Compositions and methods for glycosylation of steviol glycosides

Abstract

Disclosed are methods and compositions for glycosylation of a sugar acceptor, comprising the steps of: (i) converting an exogenous nucleoside triphosphate (NTP) to a nucleoside diphosphate (NDP); (ii) converting the NDP to NDP-sugar, comprising contacting at least one Leloir glycosyltransferase (GT) with the NDP and an exogenous sugar donor (SD) in a reaction medium; and (iii) converting a sugar acceptor (SA) to a glycosylated SA, comprising contacting the at least one GT with the NDP-sugar and the SA in the reaction medium.

Description

COMPOSITIONS AND METHODS FOR GLYCOSYLATION OF STEVIOL GLYCOSIDESCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application relates to International Patent Application No. PCT / US2023 / 077888, filed October 26, 2023, the content of which is herein incorporated by reference in its entirety. This application claims the benefit ofU.S. Provisional Patent Application No. 63 / 664,108, filed June 25, 2024, which is incorporated by reference herein in its entirety for all purposes.FIELD OF THE DISCLOSURE

[0002] The present disclosure generally relates to methods and compositions for using nucleoside triphosphates (NTP) for glycosylation of sugar acceptors. The present disclosure further relates to methods and compositions for converting NTP to nucleoside diphosphate (NDP) as well as methods and compositions for stabilizing the NDP as NDP-glucose in the composition.BACKGROUND

[0003] For synthetic and biosynthetic chemistry and for processing of natural products, it is often desirable to be able to perform glycosylation reactions. Glycosylation reactions transfer a carbohydrate moiety from a donor molecule to an acceptor molecule forming a glycosidic bond. A glycosidic bond or a glycosidic linkage joins a carbohydrate molecule to another group on the target acceptor, which may or may not be another carbohydrate. For instance, in a glycosylation reaction, a carbohydrate may be attached to a hydroxyl ("O-glycosylated") or another functional group of a sugar acceptor. The glycosylated or more highly glycosylated products can have improved solubility, improved taste, better bioavailability, improved nutritional attributes, greater resistance to proteolytic degradation, reduced aggregation, and stabilization of protein structure.

[0004] In any glycosylation reaction, the molecule that receives the glycosylation (the "sugar acceptor") can be any one of a protein, a lipid, or a biochemical such as a polyphenol, a polyketide, steviol, or a steviol glycoside. The carbohydrate group added may be one of glucose, galactose, xylose, mannose, or many others. In nature, there are over 30,000 known glycosylated compounds. Naturally occurring glycosides can be isolated from animals, plants,bacteria, and fungi. In some instances, the acceptor may already contain one or more glycosyl groups, and the glycosylation reaction involves adding additional glycosyl groups.

[0005] It is known that the human genome has around 700 genes encoding enzymes, transporters, and chaperones required for the cellular glycosylation machinery, glycan modifications, and their degradation. Almost one third of the 279 classified peptide hormones carry O-glycans. Some glycosides can be biomedically important secondary metabolites. For instance, 20 novel fungal aromatic polyketide C / O-glycosides have been identified from three selected fungi, each of which exhibit significant antiviral, antibacterial, and antidiabetic activities. Some steroids are glycosylated, including a number of important groups of drugs, for example, erythromycin, daunomycin, and amphotericin B . The sugar(s) added to the parent molecule act to change physical, chemical, and biological properties as well as biological activity.

[0006] Glycosylation as it relates to the production of steviol glycosides represents one approach to addressing a pressing healthcare challenge. Excess sugar consumption has been linked to worldwide health epidemics including diabetes and heart disease. Healthcare systems incur exorbitant costs associated with treating these diseases. Replacing added sugar in food with a low calorie, high-intensity sweetener would have significant health and economic impact.

[0007] The species Stevia rebaudiana is commonly grown for its sweet leaves, which have traditionally been used as a sweetener. Stevia extract is 200-300 times sweeter than sugar and is used commercially as a high intensity sweetener. The main glycoside components of stevia leaf are steviosides and rebaudiosides. Over ten different steviol glycosides are present in appreciable quantities in the leaf. The principal sweetening compounds are stevioside and rebaudioside A. Rebaudioside A (Reb A) is considered a higher value compared to stevioside because of its increased sweetness and decreased bitterness. The sweetness and bitterness profiles of rebaudioside D (Reb D) and rebaudioside M (Reb M) are improved compared to Reb A. However, Reb D and Reb M are present at very low quantities in the stevia leaf. Reb D and Reb M can be made by the addition of one or two glucose molecules to Reb A, respectively.

[0008] Many glycosyltransferases use a nucleotide diphosphate sugar (NDP-sugars) as the sugar donor to glycosylate the sugar acceptor. However, NDPs and NDP-sugars are expensive and limit the economic feasibility of many glycosyltransferase reactions. More economic ways to generate NDP-sugars will enable many currently economically unattractive glycosylation processes. Accordingly, the present disclosure explores lower cost methods to produce NDP- sugars for glycosyltransferase reactions.BRIEF SUMMARY

[0009] In embodiments, the present disclosure relates to a method for glycosylation of a sugar acceptor.

[0010] In embodiments, the present disclosure further relates to a method for glycosylation of a sugar acceptor, comprising the steps of: (i) converting an exogenous nucleoside triphosphate (NTP) to a nucleoside diphosphate (NDP); (ii) converting the NDP to NDP-sugar, comprising contacting at least one Leloir glycosyltransferase (GT) with the NDP and an exogenous sugar donor (SD) in a reaction medium; and (iii) converting a sugar acceptor (SA) to a glycosylated SA, comprising contacting the at least one GT with the NDP-sugar and the SA in the reaction medium.

[0011] In embodiments, the present disclosure further relates to a method for glycosylation of a steviol glycoside, comprising the steps of: (i) converting an exogenous adenosine triphosphate (ATP) to an adenosine diphosphate (ADP); (ii) converting the ADP to ADP- glucose, comprising contacting at least one Leloir glycosyltransferase (GT) with the ADP and exogenous sucrose (SD) in a reaction medium; and (iii) converting the steviol glycoside (SA) to a glycosylated SA, comprising contacting the at least one GT with the ADP-glucose and the SA in the reaction medium.

[0012] In embodiments, the present disclosure further relates to a method for glycosylation of a steviol glycoside, comprising the steps of: (i) converting an exogenous adenosine triphosphate (ATP) to adenosine diphosphate (ADP); (ii) converting the ADP to ADP-glucose, comprising contacting a sucrose synthase (SuSy) with the ADP and exogenous sucrose in a reaction medium; and (iii) converting at least one steviol glycoside (SG) to a glycosylated SG, comprising contacting the SG with the ADP-glucose and at least one glycosyltransferase (GT) selected from the group consisting of P-l,2-glycosyltransferase, P-1, 3 -glycosyltransferase, or a combination thereof in the reaction medium.

[0013] In embodiments, the present disclosure further relates to a method for glycosylation of a sugar acceptor, comprising the steps of: (i) converting an exogenous nucleoside triphosphate (NTP) to a nucleoside diphosphate (NDP); (ii) converting the NDP to NDP-sugar, comprising contacting at least one Leloir glycosyltransferase (GT) with the NDP and an exogenous sugar donor (SD); and (iii) converting a sugar acceptor (SA) to a glycosylated SA, comprising contacting the at least one GT with the NDP-sugar and the SA.BRIEF DESCRIPTION OF THE DRAWINGS

[0014] The accompanying drawings are included to provide a further understanding of the disclosure. The drawings illustrate embodiments of the disclosure and together with the description, serve to explain the principles of the embodiments of the disclosure.

[0015] FIG. 1A depicts an exemplary system of the present disclosure when NTP is employed. At reaction 1 (“(!)”), NTP is converted, or activated, to NDP. Reaction 2 (“(2)”) and Reaction 3 (“(3)”) form a consumption and recycling loop, where an NDP-sugar synthase and a glycosyltransferase cyclically conjugate NDP to a sugar and free NDP from the sugar. In recycling the NDP at (2), the NDP-sugar synthase is contacted with the NDP and a sugar donor to form NDP-sugar. At (3), the glycosyltransferase is contacted with the NDP-sugar and a sugar acceptor to glycosylate the sugar acceptor, thereby forming a target molecule. Recycling of the NDP stabilizes the NDP as NDP-sugar, preventing further dephosphorylation of the NDP to NMP. FIG. IB depicts an exemplary system of the present disclosure when the nucleoside of the NTP is adenosine. At Reaction 1 (“(1)”), ATP is converted, or activated, to ADP. Reaction 2 (“(2)”) and Reaction 3 (“(3)”) form a consumption and recycling loop, where an ADP-sugar synthase and a glycosyltransferase cyclically conjugate ADP to a sugar and free ADP from the sugar. In recycling the ADP at (2), the ADP-sugar synthase is contacted with the ADP and a sugar donor to form ADP-sugar. At (3), the glycosyltransferase is contacted with the ADP- sugar and a sugar acceptor to glycosylate the sugar acceptor, thereby forming a target molecule. Recycling of the ADP stabilizes the ADP as ADP-sugar, preventing further dephosphorylation of the ADP to AMP. In embodiments, the ATP can be provided at catalytic amounts and excess sugar donor can be used for stabilization of ADP as ADP-sugar.

[0016] FIG. 2A depicts dephosphorylation of NTP. FIG. 2B depicts dephosphorylation of ATP. In an example, as shown in FIG. 2A, dephosphorylation may occur by chemical hydrolysis.

[0017] FIG. 3A depicts a specific variation of the exemplary system shown in FIG. IB. ATP is activated at (1) using a glycerol kinase and glycerol as a phosphate acceptor. Excess sucrose is reacted (2) in the presence of a sucrose synthase enzyme and the ADP to form ADP-glucose. At (3), stevioside is glycosylated through contact with a p-l,3-glycoysltransferase in the presence of the ADP-glucose to generate rebaudioside A as the product of interest. FIG. 3B depicts the molecular structures of the acceptor, stevioside, and the product, rebaudioside A, of (3) of FIG 3A.

[0018] FIG. 4 depicts the molecular structures of three sequential glycosylation reaction products produced from the sugar acceptor stevioside to generate the product of interestrebaudioside M. A total of three glucose units are added. (3) of FIG. 3A is performed three times using a P-l,2-glycoysltransferase and P-l,3-glycoysltransferase. As shown in FIG. 4, glycosylation occurs at (3a), (3b), and (3c), generating rebaudioside M from stevioside via rebaudioside A and rebaudioside D.

[0019] FIG. 5 depicts the results of two sequential glycosylation reactions of the sugar acceptor cholesterol- -D-glucoside to generate the product of interest cholesterol-triglucoside. Two glucose units are added, as shown at (3a) and (3b).

[0020] FIG. 6 depicts O-glycosylation substrates that have been glycosylated according to the methods described herein. Glycosylation was determined by HPLC MS-QTOF analysis, as discussed in the Examples, with mass increases on one or more multiples of 162.14 g / mol. In the absence of ATP, glycosylation did not occur.

[0021] FIG. 7A depicts a steviol molecule. FIG. 7B depicts a steviol glycoside with the steviol core of FIG. 7A, with an ester bond joining a glucose to the steviol core at the C19 position (Rl) and an ether bond joining a glucose to the steviol core at the C 13 position (R2).

[0022] FIG. 8A depicts an example of activation of ATP using an acetate kinase (E9(l )) and acetate as the phosphate acceptor. Acetyl -phosphate is produced. FIG. 8B depicts an example of activation of ATP using a fructose kinase (E9(3)) and fructose as the phosphate acceptor. D- fructose-6-phosphate is produced.

[0023] FIG. 9A depicts an example of activation of ATP using acetate kinase (E9(l)) and acetate as the phosphate acceptor and stabilization of the resultant ADP using sucrose and sucrose synthase. FIG. 9B depicts an example of activation of ATP using fructose kinase and fructose as the phosphate acceptor and stabilization of the resultant ADP using sucrose and sucrose synthase. Appreciating that sucrose is a disaccharide of glucose and fructose, fructose jettisoned by the sucrose synthase may be reused as a reagent in the activation of ATP. This may result in an autocatalytic effect, where the rate of ATP activation increases with time.

[0024] FIG. 10A depicts an example of activation of ATP using acetate kinase (E9(l)) and acetate as the phosphate acceptor, stabilization of the resultant ADP using sucrose and sucrose synthase, and glycosylation of a sugar acceptor using the stabilized ADP (ADP-glucose) to generate a product of interest, R-O-glucose. FIG. 10B depicts an example of activation of ATP using fructose kinase (FRK enzyme) and fructose as the phosphate acceptor, stabilization of the resultant ADP using sucrose and sucrose synthase, and glycosylation of a sugar acceptor using the stabilized ADP (ADP-glucose) to generate a product of interest, R-O-glucose. Similar to FIG. 9B, fructose is jettisoned by the sucrose synthase during stabilization of the ADP. InFIG. 10B, the jettisoned fructose can be reused as a reagent at (1) and / or accumulates in excess, an additional mechanism to prevent unwanted activation of ADP to AMP.

[0025] FIG. 11 is a graphical comparison of the methods described herein, wherein the reaction medium comprises either ATP or ADP. Production of rebaudioside M was compared for a 2000 liter industrial reactor with ADP versus a 2 ml microreactor with ATP, demonstrating that a reaction medium comprising ATP can be used in the production of rebaudioside M.

[0026] FIG. 12 is graphical comparison of the glycosylation of stevioside according to methods of the present disclosure, wherein the reaction medium initially comprises either ATP, ADP, or AMP. The results confirm that AMP cannot be used for glycosylation while ATP and ADP are suitable reagents in the reaction medium.

[0027] FIG. 13A and FIG. 13B are graphical representations of the conversion of RA60 (which contains mixed steviol glycosides, primarily Reb A) to rebaudioside M over time. In each figure panel, catalytic levels of pure ATP, catalytic levels of a reduced purity ATP (10% ADP present), and a control case with catalytic levels of ADP were evaluated. In FIG. 13A, the catalytic levels of a reduced purity ATP comprise 2 mM ATP and 0.2 mM ADP. In FIG. 13B, the catalytic levels of a reduced purity ATP comprise 1 mM ATP and 0.1 mM ADP.

[0028] FIG. 14 is a flow diagram depicting a method for generating a partially purified ATP solution.

[0029] FIG. 15A depicts a computational model of a typical reaction for glycosylation of a feed of 11.3 g / L stevioside and 34.4 g / L Reb A, as well as traces of other rebaudiosides (stevioside, Reb AM, Reb D, Reb E, and Reb M), in a reaction that gives 50% approach to complete conversion to Reb M in 2.4 hours and has a maximum rate of Reb M formation of 17.8 g / L-hr between time 1 hour and 2 hours and was 90% complete in 6 hours with a final Reb M concentration at 9.5 hours of 64.5 g / liter. Calculations were incremental rate steps performed using a 0.5-hour increment so that the calculated concentration profiles matched experimental data for one case. FIG. 15B depicts of a computational model of a rate of ATP- glucose consumption for glycosylation reactions of rebaudiosides (including stevioside, Rebaudioside A, and Rebaudioside D, to form Rebaudioside M, as well as small amounts side reactions). In addition, computationally modeled rates of ATP-glucose consumption for the reverse reaction of fructose + ADP -glucose to give sucrose and ADP is shown.

[0030] FIG. 16 depicts the glycosylation of steroid to a steroidal glycoside.DETAILED DESCRIPTIONAbbreviations

[0031] Certain terms used herein may be referred to interchangeably by their abbreviation, as outlined in Table 1, below.Table 1Definitions

[0032] As used herein, the term “steviol glycosides” refers to a glycoside of steviol, including, but not limited to, naturally occurring steviol glycosides, e.g. steviol- 13 -O-glucoside, steviol - 19-O-glucoside, rubusoside, steviol- 1,2-bioside, steviol- 1,3 -bioside, rubusoside, dulcoside B, dulcoside A, rebaudioside B, rebaudioside G, stevioside, rebaudioside C, rebaudioside F, rebaudioside A, rebaudioside I, rebaudioside E, rebaudioside E2, rebaudioside H, rebaudioside L, rebaudioside K, rebaudioside J, rebaudioside AM, rebaudioside M, rebaudioside D, rebaudioside N, rebaudioside O, rebaudioside Q, rebaudioside X, synthetic steviol glycosides, e.g. enzymatically glycosylated steviol glycosides and combinations thereof. Table 2 provides an illustrative list of steviol glycosides. The species in Table 2 are described in International Patent Publication No. WO 2020 / 112957, which is hereby incorporated by reference in its entirety.Table 2

[0033] As used herein, unless otherwise indicated, “ESI” refers to electrospray ionization as used in mass spectroscopy. As used herein, unless otherwise indicated, “EIC” refers to an extracted-ion chromatogram. As used herein, unless otherwise indicated, “EIC (molecular weight value)” refers to an extracted-ion chromatogram with one m / z value representing one analyte molecular weight extracted from the entire data set for a chromatographic run.

[0034] As used herein, “stoichiometric levels ofNTP” and “a stoichiometric amount of NTP” are used interchangeably. “A stoichiometric amount ofNTP” relates to the total amount ofNTP added to the glycosylation reaction. When running a glycosylation reaction (e.g. Scheme la below), the glycosyltransferases must perform N moles of turnovers to convert N moles of sugar acceptor into N moles of glycosylated sugar acceptor ((3) from Fig 1A). Using “stoichiometric levels ofNTP” refers to adding NTP to the reaction at the same molar amountor more as the number of glycosyltransferase turnovers required to convert the sugar acceptor to the glycosylated sugar acceptor (target molecule). For example, if the glycosylation reaction is used to convert 80 mM of sugar acceptor to 80 mM target molecule, a stoichiometric amount of NTP would be greater than or equal to 80 mM. Note that in some glycosyltransferase reactions, multiple glycosylations are required to convert the sugar acceptor to the target molecule. For example, to convert rebaudioside A to rebaudioside M requires Reb A to be glycosylated twice. Therefore, to convert 80 mM of Reb A to 80 mM of Reb M, 160 mM of glycosyltransferase turnovers is required. In this case, a stoichiometric amount of NTP would be greater than or equal to 160 mM NTP. For some glycosylation reactions, not all of the sugar acceptor is converted to the target molecule. For example, 80 mM sugar acceptor might be added to a reaction, but only 20 mM of target molecules are made. In this case, “a stoichiometric amount of NTP” refers to adding 20 mM or greater of NTP to the reaction.

[0035] As used herein, “catalytic levels of NTP” or “a catalytic amount of NTP” are used interchangeably. “A catalytic amount of NTP” refers to adding less than a stoichiometric amount of NTP to the glycosylation reaction. For example, a glycosylation reaction requires 80 mM of glycosyltransferase turnovers to convert 80 mM of sugar acceptor to 80 mM target molecule. In this case, a catalytic amount of NTP would refer to adding less than 80 mM of NTP to the reaction. For instance, the amount of NTP may be substantially less than 80 mM. The ratio of NTP to the number of glycosyltransferase turnovers may be less than 1:5, less than 1 :10, less than 1 :20, less than 1 :40, less than 1 :80, less than 1: 120, or less than 1: 150.

[0036] As used herein, the term “polynucleotide" is used to refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides, typically DNA.

[0037] As used herein, "expression" refers to either or both steps, depending on context, of the two-step process by which polynucleotides are transcribed into mRNA and the transcribed mRNA is subsequently translated into polypeptides.

[0038] The term "encode" refers to the ability of a polynucleotide to produce an mRNA or a polypeptide if it can be transcribed to produce the mRNA and then translated to produce the polypeptide or a fragment thereof. In each case, the polynucleotide is referred to as encoding the mRNA and encoding the polypeptide. The antisense strand is the complement of such a nucleic acid, and the encoding sequence can be deduced therefrom. Similarly, a “coding sequence” refers to a region of a nucleic acid that encodes an mRNA or a polypeptide.

[0039] The term "polypeptide" is used here to refer to a molecule of two or more subunits of amino acids linked by peptide bonds. Typically, though not always, the polypeptides contain several hundred amino acids; for example, about 400 to about 900 amino acids.

[0040] A "plasmid" or “vector” is a DNA molecule that is typically separate from and capable of replicating independently of the chromosomal DNA. In many cases, it is circular and doublestranded. It is known in the art that while plasmid vectors often exist as extrachromosomal circular DNA molecules, plasmid vectors may also be designed to be stably integrated into a host chromosome either randomly or in a targeted manner. Many plasmids are commercially available for varied uses. The gene to be replicated is inserted into copies of a plasmid containing genes that make cells resistant to particular antibiotics, and a multiple cloning site (MCS, or polylinker), which is a short region containing several commonly used restriction sites allowing the easy insertion of DNA fragments at this location. Typically, the polypeptides disclosed herein are expressed from plasmids.

[0041] Enzyme preparations used herein may be crude, partially purified, or highly purified. Enzyme preparations refer to enzymes produced by a microorganism or plant culture either extracellularly or intracellularly. A crude enzyme preparation refers to an enzyme preparation resulting from extracellularly produced enzymes. A crude enzyme preparation can also refer to an enzyme preparation resulting from intracellularly produced enzymes. The cells can be optionally lysed, by mechanical, chemical, sonication, or combinations thereof, or similar technique. A partially purified enzyme preparation is the result of a crude enzyme preparation that has been further processed to remove cells, cellular debris, and unwanted proteins using one or more high-throughput methods, including but not limited to centrifugation, filtration (including microfiltration, ultrafiltration, and nanofiltration), flocculation, pasteurization, ion exchange, or combinations thereof, or other techniques with similar performance. Highly purified enzyme preparations are crude or partially purified enzyme preparations that have been further purified using a more specific, lower-throughput purification technique. Methods to produce highly-purified enzyme preparations include affinity chromatography-based techniques, such as immobilized metal affinity chromatography (IMAC), which isolate proteins based on a specific tag added to the protein. Suitable tags include, but are not limited to, a polyhistidine tag of between 4 and 10 histidine residues, preferably 6 histidine residues, a glutathione S-transferase (GST) tag, a FLAG tag, a maltose binding protein (MBP) tag, a calmodulin binding peptide (CBP) tag and a Myc tag. In addition, other methods to produce highly-purified enzyme preparations include size-exclusion chromatography. The “purity” of an enzyme preparation refers to the % of the enzyme preparation that is the target enzyme or enzyme of interest. Partially purified enzymes and enzyme preparations are generally between 1% and 85% pure. For instance, partially purified enzymes and enzyme preparation may be between 5% pure and 30% pure, between 6% pure and 29% pure, between 7% pure and 28%pure, between 8% pure and 27% pure, between 9% pure and 26% pure, between 10% pure and 25% pure, and / or between 15% pure and 20% pure. Highly purified enzyme preparations are generally at least 85% pure. For instance, highly purified enzymes and enzyme preparations may be at least 85% pure, at least 86% pure, at least 87% pure, at least 88% pure, at least 89% pure, at least 90% pure, at least 91% pure, at least 92% pure, at least 93% pure, and / or at least 95% pure.

[0042] The term “about” or “approximately” when immediately preceding a numerical value means a range (e.g., plus or minus 10% of that value). For example, “about 50” can mean 45 to 55, “about 25,000” can mean 22,500 to 27,500, etc., unless the context of the disclosure indicates otherwise, or is inconsistent with such an interpretation. For example, in a list of numerical values such as “about 49, about 50, about 55, ...”, “about 50” means a range extending to less than half the interval(s) between the preceding and subsequent values, e g., more than 49.5 to less than 52.5. Furthermore, the phrases “less than about” a value or “greater than about” a value should be understood in view of the definition of the term “about” provided herein. Similarly, the term “about” when preceding a series of numerical values or a range of values (e.g., “about 10, 20, 30” or “about 10-30”) refers, respectively to all values in the series, or the endpoints of the range.

[0043] As used herein the terms “microorganism” or “microbe” should be taken broadly. These terms are used interchangeably and include, but are not limited to, the two prokaryotic domains, Bacteria and Archaea, as well as certain eukaryotic fungi and protists. In embodiments, the disclosure refers to the “microorganisms” or “microbes” of lists and figures present in the disclosure. This characterization can refer to not only the identified taxonomic genera but also the identified taxonomic species, as well as the various novel and newly identified or designed strains of any organism in said tables or figures. The same characterization holds true for the recitation of these terms in other parts of the Specification, such as in the Examples.

[0044] Amino acids are the compounds or building blocks that make up peptides and proteins. Each amino acid is structured from an amino group (N-terminus) and a carboxyl group (C- terminus) bound to a tetrahedral carbon. This carbon is designated as the a-carbon (alphacarbon). Amino acids differ from each other with respect to their side chains, which are referred to as R groups. Though the R group for each of the amino acids will differ in structure, electrical charge, and polarity, amino acids can be grouped into several groups of amino acids having similar properties. These similar properties permit these amino acids, in certain instances, to be reasonably interchangeably within an amino acid sequence. These groups include aliphatic amino acids, aromatic amino acids, amino acids with polar neutral side chains, acidic aminoacids with electrically charged side chains, basic amino acids with electrically charged side chains, and “unique” amino acids. Aliphatic amino acids are amino acids with hydrophobic side chains and include alanine, isoleucine, leucine, methionine, and valine. Aromatic amino acids are amino acids with hydrophobic side chains and include phenylalanine, tryptophan, and tyrosine. Amino acids with polar neutral side chains include asparagine, cysteine, glutamine, serine, and threonine. Acidic amino acids with electrically charged side chains include aspartic acid and glutamic acid. Basic amino acids with electrically charged side chains include arginine, histidine, and lysine. “Unique” amino acids, which are amino acids that cannot be otherwise grouped together, include glycine and proline.

[0045] Percentage identity may be calculated using the alignment program Clustal Omega (available at / www.ebi. ac.uk / Tools / msa / clustalo / ) with default settings. The default transition matrix is Gonnet, gap opening penalty is 6 bits, and gap extension is 1 bit. Clustal Omega uses the HHalign algorithm and its default settings as its core alignment engine. The algorithm is described in Sbding, J. (2005) 'Protein homology detection by HMM-HMM comparison1. Bioinformatics 21, 951-960.

[0046] As used herein, molar ratio is used to refer to the relative molar amounts of reagents that are added to a reaction medium. For instance, the molar ratios of compounds A and B can be obtained by dividing the molar amount of compound A added to a reaction by the molar amount of compound B added to the reaction.

[0047] As used herein, nucleoside triphosphate, or “NTP,” refers to any form of NTP salts, solutions, hydrates, and free NTP. As used herein, adenosine triphosphate, or “ATP,” refers to any form of ATP salts, solutions, hydrates, and free ATP. This includes but is not limited to the following forms listed in Table 3.Table 3

[0048] NTP may be provided in various forms and at different purity levels, including as crude and / or partially purified NTP salts, solutions, or hydrates that contain NDP and / or NMP. NTPmay be produced by fermentation of a microorganism where the resulting fermentation broth contains NTP and other components. The fermentation broth may be used as is, as a source of NTP, or further purified to concentrate the level of NTP in the broth. ATP can be produced by fermentation using Corynebacterium stationis (ATCC 6872™; previously Corynebacterium ammoniagenes, Strain designation: NCTC 2399, which was deposited as Brevibacterium ammoniagenes.) At the end of the fermentation, a typical mixture is obtained containing about 4 g / L ATP, 1 g / L ADP, and 0.5 g / L AMP. This mixture could be provided as crude ATP for use in glycosylation reactions. Alternatively, the fermentation broth could be further processed, as shown in FIG. 14, to provide a partially purified ATP mixture. For example, cells can be removed using centrifugation, and the broth can be clarified with sequential application of microfiltration, ultrafiltration, and nanofiltration to remove unwanted sugars and neutrals as permeate while concentrating and retaining the ATP-, ADP-, and AMP-salts. The resultant ATP / ADP / AMP mixture can be concentrated to 20 g / L total concentration and provided as a partially purified ATP mixture for use in glycosylation reactions.

[0049] In one embodiment, the fermentation broth comprises 4 g / L ATP, 1 g / L ADP, and 0.5 g / L AMP. In one embodiment, the retentate ATP solution comprises 20 g / L ATP, 5 g / L ADP, and 2.5 g / L AMP.

[0050] Glycosylation reactions, which may be referred to herein interchangeably as glycosylation, glycosidation, or glycosyltransferase reactions, transfer a carbohydrate moiety from a donor molecule to an acceptor molecule forming a glycosidic bond. Glycosylation reactions can be catalyzed by enzymes referred to as glycosyltransferases. In any glycosylation reaction, the acceptor, or sugar acceptor, can be any one of a protein, a lipid, a carbohydrate, and a biochemical such as a polyphenol, a polyketide, or steviol glycoside. In some instances, the acceptor already contains one or more glycosyl groups, and the glycosylation reaction involves adding additional glycosyl groups. The donor, or sugar donor, is composed of a carbohydrate group that will be transferred to the acceptor and a leaving group at the carbohydrate’s anomeric carbon. The donor leaving group can be another carbohydrate, a halide, a nucleotide, a phosphate, a pyrophosphate, a lipid phosphate, trichloroacetimidate, or a thio-group, among others. The transferred carbohydrate group is typically a monosaccharide or a derivative of a monosaccharide, including but not limited to glucose, ribose, galactose, xylose, mannose, fucose, N-acetylglucosamine, N-acetylgalactosamine, glucuronic acid, N- acetylneuraminic acid (NeuAc), N-glycolylneuraminic acid (NeuGc), and deaminoneuraminic acid (KDN).

[0051] Glycosylation reactions can transfer the glycosyl group to an oxygen (O), nitrogen (N), sulfur (S), or carbon (C) atom on the acceptor molecule.

[0052] If the carbohydrate moiety is attached to an oxygen atom on the acceptor, commonly from a hydroxyl group on the acceptor, it is called O-glycosylation. This commonly occurs when the acceptor is a carbohydrate or carbohydrate derivative, as is the case during the glycosylation of rebaudioside A to rebaudioside D using an NDP -glucose as the glycosyl donor. Examples of O-glycosylation where a protein is the acceptor include the addition of the carbohydrate moiety to residues such as serine, threonine, hydroxylysine, or hydroxyproline.

[0053] If the carbohydrate is attached to a nitrogen atom on the acceptor, commonly from an amino group on the acceptor, it is called N-glycosylation. When a protein is the acceptor, examples of N-glycosylation include carbohydrate addition to the NH2 of a terminal amino acid, or a surface located asparagine NH2 residue. Examples of small molecule N-glycosides include adenosine and varenicline N-Glucoside (CAS No: 873302-31-9). N-glycosides have diverse biological and pharmacological actions and thus are useful for both research and for drug development.

[0054] If the carbohydrate is attached to a sulfur atom on the acceptor, commonly from a sulfhydryl group on the acceptor, it is called S-glycosylation. When a protein is the acceptor, an example is glycosylation of a cysteine -SH group. S-glycosylation reactions are utilized in DNA synthesis and processing. For example, S-glycosyl transformations can be useful in a DNA-encoded library (DEL)-compatible environment. One approach involves 2-chloro-l,3- dimethylimidazolidinium chloride (DMC)-mediated S-glycosylation.

[0055] If the carbohydrate is attached to a carbon atom on the acceptor, it is called C- glycosylation. For example, the enzyme UGT708C1 from buckwheat (Fagopyrum esculentum) utilizes UDP -glucose as the sugar donor to catalyze the C-glycosylation of 2- hydroxyflavanones. For example, a C-glycosyltransferase from rice (Oryza sativa; OsCGT) and the sucrose synthase from soybean (Glycine max; GmSuSy) can be used to synthesize nothofagin, the natural 3’-C-P-d-glucoside of the dihydrochalcone phloretin, with regeneration of uridine 5 ’-diphosphate (UDP) glucose from sucrose and UDP. Other examples of C- glycosylation products include narigenin-6-C-glucoside (CAS number 3682-03-9), aspalathin (CAS number 6027-43-6).

[0056] An example of a glycosylation reaction is shown in FIG. 16 in the context of a steroidal glycoside. A starting molecule that is glycosylated (the “acceptor") can be a protein, a lipid, a biochemical such as a polyphenol, a polyketide, a steroid, or a steviol glycoside, among many other biomolecules. The carbohydrate group added may be one of glucose, galactose, xylose,mannose, or many others. A target molecule of the reaction may have no glycosyl groups or may already contain one or more glycosyl groups. The addition of a glycosyl donor to an acceptor (e g., steroid) in FIG. 16 produces a steroidal glycoside as the target molecule. When one or more glycosyl groups are already present on the target molecule, glycosylation involves the addition of additional glycosyl groups. This is relevant to particular Examples herein, whereby lower order steviol glycosides may be glycosylated to form successively higher order steviol glycosides. Similarly, plant sterols, animal sterols, polyphenols, and other acceptors may be glycosylated to form successively higher order glycosides.

[0057] The extensive number of donors and acceptors that can participate in glycosylation reactions enables the synthesis of many desirable compounds. The human genome has around 700 genes encoding enzymes, transporters, and chaperones required for the cellular glycosylation machinery, glycan modifications, and their degradation. Almost one third of the 279 classified peptide hormones carry O-glycans.

[0058] Because of its broad applicability and utility, glycosylation is often a strategy used by both nature and industry to improve the water solubility and stability of beneficial compounds. Numerous flavonoids, such as fisetin, quercetin, luteolin / luteolinidin, and apigenin, have a host of health benefits along with strong senolytic activity. After glycosylation, these flavonoids maintain or improve their health benefits while improving their solubility, leading to better bioavailability. Propofol, an important anesthetic drug, can be modified to give improved solubility. Cannabinoid compounds glycosylated via yeast and plant cell suspension cultures also have improved water solubility. Similarly, glycosylated derivatives of animal and plant sterols, such as cholesterol, campesterol, sitosterol, and stigmasterol, have improved properties. Further, glycosides are biomedically important secondary metabolites. For instance, 20 fungal aromatic polyketide C / O-glycosides have been identified from three selected fungi, each of which exhibit significant antiviral, antibacterial, and antidiabetic activities. Also, several isolated glycosylated products are used as important drugs, for example, erythromycin, daunomycin, and amphotericin B.

[0059] Glycosylated steroid derivatives provide improved transport across biological membranes. Certain steroidal glycoside derivatives are useful as hypocholesterolemia agents. Plant polyphenols, some of which are glycosylated, are a ubiquitous part of animal diets as they are widely found in plants. For example, rutin is a common dietary polyphenol found in vegetables, fruits, and other plants, and exhibits a range of bioactive properties.

[0060] Numerous sterols are present in the plant and animal kingdom. Cholesterol is the main animal sterol and is essential for life. In animals, most cholesterol is either ingested orsynthesized in the blood and then transported to peripheral cells. Phytosterols are sterols naturally found in plants, including campesterol, sitosterol, and stigmasterol. The stereoselective formation of O-glycosidic bonds between carbohydrates and steroids can be difficult with relatively low yields due to the low reactivity of the secondary alcohol functions in the steroid moiety and the necessity to activate the glycosyl donors. Glycosyl donors used in chemical synthesis include glycosyl halides, trihalogenoacetimidates, thioglycosides, 1-0- sulfonyl glycosides, glycals, 1-0-acyl sugars, orthoesters or ethers, phosphate derivatives, 1- hydroxyl sugars, and 1-0-silylated glycosides.

[0061] Glycosylation can be performed by chemical synthesis methods. For example, propofol can be modified to give improved solubility by glycosylation using tri-O-acetyl glucal in dichloromethane at -78°C with boron trifluoride diethyl etherate. Sulfuric acid can be used as a catalyst with a solution of glucose, maltose and other oligomers to form branched oligosaccharides. In addition, chemical glycosylation is used to produce the N-glycosylation product nicotinamide tri O-benzyl riboside by reacting -D-ribofuranose 1-acetate 2,3,5- tribenzoate with HCL in ethanol to generate l-chloro-2,3,5-tri-O-benzyl-D-ribofuranose and then reacting the halogenated compound with nicotinamide. The energy, solvent, acid, and safety requirements of chemical glycosylation, in addition to the lack of reaction specificity make it an expensive method to perform a glycosylation reaction.

[0062] As opposed to chemical synthesis methods, such as those noted above, biochemical synthesis methods, using selective enzymes, can be deployed. These are generally preferable for selectivity and simplicity, if suitable enzymes can be found or designed, if cost-effective reagents can be used, and if a cost-effective reaction system can be operated at sufficiently large scale. Common methods for enzymatic modifications of steviol glycosides, for example, include cyclodextrin glycosyl transferase systems, a-glucosidase systems, P-glucosidase transglycosylation, a,P-galactosidase transglycosylation systems, and bioconversion using NDP-sugar.

[0063] Glycosylated biomolecules can also be used as sweeteners for food and drinks. The species Stevia rebaudiana is commonly grown for its sweet leaves, which have traditionally been used as a sweetener. Over ten different steviol glycosides are present in appreciable quantities in the leaf. These steviol glycosides are glycosylated derivates of an aglycone core molecule called steviol (shown in FIG. 7A). The plant synthesis route initially produces steviol, which is a diterpenoid core. The plant biosynthetic pathway then proceeds via a series of glycosylation steps catalyzed by uridine diphosphate (UDP)-dependent glycosyltransferases (UGTs). As suggested above, steviol glycosides can be glycosylated once or more than once,and a particular steviol glycoside comprising 2, 3, 4, or 5 glycosyl groups can have one or more additional glycosyl groups added. Stevia extract is 200-300 times sweeter than sugar and is used commercially as a high intensity sweetener. The main glycoside components of stevia leaf are mainly stevioside and rebaudioside A, with often around 1% rebaudioside M present. The sweetness and bitterness profile of stevia extract can be improved by converting stevioside and rebaudioside A to rebaudioside M, by using a glycosylation process to add 3 glucose molecules to stevioside and add 2 glucose molecules to rebaudioside A.

[0064] Glycosyltransferases (GTs) are enzymes (EC 2.4) that establish glycosidic linkages. Enzymatic glycosyl reactions involve donors with a leaving group, such as a phosphate, a pyrophosphate, a lipid phosphate, or a nucleotide. Glycosyltransferases have been classified into families according to their structural and functional similarities. The CAZy database categorizes glycosyltransferases into 137 different families. The vast majority of the glycosyltransferase families utilize sugar mono- or diphosphonucleosides and are referred to as Leloir glycosyltransferases (Schemes la and lb).{Glycosyl-NDP} + {Acceptor}-OH Glycosyl-O-Acceptor + NDP (Scheme la) {Glycosyl-NMP} + {Acceptor}-OH -> Glycosyl-O-Acceptor + NMP (Scheme lb)

[0065] Therefore, the Leloir glycosyl donor consists of a glycosyl group attached to an NMP or NDP such as UDP, guanosine diphosphate (GDP), cytidine diphosphate (CDP), ADP, thymidine diphosphate (TDP), or cytidine monophosphate (CMP). The glycosyl-NDP and glycosyl-NMP donors are called sugar nucleotides. Examples of sugar nucleotides include but are not limited to ADP-glucose, ADP-mannose, ADP -xylose, ADP-ribose, UDP -glucose, UDP -mannose, UDP -xylose, and UDP -ribose.

[0066] Leloir glycosyltransferases can proceed in the forward direction shown in Schemes la and lb or in the reverse (sugar nucleotide synthesis) direction shown in Schemes 2a and 2b.Glycosyl-“LG” + NDP{Glycosyl-NDP} + “LG” (Scheme 2a)Glycosyl-“LG” + NMP -> {Glycosyl-NMP} + “LG” (Scheme 2b)

[0067] In Schemes 2a and 2b the nucleotide is the acceptor and the glycosyl-LG is the glycosyl donor.

[0068] The present disclosure relates to reactions involving Leloir glycosyltransferase enzymes. Leloir glycosyltransferases can be used to make desirable biomolecules. For example, the Leloir P-l,2-glycosyltransferase UGT91D2 from Stevia Rebaudiana utilizes UDP -glucose and engineered P-l,2-glycosyltransferases can utilize ADP-glucose to catalyze the reactions shown in Schemes 3a and 3b:NDP-Glucose + SteviosideRebaudioside E + NDP (Scheme 3a)NDP-Glucose + Rebaudioside A -> Rebaudioside D + NDP (Scheme 3b)

[0069] Similarly, the Leloir P-1, 3 -glycosyltransferase UGT76G1 from Stevia Rebaudiana utilizes UDP -glucose and engineered P-l,3-glycosyltransferases can utilize ADP-glucose to catalyze the reactions shown in Schemes 4a-4d:NDP-glucose + SteviosideRebaudioside A + NDP (Scheme 4a)NDP-glucose + Rebaudioside ARebaudioside I + NDP (Scheme 4b) NDP-glucose + Rebaudioside ERebaudioside D + NDP (Scheme 4c) NDP-glucose + Rebaudioside DRebaudioside M + NDP (Scheme 4d)

[0070] One difficulty with using Leloir glycosyltransferases in industrial applications is that the sugar nucleotides and phosphorylated nucleosides are expensive. When performing a glycosyltransferase reaction, if a sugar nucleotide donor must be provided for each glycosyltransferase turnover, referred to as a stoichiometric amount of sugar donor, the high cost of the Leloir donor will generally make any industrial process that relies on Leloir glycosyltransferases non-economical. To make the glycosylation step more economical, nucleotide recycling can be employed to only require “catalytic” amounts of the nucleotide (i.e. much less than the number of glycosyltransferase turnovers), instead of “stoichiometric” amounts.

[0071] Activated sugar donor can be economically provided by continuously regenerating it using a glycosidic donor that either has high energy or high concentration or both. For example, the glycosyltransferase sucrose synthase (E.C. 2.4.1.13) can be run in the sugar nucleotide synthesis direction to make NDP-glucose from NDP and sucrose (Scheme 5).NDP + SucroseNDP-Glucose + Fructose (Scheme 5)

[0072] In Scheme 5, a relatively inexpensive feedstock, sucrose, is coupled with the Leloir glycosyltransferase sucrose synthase to make the expensive nucleotide sugar required for other Leloir glycosyltransferase reactions.

[0073] In embodiments, a reaction combining sucrose synthase and a B13GT (Schemes 5 and 4a) results in the overall reaction shown in Scheme 6:Sucrose + Stevioside -> Fructose + Rebaudioside A (Scheme 6)

[0074] In Scheme 6, sucrose and stevioside are used in stoichiometric amounts (approximately 1 :1), or optionally, sucrose can be used in greater amounts in the reaction compared to stevioside due to sucrose’s relatively low cost. In contrast, the expensive sugar nucleotide, NDP, is both consumed and regenerated in Scheme 6, which is why it is not explicitly listed in the scheme. This regeneration greatly reduces the required amount of NDP to levels much lower than that of sucrose and stevioside. This is referred to as using NDP at catalytic levelsand greatly improves the economics of using a Leloir glycosyltransferase in an industrial process.

[0075] Consider Scheme 4a where at least 1.00 mole of NDP-sugar (NDP -glucose) is required to glycosylate 1.00 mole of the acceptor (stevioside). This is referred to as a stochiometric amount of NDP-sugar. Typically, excess sugar donor (sucrose) is utilized to ensure all the acceptor is glycosylated to the desired target molecule (rebaudioside A). Therefore, the amount of NDP-sugar required for Scheme 4a could range from 1.01 to 2.0 mole NDP-sugar per 1.0 mole glycosylated acceptor, allowing for 1% to 100% stoichiometric excess for reaction needs. In contrast, consider Scheme 6, which doesn’t require a stoichiometric amount of NDP. In Scheme 6, NDP can be added at a level lower than a stoichiometric amount, referred to as a catalytic amount of NDP. For example, only 0.01-0.05 mole of NDP is required per 1 mole of glycosylated acceptor.

[0076] The addition of exogenous NDP is a significant cost for Leloir glycosyltransferase- based glycosylation processes. As disclosed herein, the present disclosure relates to methods for utilizing an NTP feedstock at catalytic levels in place of an NDP feedstock. The low cost of NTPs compared to NDPs, and specifically for ATP compared to ADP, provides great cost savings for Leloir glycosyltransferase processes.

[0077] However, NTP has not been used as a reagent in glycosylation because Leloir glycosyltransferases do not directly use NTP-sugars as sugar donors. Here, the present disclosure describes methods and compositions that use NTP for glycosylation reactions through the conversion of NTP to NDP-sugar. Advantageously, the methods and compositions described herein can be used with a variety of sugar acceptors as substrates, enabling the production of myriad glycosylated products.

[0078] In embodiments, the present disclosure relates to adding catalytic levels of NTP to a Leloir glycosylation system, enabling the glycosylation reaction to occur. In Examples, glycosylation of steviol glycosides is demonstrated. Selective glycosylation as it relates to the production of steviol glycosides represents one approach to addressing a pressing healthcare challenge, as discussed above. Excess sugar consumption has been linked to worldwide health epidemics including diabetes and heart disease. Replacing added sugar in food with a low calorie, high-intensity sweetener would have significant health and economic impact.

[0079] Steviol glycosides are composed of a diterpenoid steviol core (FIG. 7A) and two variable glycans. The two variable glycans are attached to the C13-hydroxyl (R2) and C19- carboxylate (Rl) of the steviol core, as shown in FIG. 7B. Steviol and a selection of its glycosides are shown below in Table 4 in the context of their variable glycans. The conversionof stevioside into Rebaudioside A is shown in FIG. 3B, whereby a glycosyl donor is interacted with an acceptor (stevioside) to produce Rebaudioside A.Table 4

[0080] Multiple glycosylation steps can give rise to linear sugar chains or branched sugar chains. In steviol chemistry, the highest level of glycosylation is generally five glucosyl groups as well as one glucose ester group for a total of six glucose molecules attached to each steviol core. The enzymatic reactions disclosed herein generate linear or branched chains of glucosyl or other sugar residues with as many as 9 residues. Steviol glycosides with glucosyl residues are shown in Table 5, where the un-glucosylated core molecule is steviol, as discussed herein with reference to FIG. 7B. In stevia leaf, levels of steviol may be very low and often are not detected by usual analytical methods. Moreover, species of stevia leaf with 5 or more glucosyl residues may not be present or present only at very low concentrations and thus may not be detected with usual measurements.Table 5NTP as a Reagent for Glycosylation of Sugar Acceptors

[0081] As disclosed herein, methods exist whereby ATP can be converted to ADP and an inorganic phosphate in sufficient quantities to permit glycosylation of e.g., steviol glycosides to readily proceed. Generally, the conversion of ATP to ADP and an inorganic phosphate, which may be referred to herein as ATP hydrolysis, is a catabolic reaction process by which chemical energy that has been stored in the high-energy phosphoanhydride bonds in ATP is released after splitting these bonds by producing work, for example in muscles, in the form of mechanical energy. The product of ATP hydrolysis is ADP and an inorganic phosphate (Pi). ADP can be further hydrolyzed to give energy, adenosine monophosphate (AMP), and another Pi. ATP can also be hydrolyzed directly to give AMP, and pyrophosphate (PPi).

[0082] The conversion of ATP to ADP and an inorganic phosphate can be performed in a number of ways, including by enzymatic hydrolysis and non-enzymatic hydrolysis, such as chemical hydrolysis. Reaction conditions, including reagents (e.g., sulfuric acid, sodium hydroxide) and temperature (>100°C), can be adjusted to accelerate the chemical hydrolysis. Further, enzymes present in living organisms may catalyze the chemical reaction: ATP + H2O = cAMP, ATP + AMP = 2 ADP, and ATP + H2O = AMP + diphosphate. Enzymes that catalyze the decomposition of ATP into ADP and a free phosphate, or the inverse reaction, may also be present. These enzymes are typically membrane bound. In one mode, they participate in charge-transferring complexes leading to ATP synthesis at the same time as moving ions through the membrane.

[0083] According to embodiments, the present disclosure provides methods and compositions for glycosylation. In embodiments, the present disclosure provides methods and compositions for using NTP in methods for enzymatic glycosylation. In particular, the present disclosure relates to the use of ATP in methods and compositions for the glycosylation of steviol glycosides using Leloir glycosyltransferases, thereby generating higher order steviol glycosides from lower order steviol glycosides. In this way, the lower cost ATP may be used in place of ADP.

[0084] According to embodiments, the present disclosure relates to a glycosylation method using NTP to perform a Leloir glycosyltransferase reaction. The method comprises converting NTP to NDP, converting the NDP to NDP-sugar by contacting at least one first Leloir glycosyltransferase with the NDP and a sugar donor to make the NDP-sugar, and converting a sugar acceptor to a glycosylated sugar acceptor by contacting the at least one second Leloir glycosyltransferase with the NDP-sugar and the sugar acceptor. In embodiments, the methodis performed in a reaction medium initially comprising the at least one first Leloir glycosyltransferase, the at least one second Leloir glycosyltransferase, the NTP, the sugar donor, and the sugar acceptor. In embodiments, the reaction medium comprising the at least one first Leloir glycosyltransferase, the at least one second Leloir glycosyltransferase, the NTP, the sugar donor, and the sugar acceptor may be a composition. In embodiments, the at least one first Leloir glycosyltransferase and the at least one second Leloir glycosyltransferase may be the same glycosyltransferase. In embodiments, the at least one first Leloir glycosyltransferase and the at least one second Leloir glycosyltransferase may be different glycosyl transferases. In embodiments, the molar ratio of NTP: SA added to the glycosylation reaction medium may be between about 0.00045: 1 and about 0.1:1. In embodiments, the molar ratio of NTP:GT in the reaction medium may be between about 0.55: 1 and about 5500:1. In embodiments, the molar ratio ofNTP:SD in the reaction medium may be between about 0.0001 : 1 and about 0.01:1.

[0085] In embodiments, the glycosylation is performed by Leloir glycosyltransferases prepared by expression in a host microorganism. Suitable host microorganisms include, but are not limited to, £ coli, Saccharomyces sp., Aspergillus sp., Pichia sp., Bacillus sp. For example, the Leloir glycosyltransferases may be expressed in L. coli. For example, the Leloir glycosyltransferases may be expressed in Pichia pastoris. In embodiments, the glycosylation is facilitated by sucrose synthase enzymes prepared by expression in a host microorganism. Suitable host microorganisms include, but are not limited to, E. coli, Saccharomyces sp., Aspergillus sp., Pichia sp., Bacillus sp. For example, the Leloir glycosyltransferases may be expressed in E. coli. For example, the Leloir glycosyltransferases may be expressed in Pichia pastoris. The glycosyltransferases may be expressed intracellularly or extracellularly.

[0086] In embodiments, recovering enzymes expressed in host microorganisms includes one or more of lysing the cells, clarifying the lysate, concentrating, and stabilizing the isolate. Clarifying the lysate may comprise centrifugation, flocculation, microfiltration, ultrafiltration, and / or nanofiltration. Concentrating may comprise removing water and an optional change of buffer or pH, such as by diafiltration. Diafiltration can also remove low molecular weight species by using a 10,000 MW cutoff ultrafiltration step. Stabilization may comprise glycerol treatment or drying, which may include spray drying or freeze drying. Enzyme preparations may have varying levels of purity, as described above. In embodiments, enzymes and enzyme preparations used herein to perform and / or facilitate glycosylation may be partially purified. In embodiments, enzymes and enzyme preparations used herein to perform and / or facilitate glycosylation may be highly purified, such as with affinity chromatography and / or sizeexclusion chromatography. In addition to other additional biomolecules, any of the crude, the partially purified, or the highly purified enzymes or enzyme preparations possibly include other enzymes produced by the host microorganism. These other enzymes, which may include kinases, phosphotransferases, or nucleoside phosphate hydrolases, may aid in catalyzing the above-mentioned activation of NTP. For instance, the other enzymes may include one or more of the enzymes shown in Table 6, which is an inexhaustive list.Table 6

[0087] In embodiments, the Leloir glycosyltransferase may be a beta-l,2-glycosyltransferase (B12GT) or a beta- 1,3 -glycosyltransferase (B13GT) provided in any suitable form, including free, immobilized, or as a whole cell system. In embodiments, the Leloir glycosyltransferase may be an engineered Leloir glycosyltransferase selected from the engineered glycosyltransferases (e.g., B12GT, B13GT) disclosed in International Patent Application No. PCT / US2023 / 073344, filed September 1, 2023, and International Patent Application No. PCT / US2024 / 032488, filed June 5, 2024, each of which is incorporated by reference herein in its entirety for all purposes. For instance, when the Leloir glycosyltransferase is a B12GT, the B12GT may have an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, and 100% identical to an amino acid sequence of the engineered glycosyltransferases disclosed in International Patent Application No. PCT / US2024 / 032488, and when the Leloir glycosyltransferase is a B13GT, the B13GT may have an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, and 100% identical to an amino acid sequence of the engineeredglycosyltransferases disclosed in International Patent Application No. PCT / US2023 / 073344. For example, when the Leloir glycosyltransferase is a B 12GT, the B12GT may have an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, and 100% identical to the amino acid sequence of SEQ ID NO: 1, as disclosed in International Patent Application No. PCT / US2024 / 032488, and when the Leloir glycosyltransferase is a B13GT, the B13GT may have an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, and 100% identical to the amino acid sequence of SEQ ID NO: 1, as disclosed in International Patent Application No. PCT / US2023 / 073344.

[0088] The degree of purity of the Leloir glycosyltransferases may vary, e.g., it may be provided as a partially purified or a highly purified (e.g., Ni- / His-tagged) enzyme preparation(s).

[0089] In embodiments, the Leloir glycosyltransferase is provided in free form. In embodiments, the Leloir glycosyltransferases are immobilized to a solid support, for example on an inorganic or organic support. The solid support may be derivatized cellulose, glass, ceramic, methacrylate, styrene, acrylic, a metal oxide, or a membrane. In embodiments, the Leloir glycosyltransferases are immobilized to the solid support by covalent attachment, adsorption, cross-linking, entrapment, or encapsulation. In embodiments, the Leloir glycosyltransferases are provided in the form of a whole cell system, for example as a living fermentative microbial cell, or as dead and stabilized microbial cell, or in the form of a cell lysate.

[0090] In embodiments, the present disclosure relates to methods and compositions for the glycosylation of a sugar acceptor. In embodiments, the methods comprise contacting a first Leloir glycosyltransferase with one or more sugar acceptors, a nucleoside triphosphate, a second Leloir glycosyltransferase (for NDP-sugar synthesis), and a sugar donor in a reaction medium. The reaction medium comprising the first Leloir glycosyltransferase, the one or more sugar acceptors, the nucleoside triphosphate, the second Leloir glycosyltransferase (for NDP- sugar synthesis), and the sugar donor may be a composition, according to embodiments. This method is shown in FIG. 1A. In FIG. 1A, as in Table 7, three reactions are shown:Table 7

[0091] Initially, NTP is converted to NDP at (1), activating the NTP for use by the glycosyltransferases of (2) and (3). A first Leloir glycosyltransferase converts the activated NDP to a nucleotide sugar, such as an NDP-sugar, at (2). Conversion of the NDP to NDP-sugar is referred to herein as stabilization, since it rescues the NDP from off target over conversion to NMP. Moreover, NDP-sugar is more stable in reaction conditions than NDP. To produce a glycosylated acceptor, a second Leloir glycosyltransferase utilizes the NDP-sugar to glycosylate the acceptor at (3), thereby generating a target molecule, or glycosylated acceptor. To ensure continuous stabilization or “regeneration” of free NDP, the first Leloir glycosyltransferase-based NDP-sugar synthesis at (2) and the second Leloir glycosyltransferase-based glycosylation at (3) operate simultaneously as an NDP cycle. In this way, free NDP formed from glycosylation of the acceptor at (3) can be “regenerated” to give additional NDP-sugar.(1) Conversion of NTP to NDP

[0092] In embodiments, the activation, or conversion, of NTP to NDP may be achieved by non-enzymatic or enzymatic means. i. Non-Enzymatic Conversion

[0093] Non-enzymatic conversion of NTP to NDP may be achieved by thermal and / or chemical means. Thermal conversion may be realized by adjusting the temperature of an aqueous solution of NTP. For example, while aqueous solutions of ATP are stable for months when frozen at -15°C, increasing the temperature of the aqueous solution increases the rate of conversion of ATP to ADP. Compared with -15°C, aqueous solutions of ATP are only stable for approximately one week at 0°C. Chemical conversion of NTP to NDP within an aqueous solution may be achieved by, for example, adjusting the pH of the aqueous solution. Chemical conversions of NTP and ATP are shown in FIG. 2A and FIG. 2B, respectively. Though aqueous solutions of ATP, for example, are stable for approximately one week at 0°C, ATP rapidly decomposes to ADP, even at 0°C, when the acidity of the aqueous solution is increased. Similarly, in alkaline solutions, ATP rapidly decomposes to inorganic pyrophosphate and adenosine 5'-phosphate, even at 0°C. Increases in temperature also increase the chemical conversion of NTP to NDP to NMP. it. Enzymatic Conversion

[0094] Enzymatic conversion of the NTP to NDP may be achieved using a hydrolase enzyme, a phosphatase enzyme, a kinase and a phosphate acceptor, or a combination thereof, In embodiments, the hydrolase enzyme may be apyrase (EC 3.6.1.5). In embodiments, the phosphatase enzyme may be a phytase (e.g., inositol -polyphosphate phosphatase (EC 3.1.3.62)). In embodiments, the kinase may be an acetate kinase and the phosphate acceptor, as the activator, may be acetate (FIG. 8A). In embodiments, the kinase may be fructose kinase and the phosphate acceptor, as the activator, may be fructose (FIG. 8B). In embodiments, the kinase may be glycerol kinase and the phosphate acceptor, as the activator, may be glycerol. A non-exhaustive list of phosphate acceptors is provided below in Table 8. In embodiments, the NTP can be converted to NDP by using native kinases from cell lysate, which may include the above kinases and / or may otherwise be configured to phosphorylate the phosphate acceptors of Table 8 or other phosphate acceptors present within the aqueous solution. In embodiments, the cell lysate could be from a microorganism such as E. coli, Saccharomyces sp., Aspergillus sp., Pichia sp., or Bacillus sp. In embodiments, the NTP can be converted to NDP using native kinases present with a partially purified enzyme or within an enzyme preparation, which may include the above kinases and / or may otherwise be configured to phosphorylate the phosphate acceptors of Table 8 or other phosphate acceptors present within the aqueous solution.Table 8

[0095] In embodiments, the glycerol phosphate acceptor, glycerol, can be present in a stabilized liquid enzyme formulation. In embodiments, the acetate phosphate acceptor, acetate, can be an acetate salt present in a buffer or ionic strength agent added to the reaction medium. In embodiments, the activation enzymes can be present in an enzyme lysate. In embodiments, the activation enzymes can be present in a partially purified enzyme preparation. In embodiments, the activation enzymes can be present in sugar cane juice. In embodiments, the activation enzymes can be present in partially purified stevia.(2) Generation of NDP-Sugar

[0096] In embodiments, stabilization of NDP as NDP-sugar may be achieved by at least one first Leloir glycosyltransferase operating in the direction of sugar-nucleotide (e g., ADP- glucose) synthesis (Schemes 2a and 2b, above). In embodiments, the sugar donor may be selected from the group consisting of sugar oligomers, sucrose, starch, maltose, lactose, alphaglucose- 1 -phosphate, beta-glucose- 1 -phosphate, cellobiose, gentiobiose, trehalose, kojibiose, nigerose, isomaltose, beta-beta-trehalose, alpha-beta-trehalose, sophorose, laminaribiose, turanose, maltulose, palatinose, gentiobiulose, nigerotriose, maltotriose, melezitose, maltotriulose, kestose, cellulose, arabinoxylan, glycogen, amylose, amylopectin, dextran, dextrin, maltodextrin, glucose syrup, cellodextrin, cyclodextrin, other disaccharides, other oligosaccharides and other polysaccharides. In embodiments, the sugar donor may be exogenous and / or may be provided in or isolated from a stevia leaf extract. In embodiments, stabilization of NDP can be catalyzed by sucrose synthase (EC 2.4.1.13), as the at least one first Leloir glycosyltransferase, where the glycosyl donor, or sugar donor, comprises sucrose. In other embodiments, the at least one first Leloir glycosyltransferase may comprise trehalose synthase (E C. 2.4.1.245) or lactose synthase (E.C. 2.4.1.22), where the respective sugar donors comprise trehalose and lactose. In embodiments, the at least one first Leloir glycosyltransferase comprises one or more of sucrose synthase, trehalose synthase, or lactose synthase. In embodiments, the at least one first Leloir glycosyltransferase comprises a B12GT and / or B13GT and the sugar donor comprises a steviol glycoside.(3) Glycosylation of a Sugar Acceptor

[0097] In embodiments, the sugar acceptor may be glycosylated by at least one second Leloir glycosyltransferase. In embodiments, the sugar acceptor may be selected from the group consisting of a protein, a lipid, steviol, a steviol glycoside, a polyketide, a steroid, a flavanone, or a polyphenol. In embodiments, the sugar acceptor may be an animal sterol and the target molecule (i.e., glycosylated sugar acceptor) may be a glycosylated sterol such as cholesterol- P-D-glucoside, cholesterol-diglucoside or cholesterol-triglucoside (FIG. 5). In other embodiments, the sugar acceptor may be a plant sterol, flavanone, or polyphenol, and the target molecule (i.e., glycosylated sugar acceptor) may be a glycosylated product such as quercetin- 3-P-D-glucoside, quercetin 3, 4'-di glucoside, prunin, spiraeoside, or P-Sitosterol- P-D- glucoside (FIG. 6). In embodiments, the sugar acceptor may be a steviol glycoside and the target molecule (i.e., glycosylated sugar acceptor) may be a steviol glycoside with one or more additional glucosyl moieties. In embodiments, the sugar acceptor may be stevioside, rebaudioside A, rebaudioside E, rebaudioside I, or rebaudioside D. In embodiments, the at leastone second Leloir glycosyltransferase may be aB12GT and / or Bl 3GT provided in any suitable form, including free, immobilized, or as a whole cell system. In an exemplary embodiment, the sugar acceptors comprise stevioside and rebaudioside A, the at least one second Leloir glycosyltransferase comprises one or more of B12GT or B13GT, and the glycosylated sugar acceptor, the target molecule, comprises rebaudioside M.Enzymatic glycosylation of sugar acceptors within a reaction medium comprising NTP

[0098] In embodiments, the reaction medium comprises NTP, at least one Leloir glycosyltransferase, a sugar donor, and a sugar acceptor. When the at least one Leloir glycosyltransferase performs NDP-sugar synthesis and glycosylation of the sugar acceptor, the at least one Leloir glycosyltransferase comprises one Leloir glycosyltransferase. When NDP- sugar synthesis and glycosylation of the sugar acceptor are performed by different Leloir glycosyltransferases, the reaction medium may comprise at least one first Leloir glycosyltransferase and at least one second Leloir glycosyltransferase. In embodiments, the above reaction mediums may define compositions of the present disclosure.

[0099] In embodiments, the conversion of NTP to NDP (1) may be performed prior to nucleotide- sugar synthesis (2) and glycosylation of the sugar acceptor (3). In embodiments, the conversion of NTP to NDP (1) and nucleoside-sugar synthesis (2) may be performed prior to glycosylation of the sugar acceptor (3). In embodiments, the reagents present during (1) and (2) (e.g., NTP, NDP-sugar synthase, sugar donor) may define a first composition and the reagents present during (2) and (3) (e.g., NDP-sugar synthase, sugar donor, sugar acceptor, glycosyltransferase) may define a second composition, according to embodiments. In embodiments, NTP to NDP conversion (1) and nucleotide-sugar synthesis (2) may be performed together in a first pot. Subsequently, nucleotide-sugar synthesis (2) and sugar acceptor glycosylation (3) may be performed simultaneously in a second pot. As in the 1-pot reaction described previously, the rate of NTP to NDP conversion (1) in the first pot may be between, for example, 0.1 mM / hr and 1 mM / hr, with a concentration of 1 mM NTP in the reaction medium being converted almost entirely to NDP-sugar (2) over a period of about 1 hour to about 10 hours. The glycosylation of the sugar acceptor (3) in the second pot may then proceed during the following about 4 hours to about 20 hours or more. In embodiments, the reagents within the first pot and the second pot define respective compositions of the present disclosure.

[0100] In embodiments, NTP to NDP conversion (1), nucleotide-sugar synthesis (2), and sugar acceptor glycosylation (3) may be performed simultaneously in a 1-pot reaction. In embodiments, when (1), (2), and (3) are performed in a 1-pot reaction, all reaction components,including NTP, sugar donor, sugar acceptor, and at least one Leloir glycosyltransferase are added at the beginning of the 1-pot reaction. In embodiments, only a portion of the reaction components are added at the beginning of the 1-pot reaction. For example, only NTP, sugar donor, and at least one Leloir glycosyltransferase are added at the beginning of the reaction, and / or only a fraction of a total amount of sugar acceptor is added at the beginning of the reaction. The remaining portion reaction components and / or fraction of the reaction components may be added to the reaction in bulk, continuously, or as fed-batch. In embodiments, any of the reaction components used for (2) or (3), e.g. nucleotide-sugar synthase, the sugar donor, the sugar acceptor glycosyltransferase, the sugar acceptor, can be absent at the start of the reaction. Each reaction component can be added at any time up to when a respective reaction is started. The composition of the reaction components at the beginning of, during, and at the end of the 1-pot reaction may define compositions of the present disclosure.

[0101] Further to the above, when NTP to NDP conversion (1), nucleotide-sugar synthesis (2), and sugar acceptor glycosylation (3) are performed simultaneously in a 1-pot reaction, the rate of NTP to NDP conversion (1) may be between, for example, about 0.1 mM / hr and about 1 mM / hr, such that a concentration of, for example, ImM NTP in the reaction medium is converted to NDP and, subsequently, to NDP-sugar (2) over a period of between, for example, about 1 hour and about 10 hours. Similarly, glycosylation of the sugar acceptor (3) proceeds during a period of between, for example, about 1 hour and about 24 hours or more and is substantially simultaneous with NTP to NDP conversion (1) and nucleotide-sugar synthesis (2), appreciating the reaction limiting delay of NDP-sugar production following NTP to NDP conversion.

[0102] In embodiments, the conversion of NTP to NDP (1), nucleotide-sugar synthesis (2), and glycosylation of the sugar acceptor (3) can be performed sequentially. In embodiments, (1) can be performed first, independently, in a first reaction medium. In embodiments, the first reaction medium from (1) can be optionally purified to isolate NDP. In embodiments, the first reaction medium or isolated NDP from the first reaction medium can be combined with a second reaction medium to perform (2) and, optionally, (1). In embodiments, the second reaction medium can be combined with a third reaction medium to perform (2) and (3) and, optionally, (1). In embodiments, reaction components used for (2) or (3) can be added to the reaction in the first reaction medium used to perform (1). In embodiments, reaction components used for (3) can be added to the reaction in the second reaction medium used to perform (1) and (2). In embodiments, reaction components used for (3) can be added to the reaction in thethird reaction medium. In embodiments, only a portion of the reaction components and / or only a fraction of each reaction component are added to respective reaction mediums at the beginning of the reaction or at the beginning of each of (1), (2), or (3). The remaining portion and / or fraction of the reaction components can be added to the reaction in bulk, continuously, or as fed-batch. The composition of each reaction medium at the beginning of, during, and at the end of the reaction may define compositions of the present disclosure.

[0103] In embodiments, the conversion of NTP to NDP (1), nucleotide-sugar synthesis (2), and glycosylation of the sugar acceptor (3) can be performed sequentially and / or combinations of two of (1), (2), and (3) can be performed sequentially. In embodiments, (1) and (2) can be performed simultaneously in a first reaction medium. In embodiments, the first reaction medium used to perform (1) and (2) can be combined with a second reaction medium to perform (1), (2), and (3). In embodiments, only a portion of the reaction components and / or only a fraction of each reaction component are added to respective reaction mediums at the beginning of the reaction or at the beginning of each of (1), (2), or (3). The remaining portion and / or fraction of each of the reaction components can be added to the reaction in bulk, continuously, or as fed-batch. In embodiments, reaction components used for (3) can be present in the first reaction medium used to perform (1) and (2). The composition of each reaction medium at the beginning of, during, and at the end of the reaction may define compositions of the present disclosure.

[0104] In embodiments, the conversion of NTP to NDP (1), nucleotide-sugar synthesis (2), and glycosylation of the sugar acceptor (3) can be performed sequentially and / or combinations of two of (1), (2), and (3) can be performed sequentially. In embodiments, (1) can be performed first, independently, in a first reaction medium. In embodiments, the first reaction medium can be optionally purified to isolate NDP. In embodiments, the first reaction medium or isolated NDP can be combined with a second reaction medium to perform (2) and (3), and optionally (1). In embodiments, only a portion of the reaction components and / or only a fraction of each reaction component are added to respective reaction mediums at the beginning of the reaction or at the beginning of each of (1), (2), or (3). The remaining portion and / or fraction of each of the reaction components can be added to the reaction in bulk, continuously, or as fed-batch. In embodiments, reaction components used for (2) or (3) can be present in the first reaction medium used to perform (1). The composition of each reaction medium at the beginning of, during, and at the end of the reaction may define compositions of the present disclosure.

[0105] In embodiments, reaction components, including NTP, sugar donor, sugar acceptor, and at least one Leloir glycosyltransferase, can be added at the beginning of the reaction and / orat the beginning of respective ones or combinations of (1), (2), and (3), or only a portion and / or fraction of the reaction components can be added at the beginning of the reaction and / or at the beginning of respective ones or combinations of (1), (2), and (3). For instance, the NTP may be added at the beginning of (1) and / or may be added during (1) such that between about 0.1 millimoles and about 10 millimoles of NTP is added in total, per liter of a final volume of the reaction medium, to the reaction. For example, the NTP may be added at between about 0.2 millimoles and about 10 millimoles, between about .25 millimoles and about 2.5 millimoles, and / or about 0.5 millimoles and about 1 millimole per liter of the final volume of the reaction medium. When sugar acceptor is added to the reaction at the beginning of and / or during (3), the sugar acceptor may be added such that between about 5 millimoles and about 200 millimoles of sugar acceptor is added in total, per liter of a final volume of the reaction medium, to the reaction. For example, the sugar acceptor may be added at between about 10 millimoles and about 150 millimoles, between about 25 millimoles and about 100 millimoles, and / or about 50 millimoles and about 75 millimoles per liter of the final volume of the reaction medium.

[0106] In embodiments, the reaction rates of NTP to NDP conversion (1), nucleotide-sugar synthesis (2), and sugar acceptor glycosylation (3), as shown in FIG. 1 A for a general case of NTP and as illustrated in FIG. IB for the case of ATP, are controlled such that the flux through the system dominates any possible off-target reactions, ensuring successful glycosylation of the sugar acceptor.

[0107] In embodiments, the conversion of ATP to ADP may be performed by an enzyme, such as a hydrolase, a phosphatase, or a kinase, and combinations thereof. In embodiments, the reaction medium comprises NTP, at least one Leloir glycosyltransferase, a sugar donor, a sugar acceptor, and a hydrolase. In embodiments, the hydrolase comprises apyrase. The hydrolase may be provided as part of an enzyme preparation of a partially purified or a highly purified Leloir glycosyltransferase, and / or the hydrolase may be provided exogenously to the reaction medium. In embodiments, the reaction medium comprises NTP, at least one Leloir glycosyltransferase, a sugar donor, a sugar acceptor, and a phosphatase. In embodiments, the phosphatase comprises phytase. In embodiments, the phosphatase may be provided as part of an enzyme preparation of a partially purified or a highly purified Leloir glycosyltransferase, and / or the phosphatase may be provided exogenously to the reaction medium. In embodiments, the reaction medium comprises NTP, at least one Leloir glycosyltransferase, a sugar donor, a sugar acceptor, a kinase, and a phosphate acceptor. In embodiments, the phosphate acceptor is a kinase substrate comprising one or more of acetate, glycerol, fructose, glucose, pyruvate, or succinate, and combinations thereof. In embodiments, the kinase may be provided to thereaction medium as part of an enzyme preparation of a partially purified or a highly purified Leloir glycosyltransferase. In embodiments, the kinase may be provided exogenously to the reaction medium. In embodiments, the kinase may be one or more of acetate kinase, glycerol kinase, fructokinase, glucokinase, pyruvate kinase, or acetyl-CoA carboxylase, and combinations thereof. A non-exhaustive listing of exemplary kinases and kinase acceptors is provided in Table 8. In embodiments, reaction mediums comprising NTP, at least one Leloir glycosyltransferase, a sugar donor, a sugar acceptor, and one or more of a hydrolase, a phosphatase, or a kinase and phosphate acceptor define compositions of the present disclosure.

[0108] In embodiments, the method of the present disclosure utilizes, as the NTP, one or more of uridine triphosphate (UTP), guanosine triphosphate (GTP), cytidine triphosphate (CTP), ATP, and / or thymidine triphosphate (TTP). In embodiments, the method may be performed with a reaction medium comprising NTP at a concentration of between about 0.01 mM and about 10 mM, based on a final volume of the reaction medium, between about 0.01 mM and about 0.05 mM, between about 0.05 mM and about 0.1 mM, between about 0.1 mM and about 0.5 mM, between about 0.5 mM and about 1 mM, between about 1 mM and about 5 mM, or between about 5 mM and about 10 mM. In embodiments, the molar ratio of NTP to sugar acceptor in the reaction medium may range from about 0.00045:1 to about 0.10: l. For example, the molar ratio of NTP to sugar acceptor may be about 0.01 : 1. In embodiments, the molar ratio of NTP to GT in the reaction medium may range from about 0.55:1 to about 5500:1.

[0109] In embodiments, the concentration of the sugar donor in the reaction medium may be between about 10 mM and about 3 M, based on a final volume of the reaction medium. For instance, the sugar donor concentration may be greater than about 10 mM, greater than about 50 mM, greater than about 100 mM, greater than about 250 mM, greater than about 500 mM, greater than about 1 M, greater than about 1.5 M, greater than about 2 M, and greater than about 2.5 M. In embodiments, the sugar donor concentration in the reaction medium may be between about 125 mM and about 2.5 M, between about 150 mM and about 2 M, or between about 175 mM and about 1.5 M. For example, the sugar donor concentration may be about 365 mM or about 1.17 M.

[0110] In embodiments, the stabilization of the NDP as an NDP-sugar (2) and the glycosylation of the sugar acceptor (3) are performed by a first Leloir glycosyltransferase and a second Leloir glycosyltransferase, respectively, and the first Leloir glycosyltransferase and the second Leloir glycosyltransferase are the same. In embodiments, the stabilization of the NDP as an NDP-sugar (2) and the glycosylation of the sugar acceptor (3) are performed by a first Leloir glycosyltransferase and a second Leloir glycosyltransferase, and the first Leloirglycosyltransferase and the second Leloir glycosyltransferase are different. In embodiments, the stabilization of the NDP as an NDP-sugar (2) is performed by a first Leloir glycosyltransferase and the glycosylation of the sugar acceptor (3) is performed by a second and a third Leloir glycosyltransferase, and the first, second, and third Leloir glycosyltransferase are different.

[0111] In embodiments, the conversion of NTP to NDP (1), NDP-sugar synthesis (2), and sugar acceptor glycosylation (3) may be performed separately or together in one or more pots and may be, separately or together, performed for a duration of time between about 1 hour and 1 week, such as, for example, between 30 minutes and 1 hours, between 1 hour and 4 hours, between 4 hours and 6 hours, between 6 hours and 12 hours, between 12 hours and 24 hours, between 1 day and 2 days, between 2 days and 3 days, 3 days and 4 days, between 4 days and 5 days, between 6 days and 7 days. In embodiments, the conversion(s) can be performed for between 0.1 hours and 96 hours such as, for example, between 0.1 hours and 23.9 hours, between 30 minutes and 9 hours, between 1 hour and 8.5 hours, between 2 hours and 8 hours, between 3 hours and 7.5 hours, between 4 hours and 7 hours, and between 5 hours and 6.5 hours. For example, the conversion(s) can be performed for about 6 hours. In embodiments, the conversion(s) can be performed for between 0.1 hours and 96 hours such as, for example, between 0.1 hours and 23.9 hours, between 30 minutes and 20 hours, between 1 hour and 18 hours, between 2 hours and 16 hours, between 3 hours and 14 hours, between 4 hours and 12 hours, and between 5 hours and 11 hours. For example, the conversion(s) can be performed for about 9 hours. In embodiments, the conversion(s) can be performed for between 0.1 hours and 96 hours such as, for example, between 1 hour and 72 hours, between 2 hours and 36 hours, between 3 hours and 30 hours, between 4 hours and 29 hours, between 5 hours and 28 hours, between 10 hours and 27 hours, between 15 hours and 26 hours, and between 20 hours and 25 hours. For example, the conversion(s) can be performed for about 24 hours.

[0112] In embodiments, the conversion of NTP to NDP (1), NDP-sugar synthesis (2), and sugar acceptor glycosylation (3) may be performed separately or together in one or more pots and may be, separately or together, performed at any temperature between 2°C and 112°C or between 4°C and 95°C, such as, for example, between 10°C to 20°C, between 20°C to 30°C, between 30°C to 40°C, between 40°C to 50°C, between 40°C and 70°C, between 50°C to 60°C, between 60°C to 70°C, or between 70°C to 80°C. For example, when all three conversions are performed in a one pot reaction, they may be performed at 60°C. In embodiments, the conversions can be performed under isothermal conditions. In embodiments, one or more of the conversions can be performed at different temperatures. For instance, if two or more potsare used to perform the conversions, each of the pots can be held at a different temperature. For instance, the NTP to NDP conversion (1) and NDP-sugar synthesis (2) may be performed in a first pot at a first temperature and NDP-sugar synthesis (2) and sugar acceptor glycosylation (3) may be performed in a second pot at a second, different temperature. If the conversions are performed in a one pot reaction, the temperature of the pot can be changed during the time course of the conversions For instance, the pot may be maintained at a first temperature for the first e g., 2 hours of the reaction and the temperature of the pot may be increased to a second temperature for the remaining e.g., 10 hours of the reaction. In embodiments, food safety is a concern, and the method is performed at a temperature of between 50°C to 75°C. In embodiments, it can be advantageous to apply pressure greater than atmospheric pressure to the bio-converter or unit used for the reaction. For instance, when the temperature of the conversions is above 80°C, additional pressure can be applied. For example, nitrogen gas can be applied to the reaction chamber at 1 psi gauge pressure.

[0113] In embodiments, the reaction mediums of the present disclosure can have a pH between 4 and 10 such as, for example, between about 4.5 and about 9, between about 5 and about 8, between about 5.5 and about 7, and between about 6 and about 6.5. In embodiments, the pH of a reaction medium can be modified to accelerate NTP activation via non-enzymatic conversion.

[0114] In embodiments, the conversions of the present disclosure are performed in one or more vessels, or “pots,” configured to hold a volume of between 1 ml and 1,000,000 liters. For instance, the conversions may be performed in one or more vessels each configured to hold a volume of about 2 ml, about 10 ml, about 25 ml, about 50 ml, about 100 ml, about 500 ml, about 1 L, about 2 L, about 3 L, about 4 L, about 5 L, about 10 L, about 15 L, about 20 L, about 25 L, about 30 L, about 45 L, about 50 L, about 60 L, about 70 L, about 80 L, about 90 L, about 100 L, about 200 L, about 300 L, about 400 L, about 500 L, about 600 L, about 700 L, about 800 L, about 900 L, about 1000 L, about 1500 L, about 2000 L, about 2500 L, about 3000 L, about 3500 L, about 4000 L, about 4500 L, about 5000 L, about 6000 L, about 7000 L, about 8000 L, about 9000 L, about 10,000 L, about 20,000 L, about 30,000 L, about 40,000 L, about 50,000 L, about 60,000 L, about 70,000 L, about 80,000 L, about 90,000 L, about 100,000 L, about 200,000 L, about 300,000 L, about 400,000 L, about 500,000 L, about 600,000 L, about 700,000 L, about 800,000 L, about 900,000 L, and / or about 1,000,000 L.

[0115] In embodiments, the reaction medium for performing the methods of the present disclosure may comprise one or more reaction mediums. For instance, as discussed elsewhere herein, the methods of the present disclosure may be performed simultaneously or sequentiallyin one reaction medium or separately and / or sequentially in multiple reaction mediums. The one or more reaction mediums may contain at least one reaction component (e.g., sucrose). Reaction mediums comprising at least one reaction component may also be referred to herein as a composition of the present disclosure. For example, a reaction medium comprising NTP may be referred to as a composition.

[0116] In embodiments, the reaction medium for performing the methods of the present disclosure can be aqueous. In embodiments, the aqueous reaction medium can include a buffer comprising one or more of acetate, citrate, lactate, succinate, or phosphate and the like to provide suitable buffering capacity. The buffer may further comprise one or more cations such as sodium, ammonium, potassium, or magnesium, and the like. In embodiments, the buffer may be an acetate buffer, a succinate buffer, a citrate buffer, HEPES, or a phosphate buffer. For example, the aqueous reaction medium may comprise phosphate buffer. The reaction medium can also be, alternatively, an organic solvent.Enzymatic glycosylation of steviol glycosides within a reaction medium comprising ATP

[0117] In embodiments, the present disclosure relates to methods and compositions for glycosylation of a steviol glycoside, the method comprising converting ATP to ADP, converting the ADP to ADP -glucose by contacting a SuSy with the ADP and sucrose, and converting the steviol glycoside to a glycosylated steviol glycoside by contacting the steviol glycoside with the ADP-glucose and at least one glycosyltransferase selected from the group consisting of P-l,2-glycosyltransferase, P-1, 3 -glycosyltransferase, or a combination thereof. In embodiments, the method is performed in a reaction medium comprising ATP, SuSy, at least one glycosyltransferase, at least one steviol glycoside, and sucrose.

[0118] In embodiments, the conversion of ATP to ADP (1) may be performed by an enzyme, such as a hydrolase, a phosphatase, or a kinase, and combinations thereof. In embodiments, the reaction medium comprises ATP, SuSy, at least one glycosyltransferase, at least one steviol glycoside, sucrose, and a hydrolase. In embodiments, the hydrolase comprises apyrase. The hydrolase may be provided as part of an enzyme preparation of a partially purified or a highly purified glycosyltransferase and / or SuSy, and / or the hydrolase may be provided exogenously to the reaction medium. In embodiments, the reaction medium comprises ATP, SuSy, at least one glycosyltransferase, at least one steviol glycoside, sucrose, and a phosphatase. In embodiments, the phosphatase comprises phytase. The phosphatase may be provided as part of an enzyme preparation of a partially purified or a highly purified glycosyltransferase and / or SuSy, and / or the phosphatase may be provided exogenously to the reaction medium. In embodiments, the reaction medium comprises ATP, SuSy, at least one glycosyltransferase, atleast one steviol glycoside, sucrose, a kinase, and a phosphate acceptor. In embodiments, the phosphate acceptor is a kinase substrate comprising one or more of acetate, glycerol, fructose, glucose, pyruvate, succinate, or AMP, and combinations thereof. In embodiments, the kinase may be provided to the reaction medium as part of an enzyme preparation of a partially purified or a highly purified glycosyltransferase and / or SuSy, and / or the kinase may be provided exogenously to the reaction medium. In embodiments, the kinase may be one or more of acetate kinase, glycerol kinase, fructokinase, glucokinase, pyruvate kinase, acetyl-CoA carboxylase, or adenylate kinase, and combinations thereof. A non-exhaustive listing of exemplary kinases and kinase acceptors is provided in Table 8. In embodiments, reaction mediums comprising ATP, SuSy, at least one glycosyltransferase, sucrose, at least one steviol glycoside, and one or more of a hydrolase, a phosphatase, or a kinase and phosphate acceptor define compositions of the present disclosure.

[0119] In embodiments, the glycosyltransferase of the method for glycosylation of a steviol glycoside may be a natural glycosyltransferase or an engineered glycosyltransferase. In embodiments, the sugar donor may be exogenous and / or may be present in a stevia leaf extract. In embodiments, the ATP may be exogenous and / or may be present in a stevia leaf extract. In embodiments, the sucrose synthase of the method for glycosylation of a steviol glycoside may be a natural sucrose synthase or an engineered sucrose synthase. In embodiments, the sucrose may be exogenous and / or may be present in a stevia leaf extract.

[0120] In embodiments, the engineered or natural glycosyltransferase (e.g., B 12GT, B13GT) and / or the engineered or natural ADP-sugar synthase (e.g. SuSy) may be a partially purified enzyme. In embodiments, the sucrose synthase may be an engineered sucrose synthase selected from the engineered sucrose synthases disclosed in International Patent Application No. PCT / US2022 / 016820, filed February 17, 2022, which is incorporated by reference herein in its entirety for all purposes. For instance, the sucrose synthase may have an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, and 100% identical to an amino acid sequence of the engineered sucrose synthases disclosed in International Patent Application No. PCT / US2022 / 016820. For example, the sucrose synthase may have an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, and 100% identical to the amino acid sequence of either SEQ ID NO: 885 or SEQ ID NO: 888, as disclosed in International Patent Application No.PCT / US2022 / 016820. In embodiments, the glycosyltransferases may be engineered glycosyltransferases selected from the engineered glycosyltransferase (e.g., B12GT, B13GT) disclosed in International Patent Application No. PCT / US2023 / 073344, filed September 1, 2023, and International Patent Application No. PCT / US2024 / 032488, filed June 5, 2024, as discussed above, each of which is incorporated by reference herein in its entirety for all purposes.

[0121] In embodiments, the method for glycosylating a steviol glycoside comprises converting stevioside and Reb A to Reb E, Reb D, Reb I, Reb AM, and Reb M. To this end, the method further comprises converting ATP to ADP (1), contacting the ADP with a SuSy and sucrose to generate ADP-glucose (2), and contacting a glycosyltransferase with each of the stevioside and the Reb A and the ADP-glucose to generate the Reb E, Reb D, Reb I, Reb AM, and Reb M (3). For instance, a glucose from ADP-glucose can be transferred to stevioside to produce Reb A. The ADP can be recycled via SuSy and sucrose (2) to replenish ADP-glucose. Separately, an additional glucose from ADP-glucose can be transferred to the Reb A to produce Reb D (3). The ADP can again be recycled via the SuSy and sucrose (2) to replenish ADP- glucose. An additional glucose from ADP-glucose can be transferred to the Reb D to produce Reb M (3). This process is shown in FIG. 4.

[0122] The enzymes used in the ATP to ADP conversion (1), when enzymatically-mediated, the stabilization of ADP in the reaction medium (2), and the glycosylation of the sugar acceptor (3), as shown in FIG. IB, which may include B12GT, sucrose synthase, and / or B13GT polypeptides, may be prepared by expression in a host microorganism. Suitable host microorganisms include, but are not limited to, E. coll, Saccharomyces sp., Aspergillus sp., Pichia sp., Bacillus sp. For example, the enzymes may be expressed in / / coli. For example, the enzymes may be expressed in Pichia pastoris. In embodiments, the enzymes are prepared by cell-free expression. The enzymes may be expressed intracellularly or extracellularly.

[0123] The B12GT, sucrose synthase, and / or B13GT polypeptides can be provided in any suitable form, including free, immobilized, as a whole cell system, for example as a living fermentative microbial cell, or as dead and stabilized microbial cell, or in the form of a cell lysate.

[0124] The degree of purity of the polypeptides may vary, e.g., they may be provided as a crude, partially purified, or highly-purified enzyme preparation(s). In embodiments, the B12GT, sucrose synthase, and / or B13GT polypeptide is free. In embodiments, the B12GT, sucrose synthase, and / or B13GT polypeptide is immobilized to a solid support, for example onan inorganic or organic support. The solid support is derivatized cellulose, glass, ceramic, methacrylate, styrene, acrylic, a metal oxide, or a membrane. In some embodiments, the B12GT, SuSy, and / or B13GT polypeptide is immobilized to the solid support by covalent attachment, adsorption, cross-linking, entrapment, or encapsulation.

[0125] In embodiments, partially purified enzyme preparations may comprise the enzyme ADP -glucose phosphorylase (E C. 2.7.7). This can lead to unwanted loss of ADP-glucose by the following reaction,ADP-glucose + PO4 -- glucose- 1 -phosphate + ADP

[0126] The ADP-glucose can then be regenerated, for example for the case of sucrose Sucrose + ADP <-- fructose + ADP-glucose Sucrose synthase

[0127] Overall, this example leads to a net reactionSucrose + PO4glucose-1 -phosphate + fructose

[0128] Thus, in systems with a significant amount of phosphate as buffer, this can lead to a reduction in available sucrose for the desired reaction. This results in a small penalty that can be addressed by increasing the amount of sucrose or substituting buffers. In embodiments where phosphate buffer is used in the reaction medium, the molar concentration of phosphate present or added should not be more than 10% of the molar concentration of sucrose present in the reaction medium.

[0129] The at least one steviol glycoside added to the reaction medium serves as a substrate for the production of a target steviol glycoside, or glycosylated steviol glycoside, as described herein. The target steviol glycoside differs chemically from its corresponding substrate steviol glycoside by the addition of one or more glucose units. As glycosylation progresses, the steviol glycoside substrate in the reaction medium may become depleted or entirely absent as glucose units are added to its structure. In this case, a steviol glycoside substrate (e g., stevioside) that is glycosylated during the reaction may become a substrate (e.g., Reb A) for a subsequent glycosylation event within the same reaction.

[0130] To this end, the at least one steviol glycoside may be at least one listed in Table 2. In embodiments, the at least one steviol glycoside is selected from the group consisting of steviol, steviol-13-O-glucoside, steviol-19-O-glucoside, rubusoside, steviol- 1,2-bioside, steviol-1,3- bioside, rubusoside, dulcoside B, dulcoside A, rebaudioside B, rebaudioside G, stevioside, rebaudioside C, rebaudioside F, rebaudioside A, rebaudioside I, rebaudioside E, rebaudioside E2, rebaudioside H, rebaudioside L, rebaudioside K, rebaudioside J, rebaudioside AM, rebaudioside M, rebaudioside D, rebaudioside N, rebaudioside O, rebaudioside Q, rebaudiosideX, an isomer thereof, a synthetic steviol glycoside or combinations thereof. In embodiments, the at least one steviol glycoside comprises stevioside and Reb A.

[0131] The at least one steviol glycoside according to the methods described herein may be synthetic or purified (partially or entirely), commercially available or prepared. In embodiments, the at least one steviol glycoside may be obtained from purification of Stevia rebaudiana plant material (e g., leaves), may be a commercially available stevia extract brought into solution with a solvent, and / or a commercially available mixture of steviol glycosides brought into solution with a solvent. Other suitable steviol glycosides include byproducts of processes to isolate and purify steviol glycosides.

[0132] In embodiments, the at least one steviol glycoside comprises a purified steviol glycoside. For example, the at least one steviol glycoside may comprise greater than about 50%, greater than about 60%, greater than about 70%, greater than about 80%, greater than about 85%, greater than about 90%, greater than about 91%, greater than about 92%, greater than about 93%, greater than about 94%, greater than about 95%, greater than about 96%, greater than about 97%, greater than about 98%, greater than about 99%, or greater than about 99.6% of at least one steviol glycoside by weight on an anhydrous basis.

[0133] In embodiments, the at least one steviol glycoside comprises a partially purified steviol glycoside composition. For example, the steviol glycoside comprises greater than about 0.5%, greater than about 1%, greater than about 2%, greater than about 3%, greater than about 4%, greater than about 5%, greater than about 10%, greater than about 20%, greater than about 30%, greater than about 40%, or greater than about 50%, of at least one steviol glycoside by weight on an anhydrous basis.

[0134] In embodiments, the at least one steviol glycoside is purified rebaudioside A, or isomers thereof. The at least one steviol glycoside may contain greater than 99% rebaudioside A, or isomers thereof, by weight on an anhydrous basis. In embodiments, the at least one steviol glycoside comprises partially purified rebaudioside A. The at least one steviol glycoside may contain greater than about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% rebaudioside A by weight on an anhydrous basis.

[0135] In embodiments, the at least one steviol glycoside comprises purified stevioside, or isomers thereof. For example, the at least one steviol glycoside may contain greater than 99% stevioside, or isomers thereof, by weight on an anhydrous basis. In embodiments, the at least one steviol glycoside comprises partially purified stevioside. For example, the at least one steviol glycoside may contain greater than about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% stevioside by weight on an anhydrous basis.

[0136] In embodiments, the at least one steviol glycoside is a combination of stevioside and rebaudioside A. The at least one steviol glycoside may contain greater than about 5% stevioside and greater than about 5% Reb A, greater than about 10% stevioside and greater than about 10% Reb A, greater than about 20% stevioside and greater than about 20% Reb A, greater than about 30% stevioside and greater than about 30% Reb A, greater than about 40% stevioside and greater than about 40% Reb A, greater than about 45% stevioside and greater than about 45% Reb A, greater than about 40% stevioside and greater than about 50% Reb A, greater than about 30% stevioside and greater than about 60% Reb A, greater than about 20% stevioside and greater than about 70% Reb A, greater than about 10% stevioside and greater than about 80% Reb A, greater than about 5% stevioside and greater than about 90% Reb A, greater than about 50% stevioside and greater than about 40% Reb A, greater than about 60% stevioside and greater than about 30% Reb A, greater than about 70% stevioside and greater than about 20% Reb A, greater than about 80% stevioside and greater than about 10% Reb A, or greater than about 90% stevioside and greater than about 5% Reb A by weight on an anhydrous basis. The at least one steviol glycoside may contain greater than about 10% stevioside and greater than about 60% Reb A, greater than about 20% stevioside and greater than about 60% Reb A, and greater than about 30% stevioside and greater than about 60% Reb A by weight on an anhydrous basis.

[0137] In embodiments, the at least one steviol glycoside may be derived from stevia leaf extract. The stevia leaf extract may be produced by a process comprising one or more steps of extracting, clarifying, and concentrating. In embodiments, the stevia leaf extract is extracted by one or more of a batch parabolic trough with a helical mixer, a batch rotating cylindrical extractor, a batch percolation extractor, horizontal continuous moving belt extractor, a horizontal counter current tubular screw extractor, a vertical counter current tubular screw extractor, a segmented rotating basket extractor, and a pressurized condensing steam extractor. In embodiments, concentrating may comprise membrane processing such as nanofiltration, evaporation, and adsorption of one or more steviol glycosides onto a solid adsorbent, elution with an alcohol, and removal of the alcohol to give a concentrated leaf extract. In embodiments, clarifying comprises one of more of thermal coagulation of proteins and impurities, diatomaceous earth treatment, ion exchange, chemical coagulation using calcium carbonate and / or ferric chloride. In embodiments, RA50, a stevia leaf extract purified to contain greater than 50% Reb A, may be used as the at least one steviol glycoside. In embodiments, RA50 is used at a concentration between about 1 and 800 g / L; for example, about 100 g / L. RA50 may be used at a concentration of about 60 g / L. In aspects, the range of RA50 is about 60 g / L toabout 100 g / L. In embodiments, RA60, stevia leaf extract purified to contain greater than 60% Reb A, is used as the at least one steviol glycoside. In embodiments, RA60 is used at a concentration between about 1 and 800 g / L. For example, RA60 may be used at a concentration of about 100 g / L. For example, RA60 may be used at a concentration of about 60 g / L. In aspects, the range of RA60 is about 60 g / L to about 100 g / L.

[0138] In embodiments, the sugar donor is sucrose and may be provided in excess For example, the sucrose may be provided in the reaction medium at 584 mM (200 g / L) relative to the steviol glycoside (e.g., at 80 g / L) and ATP (e.g., at 1 mM or 5 mM level). This drives the formation of ADP-glucose, thereby minimizing formation of AMP. In embodiments, a feed of 0.1 mM ADP and 1 mM ATP may be provided.

[0139] In embodiments, the nucleoside diphosphate cofactor can be a non-UDP nucleoside diphosphate (e.g., ADP-glucose, GDP-glucose, CDP-glucose, or TDP-glucose). In embodiments, the nucleoside diphosphate may be ADP, which may be converted from ATP available in the reaction medium. In embodiments, the method may be performed with a reaction medium comprising ATP at a concentration of between about 0.01 mM and about 10 mM, based on a final volume of the reaction medium, between about 0.01 mM and about 0.05 mM, between about 0.05 mM and about 0.1 mM, between about 0.1 mM and about 0.5 mM, between about 0.5 mM and about 1 mM, between about 1 mM and about 5 mM, or between about 5 mM and about 10 mM. In embodiments, the ATP is converted to ADP to produce an ADP concentration with the reaction medium of between about 0.01 and 10 mM, such as, for example, between 0.01 mM and 0.05 mM, between 0.05 mM and 0.1 mM, between 0.1 mM and 0.5 mM, between 0.5 mM and 1 mM, between 1 mM and 5 mM, or between 5 mM and 10 mM. For example, ATP may be used at a concentration of 0.5 mM and may be converted to ADP at a concentration of about 0.5 mM.

[0140] In embodiments, the molar ratio of ATP to at least one steviol glycoside in the reaction medium may range from about 0.00045: 1 to about 0.10:1. For example, the molar ratio of ATP to at least one steviol glycoside may be about 0.010: 1. In embodiments, the molar ratio of ATP to glycosyltransferase (e.g., B12GT, B13GT) in the reaction medium may range from about 0.55: 1 to about 5500: 1. In embodiments, the molar ratio of ATP:SG in the reaction medium is between about 0.00045:1 and about 0.10: 1. In embodiments, the molar ratio of ATP:GT in the reaction medium is between about 0.55 and about 5500. In embodiments, the molar ratio of ATP: sucrose in the reaction medium is between about 0.0001 : 1 and about 0.01 : 1. In embodiments, the glycosylated steviol glycoside comprises one or more additional glucose units than the steviol glycoside (as a sugar acceptor).

[0141] In embodiments, the concentration of the sucrose in the reaction medium may be between about 10 mM and about 3 M, based on the final volume of the reaction medium. For instance, the sucrose concentration may be greater than about 10 mM, greater than about 50 mM, greater than about 100 mM, greater than about 250 mM, greater than about 500 mM, greater than about 1 M, greater than about 1.5 M, greater than about 2 M, and greater than about 2.5 M. In embodiments, the sucrose concentration in the reaction medium may be between about 125 mM and about 2.5 M, between about 150 mM and about 2 M, or between about 175 mM and about 1.5 M. For example, the sucrose concentration may be about 365 mM or about 1.17 M.

[0142] In embodiments, the B12GT, sucrose synthase, and / or B13GT polypeptides used in the methods disclosed herein (e.g., for glycosylating at least one steviol glycoside) may be free or as immobilized to a solid support. When free, the B12GT, sucrose synthase, and / or B13GT polypeptides can be provided at a concentration of between about 100 mg / 1 and about 20 g / 1, between about 750 mg / 1 and about 9 g / 1, between about 1 g / 1 and about 8 g / 1, between about 2 g / 1 and about 7 g / 1, and / or between about 3 g / 1 and about 6 g / 1. When immobilized, the B 12GT, sucrose synthase, and / or B 13GT polypeptides can be provided at a concentration of between about 5 g / 1 and about 100 g / 1, between about 10 g / 1 and about 90 g / 1, between about 20 g / 1 and about 80 g / 1, between about 30 g / 1 and about 70 g / 1, and / or between about 40 g / 1 and about 60 g / 1.

[0143] In embodiments, and as discussed in detail above with respect to NTP, the conversions of the method disclosed herein may be performed simultaneously or in stages. For instance, the conversion of ATP to ADP (1) may be performed prior to ADP-glucose synthesis (2) and glycosylation of the at least one steviol glycoside (3). In embodiments, the reagents present during (1) and (2) (e.g., ATP, SuSy, sucrose) may define a first composition and the reagents present during (2) and (3) (e.g., SuSy, sucrose, at least one steviol glycoside, glycosyltransferase) may define a second composition, according to embodiments. In embodiments, ATP to ADP conversion (1) and ADP-glucose synthesis (2) may be performed together in a first pot. Subsequently, ADP-glucose synthesis (2) and glycosylation of the at least one steviol glycoside (3) may be performed simultaneously in a second pot. In embodiments, the reagents within the first pot and the second pot define respective compositions of the present disclosure.

[0144] In embodiments, ATP to ADP conversion (1), ADP-glucose synthesis (2), and glycosylation of the at least one steviol glycoside (3) may be performed simultaneously in a 1- pot reaction. In embodiments, when (1), (2), and (3) are performed in a 1-pot reaction, allreaction components, including ATP, SuSy, sucrose, at least one steviol glycoside, and at least one glycosyltransferase are added at the beginning of the 1-pot reaction. In embodiments, only a portion of the reaction components are added at the beginning of the 1-pot reaction. For example, only ATP, SuSy, and sucrose are added at the beginning of the reaction, and / or only a fraction of a total amount of at least one steviol glycoside is added at the beginning of the reaction. The remaining portion reaction components and / or fraction of the reaction components may be added to the reaction in bulk, continuously, or as fed-batch. In embodiments, any of the reaction components used for (2) or (3), e.g. SuSy, sucrose, the at least one glycosyltransferase, the at least one steviol glycoside, can be absent at the start of the reaction. Each reaction component can be added at any time up to when a respective reaction is started. The composition of the reaction components at the beginning of, during, and at the end of the 1-pot reaction may define compositions of the present disclosure.

[0145] In embodiments, the conversion of ATP to ADP (1), ADP -glucose synthesis (2), and glycosylation of the at least one steviol glycoside (3) can be performed sequentially. In embodiments, (1) can be performed first, independently, in a first reaction medium. In embodiments, the first reaction medium from (1) can be optionally purified to isolate NDP. In embodiments, the first reaction medium from (1) or isolated NDP from the first reaction medium can be combined with a second reaction medium to perform (2) and, optionally, (1). In embodiments, the second reaction medium comprising (1) and (2) can be combined with a third reaction medium to perform (2) and (3) and, optionally, (1). In embodiments, reaction components used for (2) or (3) can be added to the reaction in the first reaction medium used to perform (1). In embodiments, reaction components used for (3) can be added to the reaction in the second reaction medium used to perform (1) and (2). In embodiments, reaction components used for (3) can be added to the reaction in the third reaction medium. In embodiments, only a portion of the reaction components and / or only a fraction of each reaction component are added to respective reaction mediums at the beginning of the reaction or at the beginning of each of (1), (2), or (3).. The remaining portion and / or fraction of each of the reaction components can be added to the reaction in bulk, continuously, or as fed-batch. The composition of each reaction medium at the beginning of, during, and at the end of the reaction may define compositions of the present disclosure.

[0146] In embodiments, the conversion of ATP to ADP (1), ADP -glucose synthesis (2), and glycosylation of the at least one steviol glycoside (3) can be performed sequentially and / or combinations of two of (1), (2), and (3) can be performed sequentially. In embodiments, (1) and (2) can be performed simultaneously in a first reaction medium. In embodiments, the firstreaction medium used to perform (1) and (2) can be combined with a second reaction medium to perform (1), (2), and (3). In embodiments, only a portion of the reaction components and / or only a fraction of each reaction component are added to respective reaction mediums at the beginning of the reaction or at the beginning of each of (1), (2), or (3)f. The remaining portion and / or fraction of each of the reaction components can be added to the reaction in bulk, continuously, or as fed-batch. In embodiments, reaction components used for (3) can be present in the first reaction medium used to perform step (1) and (2). The composition of each reaction medium at the beginning of, during, and at the end of the reaction may define compositions of the present disclosure.

[0147] In embodiments, the conversion of ATP to ADP (1), ADP -glucose synthesis (2), and glycosylation of the at least one steviol glycoside (3) can be performed sequentially and / or combinations of two of (1), (2), and (3) can be performed sequentially. In embodiments, (1) can be performed first, independently, in a first reaction medium. In embodiments, the first reaction medium from (1) can be optionally purified to isolate NDP. In embodiments, the first reaction medium or purified reaction medium can be combined with a second reaction medium to perform (2) and (3), and optionally (1). In embodiments, only a portion of the reaction components and / or only a fraction of each reaction component are added to respective reaction mediums at the beginning of the reaction or at the beginning of each of (1), (2), or (3).. The remaining portion and / or fraction of each of the reaction components can be added to the reaction in bulk, continuously, or as fed-batch. In embodiments, reaction components used for (2) or (3) can be present in the first reaction medium used to perform (1). The composition of each reaction medium at the beginning of, during, and at the end of the reaction may define compositions of the present disclosure.

[0148] In embodiments, reaction components, including ATP, sucrose, SuSy, at least one steviol glycoside, and at least one glycosyltransferase, can be added at the beginning of the reaction and / or at the beginning of respective ones or combinations of (1), (2), and (3), or only a portion and / or fraction of the reaction components can be added at the beginning of the reaction and / or at the beginning of respective ones or combinations of (1), (2), and (3). For instance, the ATP may be added at the beginning of (1) and / or may be added during (1) such that between about 0.1 millimoles and about 10 millimoles of ATP is added in total, per liter of a final volume of the reaction medium, to the reaction. For example, the ATP may be added at between about 0.2 millimoles and about 10 millimoles, between about .25 millimoles and about 2.5 millimoles, and / or about 0.5 millimoles and about 1 millimole per liter of the final volume of the reaction medium. When at least one steviol glycoside is added to the reaction atthe beginning of and / or during (3), the at least one steviol glycoside may be added such that between about 5 millimoles and about 200 millimoles of the at least one steviol glycoside is added in total, per liter of a final volume of the reaction medium, to the reaction. For example, the at least one steviol glycoside may be added at between about 10 millimoles and about 150 millimoles, between about 25 millimoles and about 100 millimoles, and / or about 50 millimoles and about 75 millimoles per liter of the final volume of the reaction medium.

[0149] In embodiments, the conversion of ATP to ADP (1), ADP-glucose synthesis (2), and glycosylation of the at least one steviol glycoside (3) may be performed separately or together in one or more pots and may be, separately or together, performed at any temperature between 2°C and 112°C or between 4°C and 95°C, such as, for example, between 10°C to 20°C, between 20°C to 30°C, between 30°C to 40°C, between 40°C to 50°C, between 50°C to 60°C, between 60°C to 70°C, or between 70°C to 80°C. For example, when all three conversions are performed in a one pot reaction, they may be performed at 60°C. In embodiments, the conversions can be performed under isothermal conditions. In embodiments, one or more of the conversions can be performed at different temperatures. For instance, if two or more pots are used to perform the conversions, each of the pots can be held at a different temperature. For instance, the ATP to ADP conversion (1) and ADP-glucose synthesis (2) may be performed in a first pot at a first temperature and ADP-glucose synthesis (2) and glycosylation of at least one steviol glycoside (3) may be performed in a second pot, at a second, different temperature. If the conversions are performed in a one pot reaction, the temperature of the pot can be changed during the time course of the conversions. For instance, the pot may be maintained at a first temperature for the first e.g., 2 hours of the reaction and the temperature of the pot may be increased to a second temperature for the remaining e.g., 10 hours of the reaction. In embodiments, food safety is a concern and the method is performed at a temperature of between 50°C to 75°C. In embodiments, it can be advantageous to apply pressure greater than atmospheric pressure to the bio-converter or unit used for the reaction. For instance, when the temperature of the conversions is above 80°C, additional pressure can be applied. For example, nitrogen gas can be applied to the reaction chamber at 1 psi gauge pressure.

[0150] In embodiments, the conversion of ATP to ADP (1), ADP-glucose synthesis (2), and glycosylation of the at least one steviol glycoside (3) may be performed separately or together in one or more pots and may be, separately or together, performed for a duration of time between about 1 hour and 1 week, such as, for example, between 30 minutes and 1 hours, between 1 hour and 4 hours, between 4 hours and 6 hours, between 6 hours and 12 hours, between 12 hours and 24 hours, between 1 day and 2 days, between 2 days and 3 days, 3 daysand 4 days, between 4 days and 5 days, between 6 days and 7 days. In embodiments, the conversion(s) can be performed for between 0.1 hours and 96 hours such as, for example, between 0.1 hours and 23.9 hours, between 30 minutes and 9 hours, between 1 hour and 8.5 hours, between 2 hours and 8 hours, between 3 hours and 7.5 hours, between 4 hours and 7 hours, and between 5 hours and 6.5 hours. For example, the conversion(s) can be performed for about 6 hours. In embodiments, the conversion(s) can be performed for between 0.1 hours and 96 hours such as, for example, between 0.1 hours and 23.9 hours, between 30 minutes and 20 hours, between 1 hour and 18 hours, between 2 hours and 16 hours, between 3 hours and 14 hours, between 4 hours and 12 hours, and between 5 hours and 11 hours. For example, the conversion(s)can be performed for about 9 hours. In embodiments, the conversion(s) can be performed for between 0.1 hours and 96 hours such as, for example, between 1 hour and 72 hours, between 2 hours and 36 hours, between 3 hours and 30 hours, between 4 hours and 29 hours, between 5 hours and 28 hours, between 10 hours and 27 hours, between 15 hours and 26 hours, and between 20 hours and 25 hours. For example, the conversion(s) can be performed for about 24 hours.

[0151] In embodiments, the reaction medium can have a pH between 4 and 10 such as, for example, between about 4.5 and about 9, between about 5 and about 8, between about 5.5 and about 7, and between about 6 and about 6.5. In embodiments, the pH of the reaction medium can be modified to accelerate ATP activation via chemical conversion.

[0152] In embodiments, the reaction rates of ATP to ADP conversion (1), ADP-glucose synthesis (2), and glycosylation of the at least one steviol glycoside (3), as shown in FIG. IB, are controlled such that the flux through the system dominates any possible off-target reactions, ensuring successful glycosylation of the sugar acceptor. In embodiments, the reaction medium comprises ATP, sucrose synthase, a glycosyltransferase, sucrose, at least one steviol glycoside, and a phosphatase. In embodiments, the phosphatase comprises phytase.

[0153] Theoretical calculations were performed to illustrate an example of the target potential reaction rates in the early stages of the reaction. This potential reaction rate is determined by the dosage of enzymes and their activity. Consider, in the early stages of the reaction, if phytase is present at 20 mU / ml, greater than 1 mM / hr of ATP is converted to ADP. If phytase is present at a lower concentration of 2 mU / ml, 0.65 mM / hr of ATP is converted to ADP. If phytase is present at 0.2 mU / ml, 0.15 mM / hr of ATP is converted to ADP. For example, the target rate of glycosylation of at least one steviol glycoside at the early stages of the reaction may be 30 mM / hr (which may be catalyzed by B12GT and B13GT). The potential reaction rate of ADP stabilization at the early stages of the reaction is greater than ten times the glycosylation rate,e.g., 300 mM / hr. Under these conditions, increased phytase concentrations can lead to increased degradation of ADP to AMP. For instance, when phytase is present at 20 mU / ml, the rate of conversion of ADP to AMP is 0.86 mM / hr. When phytase is present at 2 mU / ml, O i l mM / hr of ADP is converted to AMP. When phytase is present at 0.2 mU / ml, 0.01 mM / hr of ADP is converted to AMP.

[0154] Additional theoretical calculations were performed to illustrate an example of the actual potential reaction rates in the early stages of the reaction. The actual reaction rate is determined by the dosage of the enzymes and their activity, as well as the levels of available substrates (i.e., steviol glycosides). In the early stages of the reaction, ATP activation occurs at a rate of 0.65 mM / hr, ADP-glucose synthesis occurs at a rate of greater than 30 mM / hr, glycosylation of at least one steviol glycoside occurs at a rate of 30 mM / hr, catalyzed by B12GT and B13GT, and minimal ADP to AMP degradation occurs (i.e., <0.01 mM / hr). At later stages of the reaction, ATP activation is completed, ADP-glucose synthesis occurs at a rate of greater than 5 mM / hr, glycosylation of at least one steviol glycoside occurs at a rate of less than about 5 mM / hr, catalyzed by B12GT and B13GT, and minimal ADP to AMP degradation occurs (i.e., <0.01 mM / hr). Notably, due to the accumulation of fructose, the SuSy (acting as a P-l,4-glycosyltransferase) now exhibits a reverse reaction rate — sucrose is reformed at a rate of greater than 100 mM / hr.

[0155] In embodiments, the conversions of the present disclosure are performed in one or more vessels, or “pots,” configured to hold a volume of between 1 ml and 1,000,000 liters. For instance, the conversions may be performed in one or more vessels each configured to hold a volume of about 2 ml, about 10 ml, about 25 ml, about 50 ml, about 100 ml, about 500 ml, about 1 L, about 2 L, about 3 L, about 4 L, about 5 L, about 10 L, about 15 L, about 20 L, about 25 L, about 30 L, about 45 L, about 50 L, about 60 L, about 70 L, about 80 L, about 90 L, about 100 L, about 200 L, about 300 L, about 400 L, about 500 L, about 600 L, about 700 L, about 800 L, about 900 L, about 1000 L, about 1500 L, about 2000 L, about 2500 L, about 3000 L, about 3500 L, about 4000 L, about 4500 L, about 5000 L, about 6000 L, about 7000 L, about 8000 L, about 9000 L, about 10,000 L, about 20,000 L, about 30,000 L, about 40,000 L, about 50,000 L, about 60,000 L, about 70,000 L, about 80,000 L, about 90,000 L, about 100,000 L, about 200,000 L, about 300,000 L, about 400,000 L, about 500,000 L, about 600,000 L, about 700,000 L, about 800,000 L, about 900,000 L, and / or about 1,000,000 L.

[0156] In embodiments, the reaction medium for performing the methods of the present disclosure may comprise one or more reaction mediums. For instance, as discussed elsewhere herein, the methods of the present disclosure may be performed simultaneously or sequentiallyin one reaction medium or separately and / or sequentially in multiple reaction mediums. The one or more reaction mediums may contain at least one reaction component (e.g., sucrose). Reaction mediums comprising at least one reaction component may also be referred to herein as a composition of the present disclosure. For example, a reaction medium comprising NTP may be referred to as a composition.

[0157] In embodiments, the reaction medium for performing the methods of the present disclosure can be aqueous. In embodiments, the aqueous reaction medium can include a buffer comprising one or more of acetate, citrate, lactate, succinate, or phosphate and the like to provide suitable buffering capacity. The buffer may further comprise one or more cations such as sodium, ammonium, potassium, or magnesium, and the like. In embodiments, the buffer may be an acetate buffer, a succinate buffer, a citrate buffer, HEPES, or a phosphate buffer. For example, the aqueous reaction medium may comprise phosphate buffer. The reaction medium can also be, alternatively, an organic solvent.

[0158] In embodiments, the reaction can be monitored by suitable methods including, but not limited to, HPLC, LCMS, TLC, IR, or NMR.

[0159] In embodiments, the target steviol glycoside, or glycosylated steviol glycoside substate, can be any steviol glycoside. In embodiments, the target steviol glycoside comprises steviol-13-O-glucoside, steviol-19-O-glucoside, rubusoside, steviol- 1,2-bioside, steviol-1,3- bioside, rubusoside, dulcoside B, dulcoside A, rebaudioside B, rebaudioside G, stevioside, rebaudioside C, rebaudioside F, rebaudioside A, rebaudioside I, rebaudioside E, rebaudioside E2, rebaudioside H, rebaudioside L, rebaudioside K, rebaudioside J, rebaudioside AM, rebaudioside M, rebaudioside D, rebaudioside N, rebaudioside O, rebaudioside Q, rebaudioside X, a rebaudioside with 7 covalently attached glucose units (e.g. rebaudioside M plus 1 glucose unit), a synthetic steviol glycoside, an isomer thereof, and / or a steviol glycoside composition. In embodiments, the target steviol glycoside comprises rebaudioside E, or isomers thereof. In embodiments, the target steviol glycoside comprises rebaudioside D, or isomers thereof. In embodiments, the target steviol glycosides comprise Reb D and Reb E. In embodiments, the target steviol glycoside comprises Reb M or isomers thereof.

[0160] In embodiments, the conversion of Reb A to Reb D and / or Reb D isomer(s) is at least about 2% complete, as determined by any of the methods mentioned above. In embodiments, conversion of Reb A to Reb D and / or Reb D isomer(s) may be at least about 10% complete, at least about 20% complete, at least about 30% complete, at least about 40% complete, at least about 50% complete, at least about 60% complete, at least about 70% complete, at least about 80% complete, or at least about 90% complete. For example, the conversion of Reb A to RebD and / or Reb D isomer(s) may be at least about 95% complete. In embodiments, at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the Reb A in the reaction medium is converted to Reb D and / or Reb D isomer(s).

[0161] In embodiments, the conversion of stevioside to Reb E and / or Reb E isomer(s) is at least about 2% complete, as determined by any of the methods mentioned above. In embodiments, the conversion of stevioside to Reb E and / or Reb E isomer(s) may be at least about 10% complete, at least about 20% complete, at least about 30% complete, at least about 40% complete, at least about 50% complete, at least about 60% complete, at least about 70% complete, at least about 80% complete, or at least about 90% complete. For example, the conversion of stevioside to Reb E and / or Reb E isomer(s) may be at least about 95% complete. In embodiments, at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the stevioside in the reaction medium is converted to Reb E and / or Reb E isomer(s).

[0162] In embodiments, the conversion of stevioside and / or Reb A to Reb M and / or Reb M isomer(s) is at least about 2% complete, as determined by any of the methods mentioned above. In embodiments, the conversion of stevioside and / or Reb A to Reb M and / or Reb M isomer(s) is at least about 10% complete, at least about 20% complete, at least about 30% complete, at least about 40% complete, at least about 50% complete, at least about 60% complete, at least about 70% complete, at least about 80% complete, or at least about 90% complete. For example, the conversion of stevioside and / or Reb A to Reb M and / or Reb M isomer(s) may be at least about 95% complete. In embodiments, at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the steviol glycosides in the reaction medium is converted to Reb M and / or Reb M isomer(s).

[0163] The target steviol glycoside(s) can be in any polymorphic or amorphous form, including hydrates, solvates, anhydrous or combinations thereof.

[0164] Optionally, the method of the present disclosure further comprises separating the target steviol glycoside from the target composition. The target steviol glycoside(s) can be separated by any suitable method, such as, for example, crystallization, filtration, separation by membranes, centrifugation, extraction, chromatographic separation or a combination of such methods.

[0165] In embodiments, the separation of target steviol glycosides produces a composition comprising greater than about 80% by weight of the target steviol glycoside(s) on an anhydrous basis, i.e., a highly purified steviol glycoside composition. In embodiments, separation produces a composition comprising greater than about 0.5%, greater than about 1%, greater than about 2%, greater than about 3%, greater than about 4%, greater than about 5%, greaterthan about 10%, greater than about 20%, greater than about 30%, greater than about 40%, greater than about 50%, greater than about 60%, greater than about 70%, greater than about 80%, greater than about 85%, greater than about 90%, greater than about 91%, greater than about 92%, greater than about 93%, greater than about 94%, greater than about 95%, greater than about 96%, greater than about 97%, greater than about 98%, greater than about 99%, or greater than about 99.6% by weight of the target steviol glycoside(s). For example, the composition may comprise greater than about 95% by weight of the target steviol glycoside(s).

[0166] Purified target steviol glycoside(s) can be used in consumable products as a sweetener. Suitable consumer products include, but are not limited to, food, beverages, pharmaceutical compositions, tobacco products, nutraceutical compositions, oral hygiene compositions, and cosmetic compositions.EXAMPLESExample 1: Glycosylation of Steviol Glycosides with ADP

[0167] Polynucleotides encoding amino sequences of a sucrose synthase (SUSY), a P-1, 2- glycosyltransferase (B12GT), and a P-1, 3 -glycosyltransferase (Bl 3 GT) were synthesized (Twist Bioscience) and inserted into expression vectors. Each recombinant vector was used to transform an E. coli strain derived from W3110. Each recombinant E. coli strain was grown by fermentation to express the protein of interest. The cells from the fermentation were harvested by centrifugation. Partially purified enzymes were obtained by mechanically lysing the cells, adding flocculant and centrifuging to precipitate cellular debris, and filtering the remaining soluble enzymes using ultrafiltration and microfiltration. The recovered soluble enzymes were formulated with 50% glycerol for storage.

[0168] A mixture was prepared comprising 60 g / L of a mixed steviol glycoside RA60, 200 g / L (580 mM) sucrose, 50mM pH 6 phosphate buffer, 250mM sodium acetate, sucrose synthase (SuSy), P-l,2-glycosyltransferase (B12GT), and P-1, 3 -glycosyltransferase (B13GT). 1 mM ADP was added to the mixture to initiate the reaction. An additional negative control reaction was conducted where 1 mM AMP was used in place of the ADP. The reactions were carried out in a 2mL well mixed isothermal microreactor at 53 °C. After 10 hours the reactions were dissolved in DMSO to stop the reaction.

[0169] The conversion of stevioside and rebaudioside A to higher order steviol glycosides was measured using a gradient HPLC method with UV detection. Mobile phase A is 0.01% trifluoroacetic acid in water, while Mobile phase B is 0.01% trifluoroacetic acid in acetonitrile. An Agilent Poroshell 120 SB-C18 2.7 micron column (150 mm x 4.6 mm) was used at 45 °C.The gradient used is as follows: 0-8 minutes hold at 25% B, 8-13 minutes ramp 25-32% B, 13- 16 minutes hold at 32% B, 16-19 minutes ramp 32-40% B, 19-23 minutes hold at 40% B, 23- 23.5 minutes ramp 40-60% B, 23.5-25 minutes hold at 60% B, 25.1 minutes step down to 25%B, 25.1-30 minutes hold at 25% B. Samples are prepared with a 3:1 vol / vol dilution in DMSO, followed by further dilution to 500 ppm Reb using 30% acetonitrile in water as the diluent.

[0170] Table 9 shows the conversion of RA60 to Reb M over 10 hours with AMP and ADP. As expected, ADP is utilized to convert RA60 to Reb M, but AMP cannot be used to make Reb M. Therefore, the SuSy, B12GT, and B13GT system represents ADP-glucose synthesis (2) and glycosylation of steviol glycosides (3), shown in FIG. IB. In the following Examples, the combination of SuSy, B12GT, and B13GT will be referred to as the steviol glycoside enzyme system.Table 9. Rebaudioside M Conversion with AMP vs ADPExample 2: Non-Enzymatic Conversion of ATP to ADP

[0171] In order to utilize ATP for glycosylation reactions, it must be converted to ADP. Both chemical and enzymatic methods exist to convert ATP to ADP. This Example demonstrates conversion of ATP to ADP using non-enzymatic methods. 37 mM ATP disodium salt hydrate was dissolved in either water, 10 mM citric acid, or 100 mM citric acid. The measured pH for each mixture was 3.2, 2.8, and 2.3 for the water, 10 mM citric acid, and 100 mM citric acid conditions, respectively. Each ATP solution was stirred at 270 RPM for 22 hours at both 20°C and 63 °C.

[0172] The conversion of ATP to ADP and AMP was measured using an isocratic HPLC method with UV detection. An aqueous mobile phase was used with 20 millimole / Liter of potassium phosphate buffer at pH 6.8 at a flowrate of 0.6 ml / minute. A column of dimensions 150 mm x 4.6 mm was used at 40’C. The packing was 2.7 micron Agilent Poroshell 120 SB- C18. Samples were clarified with a membrane filtration step to remove any enzymes or proteins present. An injection volume of 1 uL was used, and peaks recorded by UV detection at 254nm. This method separates and detects ATP, ADP, AMP, and ADP-glucose. The run times observed with this method are shown in Table 10.Table 10. Retention Times for ATP HPLC Method

[0173] For all ATP solutions incubated at 20°C, the ATP was stable and no conversion to ADP or AMP was observed. However, the ATP solutions incubated at 63°C did show conversion to ADP and AMP (Table 11). As shown, chemical conversion can be used to convert ATP to ADP, but the reaction continues to AMP. Specifically, because of the continued degradation of ADP to AMP at 63 °C, ADP was not able to reach levels above 51% via chemical conversion.Table 11. ATP, ADP, AMP conversion at 63 °C

[0174] The non-enzymatic conversion of ATP to ADP was further investigated. Specifically, 37 mM ATP disodium salt hydrate was dissolved in either 17.5 mM HC1, 10 mM citric acid, or 10 mM sodium carbonate. The measured pH for each mixture was 2.02, 6.06, and 10.13, respectively. Each ATP solution was stirred at 270 RPM for 22 hours at 63°C. The conversion of ATP to ADP was evaluated for the three ATP solutions. Activation of ATP increased with decreasing pH, as shown below in Table 12.Table 12| C | 639 | Carbonate | 10 | 34,3 | 2,4 | 0.1 | 0.0 |Example 3: Glycosylation of Steviol Glycosides with ATP using Chemical Conversion

[0175] In this Example, chemical conversion of ATP to ADP was coupled with highly purified SuSy, B12GT, and B13GT to utilize ATP to glycosylate steviol glycosides. A solution of 20 mM ATP, 10 mM succinate at pH 6, and 250 mM NaOAc was incubated at 50°C for 20 hours to convert ATP to ADP. A reaction medium containing sucrose, RA60, and highly purified SuSy, B12GT and B13GT was made such that when added to the ATP solution the final concentrations would be 200 g / L sucrose, 20 g / L RA60, 250 mM NaOAc, lOmM succinate at pH 6, and 1 mM ATP. The SuSy, B12GT, and B13GT enzymes used in this Example were expressed using the E. coll strains from Example 1. The highly purified enzyme preparations were obtained by growing the E. coll strains, mechanically lysing the cells, and purifying the protein (including enzyme) from the lysate using immobilized metal affinity chromatography (IMAC) with Ni-NTA resin. To start the glycosylation reaction, 2.5 mL of preincubated ATP solution was added to 2.5 mL of the reaction medium and the reaction medium was incubated at 63°C for 21 hours. A second reaction was also conducted where the reaction medium was combined with an ATP solution that had not been preincubated (i.e. fresh ATP) After 20 hours, the reaction with fresh ATP converted 23% of the steviol glycosides to Reb M while the reaction with preincubated ATP reached 68% Reb M. As evidenced by these data, preincubation of the ATP solution allowed for increased conversion of ATP to ADP and, accordingly, higher glycosylation rates.Example 4: Enzymatic Conversion of ATP to ADP with Phytase

[0176] We have demonstrated the use of ATP for glycosylation using chemical conversion of ATP to ADP. Enzymatic conversion of ATP to ADP can also be used to activate ATP for use in glycosylation reactions. A reaction medium consisting of 250 mM sodium acetate, 50 mM phosphate buffer at pH 6, and 0.93 mM ATP was prepared. A commercial phytase enzyme (Sigma-Aldrich P1259) was added to obtain the final activity levels of 0, 0.25, 2.5, or 25 mU / ml. The mixture was heated in a well stirred microreactor at 50°C for 1 hour. Samples were prepared and analyzed by HPLC, as in Example 2. Results are shown in Table 13. At lower phytase concentrations, ATP is not converted fully to ADP, while at higher concentrations, ADP is over-converted to AMP.Table 13. ATP Conversion to ADP and AMP with PhytaseExample 5: Glycosylation of Steviol Glycosides with Enzymatic Conversion of ATPTo demonstrate the use of phytase to activate ATP for use in glycosylation, reaction mixtures were created with and without phytase, containing RA60, sucrose, phosphate buffer, sodium acetate, SuSy, B12GT, and B13GT. The SuSy, B12GT, and B13GT enzymes were first partially purified as in Example 1 and then further purified by IMAC with Ni-NTA resin to obtain highly purified enzyme preparations. The reactions were incubated with stirring at 30°C for one hour and then incubated at 50°C for 20 hours. The reactions were initiated with the following initial components: 20 g / L RA60, 200 g / L sucrose, 250 mM sodium acetate, 100 mM phosphate buffer at pH 6, ImM ATP, SuSy, B12GT, B13GT and either 0 or 2.5 mU / mL phytase. In addition, a positive control was conducted where ImM ADP was used instead of ATP and no phytase was added. The formation of Reb M and other steviol glycosides was monitored as in Example 1. The use of phytase ATP activation made the reaction comparable to the positive control where ADP was used (Table 14).Table 14. Percent Reb M Conversion Using Phytase for ATP Activation

[0177] ATP activation was demonstrated with two commercially available kinases: acetate kinase from Escherichia coli (Sigma-Aldrich A7437) and hexokinase from Saccharomyces cerevisiae (Sigma- Aldrich H6380). Reaction mixtures were made such that the initial reaction conditions were 20 g / L RA60, 200 g / L sucrose, 250 mM sodium acetate, 100 mM phosphate buffer pH 7, ImM ATP. Additionally, 5 g / L glucose and 10 mM MgCl was added to the hexokinase condition. Highly-purified SuSy, B12GT, B13GT and either hexokinase or acetate kinase were added to the reaction mixture. In addition, a negative control with no kinase and a positive control with ImM ADP instead of ATP and no kinase were conducted. The reactions were incubated with stirring at 30 °C for one hour and then held at 50 °C for 20 hours. Bothkinases enabled the use of ATP for use in glycosylation reactions (Table 15). Specifically, acetate kinase was able to allow the use of ATP with similar efficiency as the ADP positive control.Table 15. Percent Reb M Conversion using Kinases for ATP ActivationExample 6: Enzymatic conversion of ATP to ADP with Native E. coli Enzymes from Partially Purified Enzymes

[0178] E. coli natively produces several enzymes that can convert ATP to ADP (Table 16). Specifically of interest are kinases that utilize an economical substrate, such as acetate, glycerol, glucose, fructose, pyruvate, and succinate, that can be easily included in glycosyltransferase reactions. Partially purified proteins from E. coli, such as those used in Example 1, often result in protein purities between 5-30%. Therefore, other A. coli enzymes are present in the partially purified protein preparations, as discussed previously. Native kinases and phosphatases present in partially purified protein preparations can be used to convert ATP to ADP. To demonstrate the use of partially purified protein preparations from E. coli expressions to convert ATP to ADP, partially purified enzymes were incubated with ATP. The partially purified enzymes were prepared similar to Example 1, except that as a final step, the enzymes were formulated with maltodextrin instead of glycerol, combined, and spray- dried. Seven microreactor vials were prepared as follows: 50 mM phosphate buffer, pH 6, 10 mM ATP, and spray dried enzyme blend and putative phosphate acceptor listed in Table 17. The spray dried enzyme blend comprised a SuSy, a B12GT, and a B13GT enzyme. The microreactors were held at 50°C and stirred for 1 hour to activate the ATP. The formation of ADP and AMP were monitored as in Example 2 (Table 17). Of the kinase substrates tested, acetate and glycerol show the highest activation of ATP for use in glycosylation reactions.Table 16. Native E. coli Enzymes that Convert ATP to ADPTable 17. Conversion of ADP to ADP using Native E. coli Enzymes from Partially Purified EnzymesExample 7: Enzymatic conversion of ATP to ADP with Native E. coli Enzymes from Partially Purified Enzymes

[0179] The use of native E. coli kinases from partially purified enzymes to convert ATP to ADP in different buffer conditions was also explored. Partially purified glycerol formulated enzymes obtained as in Example 1 were incubated with ATP. SuSy, B12GT, and B13GT were incubated with 1 mM ATP, 250 NaOAc and buffer. Two buffers, 50 mM pH 6 KPO4 and 10mM pH 6 citrate, were tested. The reaction mediums were incubated for 1 hour at 50°C. Negative controls were also conducted where no enzymes were added to the reaction. Incubation with partially purified enzymes enabled rapid conversion of ATP to ADP (Table 18). Therefore, the partially purified enzymes with native E. coli kinases can be used for glycosylation with ATP feedstock.Table 18. Conversion of ATP to ADP / AMP with native E. coli Enzymes from Partially Purified LysateExample 8: Glycosylation of Steviol Glycosides with Enzymatic Conversion of ATP

[0180] In this Example, SuSy, B12GT, and B13GT enzymes, partially purified as in Example 1, WQVQ used to glycosylate steviol glycosides using ATP. A 2 ml well mixed isothermal microreactor was used. A feedstock was prepared comprising 60 g / L of the mixed steviol glycoside RA60, 200 g / L (580 mM) sucrose, 50 mM phosphate buffer at pH 6, 250 mM sodium acetate, and the partially purified SuSy, B12GT and B13GT enzymes. 1 mM ATP was added to the reaction medium to initiate the reaction. The reaction was conducted at 53°C for 10 hours. Samples were acquired at time intervals over the course of the reaction. To stop the reaction, samples were dissolved in DMSO and analyzed by HPLC, as in Example 1. The HPLC analysis shows that the RA60 steviol glycosides were successfully glycosylated to form Rebaudioside M (Table 19).Table 19. Glycosylation of Steviol Glycosides with Enzymatic Conversion of ATPExample 9: Catalytic levels of ATP vs Catalytic levels of ADP for Glycosylation

[0181] As in Example 8, a feedstock was prepared comprising 60 g / L of a mixed steviol glycoside RA60, 200 g / L (580 mM) sucrose, 50 mM phosphate buffer at pH 6, 250 mM sodium acetate, and partially purified SuSy, B12GT and B13GT. Here, the reaction course for ATP and ADP were directly compared. First, 1 mM ATP was added to a 2 ml well mixed isothermal microreactor comprising the above feedstock. Second, 1 mM ADP was added to a 2000 liter well mixed isothermal reactor. Each reaction was conducted at 53°C for 10 hours. Timepoint samples were acquired over the course of the reaction. To stop the reaction, samples were dissolved in DMSO and analyzed by HPLC, as in Example 1. Results shown in FIG 11 indicate that ATP is as effective as ADP when added at catalytic levels for glycosylation.Example 10: Comparable Reaction Rates of Conversion of Stevioside using ADP and ATP at Catalytic Levels

[0182] The conversion of stevioside species based on the reaction conditions in Examples 1 and 8 was compared. Specifically, the comparison evaluated the ability of AMP, ADP, and ATP to facilitate glycosylation. A logarithmic plot (FIG. 12) was used to better illustrate the reactivity in each case. After hour 2, the slope, which represents the rate of reaction, is nearly identical for the ATP case and the positive control ADP case. Accordingly, the reaction system in Examples 8-10 successfully utilize an ATP feedstock instead of an ADP feedstock for glycosylation of steviol glycosides.Example 11: SuSy Stabilizes Mixture By Reducing Formation of AMP

[0183] Examples 2 and 4 show that methods to convert ATP to ADP can often over-convert to AMP. However, by using adequate amounts of SuSy during the ATP activation step, the SuSy can stabilize the ADP as ADP -glucose. To examine this, glycerol formulated partially purified SuSy obtained as described in Example 1 was incubated with ATP. SuSy was incubated with 1 mM ATP, 250 NaOAc, 10 mM citrate pH 6, 60 g / L RA60 and 200 g / L sucrose. The reaction medium was incubated for 1 hour at 50°C. A negative control was also conducted where no sucrose or RA60 was added to the mixture. ATP incubation with SuSy and sugar donor sucrose enabled both ADP stabilization as ADP-glucose, preventing overconversion to AMP, and increased amounts of ATP conversion so that more ADP / ADP- glucose is available for glycosylation (Table 20).Table 20. Stabilization of ADP by incubation with SuSyExample 12: Rebaudioside M Can Be Produced Using ATP With Different Buffers at Different Temperatures for Different Time PeriodsExample 12a

[0184] ATP solutions were prepared as described in recipes A, B, C, D, and E (Table 21) to perform glycosylation reactions. Two different "mastermixes" were prepared. Mastermix I contained water, sucrose, sodium acetate, succinic acid buffer, and mixture of stevioside and Rebaudioside A. Mastermix II contained water, sucrose, sodium acetate, phosphate acid buffer, and mixture of stevioside and Rebaudioside A. Both master mixes were adjusted to pH 6. In each Mastermix, the total concentration of steviol glycosides was 20 g / 1.Table 21. ATP Reaction Mediums

[0185] 2 ml reactions were conducted by combining 1.869 ml of mastermix, with 0.115 ml of an ATP reaction medium and 0.016 ml of a mixture of partially purified SuSy, B12GT and B13GT enzymes. The partially purified enzymes were obtained as described in Example 1. Specifically, the following combinations of ATP reaction mediums and mastermixes weretested: ATP from recipe A, B, or C was added to Mastermix I and ATP from recipe A, D, or E was added to Mastermix II. Reaction results, shown in Table 22 and Table 23, were measured by HPLC, as described in Example 1. These data illustrate that Reb M (%M) can successfully be produced using ATP with different buffers and by running the reactions at different temperatures and for different times.Table 22Table 23Example 12b

[0186] Additional reactions were performed. A reaction medium was prepared as follows: 40 g / L RA60, 200 g / L sucrose, 250 mM sodium acetate, 50 mM phosphate buffer at pH 6, ATP at 1 millimol e / liter was reacted with sucrose synthase and P-1, 3 -glycosyltransferase. The enzymes used were partially purified and provided as aqueous liquid formulations in 50% glycerol, as described in Example 1. The mixture was heated in a well stirred microreactor to 55°C and held for 6 hours. Samples were taken at 0, 2, 4, and 6 hours. Each sample was immediately dissolved in DMSO to quench the reaction. Samples were analyzed by HPLC, as described in Example 1. Results are shown in Table 24. In Table 24, STV means Stevioside, A D M E and I are Rebaudiosides A, D, M, E, & I, and ADPG / ADP is a ratio of ADP -glucose to ADP.Table 24

[0187] These results demonstrate that ATP can be used as a catalytic agent to convert stevioside to Rebaudioside A (via glycosylation) and onward to Rebaudioside I, using a B13GT. It also shows that the ATP is activated simultaneously with the reaction time course, at a rate of about 0.2 mM ATP per hour. Also, the onward conversion of ADP to AMP is limited as the ADP is mainly in the form ADP-glucose.

[0188] Additional reactions were conducted similar to the previous reactions above. However, B13GT was substituted with B12GT in the reaction. Results are shown in Table 25. Table 25

[0189] These data show that ATP can be used as the feedstock in the conversion of stevioside to Reb E and Reb A to Reb D using a B12GT. AMP buildup is lowered, and the ratio of ADPG / ADP is higher than the previous example.

[0190] Additional reactions were conducted similar to the reactions above, except that SuSy, B12GT, and B13GT were all added to the reaction. Results are shown in Table 26.Table 26

[0191] As shown in Table 26, stevioside and Reb A are both converted to Reb M. Here, AMP buildup is low, and the ratio of ADPG / ADP remains above 1.

[0192] Additional reactions were conducted similar to the reactions above. SuSy was omitted but B12GT and B13GT were added to the reaction. Results are shown in Table 27.Table 27

[0193] As shown in Table 27, in the absence of sucrose synthase, neither stevioside nor Reb A are converted, at least because no ADP-glucose is formed.

[0194] Additional reactions were conducted similar to the reactions above. Specifically, SuSy was added but B12GT and B13GT were omitted. Results are shown in Table 28.Table 28

[0195] As shown in Table 28, neither stevioside nor Reb A are converted. The ratio of ADPG / ADP is very high, and AMP does not form.Example 13: Glycosylation using ATP and a single Leloir glycosyl transferase

[0196] In this Example, the use of a single Leloir glycosyltransferase for both ADP-glucose synthesis and glycosylation of steviol glycosides is evaluated. Rebaudioside M is used as the sugar donor, stevioside is the sugar acceptor, and P-1, 3 -glycosyltransferase is the Leloir glycosyltransferase that can perform the reactions, as outlined below: Rebaudioside M + ADP -> Rebaudioside D + ADP-glucose Rebaudioside M + ADP -> Rebaudioside AM + ADP-glucose Stevioside + ADP-glucose -> Rebaudioside A + ADP

[0197] A reaction medium was prepared as follows: 2 gram / L Reb M, 3g / L stevioside, 250 mM sodium acetate, and 50 mM phosphate buffer at pH 6, with enzyme P-1, 3- glycosyltransferase. The enzyme used was partially purified and provided as aqueous liquid formulations in 50% glycerol, as in Example L The mixture was heated in a well stirred microreactor to 53°C and held for 6 hours. Samples were taken at 0, 2, 4, and 6 hours. Each sample was immediately dissolved in DMSO to quench the reaction. Samples were analyzed by HPLC, as in Example 1. The process successfully glycosylated stevioside to rebaudioside A using rebaudioside M as the sugar donor and B13GT as the sole glycosyltransferase (Table 29).Table 29. Glycosylation of Stevioside using Reb M as the sugar donorExample 14: Glycosylation of Fructose Using a Rebaudioside Sugar Donor

[0198] In contrast to the previous examples, this Example demonstrates the use of B 13GT as the sugar nucleotide synthase and SuSy as the glycosyltransferase for steviol glycoside glycosylation. This is achieved by using relatively high amounts of fructose as the sugar acceptor and rebaudioside M as the donor. In the presence of ATP, the following reactions should be catalyzed in the process:RebGlucose + Reb DRebGlucose + Reb AMADP-Glucose + FructoseADP + Sucrose

[0199] To give the overall glycosylation reactionReb M + FructoseSucrose + Reb D + Reb AM

[0200] Two reaction mixtures were prepared each with 4 gram / liter Reb M, 250mM sodium acetate, 50 g / L fructose and 50mM phosphate buffer, pH 6, with enzymes sucrose synthase and P-l,3-glycosyltransferase. The enzymes used were partially purified and provided as aqueous liquid formulations in 50% glycerol, as in Example 1. ATP was added and the mixture was heated in a well stirred microreactor to 53°C and held for 5 hours. Samples were analyzed by HPLC, as in Example 1. This reaction proceeds if ATP is added (Table 30), and the negative control confirms that no reaction occurs in the absence of ATP (Table 31).Table 30. Formation of Reb AM and Reb D when Reb M is used as a sugar donor.Table 31. Negative control for glycosylation with Reb M as sugar donor.Example 15: Glycosylation of p-sitosterol-|J-glucoside

[0201] To demonstrate the ability to glycosylate sugar acceptors other than steviol glycosides, 100 uL reactions were conducted comprising an initial sugar acceptor |3-sitostcrol-|3-glucosidc at 0.4 gram / liter (CAS: 474-58-8) together with 200 g / L (580 mM) sucrose, 1 mM ATP, and a buffer comprising 100 mM ammonium acetate at pH 6. The reactions were run in the presence of either 1) SuSy and B12GT, 2) SuSy, B13GT, or 3) SuSy, B12GT and B13GT. The enzymes used were partially purified as in Example 1. Negative control reactions were also run where the ATP was omitted from the reaction. The reactions were held isothermally at 60°C for 24 hours with good mixing.

[0202] Final samples at 24 hours were filtered using a 10 kD MWCO membrane and then analyzed by LCMS using an Agilent 1290 Infinity II HPLC coupled with an Agilent 6545B QTOF mass spectrometer (MS). Specifically, LCMS was used to detect glycosylation of [3- sitosterol-P-glucoside with the following parameters. Mobile phase A is 0.1% formic acid in water, and mobile phase B is 0.1% formic acid in methanol. A Hypersil GOLD™ C8 HPLC column with dimensions of 100 mm x 2.1 mm and particle size of 1.9 um was used at the flow rate of 0.3 mL / min and temperature of 40°C. The gradient used is as follows: 0-15 minutes ramp 5-70% B, 15.1-20 minutes ramp 70-92% B, step-up and hold at 99% B 20-24 minutes, stepdown and hold at 5% B 24-26 minutes. The injection volume was between 1 uL and 5 uL. The data were acquired in negative MS mode using the following settings: Vcap voltage = 3000 V, nozzle voltage = 1500 V, fragmentor = 80 V, skimmer = 50 V, and gas temperature at 250°C.

[0203] LCMS results show that for the control reaction, with no ATP, no reaction glycosylation products are formed, while for the reaction in the presence of ATP, the glycosylation reaction occurs and new peaks are formed with m / z increases of 162.03 and 324.06 compared to the substrate m / z at 621.437 (formate adduct ion), indicating addition of one and two glucosyl groups, to give p-sitosterol-diglucoside and p-sitosterol-triglucoside, respectively. This shows that ATP is effective for performing mono- and di-glycosylation.Example 16: Glycosylation of cholesterol-|3-D-glucoside

[0204] Reactions were conducted as in Example 15, however, the initial sugar acceptor was changed to 2 mg / mL cholesterol-[3-D-glucoside (CAS: 7073-61-2). Formation of glycosylationproducts was measured by LCMS as in Example 15. LCMS results show that, for the no ATP control, no reaction glycosylation products are formed. When ATP is present, new peaks were found at m / z of 755.457 and 917 509, indicating addition of one and two glucosyl groups, to give cholesterol-diglucoside and cholesterol-triglucoside, respectively. This shows that ATP is effective for mono- and di-glycosylation of cholesterol-P-D-glucoside.Example 17: Glycosylation of spiraeoside

[0205] Reactions were conducted as in Example 15, however, the initial sugar acceptor was changed to 2 mg / mL spiraeoside (CAS: 20229-56-5). Formation of glycosylation products was measured by LCMS as in Example 15. LCMS results show that, for the no ATP control, no reaction glycosylation products are formed. When ATP is present, glycosylation occurs and new peaks were found at m / z of 625.143, 787.197, 949.248, and 1111.298, indicating addition of one, two, three, and four glucosyl groups, respectively. Of note is that the LC elution resolved 4 different isomers of the m / z at 625.143 and 4 different isomers of at m / z of 787.197. This indicates glycosylation of the hydroxyl groups can occur at different positions and / or have different glycosylation linkage. This shows that ATP is effective for multi-glycosylation of polyphenols.Example 18: Glycosylation of quercetin-3-P-D-glucoside

[0206] Reactions were conducted as in Example 15, however, the initial sugar acceptor was changed to quercetin-3-P-D-glucoside (CAS 482-35-9). Formation of glycosylation products was measured by LCMS as in Example 15. LCMS data showed that no reaction glycosylation products are formed in the absence of ATP. When ATP is present, glycosylation occurs and new peaks with m / z at 625.141 and 787.191 are seen, indicating addition of one or two glycosyl groups. Of note is that the LC resolved 2 different peaks at m / z of 625.141 and resolved 2 different peaks of at m / z of 787.191. This indicates glycosylation can occur at the OH groups in different positions. This shows that ATP is effective for polyglycosylation of polyphenols.INCORPORATION BY REFERENCE

[0207] All references, articles, publications, patents, patent publications, and patent applications cited herein are incorporated by reference in their entireties for all purposes. However, mention of any reference, article, publication, patent, patent publication, and patent application cited herein is not, and should not be taken as an acknowledgment or any form of suggestion that they constitute valid prior art or form part of the common general knowledge in any country in the world.NUMBERED EMBODIMENTS OF THE INVENTION

[0208] Notwithstanding the appended claims, the disclosure sets forth the following numbered embodiments:

[0209] (Al) A method for glycosylation of a sugar acceptor, comprising the steps of: (i) converting an exogenous nucleoside triphosphate (NTP) to a nucleoside diphosphate (NDP); (ii) converting the NDP to NDP-sugar, comprising contacting at least one Leloir glycosyltransferase (GT) with the NDP and an exogenous sugar donor (SD) in a reaction medium; and (iii) converting a sugar acceptor (SA) to a glycosylated SA, comprising contacting the at least one GT with the NDP-sugar and the SA in the reaction medium.

[0210] (A2) A method for glycosylation of a steviol glycoside, comprising the steps of: (i) converting an exogenous adenosine triphosphate (ATP) to an adenosine diphosphate (ADP);(ii) converting the ADP to ADP -glucose, comprising contacting at least one Leloir glycosyltransferase (GT) with the ADP and exogenous sucrose (SD) in a reaction medium; and(iii) converting the steviol glycoside (SA) to a glycosylated SA, comprising contacting the at least one GT with the ADP-glucose and the SA in the reaction medium.

[0211] (A3) The method of any one of (Al) or (A2), wherein the reaction medium comprises the at least one GT, the SA, and the SD.

[0212] (A4) The method of any one of (A1)-(A3), wherein the total amount of the SD in the reaction medium is between about 50 mM to about 1 M based on a final volume of the reaction medium.

[0213] (A5) The method of any one of (A1)-(A4), wherein the molar ratio of the SD to the SA in the reaction medium is at least about 2:1.

[0214] (A6) The method of any one of (A3), further comprising supplementing the reaction medium with additional SA.

[0215] (A7) The method of (A6), wherein the reaction medium is supplemented with the additional SA continuously or in batches.

[0216] (A8) The method of (Al), (A2), or (A6), wherein the total amount of the SA added to the reaction medium is between about 5 millimoles and about 200 millimoles per liter of the final volume of the reaction medium.

[0217] (A9) The method of any one of (Al) and (A3)-(A8), wherein converting the NTP to NDP comprises one or more of non-enzymatic conversion or enzymatic conversion.

[0218] (Al 0) The method of any one of (A2)-(A9), wherein converting the ATP to ADP comprises one or more of non-enzymatic conversion or enzymatic conversion.

[0219] (Al l) The method of (A9) or (A10), wherein the conversion comprises enzymatic conversion and the reaction medium further comprises a phosphatase or a kinase.

[0220] (A12) The method of (Al l), wherein the phosphatase is a phytase.

[0221] (A13) The method of (A9) or (A10), wherein the conversion comprises enzymatic conversion and the reaction medium further comprises a phosphate acceptor.

[0222] (A14) The method of (A13), wherein the phosphate acceptor is a kinase substrate.

[0223] (Al 5) The method of (A13) or (A14), wherein the kinase substrate is selected from the group consisting of one or more of acetate, glycerol, glucose, fructose, pyruvate, and succinate, and combinations thereof

[0224] (Alb) The method of (Al l), wherein the reaction medium comprises a kinase.

[0225] (A17) The method of any one of (A1)-(A16), wherein the at least one GT comprises a P-l,2-glycosyltransferase, a P-l,3-glycosyltransferase, or a sucrose synthase.

[0226] (Al 8) The method of any one of (A1)-(A17), wherein the at least one GT comprises an NDP-sugar synthase.

[0227] (A19) The method of any one of (A1)-(A18), wherein the at least one GT comprises a sucrose synthase and one or more of a P-l,2-glycosyltransferase and a P-1, 3- glycosyltransferase.

[0228] (A20) The method of any one of (Al), (A3)-(A9), and (Al 1)-(A19), wherein the molar ratio ofNTP:SA is between about 0.00045:1 and about 0.1 :1.

[0229] (A21) The method of any one of (Al), (A3)-(A9), and (Al l)-(A20), wherein the molar ratio ofNTP:GT is between about 0.55:1 and about 5500: 1.

[0230] (A22) The method of any one of (A2)-(A16), wherein the molar ratio of ATP:GT is between about 0.55: 1 and about 5500: 1.

[0231] (A23) The method of any one of (A1)-(A22), wherein the method is performed under isothermal conditions.

[0232] (A24) The method of any one of (A1)-(A23), wherein the method is performed at a temperature between about 50°C and about 75°C.

[0233] (A25) The method of any one of (A1)-(A24), wherein the reaction medium further comprises a buffer.

[0234] (A26) The method of (A25), wherein the method is performed at a pH of between about 6 and about 6.5

[0235] (A27) The method of (A25) or (A26), wherein the buffer is selected from one of acetate, citrate, lactate, succinate, or phosphate.

[0236] (A28) The method of any one of (Al), (A3)-(A9), (Al l)-(A20), and (A23)-(A27), wherein the NTP is one of adenosine triphosphate, guanidine triphosphate, uridine triphosphate, cytidine triphosphate, and thymidine triphosphate.

[0237] (A29) The method of any one of (Al), (A3)-(A9), (Al l)-(A20), and (A23)-(A28), wherein the total amount of the NTP added is between about 0.1 millimoles to about 10 millimoles per liter of the final volume of the reaction medium.

[0238] (A30) The method of any one of (A2)-(A16) and (A22)-(A27), wherein the total amount of the ATP added is between about 0.1 millimoles and about 10 millimoles per liter of the final volume of the reaction medium

[0239] (A31) The method of any one of (Al)-(A30), wherein the SA is selected from the group consisting of one or more of a protein, a lipid, a flavanone, steviol, a steviol glycoside, a polyketide, a steroid, a polyphenol, and combinations thereof.

[0240] (A32) The method of any one of (A1)-(A31), wherein the SA comprises a steroid selected from the group consisting of one or more of cholesterol, cholesterol-P-D-glucoside, cholesterol-diglucoside, campesterol, stigmasterol, sitosterol, and P-sitosterol-P-D-glucoside, and combinations thereof.

[0241] (A33) The method of any one of (A1)-(A32), wherein the SA comprises a flavonoid selected from the group consisting of fisetin, luteolin, luteolinidin, apigenin, quercetin, quercetin-3-P-D-glucoside, quercetin 3,4’-diglucoside, prunin, spiraeoside.

[0242] (A34) The method of any one of (A1)-(A33), wherein the SD is a disaccharide or a polysaccharide.

[0243] (A35) The method of (A34), wherein the SD is at least one selected from the group consisting of: sucrose, starch, maltose, lactose, alpha-glucose- 1 -phosphate, beta-glucose-1- phosphate, cellobiose, gentiobiose, trehalose, kojibiose, nigerose, isomaltose, beta-beta- trehalose, alpha-beta-trehalose, sophorose, laminaribiose, turanose, maltulose, palatinose, gentiobiulose, nigerotriose, maltotriose, melezitose, maltotriulose, kestose, cellulose, glycogen, amylose, amylopectin, dextran, dextrin, arabinoxylan, maltodextrin, glucose syrup, cellodextrin, and cyclodextrin.

[0244] (A36) The method of any one of (A1)-(A35), wherein the at least one GT is derived from a natural or engineered microorganism.

[0245] (A37) The method of (A36), wherein the at least one GT is a crude enzyme or an at least partially purified enzyme.

[0246] (A38) The method of (A36) or (A37), wherein the at least one GT further comprises one or more additional enzymes produced by the natural or engineered microorganism.

[0247] (A39) The method of (A38), wherein the one or more additional enzymes comprises at least one selected from the group consisting of enzymes having phosphatase activity and enzymes having kinase activity

[0248] (A40) The method of (A38) or (A39), wherein the one or more additional enzymes comprises at least one selected from the group consisting of phosphatases and kinases.

[0249] (A41) The method of any one of (A37)-(A39), wherein the at least one GT is a highly purified enzyme.

[0250] (A42) The method of (A41), wherein the highly purified enzyme is purified by affinity chromatography and / or size exclusion chromatography.

[0251] (A43) The method of any one of (A2)-(A16), (A22)-(A27), and (A30)-(A42), wherein fewer than 10% of phosphorylated adenosines in the reaction medium are adenosine monophosphate (AMP).

[0252] (A44) The method of any one of (Al), (A3)-(A9), (Al l)-(A20), (A23)-(A29), and (A30)-(A42), wherein the molar ratio of the total amount of NTP SA added is at most 0.2:1.

[0253] (A45) The method of any one of (A2)-(A16), (A22)-(A27), and (A30)-(A43), wherein the molar ratio of ADP -glucose: ADP during the reaction is at least 1:1.

[0254] (A46) The method of any one of (A2)-(A16), (A22)-(A27), (A30)-(A43), and (A45), wherein the molar ratio of the total amount of ATP:SG added is at most 0.2:1.

[0255] (A47) The method of any one of (Al), (A3)-(A9), (Al l)-(A20), (A23)-(A29), (A30)- (A42), and (A44), wherein the molar ratio of the total amount of NTP:SG added is between about 0.00045:1 and about 0.2: 1.

[0256] (A48) The method of any one of (A2)-(A16), (A22)-(A27), (A30)-(A43), (A45), and (A46), wherein the molar ratio of the total amount of ATP:SG added is between about 0.00045:1 and about 0.2:1.

[0257] (A49) The method of any one of (A2)-(A16), (A22)-(A27), (A30)-(A43), (A45), (A46), and (A48), wherein the SA comprises one or more steviol glycosides selected from the group consisting of steviol-13-O-glucoside, steviol- 19-O-glucoside, rubusoside, steviol-1,2- bioside, steviol-l,3-bioside, rubusoside, dulcoside B, dulcoside A, rebaudioside B, rebaudioside G, stevioside, rebaudioside C, rebaudioside F, rebaudioside A, rebaudioside I, rebaudioside E, rebaudioside E2, rebaudioside AM, rebaudioside H, rebaudioside L, rebaudioside K, rebaudioside J, rebaudioside M, rebaudioside D, rebaudioside N, rebaudioside O, and rebaudioside Q, or one or more enzymatically glycosylated steviol glycosides.

[0258] (A50) The method of (A49), where the one or more steviol glycosides comprise stevioside and rebaudioside A.

[0259] (A51) The method of (A50), wherein the reaction medium comprises at least 6 g / L stevioside and at least 36 g / L rebaudioside A.

[0260] (A52) The method of (A49) or (A50), wherein the one or more steviol glycosides comprise rebaudioside A.

[0261] (A53) The method of (A52), wherein the reaction medium comprises at least 36 g / L rebaudioside A.

[0262] (A54) The method of any one of (A2)-(A16), (A22)-(A27), (A30)-(A43), (A45), (A46), and (A48)-(A53), wherein the glycosylated SA comprises one or more steviol glycosides selected from the group consisting of steviol-13-O-glucoside, steviol-19-O- glucoside, rubusoside, steviol-l,2-bioside, steviol-l,3-bioside, rubusoside, dulcoside B, dulcoside A, rebaudioside B, rebaudioside G, stevioside, rebaudioside C, rebaudioside F, rebaudioside A, rebaudioside I, rebaudioside E, rebaudioside E2, rebaudioside AM, rebaudioside H, rebaudioside L, rebaudioside K, rebaudioside J, rebaudioside M, rebaudioside D, rebaudioside N, rebaudioside O, and rebaudioside Q, or one or more enzymatically glycosylated steviol glycosides.

[0263] (A55) The method of any one of (Al), (A3)-(A9), (Al l)-(A20), (A23)-(A29), (A30)- (A42), (A44), and (A47), wherein the total amount of the NTP added is at most 10 millimoles per liter of the final volume of the reaction medium.

[0264] (A56) The method of any one of (A2)-(A16), (A22)-(A27), (A30)-(A43), (A45), (A46), and (A48)-(A54), wherein the total amount of the ATP added is at most 10 millimoles per liter of the final volume of the reaction medium.

[0265] (A57) The method of any one of (A2)-(A16), (A22)-(A27), (A30)-(A43), (A45), (A46), (A48)-(A53), and (A56), wherein the reaction medium comprises steviol glycoside in a stevia leaf extract.

[0266] (A58) The method of (A57), wherein the stevia leaf extract is produced by a process comprising one or more steps of extracting, clarifying, and concentrating.

[0267] (A59) The method of (A57) or (A58), wherein the concentrating comprises membrane processing such as nanofiltration.

[0268] (A60) The method of any one of (A57)-(A59), wherein the concentrating comprises evaporation.

[0269] (A61) The method of any one of (A57)-(A60), wherein the concentrating comprises adsorption of one or more steviol glycosides onto a solid adsorbent, elution with an alcohol, and removal of the alcohol to give a concentrated leaf extract.'ll

[0270] (A62) The method of any one of (A57)-(A61), wherein the stevia leaf extract is extracted by one or more of a batch parabolic trough with a helical mixer, a batch rotating cylindrical extractor, a batch percolation extractor, horizontal continuous moving belt extractor, a horizontal counter current tubular screw extractor, a vertical countercurrent tubular screw extractor, a segmented rotating basket extractor, and a pressurized condensing steam extractor.

[0271] (A63) The method of (A57)-(A62), wherein the clarifying comprises one of more of thermal coagulation of proteins and impurities, diatomaceous earth treatment, ion exchange, chemical coagulation using calcium carbonate and / or ferric chloride

[0272] (A64) A method for glycosylation of a steviol glycoside, comprising the steps of: (i) converting an exogenous adenosine triphosphate (ATP) to adenosine diphosphate (ADP); (ii) converting the ADP to ADP-glucose, comprising contacting a sucrose synthase (SuSy) with the ADP and exogenous sucrose in a reaction medium; and (iii) converting at least one steviol glycoside (SG) to a glycosylated SG, comprising contacting the SG with the ADP-glucose and at least one glycosyltransferase (GT) selected from the group consisting of [3-1,2- glycosyltransferase, P-l,3-glycosyltransferase, or a combination thereof in the reaction medium.

[0273] (A65) The method of (A64), wherein the reaction medium initially comprises the SuSy, the at least one GT, the SG, and the sucrose.

[0274] (A66) The method of (A64) or (A65), wherein the total amount of the sucrose in the reaction medium is between about 50 mM to about 1 M based on the final volume of the reaction medium.

[0275] (A67) The method of any one of (A64) to (A66), wherein a molar ratio of the total amount of the sucrose and the SG added is at least about 4: 1.

[0276] (A68) The method of (A65), further comprising supplementing the reaction medium with additional SA.

[0277] (A69) The method of (A68), wherein the reaction medium is supplemented with the additional SA continuously or in batches.

[0278] (A70) The method of (A64) or (A68), wherein the total amount of the SG added to the reaction medium is between about 0.5 millimoles and about 200 millimoles per liter of the final volume of the reaction medium.

[0279] (A71) The method of any one of (A64) to (A70), wherein the SG is stevioside and the glycosylated SG is at least one of rebaudioside A, rebaudioside E, or rebaudioside E2.

[0280] (A72) The method of any one of (A64) to (A71), wherein the SG is rebaudioside A and the glycosylated SG is at least one of rebaudioside D or rebaudioside I.

[0281] (A73) The method of any one of (A64) to (A72), wherein the SG is rebaudioside E and the glycosylated SG is at least one of rebaudioside D or rebaudioside AM.

[0282] (A74) The method of any one of (A64) to (A73), wherein the SG is rebaudioside E2 and the glycosylated SG is at least one of rebaudioside I or rebaudioside AM.

[0283] (A75) The method of any one of (A64) to (A74), wherein the SG is rebaudioside I and the glycosylated SG is rebaudioside M.

[0284] (A76) The method of any one of (A64) to (A75), wherein the SG is rebaudioside D and the glycosylated SG is rebaudioside M.

[0285] (A77) The method of any one of (A64) to (A76), wherein the SG is rebaudioside AM and the glycosylated SG is rebaudioside M.

[0286] (A78) The method of any one of (A64) to (A77), wherein the SG is stevioside and rebaudioside A and the glycosylated SG is rebaudioside M.

[0287] (A79) The method of any one of (A64) to (A78), wherein the SG is rebaudioside A and the glycosylated SG is rebaudioside M.

[0288] (A80) The method of any one of (A64) to (A79), wherein the SG is stevioside and the glycosylated SG is rebaudioside M.

[0289] (A81) The method of any one of (A64) to (A80), wherein converting the ADP to ADP- glucose occurs at a rate of at least about 3 mM / hr.

[0290] (A82) The method of any one of (A64) to (A81), wherein converting the ATP to the ADP initially occurs at a rate of between about 0.1 mM / hr and about 1 mM / hr.

[0291] (A83) The method of any one of (A64) to (A82), wherein converting the SG to the glycosylated SG occurs at a rate of between about 0.25 mM / hr and about 30 mM / hr.

[0292] (A84)A method for glycosylation of a sugar acceptor, comprising the steps of: (i) converting an exogenous nucleoside triphosphate (NTP) to a nucleoside diphosphate (NDP); (ii) converting the NDP to NDP-sugar, comprising contacting at least one Leloir glycosyltransferase (GT) with the NDP and an exogenous sugar donor (SD); and (iii) converting a sugar acceptor (SA) to a glycosylated SA, comprising contacting the at least one GT with the NDP-sugar and the SA.

[0293] (A85) The method of (A84), wherein (i)-(iii) are performed simultaneously in a reaction medium.

[0294] (A86) The method of (A84) or (A85), wherein (i)-(iii) are performed independently.

[0295] (A87) The method of any one of (A84) to (A86), wherein (i) is performed in a first reaction medium, and (ii) and (iii) are performed in a second reaction medium comprising the first reaction medium.

[0296] (A88) The method of (A87), wherein the first reaction medium initially comprises the NTP.

[0297] (A89) The method of (A87) or (A88), wherein the first reaction medium finally comprises the NDP, and wherein the second reaction medium further comprises the at least one GT, the SD, and the SA.

[0298] (A90) The method of any one of (A84) to (A89), wherein (i) and (ii) are performed to completion in a first reaction medium and (iii) is performed in a second reaction medium comprising the first reaction medium.

[0299] (A91) The method of (A90), wherein the first reaction medium initially comprises the NTP, at least one GT, and the SD.

[0300] (A92) The method of (A91), wherein the first reaction medium finally comprises the NDP-sugar and the at least one GT, and wherein the second reaction medium further comprises the SA.

[0301] (A93) The method of any one of (A84) to (A92), wherein (i)-(iii) are performed in a reaction medium initially comprising the NTP, the at least one GT, and the SD.

[0302] (A94) The method of any one of (A84) to (A93), wherein one or more of the at least one GT, the SD, and the SA are added to the reaction medium after (i).

[0303] (A95) The method of (A94), wherein the added one or more of the at least one GT, the SD, and the SA are added to the reaction medium continuously or in batches.

[0304] (A96) The method of any one of (A84) to (A95), wherein (i) is performed in a first reaction medium, and the method further comprises purifying the NDP from the reaction medium.

[0305] (A97) The method of (A96), wherein (ii) and (iii) are performed in a second reaction medium initially comprising the purified NDP.

[0306] (A98) The method of any one of (A84) to (A97), wherein (i) and (ii) are performed to completion in a first reaction medium and (ii) and (iii) are performed in a second reaction medium comprising the first reaction medium.

[0307] (A99) The method of (A98), wherein the first reaction medium initially comprises the NTP, at least one GT, and the SD.

[0308] (Al 00) The method of (A99), wherein the first reaction medium finally comprises the NDP-sugar, the at least one GT, and the SD, and wherein the second reaction medium further comprises the SA.

[0309] (A101) The method of any one of (Al) to (A60), wherein the NTP or ATP is provided as a purified product.

[0310] (A102) The method of any one of (Al) to (A60), wherein the NTP or ATP is provided as a crude fermentation broth.

[0311] (A103) The method of any one of (Al) to (A60), wherein the NTP or ATP is provided as a partially purified fermentation broth.

[0312] (A 104) The method of any one of (Al 01) to (A 103), wherein the NTP contains NTP, NDP, and NMP or the ATP contains ATP, ADP and AMP.

[0313] (A105) The method of any one of (A101) to (A104), wherein the NTP or ATP is a powder.

[0314] (A106) The method of any one of (A101) to (A105), wherein the NTP or ATP is a frozen aqueous solution.

[0315] (Al 07) The method of (Al 06), wherein the NTP or ATP frozen aqueous solution contains stabilizers including at least one selected from the group consisting of buffers, sodium acetate, or glycerol.

[0316] (A108) The method of any one of (A2)-(A16), (A22)-(A27), (A30)-(A43), (A45), (A46), (A48)-(A53), and (A60), wherein the ATP is provided as a clarified fermentation broth produced by Corynebacterium stationis.

[0317] (A109) The method of any one of (A2)-(A16), (A22)-(A27), (A30)-(A43), (A45), (A46), (A48)-(A53), (A60), and (Al 08), wherein the ATP is a clarified fermentation broth that has been clarified by a process including at least one ultrafiltration or microfiltration.

[0318] (Al 10) The method of any one of (A2)-(A16), (A22)-(A27), (A30)-(A43), (A45), (A46), (A48)-(A53), (A60), (A108), and (A109), wherein the ATP is a clarified fermentation broth containing ATP, ADP, and AMP that has been purified by a process that includes ion exchange to produce an ATP salt solution.

[0319] (Bl) A composition for glycosylation of a steviol glycoside, comprising exogenous adenosine triphosphate (ATP), at least one Leloir glycosyltransferase (GT), exogenous sucrose (SD), and at least one steviol glycoside (SA).

[0320] (B2) The composition of (Bl), wherein the at least one Leloir GT comprises an ADP- sugar synthase.

[0321] (B3) The composition of (Bl) or (B2), wherein the at least one Leloir GT comprises a sucrose synthase.

[0322] (B4) The composition of (Bl), wherein the at least one Leloir GT comprises a P-1, 2- glycosyltransferase, a P-l,3-glycosyltransferase, or a sucrose synthase.

[0323] (B5) The composition of (B 1), wherein the at least one Leloir GT comprises a sucrose synthase and one or more of a P-l,2-glycosyltransferase and a P-1, 3 -glycosyltransferase

[0324] (B6) The composition of any one of (B1)-(B5), further comprising a hydrolase, a phosphatase, or a kinase.

[0325] (B7) The composition of any one of (B1)-(B6), further comprising a kinase.

[0326] (B8) The composition of (B7), further comprising a kinase acceptor.

[0327] (B9) The composition of any one of (B1)-(B8), wherein the at least one SA comprises one or more steviol glycosides selected from the group consisting of steviol-13-O-glucoside, steviol-19-O-glucoside, rubusoside, steviol- 1,2-bioside, steviol-l,3-bioside, rubusoside, dulcoside B, dulcoside A, rebaudioside B, rebaudioside G, stevioside, rebaudioside C, rebaudioside F, rebaudioside A, rebaudioside I, rebaudioside E, rebaudioside E2, rebaudioside AM, rebaudioside H, rebaudioside L, rebaudioside K, rebaudioside J, rebaudioside M, rebaudioside D, rebaudioside N, rebaudioside O, and rebaudioside Q, or one or more enzymatically glycosylated steviol glycosides.

[0328] (B10) The composition of any one of (B1)-(B9), wherein the molar ratio of the SD to the SA is at least about 2:1.

[0329] (Bl 1) A composition for glycosylation of a steviol glycoside, comprising exogenous adenosine triphosphate (ATP), sucrose synthase (SuSy), exogenous sucrose (SD), at least one glycosyltransferase (GT) selected from the group consisting of P-l,2-glycosyltransferase, P- 1,3 -glycosyltransferase, or a combination thereof, and at least one steviol glycoside (SA).

[0330] (B12) A composition for glycosylation of a steviol glycoside, comprising exogenous adenosine triphosphate (ATP), ADP-sugar synthase, and exogenous sucrose.

[0331] (B13) A composition for glycosylation of a steviol glycoside, comprising exogenous adenosine triphosphate (ATP) and one or more of a hydrolase, a phosphatase, and a kinase.

[0332] (B14) A composition for glycosylation of a sugar acceptor, comprising exogenous nucleoside triphosphate (NTP), at least one Leloir glycosyltransferase (GT), exogenous sugar donor (SD), and at least one sugar acceptor (SA).

[0333] (Bl 5) A composition for glycosylation of a sugar acceptor, comprising exogenous nucleoside triphosphate (NTP), NDP-sugar synthase, exogenous sugar donor (SD), at least one glycosyltransferase, and at least one sugar acceptor (SA).

[0334] (Bl 6) A composition for glycosylation of a sugar acceptor, comprising exogenous nucleoside triphosphate (NTP), NDP-sugar synthase, and exogenous sugar donor.

[0335] (Bl 7) A composition for glycosylation of a sugar acceptor, comprising exogenous nucleoside triphosphate (NTP) and one or more of a hydrolase, a phosphatase, and a kinase.

[0336] (Cl) A method for enzymatic glycosylation of a steviol glycoside, comprising contacting a glycosyltransferase and a sucrose synthase with a starting composition comprising one or more steviol glycosides, exogenous sugar donor, and an exogenous nucleoside triphosphate (NTP).

[0337] (C2) The method of (Cl), wherein the exogenous NTP is one of adenosine triphosphate, guanidine triphosphate, uridine triphosphate, cytidine triphosphate, and thymidine triphosphate.

[0338] (C3) The method of either (Cl) or (C2), wherein the starting composition comprises the NTP at a concentration of about 0.1 mM to about 10 mM.

[0339] (C4) The method of any one of (Cl) to (C3), wherein the starting composition comprises two or more steviol glycosides, each present at a concentration of at least 5 gram per liter.

[0340] (C5) The method of any one of (Cl) to (C4), wherein the one or more steviol glycosides comprise one or more naturally-occurring steviol glycosides selected from the group consisting of steviol- 13-O-glucosi de, steviol-19-O-glucoside, rubusoside, steviol-l,2-bioside, steviol-l,3-bioside, rubusoside, dulcoside B, dulcoside A, rebaudioside B, rebaudioside G, stevioside, rebaudioside C, rebaudioside F, rebaudioside A, rebaudioside I, rebaudioside E, rebaudioside E2, rebaudioside AM, rebaudioside H, rebaudioside L, rebaudioside K, rebaudioside J, rebaudioside M, rebaudioside D, rebaudioside N, rebaudioside O, and rebaudioside Q, and one or more enzymatically glycosylated steviol glycosides, and combinations thereof.

[0341] (C6) The method of any one of (Cl) to (C5), wherein the starting composition can be synthetic or at least partially purified, commercially available or prepared.

[0342] (C7) The method of any one of (Cl) to (C6), wherein the one or more steviol glycosides in the starting composition are in a stevia leaf extract.

[0343] (C8) The method of (C7), wherein the stevia leaf extract is produced by a process comprising one or more steps of extracting, clarifying, and concentrating.

[0344] (C9) The method of (C5), where the one or more steviol glycosides comprise stevioside and rebaudioside A.

[0345] (CIO) The method of (C9), wherein the starting composition comprises at least 30 g / L stevioside and at least 50 g / L rebaudioside A.

[0346] (Cl 1) The method of (C9), wherein the starting composition comprises at least 6 g / L stevioside and at least 36 g / L rebaudioside A.

[0347] (Cl 2) The method of (C9), wherein the starting composition comprises at least 8 g / L stevioside and at least 48 g / L rebaudioside A.

[0348] (Cl 3) The method of (C9), wherein the starting composition comprises at least 10 g / L stevioside and at least 60 g / L rebaudioside A.

[0349] (Cl 4) The method of (C9), wherein the starting composition comprises at least 12 g / L stevioside and at least 72 g / L rebaudioside A.

[0350] (Cl 5) The method of (C5), wherein the one or more steviol glycosides comprise rebaudioside A.

[0351] (C16) The method of (C15), wherein the starting composition comprises at least 36 g / L rebaudioside A.

[0352] (Cl 7) The method of any one of (Cl) to (Cl 6), wherein the exogenous sugar donor is a disaccharide or a polysaccharide.

[0353] (Cl 8) The method of (C17), wherein the exogenous sugar donor is at least one selected from the group consisting of: sucrose, starch, maltose, alpha-glucose- 1 -phosphate, betaglucose- 1 -phosphate, cellobiose, gentiobiose, trehalose, kojibiose, nigerose, isomaltose, betabeta-trehalose, alpha-beta-trehalose, sophorose, laminaribiose, turanose, maltulose, palatinose, gentiobiulose, nigerotriose, maltotriose, melezitose, maltotriulose, kestose, cellulose, glycogen, amylose, amylopectin, dextran, dextrin, maltodextrin, glucose syrup, cellodextrin, and cyclodextrin.

[0354] (Cl 9) The method of (C8), wherein the concentrating comprises membrane processing such as nanofiltration.

[0355] (C20) The method of (C8), wherein the concentrating comprises evaporation.

[0356] (C21) The method of (C8), wherein the concentrating comprises adsorption of one or more steviol glycosides onto a solid adsorbent, elution with an alcohol, and removal of the alcohol to give a concentrated leaf extract.

[0357] (C22) The method of (C8), wherein the stevia leaf extract is extracted by one or more of a batch parabolic trough with a helical mixer, a batch rotating cylindrical extractor, a batch percolation extractor, horizontal continuous moving belt extractor, a horizontal countercurrent tubular screw extractor, a vertical countercurrent tubular screw extractor, a segmented rotating basket extractor, and a pressurized condensing steam extractor.

[0358] (C23) The method of (C8), wherein the clarifying comprises one of more of thermal coagulation of proteins and impurities, diatomaceous earth treatment, ion exchange, chemical coagulation using calcium carbonate and / or ferric chloride.

[0359] (C24) The method of (Cl), wherein at least one of the one or more steviol glycosides of the starting composition are a fermentation derived steviol glycoside.

[0360] (C25) The method of (Cl), wherein the enzymatic glycosylation of the steviol glycoside produces one or more glycosylated steviol glycosides.

[0361] (C26) The method of (C25), wherein the one or more glycosylated steviol glycosides comprise one or more steviol glycosides selected from the group consisting of steviol-13-O- glucoside, steviol- 19-O-glucosi de, rubusoside, steviol- 1,2-bioside, steviol-l,3-bioside, rubusoside, dulcoside B, dulcoside A, rebaudioside B, rebaudioside G, stevioside, rebaudioside C, rebaudioside F, rebaudioside A, rebaudioside I, rebaudioside E, rebaudioside E2, rebaudioside AM, rebaudioside H, rebaudioside L, rebaudioside K, rebaudioside J, rebaudioside M, rebaudioside D, rebaudioside N, rebaudioside O, and rebaudioside Q, and one or more enzymatically glycosylated steviol glycosides, and combinations thereof.

[0362] (C27) A method of glycosylating an acceptor, comprising incubating the acceptor with a glycosyltransferase in an aqueous reaction medium, wherein the aqueous reaction medium comprises adenosine triphosphate (ATP), a sugar donor, an ADP-sugar synthase, the acceptor, and a buffer.

[0363] (C28) The method of (C27), where the ATP is hydrolyzed to adenosine diphosphate (ADP).

[0364] (C29) The method of (C28), wherein the ADP-sugar synthase produces an ADP-sugar from the ADP and the sugar donor.

[0365] (C30) The method of (C29), wherein the glycosyltransferase glycosylates the acceptor with the sugar moiety of the ADP-sugar.

[0366] (C31) The method of (C28), wherein the ATP is hydrolyzed by a chemical process or an enzymatic process.

[0367] (C32) The method of (C27), wherein the concentration of ATP in the aqueous reaction medium is 0.01 and 10 mM.

[0368] (C33) The method of (C27), wherein the acceptor is selected from the group consisting of a: protein, a lipid, steviol, a steviol glycoside, a polyketide, a steroid, and a polyphenol.

[0369] (C34) The method of (C27), wherein the sugar donor is selected from the group consisting of: oligomers, sucrose, starch, maltose, alpha-glucose- 1 -phosphate, beta-glucose-1- phosphate, cellobiose, gentiobiose, trehalose, kojibiose, nigerose, isomaltose, beta-beta-trehalose, alpha-beta-trehalose, sophorose, laminaribiose, turanose, maltulose, palatinose, gentiobiulose, nigerotriose, maltotriose, melezitose, maltotriulose, kestose, cellulose, glycogen, amylose, amylopectin, dextran, dextrin, maltodextrin, glucose syrup, cellodextrin, and cyclodextrin.

Claims

CLAIMS:

1. A method for glycosylation of a sugar acceptor, comprising the steps of:(i) converting an exogenous nucleoside triphosphate (NTP) to a nucleoside diphosphate (NDP);(ii) converting the NDP to NDP-sugar, comprising contacting at least one Leloir glycosyltransferase (GT) with the NDP and an exogenous sugar donor (SD) in a reaction medium; and(iii) converting a sugar acceptor (SA) to a glycosylated SA, comprising contacting the at least one GT with the NDP-sugar and the SA in the reaction medium.

2. A method for glycosylation of a steviol glycoside, comprising the steps of:(i) converting an exogenous adenosine triphosphate (ATP) to an adenosine diphosphate (ADP);(ii) converting the ADP to ADP -glucose, comprising contacting at least one Leloir glycosyltransferase (GT) with the ADP and exogenous sucrose (SD) in a reaction medium; and(iii) converting the steviol glycoside (SA) to a glycosylated SA, comprising contacting the at least one GT with the ADP -glucose and the SA in the reaction medium.

3. The method of claim 1 or 2, wherein the reaction medium comprises the at least one GT, the SA, and the SD.

4. The method of claim 1 or 2, wherein the total amount of the SD in the reaction medium is between about 50 mM to about 1 M based on a final volume of the reaction medium.

5. The method of claim 1 or 2, wherein the molar ratio of the SD to the SA in the reaction medium is at least about 2:1.

6. The method of claim 3, further comprising supplementing the reaction medium with additional SA.

7. The method of claim 6, wherein the reaction medium is supplemented with the additional SA continuously or in batches.

8. The method of claim 1, 2, or 6, wherein the total amount of the SA added to the reaction medium is between about 5 millimoles and about 200 millimoles per liter of the final volume of the reaction medium.

9. The method of claim 1, wherein converting the NTP to NDP comprises one or more of non-enzymatic conversion or enzymatic conversion.

10. The method of claim 2, wherein converting the ATP to ADP comprises one or more of non-enzymatic conversion or enzymatic conversion.

11. The method of claim 9 or 10, wherein the conversion comprises enzymatic conversion and the reaction medium further comprises a phosphatase or a kinase.

12. The method of claim 11, wherein the phosphatase is a phytase.

13. The method of claim 9 or 10, wherein the conversion comprises enzymatic conversion and the reaction medium further comprises a phosphate acceptor.

14. The method of claim 13, wherein the phosphate acceptor is a kinase substrate.

15. The method of claim 14, wherein the kinase substrate is selected from the group consisting of one or more of acetate, glycerol, glucose, fructose, pyruvate, and succinate, and combinations thereof.

16. The method of claim 11, wherein the reaction medium comprises a kinase.

17. The method of claim 1 or 2, wherein the at least one GT comprises a P-1, 2- glycosyltransferase, a P-l,3-glycosyltransferase, or a sucrose synthase.

18. The method of claim 1 or 2, wherein the at least one GT comprises an NDP-sugar synthase.

19. The method of claim 1 or 2, wherein the at least one GT comprises a sucrose synthase and one or more of a P-l,2-glycosyltransferase and a P-l,3-glycosyltransferase.

20. The method of claim 1, wherein the molar ratio of NTP:SA is between about 0.00045:1 and about 0.1 :1.

21. The method of claim 1, wherein the molar ratio of NTP:GT is between about 0.55:1 and about 5500: 1.

22. The method of claim 2, wherein the molar ratio of ATP:GT is between about 0.55:1 and about 5500: 1.

23. The method of claim 1 or 2, wherein the method is performed under isothermal conditions.

24. The method of claim 1 or 2, wherein the method is performed at a temperature between about 50°C and about 75°C.

25. The method of claim 1 or 2, wherein the reaction medium further comprises a buffer.

26. The method of claim 25, wherein the method is performed at a pH of between about 6 and about 6.

527. The method of claim 25, wherein the buffer is selected from one of acetate, citrate, lactate, succinate, or phosphate.

28. The method of claim 1, wherein the NTP is one of adenosine triphosphate, guanidine triphosphate, uridine triphosphate, cytidine triphosphate, and thymidine triphosphate.

29. The method of claim 1, wherein the total amount of the NTP added is between about 0.1 millimoles to about 10 millimoles per liter of the final volume of the reaction medium.

30. The method of claim 2, wherein the total amount of the ATP added is between about 0.1 millimoles and about 10 millimoles per liter of the final volume of the reaction medium.

31. The method of claim 1 or 2, wherein the SA is selected from the group consisting of one or more of a protein, a lipid, a flavanone, steviol, a steviol glycoside, a polyketide, a steroid, a polyphenol, and combinations thereof.

32. The method of claim 1 or 2, wherein the SA comprises a steroid selected from the group consisting of one or more of cholesterol, cholesterol-P-D-glucoside, cholesteroldiglucoside, campesterol, stigmasterol, sitosterol, and P-sitosterol-P-D-glucoside, and combinations thereof33. The method of claim 1 or 2, wherein the SA comprises a flavonoid selected from the group consisting of fisetin, luteolin, luteolinidin, apigenin, quercetin, quercetin-3-P-D- glucoside, quercetin 3,4’-diglucoside, prunin, spiraeoside.

34. The method of claim 1 or 2, wherein the SD is a disaccharide or a polysaccharide.

35. The method of claim 34, wherein the SD is at least one selected from the group consisting of: sucrose, starch, maltose, lactose, alpha-glucose- 1 -phosphate, beta-glucose-1- phosphate, cellobiose, gentiobiose, trehalose, kojibiose, nigerose, isomaltose, beta-beta- trehalose, alpha-beta-trehalose, sophorose, laminaribiose, turanose, maltulose, palatinose, gentiobiulose, nigerotriose, maltotriose, melezitose, maltotriulose, kestose, cellulose, glycogen, amylose, amylopectin, dextran, dextrin, arabinoxylan, maltodextrin, glucose syrup, cellodextrin, and cyclodextrin.

36. The method of claim 1 or 2, wherein the at least one GT is derived from a natural or engineered microorganism.

37. The method of claim 36, wherein the at least one GT is a crude enzyme or an at least partially purified enzyme.

38. The method of claim 36, wherein the at least one GT further comprises one or more additional enzymes produced by the natural or engineered microorganism.

39. The method of claim 38, wherein the one or more additional enzymes comprises at least one selected from the group consisting of enzymes having phosphatase activity and enzymes having kinase activity40. The method of claim 38, wherein the one or more additional enzymes comprises at least one selected from the group consisting of phosphatases and kinases.

41. The method of claim 37, wherein the at least one GT is a highly purified enzyme.

42. The method of claim 41, wherein the highly purified enzyme is purified by affinity chromatography and / or size exclusion chromatography.

43. The method of claim 2, wherein fewer than 10% of phosphorylated adenosines in the reaction medium are adenosine monophosphate (AMP).

44. The method of claim 1, wherein the molar ratio of the total amount of NTP:SA added is at most 0.2: 1.

45. The method of claim 2, wherein the molar ratio of ADP-glucose:ADP during the reaction is at least 1:1.

46. The method of claim 2, wherein the molar ratio of the total amount of ATP:SG added is at most 0.2: 1.

47. The method of claim 1, wherein the molar ratio of the total amount of NTP:SG added is between about 0.00045: 1 and about 0.2:1.

48. The method of claim 2, wherein the molar ratio of the total amount of ATP:SG added is between about 0.00045: 1 and about 0.2:1.

49. The method of claim 2, wherein the SA comprises one or more steviol glycosides selected from the group consisting of steviol- 13 -O-glucoside, steviol-19-O-glucoside, rubusoside, steviol-l,2-bioside, steviol-l,3-bioside, rubusoside, dulcoside B, dulcoside A, rebaudioside B, rebaudioside G, stevioside, rebaudioside C, rebaudioside F, rebaudioside A,rebaudioside I, rebaudioside E, rebaudioside E2, rebaudioside AM, rebaudioside H, rebaudioside L, rebaudioside K, rebaudioside J, rebaudioside M, rebaudioside D, rebaudioside N, rebaudioside O, and rebaudioside Q, or one or more enzymatically glycosylated steviol glycosides.

50. The method of claim 49, where the one or more steviol glycosides comprise stevioside and rebaudioside A.

51. The method of claim 50, wherein the reaction medium comprises at least 6 g / L stevioside and at least 36 g / L rebaudioside A.

52. The method of claim 49, wherein the one or more steviol glycosides comprise rebaudioside A.

53. The method of claim 52, wherein the reaction medium comprises at least 36 g / L rebaudioside A.

54. The method of claim 2, wherein the glycosylated SA comprises one or more steviol glycosides selected from the group consisting of steviol-13-O-glucoside, steviol-19-O- glucoside, rubusoside, steviol-l,2-bioside, steviol-l,3-bioside, rubusoside, dulcoside B, dulcoside A, rebaudioside B, rebaudioside G, stevioside, rebaudioside C, rebaudioside F, rebaudioside A, rebaudioside I, rebaudioside E, rebaudioside E2, rebaudioside AM, rebaudioside H, rebaudioside L, rebaudioside K, rebaudioside J, rebaudioside M, rebaudioside D, rebaudioside N, rebaudioside O, and rebaudioside Q, or one or more enzymatically glycosylated steviol glycosides.

55. The method of claim 1, wherein the total amount of the NTP added is at most 10 millimoles per liter of the final volume of the reaction medium.

56. The method of claim 2, wherein the total amount of the ATP added is at most 10 millimoles per liter of the final volume of the reaction medium.

57. The method of claim 2, wherein the reaction medium comprises steviol glycoside in a stevia leaf extract.

58. The method of claim 57, wherein the stevia leaf extract is produced by a process comprising one or more steps of extracting, clarifying, and concentrating.

59. The method of claim 57, wherein the concentrating comprises membrane processing such as nanofiltration.

60. The method of claim 57, wherein the concentrating comprises evaporation.

61. The method of claim 57, wherein the concentrating comprises adsorption of one or more steviol glycosides onto a solid adsorbent, elution with an alcohol, and removal of the alcohol to give a concentrated leaf extract.

62. The method of claim 57, wherein the stevia leaf extract is extracted by one or more of a batch parabolic trough with a helical mixer, a batch rotating cylindrical extractor, a batch percolation extractor, horizontal continuous moving belt extractor, a horizontal countercurrent tubular screw extractor, a vertical countercurrent tubular screw extractor, a segmented rotating basket extractor, and a pressurized condensing steam extractor.

63. The method of claim 57, wherein the clarifying comprises one of more of thermal coagulation of proteins and impurities, diatomaceous earth treatment, ion exchange, chemical coagulation using calcium carbonate and / or ferric chloride.

64. A method for glycosylation of a steviol glycoside, comprising the steps of:(i) converting an exogenous adenosine triphosphate (ATP) to adenosine diphosphate (ADP);(ii) converting the ADP to ADP -glucose, comprising contacting a sucrose synthase (SuSy) with the ADP and exogenous sucrose in a reaction medium; and(iii) converting at least one steviol glycoside (SG) to a glycosylated SG, comprising contacting the SG with the ADP -glucose and at least one glycosyltransferase (GT) selected from the group consisting of P-l,2-glycosyltransferase, P-1, 3 -glycosyltransferase, or a combination thereof in the reaction medium.

65. The method of claim 64, wherein the reaction medium initially comprises the SuSy, the at least one GT, the SG, and the sucrose.

66. The method of claim 64, wherein the total amount of the sucrose in the reaction medium is between about 50 mM to about 1 M based on the final volume of the reaction medium.

67. The method of claim 64, wherein a molar ratio of the total amount of the sucrose and the SG added is at least about 4: 1.

68. The method of claim 65, further comprising supplementing the reaction medium with additional SA.

69. The method of claim 68, wherein the reaction medium is supplemented with the additional SA continuously or in batches.

70. The method of claim 64 or 68, wherein the total amount of the SG added to the reaction medium is between about 0.5 millimoles and about 200 millimoles per liter of the final volume of the reaction medium.

71. The method of claim 64, wherein the SG is stevioside and the glycosylated SG is at least one of rebaudioside A, rebaudioside E, or rebaudioside E2.

72. The method of claim 64, wherein the SG is rebaudioside A and the glycosylated SG is at least one of rebaudioside D or rebaudioside I.

73. The method of claim 64, wherein the SG is rebaudioside E and the glycosylated SG is at least one of rebaudioside D or rebaudioside AM.

74. The method of claim 64, wherein the SG is rebaudioside E2 and the glycosylated SG is at least one of rebaudioside I or rebaudioside AM.

75. The method of claim 64, wherein the SG is rebaudioside I and the glycosylated SG is rebaudioside M.

76. The method of claim 64, wherein the SG is rebaudioside D and the glycosylated SG is rebaudioside M.

77. The method of claim 64, wherein the SG is rebaudioside AM and the glycosylated SG is rebaudioside M.

78. The method of claim 64, wherein the SG is stevioside and rebaudioside A and the glycosylated SG is rebaudioside M.

79. The method of claim 64, wherein the SG is rebaudioside A and the glycosylated SG is rebaudioside M.

80. The method of claim 64, wherein the SG is stevioside and the glycosylated SG is rebaudioside M.

81. The method of claim 64, wherein converting the ADP to ADP-glucose occurs at a rate of at least about 3 mM / hr.

82. The method of claim 64, wherein converting the ATP to the ADP initially occurs at a rate of between about 0.1 mM / hr and about 1 mM / hr.

83. The method of claim 64, wherein converting the SG to the glycosylated SG occurs at a rate of between about 0.25 mM / hr and about 30 mM / hr.

84. A method for glycosylation of a sugar acceptor, comprising the steps of:(i) converting an exogenous nucleoside triphosphate (NTP) to a nucleoside diphosphate (NDP);(ii) converting the NDP to NDP-sugar, comprising contacting at least one Leloir glycosyltransferase (GT) with the NDP and an exogenous sugar donor (SD); and(iii) converting a sugar acceptor (SA) to a glycosylated SA, comprising contacting the at least one GT with the NDP-sugar and the SA.

85. The method of claim 84, wherein (i)-(iii) are performed simultaneously in a reaction medium.

86. The method of claim 84, wherein (i)-(iii) are performed independently.

87. The method of claim 84, wherein (i) is performed in a first reaction medium, and (ii) and (iii) are performed in a second reaction medium comprising the first reaction medium.

88. The method of claim 87, wherein the first reaction medium initially comprises the NTP.

89. The method of claim 87, wherein the first reaction medium finally comprises the NDP, and wherein the second reaction medium further comprises the at least one GT, the SD, and the SA.

90. The method of claim 84, wherein (i) and (ii) are performed to completion in a first reaction medium and (iii) is performed in a second reaction medium comprising the first reaction medium.

91. The method of claim 90, wherein the first reaction medium initially comprises the NTP, at least one GT, and the SD.

92. The method of claim 91, wherein the first reaction medium finally comprises the NDP-sugar and the at least one GT, and wherein the second reaction medium further comprises the SA.

93. The method of claim 84, wherein (i)-(iii) are performed in a reaction medium initially comprising the NTP, the at least one GT, and the SD.

94. The method of claim 84, wherein one or more of the at least one GT, the SD, and the SA are added to the reaction medium after (i).

95. The method of claim 94, wherein the added one or more of the at least one GT, the SD, and the SA are added to the reaction medium continuously or in batches.

96. The method of claim 84, wherein (i) is performed in a first reaction medium, and the method further comprises purifying the NDP from the reaction medium.

97. The method of claim 96, wherein (ii) and (iii) are performed in a second reaction medium initially comprising the purified NDP.

98. The method of claim 84, wherein (i) and (ii) are performed to completion in a first reaction medium and (ii) and (iii) are performed in a second reaction medium comprising the first reaction medium.

99. The method of claim 98, wherein the first reaction medium initially comprises the NTP, at least one GT, and the SD.

100. The method of claim 99, wherein the first reaction medium finally comprises the NDP-sugar, the at least one GT, and the SD, and wherein the second reaction medium further comprises the SA.

101. The method of claim 1 or 2, wherein the NTP or ATP is provided as a purified product.

102. The method of claim 1 or 2, wherein the NTP or ATP is provided as a crude fermentation broth.

103. The method of claim 1 or 2, wherein the NTP or ATP is provided as a partially purified fermentation broth.

104. The method of claims 101-103, wherein the NTP contains NTP, NDP, and NMP or the ATP contains ATP, ADP and AMP.

105. The method of claims 101-104, wherein the NTP or ATP is a powder.

106. The method of claims 101-104, wherein the NTP or ATP is a frozen aqueous solution.

107. The method of claim 106, wherein the NTP or ATP frozen aqueous solution contains stabilizers including at least one selected from the group consisting of buffers, sodium acetate, or glycerol.

108. The method of claim 2, wherein the ATP is provided as a clarified fermentation broth produced by Corynebacterium stationis.

109. The method of claim 2, wherein the ATP is a clarified fermentation broth that has been clarified by a process including at least one ultrafiltration or microfiltration.

110. The method of claim 2, wherein the ATP is a clarified fermentation broth containing ATP, ADP, and AMP that has been purified by a process that includes ion exchange to produce an ATP salt solution.

Images