Biosynthetic production of variant steviol glycosides

Abstract

The present invention relates to novel steviol glycosides R6-1, R6-2A, R6-2B, R6-4A, R6-4B and R7-2, and to the production of these novel steviol glycosides, for example, by enzymatic biotransformation. Also provided are the uses of these novel steviol glycosides as sweeteners and in orally consumable products.

Description

[0001] Related Applications

[0002] This application claims the benefit of U.S. Provisional Application No. 62 / 778,422, filed Dec. 12, 2018, and entitled "BIOSYNTHETIC PRODUCTION OF VARIANT STEVIOL GLYCOSIDES" under 35 U.S.C. § 119(e), the entire content of which is incorporated herein by reference. FIELD OF THE INVENTION

[0003] The field of the invention relates to methods and processes for the production of several specific steviol glycosides via enzymatic conversion, and related compositions. BACKGROUND OF THE INVENTION

[0004] Several steviol glycosides are found as compounds in the leaves of Stevia rebaudiana, and several of these have been widely used as high-intensity, low-calorie sweeteners in foods, feeds, and beverages. These naturally occurring steviol glycosides have the same basic diterpene structure (the steviol backbone), but differ in the number and structure of their carbohydrate residues (e.g., glucose, rhamnose, and xylose residues) at the C13 and C19 positions of the steviol backbone. Interestingly, these variations in the sugar 'ornamentation' of the basic steviol structure can affect the properties of the individual steviol glycosides themselves. These properties can include, but are not limited to: taste characteristics, crystallization point, solubility, mouthfeel, and perceived sweetness, among other differences. Steviol glycosides with known structures include stevioside, rebaudioside A, rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside E, rebaudioside I, rebaudioside M, rebaudioside D3, rebaudioside N, and rebaudioside O. In terms of commercial use, rebaudioside D and M have been recognized as generally regarded as safe (i.e., it has 'GRAS' status), and are being studied for a wide range of uses in the food and beverage markets.

[0005] On a dry weight basis, stevioside, rebaudioside A, rebaudioside C, and dulcoside A account for 9.1, 3.8, 0.6, and 0.30 percent, respectively, of the total weight of steviol glycosides found in wild-type Stevia leaves, while other steviol glycosides, such as Reb D and Reb M, are present in significantly lower amounts. Extracts from Stevia plants are commercially available, and in such extracts, stevioside and rebaudioside A are most often the major components and can be used as starting components or substrates for further enzymatic activity. In contrast, other known steviol glycosides are typically present in Stevia extracts as minor or trace components. For example, in a typical commercial preparation, the amount of rebaudioside A can range from about 20% to more than 90% of the total steviol glycoside content, while the amount of rebaudioside B is about 1 - 2%, the amount of rebaudioside C is about 7 - 15%, and the amount of rebaudioside D can be about 2% of the total steviol glycosides.

[0006] Each different steviol glycoside can have a different degree of sweetness, 'taste', and specific aftertaste associated therewith. Relative to table sugar (i.e., "sucrose"), steviol glycosides generally have a significantly higher sweetness. For example, stevioside has a sweetness that is 100 - 150 times that of sucrose but has a bitter aftertaste as noted in numerous taste tests, while rebaudioside A and E have a sweetness that is 250 - 450 times that of sucrose and have much better aftertaste characteristics than stevioside. However, these steviol glycosides still retain an obvious aftertaste and are naturally suited for those applications in the food and beverage industries where this aftertaste or the difference from sucrose can be masked or eliminated. Additionally, the overall taste characteristics of plant-derived Stevia extracts can be affected by the various steviol glycosides present in the extract, which in turn can be influenced by the environmental conditions experienced by the underlying plant and the extraction process used. These variations in plant production, weather conditions, and extraction conditions can lead to inconsistent compositions of steviol glycosides in Stevia extracts, such that the taste characteristics can vary strongly among different batches of extracted products. In such cases, these batches may not be suitable for specific uses or formulations in the food and beverage industries. The taste characteristics of Stevia extracts can also be affected by plant-derived or environment-derived contaminants (such as pigments, lipids, proteins, phenols, and sugars) remaining in the product after the extraction process. These contaminants typically have their own off-flavors and can render the resulting extract undesirable for use in consumer products. Additionally, the cost of isolating individual or specific combinations of steviol rebaudiosides that are not abundant or not present at all in Stevia extracts can be prohibitive in terms of cost and resources.

[0007] Generally, steviol glycosides are formed through a series of glycosylation reactions of steviol, which are usually catalyzed by UDP - glycosyltransferase (UGT) enzymes that use uridine 5'-diphosphate glucose (UDP - glucose) as the donor of the sugar moiety. In plants, UGTs are a very divergent group of enzymes that can transfer glucose residues from UDP - glucose to steviol. In these reactions, stevioside is often an intermediate in the biosynthesis of various rebaudioside compounds. For example, glycosylation of stevioside at the C - 3' of the C - 13 - O - glucose of stevioside yields rebaudioside A; while glycosylation at the C - 2' of the 19 - O - glucose position of stevioside yields rebaudioside E.

[0008] Previously, it has been described that Reb D3 (13 - [(2 - O - β - D - glucopyranosyl - 6 - O - β - D - glucopyranosyl - β - D - glucopyranosyloxy)] ent - kaur - 16 - ene - 19 - acid - (2 - O - β - D - glucopyranosyl - β - D - glucopyranosyl) ester) can be converted from Reb E by EUGT11 or EUS enzymes. Reb Z (Z1 and Z2) can be converted from Reb E by HV1 enzyme. A mixture of two compounds named Reb Z was characterized as 13 - [(2 - O - β - D - glucopyranosyl - 2 - O - β - D - glucopyranosyl - β - D - glucopyranosyloxy)] ent - kaur - 16 - ene - 19 - acid - 2 - O - β - D - glucopyranosyl - β - D - glucopyranosyl ester (Reb Z1), or 13 - [(2 - O - β - D - glucopyranosyl - β - D - glucopyranosyloxy)] ent - kaur - 16 - ene - 19 - acid - [(2 - O - β - D - glucopyranosyl - 2 - O - β - D - glucopyranosyl - β - D - glucopyranosyl) ester (Reb Z2).

[0009] The steviol glycoside biosynthetic pathway involves the conversion of ent - kaurenoic acid to steviol through the activity of the enzyme KAH. UGT76G1 has been shown to exhibit glycosylation activity towards steviol diglycosides to form rebaudioside B, as well as glycosylation activity towards stevioside, resulting in the production of rebaudioside A. Additionally, the interaction affinities of KAH, UGT85C2, UGT74G1, and UGT76G1 have been evaluated for ent - kaurenoic acid and steviol. The model for KAH showed the highest affinity for the ligand steviol, followed by steviol - monoglycoside and ent - kaurenoic acid. The docking results for the three - dimensional model of UGT76G1 suggested its highest binding affinity for ent - kaurenoic acid, but also suggested that these enzymes have the ability to interact with more than one ligand in the steviol glycoside biosynthetic pathway.

[0010] A practical way to improve the taste quality of stevia extracts is to increase the yield of those rebaudioside compounds that generally have more desirable taste characteristics, and to achieve this via a more productive synthetic pathway. Among the steviol glycosides tested, many consider Reb M to have the most desirable taste and chemical characteristics for use in a variety of foods and beverages. However, as stated above, the plant contains extremely small amounts of this compound in its leaves.

[0011] Furthermore, extraction processes from plants typically employ solid-liquid extraction techniques, using solvents such as hexane, chloroform, and ethanol for steviol glycoside recovery (Catchpole et al., 2003). However, solvent extraction itself is energy-intensive, leading to problems of toxic waste disposal, requires extensive growing areas for the plants themselves, and produces products that require further purification / modification or bioconversion. The use of fermentation and / or enzymatic bioconversion techniques can allow the production of steviol glycosides in microbial species, which can increase the selectivity, abundance, and purity of the desired steviol glycosides.

[0012] In addition to the above, while consumers recognize and actively seek natural and biological sources of food, feed, flavor, or medicinal components, they are also concerned with sourcing, consistent taste characteristics, and environmentally sustainable production. The microbial fermentation and production methods of the present invention can provide rebaudioside in quantities useful for a variety of industries and research, while achieving this in a more natural way than inorganic synthesis or current plant extraction techniques.

[0013] Accordingly, there is a need to develop novel steviol glycoside variants and related production methods that can be carried out economically and conveniently to further enable human and animal consumption. SUMMARY OF THE INVENTION

[0014] The present disclosure relates at least in part to the identification, synthesis, and production of several steviol glycosides from various rebaudiosides. In some aspects, the present disclosure provides the use of Reb D3 in the production of Reb R7-2 or Reb R6-2. In some aspects, the present disclosure provides the use of Reb D in the production of Reb R6-1. In some aspects, the present disclosure provides the use of Reb Z1 or Reb Z2 as a raw material for the production of Reb R6-4A. In some aspects, the present disclosure provides the use of Reb Z2 as a raw material for the production of R6-4B. The product steviol glycosides are identified by NMR analysis and are subjected to various taste tests and processing tests after production to identify their specific flavor and performance characteristics.

[0015] Some aspects of the present disclosure provide methods for producing rebaudioside R6-2A and / or R6-2B, the methods comprising:

[0016] (I) preparing a reaction mixture comprising:

[0017] (i) rebaudioside D3;

[0018] (ii) one or more substrates selected from sucrose, uridine diphosphate (UDP), uridine diphosphate-glucose (UDP-glucose), and combinations thereof; and

[0019] (iii) an enzyme selected from:

[0020] (a) UDP-glycosyltransferase (UGT);

[0021] (b) UDP-glycosyltransferase and sucrose synthase separately added to the reaction mixture; and

[0022] (c) a UDP-glycosyltransferase fusion enzyme comprising a UDP-glycosyltransferase domain coupled to a sucrose synthase domain; and (II) incubating the reaction mixture for a sufficient time to produce rebaudioside R6-2A and / or R6-2B;

[0023] wherein the rebaudioside D3 has the following structure:

[0024]

[0025] Rebaudioside R6-2A has the following structure:

[0026] and

[0027] Rebaudioside R6-2B has the following structure:

[0028]

[0029] Other aspects of the present disclosure provide methods for producing rebaudioside R7-2, the methods comprising:

[0030] (I) preparing a reaction mixture comprising:

[0031] (i) one or more of rebaudioside D3, rebaudioside R6-2A, and rebaudioside R6-2B;

[0032] (ii) one or more substrates selected from sucrose, uridine diphosphate (UDP), uridine diphosphate-glucose (UDP-glucose), and combinations thereof; and

[0033] (iii) an enzyme selected from:

[0034] (a) UDP - glycosyltransferase (UGT);

[0035] (b) UDP - glycosyltransferase and sucrose synthase separately added to the reaction mixture; and

[0036] (c) A UDP - glycosyltransferase fusion enzyme comprising a UDP - glycosyltransferase domain coupled to a sucrose synthase domain; and

[0037] (II) Incubating the reaction mixture for a sufficient time to produce rebaudioside R7 - 2;

[0038] Wherein said rebaudioside D3 has the following structure:

[0039]

[0040] Rebaudioside R6 - 2A has the following structure:

[0041]

[0042] Rebaudioside R6 - 2B has the following structure:

[0043] And

[0044] Rebaudioside R7 - 2 has the following structure:

[0045]

[0046] In some embodiments, the sucrose synthase or sucrose synthase domain is selected from Arabidopsis sucrose synthase I, Arabidopsis sucrose synthase 3, and Vigna radiate sucrose synthase. In some embodiments, the sucrose synthase or sucrose synthase domain is Arabidopsis thaliana sucrose synthase I. In some embodiments, the sucrose synthase or sucrose synthase domain has at least 80% identity with the amino acid sequence of SEQ ID NO:9. In some embodiments, the sucrose synthase or sucrose synthase domain comprises the amino acid sequence of SEQ ID NO:9.

[0047] In some embodiments, glucose is covalently coupled to rebaudioside D3 by an enzyme to produce rebaudioside R6-2A and / or R6-2B. In some embodiments, glucose is covalently coupled to sugar I of rebaudioside D3 by an enzyme to produce rebaudioside R6-2A. In some embodiments, glucose is covalently coupled to sugar II of rebaudioside D3 by an enzyme to produce rebaudioside R6-2B. In some embodiments, two glucose molecules are covalently coupled to rebaudioside D3 by an enzyme to produce rebaudioside R7-2. In some embodiments, two glucose molecules are covalently coupled to sugar I and sugar II of rebaudioside D3 by an enzyme to produce rebaudioside R7-2.

[0048] The UDP-glycosyltransferase has at least 80% identity with the amino acid sequence of SEQ ID NO:1. In some embodiments, the UDP-glycosyltransferase comprises the amino acid sequence of SEQ ID NO:1. In some embodiments, the UDP-glycosyltransferase fusion enzyme has at least 80% identity with the amino acid sequence of SEQ ID NO:5. In some embodiments, the UDP-glycosyltransferase fusion enzyme comprises the amino acid sequence of SEQ ID NO:5.

[0049] In some embodiments, the method further comprises producing rebaudioside D3 by incubating rebaudioside E with a UDP-glycosyltransferase and a substrate selected from sucrose, UDP, UDP-glucose, and combinations thereof.

[0050] Also provided herein is a method for producing rebaudioside R6-4A and / or rebaudioside R6-4B, the method comprising:

[0051] (I) preparing a reaction mixture comprising:

[0052] (i) at least one of rebaudioside Z1 and rebaudioside Z2;

[0053] (ii) one or more substrates selected from sucrose, uridine diphosphate (UDP), uridine diphosphate-glucose (UDP-glucose), and combinations thereof; and

[0054] (iii) an enzyme selected from:

[0055] (a) UDP-glycosyltransferase (UGT);

[0056] (b) UDP-glycosyltransferase and sucrose synthase separately added to the reaction mixture; and

[0057] (c) a UDP-glycosyltransferase fusion enzyme comprising a UDP-glycosyltransferase domain coupled to a sucrose synthase domain; and

[0058] (II) Incubate the reaction mixture for a sufficient time to produce rebaudioside R6-4A and / or rebaudioside R6-4B;

[0059] wherein said rebaudioside Z1 has the following structure:

[0060]

[0061] Rebaudioside Z2 has the following structure:

[0062] Rebaudioside R6-4A has the following structure:

[0063] and

[0064] Rebaudioside R6-4B has the following structure:

[0065]

[0066] In some embodiments of any of the methods or compositions provided herein, the sucrose synthase or sucrose synthase domain is selected from Arabidopsis thaliana sucrose synthase I, Arabidopsis thaliana sucrose synthase 3, and mung bean sucrose synthase. In some embodiments of any of the methods or compositions provided herein, the sucrose synthase or sucrose synthase domain is Arabidopsis thaliana sucrose synthase I. In some embodiments of any of the methods or compositions provided herein, the sucrose synthase or sucrose synthase domain has at least 80% identity with the amino acid sequence of SEQ ID NO:9. In some embodiments of any of the methods or compositions provided herein, the sucrose synthase or sucrose synthase domain comprises the amino acid sequence of SEQ ID NO:9.

[0067] In some embodiments of any of the methods or compositions provided herein, glucose is covalently coupled to rebaudioside Z1 or rebaudioside Z2 by an enzyme to produce rebaudioside R6-4A. In some embodiments of any of the methods or compositions provided herein, glucose is covalently coupled to sugar IV of rebaudioside Z1 by an enzyme to produce rebaudioside R6-4A. In some embodiments of any of the methods or compositions provided herein, glucose is covalently coupled to sugar III of rebaudioside Z2 by an enzyme to produce rebaudioside R6-4A. In some embodiments of any of the methods or compositions provided herein, glucose is covalently coupled to rebaudioside Z2 by an enzyme to produce rebaudioside R6-4B. In some embodiments of any of the methods or compositions provided herein, glucose is covalently coupled to sugar V of rebaudioside Z2 by an enzyme to produce rebaudioside R6-4B.

[0068] In some embodiments of any of the methods or compositions provided herein, the UDP - glycosyltransferase has at least 80% identity with the amino acid sequence of SEQ ID NO:3. In some embodiments of any of the methods or compositions provided herein, the UDP - glycosyltransferase comprises the amino acid sequence of SEQ ID NO:3. In some embodiments of any of the methods or compositions provided herein, the UDP - glycosyltransferase fusion enzyme has at least 80% identity with the amino acid sequence of SEQ ID NO:7. In some embodiments of any of the methods or compositions provided herein, the UDP - glycosyltransferase fusion enzyme comprises the amino acid sequence of SEQ ID NO:7.

[0069] In some embodiments of any of the methods or compositions provided herein, the method further comprises producing rebaudioside Z1 or rebaudioside Z2 by incubating rebaudioside E with a UDP - glycosyltransferase and a substrate selected from sucrose, UDP, UDP - glucose, and combinations thereof.

[0070] Other aspects of the present disclosure provide a method for producing rebaudioside R6 - 1, the method comprising:

[0071] (I) Preparing a reaction mixture comprising:

[0072] (i) Rebaudioside D;

[0073] (ii) One or more substrates selected from sucrose, uridine diphosphate (UDP), uridine diphosphate - glucose (UDP - glucose), and combinations thereof; and

[0074] (iii) An enzyme selected from:

[0075] (a) UDP - glycosyltransferase (UGT);

[0076] (b) UDP - glycosyltransferase and sucrose synthase separately added to the reaction mixture; and

[0077] (c) A UDP - glycosyltransferase fusion enzyme comprising a UDP - glycosyltransferase domain coupled to a sucrose synthase domain; and

[0078] (II) Incubating the reaction mixture for a sufficient time to produce rebaudioside R6 - 1;

[0079] wherein the rebaudioside D has the following structure:

[0080] and

[0081] Rebaudioside R6 - 1 has the following structure:

[0082]

[0083] In some embodiments of any of the methods or compositions provided herein, the sucrose synthase or sucrose synthase domain is selected from Arabidopsis thaliana sucrose synthase I, Arabidopsis thaliana sucrose synthase 3, and mung bean sucrose synthase. In some embodiments, the sucrose synthase or sucrose synthase domain is Arabidopsis thaliana sucrose synthase I. In some embodiments of any of the methods or compositions provided herein, the sucrose synthase or sucrose synthase domain has at least 80% identity to the amino acid sequence of SEQ ID NO:9. In some embodiments, the sucrose synthase or sucrose synthase domain comprises the amino acid sequence of SEQ ID NO:9. In some embodiments of any of the methods or compositions provided herein, glucose is covalently coupled to rebaudioside D by an enzyme to produce rebaudioside R6-1. In some embodiments of any of the methods or compositions provided herein, glucose is covalently coupled to sugar V of rebaudioside D by an enzyme to produce rebaudioside R6-1. In some embodiments of any of the methods or compositions provided herein, the UDP-glycosyltransferase has at least 80% identity to the amino acid sequence of SEQ ID NO:3. In some embodiments of any of the methods or compositions provided herein, the UDP-glycosyltransferase comprises the amino acid sequence of SEQ ID NO:3. In some embodiments of any of the methods or compositions provided herein, the UDP-glycosyltransferase fusion enzyme has at least 80% identity to the amino acid sequence of SEQ ID NO:7. In some embodiments of any of the methods or compositions provided herein, the UDP-glycosyltransferase fusion enzyme comprises the amino acid sequence of SEQ ID NO:7.

[0084] In some embodiments of any of the methods or compositions provided herein, the method further comprises producing rebaudioside D by incubating rebaudioside E with a UDP-glycosyltransferase and a substrate selected from sucrose, UDP, UDP-glucose, and combinations thereof. In some embodiments of any of the methods or compositions provided herein, the method further comprises producing rebaudioside D by incubating rebaudioside A with a UDP-glycosyltransferase and a substrate selected from sucrose, UDP, UDP-glucose, and combinations thereof.

[0085] In some embodiments of any of the methods or compositions provided herein, the reaction mixture is in vitro. In some embodiments of any of the methods or compositions provided herein, the reaction mixture is a cell-based reaction mixture. In some embodiments of any of the methods or compositions provided herein, the cells are selected from yeast, non-steviol glycoside-producing plants, algae, fungi, and bacteria.

[0086] Other aspects of the present disclosure provide Rebaudiosides (e.g., synthetic Rebaudiosides) selected from the following:

[0087] (i) Rebaudioside R6-2A, which has the following structure:

[0088]

[0089] (ii) Rebaudioside R6-2B, which has the following structure:

[0090]

[0091] (iii) Rebaudioside R7-2, which has the following structure:

[0092]

[0093] (iv) Rebaudioside R6-4A, which has the following structure:

[0094] (v) Rebaudioside R6-4B, which has the following structure:

[0095] and

[0096] (vi) Rebaudioside R6-1, which has the following structure:

[0097]

[0098] Compositions are also provided that contain any one of the Rebaudiosides (e.g., synthetic Rebaudiosides) provided herein.

[0099] Also provided herein is the use of any one of the Rebaudiosides (e.g., synthetic Rebaudiosides) described herein as a sweetening agent.

[0100] Other aspects of the present disclosure provide oral consumption products that contain a sweetening amount of any one of the sweetening agents provided herein, e.g., a sweetening agent selected from Rebaudioside R6-2A, R6-2B, R7-2, R6-4A, R6-4B, and / or R6-1, e.g., wherein the oral consumption product is selected from beverage products and consumption products.

[0101] In some embodiments of any one of the methods or compositions provided herein, the sweetening agent is the only sweetening agent. In some embodiments of any one of the methods or compositions provided herein, the oral consumption product contains from about 5 ppm to 100 ppm of Rebaudioside. In some embodiments of any one of the methods or compositions provided herein, the oral consumption product has a sweetness intensity equivalent to a sucrose solution of from about 1% (w / v-%) to about 4% (w / v-%).

[0102] In some embodiments of any of the methods or compositions provided herein, the oral consumption product further comprises at least one additional sweetening agent. In some embodiments of any of the methods or compositions provided herein, the at least one additional sweetening agent is selected from stevia extract, steviol glycosides, stevioside, rebaudioside A, rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside E derived from recombinant microbial biosynthesis, rebaudioside F, dulcoside A, rebaudioside M, rebaudioside V, rebaudioside W, rebaudioside D3, rebaudioside Z1, rebaudioside Z2, phyllodulcin, steviolbioside, sucrose, high fructose corn syrup, fructose, glucose, xylose, arabinose, rhamnose, erythritol, xylitol, mannitol, sorbitol, inositol, AceK, aspartame, neotame, sucralose, saccharin, naringin dihydrochalcone (NarDHC), neohesperidin dihydrochalcone (NDHC), phyllodulcin, mogroside IV, serendipide I, mogroside V, monatin, thaumatin, miraculin, sweet protein, L-alanine, glycine, Siraitia grosvenorii, hernandulcin, phyllanthin, trilobtain, and combinations thereof.

[0103] In some embodiments of any of the methods or compositions provided herein, the oral consumption product further comprises at least one additive selected, for example, from carbohydrates, polyols, amino acids or their salts, polyamino acids or their salts, sugar acids or their salts, nucleotides, organic acids, inorganic acids, organic salts, organic acid salts, organic base salts, inorganic salts, bitter compounds, edible flavors, flavoring components, astringent compounds, proteins, protein hydrolysates, surfactants, emulsifiers, flavonoids, alcohols, polymers, and combinations thereof.

[0104] In some embodiments of any of the methods or compositions provided herein, the consumer product is selected from food products, nutritional products, dietary supplements, dental hygiene compositions, edible gel compositions, cosmetics, and tabletop flavorings. In some embodiments of any of the methods or compositions provided herein, the beverage product is selected from carbonated beverage products and non-carbonated beverage products. In some embodiments of any of the methods or compositions provided herein, the beverage product is selected from soft drinks, fountain beverages, frozen beverages; ready-to-drink beverages; frozen ready-to-drink beverages, coffee, tea, milk beverages, powdered soft drinks, concentrates, flavored water, enhanced water, fruit juices, fruit juice flavored beverages, sports drinks, and energy drinks.

[0105] Any one of the steviol glycosides or compositions provided herein can be used in analgesics, pest repellents, foods or dietary supplements. Any such composition is also provided herein. Any composition provided herein can be in the form of an aerosol, liquid, gel or granule.

[0106] In any method or composition provided herein, the cell system is selected from bacteria, yeast, and combinations thereof, or any cell system that permits genetic transformation with the selected gene and subsequent biosynthetic production of the desired steviol glycoside. In any method or composition provided herein, the cell system is a microbial system, such as Escherichia coli (E. coli).

[0107] In one aspect, steviol glycosides or compositions thereof produced by any of the methods provided herein are provided.

[0108] Although the present disclosure is susceptible to various modifications and alternative forms, specific embodiments thereof are shown by way of example in the drawings and will be described in detail herein. However, it should be understood that the drawings and detailed description presented herein are not intended to limit the present disclosure to the particular embodiments disclosed, but on the contrary, are intended to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the present disclosure as defined by the appended claims.

[0109] Other features and advantages of the present invention will become apparent from the following detailed description of the preferred embodiments of the present invention with reference to the drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0110] The drawings are not necessarily to scale. In the drawings, each identical or nearly identical component shown in various figures is represented by a like numeral. For clarity, not every component may be labeled in every drawing. In the drawings:

[0111] Figure 1 . Biosynthetic pathways of R6-1, R7-2, R6-4A, and R6-4B.

[0112] Figure 2 . Biosynthetic pathways of R6-2 and R7-2.

[0113] Figure 3 . UGT76G1-catalyzed reactions for the production of R6-2 and R7-2 from Reb D3. Panel A shows the HPLC retention time of steviol glycoside D3 (“D3”) standard. Panels B-D show R6-2 and R7-2 enzymatically produced by UGT76G1 at 2 hours (Panel B), 4 hours (Panel C), and 19 hours (Panel D).

[0114] Figure 4. LC-MS analysis of the generated R6-2 compound.

[0115] Figure 5 . LC-MS analysis of the generated R7-2 compound.

[0116] Figure 6 . Structure of R7-2.

[0117] Figure 7A and 7B . Structures of R6-2A( Figure 7A ) and R6-2B( Figure 7B ).

[0118] Figure 8 . Key TOCSY and HMBC correlations of R7-2.

[0119] Figure 9 . Biosynthetic pathway of the R6-1 compound.

[0120] Figure 10 . HV1-catalyzed reaction for generating R6-1 from Reb D. Insert A shows the HPLC retention time of the rebaudioside D (“Reb D”) standard. Inserts B and C show R6-1 generated enzymatically by HV1 at 6 h (Insert B) and 24 h (Insert C).

[0121] Figure 11 . LC-MS analysis of the generated R6-1 compound.

[0122] Figure 12 . Structure of R6-1.

[0123] Figure 13 . Key TOCSY and HMBC correlations of R6-1.

[0124] Figure 14 . Biosynthetic pathways of the R6-4A and R6-4B compounds.

[0125] Figure 15 . HV1-catalyzed reaction for generating R6-4 from Reb Z. Insert A shows the HPLC retention time of the rebaudioside Z (“Z”) standard. R6-4 was generated enzymatically by HV1 at 3 h (Insert B) and 24 h (Insert C).

[0126] Figure 16 . LC-MS analysis of the generated R6-4 compound.

[0127] Figure 17 . Structures of R6-4A and R6-4B.

[0128] Definitions

[0129] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, the preferred materials and methods are described below.

[0130] The term "complementary" is used according to its ordinary and customary meaning as understood by one of ordinary skill in the art, and is used without limitation to describe the relationship between nucleotide bases that are capable of hybridizing to each other. For example, in the case of DNA, adenine is complementary to thymine, and cytosine is complementary to guanine. Accordingly, the subject technology also includes isolated nucleic acid fragments that are complementary to the complete sequences reported in the accompanying sequence listing and those nucleic acid sequences that are substantially similar.

[0131] The terms "nucleic acid" and "nucleotide" are used according to their respective ordinary and customary meanings as understood by one of ordinary skill in the art, and are used without limitation to refer to deoxyribonucleotides or ribonucleotides and their polymers in single-stranded or double-stranded form. Unless specifically restricted, the term includes nucleic acids containing known analogs of natural nucleotides that have binding properties similar to the reference nucleic acid and are metabolized in a manner similar to the naturally occurring nucleotides. Unless otherwise stated, a particular nucleic acid sequence also implicitly includes its conservatively modified or degenerate variants (e.g., degenerate codon substitutions) and complementary sequences, as well as the explicitly indicated sequences.

[0132] The term "isolated" is used according to its ordinary and customary meaning as understood by one of ordinary skill in the art, and when used in the context of an isolated nucleic acid or an isolated polypeptide, is used without limitation to refer to a nucleic acid or polypeptide that is present separately from its natural environment by human intervention and is thus not a natural product. An isolated nucleic acid or polypeptide can be in a purified form or can be present in a non-natural environment such as a transgenic host cell.

[0133] As used herein, the terms "incubating" and "incubation" refer to the process of mixing two or more chemical or biological entities (e.g., chemical compounds and enzymes) and allowing them to interact under conditions favorable for the production of steviol glycoside compositions.

[0134] The term "degenerate variant" refers to a nucleic acid sequence having a residue sequence that differs from a reference nucleic acid sequence by one or more degenerate codon substitutions. Degenerate codon substitutions can be achieved by generating a sequence in which the third position of one or more selected (or all) codons is replaced with a mixed base and / or deoxyinosine residue. A nucleic acid sequence and all of its degenerate variants will express the same amino acid or polypeptide.

[0135] The terms "polypeptide", "protein", and "peptide" are used according to their respective ordinary and customary meanings as understood by one of ordinary skill in the art; these three terms may sometimes be used interchangeably, and are used without limitation to refer to polymers of amino acids or amino acid analogs, regardless of their size or function. Although "protein" is often used to refer to relatively large polypeptides, and "peptide" is often used to refer to small polypeptides, the usage of these terms overlaps and varies in the art. Unless otherwise specified, the term "polypeptide" as used herein refers to peptides, polypeptides, and proteins. When referring to polynucleotide products, the terms "protein", "polypeptide", and "peptide" may be used interchangeably herein. Thus, exemplary polypeptides include polynucleotide products, naturally occurring proteins, homologs, orthologs, paralogs, fragments, and other equivalents, variants, and analogs of the foregoing.

[0136] When used to refer to a reference polypeptide, the terms "polypeptide fragment" and "fragment" are used according to their ordinary and customary meanings to one of ordinary skill in the art, and are used without limitation to refer to such a polypeptide in which amino acid residues are deleted compared to the reference polypeptide itself, but in which the remaining amino acid sequence is generally equivalent to the corresponding positions in the reference polypeptide. Such deletions can occur at the amino terminus or carboxyl terminus of the reference polypeptide, or alternatively at both.

[0137] A "functional fragment" of a polypeptide or protein refers to such a peptide fragment that is part of a full-length polypeptide or protein and has substantially the same biological activity as, or performs substantially the same function as, the full-length polypeptide or protein (e.g., carries out the same enzymatic reaction).

[0138] The terms "variant polypeptide", "modified amino acid sequence", or "modified polypeptide", which are used interchangeably, refer to such an amino acid sequence that differs from a reference polypeptide by one or more amino acids, for example, by one or more amino acid substitutions, deletions, and / or additions. In one aspect, the variant is a "functional variant" that retains some or all of the capabilities of the reference polypeptide.

[0139] The term "functional variant" further includes variants with conservative substitutions. The term "variant with conservative substitutions" refers to a peptide having an amino acid sequence that differs from a reference peptide by one or more conservative amino acid substitutions and that maintains some or all of the activity of the reference peptide. "Conservative amino acid substitution" is the replacement of an amino acid residue with a residue that is functionally similar. Examples of conservative substitutions include the replacement of one nonpolar (hydrophobic) residue such as isoleucine, valine, leucine, or methionine with another; the replacement of one charged or polar (hydrophilic) residue with another, such as between arginine and lysine, between glutamine and asparagine, or between threonine and serine; the replacement of one basic residue such as lysine or arginine with another; or the replacement of one acidic residue such as aspartic acid or glutamic acid with another; or the replacement of one aromatic residue such as phenylalanine, tyrosine, or tryptophan with another. Such substitutions are expected to have little or no effect on the apparent molecular weight or isoelectric point of the protein or polypeptide. The phrase "variant with conservative substitutions" also includes peptides in which a residue is replaced with a chemically derivatized residue, provided that the resulting peptide maintains some or all of the activity of the reference peptide as described herein.

[0140] The term "variant" as it pertains to a polypeptide of the subject technology further includes a functionally active polypeptide having an amino acid sequence that has at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 even 100% identity to the amino acid sequence of a reference polypeptide.

[0141] The term "homologous," in all of its grammatical forms and spelling variations, refers to the relationship between polynucleotides or polypeptides that have a "common evolutionary origin," including polynucleotides or polypeptides from a superfamily, and homologous polynucleotides or proteins from different species (Reeck et al., Cell 50:667, 1987). Such polynucleotides or polypeptides have sequence homology as reflected by their sequence similarity, whether based on the percentage of identity or the presence of specific amino acids or motifs at conserved positions. For example, two homologous polypeptides can have amino acid sequences that are at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 even 100% identical.

[0142] The "percent amino acid sequence identity (%)" of a variant polypeptide sequence with respect to the subject technology refers to the percentage of amino acid residues in the candidate sequence that are identical to the amino acid residues of the reference polypeptide after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity.

[0143] The alignment for the purpose of determining the percent amino acid sequence identity can be achieved in various ways within the skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Those skilled in the art can determine the appropriate parameters for measuring the alignment, including any algorithms required to achieve the maximum alignment over the full length of the sequences to be compared. For example, the sequence comparison program NCBI-BLAST2 can be used to determine the % amino acid sequence identity. The NCBI-BLAST2 sequence comparison program can be downloaded from ncbi.nlm.nih.gov. NCBI BLAST2 uses several search parameters, all of which are set to default values, including for example no masking = yes, strand = all, expected occurrences 10, minimum low complexity length = 15 / 5, multi-pass e-value = 0.01, multi-pass constant = 25, dropoff for final gap alignment = 25 and scoring matrix = BLOSUM62. In the case where NCBI-BLAST2 is used for amino acid sequence comparison, the % amino acid sequence identity of a given amino acid sequence A with, for, or against a given amino acid sequence B (which can alternatively be stated as a given amino acid sequence A having or containing a certain % amino acid sequence identity with, for, or against a given amino acid sequence B) is calculated as follows: 100 × fraction X / Y, where X is the number of amino acid residues scored as identical matches in the alignment of A and B by the sequence alignment program NCBI-BLAST2, and where Y is the total number of amino acid residues in B. It will be appreciated that when the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A with B will not be equal to the % amino acid sequence identity of B with A.

[0144] In this sense, techniques for determining the "similarity" of amino acid sequences are well known in the art. Generally, "similarity" refers to the comparison of the exact amino acid to amino acid at the appropriate positions of two or more polypeptides, where the amino acids are either identical or have similar chemical and / or physical properties, such as charge or hydrophobicity. The so-called "percentage similarity" between the compared polypeptide sequences can then be determined. Techniques for determining nucleic acid and amino acid sequence identity are also well known in the art and include determining the nucleotide sequence of the mRNA of the gene (usually via a cDNA intermediate) and determining the amino acid sequence encoded therein, and comparing it to a second amino acid sequence. Generally, "identity" refers to the exact nucleotide-to-nucleotide or amino acid-to-amino acid correspondence of two polynucleotide sequences or polypeptide sequences, respectively. Two or more polynucleotide sequences can be compared by determining their "percentage identity", and so can two or more amino acid sequences. Programs available in the Wisconsin Sequence Analysis Package, version 8 (available from Genetics Computer Group, Madison, Wis.), such as the GAP program, are capable of calculating the identity between two polynucleotides and the identity and similarity between two polypeptide sequences, respectively. Other programs for calculating the identity or similarity between sequences are known to those skilled in the art.

[0145] The amino acid position "corresponding to" a reference position refers to the position aligned with the reference sequence, as identified by aligning the amino acid sequences. Such alignment can be done manually or by using well-known sequence alignment programs such as ClustalW2, Blast 2, etc.

[0146] Unless otherwise specified, the percentage identity of two polypeptide sequences or polynucleotide sequences refers to the percentage of identical amino acid residues or nucleotides spanning the entire length of the shorter of the two sequences.

[0147] "Coding sequence" is used according to its ordinary and customary meaning as understood by one of ordinary skill in the art and is used, without limitation, to refer to a DNA sequence that encodes a specific amino acid sequence.

[0148] "Suitable regulatory sequences" are used according to their ordinary and customary meaning as understood by one of ordinary skill in the art and are used, without limitation, to refer to nucleotide sequences that are located upstream (5' non-coding sequence), within, or downstream (3' non-coding sequence) of the coding sequence and affect the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences can include promoters, translational leader sequences, introns, and polyadenylation recognition sequences.

[0149] "Promoter" is used according to its ordinary and customary meaning as understood by one of ordinary skill in the art, and is used without limitation to refer to a DNA sequence capable of controlling the expression of a coding sequence or functional RNA. Generally, the coding sequence is located 3' of the promoter sequence. A promoter may be derived in its entirety from a native gene, or may be composed of different elements derived from different promoters found in nature, or may even contain synthetic DNA segments. One of ordinary skill in the art understands that different promoters may direct gene expression in different cell types, or at different developmental stages, or in response to different environmental conditions. A promoter that causes a gene to be expressed in most cell types most of the time is generally referred to as a "constitutive promoter". It is further recognized that, in most cases, the exact boundaries of regulatory sequences have not been fully defined, and thus DNA fragments of different lengths may have equivalent promoter activity.

[0150] The term "operably linked" refers to the association of nucleic acid sequences on a single nucleic acid fragment such that the function of one is affected by the other. For example, a promoter is operably linked to a coding sequence when it is capable of affecting the expression of the coding sequence (i.e., the coding sequence is under the transcriptional control of the promoter). The coding sequence may be operably linked to the regulatory sequence in a sense or antisense orientation.

[0151] As used herein, the term "expression" is used according to its ordinary and customary meaning as understood by one of ordinary skill in the art, and is used without limitation to refer to the transcription and stable accumulation of sense (mRNA) or antisense RNA derived from a nucleic acid fragment of the subject technology. "Overexpression" refers to the production of a gene product in a transgenic or recombinant organism that exceeds the production level in a normal or non-transformed organism.

[0152] "Transformation" is used according to its ordinary and customary meaning as understood by one of ordinary skill in the art, and is used without limitation to refer to the transfer of a polynucleotide into a target cell. The transferred polynucleotide may integrate into the genome or chromosomal DNA of the target cell, resulting in genetically stable inheritance, or it may replicate independently of the host chromosome. A host organism containing the transformed nucleic acid fragment is referred to as a "transgenic" or "recombinant" or "transformed" organism.

[0153] When used in conjunction with a host cell herein, the terms "transformed", "transgenic", and "recombinant" are used according to their ordinary and customary meaning as understood by one of ordinary skill in the art, and are used without limitation to refer to a cell of a host organism into which a heterologous nucleic acid molecule has been introduced, such as a plant or microbial cell. The nucleic acid molecule may be stably integrated into the genome of the host cell, or the nucleic acid molecule may exist as an extrachromosomal molecule. Such an extrachromosomal molecule may be self-replicating. A transformed cell, tissue, or subject is understood to include not only the end product of the transformation process, but also its transgenic progeny.

[0154] When used in connection with polynucleotides herein, the terms "recombinant", "heterologous", and "exogenous" are used according to their ordinary and customary meanings as understood by one of ordinary skill in the art, and are used without limitation to refer to polynucleotides (e.g., DNA sequences or genes) that are derived from a source foreign to a particular host cell, or that have been modified from their original form if from the same source. Thus, a heterologous gene in a host cell includes a gene that is endogenous to the particular host cell but has been modified, e.g., by using site-directed mutagenesis or other recombinant techniques. The term also includes non-naturally occurring multiple copies of a naturally occurring DNA sequence. Thus, the term refers to a DNA segment that is foreign or heterologous to the cell, or that is homologous with the cell but in a location or form not normally found in the host cell for that element.

[0155] Similarly, when used in connection with polypeptide sequences or amino acid sequences herein, the terms "recombinant", "heterologous", and "exogenous" mean a polypeptide or amino acid sequence that is derived from a source foreign to a particular host cell, or that has been modified from its original form if from the same source. Thus, a recombinant DNA segment can be expressed in a host cell to produce a recombinant polypeptide.

[0156] The terms "plasmid", "vector", and "cassette" are used according to their ordinary and customary meanings as understood by one of ordinary skill in the art, and are used without limitation to refer to extrachromosomal elements that often carry genes that are not part of the central metabolism of the cell and that are generally in the form of circular double-stranded DNA molecules. Such elements can be linear or circular autonomously replicating sequences, genomic integration sequences, phages, or nucleotide sequences derived from any source, single-stranded or double-stranded DNA or RNA, many of which have been ligated or recombined into unique constructs that are capable of introducing into a cell a promoter fragment and the DNA sequence of a selected gene product along with appropriate 3' untranslated sequences. A "transformation cassette" refers to a particular vector that contains a foreign gene and has elements other than the foreign gene that facilitate the transformation of a particular host cell. An "expression cassette" refers to a particular vector that contains a foreign gene and has elements other than the foreign gene that permit enhanced expression of the gene in a foreign host.

[0157] The standard recombinant DNA and molecular cloning techniques used herein are well known in the art and are described, for example, by: Sambrook, J., Fritsch, E.F., and Maniatis, T. Molecular Cloning: A Laboratory Manual, 2nd ed.; Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y., 1989 (hereinafter "Maniatis"); and Silhavy, T.J., Bennan, M.L., and Enquist, L.W. Experiments with Gene Fusions; Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y., 1984; and Ausubel, F.M. et al., in Current Protocols in Molecular Biology, published by Greene Publishing and Wiley-Interscience, 1987; each of the foregoing references is hereby incorporated by reference in its entirety herein to the extent that it is consistent herewith.

[0158] As used herein, "synthetic" or "organically synthesized" or "chemically synthesized" or "organically synthesizing" or "chemically synthesizing" or "organic synthesis" or "chemical synthesis" is used to refer to the preparation of a compound by a series of chemical reactions; this does not include, for example, the extraction of a compound from a natural source.

[0159] As used herein, the term "oral consumption product" refers to any beverage, food product, dietary supplement, nutritional product, dental hygiene composition, and cosmetic that comes into contact with the mouth of a human or animal, including substances that are placed in the mouth and then spat out, as well as substances that are drunk, eaten, swallowed, or otherwise ingested; and substances that are safe for human or animal consumption when used in generally acceptable concentration ranges.

[0160] As used herein, the term "food product" refers to fruits, vegetables, fruit juices, meat products such as ham, bacon and sausage; egg products, fruit concentrates, gelatin and gelatin-like products such as jams, jellies, preserves, etc.; dairy products such as ice cream, sour cream, yogurt and sherbet; frostings, syrups including molasses; corn, wheat, rye, soy, oats, rice and barley products, cereal products, seed and nut products, cakes, cookies, confectionery such as candies, gums, fruit drops and chocolates, chewing gums, mints, creams, frostings, ice cream, pies and bread. "Food product" also refers to flavorings such as herbs, spices and seasonings, and flavor enhancers such as monosodium glutamate. "Food product" further refers to prepared and packaged products, such as dietary sweeteners, liquid sweeteners, tabletop flavorings, granular flavoring mixtures that provide non-carbonated beverages when reconstituted with water, instant pudding mixtures, instant coffee and tea, coffee whiteners, malted milk mixtures, pet foods, livestock feeds, tobacco and materials for baking applications, such as powdered baking mixtures for preparing bread, cookies, cakes, pancakes, doughnuts, etc. "Food product" also refers to dietary or low-calorie foods and beverages that contain little or no sucrose.

[0161] As used herein, the term "stereoisomer" is a general term for all isomers of a molecule that differ only in the orientation of its atoms in space. "Stereoisomers" include enantiomers, as well as isomers of compounds that are not mirror images of each other and have more than one chiral center (diastereomers).

[0162] As used herein, the term "sweetness intensity" refers to the relative intensity of the sweet taste sensation as observed or experienced by an individual such as a human, or the degree or amount of sweetness detected by a taster, for example, on a Brix scale.

[0163] As used herein, the term "enhanced sweetness" refers to the effect of rebaudioside in increasing, enhancing, intensifying, emphasizing, amplifying and / or potentiating the sensory perception of one or more sweet taste characteristics of a beverage product or consumer product of the present disclosure compared to a corresponding orally consumed product that does not contain rebaudioside of the present disclosure, without changing its nature or quality.

[0164] As used herein, the term "off-flavor" refers to the amount or degree of a flavor that is not characteristically or normally found in a beverage product or consumer product of the present disclosure. For example, an off-flavor is a flavor that is undesirable to a consumer in a sweet consumer product, such as bitterness, licorice-like flavor, metallic flavor, aversive flavor, astringency, delayed onset of sweetness, lingering sweet aftertaste, etc.

[0165] As used herein, the term "w / v-%" refers to the weight (in grams) of a compound, such as sugar, for every 100 ml of a liquid oral consumption product of the present disclosure containing such compound. As used herein, the term "w / w-%" refers to the weight (in grams) of a compound, such as sugar, for every gram of an oral consumption product of the present disclosure containing such compound.

[0166] As used herein, the term "ppm" refers to parts per million by weight, such as the weight (in milligrams) of a compound, such as steviol glycoside of the present disclosure, per kilogram of an oral consumption product of the present disclosure containing such compound (i.e., mg / kg), or the weight (in milligrams) of a compound, such as steviol glycoside of the present disclosure, per liter of an oral consumption product of the present disclosure containing such compound (i.e., mg / L); or parts per million by volume, such as the volume (in milliliters) of a compound, such as steviol glycoside of the present disclosure, per liter of an oral consumption product of the present disclosure containing such compound (i.e., ml / L).

[0167] Steviol glycosides are a class of chemical compounds responsible for the sweetness of the leaves of the South American plant Stevia rebaudiana (Asteraceae), and can be used as sweeteners in foods, feeds, and beverages either alone, in combination with each other, or in combination with other sweeteners and flavor modifiers.

[0168] A cell system is any cell that provides for the expression of a heterologous protein. It includes bacteria, yeast, plant cells, and animal cells. It includes both prokaryotic and eukaryotic cells. It also includes in vitro expression of proteins based on cell components such as ribosomes.

[0169] Grow the cell system. Growth includes providing an appropriate culture medium that allows the cells to reproduce and divide. It also includes providing resources such that the cells or cell components can translate and produce recombinant proteins.

[0170] Protein expression. Protein production can occur after gene expression. It consists of the stage after DNA has been transcribed into messenger RNA (mRNA). The mRNA is then translated into a polypeptide chain, which ultimately folds into a protein. DNA is present in the cell by transfection, which is the process of deliberately introducing nucleic acid into the cell. This term is often used for non-viral methods in eukaryotic cells. It may also refer to other methods and cell types, although other terms are preferred: "transformation" is more often used to describe non-viral DNA transfer in bacteria, non-animal eukaryotic cells including plant cells. In animal cells, transfection is the preferred term because transformation is also used to refer to the progression to a cancerous state (carcinogenesis) in these cells. Transduction is often used to describe virus-mediated DNA transfer. Transformation, transduction, and viral infection are included under the definition of transfection used in this application.

[0171] As used herein, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise.

[0172] To the extent that the terms "comprises", "comprising", etc. are used in the specification or claims, such terms are intended to be inclusive in a manner similar to the term "including" as that term is construed when used as a transitional word in a claim.

[0173] The term "exemplary" is used herein to mean "serving as an example, instance, or illustration". Any embodiment described herein as "exemplary" is not necessarily to be construed as preferred or advantageous over other embodiments. Detailed Description

[0174] The present invention relates at least in part to novel steviol glycosides R7-2, R6-2A, R6-2B, R6-1, R6-4A, and R6-4B, and methods for producing these novel steviol glycosides, for example, by enzymatic conversion. The chemical structures can be confirmed by LC MS and NMR analysis. R6-1, R6-2A, R6-2B, R6-4A, and R6-4B contain six glucosyl groups, while R7-2 contains seven glucosyl groups.

[0175] Precursor Synthesis

[0176] As previously stated, steviol glycosides are the chemical compounds responsible for the sweetness of the leaves of the South American plant Stevia rebaudiana (Asteraceae) and the plant Rubus chingii (Rosaceae). These compounds are glycosylated diterpenes. Specifically, their molecules can be regarded as steviol molecules in which the hydroxyl hydrogen atoms are replaced by glucose molecules to form esters, and the hydroxyl hydrogen is replaced by a combination of glucose and rhamnose to form acetals.

[0177] One method for preparing the compounds of interest in the present invention is to use common or inexpensive precursors, such as steviol, stevioside, Reb E, Reb D, or phyllodulcin, which are produced, for example, by chemical derivation or biosynthesis via engineered microorganisms such as bacteria and / or yeast, and to synthesize target steviol glycosides such as Reb D3 and Z, for example, by known or inexpensive methods.

[0178] Aspects of the present invention relate to methods for recombinant expression of enzymes in a microbial system capable of producing steviol. Generally, such enzymes can include: copalyl diphosphate synthase (CPS), ent-kaurene synthase (KS), and geranylgeranyl diphosphate synthase (GGPPS) enzymes. Preferably, in some embodiments, this occurs in a microbial strain that expresses an endogenous isoprenoid biosynthetic pathway, such as the non-mevalonate (MEP) pathway or the mevalonate pathway (MVA). In some embodiments of any of the methods or compositions provided herein, the cell is a bacterial cell such as Escherichia coli, or a yeast cell such as a Saccharomyces cell, a Pichia cell, or a Yarrowia cell. In some embodiments of any of the methods or compositions provided herein, the cell is an algal cell or a plant cell.

[0179] Thereafter, the precursor can be recovered from the fermentation broth and used in chemical synthesis. Typically, this is steviol, although it can be ent-kaurene or a steviol glycoside from the cell culture. In some embodiments of any of the methods or compositions provided herein, steviol, ent-kaurene, and / or steviol glycosides are recovered from the gas phase, while in other embodiments, an organic layer or a polymeric resin is added to the cell culture, and ent-kaurene, steviol, and / or steviol glycosides are recovered from the organic layer or the polymeric resin. In some embodiments of any of the methods or compositions provided herein, the steviol glycosides are selected from rebaudioside A, rebaudioside B, rebaudioside C, rebaudioside E, rebaudioside F, or dulcoside A. In some embodiments of any of the methods or compositions provided herein, the terpenoids produced are steviolbioside or stevioside. It should also be understood that in some embodiments, at least one enzymatic step, such as one or more glycosylation steps, is performed in vitro.

[0180] As described herein, the enzymes used in the methods described herein have UDP-glycosyltransferase activity and can be used to develop biosynthetic methods for preparing steviol glycosides that are either not found in nature or are typically present in low abundance in natural sources, such as rebaudioside R6-1, R2-2A, R6-2B, R6-4A, R6-4B, and R7-2, respectively.

[0181] A substrate can be any natural or synthetic compound that can be converted into a steviol glycoside compound in a reaction catalyzed by one or more UDP - glucosyltransferases. For example, the substrate can be a natural stevia extract, steviol, steviol - 13 - O - glucoside, steviol - 19 - O - glucoside, 2 - bisglycoside, rubusoside, stevioside, rebaudioside A, rebaudioside D, rebaudioside D3, rebaudioside Z1, rebaudioside Z2, or rebaudioside E. The substrate can be a pure compound or a mixture of different compounds.

[0182] Also described herein is a coupling reaction system in which the enzymes described herein (e.g., UDP transferases) can function in combination with one or more additional enzymes (e.g., sucrose synthase) to improve efficiency or modify the outcome of the overall biosynthesis of steviol glycoside compounds. For example, the additional enzyme can regenerate UDP - glucose required for the glycosylation reaction by converting UDP produced by the glycosylation reaction back to UDP - glucose (e.g., using sucrose as the donor of the glucose residue), thus improving the efficiency of the glycosylation reaction.

[0183] Sucrose synthase catalyzes the chemical reaction between UDP - glucose and D - fructose to produce UDP and sucrose. Sucrose synthase is a glycosyltransferase. The systematic name for this enzyme class is UDP - glucose:D - fructose 2 - α - D - glucosyltransferase. Other common names include UDP - glucose - fructose glucosyltransferase, sucrose synthase, sucrose - UDP - glucosyltransferase, sucrose - uridine diphosphate glucosyltransferase, and uridine diphosphate glucose - fructose glucosyltransferase. Adding sucrose synthase to a reaction mixture containing uridine diphosphate glycosyltransferase produces an "UGT - SUS coupling system". In the UGT - SUS coupling system, UDP - glucose can be regenerated from UDP and sucrose, which allows omission of adding additional UDP - glucose to the reaction mixture or using UDP in the reaction mixture.

[0184] Suitable sucrose synthases for use in the methods described herein include Arabidopsis thaliana sucrose synthase I, Arabidopsis thaliana sucrose synthase 3, and mung bean sucrose synthase. In some embodiments of any of the methods or compositions provided herein, the sucrose synthase or sucrose synthase domain is Arabidopsis sucrose synthase I.

[0185] Suitable UDP - glycosyltransferases include any UGT known in the art that is capable of catalyzing one or more reactions in the biosynthesis of steviol glycoside compounds, such as UGT85C2, UGT74G1, HV1, UGT76G1, or functional homologs thereof. In some embodiments, the UDP - glycosyltransferase used in any of the methods described herein is UGT76G1. In some embodiments, the UDP - glycosyltransferase used in any of the methods described herein is a UGT76G1 - sucrose synthase fusion enzyme. In some embodiments, the UDP - glycosyltransferase used in any of the methods described herein is HV1 UTG. In some embodiments, the UDP - glycosyltransferase used in any of the methods described herein is an HV1 UGT - sucrose synthase fusion enzyme.

[0186] Standard recombinant DNA and molecular cloning techniques used herein are well known in the art and are described, for example, by: Sambrook, J., Fritsch, E.F., and Maniatis, T. Molecular Cloning: A Laboratory Manual, 2nd ed.; Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y., 1989 (hereinafter "Maniatis"); and Silhavy, T.J., Bennan, M.L., and Enquist, L.W. Experiments with Gene Fusions; Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y., 1984; and Ausubel, F.M. et al., Current Protocols in Molecular Biology, published by Greene Publishing and Wiley - Interscience, 1987; (each of the said references is hereby incorporated by reference in its entirety).

[0187] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, the preferred materials and methods are described herein.

[0188] The present disclosure will be more fully understood after considering the following non-limiting examples. It should be understood that these examples, while indicating preferred embodiments of the subject technology, are given by way of illustration only. From the foregoing discussion and these examples, those skilled in the art can determine the basic features of the subject technology and, without departing from its spirit and scope, can make various changes and modifications to the subject technology to adapt it to various uses and conditions.

[0189] Glycosylation is often regarded as a ubiquitous reaction that controls the bioactivity and storage of plant natural products. Glycosylation of small molecules is catalyzed by a superfamily of transferases in most plant species studied to date. These glycosyltransferases (GTs) have been classified into more than 60 families. Among them, family 1 GT enzymes, also known as UDP-glycosyltransferases (UGTs) and UDP-rhamnosyltransferases, transfer sugar moieties to specific receptor molecules. These are the molecules that transfer such sugar moieties in steviol glycosides to help produce various rebaudiosides. Each of these enzymes has its own activity characteristics, as well as the preferred structural positions where it transfers its activated sugar moiety.

[0190] Synthesis of Rebaudioside R6-2A and R6-2B and Production Method

[0191] Some aspects of the present disclosure provide sweeteners (e.g., calorie-free and / or synthetic) that have been given the name "rebaudioside R6-2A (Reb R6-2A)". Rebaudioside R6-2A has a chemical formula of C 56 H 90 O 33 and has the following structure:

[0192]

[0193] Some aspects of the present disclosure provide sweeteners (e.g., calorie-free and / or synthetic) that have been given the name "rebaudioside R6-2B (Reb R6-2B)". Rebaudioside R6-2B has a chemical formula of C 56 H 90 O 33 and has the following structure:

[0194]

[0195] Some aspects of the present disclosure provide methods for producing rebaudioside R6-2A and / or rebaudioside R6-2B. In some embodiments of any of the methods or compositions provided herein, rebaudioside R6-2A and / or rebaudioside R6-2B are produced from rebaudioside D3. For example, in some embodiments, the method comprises (I) preparing a reaction mixture comprising: (i) rebaudioside D3; (ii) one or more substrates selected from sucrose, uridine diphosphate (UDP), uridine diphosphate-glucose (UDP-glucose), and combinations thereof; and (iii) an enzyme selected from: (a) UDP-glycosyltransferase (UGT); (b) UDP-glycosyltransferase and sucrose synthase added separately to the reaction mixture; and (c) a UDP-glycosyltransferase fusion enzyme comprising a UDP-glycosyltransferase domain coupled to a sucrose synthase domain; and (II) incubating the reaction mixture for a sufficient time to produce rebaudioside R6-2A and / or R6-2B.

[0196] In some embodiments of any of the methods or compositions provided herein, glucose is covalently coupled to rebaudioside D3 by an enzyme to produce rebaudioside R6-2A and / or R6-2B. In some embodiments of any of the methods or compositions provided herein, glucose is covalently coupled to sugar I of rebaudioside D3 by an enzyme to produce rebaudioside R6-2A. In some embodiments of any of the methods or compositions provided herein, glucose is covalently coupled to sugar II of rebaudioside D3 by an enzyme to produce rebaudioside R6-2B. Figure 2 The sugar numbering in rebaudioside D3, R6-2A, and R6-2B is shown.

[0197] In some embodiments of any of the methods or compositions provided herein, the enzyme used to produce rebaudioside R6-2A and / or R6-2B from rebaudioside D3 comprises a UDP-glycosyltransferase (UGT). In some embodiments of any of the methods or compositions provided herein, the UGT is a uridine diphosphate glycosyltransferase (e.g., UGT76G1 or a functional variant thereof). UGT76G1 is a UGT with 1,3-13-O-glucosylglycosylation activity. UGT76G1 has also been shown to have 1,3-19-O-glucosylglycosylation activity. In some embodiments of any of the methods or compositions provided herein, UGT76G1 has at least 70% (e.g., 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% or at least 99%) identity to the amino acid sequence of SEQ ID NO:1. In some embodiments, UGT76G1 has at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity to the amino acid sequence of SEQ ID NO:1. In some embodiments, UGT76G1 comprises the amino acid sequence of SEQ ID NO:1. In some embodiments, UGT76G1 consists essentially of the amino acid sequence of SEQ ID NO:1, or consists of the amino acid sequence of SEQ ID NO:1.

[0198] In some embodiments of any of the methods or compositions provided herein, the enzymes for producing rebaudioside R6-2A and / or R6-2B from rebaudioside D3 comprise UDP-glycosyltransferase (UGT) and sucrose synthase separately added to the reaction mixture. In some embodiments of any of the methods or compositions provided herein, the UGT is UGT76G1 and variants as described herein, and the sucrose synthase is selected from Arabidopsis thaliana sucrose synthase I, Arabidopsis thaliana sucrose synthase 3, and mung bean sucrose synthase. In some embodiments of any of the methods or compositions provided herein, the sucrose synthase or sucrose synthase domain is Arabidopsis thaliana sucrose synthase I. In some embodiments, Arabidopsis thaliana sucrose synthase I has at least 70% (e.g., 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%, or at least 99%) identity to the amino acid sequence of SEQ ID NO:9. In some embodiments, Arabidopsis thaliana sucrose synthase I has at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity to the amino acid sequence of SEQ ID NO:9. In some embodiments of any of the methods or compositions provided herein, Arabidopsis thaliana sucrose synthase I comprises the amino acid sequence of SEQ ID NO:9. In some embodiments of any of the methods or compositions provided herein, Arabidopsis thaliana sucrose synthase I consists essentially of, or consists of, the amino acid sequence of SEQ ID NO:9.

[0199] In some embodiments of any of the methods or compositions provided herein, the enzyme for producing rebaudioside R6-2A and / or R6-2B from rebaudioside D3 comprises a UDP-glycosyltransferase fusion enzyme, which comprises a UDP-glycosyltransferase domain coupled to a sucrose synthase domain. The fusion enzyme has the activity of UDP-glycosyltransferase and sucrose synthase activity. In some embodiments of any of the methods or compositions provided herein, the UDP-glycosyltransferase domain in the fusion enzyme is UGT76G1 and any of the variants as described herein, and the sucrose synthase domain is sucrose synthase (e.g., Arabidopsis thaliana sucrose synthase I, Arabidopsis thaliana sucrose synthase 3, or mung bean sucrose synthase) and any of the variants as described herein. In some embodiments of any of the methods or compositions provided herein, the fusion enzyme has at least 70% (e.g., 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%, or at least 99%) identity with the amino acid sequence of SEQ ID NO:5. In some embodiments of any of the methods or compositions provided herein, the fusion enzyme has at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity with the amino acid sequence of SEQ ID NO:5. In some embodiments of any of the methods or compositions provided herein, the fusion enzyme comprises the amino acid sequence of SEQ ID NO:5. In some embodiments of any of the methods or compositions provided herein, the fusion enzyme consists essentially of the amino acid sequence of SEQ ID NO:5 or consists of the amino acid sequence of SEQ ID NO:5.

[0200] In some embodiments, any of the methods for producing rebaudioside R6-2A and / or R6-2B described herein further comprises the step of producing rebaudioside D3. In some embodiments of any of the methods provided herein, rebaudioside D3 can be produced by incubating rebaudioside E with a UDP-glycosyltransferase and a substrate selected from sucrose, UDP, UDP-glucose, and combinations thereof. In some embodiments of any of the methods provided herein, the UDP-glycosyltransferase for producing rebaudioside D3 from rebaudioside E is EUGT11. The method for producing rebaudioside D3 from rebaudioside E is described in U.S. Patent No. US9765104, and such methods are incorporated herein by reference. Rebaudioside D3 has the following structure:

[0201]

[0202] In some embodiments, any of the methods described herein for producing rebaudioside R6-2A and / or R6-2B further comprises isolating the produced rebaudioside R6-2A and / or R6-2B.

[0203] Synthesis of Rebaudioside R7-2 and Production Method

[0204] Some aspects of the present disclosure provide sweeteners (e.g., calorie-free and / or synthetic) that have been given the name "rebaudioside R7-2 (Reb R7-2)". The Reb R7-2 compound is a steviol glycoside that has four glucosyl moieties attached at the C-13 hydroxyl group, three of which are attached as 2,3-branched glucotriosyl substituents in the form of an ether; and another 2,3-branched glucotriosyl moiety as an ester at C-19.

[0205] Rebaudioside R7-2 has the chemical formula of C 62 H 100 O 38 and has the following structure:

[0206]

[0207] Some aspects of the present disclosure provide methods for producing rebaudioside R7-2. In some embodiments of any of the provided methods, rebaudioside R7-2 is produced from one or more of rebaudioside D3, rebaudioside R6-2A, and rebaudioside R6-2B. The reaction mixture for producing rebaudioside R7-2 can be the same as the reaction mixture for producing rebaudioside R6-2A and / or R6-2B. Glucose can be covalently coupled to rebaudioside D3 by an enzyme to produce rebaudioside R6-2A and / or R6-2B. The enzyme can further covalently couple a second glucose to rebaudioside R6-2A and / or R6-2B to produce rebaudioside R7-2. In some embodiments of any of the methods or compositions provided herein, glucose is covalently coupled to each of sugar I and sugar II of rebaudioside D3 by an enzyme to produce rebaudioside R7-2. Figure 2 The sugar numbering in rebaudioside D3 and rebaudioside R7-2 is shown.

[0208] In some embodiments of any of the methods provided herein, the method comprises (I) preparing a reaction mixture comprising: (i) one or more of rebaudioside D3, rebaudioside R6-2A, and rebaudioside R6-2B; (ii) one or more substrates selected from sucrose, uridine diphosphate (UDP), uridine diphosphate-glucose (UDP-glucose), and combinations thereof; and (iii) an enzyme selected from: (a) UDP-glycosyltransferase (UGT); (b) UDP-glycosyltransferase and sucrose synthase added separately to the reaction mixture; and (c) a UDP-glycosyltransferase fusion enzyme comprising a UDP-glycosyltransferase domain coupled to a sucrose synthase domain; and (II) incubating the reaction mixture for a sufficient time to produce rebaudioside R7-2.

[0209] In some embodiments of any of the methods or compositions provided herein, the enzyme for producing rebaudioside R7-2 from rebaudioside D3 comprises UDP-glycosyltransferase (UGT). In some embodiments of any of the methods or compositions provided herein, the UGT is uridine diphosphate glycosyltransferase (e.g., UGT76G1 or a functional variant thereof). UGT76G1 is a UGT having 1,3-13-O-glucosylglycosylation activity. It has also been shown that UGT76G1 has 1,3-19-O-glucosylglycosylation activity. In some embodiments of any of the methods or compositions provided herein, UGT76G1 has at least 70% (e.g., 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%, or at least 99%) identity to the amino acid sequence of SEQ ID NO:1. In some embodiments of any of the methods or compositions provided herein, UGT76G1 has at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity to the amino acid sequence of SEQ ID NO:1. In some embodiments of any of the methods or compositions provided herein, UGT76G1 comprises the amino acid sequence of SEQ ID NO:1. In some embodiments of any of the methods or compositions provided herein, UGT76G1 consists essentially of the amino acid sequence of SEQ ID NO:1, or consists of the amino acid sequence of SEQ ID NO:1.

[0210] In some embodiments of any of the methods or compositions provided herein, the enzymes for producing rebaudioside R7-2 from rebaudioside D3 comprise UDP-glycosyltransferase (UGT) and sucrose synthase separately added to the reaction mixture. In some embodiments of any of the methods or compositions provided herein, the UGT is UGT76G1 and variants as described herein, and the sucrose synthase is selected from Arabidopsis thaliana sucrose synthase I, Arabidopsis thaliana sucrose synthase 3, and mung bean sucrose synthase. In some embodiments of any of the methods or compositions provided herein, the sucrose synthase or sucrose synthase domain is Arabidopsis thaliana sucrose synthase I. In some embodiments of any of the methods or compositions provided herein, Arabidopsis thaliana sucrose synthase I has at least 70% (e.g., 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%, or at least 99%) identity to the amino acid sequence of SEQ ID NO:9. In some embodiments of any of the methods or compositions provided herein, Arabidopsis thaliana sucrose synthase I has at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity to the amino acid sequence of SEQ ID NO:9. In some embodiments of any of the methods or compositions provided herein, Arabidopsis thaliana sucrose synthase I comprises the amino acid sequence of SEQ ID NO:9. In some embodiments of any of the methods or compositions provided herein, Arabidopsis thaliana sucrose synthase I consists essentially of, or consists of, the amino acid sequence of SEQ ID NO:9.

[0211] In some embodiments of any of the methods or compositions provided herein, the enzyme for producing rebaudioside R7-2 from rebaudioside D3 comprises a UDP-glycosyltransferase fusion enzyme, which comprises a UDP-glycosyltransferase domain coupled to a sucrose synthase domain. The fusion enzyme has the activity of UDP-glycosyltransferase and sucrose synthase activity. In some embodiments of any of the methods or compositions provided herein, the UDP-glycosyltransferase domain in the fusion enzyme is UGT76G1 and any of the variants as described herein, and the sucrose synthase domain is sucrose synthase (e.g., Arabidopsis sucrose synthase I, Arabidopsis sucrose synthase 3, or mung bean sucrose synthase) and any of the variants as described herein. In some embodiments of any of the methods or compositions provided herein, the fusion enzyme has at least 70% (e.g., 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%, or at least 99%) identity to the amino acid sequence of SEQ ID NO:5. In some embodiments of any of the methods or compositions provided herein, the fusion enzyme has at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity to the amino acid sequence of SEQ ID NO:5. In some embodiments of any of the methods or compositions provided herein, the fusion enzyme comprises the amino acid sequence of SEQ ID NO:5. In some embodiments of any of the methods or compositions provided herein, the fusion enzyme consists essentially of the amino acid sequence of SEQ ID NO:5 or consists of the amino acid sequence of SEQ ID NO:5.

[0212] In some embodiments of any of the methods or compositions provided herein, the method for producing rebaudioside R7-2 described herein further comprises the step of producing rebaudioside D3. In some embodiments, rebaudioside D3 can be produced by incubating rebaudioside E with a UDP-glycosyltransferase and a substrate selected from sucrose, UDP, UDP-glucose, and combinations thereof. In some embodiments of any of the methods or compositions provided herein, the UDP-glycosyltransferase for producing rebaudioside D3 from rebaudioside E is EUGT11. The method for producing rebaudioside D3 from rebaudioside E is described in U.S. Patent No. US9765104, and such methods are incorporated herein by reference. Rebaudioside D3 has the following structure:

[0213]

[0214] In some embodiments, any of the methods for producing rebaudioside R7-2 described herein further comprises isolating the produced rebaudioside R7-2.

[0215] Synthesis of Rebaudioside R6-4A and R6-4B and Production Method

[0216] Some aspects of the present disclosure provide a sweetener (e.g., calorie-free and / or synthetic) given the name "Rebaudioside R6-4A (Reb R6-4A)". Rebaudioside R6-4A has a molecular formula of C 56 H 90 O 33 and has the following structure:

[0217]

[0218] Some aspects of the present disclosure provide a sweetener (e.g., calorie-free and / or synthetic) given the name "Rebaudioside R6-4B (Reb R6-4B)". Rebaudioside R6-4B has a molecular formula of C 56 H 90 O 33 and has the following structure:

[0219]

[0220] Some aspects of the present disclosure provide methods for producing Rebaudioside R6-4A. In some embodiments of any of the provided methods, Rebaudioside R6-4A is produced from Rebaudioside Z1 or Rebaudioside Z2. For example, in some embodiments, the method comprises (I) preparing a reaction mixture comprising: (i) Rebaudioside Z1 or Rebaudioside Z2; (ii) one or more substrates selected from sucrose, uridine diphosphate (UDP), uridine diphosphate-glucose (UDP-glucose), and combinations thereof; and (iii) an enzyme selected from: (a) UDP-glycosyltransferase (UGT); (b) UDP-glycosyltransferase and sucrose synthase added separately to the reaction mixture; and (c) a UDP-glycosyltransferase fusion enzyme comprising a UDP-glycosyltransferase domain coupled to a sucrose synthase domain; and (II) incubating the reaction mixture for a sufficient time to produce Rebaudioside R6-4A.

[0221] In some embodiments of any of the methods or compositions provided herein, glucose is covalently coupled to Rebaudioside Z1 or Rebaudioside Z2 by an enzyme to produce Rebaudioside R6-4A. In some embodiments of any of the methods or compositions provided herein, glucose is covalently coupled to sugar IV of Rebaudioside Z1 by an enzyme to produce Rebaudioside R6-4A. In some embodiments of any of the methods or compositions provided herein, glucose is covalently coupled to sugar III of Rebaudioside Z2 by an enzyme to produce Rebaudioside R6-4A. Figure 14 The sugar numbering in Rebaudioside Z1, Z2, and R6-4A is shown.

[0222] Some aspects of the present disclosure provide methods for producing rebaudioside R6-4B. In some embodiments of any of the provided methods, rebaudioside R6-4B is produced from rebaudioside Z2. For example, in some embodiments, the method comprises (I) preparing a reaction mixture comprising: (i) rebaudioside Z2; (ii) one or more substrates selected from sucrose, uridine diphosphate (UDP), uridine diphosphate-glucose (UDP-glucose), and combinations thereof; and (iii) an enzyme selected from: (a) UDP-glycosyltransferase (UGT); (b) UDP-glycosyltransferase and sucrose synthase added separately to the reaction mixture; and (c) a UDP-glycosyltransferase fusion enzyme comprising a UDP-glycosyltransferase domain coupled to a sucrose synthase domain; and (II) incubating the reaction mixture for a sufficient time to produce rebaudioside R6-4B.

[0223] In some embodiments of any of the methods or compositions provided herein, glucose is covalently coupled to rebaudioside Z2 by an enzyme to produce rebaudioside R6-4B. In some embodiments of any of the methods or compositions provided herein, glucose is covalently coupled to sugar V of rebaudioside Z2 by an enzyme to produce rebaudioside R6-4B. Figure 14 The sugar numbering in rebaudioside Z2 and R6-4B is shown.

[0224] In some embodiments of any of the methods or compositions provided herein, the enzyme used to produce rebaudioside R6-4A or R6-4B from rebaudioside Z1 or Z2 comprises a UDP-glycosyltransferase (UGT). In some embodiments of any of the methods or compositions provided herein, the UGT is an HV1 glycosyltransferase. HV1 is a UGT with 1,2-19-O-glucosylglycosylation activity. It has also been shown that the HV1 UGT has 1,2-19-O-glucosylglycosylation activity. In some embodiments of any of the methods or compositions provided herein, the HV1 UGT has at least 70% (e.g., 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% or at least 99%) identity to the amino acid sequence of SEQ ID NO:3. In some embodiments of any of the methods or compositions provided herein, the HV1 UGT has at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity to the amino acid sequence of SEQ ID NO:3. In some embodiments of any of the methods or compositions provided herein, the HV1 UGT comprises the amino acid sequence of SEQ ID NO:3. In some embodiments of any of the methods or compositions provided herein, the HV1 UGT consists essentially of the amino acid sequence of SEQ ID NO:3, or consists of the amino acid sequence of SEQ ID NO:3.

[0225] In some embodiments of any of the methods provided herein, the enzymes for producing rebaudioside R6-4A and / or R6-4B from rebaudioside Z1 or Z2 comprise UDP-glycosyltransferase (UGT) and sucrose synthase separately added to the reaction mixture. In some embodiments of any of the methods or compositions provided herein, the UGT is HV1 UGT and variants as described herein, and the sucrose synthase is selected from Arabidopsis thaliana sucrose synthase I, Arabidopsis thaliana sucrose synthase 3, and mung bean sucrose synthase. In some embodiments of any of the methods or compositions provided herein, the sucrose synthase or sucrose synthase domain is Arabidopsis sucrose synthase I. In some embodiments, Arabidopsis sucrose synthase I has at least 70% (e.g., 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%, or at least 99%) identity to the amino acid sequence of SEQ ID NO:9. In some embodiments of any of the methods or compositions provided herein, Arabidopsis sucrose synthase I has at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity to the amino acid sequence of SEQ ID NO:9. In some embodiments of any of the methods or compositions provided herein, Arabidopsis sucrose synthase I comprises the amino acid sequence of SEQ ID NO:9. In some embodiments of any of the methods or compositions provided herein, Arabidopsis sucrose synthase I consists essentially of the amino acid sequence of SEQ ID NO:9, or consists of the amino acid sequence of SEQ ID NO:9.

[0226] In some embodiments of any of the methods provided herein, the enzyme for producing rebaudioside R6-4A or R6-4B from rebaudioside Z1 or Z2 comprises a UDP-glycosyltransferase fusion enzyme that comprises a UDP-glycosyltransferase domain coupled to a sucrose synthase domain. The fusion enzyme has UDP-glycosyltransferase activity and sucrose synthase activity. In some embodiments of any of the methods or compositions provided herein, the UDP-glycosyltransferase domain in the fusion enzyme is HV1UGT and any of the variants as described herein, and the sucrose synthase domain is a sucrose synthase (e.g., Arabidopsis thaliana sucrose synthase I, Arabidopsis thaliana sucrose synthase 3, or mung bean sucrose synthase) and any of the variants as described herein. In some embodiments of any of the methods or compositions provided herein, the fusion enzyme has at least 70% (e.g., 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%, or at least 99%) identity to the amino acid sequence of SEQ ID NO:7. In some embodiments of any of the methods or compositions provided herein, the fusion enzyme has at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity to the amino acid sequence of SEQ ID NO:7. In some embodiments of any of the methods or compositions provided herein, the fusion enzyme comprises the amino acid sequence of SEQ ID NO:7. In some embodiments of any of the methods or compositions provided herein, the fusion enzyme consists essentially of the amino acid sequence of SEQ ID NO:7 or consists of the amino acid sequence of SEQ ID NO:7.

[0227] In some embodiments, any of the methods for producing rebaudioside R6-4A and / or R6-4B described herein further comprises the step of producing rebaudioside Z1 and / or Z2. In some embodiments of any of the methods provided herein, rebaudioside Z1 and / or Z2 can be produced by incubating rebaudioside E with a UDP-glycosyltransferase and a substrate selected from sucrose, UDP, UDP-glucose, and combinations thereof. In some embodiments of any of the methods or compositions provided herein, the UDP-glycosyltransferase for producing rebaudioside Z1 and / or Z2 from rebaudioside E is UGT76G1. Methods for producing rebaudioside D3 from rebaudioside E are described in U.S. Patent No. US10081826, and such methods are incorporated herein by reference.

[0228] Rebaudioside Z1 has the following structure:

[0229]

[0230] Rebaudioside Z2 has the following structure:

[0231]

[0232] In some embodiments, any of the methods described herein for producing rebaudioside R6-4A and / or R6-4B further comprises isolating the produced rebaudioside R6-4A and / or R6-4B.

[0233] Synthesis of Rebaudioside R6-1 and Production Method

[0234] Some aspects of the present disclosure provide sweeteners (e.g., calorie-free and / or synthetic) that have been given the name "Rebaudioside R6-1 (Reb R6-1)". Rebaudioside R6-1 has a molecular formula of C 56 H 90 O 33 and has the following structure:

[0235]

[0236] Some aspects of the present disclosure provide methods for producing rebaudioside R6-1. In some embodiments of any of the methods provided herein, rebaudioside R6-1 is produced from rebaudioside D. For example, in some embodiments, the method comprises (I) preparing a reaction mixture comprising: (i) rebaudioside D; (ii) one or more substrates selected from sucrose, uridine diphosphate (UDP), uridine diphosphate-glucose (UDP-glucose), and combinations thereof; and (iii) an enzyme selected from: (a) UDP-glycosyltransferase (UGT); (b) UDP-glycosyltransferase and sucrose synthase added separately to the reaction mixture; and (c) a UDP-glycosyltransferase fusion enzyme comprising a UDP-glycosyltransferase domain coupled to a sucrose synthase domain; and (II) incubating the reaction mixture for a sufficient time to produce rebaudioside R6-1.

[0237] In some embodiments of any of the methods or compositions provided herein, glucose is covalently coupled to rebaudioside D by an enzyme to produce rebaudioside R6-1. In some embodiments of any of the methods or compositions provided herein, glucose is covalently coupled to sugar V of rebaudioside D by an enzyme to produce rebaudioside R6-1. Figure 9 The sugar numbering in rebaudioside D and R6-1 is shown.

[0238] In some embodiments of any of the methods provided herein, the enzyme for producing rebaudioside R6-1 from rebaudioside D comprises UDP-glycosyltransferase (UGT). In some embodiments of any of the methods or compositions provided herein, the UGT is HV1 glycosyltransferase. HV1 is a UGT with 1,2-19-O-glucosyl glycosylation activity. It has also been shown that HV1 UGT has 1,2-19-O-glucosyl glycosylation activity. In some embodiments of any of the methods or compositions provided herein, the HV1 UGT has at least 70% (e.g., 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% or at least 99%) identity to the amino acid sequence of SEQ ID NO:3. In some embodiments of any of the methods or compositions provided herein, the HV1 UGT has at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity to the amino acid sequence of SEQ ID NO:3. In some embodiments of any of the methods or compositions provided herein, the HV1 UGT comprises the amino acid sequence of SEQ ID NO:3. In some embodiments of any of the methods or compositions provided herein, the HV1 UGT consists essentially of, or consists of, the amino acid sequence of SEQ ID NO:3.

[0239] In some embodiments of any of the methods provided herein, the enzymes for producing rebaudioside R6-1 from rebaudioside D comprise a UDP-glycosyltransferase (UGT) and a sucrose synthase that are added separately to the reaction mixture. In some embodiments of any of the methods or compositions provided herein, the UGT is HV1 UGT and variants as described herein, and the sucrose synthase is selected from Arabidopsis thaliana sucrose synthase I, Arabidopsis thaliana sucrose synthase 3, and mung bean sucrose synthase. In some embodiments of any of the methods or compositions provided herein, the sucrose synthase or sucrose synthase domain is Arabidopsis sucrose synthase I. In some embodiments of any of the methods or compositions provided herein, Arabidopsis sucrose synthase I has at least 70% (e.g., 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%, or at least 99%) identity to the amino acid sequence of SEQ ID NO:9. In some embodiments of any of the methods or compositions provided herein, Arabidopsis sucrose synthase I has at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity to the amino acid sequence of SEQ ID NO:9. In some embodiments of any of the methods or compositions provided herein, Arabidopsis sucrose synthase I comprises the amino acid sequence of SEQ ID NO:9. In some embodiments of any of the methods or compositions provided herein, Arabidopsis sucrose synthase I consists essentially of, or consists of, the amino acid sequence of SEQ ID NO:9.

[0240] In some embodiments of any of the methods provided herein, the enzyme for producing rebaudioside R6-1 from rebaudioside D comprises a UDP-glycosyltransferase fusion enzyme comprising a UDP-glycosyltransferase domain coupled to a sucrose synthase domain. The fusion enzyme has UDP-glycosyltransferase activity and sucrose synthase activity. In some embodiments of any of the methods or compositions provided herein, the UDP-glycosyltransferase domain in the fusion enzyme is HV1 UGT and any of the variants as described herein, and the sucrose synthase domain is sucrose synthase (e.g., Arabidopsis thaliana sucrose synthase I, Arabidopsis thaliana sucrose synthase 3, or mung bean sucrose synthase) and any of the variants as described herein. In some embodiments of any of the methods or compositions provided herein, the fusion enzyme has at least 70% (e.g., 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%, or at least 99%) identity to the amino acid sequence of SEQ ID NO:7. In some embodiments of any of the methods or compositions provided herein, the fusion enzyme has at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity to the amino acid sequence of SEQ ID NO:7. In some embodiments of any of the methods or compositions provided herein, the fusion enzyme comprises the amino acid sequence of SEQ ID NO:7. In some embodiments of any of the methods or compositions provided herein, the fusion enzyme consists essentially of the amino acid sequence of SEQ ID NO:7 or consists of the amino acid sequence of SEQ ID NO:7.

[0241] In some embodiments, any of the methods for producing rebaudioside R6-1 described herein further comprises the step of producing rebaudioside D. In some embodiments of any of the methods provided, rebaudioside D can be produced by incubating rebaudioside A with a UDP-glycosyltransferase and a substrate selected from sucrose, UDP, UDP-glucose, and combinations thereof. In some embodiments of any of the methods provided, the UDP-glycosyltransferase for producing rebaudioside D from rebaudioside A is EUGT11. The method for producing rebaudioside D from rebaudioside A is described in U.S. Patent Application Publication No. US20180037600, and such methods are incorporated herein by reference.

[0242] In some embodiments of any of the methods provided herein, rebaudioside D can be produced by incubating rebaudioside E with a UDP - glycosyltransferase and a substrate selected from sucrose, UDP, UDP - glucose, and combinations thereof. In some embodiments of any of the methods provided, the UDP - glycosyltransferase for producing rebaudioside D from rebaudioside E is HV1 UGT or UGT76G1. Methods for producing rebaudioside D from rebaudioside E are described in U.S. Patent Application Publication No. 10253344, and such methods are incorporated herein by reference.

[0243] Rebaudioside D has the following structure:

[0244]

[0245] In some embodiments, any of the methods for producing rebaudioside R6 - 1 described herein further comprises isolating the produced rebaudioside R6 - 1.

[0246] Reaction Mixtures, Nucleic Acids, and Cellular Systems

[0247] In some embodiments of any of the methods provided, the reaction mixture is in vitro, i.e., the methods described herein are performed in vitro. For in vitro reactions, isolated enzymes (e.g., UDP - glycosyltransferase, sucrose synthase, and / or UDP - glycosyltransferase fusion enzyme) can be added to the in vitro reaction mixture.

[0248] In some embodiments of any of the methods provided, the reaction mixture is a cell - based reaction mixture, i.e., the reaction is performed in cells. For cell - based reactions, the enzymes (e.g., UDP - glycosyltransferase, sucrose synthase, and / or UDP - glycosyltransferase fusion enzyme) are expressed in a host cell.

[0249] In some embodiments of any of the provided methods, the enzymes (e.g., UDP - glycosyltransferase, sucrose synthase, and / or UDP - glycosyltransferase fusion enzyme) are expressed from the nucleotide sequences encoding them. Accordingly, nucleic acids encoding any of the enzymes described herein are provided. The present disclosure further provides host cells comprising a nucleotide sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identity to any one of SEQ ID NO: 2, 4, 6, 8, and 10. In some embodiments of any of the provided methods, the host cell comprises a nucleotide sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identity to SEQ ID NO: 2, and a nucleotide sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identity to SEQ ID NO: 10. In some embodiments of any of the provided methods, the host cell comprises a nucleotide sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identity to SEQ ID NO: 4, and a nucleotide sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identity to SEQ ID NO: 10.

[0250] In some embodiments of any of the provided methods, the host cells are selected from yeast, non - steviol glycoside - producing plants, algae, fungi, and bacteria.

[0251] In some embodiments of any of the provided methods, the host cell is selected from the group consisting of Escherichia; Salmonella; Bacillus; Acinetobacter; Streptomyces; Corynebacterium; Methylosinus; Methylomonas; Rhodococcus; Pseudomonas; Rhodobacter; Synechocystis; yeast; Zygosaccharomyces; Kluyveromyces; Candida; Hansenula; Debaryomyces; Mucor; Pichia; Torulopsis; Aspergillus; Arthrobotrys; Brevibacteria; Microbacterium; Arthrobacter; Citrobacter; Klebsiella; Pantoea; and Clostridium. In some embodiments of any of the provided methods, the host cell is a bacterial cell (e.g., an Escherichia coli cell). In some embodiments of any of the provided methods, the host cell is a yeast cell (e.g., a Saccharomyces cerevisiae cell).

[0252] In some embodiments of any of the provided methods, the host cell is a cell isolated from a plant selected from the group consisting of soybean; rapeseed; sunflower; cotton; corn; tobacco; alfalfa; wheat; barley; oats; sorghum; rice; broccoli; cauliflower; cabbage; parsnip; melon; carrot; celery; parsley; tomato; potato; strawberry; peanut; grape; forage crop; sugar beet; sugar cane; bean; pea; rye; flax; broadleaf tree; coniferous tree; forage grass; Arabidopsis; rice (Oryza sativa); barley (Hordeum vulgare); switchgrass (Panicum virgatum); Brachypodium species; Brassica species; and Crambe abyssinica.

[0253] Protein expression in prokaryotes is most often carried out in bacterial host cells using vectors that contain constitutive or inducible promoters that direct the expression of fusion or non-fusion proteins. Fusion vectors add a number of amino acids to the protein encoded therein, usually to the amino terminus of the recombinant protein. Such fusion vectors generally serve three purposes: 1) to increase the expression of the recombinant protein; 2) to increase the solubility of the recombinant protein; and 3) to aid in the purification of the recombinant protein by acting as a ligand in affinity purification. Often, a proteolytic cleavage site is introduced at the junction between the fusion moiety and the recombinant protein so that the recombinant protein can be separated from the fusion moiety after purification of the fusion protein. Such vectors are within the scope of the present disclosure.

[0254] In one embodiment, the expression vector includes those genetic elements for expressing a recombinant polypeptide in a bacterial cell. Elements for transcription and translation in a bacterial cell can include a promoter, a coding region of a protein complex, and a transcription terminator.

[0255] Those of ordinary skill in the art will know the molecular biology techniques available for preparing expression vectors. As described above, the polynucleotides to be incorporated into the expression vectors of the subject technology can be prepared by conventional techniques such as polymerase chain reaction (PCR).

[0256] Several molecular biology techniques can be developed to operably link DNA to a vector via complementary sticky ends. In one embodiment, complementary homopolymer tails can be added to the nucleic acid molecule to be inserted into the vector DNA. The vector and the nucleic acid molecule are then joined by hydrogen bonding between the complementary homopolymer tails to form a recombinant DNA molecule.

[0257] In an alternative embodiment, synthetic linkers containing one or more restriction sites are provided for operably linking the polynucleotides of the subject technology to an expression vector. In one embodiment, the polynucleotides are generated by restriction endonuclease digestion. In one embodiment, the nucleic acid molecule is treated with bacteriophage T4 DNA polymerase or Escherichia coli DNA polymerase I, which removes protruding 3'-single-stranded ends with its 3'-5'-exonuclease activity and fills in recessed 3' ends with its polymerase activity, thereby generating blunt-ended DNA segments. The blunt-ended segments are then incubated with a large molar excess of linker molecules in the presence of an enzyme that can catalyze the ligation of blunt-ended DNA molecules, such as bacteriophage T4 DNA ligase. Thus, the product of the reaction is a polynucleotide that carries a polymeric linker sequence at its ends. These polynucleotides are then cut with an appropriate restriction enzyme and ligated to an expression vector that has been cut with an enzyme that produces ends compatible with the ends of the polynucleotides.

[0258] Alternatively, a vector having a ligation-independent cloning (LIC) site can be employed. Then, without the need for restriction digestion or ligation, the desired PCR-amplified polynucleotide can be cloned into the LIC vector (Aslanidis and de Jong, Nucl. Acid. Res. 18, 6069-74, (1990), Haun et al., Biotechniques 13, 515-18 (1992), the disclosures of which are incorporated herein by reference to the extent that they are consistent herewith).

[0259] In one embodiment, PCR is suitable for isolating and / or modifying a polynucleotide of interest for insertion into a selected plasmid. Appropriate primers can be designed for PCR preparation of the sequence to isolate the coding region of the desired nucleic acid molecule, add a restriction endonuclease or LIC site, and place the coding region in the desired reading frame.

[0260] In one embodiment, a polynucleotide for incorporation into an expression vector of the subject technology is prepared using PCR with appropriate oligonucleotide primers. The coding region is amplified and the primers themselves become incorporated into the amplified sequence product. In one embodiment, the amplification primers contain restriction endonuclease recognition sites that allow the amplified sequence product to be cloned into an appropriate vector.

[0261] An expression vector can be introduced into a plant or microbial host cell by conventional transformation or transfection techniques. Transformation of appropriate cells with the expression vector of the subject technology is accomplished by methods known in the art and generally depends on both the type of vector and the type of cell. Suitable techniques include calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, chemoporation, or electroporation.

[0262] Successfully transformed cells, i.e., those containing the expression vector, can be identified by techniques well known in the art. For example, cells transfected with the expression vector of the subject technology can be cultured to produce the polypeptides described herein. The cells can be examined for the presence of the expression vector DNA by techniques well known in the art.

[0263] The host cell can contain a single copy of the previously described expression vector or, alternatively, multiple copies of the expression vector.

[0264] In some embodiments, the transformed cell is an animal cell, an insect cell, a plant cell, an algal cell, a fungal cell, or a yeast cell. In some embodiments, the cell is a plant cell selected from the group consisting of canola plant cells, rapeseed plant cells, palm plant cells, sunflower plant cells, cotton plant cells, corn plant cells, peanut plant cells, flax plant cells, sesame plant cells, soybean plant cells, and petunia plant cells.

[0265] Microbial host cell expression systems and expression vectors containing regulatory sequences that direct high-level expression of foreign proteins are well known to those skilled in the art. Any of these can be used to construct vectors for expressing the recombinant polypeptides of the subject technology in microbial host cells. These vectors can then be introduced into the appropriate microorganism via transformation to permit high-level expression of the recombinant polypeptides of the subject technology.

[0266] Vectors or cassettes that can be used to transform suitable microbial host cells are well known in the art. Generally, the vector or cassette contains sequences that direct transcription and translation of the relevant polynucleotide, a detectable marker, and sequences that permit autonomous replication or chromosomal integration. Suitable vectors contain a region of the polynucleotide 5' that contains transcriptional initiation control and a DNA fragment 3' that controls transcriptional termination. In some embodiments, it is preferred that both control regions are derived from genes homologous to the transformed host cell, although it should be understood that such control regions need not be derived from genes native to the particular species selected as the host.

[0267] The initiation control regions or promoters that can be used to drive expression of the recombinant polypeptides in the desired microbial host cells are numerous and familiar to those skilled in the art. In fact, any promoter capable of driving these genes is suitable for the subject technology, including but not limited to CYCI, HIS3, GALI, GALIO, ADHI, PGK, PH05, GAPDH, ADCI, TRPI, URA3, LEU2, ENO, TPI (for expression in Saccharomyces); AOXI (for expression in Pichia); and lac, trp, JPL, IPR, T7, tac, and trc (for expression in Escherichia coli).

[0268] The termination control regions can also be derived from a variety of genes native to the microbial host. Termination sites can optionally be included for the microbial hosts described herein.

[0269] In plant cells, the expression vector of the subject technology may include a coding region operably linked to a promoter that is capable of directing the expression of the recombinant polypeptide of the subject technology in the desired tissue at the desired developmental stage. For convenience, the polynucleotide to be expressed may contain a promoter sequence and a translation leader sequence derived from the same polynucleotide. A 3' non-coding sequence encoding a transcription termination signal should also be present. The expression vector may also contain one or more introns to enhance polynucleotide expression.

[0270] For plant host cells, any combination of any promoter and any terminator capable of inducing the expression of the coding region can be used in the vector sequence of the subject technology. Some suitable examples of promoters and terminators include those from the nopaline synthase (nos), octopine synthase (ocs), and cauliflower mosaic virus (CaMV) genes. One class of effective plant promoters that can be used is high-level plant promoters. Such a promoter operably linked to the expression vector of the subject technology should be capable of promoting the expression of the vector. High-level plant promoters that can be used in the subject technology include, for example, the promoter of the small subunit (ss) of ribulose-1,5-bisphosphate carboxylase from soybean (Berry-Lowe et al., J. MOLECULAR AND APP. GEN., 1:483 498 (1982), the entire content of which is incorporated herein by reference to the extent that it is consistent herewith), and the promoter of the chlorophyll a / b binding protein. Both of these promoters are known to be light-induced in plant cells (see, for example, GENETIC ENGINEERING OF PLANTS, AN AGRICULTURAL PERSPECTIVE, A. Cashmore, Plenum, N.Y. (1983), pp. 29-38; Coruzzi, G. et al., The Journal of Biological Chemistry, 258:1399 (1983), and Dunsmuir, P. et al., Journal of Molec. Appl. Gen., 2:285 (1983), the respective references of which are each incorporated herein by reference to the extent that they are consistent herewith.

[0271] Oral Consumer Products

[0272] Rebaudiosides produced by any of the methods described herein (e.g., rebaudioside R6-1, R6-2A, R6-2B, R6-4A, R6-4B, and R7-2) can be used as sweeteners. Other aspects of the present disclosure provide oral consumption products, such as those selected from beverage products and consumer products, having a sweetening amount of rebaudioside R6-1, rebaudioside R6-2A, rebaudioside R6-2B, rebaudioside R6-4A, rebaudioside R6-4B, and / or rebaudioside R7-2.

[0273] Any oral consumption product can have a sweetening intensity equivalent to that of a sucrose solution of about 1% (w / v-%) to about 4% (w / v-%).

[0274] Any oral consumption product can have about 5 ppm to about 100 ppm of rebaudioside R6-1. Any oral consumption product can have about 5 ppm to about 100 ppm of rebaudioside R6-2A. Any oral consumption product can have about 5 ppm to about 100 ppm of rebaudioside R6-2B. Any oral consumption product can have about 5 ppm to about 100 ppm of rebaudioside R6-4A. Any oral consumption product can have about 5 ppm to about 100 ppm of rebaudioside R6-4B. Any oral consumption product can have about 5 ppm to about 100 ppm of rebaudioside R7-2.

[0275] Rebaudioside R6-1 can be the sole sweetener in an oral consumption product. Rebaudioside R6-2A can be the sole sweetener in an oral consumption product. Rebaudioside R6-2B can be the sole sweetener in an oral consumption product. Rebaudioside R6-4A can be the sole sweetener in an oral consumption product. Rebaudioside R6-4B can be the sole sweetener in an oral consumption product. Rebaudioside R7-2 can be the sole sweetener in an oral consumption product.

[0276] Any oral consumption product may also have at least one additional sweetener. The at least one additional sweetener may be, for example, a natural high-intensity sweetener. The additional sweetener may be selected from stevia extract, steviol glycosides, stevioside, rebaudioside A, rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside D2, rebaudioside E, rebaudioside F, rebaudioside M, rebaudioside V, rebaudioside W, rebaudioside Z1, rebaudioside Z2, rebaudioside D3, dulcoside A, phyllodulcin, steviolbioside, sucrose, high fructose corn syrup, fructose, glucose, xylose, arabinose, rhamnose, erythritol, xylitol, mannitol, sorbitol, inositol, AceK, aspartame, neotame, sucralose, saccharin, naringin dihydrochalcone (NarDHC), neohesperidin dihydrochalcone (NDHC), phyllodulcin, mogroside IV, serendipide I, mogroside V, monatin, thaumatin, miraculin, sweet protein, L-alanine, glycine, Siraitia grosvenorii, hernandulcin, phyllanthin, trilobatin, and combinations thereof.

[0277] Any oral consumption product may also have at least one additive. The additive may be, for example, a carbohydrate, a polyol, an amino acid or its salt, a polyamino acid or its salt, a sugar acid or its salt, a nucleotide, an organic acid, an inorganic acid, an organic salt, an organic acid salt, an organic base salt, an inorganic salt, a bitter compound, a food flavor, a flavoring ingredient, an astringent compound, a protein, a protein hydrolysate, a surfactant, an emulsifier, a flavonoid, an alcohol, a polymer, and combinations thereof.

[0278] In some embodiments, the present disclosure provides a beverage product comprising a sweetening amount of rebaudioside R6-1. In some embodiments, the present disclosure provides a beverage product comprising a sweetening amount of rebaudioside R6-2A. In some embodiments, the present disclosure provides a beverage product comprising a sweetening amount of rebaudioside R6-2B. In some embodiments, the present disclosure provides a beverage product comprising a sweetening amount of rebaudioside R6-4A. In some embodiments, the present disclosure provides a beverage product comprising a sweetening amount of rebaudioside R6-4B. In some embodiments, the present disclosure provides a beverage product comprising a sweetening amount of rebaudioside R7-2.

[0279] Any beverage product may be, for example, a carbonated beverage product and a non-carbonated beverage product. Any beverage product may also be, for example, a soft drink, a fountain drink, a frozen drink; a ready-to-drink beverage; a frozen ready-to-drink beverage, coffee, tea, a milk beverage, a powdered soft drink, a concentrate, a flavored water, a fortified water, a fruit juice, a fruit juice flavored beverage, a sports drink, and an energy drink.

[0280] In some embodiments, any of the beverage products of the present disclosure can include one or more beverage ingredients, such as acidulants, fruit and / or vegetable juices, pulps, etc., flavorings, colorants, preservatives, vitamins, minerals, electrolytes, erythritol, tagatose, glycerol, and carbon dioxide. Such beverage products can be provided in any suitable form, such as beverage concentrates and carbonated ready-to-drink beverages.

[0281] In certain embodiments, any of the beverage products of the present disclosure can have any of a number of different specific formulations or compositions. The formulation of the beverage products of the present disclosure can vary to some extent depending on such factors as the intended market segment of the product, its desired nutritional profile, flavor characteristics, and the like. For example, in certain embodiments, further ingredients can generally be selected to be added to the formulation of a particular beverage product. For example, additional (i.e., more and / or other) sweeteners can be added, and flavorings, electrolytes, vitamins, fruit juices or other fruit products, tastents, masking agents, etc., flavor enhancers, and / or carbonation can generally be added to any such formulation to alter the taste, mouthfeel, nutritional profile, etc. In an embodiment, any of the beverage products can be a cola beverage that contains water, from about 5 ppm to about 100 ppm of rebaudioside R6-1, an acidulant, and a flavoring. In an embodiment, any of the beverage products can be a cola beverage that contains water, from about 5 ppm to about 100 ppm of rebaudioside R6-2A, an acidulant, and a flavoring. In an embodiment, any of the beverage products can be a cola beverage that contains water, from about 5 ppm to about 100 ppm of rebaudioside R6-2B, an acidulant, and a flavoring. In an embodiment, any of the beverage products can be a cola beverage that contains water, from about 5 ppm to about 100 ppm of rebaudioside R6-4A, an acidulant, and a flavoring. In an embodiment, any of the beverage products can be a cola beverage that contains water, from about 5 ppm to about 100 ppm of rebaudioside R6-4B, an acidulant, and a flavoring. In an embodiment, any of the beverage products can be a cola beverage that contains water, from about 5 ppm to about 100 ppm of rebaudioside R7-2, an acidulant, and a flavoring.

[0282] Exemplary flavorings can be, for example, cola flavorings, citrus flavorings, and spice flavorings. In some embodiments, carbonation in the form of carbon dioxide can be added for effervescence. In other embodiments, preservatives can be added, depending on other ingredients, production techniques, desired shelf life, etc. In certain embodiments, caffeine can be added. In some embodiments, the beverage product can be a cola-flavored carbonated beverage that characteristically contains carbonated water, a sweetener, cola nut extract and / or other flavorings, caramel color, one or more acids, and optionally other ingredients.

[0283] A suitable amount of rebaudioside R6-1, rebaudioside R6-2A, rebaudioside R6-2B, rebaudioside R6-4A, rebaudioside R6-4B, or rebaudioside R7-2 present in a beverage product can be, for example, from about 5 ppm to about 100 ppm. In some embodiments, a low concentration of rebaudioside R6-1, rebaudioside R6-2A, rebaudioside R6-2B, rebaudioside R6-4A, rebaudioside R6-4B, or rebaudioside R7-2, such as less than 100 ppm, has a sweetness equivalent to a sucrose solution having a concentration between 10,000 ppm and 30,000 ppm. The final concentration ranges from about 5 ppm to about 100 ppm, from about 5 ppm to about 95 ppm, from about 5 ppm to about 90 ppm, from about 5 ppm to about 85 ppm, from about 5 ppm to about 80 ppm, from about 5 ppm to about 75 ppm, from about 5 ppm to about 70 ppm, from about 5 ppm to about 65 ppm, from about 5 ppm to about 60 ppm, from about 5 ppm to about 55 ppm, from about 5 ppm to about 50 ppm, from about 5 ppm to about 45 ppm, from about 5 ppm to about 40 ppm, from about 5 ppm to about 35 ppm, from about 5 ppm to about 30 ppm, from about 5 ppm to about 25 ppm, from about 5 ppm to about 20 ppm, from about 5 ppm to about 15 ppm, or from about 5 ppm to about 10 ppm. Alternatively, rebaudioside R6-1, rebaudioside R6-2A, rebaudioside R6-2B, rebaudioside R6-4A, rebaudioside R6-4B, or rebaudioside R7-2 can be present in the beverage products of the present disclosure at a final concentration ranging from about 5 ppm to about 100 ppm, from about 10 ppm to about 100 ppm, from about 15 ppm to about 100 ppm, from about 20 ppm to about 100 ppm, from about 25 ppm to about 100 ppm, from about 30 ppm to about 100 ppm, from about 35 ppm to about 100 ppm, from about 40 ppm to about 100 ppm, from about 45 ppm to about 100 ppm, from about 50 ppm to about 100 ppm, from about 55 ppm to about 100 ppm, from about 60 ppm to about 100 ppm, from about 65 ppm to about 100 ppm, from about 70 ppm to about 100 ppm, from about 75 ppm to about 100 ppm, from about 80 ppm to about 100 ppm, from about 85 ppm to about 100 ppm, from about 90 ppm to about 100 ppm, or from about 95 ppm to about 100 ppm.

[0284] In some embodiments, the present disclosure provides consumer products comprising a sweetening amount of rebaudioside R6-1. In some embodiments, the present disclosure provides consumer products comprising a sweetening amount of rebaudioside R6-2A. In some embodiments, the present disclosure provides consumer products comprising a sweetening amount of rebaudioside R6-2B. In some embodiments, the present disclosure provides consumer products comprising a sweetening amount of rebaudioside R6-4A. In some embodiments, the present disclosure provides consumer products comprising a sweetening amount of rebaudioside R6-4B. In some embodiments, the present disclosure provides consumer products comprising a sweetening amount of rebaudioside R7-2. Consumer products can be, for example, food products, nutritional products, dietary supplements, dental hygiene compositions, edible gel compositions, cosmetics, and tabletop flavorings.

[0285] As used herein, "dietary supplement" refers to a compound that is intended to supplement the diet and provide nutrients that may be missing or may not be consumed in sufficient amounts in the diet, such as vitamins, minerals, fiber, fatty acids, amino acids, etc. Any suitable dietary supplement known in the art can be used. Examples of suitable dietary supplements can be, for example, nutrients, vitamins, minerals, fiber, fatty acids, herbs, botanicals, amino acids, and metabolites.

[0286] As used herein, "nutritional product" refers to a compound that includes any food or portion of a food that may provide a medicinal or health benefit, such as preventing and / or treating a disease or disorder (e.g., fatigue, insomnia, effects of aging, memory loss, mood disorders, cardiovascular disease and high levels of cholesterol in the blood, diabetes, osteoporosis, inflammation, autoimmune disorders, etc.). Any suitable nutritional product known in the art can be used. In some embodiments, the nutritional product can be used as a supplement to food and beverages.

[0287] In some embodiments, the dietary supplement and the nutritional product can further contain protective hydrocolloids (such as gums, proteins, modified starches), binders, film formers, encapsulating agents / materials, wall / shell materials, matrix compounds, coatings, emulsifiers, surfactants, solubilizers (oils, fats, waxes, lecithin, etc.), adsorbents, carriers, fillers, co-compounds, dispersants, wetting agents, processing aids (solvents), flow agents, taste masking agents, weight increasing agents, jellyfying agents, gel forming agents, antioxidants, and antimicrobial agents.

[0288] As used herein, "gel" refers to a colloidal system in which a network of particles spans the volume of a liquid medium. Although gels are composed primarily of liquid and thus exhibit a density similar to that of a liquid, gels have the structural cohesion of a solid due to the network of particles spanning the liquid medium. For this reason, gels generally appear as solid, jelly-like materials. Gels can be used in a variety of applications. For example, gels can be used in foods, paints, and adhesives. Edible gels are referred to as "edible gel compositions". Edible gel compositions are typically consumed as snacks, desserts, as part of a staple food, or together with a staple food. Examples of suitable edible gel compositions can be, for example, gel desserts, puddings, jams, jellies, pastes, trifles, aspics, marshmallows, fudges, and the like. In some embodiments, the edible gel mixture is generally a powdery or granular solid to which a fluid can be added to form an edible gel composition. Examples of suitable fluids can be, for example, water, milk fluids, milk-like fluids, fruit juices, alcohols, alcoholic beverages, and combinations thereof. Examples of suitable milk fluids can be, for example, milk, cultured milk, cream, fluid whey, and mixtures thereof. Examples of suitable milk-like fluids can be, for example, soy milk and non-dairy coffee whiteners.

[0289] As used herein, the term "gelling component" refers to any material that can form a colloidal system within a liquid medium. Examples of suitable gelling components can be, for example, gelatin, alginate, carrageenan, gums, pectin, konjac, agar, food acids, rennet, starches, starch derivatives, and combinations thereof. It is well known to those skilled in the art that the amount of gelling component used in an edible gel mixture or edible gel composition can vary significantly depending on a number of factors, such as the specific gelling component used, the specific fluid matrix used, and the desired gel properties.

[0290] The gel mixtures and gel compositions of the present disclosure can be prepared by any suitable method known in the art. In some embodiments, the edible gel mixtures and edible gel compositions of the present disclosure can be prepared using other ingredients in addition to gelling agents. Examples of other suitable ingredients can be, for example, food acids, salts of food acids, buffer systems, fillers, chelating agents, cross-linking agents, one or more flavorings, one or more colorants, and combinations thereof.

[0291] Rebaudioside R6-1, rebaudioside R6-2A, rebaudioside R6-2B, rebaudioside R6-4A, rebaudioside R6-4B or rebaudioside R7-2 can be used with any suitable dental and oral hygiene compositions known in the art. Examples of suitable dental and oral hygiene compositions can be, for example, toothpaste, tooth polish, dental floss, mouthwash, gargle, dentifrice, oral spray, oral freshener, plaque gargle, tooth analgesic, and the like. Also provided are dental and oral hygiene compositions comprising any one of the rebaudiosides provided herein.

[0292] A suitable amount of rebaudioside R6-1, rebaudioside R6-2A, rebaudioside R6-2B, rebaudioside R6-4A, rebaudioside R6-4B, or rebaudioside R7-2 present in any of the compositions provided herein, such as consumer products, can be, for example, from about 5 parts per million (ppm) to about 100 parts per million (ppm). In some embodiments of any of the compositions provided, a low concentration of rebaudioside R6-1, rebaudioside R6-2A, rebaudioside R6-2B, rebaudioside R6-4A, rebaudioside R6-4B, or rebaudioside R7-2, such as less than 100 ppm, has a sweetness equivalent to that of a sucrose solution having a concentration between 10,000 ppm and 30,000 ppm. The final concentration range can be from about 5 ppm to about 100 ppm, from about 5 ppm to about 95 ppm, from about 5 ppm to about 90 ppm, from about 5 ppm to about 85 ppm, from about 5 ppm to about 80 ppm, from about 5 ppm to about 75 ppm, from about 5 ppm to about 70 ppm, from about 5 ppm to about 65 ppm, from about 5 ppm to about 60 ppm, from about 5 ppm to about 55 ppm, from about 5 ppm to about 50 ppm, from about 5 ppm to about 45 ppm, from about 5 ppm to about 40 ppm, from about 5 ppm to about 35 ppm, from about 5 ppm to about 30 ppm, from about 5 ppm to about 25 ppm, from about 5 ppm to about 20 ppm, from about 5 ppm to about 15 ppm, or from about 5 ppm to about 10 ppm. Alternatively, rebaudioside R6-1, rebaudioside R6-2A, rebaudioside R6-2B, rebaudioside R6-4A, rebaudioside R6-4B, or rebaudioside R7-2 can be present in any of the compositions provided, such as any consumer product of the present disclosure, in a final concentration range from about 5 ppm to about 100 ppm, from about 10 ppm to about 100 ppm, from about 15 ppm to about 100 ppm, from about 20 ppm to about 100 ppm, from about 25 ppm to about 100 ppm, from about 30 ppm to about 100 ppm, from about 35 ppm to about 100 ppm, from about 40 ppm to about 100 ppm, from about 45 ppm to about 100 ppm, from about 50 ppm to about 100 ppm, from about 55 ppm to about 100 ppm, from about 60 ppm to about 100 ppm, from about 65 ppm to about 100 ppm, from about 70 ppm to about 100 ppm, from about 75 ppm to about 100 ppm, from about 80 ppm to about 100 ppm, from about 85 ppm to about 100 ppm, from about 90 ppm to about 100 ppm, or from about 95 ppm to about 100 ppm.

[0293] In certain embodiments, about 5 ppm to about 100 ppm of rebaudioside R6-1, rebaudioside R6-2A, rebaudioside R6-2B, rebaudioside R6-4A, rebaudioside R6-4B, or rebaudioside R7-2 is present in a food product composition. Such compositions are also provided. As used herein, "food product composition" refers to any solid or liquid ingestible material that may but need not have nutritional value and is intended for consumption by humans and animals.

[0294] Examples of suitable food product compositions can be, for example, confectionery compositions such as candies, mints, fruit drops, cocoa products, chocolates, etc.; condiments such as ketchup, mustard, mayonnaise, etc.; chewing gum; cereal compositions; baked foods such as bread, cakes, pies, cookies, etc.; dairy products such as milk, cheese, cream, ice cream, sour cream, yogurt, fruit curds, etc.; tabletop sweetener compositions; soups; stews; convenience foods; meats such as ham, bacon, sausage, jerky, etc.; gelatin and gelatin-like products such as jams, jellies, preserves, etc.; fruits; vegetables; egg products; frostings; syrups including molasses; snacks; nut and nut products; and animal feeds.

[0295] The food product composition can also be herbs, spices and seasonings, natural and synthetic flavorings, and flavor enhancers such as monosodium glutamate. In some embodiments, any one of the food product compositions can be, for example, a prepared packaged product such as a dietary sweetener, a liquid sweetener, a granular seasoning mixture, pet food, livestock feed, tobacco, and materials for baking applications such as a powdered baking mixture for preparing bread, cookies, cakes, pancakes, doughnuts, etc. In other embodiments, any one of the food product compositions can also be a diet containing little or no sucrose and low-calorie foods and beverages.

[0296] In certain embodiments, which may or may not be combined with any of the foregoing embodiments, rebaudioside R6-1, rebaudioside R6-2A, rebaudioside R6-2B, rebaudioside R6-4A, rebaudioside R6-4B, or rebaudioside R7-2 is the sole sweetener and the product has a sweetness intensity equivalent to a sucrose solution of about 1% to about 4% (w / v-%). In certain embodiments, which may or may not be combined with any of the foregoing embodiments, the consumer product and the beverage product may further comprise an additional sweetener, wherein the product has a sweetness intensity equivalent to a sucrose solution of about 1% to about 10% (w / v-%). In certain embodiments, which may or may not be combined with any of the foregoing embodiments, each sweetening component in the product is a high-intensity sweetener. In certain embodiments, which may or may not be combined with any of the foregoing embodiments, each sweetening component in the product may be a natural high-intensity sweetener. In certain embodiments, which may or may not be combined with any of the foregoing embodiments, the additional sweetener comprises one or more sweeteners selected from: stevia extract, steviol glycosides, stevioside, rebaudioside A, rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside D2, rebaudioside F, rebaudioside D3, rebaudioside Z1, rebaudioside Z2, rebaudioside M, rebaudioside W, rebaudioside V, dulcoside A, phyllodulcin, steviolbioside, sucrose, high fructose corn syrup, fructose, glucose, xylose, arabinose, rhamnose, erythritol, xylitol, mannitol, sorbitol, inositol, AceK, aspartame, neotame, sucralose, saccharin, naringin dihydrochalcone (NarDHC), neohesperidin dihydrochalcone (NDHC), phyllodulcin, mogroside IV, thaumatin I, mogroside V, monatin, thaumatin, brazzein, sweet protein, L-alanine, glycine, momordica grosvenori, hernandulcin, phyllanthin, trilobatin, and combinations thereof. In certain embodiments, which may or may not be combined with any of the foregoing embodiments, the consumer product and the beverage product may further comprise one or more additives selected from: carbohydrates, polyols, amino acids or their salts, polyamino acids or their salts, sugar acids or their salts, nucleotides, organic acids, inorganic acids, organic salts, organic acid salts, organic base salts, inorganic salts, bitter compounds, edible flavors, flavoring components, astringent compounds, proteins, protein hydrolysates, surfactants, emulsifiers, flavonoids, alcohols, polymers, and combinations thereof. In certain embodiments, which may or may not be combined with any of the foregoing embodiments, rebaudioside R6-1, rebaudioside R6-2A, rebaudioside R6-2B, rebaudioside R6-4A, rebaudioside R6-4B, or rebaudioside R7-2 has a purity of about 50% to about 100% by weight before being added to the product.

[0297] Example

[0298] Example 1: Production of Novel Steviol Glycosides R6-2 and R7-2 by Enzymatic Biotransformation

[0299] According to the present invention, full-length DNA fragments of all candidate UDP-glucosyltransferase (UGT) genes were commercially synthesized. Almost all cDNA codons were changed to codons preferred by Escherichia coli (Genscript, NJ). The synthesized DNA was cloned into the bacterial expression vector pETite N-His SUMO Kan Vector (Lucigen).

[0300] Each expression construct was transformed into Escherichia coli BL21(DE3), which was then grown in LB medium containing 50 μg / mL kanamycin at 37 °C until an OD600 of 0.8 - 1.0 was reached. Protein expression was induced by adding 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG), and the culture was further grown at 16 °C for 22 h. Cells were harvested by centrifugation (3,000 x g; 10 min; 4 °C). The cell pellet was collected and used immediately or stored at -80 °C.

[0301] The cell pellet was usually resuspended in lysis buffer (50 mM potassium phosphate buffer pH 7.2, 25 μg / ml lysozyme, 5 μg / ml DNase I, 20 mM imidazole, 500 mM NaCl, 10% glycerol, and 0.4% Triton X-100). Cells were disrupted by sonication at 4 °C, and cell debris was clarified by centrifugation (18,000 x g; 30 min). The supernatant was loaded onto a pre-equilibrated (equilibration buffer: 50 mM potassium phosphate buffer pH 7.2, 20 mM imidazole, 500 mM NaCl, 10% glycerol) Ni-NTA (Qiagen) affinity column. After loading the protein sample, the column was washed with equilibration buffer to remove unbound contaminating proteins. The His-tagged UGT recombinant polypeptide was eluted with equilibration buffer containing 250 mM imidazole.

[0302] The glycosylation activity of the purified candidate UGT recombinant polypeptide was determined by using rebaudioside D3 (Reb D3) as a substrate. Generally, the recombinant polypeptide (10 - 50 μg) was tested in a 200 μl in vitro reaction system. The reaction system contained 50 mM potassium phosphate buffer pH 7.2, 3 mM MgCl 2, 1 mg / ml Reb D3 substrate, 1 mM UDP-glucose or UDP and / or sucrose synthase (SUS), and 250 mM sucrose. The reaction was carried out at 30 - 37 °C and terminated by adding 200 μL of 1-butanol at different time points to a 50 μL reaction. The samples were extracted three times with 200 μL of 1-butanol. The combined fractions were dried and dissolved in 100 μL of 80% methanol for high performance liquid chromatography (HPLC) analysis.

[0303] Then HPLC analysis was performed using a Dionex UPLC ultimate 3000 system (Sunnyvale, CA), which included a quaternary pump, a temperature-controlled column compartment, an autosampler, and a UV absorbance detector. A Synergi Hydro-RP column (Phenomenex) with a guard column was used to characterize the steviol glycosides in the combined samples. An aqueous solution of acetonitrile was used for isocratic elution in the HPLC analysis. The detection wavelength used in the HPLC analysis was 210 nm.

[0304] After activity screening, UGT76G1 (SEQ ID NO:1) was found to have strong activity in producing R6-2 and R7-2 steviol glycosides ( Figure 2 ).

[0305] As Figure 3 shown, UGT76G1 can convert Reb D3 to R6-2, and the resulting R6-2 can be subsequently (continentally) converted to R7-2. R6-2 and R7-2 were produced at an earlier reaction time (2 h, Figure 3 , inset B). The resulting R6-2 can be converted to R7-2 at a later time point (4 h, Figure 3 , inset C). Eventually, all Reb D3 and the resulting R6-2 can be completely converted to the R7-2 compound (19 h, Figure 3 , inset D).

[0306] Example 2: Identification of the Production of R6-2 and R7-2 by LC-MS Analysis

[0307] To confirm the resulting compounds, the resulting compounds were analyzed by LC-MS analysis in comparison with standards, and their identities were confirmed.

[0308] The same sample from the above enzymatic biotransformation was analyzed by LC-MS using a Synergy Hydro-RP column. Mobile phase A was an aqueous solution of 0.1% formic acid, while mobile phase B was an acetonitrile solution of 0.1% formic acid. The flow rate was 0.6 ml / min. The mass spectrometry analysis of the sample was completed on a QExactive Hybrid Quadrupole-Orbitrap Mass Spectrometer (Thermo Fisher Scientific) using an optimized method in the positive ion mode.

[0309] The molecular formula of compound R6-2 was deduced as C 56 H 90 O 33 based on its positive high-resolution (HR) mass spectrum, which showed an adduct ion corresponding to [M+H] + at m / z 1291.5401 ( Figure 4 ). The predicted structure of R6-2 is presented in Figure 2 .

[0310] The molecular formula of compound R7-2 was deduced as C 62 H 100 O 38 based on its positive high-resolution (HR) mass spectrum, which showed an adduct ion corresponding to [M+H] + at m / z 1453.5921 ( Figure 5 ). The predicted structure of R7-2 is presented in Figure 2 , 6 and 7.

[0311] Example 3: Structure Analysis of R7-2 by NMR:

[0312] The resulting compound R7-2 was purified by semi-preparative chromatography as described above.

[0313] HRMS data were generated using an LTQ Orbitrap Discovery HRMS instrument with a resolution set to 30k. Data were scanned from m / z 150 to 1500 in the positive ion electrospray mode. The needle voltage was set to 4 kV; other source conditions were sheath gas = 25, auxiliary gas = 0, sweep gas = 5 (all gas flows are in arbitrary units), capillary voltage = 30 V, capillary temperature = 300 °C, and tube lens voltage = 75. The sample was diluted with pyridine and injected at 50 μl.

[0314] NMR spectra were obtained using standard pulse sequences on a Bruker Avance DRX 500 MHz or Varian INOVA 600 MHz instrument. 1D ( 1 H and13 C) and 2D (TOCSY, HSQC, ROESY, and HMBC) NMR spectra were performed in CDCl₃ 5 D 5 N.

[0315] The molecular formula of compound R7-2 was deduced to be C₅₅H₈₄O₂₀ based on its positive high-resolution (HR) mass spectrum, which showed adduct ions corresponding to [M + NH₄]⁺ at m / z 1470.6222 and [M + Na]⁺ at m / z 1475.5774; this composition was supported by ¹³C NMR and HSQC spectral data. The ¹H NMR spectrum of R7-2 showed the presence of two sp³ methyl singlets at δ 1.33 and 1.39; two olefinic protons as singlets at δ 5.00 and 5.74 for an exocyclic double bond; nine sp³ methylene and two sp³ methine protons between δ 0.75 - 2.74 characteristic of ent-kaurane diterpenoids isolated earlier from Stevia. Enzymatic hydrolysis of R7-2 provided a compound that was found to be identical to steviol based on NMR spectral data (Ohtani et al., 1992). The ¹H NMR spectrum of R7-2 also showed the presence of anomeric protons resonating at δ 4.99, 5.05, 5.35, 5.38, 5.43, 5.82, and 6.40, indicating seven sugar units in its molecular structure. R7-2 was treated with 5% H₂SO₄ 62 H 100 O 38 , the mass spectrum showing adduct ions corresponding to [M + NH₄]⁺ at m / z 1470.6222 and [M + Na]⁺ at m / z 1475.5774; this composition was supported by ¹³C NMR and HSQC spectral data. The ¹H NMR spectrum of R7-2 showed the presence of two sp³ methyl singlets at δ 1.33 and 1.39; two olefinic protons as singlets at δ 5.00 and 5.74 for an exocyclic double bond; nine sp³ methylene and two sp³ methine protons between δ 0.75 - 2.74 characteristic of ent-kaurane diterpenoids isolated earlier from Stevia. Enzymatic hydrolysis of R7-2 provided a compound that was found to be identical to steviol based on NMR spectral data (Ohtani et al., 1992). The ¹H NMR spectrum of R7-2 also showed the presence of anomeric protons resonating at δ 4.99, 5.05, 5.35, 5.38, 5.43, 5.82, and 6.40, indicating seven sugar units in its molecular structure. R7-2 was treated with 5% H₂SO₄ 4 + and [M + Na]⁺ at m / z 1475.5774; this composition was supported by ¹³C NMR and HSQC spectral data. The ¹H NMR spectrum of R7-2 showed the presence of two sp³ methyl singlets at δ 1.33 and 1.39; two olefinic protons as singlets at δ 5.00 and 5.74 for an exocyclic double bond; nine sp³ methylene and two sp³ methine protons between δ 0.75 - 2.74 characteristic of ent-kaurane diterpenoids isolated earlier from Stevia. Enzymatic hydrolysis of R7-2 provided a compound that was found to be identical to steviol based on NMR spectral data (Ohtani et al., 1992). The ¹H NMR spectrum of R7-2 also showed the presence of anomeric protons resonating at δ 4.99, 5.05, 5.35, 5.38, 5.43, 5.82, and 6.40, indicating seven sugar units in its molecular structure. R7-2 was treated with 5% H₂SO₄ + ; this composition was supported by ¹³C NMR and HSQC spectral data. The ¹H NMR spectrum of R7-2 showed the presence of two sp³ methyl singlets at δ 1.33 and 1.39; two olefinic protons as singlets at δ 5.00 and 5.74 for an exocyclic double bond; nine sp³ methylene and two sp³ methine protons between δ 0.75 - 2.74 characteristic of ent-kaurane diterpenoids isolated earlier from Stevia. Enzymatic hydrolysis of R7-2 provided a compound that was found to be identical to steviol based on NMR spectral data (Ohtani et al., 1992). The ¹H NMR spectrum of R7-2 also showed the presence of anomeric protons resonating at δ 4.99, 5.05, 5.35, 5.38, 5.43, 5.82, and 6.40, indicating seven sugar units in its molecular structure. R7-2 was treated with 5% H₂SO₄ 13 ¹³C NMR and HSQC spectral data. The ¹H NMR spectrum of R7-2 showed the presence of two sp³ methyl singlets at δ 1.33 and 1.39; two olefinic protons as singlets at δ 5.00 and 5.74 for an exocyclic double bond; nine sp³ methylene and two sp³ methine protons between δ 0.75 - 2.74 characteristic of ent-kaurane diterpenoids isolated earlier from Stevia. Enzymatic hydrolysis of R7-2 provided a compound that was found to be identical to steviol based on NMR spectral data (Ohtani et al., 1992). The ¹H NMR spectrum of R7-2 also showed the presence of anomeric protons resonating at δ 4.99, 5.05, 5.35, 5.38, 5.43, 5.82, and 6.40, indicating seven sugar units in its molecular structure. R7-2 was treated with 5% H₂SO₄ 1 ¹H NMR spectrum showed the presence of two sp³ methyl singlets at δ 1.33 and 1.39; two olefinic protons as singlets at δ 5.00 and 5.74 for an exocyclic double bond; nine sp³ methylene and two sp³ methine protons between δ 0.75 - 2.74 characteristic of ent-kaurane diterpenoids isolated earlier from Stevia. Enzymatic hydrolysis of R7-2 provided a compound that was found to be identical to steviol based on NMR spectral data (Ohtani et al., 1992). The ¹H NMR spectrum of R7-2 also showed the presence of anomeric protons resonating at δ 4.99, 5.05, 5.35, 5.38, 5.43, 5.82, and 6.40, indicating seven sugar units in its molecular structure. R7-2 was treated with 5% H₂SO₄ 1 ¹H NMR spectrum also showed the presence of anomeric protons resonating at δ 4.99, 5.05, 5.35, 5.38, 5.43, 5.82, and 6.40, indicating seven sugar units in its molecular structure. R7-2 was treated with 5% H₂SO₄ 2 SO 4 ​Acid hydrolysis of

[0316] gave D-glucose, which was identified by direct comparison with an authentic sample by TLC, suggesting the presence of six glucopyranosyl moieties in its molecular structure (Bedir et al., 2001; Chaturvedula et al., 2003; and Huan et al., 1998). Further, the configuration of D-glucose was identified by preparing its corresponding thiocarbamoyl-thiazolidinecarboxylate derivative with L-cysteine methyl ester and o-tolyl isothiocyanate and comparing its retention time with that of standard sugars as described in the literature comparison (Tanaka et al., 2007). Based on the results of NMR spectral data from R7-2, it was concluded that seven D-glucosyl units attached to the steviol moiety were present in its structure. The basic skeleton of steviol in R7-2 was supported by TOCSY (H-1 / H-2; H-2 / H-3; H-5 / H-6; H-6 / H-7; H-9 / H-11; H-11 / H-12) and HMBC (H-1 / C-2, C-10; H-3 / C-1, C-2, C-4, C-5, C-18, C-19; H-5 / C-4, C-6, C-7, C-9, C-10, C-18, C-19, C-20; H-9 / C-8, C-10, C-11, C-12, C-14, C-15; H-14 / C-8, C-9, C-13, C-15, C-16 and H-17 / C-13, C-15, C-16) correlations.

[0316] For all protons and carbons in R7-2 1 H and 13 C NMR values were assigned based on TOCSY, HMQC and HMBC correlations and are given in Table 1.

[0317] A close comparison of the 1 H and 13 C NMR spectra of R7-2 with those of rebaudioside D3 (Mao et al. 2017) suggested that this compound was also a steviol glycoside, which had four glucosyl moieties attached as an ether at the C-13 hydroxyl group, three of which were attached as 2,3-branched glucotriosyl substituents; and another 2,3-branched glucotriosyl moiety as an ester at C-19. The above data explained six glucosyl units in the molecular structure of R7-2, leaving the identification of an additional glucosyl moiety on the 2,3-branched glucotriosyl substituent at the C-13 position. Based on Figure 8 the key TOCSY and HMBC correlations shown in

[0318] The large coupling constants observed for all seven anomeric protons of the glucosyl moieties at δ 4.99 (d, J = 7.6 Hz), 5.05 (d, J = 7.4 Hz), 5.35 (d, J = 8.1 Hz), 5.38 (d, J = 7.7 Hz), 5.43 (d, J = 7.7 Hz), 5.82 (d, J = 6.6 Hz) and 6.40 (d, J = 8.1 Hz) suggest a β-orientation as reported for steviol glycosides.

[0319] Based on NMR and HR mass spectrometry data and hydrolysis studies, the structure of R7-2 produced by the enzymatic conversion of rebaudioside D3 was deduced to be 13-[(2-O-β-D-glucopyranosyl-3-O-β-D-glucopyranosyl-6-O-β-D-glucopyranosyl-β-D-glucopyranosyl)oxy] ent-kaur-16-ene-19-oic acid-[(2-O-β-D-glucopyranosyl-3-O-β-D-glucopyranosyl-β-D-glucopyranosyl) ester.

[0320] By maintaining the pH at 4.5, R7-2 (500 μg) was dissolved in 5.0 ml of 0.1 M sodium acetate buffer and 100 μL of crude pectinase from Aspergillus niger (Sigma-Aldrich) was added. The mixture was stirred at 50 °C for 96 h and the product precipitated during the reaction was filtered and then solidified. By comparing its 1 1H NMR spectral data, the obtained product was identified as steviol.

[0321] R7-2 (1 mg) was dissolved in MeOH (8 ml) and 5% H 2 2 4 SO 2 4 2 (25 mL) was added. The mixture was refluxed for 16 h, cooled to room temperature and then neutralized with saturated sodium carbonate. The aqueous phase was extracted with ethyl acetate (EtOAc, 2 x 25 ml) and the aqueous layer was concentrated and compared with standard sugars using a TLC system of EtOAc / n-butanol / water (2:7:1) and CH

[0322] R7-2 (1 mg) was hydrolyzed with 0.5 M HCl (1.5 mL) for 1.5 h. After cooling, the mixture was passed through an Amberlite IRA400 column, and the eluate was lyophilized. The residue was dissolved in pyridine (0.75 mL) and heated with L-cysteine methyl ester HCl (7.5 mg) at 60 °C for 1.5 h, then o-tolyl isothiocyanate (30 μL) was added to the mixture and heated at 60 °C for an additional 1.5 h. HPLC analysis of the reaction mixture was carried out on a Phenomenex Luna column [C18, 150 x 4.6 mm (5 μm)], using a mobile phase of 25% acetonitrile - 0.2% TFA in water, at 1 mL / min with UV detection at 250 nm. The sugar was identified as D-glucose (tR, 12.72) [authentic sample, D-glucose (tR, 12.64) and L-glucose (tR, 11.48 min)] (Tanaka et al., 2007).

[0323] The compound designated as R7-2 was obtained from the enzymatic biotransformation of rebaudioside D3. The structural elucidation and complete NMR spectral assignment ( 1 H and 13 C) of R7-2 were carried out on the basis of extensive 1D and 2D NMR as well as high-resolution mass spectrometry data and hydrolysis studies, which suggested the structure as 13-[(2-O-β-D-glucopyranosyl-3-O-β-D-glucopyranosyl-6-O-β-D-glucopyranosyl-β-D-glucopyranosyl)oxy] ent-kaur-16-ene-19-oic acid - [(2-O-β-D-glucopyranosyl-3-O-β-D-glucopyranosyl-β-D-glucopyranosyl) ester ( Figure 6 ).

[0324] Example 5: Production of Novel Steviol Glycoside R6-1 by Enzymatic Biotransformation

[0325] Full-length DNA fragments of all candidate UDP-glucosyltransferase (UGT) genes were commercially synthesized. Almost all cDNA codons were changed to E. coli-preferred codons (Genscript, NJ). The synthesized DNA was cloned into the bacterial expression vector pETite N-His SUMO Kan Vector (Lucigen).

[0326] Each expression construct was transformed into E. coli BL21(DE3), which was then grown in LB medium containing 50 μg / mL kanamycin at 37 °C until an OD of 0.8 - 1.0 was reached 600Protein expression was induced by adding 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG), and the culture was further grown at 16 °C for 22 h. Cells were harvested by centrifugation (3,000 x g; 10 min; 4 °C). The cell pellet was collected and used immediately or stored at -80 °C.

[0327] The cell pellet was usually resuspended in lysis buffer (50 mM potassium phosphate buffer pH 7.2, 25 μg / ml lysozyme, 5 μg / ml DNase I, 20 mM imidazole, 500 mM NaCl, 10% glycerol and 0.4% Triton X-100). Cells were disrupted by sonication at 4 °C, and cell debris was clarified by centrifugation (18,000 x g; 30 min). The supernatant was loaded onto a Ni-NTA (Qiagen) affinity column equilibrated with (equilibration buffer: 50 mM potassium phosphate buffer pH 7.2, 20 mM imidazole, 500 mM NaCl, 10% glycerol). After loading the protein sample, the column was washed with equilibration buffer to remove unbound contaminating proteins. The His-tagged UGT recombinant polypeptide was eluted with equilibration buffer containing 250 mM imidazole.

[0328] The glycosylation activity of the purified candidate UGT recombinant polypeptide was determined using rebaudioside D (Reb D) as a substrate. Typically, the recombinant polypeptide (50 - 100 μg) was tested in a 200 μl in vitro reaction system. The reaction system contained 50 mM potassium phosphate buffer pH 7.2, 3 mM MgCl 2 , 0.5 mg / ml Reb D substrate, 1 mM UDP-glucose or UDP and / or sucrose synthase (SUS) and 250 mM sucrose. The reaction was carried out at 30 - 37 °C, and 50 μl of the reaction was terminated by adding 200 μL of 1-butanol at different time points. The samples were extracted 3 times with 200 μL of 1-butanol. The combined fractions were dried and dissolved in 100 μL of 80% methanol for high performance liquid chromatography (HPLC) analysis.

[0329] HPLC analysis was then performed using a Dionex UPLC ultimate 3000 system (Sunnyvale, CA), which included a quaternary pump, a thermostatted column compartment, an autosampler and a UV absorbance detector. A SynergiHydro-RP column (Phenomenex) with a guard column was used to characterize the steviol glycosides in the combined samples. Mobile phase A was water, while mobile phase B was acetonitrile. The detection wavelength used in the HPLC analysis was 210 nm.

[0330] After activity screening, HV1 (SEQ ID NO:3) was found to have strong activity in producing R6-1 steviol glycoside ( Figure 9 ). As shown in Figure 10 , HV1 can convert Reb D to R6-1. After a longer reaction time (24 hours, Figure 10 , small panel C), more R6-1 can be produced.

[0331] Example 6: Identification of the Production of R6-1 by LC-MS Analysis

[0332] To confirm the produced compound, the produced compound was analyzed by LC-MS analysis comparing with the standard, and its identity was confirmed.

[0333] Using a Synergy Hydro-RP column, the same sample from the above enzymatic biotransformation was analyzed by LC-MS. Mobile phase A was an aqueous solution of 0.1% formic acid, while mobile phase B was an acetonitrile solution of 0.1% formic acid. The flow rate was 0.6 ml / min. On a QExactive Hybrid Quadrupole-Orbitrap Mass Spectrometer (Thermo Fisher Scientific), the mass spectrometry analysis of the sample was completed using an optimized method in positive ion mode.

[0334] The molecular formula of compound R6-1 was deduced to be C 56 H 90 O 33 based on its positive high-resolution (HR) mass spectrum, which showed adduct ions corresponding to [M+H] at m / z 1291.5430 + and [M+Na] at m / z 1313.5245 + ( Figure 11 ). The predicted structure of R6-1 is presented in Figure 9 and 12 .

[0335] Example 7: Structure Analysis of R6-1 by NMR:

[0336] The produced R6-1 compound was purified by semi-preparative chromatography as described above.

[0337] High-resolution mass data were generated using an LTQ Orbitrap Discovery HRMS instrument with a resolution set at 30k. Data were scanned from m / z 150 to 1500 in positive ion electrospray mode. The needle voltage was set at 4 kV; other source conditions were sheath gas = 25, auxiliary gas = 0, sweep gas = 5 (all gas flows in arbitrary units), capillary voltage = 30 V, capillary temperature = 300 °C, and tube lens voltage = 75. Samples were diluted with pyridine and injected at 50 μL.

[0338] NMR spectra were obtained using standard pulse sequences on a Bruker Avance DRX 500 MHz or Varian INOVA 600 MHz instrument. 1D ( 1 H and 13 C) and 2D (TOCSY, HSQC, ROESY, and HMBC) NMR spectra were performed in C 5 D 5 N.

[0339] The molecular formula of R6-1 was deduced as C 56 H 90 O 33 based on its positive high-resolution (HR) mass spectrum, which showed adducts corresponding to [M+NH 4 + and [M+Na] + ions at m / z 1308.5693 and 1313.5236, respectively; this composition was supported by 13 C NMR spectral data. The 1 H NMR spectrum of R6-1 showed the presence of two sp3 methyl singlets at δ 1.12 and 1.48; two olefinic protons as singlets at δ 5.01 and 5.64 for exocyclic double bonds; nine sp3 methylene and two sp3 methine protons between δ 0.74 - 2.87 characteristic of ent-kaurane diterpenoids previously isolated from Stevia. The basic skeleton of the ent-kaurane diterpenoid was supported by TOCSY (H-1 / H-2; H-2 / H-3; H-5 / H-6; H-6 / H-7; H-9 / H-11; H-11 / H-12) and HMBC (H-1 / C-2, C-10; H-3 / C-1, C-2, C-4, C-5, C-18, C-19; H-5 / C-4, C-6, C-7, C-9, C-10, C-18, C-19, C-20; H-9 / C-8, C-10, C-11, C-12, C-14, C-15; H-14 / C-8, C-9, C-13, C-15, C-16 and H-17 / C-13, C-15, C-16) correlations.

[0340] ​D-Glucose was obtained by acid hydrolysis of R6-1 with 5% H 2 SO 4 and was identified by direct comparison with an authentic sample by TLC, suggesting the presence of six glucopyranosyl moieties in its molecular structure (Bedir et al., 2001; Chaturvedula et al., 2003; and Huan et al., 1998). Enzymatic hydrolysis of R6-1 provided a compound which was found to be identical to steviol based on NMR spectral data (Ohtani et al., 1992). The configuration of D-glucose was identified by preparing its corresponding thiocarbamoyl-thiazolidinecarboxylate derivative with L-cysteine methyl ester and o-tolyl isothiocyanate and comparing its retention time with that of a standard sugar as described in the literature comparison (Tanaka et al., 2007). Based on the results from the NMR spectral data of R6-1, it was concluded that six glucosyl units were present in its structure, which was supported by the 1 1H NMR spectrum which showed the presence of anomeric protons resonating at δ 5.04, 5.05, 5.24, 5.37, 5.57 and 6.36; suggesting six sugar units in its molecular structure. 1 1H and 13 13C NMR spectra in close comparison with those of rebaudioside D suggested that compound R6-1 was also a steviol glycoside which had three glucose residues attached as an ether as a 2,3-branched glucotriosyl substituent at the C-13 hydroxyl and a 2-substituted glucobiosyl moiety as an ester at C-19, leaving the assignment of the additional glucosyl moiety. Figure 13 The key TOCSY and HMBC correlations shown in

[0341] suggested the placement of the sixth glucosyl moiety at the C-2 position of sugar V. The large coupling constants observed for the six anomeric protons of the glucose moieties at δ 5.04 (d, J = 7.6 Hz), 5.05 (d, J = 7.4 Hz), 5.24 (d, J = 7.6 Hz), 5.37 (d, J = 7.4 Hz), 5.57 (d, J = 7.7 Hz) and 6.36 (d, J = 7.6 Hz) suggested its β-orientation as reported for steviol glycosides.

[0342] 1H and 13C NMR values for all protons and carbons in R6-1 were assigned based on TOCSY, HMQC and HMBC correlations and are given in Table 2. 1 1H and 13 13C NMR values for all protons and carbons in R6-1 were assigned based on TOCSY, HMQC and HMBC correlations and are given in Table 2.

[0343] Based on the results of NMR and HR mass spectrometry data and hydrolysis studies, the structure of R6-1 produced by the enzymatic conversion of rebaudioside D was deduced to be 13-[(2-O-β-D-glucopyranosyl-3-O-β-D-glucopyranosyl-β-D-glucopyranosyl)oxy]ent-kaur-16-ene-19-oic acid-[(2-O-{2-O-β-D-glucopyranosyl}-β-D-glucopyranosyl-β-D-glucopyranosyl) ester Figure 12 ).

[0344] By maintaining the pH at 4.5, R6-1 (250 μg) was dissolved in 2.5 ml of 0.1 M sodium acetate buffer, and 50 μL of crude pectinase from Aspergillus niger (Sigma-Aldrich) was added. The mixture was stirred at 50 °C for 48 h, and the product precipitated during the reaction was filtered and then crystallized. By comparing its 1 1H NMR spectral data, the obtained product was identified as steviol (Ohtani et al., 1992).

[0345] R6-1 (500 μg) was dissolved in MeOH (3 ml), and 5% H 2 2 4 SO 2 4 2 (10 mL) was added. The mixture was refluxed for 16 h, cooled to room temperature, and then neutralized with saturated sodium carbonate. The aqueous phase was extracted with ethyl acetate (EtOAc, 2 x 15 ml), and the aqueous layer was concentrated, and using a TLC system of EtOAc / n-butanol / water (2:7:1) and CH

[0346] R6-1 (500 μg) was hydrolyzed with 0.5 M HCl (0.5 mL) for 1.5 h. After cooling, the mixture was passed through an Amberlite IRA400 column and the eluate was lyophilized. The residue was dissolved in pyridine (0.25 mL) and heated with L-cysteine methyl ester HCl (2.5 mg) at 60 °C for 1.5 h, then o-tolyl isothiocyanate (12.5 μL) was added to the mixture and heated at 60 °C for an additional 1.5 h. HPLC analysis of the reaction mixture was carried out on a Phenomenex Luna column [C18, 150 x 4.6 mm (5 μm)], using a mobile phase of 25% acetonitrile - 0.2% TFA in water, at 1 mL / min with UV detection at 250 nm. The sugar was identified as D-glucose (tR, 12.64) [authentic sample, D-glucose (tR, 12.54) and L-glucose (tR, 11.42 min)] (Tanaka et al., 2007).

[0347] The complete 1 H and 13 C NMR spectral assignments were made on the basis of extensive 1D and 2D NMR as well as high-resolution mass spectrometry data and hydrolysis, which suggested the structure as 13-[(2-O-β-D-glucopyranosyl-3-O-β-D-glucopyranosyl-β-D-glucopyranosyl)oxy] ent-kaur-16-ene-19-oic acid-[(2-O-{2-O-β-D-glucopyranosyl}-β-D-glucopyranosyl-β-D-glucopyranosyl) ester ( Figure 12 ).

[0348] Example 8: Production of steviol glycosides rebaudioside R6-4A and rebaudioside R6-4B by enzymatic biotransformation.

[0349] Full-length DNA fragments of all candidate UDP-glucosyltransferase (UGT) genes were commercially synthesized. Almost all cDNA codons were changed to E. coli-preferred codons (Genscript, NJ). The synthesized DNA was cloned into the bacterial expression vector pETite N-His SUMO Kan Vector (Lucigen).

[0350] Each expression construct was transformed into E. coli BL21(DE3), which was then grown in LB medium containing 50 μg / mL kanamycin at 37 °C until an OD of 0.8 - 1.0 was reached 600Protein expression was induced by adding 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG), and the culture was further grown at 16 °C for 22 h. Cells were harvested by centrifugation (3,000 x g; 10 min; 4 °C). The cell pellet was collected and used immediately or stored at -80 °C.

[0351] The cell pellet was usually resuspended in lysis buffer (50 mM potassium phosphate buffer pH 7.2, 25 μg / ml lysozyme, 5 μg / ml DNase I, 20 mM imidazole, 500 mM NaCl, 10% glycerol and 0.4% Triton X-100). Cells were disrupted by sonication at 4 °C, and cell debris was clarified by centrifugation (18,000 x g; 30 min). The supernatant was loaded onto a Ni-NTA (Qiagen) affinity column equilibrated with equilibration buffer (50 mM potassium phosphate buffer pH 7.2, 20 mM imidazole, 500 mM NaCl, 10% glycerol). After loading the protein sample, the column was washed with equilibration buffer to remove unbound contaminating proteins. The His-tagged UGT recombinant polypeptide was eluted with equilibration buffer containing 250 mM imidazole.

[0352] The glycosylation activity of the purified candidate UGT recombinant polypeptide was determined using rebaudioside Z (a mixture of Reb Z1 and Reb Z2) as a substrate. Generally, the recombinant polypeptide (10 - 50 μg) was tested in a 200 μl in vitro reaction system. The reaction system contained 50 mM potassium phosphate buffer pH 7.2, 3 mM MgCl 2 2, 1 mg / ml Reb Z substrate, 1 mM UDP-glucose or UDP and / or sucrose synthase (SUS) and 250 mM sucrose. The reaction was carried out at 30 - 37 °C, and 50 μl of the reaction was terminated by adding 200 μL of 1-butanol at different time points. The samples were extracted 3 times with 200 μL of 1-butanol. The combined fractions were dried and dissolved in 100 μL of 80% methanol for high performance liquid chromatography (HPLC) analysis.

[0353] HPLC analysis was then performed using a Dionex UPLC ultimate 3000 system (Sunnyvale, CA), which included a quaternary pump, a thermostatted column compartment, an autosampler and a UV absorbance detector. A SynergiHydro-RP column (Phenomenex) with a guard column was used to characterize the steviol glycosides in the combined samples. Acetonitrile and water were used as the mobile solutions in the HPLC analysis. The detection wavelength used in the HPLC analysis was 210 nm.

[0354] After activity screening, HV1 (SEQ ID NO:3) was found to have strong activity in producing R6-4 steviol glycoside ( Figure 14 ). As shown in Figure 15 , HV1 can convert Reb Z to R6-4. More R6-4 can be produced at a later reaction time ( Figure 15 , small panel C).

[0355] Example 9: Identification of the Production of R6-4 by LC-MS Analysis

[0356] To confirm the produced compound, the produced compound was analyzed by LC-MS analysis in comparison with the standard, and its identity was confirmed.

[0357] Using a Synergy Hydro-RP column, the same sample from the above enzymatic biotransformation was analyzed by LC-MS. Mobile phase A was an aqueous solution of 0.1% formic acid, while mobile phase B was an acetonitrile solution of 0.1% formic acid. The flow rate was 0.6 ml / min. On a QExactive Hybrid Quadrupole-Orbitrap Mass Spectrometer (Thermo Fisher Scientific), the mass spectrometry analysis of the sample was completed using an optimized method in the positive ion mode.

[0358] The molecular formula of compound R6-4 was deduced to be C 56 H 90 O 33 based on its positive high-resolution (HR) mass spectrum, and the mass spectrum showed an adduct ion corresponding to [M+Na]+ at m / z 1313.5242 ( Figure 16 ). The predicted structures of R6-4A and R6-4B are presented in Figure 14 and 17 .

[0359] Example 10: Structure analysis of R6-4 by NMR:

[0360] The produced R6-4 compound was purified by semi-preparative chromatography as described above.

[0361] HRMS data was generated using an LTQ Orbitrap Discovery HRMS instrument with a resolution set to 30k. Data was scanned from m / z 150 to 1500 in the positive ion electrospray mode. The needle voltage was set to 4 kV; other source conditions were sheath gas = 25, auxiliary gas = 0, sweep gas = 5 (all gas flows were in arbitrary units), capillary voltage = 30 V, capillary temperature = 300 °C, and tube lens voltage = 75. The sample was diluted with pyridine and 50 μl was injected.

[0362] NMR spectra were obtained using standard pulse sequences on a Bruker Avance DRX 500 MHz or a Varian INOVA 600 MHz instrument. 1D ( 1 H and 13 C) and 2D (TOCSY, HSQC, ROESY and HMBC) NMR spectra were performed in C 5 D 5 N.

[0363] The molecular formula of the R6-4 compound was deduced to be C 56 H 90 O 33 based on its positive high-resolution (HR) mass spectrum, which showed adducts corresponding to [M+NH 4 + and [M+Na] + ions at m / z 1308.5692 and 1313.5237, respectively, and this composition was supported by 13 C NMR spectral data. The NMR spectral data and HPLC of R6-4 indicated that the compound was a mixture of two major compounds, R6-4A and R6-4B, in a ratio of approximately 70:30. Thus, the 1 H and 13 C NMR spectral data of R6-4 showed two peaks for most of the protons and carbons present in its molecular structure. The 1 H NMR spectrum of R6-4 showed the presence of two sp3 methyl singlets, two vinylic protons as singlets for exocyclic double bonds, nine sp3 methylene and two sp3 methine protons characteristic of ent-kaurane diterpenes previously isolated from Stevia species. Enzymatic hydrolysis of R6-4 provided a compound that was found to be identical to steviol based on NMR spectral data (Ohtani et al., 1992). Acid hydrolysis of R6-4 with 5% H 2 SO 4 gave D-glucose, which was identified by direct comparison with an authentic sample by TLC, suggesting the presence of six glucopyranosyl moieties in its molecular structure (Bedir et al., 2001; Chaturvedula et al., 2003; and Huan et al., 1998). The configuration of D-glucose was identified by preparing its corresponding thiocarbamoyl-thiazolidinecarboxylic acid ester derivative with L-cysteine methyl ester and o-tolyl isothiocyanate and comparing its retention time with that of a standard sugar as described in the literature (Tanaka et al., 2007). Based on the results from NMR spectral data and hydrolysis studies, it was concluded that six glucosyl units were present in the molecular structure of R6-4, which was supported by 1 ​Supported by the ¹H NMR spectrum which shows six anomeric protons resonating between δ 5.03 - 6.44. Further, the large coupling constants observed for all six anomeric protons of the glucosyl moiety suggest a β-orientation as reported for steviol glycosides.

[0364] For all protons and carbons of compounds R6-4A and R6-4B 1 ¹H and 13 ¹³C NMR values were assigned based on TOCSY, HMQC, HMBC and ROESY correlations and are given in Table 3.

[0365] With that of rebaudioside Z (a mixture of Reb Z1 and Reb Z2) 1 ¹H and 13 ¹³C NMR spectra and close comparison from key TOCSY, ROESY and HMBC correlations suggest that the R6-4 compound is a mixture of two steviol glycosides, where the major compound has three glycosyl units attached as an ether at the C-13 hydroxyl and another three glycosyl units as an ester at C-19, as shown in R6-4A, while the minor compound has two glycosyl units attached as an ether at the C-13 hydroxyl and four glycosyl units as an ester at C-19 as represented in R6-4B.

[0366] Based on the NMR and HR mass spectrometry data and the results of hydrolysis studies, the structure of R6-4 produced by the enzymatic conversion of rebaudioside Z (which is a mixture of two major compounds) was deduced to be 13-[(2-O-{2-O-β-D-glucopyranosyl-β-D-glucopyranosyl}-β-D-glucopyranosyl)oxy] ent-kaur-16-ene-19-oic acid-[(2-O-{2-O-β-D-glucopyranosyl}-β-D-glucopyranosyl-β-D-glucopyranosyl) ester (R6-4A), or 13-[(2-O-β-D-glucopyranosyl-β-D-glucopyranosyl)oxy] ent-kaur-16-ene-19-oic acid-[(2-O-[(2-O-{2-O-β-D-glucopyranosyl-β-D-glucopyranosyl}-β-D-glucopyranosyl)-β-D-glucopyranosyl) ester (R6-4B)( Figure 17 )

[0367] By maintaining the pH at 4.5, the R6-4 compound (1 mg) was dissolved in 10 ml of 0.1 M sodium acetate buffer and 250 μL of crude pectinase from Aspergillus niger (Sigma-Aldrich) was added. The mixture was stirred at 50 °C for 48 h and the product precipitated during the reaction was filtered and then solidified. By comparing its 1The obtained product was identified as steviol by ¹H NMR spectroscopic data and co-TLC (Ohtani et al., 1992).

[0368] Compound R6-4 (500 μg) was dissolved in MeOH (5 ml), and 5% H 2 SO 4 (15 mL) was added. The mixture was refluxed for 24 h, cooled to room temperature, and then neutralized with saturated sodium carbonate. The aqueous phase was extracted with ethyl acetate (EtOAc, 2 x 25 ml), and the aqueous layer was concentrated and analyzed using TLC systems EtOAc / n-butanol / water (2:7:1) and CH 2 Cl 2 / MeOH / water (10:6:1) (Bedir et al., 2001; Chaturvedula et al., 2003; Huan et al., 1998), comparing with standard sugars; the sugar in R6-4 was identified as D-glucose.

[0369] Compound R6-4 (1 mg) was hydrolyzed with 0.5 M HCl (2 mL) for 1.5 h. After cooling, the mixture was passed through an Amberlite IRA400 column and the eluate was lyophilized. The residue was dissolved in pyridine (0.75 mL) and heated with L-cysteine methyl ester HCl (5 mg) at 60 °C for 1.5 h, then o-tolyl isothiocyanate (30 μL) was added to the mixture and heated at 60 °C for an additional 1.5 h. HPLC analysis of the reaction mixture was performed on a Phenomenex Luna column [C18, 150 x 4.6 mm (5u)], using a mobile phase of 25% acetonitrile - 0.2% TFA in water, at 1 mL / min with UV detection at 250 nm. The sugar was identified as D-glucose (tR, 12.38) [authentic sample, D-glucose (tR, 12.44) and L-glucose (tR, 11.30 min)] (Tanaka et al., 2007).

[0370] Sequence:

[0371] UGT76G1: Amino acid sequence (SEQ ID NO:1)

[0372] MENKTETTVRRRRRIILFPVPFQGHINPILQLANVLYSKGFSITIFHTNFNKPKTSNYPHFTFRFILDNDPQDERISNLPTHGPLAGMRIPIINEHGADELRRELELLMLASEEDEEVSCLITDALWYFAQSVADSLNLRRLVLMTSSLFNFHAHVSLPQFDELGYLDPDDKTRLEEQASGFPMLKVKDIKSAYSNWQILKEILGKMIKQTKASSGVIWNSFKELEESELETVIREIPAPSFLIPLPKHLTASSSSLLDHDRTVFQWLDQQPPSSVLYVSFGSTSEVDEKDFLEIARGLVDSKQSFLWVVRPGFVKGSTWVEPLPDGFLGERGRIVKWVPQQEVLAHGAIGAFWTHSGWNSTLESVCEGVPMIFSDFGLDQPLNARYMSDVLKVGVYLENGWERGEIANAIRRVMVDEEGEYIRQNARVLKQKADVSLMKGGSSYESLESLVSYISSL

[0373] UGT76G1: DNA sequence (SEQ ID NO: 2)

[0374]

[0375] HV1 UDP-glycosyltransferase: Amino acid sequence (SEQ ID NO: 3)

[0376] MDGNSSSSPLHVVICPWLALGHLLPCLDIAERLASRGHRVSFVSTPRNIARLPPLRPAVAPLVDFVALPLPHVDGLPEGAESTNDVPYDKFELHRKAFDGLAAPFSEFLRAACAEGAGSRPDWLIVDTFHHWAAAAAVENKVPCVMLLLGAATVIAGFARGVSEHAAAAVGKERPAAEAPSFETERRKLMTTQNASGMTVAERYFLTLMRSDLVAIRSCAEWEPESVAALTTLAGKPVVPLGLLPPSPEGGRGVSKEDAAVRWLDAQPAKSVVYVALGSEVPLRAEQVHELALGLELSGARFLWALRKPTDAPDAAVLPPGFEERTRGRGLVVTGWVPQIGVLAHGAVAAFLTHCGWNSTIEGLLFGHPLIMLPISSDQGPNARLMEGRKVGMQVPRDESDGSFRREDVAATVRAVAVEEDGRRVFTANAKKMQEIVADGACHERCIDGFIQQLRSYKA

[0377] HV1 UDP-glycosyltransferase: DNA sequence (SEQ ID NO: 4)

[0378]

[0379] UGT76G1-Sucrose synthase (SUS) fusion enzyme: Amino acid sequence (SEQ ID NO: 5)

[0380]

[0381] UGT76G1-Sucrose synthase (SUS) fusion enzyme: DNA sequence (SEQ ID NO: 6)

[0382]

[0383] HV1-SUS fusion enzyme: Amino acid sequence (SEQ ID NO: 7)

[0384]

[0385] HV1-SUS fusion enzyme: DNA sequence (SEQ ID NO: 8)

[0386]

[0387] Arabidopsis thaliana sucrose synthase I: Amino acid sequence (SEQ ID NO: 9)

[0388] MANAERMITRVHSQRERLNETLVSERNEVLALLSRVEAKGKGILQQNQIIAEFEALPEQTRKKLEGGPFFDLLKSTQEAIVLPPWVALAVRPRPGVWEYLRVNLHALVVEELQPAEFLHFKEELVDGVKNGNFTLELDFEPFNASIPRPTLHKYIGNGVDFLNRHLSAKLFHDKESLLPLLKFLRLHSHQGKNLMLSEKIQNLNTLQHTLRKAEEYLAELKSETLYEEFEAKFEEIGLERGWGDNAERVLDMIRLLLDLLEAPDPCTLETFLGRVPMVFNVVILSPHGYFAQDNVLGYPDTGGQVVYILDQVRALEIEMLQRIKQQGLNIKPRILILTRLLPDAVGTTCGERLERVYDSEYCDILRVPFRTEKGIVRKWISRFEVWPYLETYTEDAAVELSKELNGKPDLIIGNYSDGNLVASLLAHKLGVTQCTIAHALEKTKYPDSDIYWKKLDDKYHFSCQFTADIFAMNHTDFIITSTFQEIAGSKETVGQYESHTAFTLPGLYRVVHGIDVFDPKFNIVSPGADMSIYFPYTEEKRRLTKFHSEIEELLYSDVENKEHLCVLKDKKKPILFTMARLDRVKNLSGLVEWYGKNTRLRELANLVVVGGDRRKESKDNEEKAEMKKMYDLIEEYKLNGQFRWISSQMDRVRNGELYRYICDTKGAFVQPALYEAFGLTVVEAMTCGLPTFATCKGGPAEIIVHGKSGFHIDPYHGDQAADTLADFFTKCKEDPSHWDEISKGGLQRIEEKYTWQIYSQRLLTLTGVYGFWKHVSNLDRLEARRYLEMFYALKYRPLAQAVPLAQDD

[0389] Arabidopsis thaliana sucrose synthase I: DNA sequence (SEQ ID NO: 10)

[0390]

[0391] Table 1. 1 H and 13 C NMR spectroscopic data (chemical shifts and coupling constants) a-c .

[0392]

[0393]

[0394]

[0395] a Assignments were made based on TOCSY, HSQC, ROESY, and HMBC correlations; b chemical shift values are in δ (ppm); c coupling constants are in Hz.

[0396] Table 2. 1 H and 13 C NMR spectroscopic data (chemical shifts and coupling constants) a-c .

[0397]

[0398]

[0399] a Assignments were made based on TOCSY, HSQC, ROESY, and HMBC correlations; b chemical shift values are in δ (ppm); c coupling constants are in Hz.

[0400] Table 3. 1 H and 13 C NMR spectroscopic data (chemical shifts and coupling constants) a-c .

[0401]

[0402]

[0403]

[0404] a Assignments were made based on TOCSY, HSQC, ROESY, and HMBC correlations; b chemical shift values are in δ (ppm); c coupling constants are in Hz.

[0405] Industrial applicability / Field of technology statement This disclosure has applicability in the food, feed, beverage, and pharmacological industries. This disclosure generally relates to methods for the biosynthesis of certain steviol glycosides via enzymes and / or modified microbial strains, and to their potential use as sweeteners for food products and beverages, and related compositions.

[0406] References:

[0407] 1. Bedir, E. et al., (2001), A new dammarane type triterpene glycoside from Polyscias fulva. J. Natural Products, (64):95–97.

[0408] 2. Brandle, J.E. et al., (1998). Stevia Rebaudiana: Its Agricultural, Biological, and Chemical Properties, Canadian J. Plant Science. 78(4):527–36.

[0409] 3. Ceunen, S. and J.M.C. Geuns, Steviol Glycosides: Chemical Diversity, Metabolism, and Function, J. Nat. Prod., 2013, 76(6), pp 1201–28 (2013).

[0410] 4. Chaturvedula, V.S.P. et al., (2003), New cytotoxic oleanane saponis from the infructescences of Polyscias amplifolia from the Madagascar rainforest. Planta Medica, (69):440–44.

[0411] 5. Daugherty, A.B. et al., Structural and Functional Consequences of Circular Permutation on the Active Site of Old Yellow Enzyme, ACS Catal. (2015) 5:892-99.

[0412] 6. Du J et al., (2011), Engineering microbial factories for synthesis of value-added products, J Ind Microbiol. Biotechnol. 38:873-90.

[0413] 7. GRAS Notices, USA Food and Drug Administration, United States Health & Human Services. (2016) (related to steviol glycosides and polyglycosides).

[0414] 8. Huan, V.D. et al., (1998). Oleanane saponins from Polyscias fructicosa. Phytochemistry, (47):451–57.

[0415] 9. A and Münch T., (1997), Microbial Production of Natural Flavors, ASM News (63):551-59.

[0416] 10. Mao, G. et al., (2017), Enzymatic Synthesis and Structural Characterization of Rebaudioside D3, a Minor Steviol Glycoside of Stevia rebaudiana Bertoni, Amer. J. Plant Sciences, (8):441-50.

[0417] 11. Ohtani, K. et al., (1992). Minor diterpene glycosides from sweet leaves of Rubus Suavissimus, Phytochemistry, (31):1553-59.

[0418] 12. Prakash I. et al.; Isolation and Characterization of a Novel Rebaudioside M Isomer from a Bio conversion Reaction of Rebaudioside A and NMR Comparison Studies of Rebaudioside M Isolated from Stevia rebaudiana Bertoni and Stevia rebaudiana Morita, Biomolecules, 2014 Jun; 4(2):374–89. (Online published March 31, 2014. 2014).

[0419] 13. Prakash I. et al., Development of Next Generation Stevia Sweetener: Rebaudioside M, Foods, (2014), 3:162-175.

[0420] 14. Qian, Z. et al., Improving the catalytic activity of Candida antarctica lipase B by circular permutation, J. of the American Chemical Society. (2005) 127(39):13466–13467.

[0421] 15. Richman A. et al., Functional genomics uncovers three glucosyltransferases involved in the synthesis of the major sweet glucosides of Stevia rebaudiana, Plant J. (2005) Jan; 41(1):56-67.

[0422] 16. Shockey J.M. et al., (2003), Arabidopsis contains a large superfamily of acyl-activating enzymes: phylogenetic and biochemical analysis reveals a new class of acyl-coenzyme A synthetases. Plant Physiol. 132 1065–76.

[0423] 17. Tanaka, T. et al., (2007), Facile discrimination of aldose enantiomers by reversed-phase HPLC, Chem. Pharm. Bull., (55):899-901.

[0424] 18. Topell, S. et al., Circularly permuted variants of the green fluorescent protein, FEBS Letters. (1999) 457(2):283–89.

[0425] 19. Wang J. et al., Pathway mining-based integration of critical enzyme parts for de novo biosynthesis of steviol glycosides sweetener in Escherichia coli, Cell Research (2016) 26:258–61.

[0426] All publications, patents, patent applications, publications, and database entries (e.g., sequence database entries) mentioned in this document, for example, in the Background Art, Summary of the Invention, Detailed Description of the Invention, Examples, and / or References sections, are hereby incorporated by reference in their entirety as if each individual publication, patent, patent application, patent application publication, and database entry were specifically and individually incorporated by reference herein. In case of conflict, the present application (including any definitions herein) shall prevail.

Claims

1. A method for producing rebaudioside R6-4A and / or rebaudioside R6-4B, the method comprising: (I) preparing a reaction mixture comprising: (i) at least one of rebaudioside Z1 and rebaudioside Z2; (ii) one or more substrates selected from sucrose, uridine diphosphate (UDP), uridine diphosphate-glucose (UDP-glucose), and combinations thereof; and (iii) an enzyme selected from: (a) UDP-glycosyltransferase (UGT); (b) UDP-glycosyltransferase and sucrose synthase added separately to the reaction mixture; and (c) a UDP-glycosyltransferase fusion enzyme comprising a UDP-glycosyltransferase domain coupled to a sucrose synthase domain; and (II) Incubate the reaction mixture in a reaction system containing 50 mM potassium phosphate buffer pH 7.2, 3 mM MgCl 2 at 30-37 °C for a sufficient time to produce rebaudioside R6-4A and / or rebaudioside R6-4B; wherein the rebaudioside Z1 has the following structure: The rebaudioside Z2 has the following structure: The rebaudioside R6-4A has the following structure: and the rebaudioside R6-4B has the following structure: wherein the UDP-glycosyltransferase has the amino acid sequence of SEQ ID NO:3; or wherein the UDP-glycosyltransferase fusion enzyme has the amino acid sequence of SEQ ID NO:

7.

2. The method of claim 1, wherein the sucrose synthase or sucrose synthase domain is selected from Arabidopsis thaliana sucrose synthase I, Arabidopsis thaliana sucrose synthase 3, and mung bean sucrose synthase.

3. The method of claim 2, wherein the sucrose synthase or sucrose synthase domain is Arabidopsis thaliana sucrose synthase I.

4. The method of claim 3, wherein the sucrose synthase or sucrose synthase domain has at least 80% identity with the amino acid sequence of SEQ ID NO:

9.

5. The method of claim 4, wherein the sucrose synthase or sucrose synthase domain comprises the amino acid sequence of SEQ ID NO:

9.

6. The method of any one of claims 1-5, wherein glucose is covalently coupled to rebaudioside Z1 or rebaudioside Z2 by the enzyme to produce rebaudioside R6-4A.

7. The method of claim 6, wherein the glucose is covalently coupled to sugar IV of rebaudioside Z1 by the enzyme to produce rebaudioside R6-4A.

8. The method of claim 6, wherein the glucose is covalently coupled to sugar III of rebaudioside Z2 by the enzyme to produce rebaudioside R6-4A.

9. The method of any one of claims 1-5, wherein the glucose is covalently coupled to rebaudioside Z2 by the enzyme to produce rebaudioside R6-4B.

10. The method of claim 9, wherein the glucose is covalently coupled to sugar V of rebaudioside Z2 by the enzyme to produce rebaudioside R6-4B.

11. The method of claim 10, wherein the UDP-glycosyltransferase comprises the amino acid sequence of SEQ ID NO:

3.

12. The method of claim 11, wherein the UDP-glycosyltransferase fusion enzyme comprises the amino acid sequence of SEQ ID NO:

7.

13. The method of any one of claims 1 - 8, further comprising producing rebaudioside Z1 or rebaudioside Z2 by incubating rebaudioside E with UDP - glycosyltransferase and a substrate selected from sucrose, UDP, UDP - glucose, and combinations thereof.

14. A method for producing rebaudioside R6 - 1, the method comprising: (I) preparing a reaction mixture comprising: (i) rebaudioside D; (ii) one or more substrates selected from sucrose, uridine diphosphate (UDP), uridine diphosphate - glucose (UDP - glucose), and combinations thereof; and (iii) an enzyme selected from: (a) UDP - glycosyltransferase (UGT); (b) UDP - glycosyltransferase and sucrose synthase separately added to the reaction mixture; and (c) a UDP - glycosyltransferase fusion enzyme comprising a UDP - glycosyltransferase domain coupled to a sucrose synthase domain; and (II) Incubate the reaction mixture in a reaction system containing 50 mM potassium phosphate buffer pH 7.2, 3 mM MgCl 2 at 30 - 37 °C for a sufficient time to produce rebaudioside R6-1; wherein the rebaudioside D has the following structure: and The rebaudioside R6 - 1 has the following structure: wherein the UDP - glycosyltransferase is the amino acid sequence of SEQ ID NO:3; or wherein the UDP - glycosyltransferase fusion enzyme is the amino acid sequence of SEQ ID NO:

7.

15. The method of claim 14, wherein the sucrose synthase or sucrose synthase domain is selected from Arabidopsis sucrose synthase I, Arabidopsis sucrose synthase 3, and mung bean sucrose synthase.

16. The method of claim 15, wherein the sucrose synthase or sucrose synthase domain is Arabidopsis sucrose synthase I.

17. The method of claim 16, wherein the sucrose synthase or sucrose synthase domain has at least 80% identity with the amino acid sequence of SEQ ID NO:

9.

18. The method of claim 17, wherein the sucrose synthase or sucrose synthase domain comprises the amino acid sequence of SEQ ID NO:

9.

19. The method of any one of claims 15 - 18, wherein glucose is covalently coupled to rebaudioside D by the enzyme to produce rebaudioside R6 - 1.

20. The method of claim 19, wherein the glucose is covalently coupled to sugar V of rebaudioside D by the enzyme to produce rebaudioside R6 - 1.

21. The method of claim 20, wherein the UDP - glycosyltransferase comprises the amino acid sequence of SEQ ID NO:

3.

22. The method of claim 20, wherein the UDP - glycosyltransferase fusion enzyme comprises the amino acid sequence of SEQ ID NO:

7.

23. The method of any one of claims 15 - 18, further comprising producing rebaudioside D by incubating rebaudioside E with UDP - glycosyltransferase and a substrate selected from sucrose, UDP, UDP - glucose, and combinations thereof.

24. The method of any one of claims 15 - 18, further comprising producing rebaudioside D by incubating rebaudioside A with UDP - glycosyltransferase and a substrate selected from sucrose, UDP, UDP - glucose, and combinations thereof.

25. The method according to any one of claims 1-8 and 14-18, wherein the reaction mixture is in vitro.

26. The method according to any one of claims 1-8 and 14-18, wherein the reaction mixture is a cell-based reaction mixture.

27. The method according to claim 26, wherein the cells are selected from yeast, non-steviol glycoside-producing plants, algae, fungi, and bacteria.

28. A synthesized steviol glycoside selected from: (i) Rebaudioside R6-4A having the following structure: (ii) Rebaudioside R6-4B having the following structure: and (iii) Rebaudioside R6-1 having the following structure:

29. A composition comprising the synthesized steviol glycoside according to claim 28.

30. The synthesized steviol glycoside according to claim 28, which is used as a sweetener.

31. An orally consumable product comprising a sweetening amount of a sweetener selected from Rebaudioside R6-4A, R6-4B, and / or R6-1, wherein the orally consumable product is selected from beverage products and consumer products. Among them, the structure of rebaudioside R6-4A is as follows: The structure of rebaudioside R6-4B is as follows: The structure of rebaudioside R6-1 is as follows:

32. The orally consumable product according to claim 31, wherein the sweetener is the only sweetener.

33. The orally consumable product according to claim 31 or claim 32, which comprises about 5 ppm to 100 ppm of rebaudioside.

34. The orally consumable product according to claim 31 or claim 32, wherein the orally consumable product has a sweetness intensity equivalent to that of a sucrose solution of about 1% (w / v-%) to about 4% (w / v-%).

35. The orally consumable product according to claim 31 or claim 32, which further comprises at least one additional sweetener.

36. The orally consumable product according to claim 35, wherein the at least one additional sweetener is selected from stevioside, rebaudioside A, rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside E derived from recombinant microbial biosynthesis, rebaudioside F, dulcoside A, rebaudioside M, rebaudioside V, rebaudioside W, rebaudioside D3, rebaudioside Z1, rebaudioside Z2, phyllodulcin, steviolbioside, sucrose, high fructose corn syrup, fructose, glucose, xylose, arabinose, rhamnose, erythritol, xylitol, mannitol, sorbitol, inositol, AceK, aspartame, neotame, sucralose, saccharin, naringin dihydrochalcone (NarDHC), neohesperidin dihydrochalcone (NDHC), phyllodulcin, mogroside IV, synephrine glycoside I, mogroside V, monatin, thaumatin, miraculin, sweet protein, L-alanine, glycine, hernandulcin, phyllanthin, trilobatin, and combinations thereof.

37. The orally consumable product according to claim 31 or claim 32, which further comprises at least one additive selected from the following: carbohydrates, polyols, amino acids or their salts, polyamino acids or their salts, sugar acids or their salts, nucleotides, organic acids, inorganic acids, organic salts, organic acid salts, organic base salts, inorganic salts, flavoring components, astringent compounds, surfactants, emulsifiers, flavonoids, alcohols, polymers, and combinations thereof.

38. An orally consumable product according to claim 31 or 32, wherein the consumable product is selected from food products, dental hygiene compositions and cosmetics.

39. An orally consumable product according to claim 31 or 32, wherein the beverage product is selected from carbonated beverage products and non-carbonated beverage products.

40. The beverage product according to claim 39, wherein the beverage product is a soft drink.

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