g., the MVA, MEP, or SG pathway as described herein).

[0134] Exemplary examples of manipulated cells include engineered yeast, bacteria, and fungi.

[0135] "Total steviol glycosides" refers to all steviol glycosides present in the culture medium after the period of cell culture, including the amount of steviol glycosides, including the xylosylated steviol glycosides of this disclosure, in a liquid medium, and can be obtained from genetically modified yeast. The steviol glycoside content can be expressed in terms of the total amount of 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 the xylosylated steviol glycoside, in the composition can be expressed in weight percentage or mole percentage, for example, as a weight percentage of the total amount of steviol glycosides, or as a ratio or range of ratios, either with respect to each other or with respect to the total amount of steviol glycosides.

[0136] To recover the xylosylated steviol glycoside, the culture medium can then be centrifuged or filtered to remove the manipulated cells. The culture medium may optionally be treated to remove low molecular weight components (glucose, basal nutrients, and salts), for example, by membrane dialysis. Depending on the desired application, a composition containing one or more steviol glycoside compounds can be used.

[0137] If it is desired to provide a composition containing xylosylated steviol glycosides in a concentrated or purified form, or if certain steviol glycosides are to be separated from each other, further purification may be performed. Such concentration or purification of steviol glycoside components may be carried out on a culture medium in which fermentation is occurring, or the medium may be dried before purification. For example, the medium may be dried by freeze-drying to form a dried composition (e.g., powder or flakes) containing steviol glycosides that can be processed later.

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

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

[0140] The concentrated steviol glycoside compound in the dried fermentation broth can be purified or refined, for example, by methods such as crystallization or by reversed-phase liquid chromatography. Known techniques 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 ultrafiltration and nanomembrane filtration (see, for example, 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 an anhydrous methanol solution to obtain 80% pure rebaudioside A. U.S. Patent Publication 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 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. International Publication Nos. 2007 / 149672 and International Publication Nos. 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 compound on the column while removing hydrophilic compounds that are washed through the column with a liquid such as water. Elution of the steviol glycoside from the column can be achieved 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). Steviol glycoside compositions 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). This composition may also be heated to promote the dissolution of the steviol glycoside material, such as by heating to about 50°C. The solution may also be filtered before injection into the column, for example, using a 0.2 μm filter. The stationary phase is C18 with a Phenomenex Kinetex XB-C18 5 μm core-shell silica solid support, and isobutyl side chains and TMS end caps. The flow rate through the column is determined based on the column characteristics (e.g., about 20 mL / min), and the maximum pressure is 400 bar. Various xylosylated steviol glycosides can be identified by their elution time from the column. Those involved will understand that the elution time of xylosylated steviol glycosides can change with changes in solvent and / or equipment.

[0143] Elution of xylosylated steviol glycosides from a reversed-phase column can yield compositions that may be useful for any one of several purposes. For example, a purified xylosylated steviol glycoside composition may be used as a sweetener composition for oral ingestion or use. This composition may be defined in relation to the steviol glycosides in the composition.

[0144] As used herein, a sweetener composition (also called 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, the sweetener composition may contain one or more steviol glycosides of formula II (SG of formula II) together with other steviol glycosides other than those of formula II (SG of non-formula II). In some embodiments, when multiple steviol glycosides are present in the sweetener composition, the non-formula II SV may be present in small amounts in the composition, or in amounts equal to the total amount of steviol glycosides in the composition (e.g., less than about 25%, less than about 20%, less than about 15%, or less than about 10%). One or more formula II SGs may be present in the composition in major amounts such as over 75%, over 80%, over 85%, over 90%, or over 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 in the sweetener composition, meaning it is present in greater amounts than any other SG in the composition. In some embodiments, compound E is present in a greater amount than the total 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, compound E is present in an amount greater than 60%, 70%, 80%, 90%, 95%, 98%, or 99% (by weight) of the total amount of steviol glycosides in the composition.

[0145] The sweetener composition may optionally contain other sweeteners, additives, liquid carriers, or combinations thereof. The sweetener composition is used to sweeten other compositions (compositions that can be sweetened), such as foods, beverages, pharmaceuticals, oral hygiene compositions, and nutritional supplements.

[0146] As used herein, “sweetened composition” means a substance that comes into contact with the oral cavity of a human or animal, and includes substances that are put in the mouth but later spit out (such as mouthwash and rinse) and substances that are ingested, eaten, swallowed, or digested and are suitable for consumption by humans or animals when used within normally acceptable limits. A sweetened composition is a precursor composition of a sweetened composition, and is converted into a sweetened composition by combining a sweetened composition with at least one sweetened composition containing one or more xylosylated steviol glycosides of formula II. For example, a beverage that does not contain sweetener components is a type of sweetened composition. A sweetener composition containing one or more xylosylated steviol glycosides of formula II can be added to an unsweetened beverage to provide a sweetened beverage. A sweetened beverage is a type of sweetened composition.

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

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

[0149] The amount of xylosylated steviol glycoside of formula II in the sweetener composition may vary. One or more xylosylated steviol glycosides of formula II may 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, if one or more xylosylated steviol glycosides of formula II are present in the sweetener composition in an amount effective to provide a total steviol glycoside concentration of about 1 ppm to about 10,000 ppm, 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 about 10 ppm to about 2,500 ppm, more specifically, about 10 ppm to about 2,000 ppm, about 10 ppm to about 1,500 ppm, about 10 ppm to about 1,250 ppm, about 10 ppm to about 1,000 ppm, about 10 ppm to about 800 ppm, about 50 ppm to about 800 ppm, about 50 ppm to about 600 ppm, or about 200 ppm to about 500 ppm. Unless otherwise specified, ppm is based on weight.

[0150] Optionally, the sweetener composition may also contain one or more additional non-steviol glycoside sweetener compounds, such as natural sweeteners like sucrose, fructose, glucose, and erythritol, or one or more synthetic sweeteners like 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 a liquid carrier, a binder matrix, 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 and organic base salts, inorganic salts, bitter compounds, flavorings and fragrances, astringent compounds, proteins or protein hydrolysates, surfactants, emulsifiers, fillers, gums, antioxidants, colorants, flavonoids, alcohols, polymers, and combinations thereof. In some embodiments, the additives act to improve the temporal and flavor profiles to provide a sweetener composition having a desirable taste, such as a sucrose-like taste.

[0152] A sweetener composition may contain one or more functional materials that provide the composition with actual or perceived health benefits. Examples of functional materials include, but are not limited to, saponins, antioxidants, dietary fiber sources, fatty acids, vitamins, glucosamine, minerals, preservatives, wettable powders, probiotics, prebiotics, weight management agents, osteoporosis management agents, phytoestrogens, long-chain primary aliphatic saturated alcohols, phytosterols, and combinations thereof.

[0153] Generally, the amount of functional ingredients in a sweetener composition or sweetened composition varies widely depending on the specific sweetener composition or sweetened composition and the desired functional ingredients. Those skilled in the art will readily be able to determine the appropriate amount of functional ingredients for each sweetener composition or sweetened composition.

[0154] One or more xylosylated steviol glycosides of formula II, or sweetener compositions containing these steviol glycosides, can be incorporated into any known food ingredient (hereinafter referred to as “sweetener-granting composition”), or other compositions intended for oral digestion and / or ingestion by humans or animals, such as pharmaceutical compositions, edible gel mixtures and compositions, dental and oral hygiene compositions, foods (sugars, spices, chewing gum, cereal compositions, baked goods, semi-cooked foods, food additives, dairy products, and table sweetener compositions), beverages, and other beverage products (e.g., beverage mixtures, 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, “beverage product” refers to an instant beverage, beverage concentrate, beverage syrup, frozen beverage, or powdered beverage. Suitable ready-to-drink beverages include carbonated and non-carbonated beverages. Examples of carbonated beverages include, but are not limited to, highly carbonated soft drinks, cola, lemon-lime flavored carbonated soft drinks, orange flavored carbonated soft drinks, grape flavored carbonated soft drinks, strawberry flavored carbonated soft drinks, pineapple flavored carbonated 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 natural or synthetic flavorings added), coconut water, tea-type beverages (e.g., black tea, green tea, black tea, oolong tea), coffee, cocoa drinks, beverages containing dairy ingredients (e.g., dairy drinks, coffee containing dairy ingredients, café latte, milk tea, fruit juice milk drinks), beverages containing grain extracts, smoothies, and combinations thereof.

[0156] Examples of frozen beverages include, but are not limited to, icys, frozen cocktails, daiquiris, piña coladas, margaritas, milkshakes, frozen coffee, frozen lemonade, granitas, and slushies.

[0157] Beverage concentrates and beverage syrups can be prepared with an initial volume of liquid matrix (e.g., water) and the desired beverage components. The final concentration of the beverage is then prepared by adding further volumes of water. Powdered beverages are prepared by dry mixing all beverage ingredients in the absence of a liquid matrix. The beverage of full strength is then prepared by adding the total volume of water.

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

[0159] In another embodiment, the 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 amounts ranging from about 1 ppm to about 10,000 ppm, for example, about 25 ppm to about 800 ppm. In yet another embodiment, the steviol glycosides are present in the beverage in amounts ranging from about 100 ppm to about 600 ppm. In yet another embodiment, the steviol glycosides are present in the beverage in amounts ranging from about 100 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 embodiment, the steviol glycosides are present in the beverage in amounts ranging from about 300 to about 700 ppm, for example, about 400 ppm to about 600 ppm. In certain embodiments, steviol glycosides are present in beverages at an amount of approximately 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, and mechanically strong granules of a steviol glycoside sweetener composition. Granulation methods are known to those skilled in the art and are described in detail in PCT Publication WO01 / 60842.

[0161] Preparation of UGT enzymes The amino acid sequence of Sequence ID No. 1 was fused to the C-terminus of the GST protein, and the sequence was codon-optimized for expression in E. coli (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 plasmid was transformed into E. coli. 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 following morning, the seed cultures had an OD600 of 2.4 ± 0.5. The following morning, 16 mL of the overnight culture was inoculated into a 3 L Fernbach flask containing either LB or Terrific Broth + 50 mg / mL kanamycin, targeting an initial OD600 of 0.04. The 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, the cultures were incubated overnight at 250 RPM at the temperatures shown in Table 1. The biomass was pelleted in a 1-liter centrifuge bottle at 8000 RPM for 15 minutes at 4°C. The cells were resuspended in lysis buffer and lysed by sonication. The cell suspension was sonicated for 2 minutes with a 20-second pulse, followed by standing on ice for 20 seconds. The cells were transferred to a 30 mL Oak Ridge tube and centrifuged at 15,000 RPM for 15 minutes. The 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) and activated xylose (UDP-xylose), which are known to play important roles in SG biosynthesis in stevia leaves.

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

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

[0165] SG materials were obtained from various sources with a purity of over 90%. All SG materials were prepared at 20 mM using 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 has no effect on biotransformation.

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

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

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

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

[0170] As shown in Table 1 and Figure 5, the polypeptide of Sequence ID No. 1 was more effective than UGT76G1 in transferring xylose to the SG acceptor. This result was rather unexpected, as others had assumed that UGT76G1 had broader activity to acceptors and donors than Sequence ID No. 1. When using the polypeptide of Sequence ID No. 1, stevioside, Reb A, and Reb C all showed very high conversion rates, and one of the Reb D replicas showed approximately 10% conversion. When using UGT76G1, stevioside showed a higher conversion rate than Reb A and Reb C.

[0171] [Table 1]

[0172] Example 2: Time-dependent analysis of glycosylation of Reb A using SEQ ID NO: 1 and activated sugars The ability of Sequence ID No. 1 to glycosylate Reb A was investigated using different activated sugars at different concentrations, and the reaction products formed were measured throughout the reaction process.

[0173] The purified protein of Sequence ID No. 1 was used together 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 effectively transfer glucose and xylose from UDP-glucose and USP-xylose, respectively, to RebA. In the presence of purified SEQ ID NO: 1 protein and 1 mM activated sugar, glucosylation and xylosylation of RebA reached a plateau after 20 minutes, and RebA had a slightly higher level of glucosylation than xylosylation (see Table 2 and Figure 8A). In the presence of purified SEQ ID NO: 1 protein and 2 mM activated sugar, glucosylation and xylosylation of RebA reached a plateau after 20 minutes and were at similar levels, but rhamnosylation using 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 Sequence ID No. 1 to glycosylate the -19C(O)-β-Glc residue of RebF, RebG, and dulcoside A was investigated using different activated sugars at different concentrations, and the reaction products formed were measured throughout the reaction process.

[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] Sequence ID 1 effectively transferred glucose and xylose from UDP-glucose and UDP-xylose, respectively, to the -19C(O)-βGlc residue of Reb F, Reb G, and dulcoside A, with glucosylation and xylosylation reaching a plateau after 20 minutes. Rhamnosylation using UDP-rhamnose was significantly lower (see Tables 4-6 and Figures 9A-C).

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

[0184] Glycosyltransferase assay for enzymes produced by in vitro protein synthesis The steviol glycoside (SG) "acceptor" Reb A was tested using four glucosyltransferase enzymes (SEQ ID NOs: 1-4) homologous to glucosyltransferase in SG biosynthesis in stevia leaves. The identity of SEQ ID NOs: 2-4 to SEQ ID NO: 1 is listed in Table 7.

[0185] [Table 7]

[0186] UDP-xylose (98.9% purity) was purchased from BOC Sciences. The activated sugar donor was prepared to a concentration of 10 mM in 0.05 M Tris buffer and stored according to the manufacturer's recommendations before use. SG materials were obtained from various sources with a purity of 90% or higher. All SG materials were prepared to 20 mM using 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 has no effect on biotransformation.

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

[0188] The enzyme was produced via in vitro transcription-translation (IVTT), and its activity was confirmed in a preliminary test using UDP-glucose. The bioconversion reaction was carried out in a total of 100 μL sealed vials containing Tris buffer, 0.1 μmol of SG acceptor, 0.2 μmol of activated sugar donor, 1 μmol of sucrose, and 2 μg of enzyme protein. Buffer, UDP-sugar donor, and SG acceptor were added to each vial in that order, followed by the enzyme. Each SG donor was included as a control containing the same amount of enzyme without UDP-sugar donor. After manual mixing, the vials were placed in a temperature-controlled shaker set to 30°C and 100 rpm and rotated for 96 hours (4 days).

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

[0190] The activity of SEQ ID NOs: 2-4 is 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 NOs: 3 or 4 in the transfer of xylose to the SG acceptor. The substantially 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 its xylosylated product occurred in the presence of both UDP-glucose and UDP-xylose. [SEQ ID NO: 3 was performed more than twice in this experiment.]

[0191] [Table 8]

Claims

1. A method for forming a glycosylated steviol glycoside, wherein the method is The method involves forming a compound of formula II from a compound of formula I in vitro or in manipulated cells using a UDP-xylose:19-steviol xylosyltransferase having glycosyltransferase activity and activated xylose. Equation I is as follows: 【Chemistry 1】 In the formula, R １ However, it contains a glucose residue, R ２ However, it contains one or more sugar residues, or is hydrogen. The glycosyltransferase transfers xylose from activated xylose to the compound of formula I, Equation II is as follows: 【Chemistry 2】 In the formula, R ３ However, it contains one or more xylose residues added by the glycosyltransferase, R ４ However, R ２ It is the same as, or contains one or more additional sugar residues, The compound of formula I is selected from the group consisting of stevioside, rebaudioside A, rebaudioside C, rebaudioside F, rebaudioside G, and dulcoside-A. The activated xylose is UDP-xylose, A method wherein the glycosyltransferase has 90% or more, 92.5% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity with respect to SEQ ID NO:

1.

2. (i) R in equation II ３ but is an oligosaccharide moiety containing one or more xylose residues; and / or (ii) The oligosaccharide portion is -β-Glu-β-Xyl or -β-Glu-β-Xyl-R ５ And in the formula, R ５ contains one or more sugar residues; and / or (iii) R of formula II ３ is composed of -Glu-β-Xyl, the method according to claim 1.

3. The method according to claim 1 or 2, wherein the compound of formula I is selected from the group consisting of stevioside, rebaudioside A, and rebaudioside C.

4. The method according to claim 1 or 2, wherein the compound of formula I is rebaudioside A.

5. The glycosyltransferase described above has the following amino acids compared to SEQ ID NO: (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 4, comprising both (A) and (B).

6. The method according to any one of claims 1 to 5, wherein the glycosyltransferase comprises, in comparison to SEQ ID NO: 1, one or more of the following amino acid motifs: 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).

7. The method according to any one of claims 1 to 6, which 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 the UDP-xylose:19-steviol xylosyltransferase activity.

8. (i) The compound of formula I constitutes more than 50% (mol) of the steviol glycoside in the reaction composition, 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 it; and / or (ii) The method according to claim 7, wherein in the reaction composition, more than 10%, more than 20%, more than 30%, more than 40%, more than 50%, more than 60%, more than 65%, more than 75%, or more than 80% of the steviol glycoside compound of formula I is converted to the xylosylated steviol glycoside compound of formula II.

9. The compound of formula II is 【Transformation 3】 The method according to any one of claims 1 to 8, wherein the material is rebaudioside DG.

10. The reaction composition is as follows: (a) magnesium and / or manganese salt, (b) pH in the range of 3 to 9, 4 to 8, 6.8 to 7.8, or 7.1 to 7.5, (c) UDP-xylose (UDP-X) in a molar excess relative to the steviol glycoside compound (SGI) of formula I, or a UDP-X:SGI molar ratio 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, wherein the activated xylose is the UDP-xylose (UDP-X) The method according to any one of claims 7 to 9, comprising: (d) a molar excess of UDP-xylose (UDP-X), or a UDP-X:SGI molar ratio; (d) a polypeptide (weight) to UDP-X (mol) ratio in the range of 50 μg:1 μmol to 10 μg:1 μmol, 25 μg:1 μmol to 15 μg:1 μmol, or 20 μg:1 μmol; (e) a polypeptide (weight) to SGI (mol) ratio in the range of 25 μg:1 μmol to 5 μg:1 μmol, 15 μg:1 μmol to 7.5 μg:1 μmol, or 10 μg:1 μmol.

11. The method according to any one of claims 7 to 10, wherein the reaction is carried out under one or more of the following conditions: (a) a time in the range of 1 hour to 10 days, 1 hour to 24 hours, 1 hour to 12 hours, 12 hours to 7 days, or 1 day to 5 days; or (b) 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.

12. The formation is carried out in manipulated cells capable of producing the steviol glycoside compound of formula I. The method according to any one of claims 1 to 6, comprising the following: the activated xylose is supplied to the cell (from an external source), or the manipulated cell is capable of producing the activated xylose, and the xylose is transferred from activation to the compound of formula I to form a xylosylated steviol glycoside compound of formula II.

13. Manipulated cells, A pathway for forming the steviol glycoside compound of formula I, 【Chemistry 4】 In the formula, R １ However, it contains a glucose residue, R ２ However, it contains one or more sugar residues, or is hydrogen. The aforementioned pathway provides an amount of the steviol glycoside compound of formula I greater than the amount of steviol glycoside other than formula I that can be formed within the cell, A glycosyltransferase having UDP-xylose:19-steviol xylosyltransferase activity, wherein the glycosyltransferase can transfer one or more xylose residues to the compound of formula I to form a glycosylated steviol glycoside compound of formula II. 【Transformation 5】 In the formula, R in formula II ３ However, it contains one or more xylose residues, R ４ However, R ２ A glycosyltransferase that is the same as or contains one or more additional sugar residues, The compound of formula I is selected from the group consisting of stevioside, rebaudioside A, rebaudioside C, rebaudioside F, rebaudioside G, and dulcoside-A. Manipulated cells in which the glycosyltransferase has 90% or more, 92.5% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity with SEQ ID NO:

1.

14. The manipulated cell according to claim 13, wherein the manipulated cell includes a pathway for the production of UDP-xylose.

15. The manipulated cell according to claim 14, wherein the pathway for the production of UDP-xylose in the manipulated cell comprises UDP-glucose dehydrogenase, UDP-glucuronide decarboxylase, UDP-xylose synthase, or a combination thereof.

16. The manipulated cell according to any one of claims 13 to 15, wherein the manipulated cell comprises one or more UDP-glucose recyclases and / or one or more UGTs under heterogeneous control.

17. The manipulated cells are manipulated yeasts selected from the group consisting of species of the genera Candida, Kloeckera (Hanseniaspora), Issatchenkia, Kluyveromyces, Lipomyces, Pichia (Hansenula), Rhodotorula, Saccharomycete, Saccharomyces, Schizosaccharomyces, Torulopsis, Torulaspora, Yarrowia, and Zygosaccharomyces; or The manipulated cell according to any one of claims 13 to 16, wherein the manipulated cell is a manipulated bacterial cell selected from the group consisting of species of the genera Escherichia and Bacillus.

18. The manipulated cell according to any one of claims 13 to 17, wherein the manipulated cell expresses one or more exogenous nucleic acids encoding one or more of the following heterogeneous proteins: GGPPS polypeptide, ent-coparyl diphosphate synthase (CDPS) polypeptide, kauren oxidase (KO) polypeptide, kauren synthase (KS) polypeptide, steviol synthase (KAH) polypeptide, cytochrome P450 reductase (CPR) polypeptide, UGT74G1 polypeptide, and UGT76G1 polypeptide.

19. A method for forming a xylosylated steviol glycoside, wherein the method is (i) one or more of stevioside, rebaudioside A, rebaudioside C, rebaudioside F, rebaudioside G, or dulcoside A, (ii) UDP-xylose, (iii) polypeptide of SEQ ID NO: 1 or SEQ ID NO: 2, (iv) inactivated sugar, and (v) magnesium and / or manganese salt, forming a xylosylated steviol glycoside product from a steviol glycoside reaction product in a reaction composition comprising: The reaction composition has a pH in the range of 4.8 to 7.

8. One or more of the aforementioned stevioside, rebaudioside A, rebaudioside C, rebaudioside F, rebaudioside G, or dulcoside A are present in the reaction composition at a total amount of 80% (mol) or more of all steviol glycosides. The ratio of the polypeptide (by weight) to one or more of the stevioside, rebaudioside A, rebaudioside C, rebaudioside F, rebaudioside G, or dulcoside A (mol) is in the range of 15 μg:1 μmol to 7.5 μg:1 μmol, and the compound is formed accordingly. A method comprising 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 containing one or more compounds D, E, G, H, I, or J. 【Transformation 6】 【Transformation 7】 【Transformation 8】

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