阅读说明：本技术 无热量甜味剂及合成方法 (Non-caloric sweetener and its synthesis method ) 是由 毛国红 X·于 于 2015-10-02 设计创作，主要内容包括：本申请涉及无热量甜味剂及合成方法。公开了称为莱鲍迪苷V和莱鲍迪苷W的甜菊醇糖苷。还公开了生成莱鲍迪苷M(RebM)、莱鲍迪苷G(RebG)、莱鲍迪苷KA(RebKA)、莱鲍迪苷V(RebV)和莱鲍迪苷(RebW)的方法。本公开总体上涉及天然甜味剂。更具体地,本公开涉及无热量甜味剂和合成所述无热量甜味剂的方法。(The present application relates to non-caloric sweeteners and methods of synthesis. Steviol glycosides, designated rebaudioside V and rebaudioside W, are disclosed. Also disclosed are methods of producing rebaudioside m (rebm), rebaudioside g (rebg), rebaudioside ka (rebka), rebaudioside v (rebv), and rebaudioside (RebW). The present disclosure relates generally to natural sweeteners. More particularly, the present disclosure relates to non-caloric sweeteners and methods for synthesizing the non-caloric sweeteners.)

1. A synthetic rebaudioside consisting of the following chemical structure:

。

2. a synthetic rebaudioside consisting of the following chemical structure:

。

3. a sweetener comprising a synthetic rebaudioside, the synthetic rebaudioside consisting of the chemical structure:

。

4. a sweetener as claimed in claim 3 further comprising at least one of a filler, a bulking agent and an anti-caking agent.

5. A sweetener comprising a synthetic rebaudioside, the synthetic rebaudioside consisting of the chemical structure:

。

6. a sweetener as claimed in claim 5 further comprising at least one of a filler, a bulking agent and an anti-caking agent.

BACKGROUND OF THE DISCLOSURE

The present disclosure relates generally to natural sweeteners. More particularly, the present disclosure relates to non-caloric sweeteners and methods for synthesizing non-caloric sweeteners.

Steviol glycosides are natural products isolated from the leaves of Stevia rebaudiana (Stevia rebaudiana). Steviol glycosides are widely used as high intensity, low calorie sweeteners and are significantly sweeter than sucrose. As a natural sweetener, different steviol glycosides have different sweetness and aftertaste. The sweetness of steviol glycosides is significantly higher than that of sucrose. For example, stevioside is 100-fold more sweet than sucrose, with a bitter aftertaste. Rebaudioside C (rebaudioside C) is 40-60 times sweeter than sucrose. Dulcoside a (dulcoside a) is about 30 times sweeter than sucrose.

Naturally occurring steviol glycosides share the same basic steviol structure, but differ in the content of carbohydrate residues (e.g., glucose, rhamnose, and xylose residues) at positions C13 and C19. Steviol glycosides having known structures include steviol, stevioside, rebaudioside a, rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside E, rebaudioside F, and dulcoside a (see, e.g., table 1). Other steviol glycosides are rebaudioside M, rebaudioside N, and rebaudioside O.

TABLE 1 Steviol glycosides.

Stevioside, rebaudioside a, rebaudioside C and dulcoside a make up 9.1, 3.8, 0.6 and 0.3% of the total weight of steviol glycosides in the leaves, respectively, by dry weight, while other steviol glycosides are present in much lower amounts. Extracts from stevia plants are commercially available, and typically contain stevioside and rebaudioside a as the major compounds. Other steviol glycosides are present in stevia extracts as minor components. For example, the amount of rebaudioside a in a commercial formulation may vary from about 20% to greater than 90% of the total steviol glycoside content, while the amount of rebaudioside B may be about 1-2%, the amount of rebaudioside C may be about 7-15%, and the amount of rebaudioside D may be about 2% of the total steviol glycoside.

Most steviol glycosides are formed by several glycosylation reactions of steviol, usually catalyzed by UDP-glycosyltransferase (UGT), using uridine 5' -diphosphoglucose (UDP-glucose) as the donor of the sugar moiety. UGTs in plants constitute a very different class of enzymes that transfer glucose residues from UDP-glucose to steviol. For example, glycosylation of C-3' of stevioside C-13-O-glucose produces rebaudioside A; and glycosylation of stevioside at C-2' of 19-O-glucose produces rebaudioside E. Further glycosylation of rebaudioside A (at C-2 '-19-O-glucose) or rebaudioside E (at C-3' -13-O-glucose) produces rebaudioside D. (FIG. 1).

Alternative sweeteners are receiving increasing attention because of the awareness that many diseases are associated with the consumption of high-sugar foods and beverages. Although artificial sweeteners are available, many artificial sweeteners such as dulcin, sodium cyclamate and saccharin have been banned or limited by some countries due to safety concerns. Thus, non-caloric sweeteners of natural origin are becoming increasingly popular. One of the major obstacles to the widespread use of stevia sweeteners is their poor taste profile. Accordingly, there is a need to develop alternative sweeteners and methods for their production to provide an optimal combination of sweetness potential and flavor properties.

Summary of The Invention

The present disclosure relates generally to natural sweeteners. More particularly, the present disclosure relates to non-caloric sweeteners and methods for synthesizing non-caloric sweeteners.

Rebaudioside V was synthesized. In one aspect, the present disclosure relates to a synthetic rebaudioside (rebaudioside V) consisting of the following chemical structure:

rebaudioside W was synthesized. In one aspect, the present disclosure relates to a synthetic rebaudioside (rebaudioside W) consisting of the following chemical structure:

methods for producing rebaudioside V from rebaudioside G. In another aspect, the present disclosure relates to a method for synthesizing rebaudioside V from rebaudioside G. The method comprises preparing a reaction mixture comprising rebaudioside G, substrates selected from the group consisting of sucrose, Uridine Diphosphate (UDP) and uridine diphosphate-glucose (UDP-glucose), and HV1 UDP-glycosyltransferase, with or without sucrose synthase (SUS); and incubating the reaction mixture for a sufficient time to produce rebaudioside V, wherein a glucose is covalently coupled to rebaudioside G to produce rebaudioside V.

Methods for producing rebaudioside V from rebaudioside G. In another aspect, the present disclosure relates to a method for synthesizing rebaudioside V from rebaudioside G. The method comprises preparing a reaction mixture comprising rebaudioside G, substrates selected from the group consisting of sucrose, Uridine Diphosphate (UDP) and uridine diphosphate-glucose (UDP-glucose), uridine diphosphate glycosyltransferase (UDP-glycosyltransferase) selected from the group consisting of uridine diphosphate glycosyltransferase (EUGT11), UDP-glycosyltransferase-sucrose synthase (SUS) fusion enzymes, with or without sucrose synthase (SUS); and incubating the reaction mixture for a sufficient time to produce rebaudioside V, wherein a glucose is covalently coupled to rebaudioside G to produce rebaudioside V.

A method for producing rebaudioside V from rebaudioside KA. In another aspect, the disclosure relates to a method for synthesizing rebaudioside V from rebaudioside KA. The method comprises preparing a reaction mixture comprising rebaudioside KA, a substrate selected from the group consisting of sucrose, Uridine Diphosphate (UDP), and uridine diphosphate-glucose (UDP-glucose), a uridine diphosphate glycosyltransferase (UDP-glycosyltransferase) selected from the group consisting of a UDP-glycosyltransferase (UGT76G 1; SEQ ID NO:1) and a UDP-glycosyltransferase-sucrose synthase fusion enzyme, with or without sucrose synthase (SUS); and incubating the reaction mixture for a sufficient time to produce rebaudioside V, wherein a glucose is covalently coupled to rebaudioside KA to produce rebaudioside V.

Methods for producing rebaudioside V from rubusoside. In another aspect, the disclosure relates to a method of synthesizing rebaudioside V from rubusoside. The method comprises preparing a reaction mixture comprising rubusoside, a substrate selected from the group consisting of sucrose, Uridine Diphosphate (UDP) and uridine diphosphate-glucose (UDP-glucose), a uridine diphosphate glycosyltransferase (UDP-glycosyltransferase) selected from the group consisting of UDP-glycosyltransferase (UGT76G1), HV1, and a UDP-glycosyltransferase-sucrose synthase fusion enzyme, with or without sucrose synthase (SUS); and incubating the reaction mixture for a sufficient time to produce rebaudioside V, wherein a glucose is covalently coupled to rubusoside to produce rebaudioside KA. Continuously, glucose is covalently coupled to rebaudioside KA to produce rebaudioside V. Glucose is covalently coupled to rubusoside to produce rebaudioside G. Continuously, glucose was covalently coupled to rebaudioside G to produce rebaudioside V.

Methods for producing rebaudioside V from rubusoside. In another aspect, the disclosure relates to a method of synthesizing rebaudioside V from rubusoside. The method comprises preparing a reaction mixture comprising rubusoside, a substrate selected from sucrose, Uridine Diphosphate (UDP) and uridine diphosphate-glucose (UDP-glucose), a uridine diphosphate glycosyltransferase (UDP-glycosyltransferase) selected from the group consisting of a UDP-glycosyltransferase (UGT76G1), an EUGT11, and a UDP-glycosyltransferase-sucrose synthase fusion enzyme, with or without sucrose synthase; and incubating the reaction mixture for a sufficient time to produce rebaudioside V, wherein a glucose is covalently coupled to rubusoside to produce rebaudioside KA and a glucose is covalently coupled to rebaudioside KA to produce rebaudioside V. Glucose is covalently coupled to rubusoside to produce rebaudioside G and glucose is covalently coupled to rebaudioside G to produce rebaudioside V.

Methods for producing rebaudioside W from rebaudioside V. In another aspect, the present disclosure relates to a method for synthesizing rebaudioside W from rebaudioside V. The method comprises preparing a reaction mixture comprising rebaudioside V, a substrate selected from the group consisting of sucrose, Uridine Diphosphate (UDP) and uridine diphosphate-glucose (UDP-glucose), a uridine diphosphate glycosyltransferase (UDP-glycosyltransferase) selected from the group consisting of a UDP-glycosyltransferase (UGT76G1) and a UDP-glycosyltransferase-sucrose synthase fusion enzyme, with or without a sucrose synthase; and incubating the reaction mixture for a sufficient time to produce rebaudioside W, wherein a glucose is covalently coupled to rebaudioside V to produce rebaudioside W.

A method for producing rebaudioside W from rebaudioside G. In another aspect, the present disclosure relates to a method for synthesizing rebaudioside G from rebaudioside G. The method comprises preparing a reaction mixture comprising rebaudioside G, substrates selected from the group consisting of sucrose, Uridine Diphosphate (UDP) and uridine diphosphate-glucose (UDP-glucose), a uridine diphosphate glycosyltransferase (UDP-glycosyltransferase) selected from the group consisting of a UDP-glycosyltransferase (UGT76G1), a UDP-glycosyltransferase-sucrose synthase fusion enzyme, and HV 1; with or without sucrose synthase; and incubating the reaction mixture for a sufficient time to produce rebaudioside W, wherein glucose is covalently coupled to rebaudioside G by HV1 to produce rebaudioside V. Continuously, glucose was covalently coupled to rebaudioside V via UGT76G1 to produce rebaudioside W.

A method for producing rebaudioside W from rebaudioside G. In another aspect, the present disclosure relates to a method for synthesizing rebaudioside G from rebaudioside G. The method comprises preparing a reaction mixture comprising rebaudioside G, substrates selected from the group consisting of sucrose, Uridine Diphosphate (UDP) and uridine diphosphate-glucose (UDP-glucose), a uridine diphosphate glycosyltransferase (UDP-glycosyltransferase) selected from the group consisting of UGT76G1, EUGT11, and a UDP-glycosyltransferase-sucrose synthase fusion enzyme; and incubating the reaction mixture for a sufficient time to produce rebaudioside W, wherein glucose is covalently coupled to rebaudioside G through EUGT11 to produce rebaudioside V. Continuously, glucose was covalently coupled to rebaudioside V via UGT76G1 to produce rebaudioside W.

A method for producing rebaudioside W from rebaudioside KA. In another aspect, the disclosure relates to a method for synthesizing rebaudioside W from rebaudioside KA. The method includes preparing a reaction mixture comprising rebaudioside KA; a substrate selected from sucrose, Uridine Diphosphate (UDP) and uridine diphosphate-glucose (UDP-glucose); a uridine diphosphate glycosyltransferase (UDP-glycosyltransferase) selected from the group consisting of a UDP-glycosyltransferase (UGT76G1) and a UDP-glycosyltransferase-sucrose synthase fusion enzyme, with or without a sucrose synthase; and incubating the reaction mixture for a sufficient time to produce rebaudioside W, wherein a glucose is covalently coupled to rebaudioside KA to produce rebaudioside V. Continuously, glucose was covalently coupled to rebaudioside V to produce rebaudioside W.

A method for producing rebaudioside W from rubusoside. In another aspect, the disclosure relates to a method for synthesizing rebaudioside D from rubusoside. The method comprises preparing a reaction mixture comprising rubusoside, a substrate selected from sucrose, Uridine Diphosphate (UDP) and uridine diphosphate-glucose (UDP-glucose), a uridine diphosphate glycosyltransferase (UDP-glycosyltransferase) selected from UGT76G1, HV1 and a UDP-glycosyltransferase-sucrose synthase fusion enzyme, with or without sucrose synthase; and incubating the reaction mixture for a sufficient time to produce rebaudioside W.

A method for producing rebaudioside W from rubusoside. In another aspect, the disclosure relates to a method of synthesizing rebaudioside W from rubusoside. The method comprises preparing a reaction mixture comprising rubusoside, a substrate selected from sucrose, Uridine Diphosphate (UDP) and uridine diphosphate-glucose (UDP-glucose), a uridine diphosphate glycosyltransferase (UDP-glycosyltransferase) selected from UGT76G1, EUGT11 and a UDP-glycosyltransferase-sucrose synthase fusion enzyme, with or without sucrose synthase; and incubating the reaction mixture for a sufficient time to produce rebaudioside W.

A method for producing a mixture of rubusoside and rebaudioside KA from rubusoside. In another aspect, the disclosure relates to a method of synthesizing a mixture of stevioside and rebaudioside KA from rubusoside. The method comprises preparing a reaction mixture comprising rubusoside, a substrate selected from sucrose, Uridine Diphosphate (UDP) and uridine diphosphate-glucose (UDP-glucose), a UDP-glycosyltransferase selected from EUGT11 and a UDP-glycosyltransferase-sucrose synthase fusion enzyme, with or without sucrose synthase; and incubating the reaction mixture for a sufficient time to produce a mixture of stevioside and rebaudioside KA, wherein a glucose is covalently coupled to the C2' -19-O-glucose of rubusoside to produce rebaudioside KA; glucose is covalently coupled to C2' -13-O-glucose of rubusoside to generate stevioside.

A method for producing rebaudioside KA from rubusoside. In another aspect, the disclosure relates to a method for synthesizing rebaudioside KA from rubusoside. The method comprises preparing a reaction mixture comprising rubusoside, a substrate selected from sucrose, Uridine Diphosphate (UDP) and uridine diphosphate-glucose (UDP-glucose) and HV1, with or without sucrose synthase; and incubating the reaction mixture for a sufficient time to produce rebaudioside KA, wherein a glucose is covalently coupled to the C2' -19-O-glucose of rubusoside to produce rebaudioside KA.

A method for producing rebaudioside G from rubusoside. In another aspect, the disclosure relates to a method for synthesizing rebaudioside G from rubusoside. The method comprises preparing a reaction mixture comprising rubusoside, a substrate selected from the group consisting of sucrose, Uridine Diphosphate (UDP) and uridine diphosphate-glucose (UDP-glucose), a UDP-glycosyltransferase selected from the group consisting of UGT76G1 and a UDP-glycosyltransferase-sucrose synthase fusion enzyme, with or without sucrose synthase; and incubating the reaction mixture for a sufficient time to produce rebaudioside G, wherein the glucose is covalently coupled to the C3' -13-O-glucose of rubusoside to produce rebaudioside G.

A method for producing rebaudioside E from rebaudioside KA. In another aspect, the disclosure relates to a method for synthesizing rebaudioside E from rebaudioside KA. The method comprises preparing a reaction mixture comprising rebaudioside KA, substrates selected from the group consisting of sucrose, Uridine Diphosphate (UDP) and uridine diphosphate-glucose (UDP-glucose), and HV1 UDP-glycosyltransferase, with or without a sucrose synthase; and incubating the reaction mixture for a sufficient time to produce rebaudioside E, wherein a glucose is covalently coupled to the C2' 13-O-glucose of rebaudioside KA to produce rebaudioside E.

A method for producing rebaudioside E from rebaudioside KA. In another aspect, the disclosure relates to a method for synthesizing rebaudioside E from rebaudioside KA. The method comprises preparing a reaction mixture comprising rebaudioside KA, a substrate selected from the group consisting of sucrose, Uridine Diphosphate (UDP) and uridine diphosphate-glucose (UDP-glucose), a UDP-glycosyltransferase selected from the group consisting of EUGT11 and a UDP-glycosyltransferase-sucrose synthase fusion enzyme, with or without sucrose synthase; and incubating the reaction mixture for a sufficient time to produce rebaudioside E, wherein a glucose is covalently coupled to the C2' 13-O-glucose of rebaudioside KA to produce rebaudioside E.

A method for producing rebaudioside E from rubusoside. In another aspect, the disclosure relates to a method of synthesizing rebaudioside E from rubusoside. The method comprises preparing a reaction mixture comprising rubusoside, a substrate selected from sucrose, Uridine Diphosphate (UDP) and uridine diphosphate-glucose (UDP-glucose), and a UDP-glycosyltransferase selected from EUGT11 and a UDP-glycosyltransferase-sucrose synthesis fusion enzyme, with or without sucrose synthase; and incubating the reaction mixture for a sufficient time to produce rebaudioside E, wherein a glucose is covalently coupled to rubusoside to produce a mixture of rebaudioside KA and stevioside. Continuously, glucose is covalently coupled to rebaudioside KA and stevioside to produce rebaudioside E.

A method for producing rebaudioside E from rubusoside. In another aspect, the disclosure relates to a method of synthesizing rebaudioside E from rubusoside. The method comprises preparing a reaction mixture comprising rubusoside, a substrate selected from the group consisting of sucrose, Uridine Diphosphate (UDP) and uridine diphosphate-glucose (UDP-glucose), and HV1 UDP-glycosyltransferase, with or without a sucrose synthase; incubating the reaction mixture for a sufficient time to produce rebaudioside E, wherein a glucose is covalently coupled to rubusoside to produce rebaudioside KA; and further incubating rebaudioside KA with HV1 to produce rebaudioside E.

A method for producing rebaudioside D3 from rubusoside. In another aspect, the disclosure relates to a method for synthesizing rebaudioside D3 from rubusoside. The method comprises preparing a reaction mixture comprising rubusoside, a substrate selected from sucrose, Uridine Diphosphate (UDP) and uridine diphosphate-glucose (UDP-glucose), a UDP-glycosyltransferase selected from EUGT11 and a UDP-glycosyltransferase-sucrose synthase fusion enzyme, with or without sucrose synthase; incubating the reaction mixture for a sufficient time to produce a mixture of stevioside and rebaudioside D3, wherein a glucose is covalently coupled to rubusoside to produce a mixture of stevioside and rebaudioside KA; further incubating the mixture of stevioside and rebaudioside KA with EUGT11 to produce rebaudioside E, wherein a glucose is covalently coupled to the stevioside and rebaudioside KA to produce rebaudioside E; and further incubating rebaudioside E with EUGT11 to produce rebaudioside D3, wherein a glucose is covalently coupled to rebaudioside E to produce rebaudioside D3.

Method for producing rebaudioside D3 from rebaudioside KA. In another aspect, the disclosure relates to a method for synthesizing rebaudioside D3 from rebaudioside KA. The method comprises preparing a reaction mixture comprising rebaudioside KA, a substrate selected from the group consisting of sucrose, Uridine Diphosphate (UDP) and uridine diphosphate-glucose (UDP-glucose), a UDP-glycosyltransferase selected from the group consisting of EUGT11 and a UDP-glycosyltransferase-sucrose synthase fusion enzyme, with or without sucrose synthase; incubating the reaction mixture for a sufficient time to produce rebaudioside D3, wherein a glucose is covalently coupled to rebaudioside KA to produce rebaudioside E; and further incubating the mixture of rebaudioside E and EUGT11 to produce rebaudioside D3, wherein a glucose is covalently coupled to rebaudioside E to produce rebaudioside D3.

Methods for producing rebaudioside Z from rebaudioside E. In another aspect, the disclosure relates to a method for synthesizing rebaudioside Z from rebaudioside E. The method comprises preparing a reaction mixture comprising rebaudioside E, substrates selected from the group consisting of sucrose, Uridine Diphosphate (UDP) and uridine diphosphate-glucose (UDP-glucose), HV1 and a sucrose synthase; incubating the reaction mixture for a sufficient time to produce rebaudioside Z, wherein a glucose is covalently coupled to the C2' -13-O-glucose of rebaudioside E to produce rebaudioside Z1. Glucose was covalently coupled to rebaudioside E C2' -19-O-glucose to produce rebaudioside Z2.

Methods for producing rebaudioside M from rebaudioside D. In another aspect, the present disclosure relates to a method for synthesizing rebaudioside M from rebaudioside D. The method comprises preparing a reaction mixture comprising rebaudioside D, a substrate selected from the group consisting of sucrose, Uridine Diphosphate (UDP), uridine diphosphate-glucose (UDP-glucose), and combinations thereof, and a UDP-glycosyltransferase selected from the group consisting of UGT76G1, UDP-glycosyltransferase-sucrose synthase fusion enzyme, and combinations thereof, with or without sucrose synthase; and incubating the reaction mixture for a sufficient time to produce rebaudioside M, wherein a glucose is covalently coupled to rebaudioside D to produce rebaudioside M.

Methods for producing rebaudioside D and rebaudioside M from stevioside. In another aspect, the disclosure relates to a method of synthesizing rebaudioside D and rebaudioside M from stevioside. The method comprises preparing a reaction mixture comprising stevioside, a substrate selected from the group consisting of sucrose, Uridine Diphosphate (UDP), uridine diphosphate-glucose (UDP-glucose), and combinations thereof, and a UDP-glycosyltransferase selected from the group consisting of HV1, UGT76G1, UDP-glycosyltransferase-sucrose synthase fusion enzyme, and combinations thereof, with or without sucrose synthase; and incubating the reaction mixture for a sufficient time to produce rebaudioside M. In certain embodiments, the glucose is covalently coupled to stevioside to produce rebaudioside a and/or rebaudioside E. Continuously, a glucose is covalently coupled to rebaudioside a and/or rebaudioside E to produce rebaudioside D, and a glucose is covalently coupled to rebaudioside D to produce rebaudioside M.

Methods for producing rebaudioside D and rebaudioside M from rebaudioside a. In another aspect, the present disclosure relates to a method for synthesizing rebaudioside D and rebaudioside M from rebaudioside a. The method comprises preparing a reaction mixture comprising rebaudioside a, a substrate selected from the group consisting of sucrose, Uridine Diphosphate (UDP), uridine diphosphate-glucose (UDP-glucose), and combinations thereof, and a UDP-glycosyltransferase selected from the group consisting of HV1, UGT76G1, UDP-glycosyltransferase-sucrose synthase fusion enzyme, and combinations thereof, with or without sucrose synthase; and incubating the reaction mixture for a sufficient time to produce rebaudioside M, wherein a glucose is covalently coupled to rebaudioside a to produce rebaudioside D and a glucose is covalently coupled to rebaudioside D to produce rebaudioside M.

Methods for producing rebaudioside D and rebaudioside M from rebaudioside E. In another aspect, the present disclosure relates to a method for synthesizing rebaudioside D and rebaudioside M from rebaudioside E. The method comprises preparing a reaction mixture comprising rebaudioside E, a substrate selected from the group consisting of sucrose, Uridine Diphosphate (UDP), uridine diphosphate-glucose (UDP-glucose), and combinations thereof, and a UDP-glycosyltransferase selected from the group consisting of UGT76G1, UDP-glycosyltransferase-sucrose synthase fusion enzyme, and combinations thereof, with or without sucrose synthase; and incubating the reaction mixture for a sufficient time to produce rebaudioside M, wherein a glucose is covalently coupled to rebaudioside E to produce rebaudioside D, and wherein a glucose is covalently coupled to rebaudioside D to produce rebaudioside M.

In another aspect, the present disclosure relates to an orally consumable product comprising a sweetening amount of a rebaudioside selected from the group consisting of rebaudioside V, rebaudioside W, rebaudioside G, rebaudioside KA, rebaudioside M, and combinations thereof, wherein the orally consumable product is selected from the group consisting of a beverage product and a consumable product.

In another aspect, the present disclosure relates to a beverage product comprising a sweetening amount of a rebaudioside selected from the group consisting of rebaudioside V, rebaudioside W, rebaudioside G, rebaudioside KA, rebaudioside M, and combinations thereof. The rebaudioside is present in the beverage product in a concentration from about 5ppm to about 100 ppm. In some embodiments, low concentrations of rebaudioside, e.g., less than 100ppm, have a sweetness comparable to sucrose solutions at concentrations between 10,000 and 30,000 ppm.

In another aspect, the present disclosure relates to a consumable product comprising a sweetening amount of a rebaudioside selected from the group consisting of rebaudioside V, rebaudioside W, rebaudioside G, rebaudioside KA, rebaudioside M, and combinations thereof. The rebaudioside is present in the consumable product at a concentration of about 5ppm to about 100 ppm. In some embodiments, low concentrations of rebaudioside, e.g., less than 100ppm, have a sweetness comparable to sucrose solutions at concentrations between 10,000 and 30,000 ppm.

In another aspect, the present disclosure relates to a sweetener consisting of the following chemical structure:

in another aspect, the present disclosure relates to a sweetener consisting of the following chemical structure:

in certain embodiments that may be combined with any of the preceding embodiments, rebaudioside V or rebaudioside W or rebaudioside G or rebaudioside KA or rebaudioside M may be the only sweetener, and the product has a sweetness intensity equivalent to about 1% to about 4% (W/V-%) sucrose solution. In certain embodiments that may be combined with any of the preceding embodiments, the orally consumable product may further comprise an additional sweetener, wherein the product has a sweetness intensity equivalent to about 1% to about 10% (w/v-%) sucrose solution. In certain embodiments that may be combined with any of the preceding embodiments, each sweetening ingredient in the product may be a high intensity sweetener. In certain embodiments that may be combined with any of the preceding embodiments, each sweetening ingredient in the product may be a natural high intensity sweetener. In certain embodiments that may be combined with any of the preceding embodiments, the additional sweetener may be one or more sweeteners selected from the group consisting of: stevia extract, steviol glycoside, stevioside, rebaudioside a, rebaudioside B, rebaudioside C, rebaudioside D3, rebaudioside E, rebaudioside F, rebaudioside G, rebaudioside KA, rebaudioside M, dulcoside a, rubusoside, steviolbioside, sucrose, high fructose corn syrup, fructose, glucose, xylose, arabinose, rhamnose, erythritol, xylitol, mannitol, sorbitol, inositol, AceK, aspartame, neotame, sucralose (sucromame), saccharin, naringin dihydrochalcone (NarDHC), Neohesperidin Dihydrochalcone (NDHC), rubusoside, mogroside iv (mogroside iv), siamenoside i (siamenoside i), mogroside V, monatin, thaumatin (thaumatin), nemulin (zebrazzein), and brazzein (brazzein), L-alanine, glycine, Lo Han Guo, Henan Dexin (hernandulcin), phyllodulcin (phylloddulcin), trilobatin (trilobtain), and combinations thereof. In certain embodiments that may be combined with any of the preceding embodiments, the beverage product and consumable product may further comprise one or more additives selected from the group consisting of: carbohydrates, polyols, amino acids or salts thereof, polyamino acids or salts thereof, sugar acids or salts thereof, nucleotides, organic acids, inorganic acids, organic salts, organic acid salts, organic base salts, inorganic salts, bitter compounds, flavorants, flavoring ingredients, astringent compounds, proteins, protein hydrolysates, surfactants, emulsifiers, flavonoids, alcohols, polymers, and combinations thereof. In certain embodiments that may be combined with any of the preceding embodiments, rebaudioside V has a purity of about 50% to about 100% by weight prior to its addition to the product. In certain embodiments that may be combined with any of the preceding embodiments, W has a purity of about 50% to about 100% by weight prior to its addition to the product. In certain embodiments that may be combined with any of the preceding embodiments, the rebaudioside V in the product is a rebaudioside V polymorph or amorphous rebaudioside V. In certain embodiments that may be combined with any of the preceding embodiments, the rebaudioside V in the product is a rebaudioside V stereoisomer. In certain embodiments that may be combined with any of the preceding embodiments, the rebaudioside W in the product is a rebaudioside W polymorph or amorphous rebaudioside W. In certain embodiments that may be combined with any of the preceding embodiments, the rebaudioside W in the product is a rebaudioside W stereoisomer.

Other aspects of the present disclosure relate to a method of preparing a beverage product and a consumable product by including a synthetic rebaudioside selected from the group consisting of rebaudioside V, rebaudioside W, rebaudioside KA, rebaudioside M and rebaudioside G into the product or ingredients used to prepare the beverage product and consumable product, wherein the rebaudioside selected from the group consisting of rebaudioside V, rebaudioside W, rebaudioside KA, rebaudioside M and rebaudioside G is present in the product in a concentration from about 5ppm to about 100 ppm. Other aspects of the present disclosure relate to a method of enhancing the sweetness of a beverage product and a consumable product by adding from about 5ppm to about 100ppm of a synthetic rebaudioside selected from the group consisting of rebaudioside V, rebaudioside W, rebaudioside KA, rebaudioside M, and rebaudioside G to the beverage product and the consumable product, wherein the added synthetic rebaudioside selected from the group consisting of rebaudioside V, rebaudioside W, rebaudioside KA, rebaudioside M, and rebaudioside G enhances the sweetness of the beverage product and the consumable product as compared to a corresponding beverage product and consumable product lacking the synthetic rebaudioside selected from the group consisting of rebaudioside V, rebaudioside W, rebaudioside KA, rebaudioside M, and rebaudioside G.

In certain embodiments that may be combined with any of the preceding embodiments, rebaudioside V is the only sweetener, and the product has a sweetness intensity equivalent to about 1% to about 4% (w/V-%) sucrose solution. In certain embodiments that may be combined with any of the preceding embodiments, rebaudioside KA is the only sweetener, and the product has a sweetness intensity equivalent to about 1% to about 4% (w/v-%) sucrose solution. In certain embodiments that may be combined with any of the preceding embodiments, rebaudioside G is the only sweetener, and the product has a sweetness intensity equivalent to about 1% to about 4% (w/v-%) sucrose solution. In certain embodiments that may be combined with any of the preceding embodiments, rebaudioside W is the only sweetener, and the product has a sweetness intensity equivalent to about 1% to about 4% (W/v-%) sucrose solution. In certain embodiments that may be combined with any of the preceding embodiments, rebaudioside M is the only sweetener, and the product has a sweetness intensity equivalent to about 1% to about 4% (w/v-%) sucrose solution. In certain embodiments that may be combined with any of the preceding embodiments, the method further comprises adding an additional sweetener, wherein the product has a sweetness intensity equivalent to about 1% to about 10% (w/v-%) sucrose solution.

Other aspects of the present disclosure relate to a method of preparing a sweetened beverage product or a sweetened consumable product by: a) providing a beverage product or consumable product comprising one or more sweeteners; and b) adding about 5ppm to about 100ppm of a synthetic rebaudioside selected from the group consisting of rebaudioside V, rebaudioside W, rebaudioside KA, rebaudioside M and rebaudioside G and combinations thereof to the beverage product or consumable product.

In certain embodiments that may be combined with any of the preceding embodiments, the method further comprises adding one or more additives to the beverage product or consumable product. In certain embodiments that may be combined with any of the preceding embodiments, the orally consumable product further comprises one or more additives. In certain embodiments that may be combined with any of the preceding embodiments, the one or more additives are selected from the group consisting of carbohydrates, polyols, amino acids or salts thereof, polyamino acids or salts thereof, sugar acids or salts thereof, nucleotides, organic acids, inorganic acids, organic salts, organic acid salts, organic base salts, inorganic salts, bitter compounds, flavorants, flavoring ingredients, astringent compounds, proteins, protein hydrolysates, surfactants, emulsifiers, flavonoids, alcohols, polymers, and combinations thereof. In certain embodiments that may be combined with any of the preceding embodiments, each sweetening ingredient in the product may be a high intensity sweetener. In certain embodiments that may be combined with any of the preceding embodiments, each sweetening ingredient in the product may be a natural high intensity sweetener. In certain embodiments that may be combined with any of the preceding embodiments, the sweetener is selected from the group consisting of stevia extract, steviol glycosides, stevioside, rebaudioside a, rebaudioside B, rebaudioside C, rebaudioside D3, rebaudioside E, rebaudioside F, rebaudioside G, rebaudioside KA, rebaudioside M, dulcoside a, rubusoside, steviol bioside, 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), rubusoside, mogroside IV, siamenoside I, mogroside V, monatin, thaumatin, monellin, brazzein, L-alanine, rebaudioside a, rebaudioside B, rebaudioside C, xylose, arabinose, xylitol, mannitol, sorbitol, stevioside, L-alanine, sucrose, fructose, a mixture, a, Glycine, momordica grosvenori, hernandenosine, phyllodulcin, trilobatin and combinations thereof. In certain embodiments that may be combined with any of the preceding embodiments, rebaudioside V has a purity of about 50% to about 100% by weight prior to its addition to the product. In certain embodiments that may be combined with any of the preceding embodiments, the rebaudioside V in the product is a rebaudioside V polymorph or amorphous rebaudioside V. In certain embodiments that may be combined with any of the preceding embodiments, rebaudioside W has a purity of about 50% to about 100% by weight prior to its addition to the product. In certain embodiments that may be combined with any of the preceding embodiments, the rebaudioside W in the product is a rebaudioside W polymorph or amorphous rebaudioside W.

Brief Description of Drawings

The present disclosure will be better understood, and features, aspects, and advantages over those mentioned above will become apparent from consideration of the following detailed description thereof. This detailed description makes reference to the following drawings, in which:

FIG. 1 depicts the steviol glycoside biosynthesis pathway starting from steviol.

Figure 2 depicts SDS-PAGE analysis of purified recombinant protein indicated by arrows: a: HV1, B: UGT76G1, C: EUGT11, D: AtSUS1, E: UGT76G1-SUS1(GS), F: EUGT11-SUS1 (EUS).

Fig. 3 depicts HV1 catalyzed reactions to produce rebaudioside KA ("Reb KA") and rebaudioside E ("Reb E") from rubusoside. A-C: HPLC retention times of rubusoside ("Rub"), stevioside ("Ste"), and rebaudioside E ("Reb E") standards are shown. Enzymatic production of Reb KA by HV1 alone at 6 hours (D), 12 hours (F), and 24 hours (H); reb KA and Reb E were enzymatically generated by UGT-SUS (HV1-AtSUS1) coupling system at 6 hours (E), 12 hours (G) and 24 hours (I).

Fig. 4 depicts the conversion of Reb E to rebaudioside Z by HV 1. (A) The method comprises the following steps HPLC retention times for rebaudioside E ("Reb E") are shown. Rebaudioside Z ("Reb Z") was enzymatically produced by HV1 in HV1-AtSUS1 coupling system at 3 hours (B), 7 hours (C), 24 hours (D), and 44 hours (E).

Fig. 5 depicts the conversion of Reb KA to Reb E by HV 1. (A-B): HPLC retention times for rebaudioside KA ("Reb KA") and rebaudioside E ("Reb E") standards are shown. Enzymatic production of Reb E by HV1 alone at 12 hours (C); reb E was enzymatically generated at 12 hours (D) by a UGT-SUS (HV1-AtSUS1) coupling system.

FIG. 6 depicts the EUGT11 catalyzed reaction of rubusoside to Reb KA and stevioside. (A-F): the HPLC retention times of rubusoside ("Rub"), stevioside ("Ste"), rebaudioside G ("Reb G"), rebaudioside E ("Reb E"), rebaudioside D ("Reb D"), and rebaudioside D3 ("Reb D3") standards are shown. Enzymatic reaction by EUGT11 alone at 12 hours (G) and 48 hours (J); enzymatic reaction by UGT-SUS (EUGT11-AtSUS1) coupling system at 12 hours (H) and 48 hours (K); enzymatic reaction by EUS fusion protein was carried out at 12 hours (I) and 48 hours (L).

FIG. 7 depicts the conversion of Reb KA to Reb E and Reb D3 via EUGT11 and EUS fusion proteins. (A-C): HPLC retention times for rebaudioside KA ("Reb KA"), rebaudioside E ("Reb E"), and rebaudioside D3 ("Reb D3") standards are shown. Enzymatic reaction by EUGT11 alone at 12 hours (D) and 48 hours (G); enzymatic reaction by UGT-SUS (EUGT11-AtSUS1) coupling system at 12 hours (E) and 48 hours (H); enzymatic reaction by EUS fusion protein was carried out at 12 hours (F) and 48 hours (I).

FIG. 8 depicts UGT76G1 production of rebaudioside G in vitro. (A-B): HPLC retention times for rubusoside ("Rub") and rebaudioside G ("Reb G") standards are shown. Enzymatic reaction by UGT76G1 alone at 12 hours (C) and 24 hours (F); enzymatic reaction by UGT-SUS (EUGT11-AtSUS1) coupling system at 12 hours (D) and 24 hours (G); enzymatic reaction by GS fusion protein at 12 hours (E) and 48 hours (H).

Fig. 9 depicts UGT76G1 catalyzed reaction of rebaudioside KA to the steviol glycosides Reb V and Reb W. (A-D): HPLC retention times of rubusoside ("Rub"), rebaudioside D ("Reb D"), rebaudioside E ("Reb E"), and rebaudioside KA ("Reb KA") standards are shown. Enzymatic reaction by UGT76G1 alone at 6 hours (E) and 12 hours (H); enzymatic reaction by UGT-SUS (UGT76G1-AtSUS1) coupling system at 6 hours (F) and 12 hours (I); enzymatic reaction by the GS fusion protein at 6 hours (G) and 12 hours (J).

FIG. 10 depicts UGT76G1 conversion of Reb V to Reb W in vitro. (A-B): HPLC retention times for Reb V and Reb W are shown. (C) The method comprises the following steps Enzymatic reaction by UGT76G1-AtSUS1 coupling system at 6 hours.

Fig. 11 depicts HV1 conversion of Reb G to Reb V. (A-C): HPLC retention times for rebaudioside G ("Reb G"), rebaudioside a ("RebA"), and rebaudioside E ("Reb E") standards are shown. Enzymatic reaction by HV1 alone at 12 hours (D) and 24 hours (F); enzymatic reaction by UGT-SUS (HV1-AtSUS1) coupling system at 12 hours (E) and 24 hours (G).

FIG. 12 depicts the conversion of Reb G to EUGT11 of Reb V. (A-D): HPLC retention times for rebaudioside G ("Reb G"), rebaudioside a ("RebA"), rebaudioside E ("Reb E"), and rebaudioside D ("Reb D") standards are shown. Enzymatic reaction by EUGT11 alone at 12 hours (E) and 24 hours (H); enzymatic reaction by UGT-SUS (EUGT11-AtSUS1) coupling system at 12 hours (F) and 24 hours (I); enzymatic reaction by EUS fusion enzyme was carried out at 12 hours (G) and 24 hours (J).

Fig. 13 depicts the in vitro production of Reb W from rubusoside catalyzed by a combination of recombinant HV1 polypeptide, recombinant UGT76G1, GS fusion enzyme, and recombinant atasus 1. (A-F): standards for rubusoside ("Rub"), stevioside ("Ste"), rebaudioside G ("Reb G"), rebaudioside a ("Reb a"), rebaudioside D ("Reb D"), and rebaudioside E ("Reb E") are shown. Enzymatic production of Reb W by HV1, UGT76G1, and AtSUS1 at 6 hours (G), 12 hours (I), and 24 hours (K); reb W was enzymatically generated by HV1 and GS fusion protein at 6 hours (H), 12 hours (J), and 24 hours (L).

Figure 14 depicts the in vitro production of Reb W from rubusoside catalyzed by a combination of recombinant EUGT11 polypeptide, recombinant UGT76G1, GS fusion enzyme, and recombinant atasus 1. (A-E): standards for rubusoside ("Rub"), stevioside ("Ste"), rebaudioside G ("Reb G"), rebaudioside E ("Reb E"), and rebaudioside D ("Reb D") are shown. Enzymatically producing Reb W at 12 hours (F) and 48 hours (H) by EUGT11, UGT76G1, and AtSUS 1; reb W was enzymatically generated by EUGT11 and GS fusion protein at 12 hours (G) and 48 hours (I).

Fig. 15 depicts the in vitro production of Reb W from Reb G catalyzed by a combination of recombinant HV1 polypeptide, recombinant UGT76G1, GS fusion enzyme, and recombinant atasus 1. a-D show standards for rebaudioside G ("Reb G"), rebaudioside a ("Reb a"), rebaudioside D ("Reb D"), rebaudioside, and rebaudioside E ("Reb E"). Enzymatically producing Reb V and Reb W by HV1, UGT76G1, and AtSUS1 at 6 hours (E), 12 hours (G), and 36 hours (I); reb V and Reb W were enzymatically generated by HV1 and GS fusion protein at 6 hours (F), 12 hours (H), and 36 hours (J).

Fig. 16 depicts the in vitro production of Reb W from Reb G catalyzed by a combination of recombinant EUGT11 polypeptide, recombinant UGT76G1, GS fusion enzyme, and recombinant AtSUS 1. (A-D): standards for rebaudioside G ("Reb G"), rebaudioside a ("RebA"), rebaudioside E ("Reb E"), and rebaudioside D ("Reb D") are shown. Enzymatic production of Reb W by EUGT11, UGT76G1 and AtSUS1 at 12 hours (E) and 48 hours (G); reb W was enzymatically generated by EUGT11 and GS fusion protein at 12 hours (F) and 48 hours (H).

FIG. 17 depicts the structures of Reb V and Reb G.

Fig. 18 depicts key TOCSY and HMBC correlations for Reb V.

FIG. 19 depicts the structures of Reb W and Reb V.

Fig. 20 depicts key TOCSY and HMBC correlations for Reb W.

FIG. 21 depicts the biosynthetic pathway of steviol glycosides.

Fig. 22 depicts the in vitro production of Reb M from Reb D catalyzed by UGT76G1 and GS fusogenic enzymes. (A-B): HPLC retention times for rebaudioside D ("Reb D") and rebaudioside M ("Reb M") standards are shown. Enzymatic reaction by UGT76G1 alone at 3 hours (C) and 6 hours (F); enzymatic reaction by UGT-SUS (UGT76G1-AtSUS1) coupling system at 3 hours (D) and 6 hours (G); enzymatic reaction by GS fusion enzyme was performed at 3 hours (E) and 6 hours (H).

Fig. 23 depicts the in vitro production of Reb D and Reb M from Reb E catalyzed by UGT76G1 and GS fusogenic enzymes. (A-C): HPLC retention times for rebaudioside E ("Reb E"), rebaudioside D ("Reb D"), and rebaudioside M ("Reb M") standards are shown. Enzymatic reactions by UGT76G1 alone at 3 hours (D), 12 hours (G) and 24 hours (J); enzymatic reactions by UGT-SUS (UGT76G1-AtSUS1) coupling system at 3 hours (E), 12 hours (H) and 24 hours (K); enzymatic reaction by GS fusion enzyme was performed at 3 hours (F), 12 hours (I) and 24 hours (L).

Fig. 24 depicts the in vitro production of Reb D and Reb M from stevioside catalyzed by a combination of recombinant HV1, recombinant UGT76G1, GS fusion enzyme, and/or recombinant AtSUS 1. (A-D): HPLC retention times for stevioside ("Ste"), rebaudioside a ("RebA"), rebaudioside D ("Reb D"), and rebaudioside M ("Reb M") standards are shown. Enzymatic reactions by HV1 and UGT76G1 in UGT-SUS coupling system at 6 hours (E), 12 hours (H) and 24 hours (K); enzymatic reaction by HV1 and GS fusion enzyme at 6 hours (F), 12 hours (I) and 24 hours (L); enzymatic reactions by UGT76G1 and HV1 at 6 hours (G), 12 hours (J) and 24 hours (M).

Fig. 25 depicts the in vitro production of Reb D and Reb M from rebaudioside a catalyzed by recombinant HV1, recombinant UGT76G1, GS fusion enzyme, and/or recombinant AtSUS 1. (A-C): HPLC retention times for rebaudioside a ("RebA"), rebaudioside D ("Reb D"), and rebaudioside M ("Reb M") standards are shown. Enzymatic reactions by HV1 and UGT76G1 in UGT-SUS coupling system at 6 hours (D), 12 hours (G) and 24 hours (J); enzymatic reactions by HV1 and GS fusion enzyme at 6 hours (E), 12 hours (H) and 24 hours (K). Enzymatic reactions by UGT76G1 and HV1 at 6 hours (F), 12 hours (I) and 24 hours (J).

Fig. 26 depicts the structure of Reb M.

Fig. 27 depicts key TOCSY and HMBC correlations for Reb M.

While the disclosure is susceptible to various modifications and alternative forms, specific embodiments thereof have been shown by way of example in the drawings and are herein described in detail. It should be understood, however, that the description of specific embodiments is not intended to limit the disclosure to the particular forms disclosed, but on the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the disclosure as defined by the appended claims.

Detailed description of the invention

Unless defined otherwise, 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.

The term "complementary" is used according to its ordinary and customary meaning as understood by those of ordinary skill in the art and is not limited to being used to describe relationships between nucleotide bases capable of hybridizing to one another. For example, in the case of DNA, adenosine is complementary to thymine and cytosine is complementary to guanine. Thus, the subject technology also includes isolated nucleic acid fragments that are complementary to the complete sequences reported in the accompanying sequence listing, as well as those substantially similar nucleic acid sequences.

The terms "nucleic acid" and "nucleotide" are used according to their respective ordinary and customary meaning as understood by those of ordinary skill in the art and are not limited to use in reference to deoxyribonucleotides or ribonucleotides and polymers thereof in either single-or double-stranded form. Unless specifically limited, the term encompasses known analogs containing natural nucleotides, nucleic acids that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also encompasses conservatively modified or degenerate variants thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated.

The term "isolated" is used in accordance with its ordinary and customary meaning as understood by those of ordinary skill in the art and when used in the context of an isolated nucleic acid or isolated polypeptide, is not limited to use in reference to a nucleic acid or polypeptide that has been artificially created, exists apart from its natural environment, and is therefore not a natural product. An isolated nucleic acid or polypeptide may exist in a purified form or may exist in a non-natural environment, such as a transgenic host cell.

The term "incubating" as used herein refers to the process of mixing and allowing two or more chemical or biological entities (such as chemical compounds and enzymes) to interact under conditions conducive to the production of a steviol glycoside composition.

The term "degenerate variant" refers to a nucleic acid sequence that has a sequence of residues that differs from a reference nucleic acid sequence by substitution of one or more degenerate codons. Degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed base and/or deoxyinosine residues. Nucleic acid sequences and all degenerate variants thereof will express the same amino acid or polypeptide.

The terms "polypeptide", "protein" and "peptide" are used according to their respective ordinary and customary meaning as understood by those of ordinary skill in the art; the three terms are sometimes used interchangeably and are not limited to being used to refer to amino acid polymers or amino acid analogs regardless of their size or function. Although "protein" is often used in reference to relatively large polypeptides and "peptide" is often used in reference to smaller polypeptides, there are overlaps and variations in the use of these terms in the art. The term "polypeptide" as used herein refers to peptides, polypeptides and proteins, unless otherwise indicated. The terms "protein," "polypeptide," and "peptide" are used interchangeably when referring to a polynucleotide product. Thus, exemplary polypeptides include polynucleotide products, naturally occurring proteins, homologs, orthologs, paralogs, fragments, and other equivalents, variants and analogs of the foregoing.

The terms "polypeptide fragment" and "fragment" when used in reference to a reference polypeptide are used according to their ordinary and customary meaning as understood by those of ordinary skill in the art, and are not limited to use in reference to polypeptides in which amino acid residues are deleted as compared to the reference polypeptide itself, but in which the remaining amino acid sequence is generally identical to the corresponding position in the reference polypeptide. Such deletions may occur at the amino-terminus or the carboxy-terminus of the reference polypeptide, or alternatively at both.

The term "functional fragment" of a polypeptide or protein refers to a peptide fragment that is a portion of a full-length polypeptide or protein and has substantially the same biological activity, or performs substantially the same function (e.g., performs the same enzymatic reaction), as the full-length polypeptide or protein.

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

The term "functional variant" also includes variants that are conservatively substituted. The term "conservatively substituted variant" refers to a peptide having an amino acid sequence that differs from a reference peptide by one or more conservative amino acid substitutions and retains some or all of the activity of the reference peptide. A "conservative amino acid substitution" is a substitution of an amino acid residue with a functionally similar residue. Examples of conservative substitutions include the substitution of one nonpolar (hydrophobic) residue such as isoleucine, valine, leucine or methionine for another; substitution of one charged or polar (hydrophilic) residue for another such as between arginine and lysine, between glutamine and asparagine, and between threonine and serine; substitution of one basic residue, such as lysine or arginine, for the other; or substitution of one acidic residue, such as aspartic acid or glutamic acid, for the other; or substitution of one aromatic residue, such as phenylalanine, tyrosine or tryptophan, for 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 "conservatively substituted variant" also includes peptides in which one residue is replaced with a chemically derivatized residue, provided that the resulting peptide retains some or all of the activity of the reference peptide as described herein.

The term "variant" in relation to a polypeptide of the subject technology also includes functionally active polypeptides having an amino acid sequence that is 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 to the amino acid sequence of the reference polypeptide.

The term "homologous" in all its grammatical forms and spelling variants refers to polynucleotides or polypeptides having "common evolutionary origin", including the relationship between polynucleotides or polypeptides from the superfamily and homologous polynucleotides or proteins from different species (Reeck et al, Cell 50:667,1987). Such polynucleotides or polypeptides, whether in the presence of specific amino acids or motifs at percent identity or conserved positions, have sequence homology reflected by their sequence similarity. For example, two homologous polypeptides may have an amino acid sequence that is 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.

"percent (%) amino acid sequence identity" with respect to a variant polypeptide sequence of the subject technology refers to the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues of a reference polypeptide, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, without considering any conservative substitutions as part of the sequence identity.

Alignment to determine percent amino acid sequence identity can be accomplished in a variety of ways within the skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN-2, or Megalign (DNASTAR) software. One skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms necessary to achieve maximal alignment over the full length of the sequences being compared. For example, the% amino acid sequence identity can be determined using the sequence comparison program NCBI-BLAST 2. NCBI-BLAST2 sequence comparison programs are available from NCBI. NCBI BLAST2 uses several search parameters, where those search parameters are all set to default values, including, for example, unmasked is, chain full, expected to occur 10, minimum low complexity length 15/5, multi-pass e value 0.01, multi-pass constant 25, final reduction of gap alignment 25 and scoring matrix BLOSUM 62. In the case of amino acid sequence comparisons using NCBI-BLAST2, the amino acid sequence identity% (which may optionally be expressed as, have or comprise some% amino acid sequence identity with or against a given amino acid sequence B) for a given amino acid sequence a pair, with or against a given amino acid sequence B is calculated as follows: 100X score X/Y, wherein X is the number of amino acid residues scored as identical matches in the alignment of the program to a and B by the sequence alignment program NCBI-BLAST2, and wherein Y is the total number of amino acid residues in B. It will be appreciated that where 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 to B will not be equal to the% amino acid sequence identity of B to A.

In this sense, techniques for determining the "similarity" of amino acid sequences are well known in the art. In general, "similarity" refers to the exact comparison of amino acids to amino acids of two or more polypeptides at appropriate positions where the amino acids are the same or have similar chemical and/or physical properties, such as charge or hydrophobicity. The so-called "percent 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 for that gene (typically via a cDNA intermediate) and determining the amino acid sequence encoded therein, and comparing this to a second amino acid sequence. In general, "identity" refers to the exact nucleotide-to-nucleotide or amino acid-to-amino acid identity of two polynucleotide or polypeptide sequences, respectively. Just as with two or more amino acid sequences, two or more polynucleotide sequences can be compared by determining their "percent identity". The programs available in the Wisconsin sequence analysis Package, version 8 (available from Genetics Computer Group, Madison, Wis.), such as the GAP program, enable the calculation of identity between two polynucleotides and identity and similarity, respectively, between two polypeptide sequences. Other procedures for calculating identity or similarity between sequences are known to those skilled in the art.

An amino acid position "corresponding" to a reference position refers to a position that matches the reference sequence, as identified by alignment of the amino acid sequences. Such alignment may be performed manually or by using well known sequence alignment programs such as ClustalW2, Blast2, and the like.

Unless otherwise indicated, percent identity of two polypeptide or polynucleotide sequences refers to the percentage of identical amino acid residues or nucleotides over the entire length of the shorter of the two sequences.

"coding sequence" is used in accordance with its ordinary and customary meaning as understood by those of ordinary skill in the art and is not limited to being used to refer to a DNA sequence encoding a particular amino acid sequence.

"suitable control sequences" are used in their ordinary and customary meaning as understood by those of ordinary skill in the art and are not limited to use in reference to nucleotide sequences located upstream (5 'non-coding sequences), within, or downstream (3' non-coding sequences) of a coding sequence, and which affect transcription, RNA processing or stability or translation of the relevant coding sequence. Regulatory sequences may include promoters, translation leader sequences, introns, and polyadenylation recognition sequences.

"promoter" is used according to its ordinary and customary meaning as understood by those of ordinary skill in the art and is not limited to being used to refer to a DNA sequence capable of controlling the expression of a coding sequence or functional RNA. Generally, a coding sequence is located 3' to a promoter sequence. Promoters may be derived in their entirety from a native gene, or be composed of different elements derived from different promoters found in nature, or even comprise synthetic DNA fragments. It will be appreciated by those skilled in the art that different promoters may direct the expression of genes in different cell types or at different stages of development or in response to different environmental conditions. Promoters which allow genes to be expressed in most cell types most of the time are commonly referred to as "constitutive promoters". It is further recognized that since the exact boundaries of regulatory sequences are not fully defined in most cases, DNA fragments of different lengths may have the same promoter activity.

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

The term "expression" as used herein is used according to its ordinary and customary meaning as understood by those of ordinary skill in the art, and is not limited to being used to refer to the transcription and stable accumulation of sense (mRNA) or antisense RNA of a nucleic acid fragment derived from the subject technology. "overexpression" refers to the production of a gene product in a transgenic or recombinant organism above the level of production in a normal or non-transformed organism.

"transformation" is used according to its ordinary and customary meaning as understood by those of ordinary skill in the art and is not limited to being used to refer to the transfer of a polynucleotide into a target cell. The transferred polynucleotide may be incorporated into the genomic or chromosomal DNA of the target cell, resulting in gene-stable inheritance, or it may replicate independently of the host chromosome. Host organisms containing the transformed nucleic acid fragments are referred to as "transgenic" or "recombinant" or "transformed" organisms.

The terms "transformation", "transgene" and "recombinant", when used herein in connection with a host cell, are used according to their ordinary and customary meaning as understood by those of ordinary skill in the art, and are not limited to being used to refer to a cell of a host organism, such as a plant or microbial cell, into which a heterologous nucleic acid molecule has been introduced. The nucleic acid molecule may be stably integrated into the genome of the host cell, or the nucleic acid molecule may be present as an extrachromosomal molecule. Such extrachromosomal molecules can replicate autonomously. Transformed cells, tissues or subjects are understood to encompass not only the end product of the transformation process, but also transgenic progeny thereof.

The terms "recombinant," "heterologous," and "exogenous," when used herein in connection with a polynucleotide, are used in accordance with their ordinary and customary meaning as understood by those of ordinary skill in the art, and are not limited to use in reference to a polynucleotide (e.g., a DNA sequence or gene) that is foreign to a particular host cell or, if from the same source, is modified from its original form. Thus, a heterologous gene in a host cell includes a gene that is endogenous to the particular host cell but has been modified, for example, 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 homologous to the cell but in a position or form within the host cell in which the element is not normally found.

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

The terms "plasmid", "vector" and "cassette" are used according to their ordinary and customary meaning as understood by those of ordinary skill in the art and are not limited to use in reference to extrachromosomal elements that often carry genes that are not part of the central metabolism of the cell and are usually in the form of circular double stranded DNA molecules. Such elements may be autonomously replicating sequences, genome integrating sequences, phage or nucleotide sequences, linear or circular, of single-or double-stranded DNA or RNA, derived from any source, many of which have been joined or recombined into unique configurations capable of introducing a promoter fragment of a selected gene product and a DNA sequence into a cell, along with appropriate 3' untranslated sequences. "transformation cassette" refers to a particular vector that contains a foreign gene and has elements in addition to the foreign gene that facilitate transformation of a particular host cell. "expression cassette" refers to a specific vector that contains a foreign gene and has elements in addition to the foreign gene that allow for enhanced expression of the gene in a foreign host.

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 manitis, t.molecular Cloning: laboratory Manual, 2 nd edition; cold Spring Harbor Laboratory Cold Spring Harbor, N.Y.,1989 (hereinafter referred to as "Maniatis"); and by 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 Current Protocols in Molecular Biology, published in 1987 by Greene Publishing and Wiley-Interscience, by Ausubel, f.m., et al, each of which is hereby incorporated by reference in its entirety to the extent it is consistent therewith.

As used herein, "synthesis" 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 extracting the compound, for example, from a natural source.

The term "orally consumable product" as used herein refers to any beverage, food product, dietary supplement, nutraceutical, pharmaceutical, oral hygiene, and cosmetic product that comes into contact with the oral cavity of a human or animal, including substances that are ingested into the oral cavity and subsequently excreted by the oral cavity and substances that are drunk, chewed, swallowed, or otherwise digested; and substances that are safe for human or animal consumption when used within the generally acceptable concentration range.

The term "food product" as used herein refers to fruits, vegetables, fruit juices, meat products such as ham, bacon (bacon), sausages; egg products, fruit concentrates, gelatin and gelatin-like products such as jams, jellies, preserves and the like; dairy products such as ice cream, sour cream, yogurt and frozen fruit juice; frosting, syrups, including molasses; corn, wheat, rye, soybean, oats, rice and barley products, cereal products, nut kernels and nut products, cakes, cookies, confectionery (e.g., candies), gums, fruit flavored hard candies (fruit flavored drops) and chocolate, chewing gum, mints, creams, frostings, ice creams, pies, and breads. "food product" also refers to flavorings such as herbs, spices and condiments, and flavoring agents such as monosodium glutamate. "food product" further refers to packaged products that also include preparations, such as dietary sweeteners, liquid sweeteners, table top flavorings, particulate flavor mixes that are reconstituted with water to obtain non-carbonated beverages, instant pudding mixes, instant coffee and tea, coffee creamer, malted milk mixes, pet food, livestock feed, tobacco, and materials for baking applications, such as powdered baking mixes for making bread, cookies, cakes, pancakes, donuts, and the like. "food product" also refers to diet or low calorie foods and beverages containing little or no sucrose.

As used herein, the term "stereoisomer" is a collective term for all isomers of individual molecules that differ only in the orientation of their atoms in space. "stereoisomers" include enantiomers and isomers of compounds having more than one chiral center that are not mirror images of each other (diastereomers).

The term "amorphous rebaudioside V" as used herein refers to a non-crystalline solid form of rebaudioside V. The term "amorphous rebaudioside W" as used herein refers to a non-crystalline solid form of rebaudioside W.

As used herein, the term "sweetness intensity" refers to the relative intensity of a perception of sweetness observed or experienced by an individual (e.g., a human), or the degree or amount of sweetness detected by a taster, e.g., based on the Brix scale.

As used herein, the term "sweetness enhancing" refers to the effect of rebaudioside V and/or rebaudioside W to increase, augment, enhance, exacerbate, magnify and/or enhance the sensory perception of one or more sweetness profiles of a beverage product or consumable product of the present disclosure without altering its nature or quality as compared to a corresponding orally consumable product that does not contain rebaudioside V and/or rebaudioside W.

As used herein, the term "off-flavor" refers to the amount or degree of taste that is atypical or not commonly present in the beverage products or consumable products of the present disclosure. For example, off-tastes are tastes that are undesirable to consumers for sweetening consumables, e.g., bitter, licorice-like, metallic, objectionable, astringent, delayed sweetness production, lingering sweet aftertaste, and the like, among others.

As used herein, the term "w/v-%" refers to the weight (in grams) of a compound, such as sugar, per 100ml of a liquid orally consumable product of the disclosure containing such compound. As used herein, the term "w/w-%" refers to the weight (in grams) of a compound, such as a sugar, per gram of an orally consumable product of the disclosure containing such compound.

As used herein, the term "ppm" refers to parts per million by weight, e.g., the weight (in milligrams) of such compounds, such as rebaudioside V and/or rebaudioside W per kilogram (i.e., mg/kg) of an orally consumable product of the present disclosure containing the compound or the weight (in milligrams) of such compounds, such as rebaudioside V and/or rebaudioside W per liter (i.e., mg/L) of an orally consumable product of the present disclosure containing the compound; or parts per million by volume, such as the weight (in milliliters) of such compounds, such as rebaudioside V and/or rebaudioside W, per liter of an orally consumable product of the present disclosure containing such compounds (i.e., ml/L).

In accordance with the present disclosure, non-caloric sweeteners and methods of synthesizing non-caloric sweeteners are disclosed. Also in accordance with the present disclosure, an enzyme and a method of using the enzyme for making non-caloric sweeteners are disclosed.

Synthetic non-caloric sweeteners: synthesis of rebaudioside V

In one aspect, the present disclosure relates to a synthetic non-caloric sweetener. The synthetic non-caloric sweetener is a synthetic rebaudioside-type steviol glycoside and has been designated "rebaudioside V". Rebaudioside V ("Reb V") is a steviol glycoside having in its structure four β -D-glucosyl units linked to the aglycone steviol, the steviol aglycone part having Glc β 1-3-Glc β 1 units as ether linkages at C-13 and another Glc β 1-2-Glc β 1 units as ester linkages at the C-19 position.

Rebaudioside V has the formula C based on extensive 1D and 2D NMR and high resolution mass spectral data and hydrolysis studies₄₄H₇₀O₂₃And IUPAC name, 13- [ (3-O-beta-D-glucopyranosyl) oxy]Ent-kauri-16-en-19-enoic acid- (2-O- β -D-glucopyranosyl) ester.

Synthetic non-caloric sweeteners: synthesis of rebaudioside W

In one aspect, the present disclosure relates to a synthetic non-caloric sweetener. The synthetic non-caloric sweetener is a synthetic rebaudioside-type steviol glycoside and has been designated "rebaudioside W". Rebaudioside W ("Reb W") is a steviol glycoside having in its structure five β -D-glucosyl units linked to the aglycone steviol, the steviol aglycone part having Glc β 1-3-Glc β 1 units as ether linkages at C-13 and Glc β 1-2(Glc β 1-3) -Glc β 1 units as ester linkages at the C-19 position.

Rebaudioside W has the formulaC₅₀H₈₀O₂₈And IUPAC name, 13- [ (3-O-beta-D-glucopyranosyl) oxy]Ent-kauri-16-en-19-enoic acid- [ (2-O- β -D-glucopyranosyl-3-O- β -D-glucopyranosyl) ester.

Synthetic non-caloric sweeteners: synthesis of rebaudioside KA

In one aspect, the present disclosure relates to a synthetic non-caloric sweetener. The synthetic non-caloric sweetener is a synthetic rebaudioside-type steviol glycoside and has been designated "rebaudioside KA". Rebaudioside KA ("Reb KA") is a steviol glycoside having in its structure three β -D-glucosyl units linked to the aglycone steviol, the steviol aglycone part having Glc β 1 units as ether linkages at C-13 and Glc β 1-2-Glc β 1 units as ether linkages at C-19. Rebaudioside KA has the molecular formula C based on extensive 1D and 2D NMR and high resolution mass spectral data and hydrolysis studies₃₈H₆₀O₁₈And IUPAC name, 13-beta-D-glucopyranosyloxy]Ent-kauri-16-en-19-enoic acid- (2-O- β -D-glucopyranosyl) ester.

Synthetic non-caloric sweeteners: synthesis of rebaudioside G

In one aspect, the present disclosure relates to a synthetic non-caloric sweetener. The synthetic non-caloric sweetener is a synthetic rebaudioside-type steviol glycoside and has been designated "rebaudioside G". Rebaudioside G ("Reb G") is a steviol glycoside having in its structure three β -D-glucosyl units linked to the aglycone steviol, the steviol aglycone part having Glc β 1-3-Glc β 1 units as ether linkages at C-13 and Glc β 1 units as ether linkages at C-19.

Rebaudioside G has the molecular formula C based on extensive 1D and 2D NMR and high resolution mass spectral data and hydrolysis studies₃₈H₆₀O₁₈And IUPAC name, 13- [ (3-O-beta-D-glucopyranosyl) oxy]Ent-kauri-16-en-19-enoic acid-beta-D-glucopyranosyl) ester.

Synthetic non-caloric sweeteners: synthesis of rebaudioside M

In one aspect, the present disclosure relates to a synthetic non-caloric sweetener. The synthetic non-caloric sweetener is a synthetic rebaudioside-type steviol glycoside and has been designated "rebaudioside M". Rebaudioside M ("Reb M") is a steviol glycoside having six β -D-glucosyl units linked in its structure to the aglycone steviol, the steviol aglycone part having Glcb1-2 (Glcb1-3) -Glcb1 units in the form of an ether linkage at the C-13 position and Glcb1-2 (Glcb1-3) -Glcb1 units in the form of an ester linkage at the C-19 position.

Rebaudioside M has the formula C based on extensive 1D and 2D NMR and high resolution mass spectral data and hydrolysis studies₅₆H₉₀O₃₃And the IUPAC name, 13- [ (2-O-beta-D-glucopyranosyl-3-O-beta-D-glucopyranosyl) oxy]Ent-kauri-16-en-19-enoic acid- [ (2-O- β -D-glucopyranosyl-3-O- β -D-glucopyranosyl) ester.

Method for synthesizing steviol glycoside

Methods for producing rebaudioside V from rebaudioside G. In another aspect, the present disclosure relates to a method for synthesizing rebaudioside V from rebaudioside G. The method includes preparing a reaction mixture comprising rebaudioside G; a substrate selected from sucrose, Uridine Diphosphate (UDP) and uridine diphosphate-glucose (UDP-glucose); and HV1 UDP-glycosyltransferase; with or without sucrose synthase (SUS) and incubating the reaction mixture for a sufficient time to produce rebaudioside V, wherein a glucose is covalently coupled to rebaudioside G to produce rebaudioside V.

Methods for producing rebaudioside V from rebaudioside G. In another aspect, the present disclosure relates to a method for synthesizing rebaudioside V from rebaudioside G. The method includes preparing a reaction mixture comprising rebaudioside G; a substrate selected from sucrose, Uridine Diphosphate (UDP) and uridine diphosphate-glucose (UDP-glucose); a uridine diphosphate glycosyltransferase (UDP-glycosyltransferase) selected from the group consisting of EUGT11, UDP-glycosyltransferase-sucrose synthase (SUS) fusion enzyme; with or without sucrose synthase (SUS) and incubating the reaction mixture for a sufficient time to produce rebaudioside V, wherein a glucose is covalently coupled to rebaudioside G to produce rebaudioside V.

A method for producing rebaudioside V from rebaudioside KA.

In another aspect, the disclosure relates to a method for synthesizing rebaudioside V from rebaudioside KA. The method includes preparing a reaction mixture comprising rebaudioside KA; a substrate selected from sucrose, Uridine Diphosphate (UDP) and uridine diphosphate-glucose (UDP-glucose); a uridine diphosphate glycosyltransferase (UDP-glycosyltransferase) selected from the group consisting of a UDP-glycosyltransferase (UGT76G1) and a UDP-glycosyltransferase-sucrose synthase fusion enzyme; with or without sucrose synthase (SUS) and incubating the reaction mixture for a sufficient time to produce rebaudioside V, wherein a glucose is covalently coupled to rebaudioside KA to produce rebaudioside V.

Methods for producing rebaudioside V from rubusoside. In another aspect, the disclosure relates to a method of synthesizing rebaudioside V from rubusoside. The method comprises preparing a reaction mixture comprising rubusoside; a substrate selected from sucrose, Uridine Diphosphate (UDP) and uridine diphosphate-glucose (UDP-glucose); a uridine diphospho glycosyltransferase (UDP-glycosyltransferase) selected from the group consisting of UDP-glycosyltransferase (UGT76G1), HV1, and UDP-glycosyltransferase-sucrose synthase fusion enzyme; with or without sucrose synthase (SUS) and incubating the reaction mixture for a sufficient time to produce rebaudioside V, wherein a glucose is covalently coupled to rubusoside to produce rebaudioside KA. Continuously, glucose is covalently coupled to rebaudioside KA to produce rebaudioside V.

Methods for producing rebaudioside V from rubusoside. In another aspect, the present disclosure relates to a method of synthesizing a mixture of rebaudioside a and rebaudioside V from rubusoside. The method comprises preparing a reaction mixture comprising rubusoside; a substrate selected from sucrose, Uridine Diphosphate (UDP) and uridine diphosphate-glucose (UDP-glucose); a uridine diphospho glycosyltransferase (UDP-glycosyltransferase) selected from the group consisting of UDP-glycosyltransferase (UGT76G1), EUGT11, and UDP-glycosyltransferase-sucrose synthase fusion enzyme; with or without sucrose synthase (SUS); and incubating the reaction mixture for a sufficient time to produce rebaudioside V, wherein a glucose is covalently coupled to rubusoside to produce rebaudioside KA and a glucose is covalently coupled to rebaudioside KA to produce rebaudioside V. Glucose is covalently coupled to rubusoside to produce rebaudioside G. Continuously, glucose was covalently coupled to rebaudioside G to produce rebaudioside V.

Methods for producing rebaudioside W from rebaudioside V. In another aspect, the present disclosure relates to a method for synthesizing rebaudioside W from rebaudioside V. The method includes preparing a reaction mixture comprising rebaudioside V; a substrate selected from sucrose, Uridine Diphosphate (UDP) and uridine diphosphate-glucose (UDP-glucose); a uridine diphosphate glycosyltransferase (UDP-glycosyltransferase) selected from the group consisting of a UDP-glycosyltransferase (UGT76G1) and a UDP-glycosyltransferase-sucrose synthase fusion enzyme; with or without sucrose synthase (SUS) and incubating the reaction mixture for a sufficient time to produce rebaudioside W, wherein a glucose is covalently coupled to rebaudioside V to produce rebaudioside W.

A method for producing rebaudioside W from rebaudioside G. In another aspect, the present disclosure relates to a method for synthesizing rebaudioside G from rebaudioside G. The method includes preparing a reaction mixture comprising rebaudioside G; a substrate selected from sucrose, Uridine Diphosphate (UDP) and uridine diphosphate-glucose (UDP-glucose); a uridine diphosphate glycosyltransferase (UDP-glycosyltransferase) selected from the group consisting of uridine diphosphate glycosyltransferase (UGT76G1), UDP-glycosyltransferase-sucrose synthase fusion enzyme, and HV 1; with or without sucrose synthase (SUS); and incubating the reaction mixture for a sufficient time to produce rebaudioside W, wherein glucose is covalently coupled to rebaudioside G by HV1 to produce rebaudioside V. Continuously, glucose was covalently coupled to rebaudioside V via UGT76G1 to produce rebaudioside W.

A method for producing rebaudioside W from rebaudioside G. In another aspect, the present disclosure relates to a method for synthesizing rebaudioside G from rebaudioside G. The method includes preparing a reaction mixture comprising rebaudioside G; a substrate selected from sucrose, Uridine Diphosphate (UDP) and uridine diphosphate-glucose (UDP-glucose); a uridine diphosphate glycosyltransferase (UDP-glycosyltransferase) selected from the group consisting of UGT76G1, EUGT11, and a UDP-glycosyltransferase-sucrose synthase fusion enzyme; and incubating the reaction mixture for a sufficient time to produce rebaudioside W, wherein glucose is covalently coupled to rebaudioside G through EUGT11 to produce rebaudioside V. Continuously, glucose was covalently coupled to rebaudioside V via UGT76G1 to produce rebaudioside W.

A method for producing rebaudioside W from rebaudioside KA. In another aspect, the disclosure relates to a method for synthesizing rebaudioside W from rebaudioside KA. The method includes preparing a reaction mixture comprising rebaudioside KA; a substrate selected from sucrose, Uridine Diphosphate (UDP) and uridine diphosphate-glucose (UDP-glucose); a uridine diphosphate glycosyltransferase (UDP-glycosyltransferase) selected from the group consisting of a uridine diphosphate glycosyltransferase (UGT76G1) and a UDP-glycosyltransferase-sucrose synthase fusion enzyme; with or without sucrose synthase; and incubating the reaction mixture for a sufficient time to produce rebaudioside W, wherein a glucose is covalently coupled to rebaudioside KA to produce rebaudioside V. Continuously, glucose was covalently coupled to rebaudioside V to produce rebaudioside W.

A method for producing rebaudioside W from rubusoside. In another aspect, the disclosure relates to a method of synthesizing rebaudioside W from rubusoside. The method comprises preparing a reaction mixture comprising rubusoside; a substrate selected from sucrose, Uridine Diphosphate (UDP) and uridine diphosphate-glucose (UDP-glucose); a uridine diphosphate glycosyltransferase selected from the group consisting of UGT76G1, HV1, and a UDP-glycosyltransferase-sucrose synthase fusion enzyme; with or without sucrose synthase; and incubating the reaction mixture for a sufficient time to produce a mixture of rebaudioside W.

A method for producing rebaudioside W from rubusoside. In another aspect, the disclosure relates to a method of synthesizing rebaudioside W from rubusoside. The method comprises preparing a reaction mixture comprising rubusoside; a substrate selected from sucrose, Uridine Diphosphate (UDP) and uridine diphosphate-glucose (UDP-glucose); a uridine diphosphate glycosyltransferase selected from the group consisting of UGT76G1, EUGT11, and a UDP-glycosyltransferase-sucrose synthase fusion enzyme; with or without sucrose synthase; and incubating the reaction mixture for a sufficient time to produce rebaudioside W.

A method for producing a mixture of stevioside and rebaudioside KA from rubusoside. In another aspect, the disclosure relates to a method of synthesizing a mixture of stevioside and rebaudioside KA from rubusoside. The method comprises preparing a reaction mixture comprising rubusoside; a substrate selected from sucrose, Uridine Diphosphate (UDP) and uridine diphosphate-glucose (UDP-glucose); a UDP-glycosyltransferase selected from the group consisting of EUGT11 and a UDP-glycosyltransferase-sucrose synthase fusion enzyme; with or without sucrose synthase; and incubating the reaction mixture for a sufficient time to produce a mixture of stevioside and rebaudioside KA, wherein a glucose is covalently coupled to the C2' -19-O-glucose of rubusoside to produce rebaudioside KA; wherein glucose is covalently coupled to C2' -13-O-glucose of rubusoside to produce stevioside.

A method for producing rebaudioside KA from rubusoside. In another aspect, the disclosure relates to a method for synthesizing rebaudioside KA from rubusoside. The method comprises preparing a reaction mixture comprising rubusoside; a substrate selected from sucrose, Uridine Diphosphate (UDP) and uridine diphosphate-glucose (UDP-glucose); and HV1 UDP-glycosyltransferase; with or without sucrose synthase; and incubating the reaction mixture for a sufficient time to produce rebaudioside KA, wherein a glucose is covalently coupled to the C2' -19-O-glucose of rubusoside to produce rebaudioside KA.

A method for producing rebaudioside G from rubusoside. In another aspect, the disclosure relates to a method for synthesizing rebaudioside G from rubusoside. The method comprises preparing a reaction mixture comprising rubusoside; a substrate selected from sucrose, Uridine Diphosphate (UDP) and uridine diphosphate-glucose (UDP-glucose); a UDP-glycosyltransferase selected from UGT76G1 and a UDP-glycosyltransferase-sucrose synthase fusion enzyme; with or without sucrose synthase; and incubating the reaction mixture for a sufficient time to produce rebaudioside G, wherein the glucose is covalently coupled to the C3' -13-O-glucose of rubusoside to produce rebaudioside G.

A method for producing rebaudioside E from rebaudioside KA. In another aspect, the disclosure relates to a method for synthesizing rebaudioside E from rebaudioside KA. The method includes preparing a reaction mixture comprising rebaudioside KA; a substrate selected from sucrose, Uridine Diphosphate (UDP) and uridine diphosphate-glucose (UDP-glucose); and HV1 UDP-glycosyltransferase; with or without sucrose synthase; and incubating the reaction mixture for a sufficient time to produce rebaudioside E, wherein a glucose is covalently coupled to the C2' 13-O-glucose of rebaudioside KA to produce rebaudioside E.

A method for producing rebaudioside E from rebaudioside KA. In another aspect, the disclosure relates to a method for synthesizing rebaudioside E from rebaudioside KA. The method includes preparing a reaction mixture comprising rebaudioside KA; a substrate selected from sucrose, Uridine Diphosphate (UDP) and uridine diphosphate-glucose (UDP-glucose); a UDP-glycosyltransferase selected from the group consisting of EUGT11 and a UDP-glycosyltransferase-sucrose synthase fusion enzyme; with or without sucrose synthase; and incubating the reaction mixture for a sufficient time to produce rebaudioside E, wherein a glucose is covalently coupled to the C2' 13-O-glucose of rebaudioside KA to produce rebaudioside E.

A method for producing rebaudioside E from rubusoside. In another aspect, the disclosure relates to a method of synthesizing rebaudioside E from rubusoside. The method comprises preparing a reaction mixture comprising rubusoside; a substrate selected from sucrose, Uridine Diphosphate (UDP) and uridine diphosphate-glucose (UDP-glucose); and a UDP-glycosyltransferase selected from the group consisting of EUGT11 and a UDP-glycosyltransferase-sucrose synthesis fusion enzyme; with or without sucrose synthase; and incubating the reaction mixture for a sufficient time to produce rebaudioside E, wherein a glucose is covalently coupled to rubusoside to produce a mixture of rebaudioside KA and stevioside. Continuously, glucose is covalently coupled to rebaudioside KA and stevioside to produce rebaudioside E.

A method for producing rebaudioside E from rubusoside. In another aspect, the disclosure relates to a method of synthesizing rebaudioside E from rubusoside. The method comprises preparing a reaction mixture comprising rubusoside; a substrate selected from sucrose, Uridine Diphosphate (UDP) and uridine diphosphate-glucose (UDP-glucose); and HV1 UDP-glycosyltransferase; with or without sucrose synthase; incubating the reaction mixture for a sufficient time to produce rebaudioside E, wherein a glucose is covalently coupled to rubusoside to produce rebaudioside KA; and further incubating rebaudioside KA with HV1 to produce rebaudioside E.

A method for producing rebaudioside D3 from rubusoside. In another aspect, the disclosure relates to a method for synthesizing rebaudioside D3 from rubusoside. The method comprises preparing a reaction mixture comprising rubusoside; a substrate selected from sucrose, Uridine Diphosphate (UDP) and uridine diphosphate-glucose (UDP-glucose); a UDP-glycosyltransferase selected from the group consisting of EUGT11 and a UDP-glycosyltransferase-sucrose synthesis fusion enzyme; with or without sucrose synthase; incubating the reaction mixture for a sufficient time to produce rebaudioside D3, wherein a glucose is covalently coupled to rubusoside to produce a mixture of stevioside and rebaudioside KA; further incubating the mixture of stevioside and rebaudioside KA with EUGT11 to produce rebaudioside E, wherein a glucose is covalently coupled to the stevioside and rebaudioside KA to produce rebaudioside E; and further incubating rebaudioside E with EUGT11 to produce rebaudioside D3, wherein a glucose is covalently coupled to rebaudioside E to produce rebaudioside D3.

Method for producing rebaudioside D3 from rebaudioside KA. In another aspect, the disclosure relates to a method for synthesizing rebaudioside D3 from rebaudioside KA. The method comprises preparing a reaction mixture comprising rebaudioside KA, a substrate selected from the group consisting of sucrose, Uridine Diphosphate (UDP) and uridine diphosphate-glucose (UDP-glucose), a UDP-glycosyltransferase selected from the group consisting of EUGT11 and a UDP-glycosyltransferase-sucrose synthase fusion enzyme, with or without sucrose synthase; incubating the reaction mixture for a sufficient time to produce rebaudioside D3, wherein a glucose is covalently coupled to rebaudioside KA to produce rebaudioside E; and further incubating the mixture of rebaudioside E and EUGT11 to produce rebaudioside D3, wherein a glucose is covalently coupled to rebaudioside E to produce rebaudioside D3.

Methods for producing rebaudioside Z from rebaudioside E. In another aspect, the disclosure relates to a method for synthesizing rebaudioside Z from rebaudioside E. The method includes preparing a reaction mixture comprising rebaudioside E; a substrate selected from sucrose, Uridine Diphosphate (UDP) and uridine diphosphate-glucose (UDP-glucose); and HV1 UDP-glycosyltransferase; and a sucrose synthase; incubating the reaction mixture for a sufficient time to produce rebaudioside Z, wherein a glucose is covalently coupled to rebaudioside E to produce rebaudioside Z, wherein a glucose is covalently coupled to the C2' -13-O-glucose of rebaudioside E to produce rebaudioside Z1. Glucose was covalently coupled to rebaudioside E C2' -19-O-glucose to produce rebaudioside Z2.

Methods for producing rebaudioside M from rebaudioside D. In another aspect, the present disclosure relates to a method for synthesizing rebaudioside M from rebaudioside D. The method comprises preparing a reaction mixture comprising rebaudioside D, a substrate selected from the group consisting of sucrose, Uridine Diphosphate (UDP), uridine diphosphate-glucose (UDP-glucose), and combinations thereof, and a UDP-glycosyltransferase selected from the group consisting of UGT76G1, UDP-glycosyltransferase-sucrose synthase fusion enzyme, and combinations thereof, with or without sucrose synthase; and incubating the reaction mixture for a sufficient time to produce rebaudioside M, wherein a glucose is covalently coupled to rebaudioside D to produce rebaudioside M.

Methods for producing rebaudioside D and rebaudioside M from stevioside. In another aspect, the disclosure relates to a method of synthesizing rebaudioside D and rebaudioside M from stevioside. The method comprises preparing a reaction mixture comprising stevioside, a substrate selected from the group consisting of sucrose, Uridine Diphosphate (UDP), uridine diphosphate-glucose (UDP-glucose), and combinations thereof, and a UDP-glycosyltransferase selected from the group consisting of HV1, UGT76G1, UDP-glycosyltransferase-sucrose synthase fusion enzyme, and combinations thereof, with or without sucrose synthase; and incubating the reaction mixture for a sufficient time to produce rebaudioside D and/or rebaudioside M. For example, in embodiments, the reaction mixture can be incubated for a sufficient time to produce rebaudioside D and the reaction mixture comprising rebaudioside D is further incubated (e.g., with UGT76G1 and/or a fusogenic enzyme) to produce rebaudioside M. In certain embodiments, the reaction mixture will comprise HV1 and UGT76G 1. In other embodiments, the reaction mixture will comprise HV1 and a fusion enzyme.

In certain embodiments, the glucose is covalently coupled to stevioside to produce rebaudioside a and/or rebaudioside E. For example, glucose may be covalently coupled to stevioside via UGT76G1 or a fusion enzyme to produce rebaudioside a and/or glucose may be covalently coupled to stevioside via HV1 to produce rebaudioside E. Continuously, glucose can be covalently coupled to rebaudioside a by HV1 to produce rebaudioside D and/or glucose can be covalently coupled to rebaudioside E by UGT76G1 or a fusion enzyme to produce rebaudioside D. Glucose can be further covalently coupled to rebaudioside D by UGT76G1 or a fusion enzyme to produce rebaudioside M.

Methods for producing rebaudioside D and rebaudioside M from rebaudioside a. In another aspect, the present disclosure relates to a method for synthesizing rebaudioside D and rebaudioside M from rebaudioside a. The method comprises preparing a reaction mixture comprising rebaudioside a, a substrate selected from the group consisting of sucrose, Uridine Diphosphate (UDP), uridine diphosphate-glucose (UDP-glucose), and combinations thereof, and a UDP-glycosyltransferase selected from the group consisting of HV1, UGT76G1, UDP-glycosyltransferase-sucrose synthase fusion enzyme, and combinations thereof, with or without sucrose synthase; and incubating the reaction mixture for a sufficient time to produce rebaudioside D and/or rebaudioside M. For example, in embodiments, a reaction mixture (e.g., comprising HV1) can be incubated for a sufficient time to produce rebaudioside D and the reaction mixture comprising rebaudioside D (e.g., with UGT76G1 and/or a fusogenic enzyme) can be further incubated to produce rebaudioside M. In certain embodiments, the reaction mixture will comprise HV1 and UGT76G 1. In other embodiments, the reaction mixture will comprise HV1 and a fusion enzyme.

Glucose is covalently coupled to rebaudioside a to produce rebaudioside D. For example, glucose can be covalently coupled to rebaudioside a by HV1 to produce rebaudioside D. Continuously, glucose can be covalently coupled to rebaudioside D by UGT76G1 or a fusion enzyme to produce rebaudioside M.

Methods for producing rebaudioside D and rebaudioside M from rebaudioside E. In another aspect, the present disclosure relates to a method for synthesizing rebaudioside D and rebaudioside M from rebaudioside E. The method comprises preparing a reaction mixture comprising rebaudioside E, a substrate selected from the group consisting of sucrose, Uridine Diphosphate (UDP), uridine diphosphate-glucose (UDP-glucose), and combinations thereof, and a UDP-glycosyltransferase selected from the group consisting of UGT76G1, UDP-glycosyltransferase-sucrose synthase fusion enzyme, and combinations thereof, with or without sucrose synthase; and incubating the reaction mixture for a sufficient time to produce rebaudioside D and/or rebaudioside M. For example, in embodiments, the reaction mixture (e.g., comprising UGT76G1 and/or a fusion enzyme) can be incubated for a sufficient time to produce rebaudioside D and the reaction mixture comprising rebaudioside D can be further incubated to produce rebaudioside M.

Glucose is covalently coupled to rebaudioside E to produce rebaudioside D. For example, glucose can be covalently coupled to rebaudioside E by UGT76G1 or a fusion enzyme to produce rebaudioside D. Continuously, glucose can be covalently coupled to rebaudioside D by UGT76G1 or a fusion enzyme to produce rebaudioside M.

Most steviol glycosides are formed by several glycosylation reactions of steviol, usually catalyzed by UDP-glycosyltransferase (UGT), using uridine 5' -diphosphoglucose (UDP-glucose) as the donor of the sugar moiety. In plants, UGTs are a very different class of enzymes that transfer glucose residues from UDP-glucose to steviol.

Uridine diphosphate glycosyltransferase (UGT76G1) is a UGT with 1, 3-13-O-glucosylation activity that produces related glycosides (rebaudioside a and D). Surprisingly and unexpectedly, it was found that UGT76G1 also has 1, 3-19-O-glucose glycosylation activity for producing rebaudioside G from rubusoside and rebaudioside M from rebaudioside D. UGT76G1 can convert rebaudioside KA to Reb V and proceed to form Reb W. A particularly suitable UGT76G1 has the amino acid sequence of SEQ ID NO. 1.

EUGT11 (described in WO 2013022989) is UGT having 1, 2-19-O-glucose and 1, 2-13-O-glucose glycosylation activities. EUGT11 is known to catalyze the production of rebaudioside E from stevioside and rebaudioside D from rebaudioside a. Surprisingly and unexpectedly, it was found that EUGT11 can be used in vitro to synthesize rebaudioside D3 from rebaudioside E by a novel enzymatic activity (β 1, 6-13-O-glucose glycosylation activity) (U.S. patent application serial No. 14/269,435 assigned to connegen, inc. EUGT11 has 1, 2-19-O-glucosylation activity for producing rebaudioside KA from rubusoside. A particularly suitable EUGT11 has the amino acid sequence of SEQ ID NO. 3.

HV1 is UGT with 1, 2-19-O-glucose glycosylation activity that produces the relevant steviol glycosides (rebaudiosides E, D and Z). Surprisingly and unexpectedly, HV1 was also found to have 1, 2-19-O-glucosylation activity for rebaudioside KA from rubusoside. HV1 can also convert Reb G to Reb V and Reb KA to Reb E. A particularly suitable HV1 has the amino acid sequence of SEQ ID NO 5.

The method may further comprise adding a sucrose synthase to the reaction mixture comprising a Uridine Diphosphate (UDP) glycosyltransferase. Sucrose synthase catalyzes a chemical reaction between NDP-glucose and D-fructose to produce NDP and sucrose. Sucrose synthase is a glycosyltransferase. The system name for this enzyme class is NDP-glucose D-fructose 2-alpha-D-glucosyltransferase. Other names commonly used include UDP glucose-fructose glucosyltransferase, sucrose synthase, sucrose-UDP glucosyltransferase, sucrose-uridine diphosphate glucosyltransferase, and uridine diphosphate glucose-fructose glucosyltransferase. The addition of sucrose synthase to the reaction mixture comprising uridine diphosphate glycosyltransferase produces a "UGT-SUS coupling system". In UGT-SUS coupled systems, UDP-glucose can be regenerated from UDP and sucrose, which allows for the omission of adding additional UDP-glucose to the reaction mixture or the use of UDP in the reaction mixture. Suitable sucrose synthases may be, for example, the Arabidopsis thaliana (Arabidopsis) sucrose synthase 1; an arabidopsis sucrose synthase 3; and mung bean (Vigna radiate) sucrose synthase. A particularly suitable sucrose synthase may be, for example, arabidopsis sucrose synthase 1. A particularly suitable Arabidopsis sucrose synthase 1 is Arabidopsis sucrose synthase 1(AtSUS1) having the amino acid sequence of SEQ ID NO: 7.

In another aspect, a uridine diphosphate glycosyltransferase fusion enzyme may be used in the method. A particularly suitable uridine diphosphate glycosyltransferase fusion enzyme may be UGT-SUS1 fusion enzyme. The UDP-glycosyltransferase can be a UDP-glycosyltransferase fusion enzyme comprising a uridine diphosphate glycosyltransferase domain coupled to a sucrose synthase domain. In particular, the UDP-glycosyltransferase fusion enzyme includes a uridine diphospho glycosyltransferase domain coupled to a sucrose synthase domain. In addition, UGT-SUS1 fusion enzyme has sucrose synthase activity, and therefore, UDP-glucose can be regenerated from UDP and sucrose. A particularly suitable UGT-SUS1 fusion enzyme can be, for example, UGT76G1-AtSUS1 fusion enzyme (designated: "GS") having the amino acid sequence of SEQ ID NO: 9. Another particularly suitable UGT-SUS1 fusion enzyme can be, for example, EUGT11-SUS1 fusion enzyme having the amino acid sequence of SEQ ID NO:11 (designated: "EUS").

Suitable sucrose synthase domains can be, for example, an arabidopsis sucrose synthase 1; an arabidopsis sucrose synthase 3; and mung bean sucrose synthase. A particularly suitable sucrose synthase domain can be, for example, arabidopsis sucrose synthase 1. A particularly suitable Arabidopsis sucrose synthase 1 is Arabidopsis sucrose synthase 1(AtSUS1) having the amino acid sequence of SEQ ID NO. 7.

The UGT76G1-AtSUS1 ("GS") fusion enzyme may have a polypeptide sequence that is at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, and even 100% identical to the amino acid sequence set forth in SEQ ID No. 9. Suitably, the amino acid sequence of the UGT-AtSUS1 fusion enzyme has at least 80% identity to SEQ ID NO. 9. More suitably, the amino acid sequence of the UGT-AtSUS1 fusion enzyme has at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, and even 100% amino acid sequence identity to the amino acid sequence set forth in SEQ ID No. 9.

An isolated nucleic acid having a nucleic acid sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, and even 100% sequence homology to the nucleic acid sequence set forth in SEQ ID No. 10 may include a nucleotide sequence encoding a polypeptide of UGT-AtSUS1 fusion enzyme. Suitably, the isolated nucleic acid comprises a nucleotide sequence encoding a polypeptide of a UDP-glycosyltransferase fusion enzyme having an amino acid sequence with at least 80% sequence identity to the amino acid sequence set forth in SEQ ID No. 9. More suitably, the isolated nucleic acid comprises a nucleotide sequence encoding a polypeptide of a UDP-glycosyltransferase fusion enzyme having an amino acid sequence with at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, and even 100% sequence identity to the amino acid sequence set forth in SEQ ID No. 9. Isolated nucleic acids thus include those nucleotide sequences that encode a functional fragment of SEQ ID NO. 10, a functional variant of SEQ ID NO. 9, or other homologous polypeptides having, for example, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, and even 100% sequence identity to SEQ ID NO. 9. As is known to those skilled in the art, a nucleic acid sequence encoding a UDP-glycosyltransferase can be codon-optimized for expression in a suitable host organism, such as bacteria and yeast.

The EUGT11-SUS1 ("EUS") fusion enzyme may have a polypeptide sequence that is at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% and even 100% identical to the amino acid sequence set forth in SEQ ID NO. 11. Suitably, the amino acid sequence of the EUGT11-SUS1 fusion enzyme has at least 80% identity to SEQ ID NO. 11. More suitably, the amino acid sequence of the EUGT11-SUS1 fusion enzyme has at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% and even 100% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO. 11.

Isolated nucleic acids having a nucleic acid sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% and even 100% sequence homology to the nucleic acid sequence set forth in SEQ ID NO. 12 may include nucleotide sequences encoding polypeptides of the EUGT11-SUS1 fusion enzyme. Suitably, the isolated nucleic acid comprises a nucleotide sequence encoding a polypeptide of EUGT11-SUS1 fusion enzyme having an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO. 11. More suitably, the isolated nucleic acid comprises a nucleotide sequence encoding a polypeptide of an EUGT11-SUS1 fusion enzyme having an amino acid sequence with at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% and even 100% sequence identity to the amino acid sequence set forth in SEQ ID No. 11. Isolated nucleic acids thus include those nucleotide sequences that encode a functional fragment of SEQ ID NO. 11, a functional variant of SEQ ID NO. 11, or other homologous polypeptides having, for example, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, and even 100% sequence identity to SEQ ID NO. 11. As will be appreciated by those skilled in the art, the nucleic acid sequence encoding EUGT11-SUS1 may be codon optimized for expression in a suitable host organism, such as bacteria and yeast.

Orally consumable product

In another aspect, the present disclosure relates to an orally consumable product having a sweetening amount of rebaudioside V selected from the group consisting of beverage products and consumable products. In another aspect, the present disclosure relates to an orally consumable product having a sweetening amount of rebaudioside W selected from the group consisting of beverage products and consumable products. In another aspect, the present disclosure relates to an orally consumable product selected from the group consisting of a beverage product and a consumable product having a sweetening amount of rebaudioside KA. In another aspect, the present disclosure relates to an orally consumable product having a sweetening amount of rebaudioside G selected from the group consisting of beverage products and consumable products. In another aspect, the present disclosure relates to an orally consumable product having a sweetening amount of rebaudioside M selected from the group consisting of beverage products and consumable products.

The orally consumable product can have a sweetness intensity equivalent to about 1% (w/v-%) to about 4% (w/v-%) sucrose solution.

The orally consumable product can have about 5ppm to about 100ppm rebaudioside V. The orally consumable product can have about 5ppm to about 100ppm rebaudioside W. The orally consumable product can have about 5ppm to about 100ppm rebaudioside KA. The orally consumable product can have about 5ppm to about 100ppm rebaudioside G. The orally consumable product can have about 5ppm to about 100ppm rebaudioside M.

Rebaudioside V may be the only sweetener in the orally consumable product. Rebaudioside W may be the only sweetener in the orally consumable product. Rebaudioside KA may be the only sweetener in an orally consumable product. Rebaudioside G may be the only sweetener in the orally consumable product. Rebaudioside M may be the only sweetener in the orally consumable product.

The orally consumable product can 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 D3, rebaudioside E, rebaudioside F, dulcoside a, rubusoside, 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), rubusoside, mogroside IV, siamenoside I, mogroside V, monatin, thaumatin, monellin, brazzein, L-alanine, glycine, lo han guo, hernandinin, phyllodulcin, trilobatin, and combinations thereof.

The orally consumable product may also have at least one additive. The additive can be, for example, a carbohydrate, a polyol, an amino acid or salt thereof, a polyamino acid or salt thereof, a sugar acid or salt thereof, 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 flavorant, a flavoring ingredient, an astringent compound, a protein hydrolysate, a surfactant, an emulsifier, a flavonoid, an alcohol, a polymer, and combinations thereof.

In one aspect, the present disclosure relates to a beverage product comprising a sweetening amount of rebaudioside V. In one aspect, the present disclosure relates to a beverage product comprising a sweetening amount of rebaudioside W. In one aspect, the disclosure relates to a beverage product comprising a sweetening amount of rebaudioside KA. In one aspect, the present disclosure is directed to a beverage product comprising a sweetening amount of rebaudioside G. In one aspect, the present disclosure relates to a beverage product comprising a sweetening amount of rebaudioside M.

The beverage products can be, for example, carbonated beverage products and non-carbonated beverage products. The beverage product can also be, for example, a soft drink, fountain drink, frozen drink; a ready-to-drink beverage; frozen ready-to-drink beverages, coffee, tea, dairy beverages, powdered soft drinks, liquid concentrates, flavored waters, fortified waters, fruit juices, fruit juice flavored drinks, sports drinks, and energy drinks.

In some embodiments, the beverage products of the present disclosure can include one or more beverage ingredients (e.g., acidulants, fruit and/or vegetable juices, pulp, 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.

In certain embodiments, 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 may vary to some extent depending on factors such as the product's intended market segment, its desired nutritional characteristics, flavor characteristics, and the like. For example, in certain embodiments, the addition of additional ingredients to the formulation of a particular beverage product may generally be an option. For example, additional (i.e., more and/or other) sweeteners may be added to any such formulation, flavorings, electrolytes, vitamins, fruit juices or other fruit products, tastants, masking agents and the like, flavor enhancers and/or carbonators may typically be added to alter the taste, mouthfeel, nutritional characteristics, and the like. In embodiments, the beverage product can be a cola beverage comprising water, about 5ppm to about 100ppm rebaudioside V, an acidulant and a flavoring. In an embodiment, the beverage product can be a cola beverage comprising water, about 5ppm to about 100ppm rebaudioside W, an acidulant and a flavoring. In embodiments, the beverage product can be a cola beverage comprising water, about 5ppm to about 100ppm rebaudioside M, an acidulant and a flavoring. Exemplary flavors may be, for example, cola flavors, citrus flavors, and spice flavors. In some embodiments, a carbonating agent in the form of carbon dioxide may be added for foaming. In other embodiments, preservatives may be added depending on other ingredients, manufacturing techniques, desired shelf life, and the like. In certain embodiments, caffeine may be added. In some embodiments, the beverage product may be a cola-flavored carbonated beverage, typically containing soda, sweetener, cola nut extract and/or other flavoring, caramel coloring, one or more acids, and optionally other ingredients.

A suitable amount of rebaudioside V, rebaudioside W, rebaudioside KA, rebaudioside M, or rebaudioside G present in the beverage product may be, for example, about 5ppm to about 100 ppm. In some embodiments, low concentrations of rebaudioside V, rebaudioside W, rebaudioside KA, rebaudioside M, or rebaudioside G, for example, are below 100ppm and have a sweetness comparable to sucrose solutions at concentrations between 10,000ppm and 30,000 ppm. The final concentration ranges from about 5ppm to about 100ppm, about 5ppm to about 95ppm, about 5ppm to about 90ppm, about 5ppm to about 85ppm, about 5ppm to about 80ppm, about 5ppm to about 75ppm, about 5ppm to about 70ppm, about 5ppm to about 65ppm, about 5ppm to about 60ppm, about 5ppm to about 55ppm, about 5ppm to about 50ppm, about 5ppm to about 45ppm, about 5ppm to about 40ppm, about 5ppm to about 35ppm, about 5ppm to about 30ppm, about 5ppm to about 25ppm, about 5ppm to about 20ppm, about 5ppm to about 15ppm, or about 5ppm to about 10 ppm. Alternatively, rebaudioside V or rebaudioside W can be present in the beverage products of the present disclosure in a final concentration in the following ranges: about 5ppm to about 100ppm, about 10ppm to about 100ppm, about 15ppm to about 100ppm, about 20ppm to about 100ppm, about 25ppm to about 100ppm, about 30ppm to about 100ppm, about 35ppm to about 100ppm, about 40ppm to about 100ppm, about 45ppm to about 100ppm, about 50ppm to about 100ppm, about 55ppm to about 100ppm, about 60ppm to about 100ppm, about 65ppm to about 100ppm, about 70ppm to about 100ppm, about 75ppm to about 100ppm, about 80ppm to about 100ppm, about 85ppm to about 100ppm, about 90ppm to about 100ppm, or about 95ppm to about 100 ppm.

In another aspect, the present disclosure relates to a consumable comprising a sweetening amount of rebaudioside V. In another aspect, the present disclosure is directed to a consumable comprising a sweetening amount of rebaudioside W. In another aspect, the disclosure relates to a consumable comprising a sweetening amount of rebaudioside KA. In another aspect, the present disclosure is directed to a consumable comprising a sweetening amount of rebaudioside G. In another aspect, the present disclosure relates to a consumable comprising a sweetening amount of rebaudioside M. The consumable may be, for example, a food product, a nutraceutical, a pharmaceutical, a dietary supplement, an oral hygiene composition, an edible gel composition, a cosmetic product, and a table top flavoring.

As used herein, "dietary supplement" refers to a compound intended to supplement the diet and provide nutrients (e.g., vitamins, minerals, fiber, fatty acids, amino acids, etc.), which may be absent or not consumed in large amounts in the diet. Any suitable dietary supplement known in the art may be used. Examples of suitable dietary supplements may be, for example, nutrients, vitamins, minerals, fiber, fatty acids, herbs, botanicals, amino acids, and metabolites.

As used herein, "nutraceutical" refers to a compound that includes any food or food portion that can provide a medicinal or health benefit, including the prevention and/or treatment of diseases or disorders (e.g., fatigue, insomnia, aging effects, amnesia, mood disorders, cardiovascular disease and high cholesterol levels in the blood, diabetes, osteoporosis, inflammation, autoimmune disorders, etc.). Any suitable nutritional formulation known in the art may be used. In some embodiments, the nutritional formulations may be used as supplements for food and beverages and as pharmaceutical formulations for enteral or parenteral application, which may be solid formulations such as capsules or tablets, or liquid formulations such as solutions or suspensions.

In some embodiments, the dietary supplements and nutritional formulations may further contain protective hydrocolloids (such as gums, proteins, modified starches), binders, film forming agents, encapsulating agents/materials, wall/shell materials, matrix compounds, coatings, emulsifiers, surfactants, solubilizing agents (oils, fats, waxes, lecithins, etc.), adsorbents, carriers, fillers, co-compounds, dispersing agents, wetting agents, processing aids (solvents), fluidizing agents, taste masking agents, weighting agents, gel forming agents, gelling agents, antioxidants, and antimicrobials.

As used herein, "gel" refers to a colloidal system in which a network of particles spans the volume of a liquid medium. While gels are primarily composed of liquids and therefore exhibit liquid-like densities, gels have structural consistency as solids due to the network of particles that span the liquid medium. For this reason, gels generally behave as solid, gel-like materials. Gels are useful in many applications. For example, gels can be used in foods, paints and adhesives. Edible gels are referred to as "edible gel compositions". The edible gel composition is typically consumed as a snack, as a dessert, as part of a staple food, or with a staple food. Examples of suitable edible gel compositions may be, for example, gel desserts, puddings, jams, jellies, pastes, sponge cakes, aspics, marshmallows, gummies, and the like. In some embodiments, the edible gel mixture is generally a powdered or granular solid to which a fluid may be added to form the edible gel composition. Examples of suitable fluids may be, for example, water, dairy fluids, dairy-like fluids, juices, alcohols, alcoholic beverages, and combinations thereof. Examples of suitable dairy fluids may be, for example, milk, fermented milk, cream, liquid whey, and mixtures thereof. Examples of suitable dairy-like fluids may be, for example, soy milk and non-dairy coffee creamer.

As used herein, the term "gelling component" refers to any material that can form a colloidal system in a liquid medium. Examples of suitable gelling ingredients may be, for example, gelatin, alginates, carageenan, gums, pectins, konjac, agar, food acids, rennet, starch derivatives, and combinations thereof. It is well known to those of ordinary skill in the art that the amount of gelling ingredient used in an edible gel mixture or edible gel composition can vary significantly depending on a number of factors, such as the particular gelling ingredient used, the particular liquid base used, and the desired gel properties.

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 may be prepared using other ingredients in addition to the gelling agent. Examples of other suitable ingredients may be, for example, an edible acid, a salt of an edible acid, a buffering system, a bulking agent, a chelating agent, a crosslinking agent, one or more flavorings, one or more colorants, and combinations thereof.

Any suitable pharmaceutical composition known in the art may be used. In certain embodiments, the pharmaceutical compositions of the present disclosure may contain about 5ppm to about 100ppm rebaudioside V and one or more pharmaceutically acceptable excipients. In certain embodiments, the pharmaceutical compositions of the present disclosure may contain about 5ppm to about 100ppm rebaudioside W and one or more pharmaceutically acceptable excipients. In certain embodiments, the pharmaceutical compositions of the present disclosure can contain about 5ppm to about 100ppm rebaudioside KA and one or more pharmaceutically acceptable excipients. In certain embodiments, the pharmaceutical compositions of the present disclosure may contain about 5ppm to about 100ppm rebaudioside G and one or more pharmaceutically acceptable excipients. In certain embodiments, the pharmaceutical compositions of the present disclosure may contain about 5ppm to about 100ppm rebaudioside M and one or more pharmaceutically acceptable excipients. In some embodiments, the pharmaceutical compositions of the present disclosure may be used to formulate medicaments containing one or more biologically active agents. Thus, in some embodiments, a pharmaceutical composition of the present disclosure may contain one or more active agents that exert a biological effect. Suitable active agents are well known in the art (e.g., physician's desk reference). Such compositions may be prepared according to procedures well known in the art, for example, as described in Remington's Pharmaceutical Sciences, Mack Publishing co., Easton, Pa., USA.

Rebaudioside V, rebaudioside W, rebaudioside KA, rebaudioside M, or rebaudioside G may be used with any suitable dental and oral hygiene composition known in the art. Examples of suitable dental and oral hygiene compositions may be, for example, toothpaste, tooth polishing agents (tooth polish), dental floss, mouthrinse, mouth rinse, dentifrice (dentrifices), mouth spray, mouth freshener, plaque rinse, dental pain killer, and the like.

Suitable amounts of rebaudioside V, rebaudioside W, rebaudioside KA, rebaudioside M, or rebaudioside G present in the consumable can be, for example, about 5 parts per million (ppm) to about 100 parts per million (ppm). In some embodiments, low concentrations of rebaudioside V, rebaudioside W, rebaudioside KA, rebaudioside M, or rebaudioside G, e.g., below 100ppm, have a sweetness comparable to sucrose solutions at concentrations between 10,000ppm to 30,000 ppm. The final concentration ranges from about 5ppm to about 100ppm, about 5ppm to about 95ppm, about 5ppm to about 90ppm, about 5ppm to about 85ppm, about 5ppm to about 80ppm, about 5ppm to about 75ppm, about 5ppm to about 70ppm, about 5ppm to about 65ppm, about 5ppm to about 60ppm, about 5ppm to about 55ppm, about 5ppm to about 50ppm, about 5ppm to about 45ppm, about 5ppm to about 40ppm, about 5ppm to about 35ppm, about 5ppm to about 30ppm, about 5ppm to about 25ppm, about 5ppm to about 20ppm, about 5ppm to about 15ppm, or about 5ppm to about 10 ppm. Alternatively, rebaudioside V or rebaudioside W may be present in the consumable product of the present disclosure in a final concentration in the following ranges: about 5ppm to about 100ppm, about 10ppm to about 100ppm, about 15ppm to about 100ppm, about 20ppm to about 100ppm, about 25ppm to about 100ppm, about 30ppm to about 100ppm, about 35ppm to about 100ppm, about 40ppm to about 100ppm, about 45ppm to about 100ppm, about 50ppm to about 100ppm, about 55ppm to about 100ppm, about 60ppm to about 100ppm, about 65ppm to about 100ppm, about 70ppm to about 100ppm, about 75ppm to about 100ppm, about 80ppm to about 100ppm, about 85ppm to about 100ppm, about 90ppm to about 100ppm, or about 95ppm to about 100 ppm.

In certain embodiments, about 5ppm to about 100ppm rebaudioside V, rebaudioside W, rebaudioside KA, rebaudioside M, or rebaudioside G is present in the food product composition. 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 human and animal consumption.

Examples of suitable food product compositions can be, for example, confectionery compositions such as candies, mints, fruit flavored hard candies, cocoa products, chocolates, and the like; condiments, such as ketchup, mustard, mayonnaise, and the like; a chewing gum; a cereal composition; baked goods such as bread, cake, pie, cookies, etc.; dairy products such as milk, cheese, cream, ice cream, sour cream, yoghurt, sherbet and the like; a tabletop sweetener composition; soup; stewing the vegetables; instant food; meat, such as ham, bacon, sausage, jerky, etc.; gelatin and gelatin-like products such as jams, jellies, preserves, and the like; fruits; vegetables; an egg product; sugar frost; syrups, including molasses; snacks; nut kernels and nut products; and animal feed.

The food product composition may also be herbs, spices and seasonings, natural and synthetic seasonings, and flavoring agents such as monosodium glutamate. In some embodiments, the food product composition can be, for example, a prepared packaged product, such as a dietary sweetener, a liquid sweetener, a granular flavoring mixture, a pet food, a livestock feed, tobacco, and a material for baking applications, such as a powdered baking mixture for preparing bread, cookies, cakes, pancakes, donuts, and the like. In other embodiments, the food product composition may also be diet or low calorie foods and beverages with little or no sucrose.

In certain embodiments that may be combined with any of the preceding embodiments, rebaudioside V, rebaudioside W, rebaudioside KA, rebaudioside M, or rebaudioside G may be the only sweetener, and the product has a sweetness intensity equivalent to about 1% to about 4% (W/V-%) sucrose solution. In certain embodiments that may be combined with any of the preceding embodiments, the consumable product and beverage product may further comprise additional sweeteners, wherein the product has a sweetness intensity equivalent to about 1% to about 10% (w/v-%) sucrose solution. In certain embodiments that may be combined with any of the preceding embodiments, each sweetening ingredient in the product may be a high intensity sweetener. In certain embodiments that may be combined with any of the preceding embodiments, each sweetening ingredient in the product may be a natural high intensity sweetener. In certain embodiments that may be combined with any of the preceding embodiments, the additional sweetener comprises one or more sweeteners selected from the group consisting of: stevia extract, steviol glycosides, stevioside, rebaudioside a, rebaudioside B, rebaudioside C, rebaudioside D3, rebaudioside F, dulcoside a, rubusoside, 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), rubusoside, mogroside IV, siamenoside I, mogroside V, monatin, thaumatin, monellin, brazzein, L-alanine, glycine, lo han guo, hernandinine, phyllodulcin, trilobatin, and combinations thereof. In certain embodiments that may be combined with any of the preceding embodiments, the consumable product and the beverage product may further comprise one or more additives selected from the group consisting of: carbohydrates, polyols, amino acids or salts thereof, polyamino acids or salts thereof, sugar acids or salts thereof, nucleotides, organic acids, inorganic acids, organic salts, organic acid salts, organic base salts, inorganic salts, bitter compounds, flavorants, flavoring ingredients, astringent compounds, proteins, protein hydrolysates, surfactants, emulsifiers, flavonoids, alcohols, polymers, and combinations thereof. In certain embodiments that may be combined with any of the preceding embodiments, rebaudioside D3 has a purity of about 50% to about 100% by weight prior to its addition to the product.

Sweetening agent

In another aspect, the present disclosure relates to a sweetener consisting of the following chemical structure:

in another aspect, the present disclosure relates to a sweetener consisting of the following chemical structure:

in another aspect, the present disclosure relates to a sweetener consisting of the following chemical structure:

in another aspect, the present disclosure relates to a sweetener consisting of the following chemical structure:

in another aspect, the present disclosure relates to a sweetener consisting of the following chemical structure:

in certain embodiments, the sweetener may further comprise at least one of a filler, a bulking agent, and an anti-caking agent. Suitable fillers, bulking agents and anti-caking agents are known in the art.

In certain embodiments, rebaudioside V, rebaudioside W, rebaudioside KA, rebaudioside M, or rebaudioside G sweeteners may be included and/or added in final concentrations sufficient to sweeten and/or enhance the sweetness of consumable products and beverage products. By "final concentration" of rebaudioside V, rebaudioside W, rebaudioside KA, rebaudioside M, or rebaudioside G is meant the concentration of rebaudioside V, rebaudioside W, rebaudioside KA, rebaudioside M, or rebaudioside G present in the final consumable product and beverage product (i.e., after all ingredients and/or compounds have been added to produce the consumable product and beverage product). Thus, in certain embodiments, rebaudioside V, rebaudioside W, rebaudioside KA, rebaudioside M, or rebaudioside G is included and/or added to compounds or ingredients used to prepare consumable products and beverage products. Rebaudioside V, rebaudioside W, rebaudioside KA, rebaudioside M, or rebaudioside G may be present in a single compound or ingredient, or in multiple compounds and ingredients. In other embodiments, rebaudioside V, rebaudioside W, rebaudioside KA, rebaudioside M, or rebaudioside G may be included and/or added to consumable products and beverage products. In certain preferred embodiments, rebaudioside V, rebaudioside W, rebaudioside KA, rebaudioside M, or rebaudioside G are included and/or added at final concentrations in the following ranges: about 5ppm to about 100ppm, about 5ppm to about 95ppm, about 5ppm to about 90ppm, about 5ppm to about 85ppm, about 5ppm to about 80ppm, about 5ppm to about 75ppm, about 5ppm to about 70ppm, about 5ppm to about 65ppm, about 5ppm to about 60ppm, about 5ppm to about 55ppm, about 5ppm to about 50ppm, about 5ppm to about 45ppm, about 5ppm to about 40ppm, about 5ppm to about 35ppm, about 5ppm to about 30ppm, about 5ppm to about 25ppm, about 5ppm to about 20ppm, about 5ppm to about 15ppm, or about 5ppm to about 10 ppm. Optionally, rebaudioside V or rebaudioside W is included and/or added at a final concentration in the following ranges: about 5ppm to about 100ppm, about 10ppm to about 100ppm, about 15ppm to about 100ppm, about 20ppm to about 100ppm, about 25ppm to about 100ppm, about 30ppm to about 100ppm, about 35ppm to about 100ppm, about 40ppm to about 100ppm, about 45ppm to about 100ppm, about 50ppm to about 100ppm, about 55ppm to about 100ppm, about 60ppm to about 100ppm, about 65ppm to about 100ppm, about 70ppm to about 100ppm, about 75ppm to about 100ppm, about 80ppm to about 100ppm, about 85ppm to about 100ppm, about 90ppm to about 100ppm, or about 95ppm to about 100 ppm. For example, rebaudioside V or rebaudioside W may be included and/or added at the following final concentrations: about 5ppm, about 10ppm, about 15ppm, about 20ppm, about 25ppm, about 30ppm, about 35ppm, about 40ppm, about 45ppm, about 50ppm, about 55ppm, about 60ppm, about 65ppm, about 70ppm, about 75ppm, about 80ppm, about 85ppm, about 90ppm, about 95ppm, or about 100ppm, including any range between these values.

In certain embodiments, rebaudioside V, rebaudioside W, rebaudioside KA, rebaudioside M, or rebaudioside G is the only sweetener included and/or added to the consumable products and beverage products. In such embodiments, the consumable products and beverage products have a sweetness intensity equivalent to about 1% to about 4% (w/v-%) sucrose solution, about 1% to about 3% (w/v-%) sucrose solution, or about 1% to about 2% (w/v-%) sucrose solution. Alternatively, consumable products and beverage products have a sweetness intensity equivalent to about 1% to about 4% (w/v-%) sucrose solution, about 2% to about 4% (w/v-%) sucrose solution, about 3% to about 4% (w/v-%) sucrose solution, or about 4% sucrose solution. For example, consumable products and beverage products can have sweetness intensities equivalent to about 1%, about 2%, about 3%, or about 4% (w/v-%) sucrose solutions, including any range between these values.

Consumable products and beverage products of the present disclosure can include a mixture of rebaudioside V, rebaudioside W, rebaudioside KA, rebaudioside M, or rebaudioside G and one or more sweeteners of the present disclosure in a ratio sufficient to achieve the desired sweetness intensity, nutritional characteristics, taste characteristics, mouthfeel, or other organoleptic factors.

The present disclosure will be more fully understood upon consideration of the following non-limiting examples.

Examples

Example 1

In this example, full-length DNA fragments of all candidate UGT genes were synthesized.

In particular, codon optimized cDNA was used for e.coli (e.coli) expression (Genscript, Piscataway, NJ). The synthesized DNA was cloned into the bacterial expression Vector pETite N-His SUMO Kan Vector (Lucigen). For the nucleotide sequences encoding UDP-glycosyltransferase fusion enzymes (UGT76G1-AtSUS1 and EUGT11-AtSUS1), a GSG-linker (encoded by the nucleotide sequence: ggttctggt) was inserted in-frame between the nucleotide sequence encoding the uridine diphosphate glycosyltransferase domain from Arabidopsis thaliana (AtSUS1) and the nucleotide sequence encoding sucrose synthase 1. Table 2 summarizes the protein and sequence identification numbers.

Table 2 sequence identification numbers.

Each expression construct was transformed into E.coli BL21(DE3), which was subsequently grown at 37 ℃ in LB medium containing 50. mu.g/mL kanamycin (kanamycin) until an OD of 0.8-1.0 was reached₆₀₀. Protein expression was induced by addition of 1mM isopropyl β -D-1-thiogalactopyranoside (IPTG) and the culture was further grown for 22 hours at 16 ℃. The cells were harvested by centrifugation (3,000x g; 10 min; 4 ℃). The cell pellet was collected and used immediately or stored at-80 ℃.

The cell pellet was resuspended in lysis buffer (50mM potassium phosphate buffer (pH 7.2), 25. mu.g/ml lysozyme, 5. mu.g/ml DNase I, 20mM imidazole, 500mM NaCl, 10% glycerol and 0.4% TRITON X-100). The cells were disrupted by sonication at 4 ℃ and the cell debris was clarified by centrifugation (18,000x g; 30 min). The supernatant was loaded onto an equilibrated (equilibration buffer: 50mM potassium phosphate buffer (pH 7.2), 20mM imidazole, 500mM 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 250mM imidazole. Purified HV1(61.4kD), UGT76G1(65.4kD), AtSUS1(106.3kD), EUGT11(62kD), UGT76G1-SUS1(GS) (157.25kD), and EUGT11-AtSUS1(155kD) fusion proteins are shown in FIG. 2.

Example 2

In this example, glycosyltransferase activity of a candidate UGT recombinant polypeptide is determined by using the test steviol glycoside as a substrate.

Typically, the recombinant polypeptide (10. mu.g) is tested in an in vitro reaction system at 200. mu.l. The reaction system contained 50mM potassium phosphate buffer (pH 7.2), 3mM MgCl₂1mg/ml steviol glycoside substrate, 1mM UDP-glucose. The reaction was carried out at 30 ℃ and stopped by the addition of 200. mu.L of 1-butanol. The sample was extracted three times with 200. mu.L of 1-butanol. The pooled fractions were dried and dissolved in 70 μ L of 80% methanol for High Performance Liquid Chromatography (HPLC) analysis. Rubusoside (99%, Blue California, CA), purified Reb G (98.8%), Reb KA (98.4%) and Reb V (80%) were used as substrates in vitro reactions.

The glycosylation reaction catalyzed by UGT is coupled with the UDP-glucose generation reaction catalyzed by sucrose synthase (such as AtSUS 1). In this method, UDP-glucose is generated from sucrose and UDP, so that the addition of additional UDP-glucose can be omitted. In the assay, recombinant AtSUS1 was added to the UGT reaction system and UDP-glucose was regenerated from UDP. The AtSUS1 sequence (Bieniowska et al, Plant J.2007,49:810-828) was synthesized and inserted into a bacterial expression vector. Recombinant AtSUS1 protein was expressed and purified by affinity chromatography.

HPLC analysis was performed using a Dionex UPLC ultmate 3000 system (Sunnyvale, CA) including a quaternary pump, a temperature-controlled column chamber, an autosampler, and a uv absorbance detector. Phenomenex LunaNH was used₂And the Luna C18 or Synergi Hydro-RP and the protective column are subjected to steviol glycoside characterization. In water or in Na₃PO₄Acetonitrile in buffer was used for isocratic elution in HPLC analysis. The detection wavelength was 210 nm.

Example 3

In this example, recombinant HV1 polypeptides were analyzed and the sugar moieties were transferred to rubusoside under all reaction conditions with or without AtSUS1 to produce rebaudioside KA ("Minor dimension glycosides from the leaves of Stevia rebaudiana". Journal of Natural Products (2014),77(5), 1231-1235).

As shown in fig. 3, recombinant HV1 polypeptide transferred the sugar moiety to rubusoside to produce Reb KA under all reaction conditions with or without AtSUS 1. Rubusoside is completely converted to Reb KA and Reb E by recombinant HV1 in UGT-SUS coupling reaction system (G, I). However, after 24 hours (H) only part of rubusoside was converted to Reb KA by recombinant HV1 polypeptide alone, which was not coupled to AtSUS1, indicating that AtSUS1 increased the conversion efficiency in the UGT-SUS coupling system. In the HV1-AtSUS1 coupling reaction system, the produced Reb KA can be continuously converted into Reb E.

Example 4

In this example, HV1 activity was analyzed using Reb E as a substrate.

Reb E substrate (0.5mg/ml) was incubated with recombinant HV1 polypeptide (20. mu.g) and AtSUS1 (20. mu.g) in a UGT-SUS coupling reaction system (200. mu.L) under conditions similar to those used in the above examples. As shown in fig. 4, Reb Z was generated from the combination of recombinant HV1 polypeptide and AtSUS 1. These results indicate that HV1 can transfer the glucose moiety to Reb E to form RZ. Fig. 4 shows that rebaudioside Z ("Reb Z") can be produced from rebaudioside E ("Reb E"), catalyzed by recombinant HV1 polypeptide and recombinant AtSUS1 in a HV1-AtSUS1 coupled reaction system. HV1 may transfer glucose to Reb E to produce Reb Z, a mixture of Reb Z1 and Reb Z2 at a ratio between 60:40 and 70:30 (U.S. provisional application No. 61/898,571 assigned to Conagen inc.

Example 5

In this example, to confirm the conversion of Reb KA to Reb E, purified Reb KA substrate was incubated with recombinant HV1, with or without AtSUS 1. As shown in fig. 5, Reb E was produced from recombinant HV1 polypeptide under two reaction conditions. However, AtSUS1 polypeptide in the UGT-SUS coupling reaction system can improve the reaction efficiency. All Reb KA substrates can be completely converted to Reb E in the UGT-SUS coupling system (D).

Example 6

In this example, EUGT11 activity was analyzed using rubusoside as a substrate.

As shown in fig. 6, EUGT11 can transfer the sugar moiety to rubusoside under all reaction conditions with or without AtSUS1 to produce Reb KA and stevioside. AtSUS1 improved the conversion efficiency in the UGT-SUS coupling system. Reb E can be continuously converted by EUGT11 in the HV1-AtSUS1 coupling reaction system. The EUS fusion protein showed higher activity under the same reaction conditions. All the Reb KA and stevioside produced at 48 hours were completely converted to Reb E by EUS. Reb E can be continuously converted to Reb D3.

Example 7

In this example, EUGT11 activity was analyzed using Reb KA as a substrate.

EUGT11 is UGT having 1, 2-19-O-glucose glycosylation activity that produces steviol glycosides (PCT published application WO2013/022989 assigned to Evolva SA). For example, EUGT11 may catalyze the reaction of stevioside to Reb E. EUGT11 also has 1, 6-13-O-glucosylation activity that can transfer glucose molecules to rebaudioside E to form rebaudioside D3 (U.S. patent application Ser. No. 14/269,435 assigned to Conagen, Inc.). In experiments, we found that EUGT11 can transfer glucose residues to Reb KA to form Reb E. As shown in fig. 7, EUGT11 can transfer the sugar moiety to Reb KA to produce Reb E under all reaction conditions with AtSUS1(E, H) or without AtSUS1(D, G). AtSUS1 improved the conversion efficiency in the UGT-SUS coupling system (E, H). In the EUGT11-AtSUS1 coupling reaction system (E, H) and EUS fusion reaction system (F, I), all Reb KA was completely converted and the produced Reb E could be continuously converted to Reb D3.

Example 8

In this example, UGT76G1 activity was analyzed using rubusoside as a substrate.

UGT76G1 has 1, 3-13-O-glucose glycosylation activity that can transfer a glucose molecule to stevioside to form rebaudioside a and to Reb E to form rebaudioside D. In this example, we found that UGT76G1 can transfer a glucose residue to rubusoside to form rebaudioside G.

As shown in fig. 8, UGT76G1 can transfer sugar moieties to rubusoside to produce Reb G under all reaction conditions with AtSUS1(D, G) or without AtSUS1(C, F). AtSUS1 improved the conversion efficiency in the UGT-SUS coupling system. The GS fusion protein showed higher activity under the same reaction conditions (E, H). At 12 hours all rubusoside was completely converted to Reb G (E).

Example 9

In this example, UGT76G1 activity was analyzed using rebaudioside KA as a substrate.

To further characterize the enzyme activity of UGT76G1, in vitro assays were performed using rebaudioside KA as a substrate. Surprisingly, a novel steviol glycoside (rebaudioside V "Reb V") was produced at an early time point. At a later point in time, Reb V produced in the reaction is converted to another novel steviol glycoside (rebaudioside W "Reb W").

As shown in fig. 9, UGT76G1 can transfer the sugar moiety to Reb KA to produce Reb V under all reaction conditions with AtSUS1(F, I) or without AtSUS1(E, H). AtSUS1 improved the conversion efficiency in the UGT-SUS coupling system (F, I). In the UGT76G1-AtSUS1 coupling reaction system (I) and the GS fusion reaction system (J), Reb V generated at 12 hours was completely converted into Reb W.

Example 10

In this example, UGT76G1 activity was analyzed using Reb V as a substrate.

Purified Reb V as a substrate was introduced into the reaction system. As shown in fig. 10C, Reb V was surprisingly completely converted to Reb W by UGT76G1 recombinant polypeptide in UGT-SUS1 coupled system at 6 hours.

Example 11

In this example, HV1 activity was analyzed using Reb G as a substrate.

As shown in fig. 11, recombinant HV1 polypeptide transferred the sugar moiety to rebaudioside G to produce Reb V under all reaction conditions with or without AtSUS 1. Reb G was completely converted to Reb V by recombinant HV1 in UGT-SUS coupling reaction system (E, G). However, after 24 hours (F) only part of Reb G was converted to Reb V by recombinant HV1 polypeptide alone, which was not coupled to AtSUS1, indicating that AtSUS1 improved the conversion efficiency in the UGT-SUS coupling system.

Example 12

In this example, the activity of EUGT11 was analyzed using Reb G as substrate.

As shown in fig. 12, the recombinant EUGT11 polypeptide transferred the sugar moiety to rebaudioside G to produce Reb V under all reaction conditions with AtSUS1(F, I) or without AtSUS1(E, H). More Reb G was converted to Reb V by recombinant EUGT11 in UGT-SUS coupling reaction system (F, I). However, only part of Reb G was converted to Reb V (E, H) by a separate recombinant EUGT11 polypeptide not coupled to AtSUS1, indicating that AtSUS1 increased the conversion efficiency in the UGT-SUS coupling system. EUS fusion proteins showed higher activity under the same reaction conditions (G, J). 24 hours (J) all Reb G in the reaction was completely converted to Reb V by EUS.

Example 13

In this example, the activity of HV1 in combination with UGT76G1 was analyzed using rubusoside as a substrate.

The rubusoside substrate was incubated with recombinant HV1 polypeptide, UGT76G1, and AtSUS1 in a UGT-SUS coupling reaction system under conditions similar to those used in the above examples. The product was analyzed by HPLC. As shown in fig. 13, Reb V and Reb W were generated from the combination of recombinant HV1 polypeptide, UGT76G1, and atasus 1. Thus, recombinant HV1 polypeptides exhibiting 1, 2-19-O-glucose and 1, 2-13-O-glucose glycosylation activity can be used in combination with other UGT enzymes (such as UGT76G1, which exhibits 1, 3-13-O-glucose and 1, 3-19-O-glucose glycosylation activity) for complex, multi-step biosynthesis of steviol glycosides. Reb V and Reb W were also produced from these UGT enzymes if the HV1 recombinant protein was combined with the GS fusion protein in the reaction system, indicating that UGT-SUS coupling reactions could be produced from the GS fusion protein.

Example 14

In this example, the activity of EUGT11 in combination with UGT76G1 was analyzed using rubusoside as a substrate.

The rubusoside substrate was incubated with recombinant EUGT11 polypeptide, UGT76G1 and AtSUS1 in a UGT-SUS coupling reaction system under conditions similar to those used in the above examples. The product was analyzed by HPLC. As shown in fig. 14, Reb W was produced from the combination of recombinant EUGT11 polypeptide, UGT76G1, and atasus 1. Thus, recombinant EUGT11 polypeptide exhibiting 1, 2-19-O-glucose and 1, 2-13-O-glucose glycosylation activity can be used in combination with other UGT enzymes (e.g., UGT76G1, which exhibits 1, 3-13-O-glucose and 1, 3-19-O-glucose glycosylation activity) for complex, multi-step biosynthesis of steviol glycosides. If the EUGT11 recombinant protein was combined with the GS fusion protein in the reaction system, Reb W was also produced by these UGT enzymes, indicating that UGT-SUS coupling reactions could be produced from the GS fusion protein.

Example 15

In this example, the activity of HV1 in combination with UGT76G1 was analyzed using Reb G as a substrate.

The Reb G substrate was incubated with recombinant HV1 polypeptide, UGT76G1, and AtSUS1 in a UGT-SUS coupled reaction system under conditions similar to those used in the above examples. The product was analyzed by HPLC. As shown in fig. 15, Reb V and Reb W were generated from the combination of recombinant HV1 polypeptide, UGT76G1, and atasus 1. After 12 hours, all rubusoside substrates were converted to Reb V, and after 36 hours, all Reb V produced was converted to Reb W. Thus, recombinant HV1 polypeptides exhibiting 1, 2-19-O-glucose and 1, 2-13-O-glucose glycosylation activity can be used in combination with other UGT enzymes (such as UGT76G1, which exhibits 1, 3-13-O-glucose and 1, 3-19-O-glucose glycosylation activity) for complex, multi-step biosynthesis of steviol glycosides. Reb V and Reb W were also produced from these UGT enzymes if the HV1 recombinant protein was combined with the GS fusion protein in the reaction system, indicating that UGT-SUS coupling reactions could be produced from the GS fusion protein.

Example 16

In this example, the activity of EUGT11 in combination with UGT76G1 was analyzed using Reb G as a substrate.

The Reb G substrate was incubated with recombinant EUGT11 polypeptide, UGT76G1, and AtSUS1 in a UGT-SUS coupling reaction system under conditions similar to those used in the above examples. The product was analyzed by HPLC. As shown in fig. 16, the product was produced from the combination of recombinant EUGT11 polypeptide, UGT76G1 and atasus 1, and Reb W. Thus, recombinant EUGT11 polypeptide exhibiting 1, 2-19-O-glucose and 1, 2-13-O-glucose glycosylation activity can be used in combination with other UGT enzymes (e.g., UGT76G1, which exhibits 1, 3-13-O-glucose and 1, 3-19-O-glucose glycosylation activity) for complex, multi-step biosynthesis of steviol glycosides. If the EUGT11 recombinant protein was combined with the GS fusion protein in the reaction system, Reb W was also produced by these UGT enzymes, indicating that UGT-SUS coupling reactions could be produced from the GS fusion protein.

Example 17

In this example, UGT76G1 and GS fusion enzyme activities were analyzed using Reb D as a substrate.

Reb D substrate was incubated with recombinant UGT76G1 under conditions similar to those used in the above examples. The product was analyzed by HPLC. As shown in fig. 22, Reb M was generated from UGT76G1 with AtSUS1 (fig. 22D and G) or without AtSUS1 (fig. 22C and F) in the reaction. Thus, recombinant UGT76G1 polypeptides exhibiting 1, 3-19-O-glucosylation activity are useful for the biosynthesis of rebaudioside M. In the UGT-SUS coupling reaction system (FIG. 22G), Reb D was completely converted into Reb M by recombinant UGT76G 1. However, after 6 hours (F) only part of Reb D was converted to Reb M by recombinant UGT76G1 polypeptide alone, which was not coupled to AtSUS1, indicating that AtSUS1 improved the conversion efficiency in the UGT-SUS coupling system. The GS fusion protein showed similar activity to the UGT76G1-AtSUS1 coupling reaction under the same reaction conditions (E, H). At 6 hours (H) all Reb D was completely converted to Reb M by GS, indicating that UGT-SUS coupling reactions could be generated from the GS fusion protein.

Example 18

In this example, UGT76G1 and GS fusion enzyme activities were analyzed using Reb E as a substrate.

Reb E substrate was incubated with recombinant UGT76G1 or GS fusion enzyme under conditions similar to those used in the above examples. The product was analyzed by HPLC. As shown in fig. 23, Reb D was produced from UGT76G1 and GS fusion enzyme (fig. 23F, I and L) with AtSUS1 (fig. 23E, H and K) or without AtSUS1 (fig. 22D, G and J) in the reaction. Furthermore, Reb M is formed from Reb D generated in the reaction. Thus, recombinant UGT76G1 polypeptides that exhibit 1, 3-13-O-glucose and 1, 3-19-O-glucose glycosylation activity are useful for the biosynthesis of rebaudioside D and rebaudioside M. Reb E was completely converted to Reb M by recombinant UGT76G1 in the UGT-SUS coupling reaction system after 24 hours (fig. 23K). However, (J) only Reb D was converted from Reb E by a separate recombinant UGT76G1 polypeptide not coupled to AtSUS1 after 24 hours, indicating that AtSUS1 improved the conversion efficiency in the UGT-SUS coupling system by continuous UDPG production. The GS fusion protein exhibited similar activity to the UGT76G1-AtSUS1 coupling reaction under the same reaction conditions (FIGS. 23F, I and L), indicating that the UGT-SUS coupling reaction could be generated from the GS fusion protein.

Example 19

In this example, the activity of HV1 in combination with UGT76G1 was analyzed using stevioside as a substrate.

The rubusoside substrate was incubated with recombinant HV1 polypeptide and UGT76G1 or GS fusion enzyme under conditions similar to those used in the above examples. The product was analyzed by HPLC. As shown in fig. 24, Reb a was produced in all reactions from the combination of recombinant HV1 polypeptide and UGT76G 1. In addition, Reb D and Reb M were detected in the reaction using a combination of recombinant HV1 polypeptide, UGT76G1 polypeptide, and atasus 1 (fig. 24E, H and K) or a combination of recombinant GS fusion enzyme and HV1 polypeptide (fig. 24F, I and L). Recombinant HV1 polypeptide exhibiting 1, 2-19-O-glucosylation activity, can be used in combination with other UGT enzymes (such as UGT76G1, which exhibits 1, 3-13-O-glucose and 1, 3-19-O-glucosylation activity) for complex, multi-step biosynthesis of rebaudioside D and rebaudioside M. The results also show that AtSUS1 improved the conversion efficiency in the UGT-SUS coupling system by continuous UDPG production (fig. 24E, H and K). The GS fusion protein exhibited similar activity to the UGT76G1-AtSUS1 coupling reaction under the same reaction conditions (FIGS. 24F, I and L), indicating that the UGT-SUS coupling reaction could be generated from the GS fusion protein.

Example 20

In this example, the activity of HV1 in combination with UGT76G1 was analyzed using Reb a as a substrate.

The RebA substrate is incubated with a recombinant HV1 polypeptide and UGT76G1 or a GS fusion enzyme under conditions similar to those used in the above examples. The product was analyzed by HPLC. As shown in fig. 25, Reb D was produced in all reactions from the combination of recombinant HV1 polypeptide and UGT76G 1. Furthermore, Reb M was detected in the reaction using a combination of recombinant HV1 polypeptide, UGT76G1 polypeptide and atasus 1 (fig. 25D, G and J) or a combination of recombinant GS fusion enzyme and HV1 polypeptide (fig. 25E, H and K). Recombinant HV1 polypeptide exhibiting 1, 2-19-O-glucosylation activity, can be used in combination with other UGT enzymes (such as UGT76G1, which exhibits 1, 3-19-O-glucosylation activity) for complex, multi-step biosynthesis of rebaudioside D and rebaudioside M. The results also show that AtSUS1 improved the conversion efficiency in the UGT-SUS coupling system by continuous UDPG production (fig. 25D, G and J). The GS fusion protein showed similar activity to the UGT76G1-AtSUS1 coupling reaction under the same reaction conditions (fig. 25E, H and K), indicating that the UGT-SUS coupling reaction could be generated from the GS fusion protein.

Example 21

In this example, the structure of Reb V was analyzed by NMR.

The material for Reb V characterization was generated by enzymatic conversion using Reb G and purified by HPLC. HRMS data was generated with an LTQ Orbitrap discovery HRMS instrument setting its resolution to 30 k. The data was scanned from m/z 150 to 1500 in positive ion electrospray mode. The pin voltage was set to 4 kV; other source conditions were sheath gas 25, assist gas 0, purge gas 5 (all gas flows in arbitrary units), capillary voltage 30V, capillary temperature 300 ℃ and tube lens voltage 75. The sample was diluted with 2:2:1 acetonitrile: methanol: water (same as the infusion eluent) and injected with 50 microliters. NMR spectra were obtained on a Bruker Avance DRX 500MHz or Varian INOVA600MHz instrument using standard pulse sequences. 1D (¹H and¹³C) and 2D (TOCSY, HMQC and HMBC) NMR spectra at C₅D₅And (4) performing in N.

Has been based on that it shows a correspondence to [ M + Na ] at M/z 989.4198]⁺Positive High Resolution (HR) Mass Spectrometry of adducted ions of (A) deduces the molecular formula of Reb V as C₄₄H₇₀O₂₃(ii) a The component is composed of¹³C NMR-spectrum data support. Of Reb V¹The H NMR spectroscopic data showed the presence of two methyl singlet states at δ 0.97 and 1.40, two olefinic protons in singlet states at δ 5.06 and 5.71 at the exocyclic double bond, nine sp3 methylene groups and two sp3 methine protons at δ 0.74-2.72, characteristic of the ent-kauran diterpenes isolated earlier from stevia. The basic skeleton of ent-kaurane diterpenes is supported by COSY and TOCSY studies showing key correlations: H-1/H-2; H-2/H-3; H-5/H-6; H-6/H-7; H-9/H-11; H-11/H-12. Of Reb V¹The H NMR spectrum also showed the presence of four anomeric protons resonating at δ 5.08, 5.38, 5.57 and 6.23; indicating the presence of four saccharide units in its structure. With 5% H₂SO₄Acid hydrolysis of Reb V provided D-glucose identified by direct comparison of TLC to authentic samples. Enzymatic hydrolysis of Reb V by reaction with a standard compound¹Comparison of H NMR and co-TLC identified the aglycone of steviol. The large coupling constants observed for the four anomeric protons of the glucose moiety at δ 5.08(d, J ═ 7.8Hz), 5.38(d, J ═ 8.1Hz), 5.57(d, J ═ 8.0Hz), and 6.23(d, J ═ 7.8Hz) indicate that their β -orientation is the same as reported for steviol glycosides. Reb V assignment based on TOCSY, HMQC, and HMBC data¹H and¹³c NMR values and are given in table 3.

TABLE 3 of Reb V and Reb G¹H and¹³c NMR spectroscopic data (chemical shifts and coupling constants)^a-c

^aAssignment based on TOCSY, HMQC and HMBC correlations;^bchemical shift values in δ (ppm);^ccoupling constants are in Hz.

Based on the NMR spectroscopic data from Reb V and the results of the hydrolysis experiments, it was concluded that there are four β -D-glucosyl units linked to the aglycone steviol in its structure. For Reb V and Reb G¹H and¹³a close comparison of the C NMR values indicated the presence of a steviol aglycone with a 3-O- β -D-glucopyranosyl unit in the form of an ether linkage at C-13 and another β -D-glucosyl unit in the form of an ester linkage at the C-19 position, leaving a distribution of a fourth β -D-glucosyl moiety (FIG. 17). At the 2-position of the sugar I of the beta-D-glucosyl moiety¹H and¹³the low field shift of the C chemical shift supports the presence of a β -D-glucosyl unit at this position. This structure is also supported by the critical TOCSY and HMBC correlations as shown in fig. 18. Based on NMR and mass spectral data and the results of hydrolysis studies, the structure of Reb V generated by the enzymatic conversion of Reb G was deduced to be 13- [ (3-O- β -D-glucopyranosyl) oxy]Ent-kauri-16-en-19-enoic acid- (2-O- β -D-glucopyranosyl) ester.

Acid hydrolysis of Reb V. To a solution of Reb V (5mg) in MeOH (10ml) was added 3ml of 5% H₂SO₄And the mixture was refluxed for 24 hours. The reaction mixture was then neutralized with saturated sodium carbonate and extracted with ethyl acetate (EtOAc) (2 × 25ml) to give an aqueous fraction containing sugars and an EtOAc fraction containing the aglycone moiety. The aqueous phase was concentrated and the TLC system EtOAc/n-butanol/water (2:7:1) and CH were used₂Cl₂Comparison of/MeOH/water (10:6:1) with standard sugars; the sugar was identified as D-glucose.

Enzymatic hydrolysis of Reb V. Reb V (1mg) was dissolved in 10ml of 0.1M sodium acetate buffer (pH 4.5) and crude pectinase from Aspergillus niger (50uL, Sigma-Aldrich, P2736) was added. The mixture was stirred at 50 ℃ for 96 hours. The product precipitated by hydrolysis of 1 during the reaction was purified by comparing its co-TLC and standard compounds¹HNMR spectroscopic data were identified as steviol. Based on extensive 1D and 2D NMR and high resolution mass spectral data and hydrolysis studies, the compound designated Reb V was confirmed to be 13- [ (3-O-beta-D-glucopyranosyl-beta)-D-glucopyranosyl) oxy]Ent-kauri-16-en-19-enoic acid- (2-O- β -D-glucopyranosyl) ester.

Example 22

In this example, the structure of Reb W was analyzed by NMR.

The material for Reb W characterization was generated by enzymatic conversion using Reb V and purified by HPLC. HRMS data was generated with an LTQ Orbitrap discovery HRMS instrument setting its resolution to 30 k. The data was scanned from m/z 150 to 1500 in positive ion electrospray mode. The pin voltage was set to 4 kV; other source conditions are sheath gas 25, assist gas 0, purge gas 5 (for all gas flows in arbitrary units), capillary voltage 30V, capillary temperature 300C and tube lens voltage 75. The sample was diluted with 2:2:1 acetonitrile: methanol: water (same as the infusion eluent) and injected with 50 microliters. NMR spectra were obtained on a Bruker Avance DRX 500MHz or Varian INOVA600MHz instrument using standard pulse sequences. 1D (¹H and¹³C) and 2D (TOCSY, HMQC and HMBC) NMR spectra at C₅D₅And (4) performing in N.

Has been based on that it shows a correspondence to [ M + Na ] at M/z 1151.4708]⁺Positive High Resolution (HR) Mass Spectrometry of adducted ions of (A) deduces the molecular formula of Reb W as C₅₀H₈₀O₂₈(ii) a The component is composed of¹³C NMR spectroscopic data. Of Reb W¹The H NMR spectroscopic data showed the presence of two methyl singlet states at δ 0.92 and 1.39, two olefinic protons in singlet states at δ 5.10 and 5.73 of the exocyclic double bond, nine sp3 methylene groups and two sp3 methine protons at δ 0.72-2.72, characteristic of the ent-kauran diterpenes isolated earlier from stevia. The basic skeleton of ent-kaurane diterpenes is supported by TOCSY studies showing key correlations: H-1/H-2; H-2/H-3; H-5/H-6; H-6/H-7; H-9/H-11; H-11/H-12. Of Reb W¹HNMR spectra also showed the presence of five anomeric protons resonating at δ 5.10, 5.34, 5.41, 5.81 and 6.14; indicating the presence of five sugar units in its structure. With 5% H₂SO₄Acid hydrolysis of Reb W provided D-glucose identified by direct TLC comparison to authentic samples. EnzymeHydrolysis of Reb W by reaction with a standard compound¹Comparison of HNMR and co-TLC identified the aglycone of steviol. The large coupling constants observed for the five anomeric protons of the glucose moiety at δ 5.10(d, J ═ 7.4Hz), 5.34(d, J ═ 7.9Hz), 5.41(d, J ═ 7.9Hz), 5.89(d, J ═ 7.9Hz), and 6.14(d, J ═ 7.9Hz) indicate the same β -orientation as reported for steviol glycosides [1-5,9-13 ═ 9 ═ 1-5]. Allocating Reb W based on TOCSY, HMQC and HMBC data¹H and¹³c NMR values are given in Table 4.

TABLE 4 of Reb W and Reb V¹H and¹³c NMR spectroscopic data (chemical shifts and coupling constants)^a-c

^aAssignment based on TOCSY, HMQC and HMBC correlations;^bchemical shift values in δ (ppm);^ccoupling constants are in Hz.

Based on the NMR spectroscopic data from Reb W and the results of the hydrolysis experiments, it was concluded that there are five β -D-glucosyl units linked to the aglycone steviol in its structure. For Reb W and Reb V¹H and¹³a close comparison of the C NMR values indicated the presence of steviol aglycone with a 3-O- β -D-glucopyranosyl unit in the form of an ether linkage at C-13 and a 2-O- β -D-glucopyranosyl unit in the form of an ester linkage at the C-19 position, leaving a fifth assignment of β -D-glucosyl moieties (FIG. 19). At the 3-position of the sugar I of the beta-D-glucosyl moiety¹H and¹³the low field shift of the C chemical shift supports the presence of a β -D-glucosyl unit at this position. This structure is also supported by the critical TOCSY and HMBC correlations as shown in fig. 20. Based on NMR and mass spectral data and the results of hydrolysis studies, the structure of Reb W produced by enzymatic conversion of Reb V was deduced to be 13- [ (3-O- β -D-glucopyranosyl- β -D-pyranGlucosyl) oxy]Ent-kauri-16-en-19-enoic acid- [ (2-O- β -D-glucopyranosyl-3-O- β -D-glucopyranosyl) ester.

Acid hydrolysis of Reb W. To a solution of Reb W (5mg) in MeOH (10ml) was added 3ml of 5% H₂SO₄And the mixture was refluxed for 24 hours. The reaction mixture was then neutralized with saturated sodium carbonate and extracted with ethyl acetate (EtOAc) (2 × 25ml) to give an aqueous fraction containing sugars and an EtOAc fraction containing the aglycone moiety. The aqueous phase was concentrated and the TLC system EtOAc/n-butanol/water (2:7:1) and CH were used₂Cl₂Comparison of/MeOH/water (10:6:1) with standard sugars; the sugar was identified as D-glucose.

Enzymatic hydrolysis of Reb W. Reb W (1mg) was dissolved in 10ml of 0.1M sodium acetate buffer (pH 4.5) and crude pectinase from Aspergillus niger (50uL, Sigma-Aldrich, P2736) was added. The mixture was stirred at 50 ℃ for 96 hours. The product precipitated during the reaction and was filtered and then crystallized. The resulting product obtained from Reb W hydrolysis was compared to the co-TLC and standard compounds¹HNMR spectroscopic data were identified as steviol. Based on extensive 1D and 2D NMR and high resolution mass spectral data and hydrolysis studies, the compound designated Reb W was confirmed to be 13- [ (3-O- β -D-glucopyranosyl) oxy]Ent-kauri-16-en-19-enoic acid- [ (2-O- β -D-glucopyranosyl-3-O- β -D-glucopyranosyl) ester.

Following NMR analysis, the structures of Reb V and Reb W were identified as novel steviol glycosides. The above results further demonstrate that UGT76G1 has not only 1, 3-13-O-glucose glycosylation activity, but also 1, 3-19-O-glucose glycosylation activity.

Example 23

In this example, the structure of Reb M was analyzed by NMR.

The material for Reb M characterization was generated by enzymatic conversion using Reb D and purified by HPLC. HRMS data was generated with an LTQ Orbitrap discovery HRMS instrument setting its resolution to 30 k. The data was scanned from m/z 150 to 1500 in positive ion electrospray mode. The pin voltage was set to 4 kV; other source conditions are sheath gas 25, assist gas 0, purge gas 5 (for all gas flows in arbitrary units), capillary voltage 30V, capillary temperature 300C and tube lens voltage 75. The sample was diluted with 2:2:1 acetonitrile: methanol: water (same as the infusion eluent) and injected with 50 microliters.

NMR spectra were obtained on a Bruker Avance DRX 500MHz or Varian INOVA600MHz instrument using standard pulse sequences. 1D (¹H and¹³C) and 2D (TOCSY, HMQC and HMBC) NMR spectra at C₅D₅And (4) performing in N.

Has been based on that it shows [ M + NH ] at M/z 1349.5964₄+CH₃CN]⁺Positive electric High Resolution (HR) Mass Spectrometry of ions the molecular formula of Reb M was deduced as C₅₆H₉₀O₃₃(ii) a The component is composed of¹³C NMR spectroscopic data. Of Reb M¹The H NMR spectrum showed the presence of two methyl singlet states at δ 1.35 and 1.42, two olefin protons in singlet states at δ 4.92 and 5.65 of the exocyclic double bond, nine methylene groups and two methine protons at δ 0.77-2.77, characteristic of the ent-kaurane diterpene isolated earlier from stevia. The basic skeleton of ent-kaurane diterpene is composed of COSY (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-10, C-6, C-7, C-9, C-10, C-18, C-19, C-20, C-9/C-8, C-10, C-11, C-12, C-10, C-3, C-1, C-4, C-9, C-9, C-, 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). Of Reb M¹HNMR spectra also showed the presence of anomeric protons resonating at δ 5.33, 5.47, 5.50, 5.52, 5.85 and 6.43; indicating the presence of six sugar units in its structure. Enzymatic hydrolysis of Reb M supplied the aglycone identified as steviol by comparison to co-TLC of standard compounds. With 5% H₂SO₄Acid hydrolysis of Reb M provided glucose identified by direct TLC comparison to authentic samples. Assignment of selected protons and carbons in Reb M based on TOCSY, HMQC and HMBC correlations¹H and¹³c NMR values (Table 5).

Based on the results of NMR spectroscopic data from Reb M, it was concluded that six glucosyl units were present in its structure (fig. 26). For Reb MWith rebaudioside D¹H and¹³a close comparison of the C NMR spectra shows that Reb M is also a steviol glycoside, having three glucose residues attached at the C-13 hydroxyl group as a 2, 3-branched glucopyranosyl substituent and a 2-substituted glucopyranosyl moiety in the form of an ester at C-19, leaving a partition of another glucosyl moiety. The key TOCSY and HMBC correlations shown in fig. 27 indicate that the sixth glucosyl moiety is placed at the C-3 position of sugar I. The large coupling constants observed at δ 5.33(d, J ═ 8.4Hz), 5.47(d, J ═ 7.8Hz), 5.50(d, J ═ 7.4Hz), 5.52(d, J ═ 7.4Hz), 5.85(d, J ═ 7.4Hz), and 6.43(d, J ═ 7.8Hz) for the six anomeric protons of the glucose moiety indicate that their β -orientation is the same as reported for steviol glycosides. Based on the results of NMR and mass spectrometry studies and in comparison with the spectral values of rebaudioside M reported from the literature, the structure of Reb M generated by the enzymatic reaction was assigned to 13- [ (2-O- β -D-glucopyranosyl-3-O- β -D-glucopyranosyl) oxy]Ent-kauri-16-en-19-enoic acid- [ (2-O- β -D-glucopyranosyl-3-O- β -D-glucopyranosyl) ester.

TABLE 5 production of Reb M by enzymatic reaction¹H and¹³c NMR spectroscopic data (chemical shifts and coupling constants)^a-c。

^aAssignment based on TOCSY, HSQC and HMBC correlations;^bchemical shift values in δ (ppm);^ccoupling constants are in Hz.

Acid hydrolysis of compound 1: to a solution of the formed Reb M (5mg) in MeOH (10ml)3ml of 5% H were added₂SO₄And the mixture was refluxed for 24 hours. The reaction mixture was then neutralized with saturated sodium carbonate and extracted with ethyl acetate (EtOAc) (2 × 25ml) to give an aqueous fraction containing sugars and an EtOAc fraction containing the aglycone moiety. The aqueous phase was concentrated and the TLC system EtOAc/n-butanol/water (2:7:1) and CH were used₂Cl₂Comparison of/MeOH/water (10:6:1) with standard sugars; the sugar was identified as D-glucose.

Enzymatic hydrolysis of the compound: the generated Reb M (1mg) was dissolved in 10ml of 0.1M sodium acetate buffer (pH 4.5) and crude pectinase from Aspergillus niger (50uL, Sigma-Aldrich, P2736) was added. The mixture was stirred at 50 ℃ for 96 hours. The product precipitated by hydrolysis of 1 during the reaction was purified by comparing its co-TLC and standard compounds¹HNMR spectroscopic data were identified as steviol.

The obtained compound designated rebaudioside m (reb m) was produced by biotransformation. For rebaudioside M (Reb M)¹H and¹³the C NMR spectral distribution was based on extensive 1D and 2D NMR and high resolution mass spectral data, which indicated the structure 13- [ (2-O- β -D-glucopyranosyl-3-O- β -D-glucopyranosyl) oxy]Ent-kauri-16-en-19-enoic acid- [ (2-O- β -D-glucopyranosyl-3-O- β -D-glucopyranosyl) ester.

Example 24

In this example, the biosynthetic pathway of steviol glycosides is discussed.

FIG. 21 is a diagram illustrating a novel pathway for the biosynthesis of steviol glycosides from rubusoside. As described herein, a recombinant HV1 polypeptide ("HV 1") contains 1, 2-O-glucosylation activity that transfers a second glucoside moiety to C-2' of rubusoside 19-O-glucose to produce rebaudioside KA ("Reb KA"); the recombinant EUGT11 polypeptide ("EUGT 11") contains a C-2' that transfers a second glucose moiety to rubusoside 19-O-glucose to produce rebaudioside KA; or transferring the second glucose moiety to C-2' of 13-O-glucose of rubusoside to generate 1, 2-O-glucose glycosylation activity of stevioside; the recombinant UGT76G1 enzyme ("UGT 76G 1") contains 1, 3-O-glucosylation activity that transfers a second glucose moiety to the C-3' of the 13-O-glucose of rubusoside to produce rebaudioside G ("Reb G"). Both HV1 and EUGT11 transferred the second sugar moiety to C-2 'of the 19-O-glucose of rebaudioside G to produce rebaudioside V ("Reb V"), or transferred the second glucose moiety to C-2' of the 13-O-glucose of rebaudioside KA to produce rebaudioside E ("Reb E"). Fig. 21 also shows that recombinant UGT76G1 catalyzes the reaction in which the third glucose moiety is transferred to C-3 'of the C-19-O-glucose of rebaudioside V to produce rebaudioside W ("Reb W") and EUGT11 can continuously transfer the third glucose moiety to C-6' of the C-13-O-glucose of rebaudioside E to produce rebaudioside D3. HV1 may transfer the third glucose moiety to C-2 'of the C-13-O-glucose of rebaudioside E to produce rebaudioside Z1 ("Reb Z1") and may transfer the third glucose moiety to C-2' of the C-19-O-glucose of rebaudioside E to produce rebaudioside Z2 ("Reb Z2"). Both HV1 and EUGT11 can catalyze the conversion of stevioside to Reb E and rebaudioside a ("Reb a") to rebaudioside D ("Reb D"). The UGT76G1 can transfer a third glucose moiety to C-3' of the C-13-O-glucose of rebaudioside E ("Reb E") to form rebaudioside D ("Reb D"). UGT76G1 also catalyzes the conversion of stevioside to rebaudioside ("RebA") and rebaudioside D ("Reb D") to rebaudioside M ("Reb M").

In view of the above, it will be seen that the several advantages of the present disclosure are achieved and other advantageous results attained. As various changes could be made in the above methods and systems without departing from the scope of the disclosure, it is intended that all matter contained in the above description and shown in the accompanying drawings shall be interpreted as illustrative and not in a limiting sense.

When introducing elements of the present disclosure or versions, embodiments, or aspects thereof, the articles "a," "an," "the," and "said" are intended to mean that there are one or more of the elements. The terms "comprising," "including," and "having" are intended to be inclusive and mean that there may be additional elements other than the listed elements.

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1 5 10 15

Val Val Ile Cys Pro Trp Leu Ala Phe Gly His Leu Leu Pro Cys Leu

20 25 30

Asp Leu Ala Gln Arg Leu Ala Ser Arg Gly His Arg Val Ser Phe Val

35 40 45

Ser Thr Pro Arg Asn Ile Ser Arg Leu Pro Pro Val Arg Pro Ala Leu

50 55 60

Ala Pro Leu Val Ala Phe Val Ala Leu Pro Leu Pro Arg Val Glu Gly

65 70 75 80

Leu Pro Asp Gly Ala Glu Ser Thr Asn Asp Val Pro His Asp Arg Pro

85 90 95

Asp Met Val Glu Leu His Arg Arg Ala Phe Asp Gly Leu Ala Ala Pro

100 105 110

Phe Ser Glu Phe Leu Gly Thr Ala Cys Ala Asp Trp Val Ile Val Asp

115 120 125

Val Phe His His Trp Ala Ala Ala Ala Ala Leu Glu His Lys Val Pro

130 135 140

Cys Ala Met Met Leu Leu Gly Ser Ala His Met Ile Ala Ser Ile Ala

145 150 155 160

Asp Arg Arg Leu Glu Arg Ala Glu Thr Glu Ser Pro Ala Ala Ala Gly

165 170 175

Gln Gly Arg Pro Ala Ala Ala Pro Thr Phe Glu Val Ala Arg Met Lys

180 185 190

Leu Ile Arg Thr Lys Gly Ser Ser Gly Met Ser Leu Ala Glu Arg Phe

195 200 205

Ser Leu Thr Leu Ser Arg Ser Ser Leu Val Val Gly Arg Ser Cys Val

210 215 220

Glu Phe Glu Pro Glu Thr Val Pro Leu Leu Ser Thr Leu Arg Gly Lys

225 230 235 240

Pro Ile Thr Phe Leu Gly Leu Met Pro Pro Leu His Glu Gly Arg Arg

245 250 255

Glu Asp Gly Glu Asp Ala Thr Val Arg Trp Leu Asp Ala Gln Pro Ala

260 265 270

Lys Ser Val Val Tyr Val Ala Leu Gly Ser Glu Val Pro Leu Gly Val

275 280 285

Glu Lys Val His Glu Leu Ala Leu Gly Leu Glu Leu Ala Gly Thr Arg

290 295 300

Phe Leu Trp Ala Leu Arg Lys Pro Thr Gly Val Ser Asp Ala Asp Leu

305 310 315 320

Leu Pro Ala Gly Phe Glu Glu Arg Thr Arg Gly Arg Gly Val Val Ala

325 330 335

Thr Arg Trp Val Pro Gln Met Ser Ile Leu Ala His Ala Ala Val Gly

340 345 350

Ala Phe Leu Thr His Cys Gly Trp Asn Ser Thr Ile Glu Gly Leu Met

355 360 365

Phe Gly His Pro Leu Ile Met Leu Pro Ile Phe Gly Asp Gln Gly Pro

370 375 380

Asn Ala Arg Leu Ile Glu Ala Lys Asn Ala Gly Leu Gln Val Ala Arg

385 390 395 400

Asn Asp Gly Asp Gly Ser Phe Asp Arg Glu Gly Val Ala Ala Ala Ile

405 410 415

Arg Ala Val Ala Val Glu Glu Glu Ser Ser Lys Val Phe Gln Ala Lys

420 425 430

Ala Lys Lys Leu Gln Glu Ile Val Ala Asp Met Ala Cys His Glu Arg

435 440 445

Tyr Ile Asp Gly Phe Ile Gln Gln Leu Arg Ser Tyr Lys Asp

450 455 460

<210> 4

<211> 1389

<212> DNA

<213> Artificial sequence

<220>

<223> synthetic

<400> 4

atggattcgg gttactcttc ctcctatgcg gcggctgcgg gtatgcacgt tgttatctgt 60

ccgtggctgg cttttggtca cctgctgccg tgcctggatc tggcacagcg tctggcttca 120

cgcggccatc gtgtcagctt cgtgtctacc ccgcgcaata tttcgcgtct gccgccggtt 180

cgtccggcac tggctccgct ggttgcattt gtcgctctgc cgctgccgcg cgtggaaggt 240

ctgccggatg gtgcggaaag taccaacgac gtgccgcatg atcgcccgga catggttgaa 300

ctgcaccgtc gtgcattcga tggtctggca gcaccgtttt ccgaatttct gggtacggcg 360

tgcgccgatt gggtgatcgt tgacgtcttt catcactggg cggcggcggc ggcgctggaa 420

cataaagttc cgtgtgcaat gatgctgctg ggctcagctc acatgattgc gtcgatcgca 480

gaccgtcgcc tggaacgtgc agaaaccgaa agtccggctg cggccggcca gggtcgcccg 540

gcagctgcgc cgaccttcga agtggcccgc atgaaactga ttcgtacgaa aggcagctct 600

ggtatgagcc tggcagaacg ctttagtctg accctgtccc gtagttccct ggtggttggt 660

cgcagttgcg ttgaatttga accggaaacc gtcccgctgc tgtccacgct gcgtggtaaa 720

ccgatcacct ttctgggtct gatgccgccg ctgcatgaag gccgtcgcga agatggtgaa 780

gacgcaacgg tgcgttggct ggatgcacag ccggctaaaa gcgtcgtgta tgtcgccctg 840

ggctctgaag tgccgctggg tgtggaaaaa gttcacgaac tggcactggg cctggaactg 900

gctggcaccc gcttcctgtg ggcactgcgt aaaccgacgg gtgtgagcga tgcggacctg 960

ctgccggccg gttttgaaga acgtacccgc ggccgtggtg ttgtcgcaac gcgttgggtc 1020

ccgcaaatga gcattctggc gcatgccgca gtgggcgcct ttctgaccca ctgtggttgg 1080

aacagcacga tcgaaggcct gatgtttggt cacccgctga ttatgctgcc gatcttcggc 1140

gatcagggtc cgaacgcacg tctgattgaa gcgaaaaatg ccggcctgca agttgcgcgc 1200

aacgatggcg acggttcttt cgaccgtgag ggtgtggctg cggccattcg cgcagtggct 1260

gttgaagaag aatcatcgaa agtttttcag gcgaaagcca aaaaactgca agaaatcgtc 1320

gcggatatgg cctgccacga acgctacatt gatggtttca ttcagcaact gcgctcctac 1380

aaagactaa 1389

<210> 5

<211> 459

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic

<400> 5

Met Asp Gly Asn Ser Ser Ser Ser Pro Leu His Val Val Ile Cys Pro

1 5 10 15

Trp Leu Ala Leu Gly His Leu Leu Pro Cys Leu Asp Ile Ala Glu Arg

20 25 30

Leu Ala Ser Arg Gly His Arg Val Ser Phe Val Ser Thr Pro Arg Asn

35 40 45

Ile Ala Arg Leu Pro Pro Leu Arg Pro Ala Val Ala Pro Leu Val Asp

50 55 60

Phe Val Ala Leu Pro Leu Pro His Val Asp Gly Leu Pro Glu Gly Ala

65 70 75 80

Glu Ser Thr Asn Asp Val Pro Tyr Asp Lys Phe Glu Leu His Arg Lys

85 90 95

Ala Phe Asp Gly Leu Ala Ala Pro Phe Ser Glu Phe Leu Arg Ala Ala

100 105 110

Cys Ala Glu Gly Ala Gly Ser Arg Pro Asp Trp Leu Ile Val Asp Thr

115 120 125

Phe His His Trp Ala Ala Ala Ala Ala Val Glu Asn Lys Val Pro Cys

130 135 140

Val Met Leu Leu Leu Gly Ala Ala Thr Val Ile Ala Gly Phe Ala Arg

145 150 155 160

Gly Val Ser Glu His Ala Ala Ala Ala Val Gly Lys Glu Arg Pro Ala

165 170 175

Ala Glu Ala Pro Ser Phe Glu Thr Glu Arg Arg Lys Leu Met Thr Thr

180 185 190

Gln Asn Ala Ser Gly Met Thr Val Ala Glu Arg Tyr Phe Leu Thr Leu

195 200 205

Met Arg Ser Asp Leu Val Ala Ile Arg Ser Cys Ala Glu Trp Glu Pro

210 215 220

Glu Ser Val Ala Ala Leu Thr Thr Leu Ala Gly Lys Pro Val Val Pro

225 230 235 240

Leu Gly Leu Leu Pro Pro Ser Pro Glu Gly Gly Arg Gly Val Ser Lys

245 250 255

Glu Asp Ala Ala Val Arg Trp Leu Asp Ala Gln Pro Ala Lys Ser Val

260 265 270

Val Tyr Val Ala Leu Gly Ser Glu Val Pro Leu Arg Ala Glu Gln Val

275 280 285

His Glu Leu Ala Leu Gly Leu Glu Leu Ser Gly Ala Arg Phe Leu Trp

290 295 300

Ala Leu Arg Lys Pro Thr Asp Ala Pro Asp Ala Ala Val Leu Pro Pro

305 310 315 320

Gly Phe Glu Glu Arg Thr Arg Gly Arg Gly Leu Val Val Thr Gly Trp

325 330 335

Val Pro Gln Ile Gly Val Leu Ala His Gly Ala Val Ala Ala Phe Leu

340 345 350

Thr His Cys Gly Trp Asn Ser Thr Ile Glu Gly Leu Leu Phe Gly His

355 360 365

Pro Leu Ile Met Leu Pro Ile Ser Ser Asp Gln Gly Pro Asn Ala Arg

370 375 380

Leu Met Glu Gly Arg Lys Val Gly Met Gln Val Pro Arg Asp Glu Ser

385 390 395 400

Asp Gly Ser Phe Arg Arg Glu Asp Val Ala Ala Thr Val Arg Ala Val

405 410 415

Ala Val Glu Glu Asp Gly Arg Arg Val Phe Thr Ala Asn Ala Lys Lys

420 425 430

Met Gln Glu Ile Val Ala Asp Gly Ala Cys His Glu Arg Cys Ile Asp

435 440 445

Gly Phe Ile Gln Gln Leu Arg Ser Tyr Lys Ala

450 455

<210> 6

<211> 1380

<212> DNA

<213> Artificial sequence

<220>

<223> synthetic

<400> 6

atggatggta actcctcctc ctcgccgctg catgtggtca tttgtccgtg gctggctctg 60

ggtcacctgc tgccgtgtct ggatattgct gaacgtctgg cgtcacgcgg ccatcgtgtc 120

agttttgtgt ccaccccgcg caacattgcc cgtctgccgc cgctgcgtcc ggctgttgca 180

ccgctggttg atttcgtcgc actgccgctg ccgcatgttg acggtctgcc ggagggtgcg 240

gaatcgacca atgatgtgcc gtatgacaaa tttgaactgc accgtaaggc gttcgatggt 300

ctggcggccc cgtttagcga atttctgcgt gcagcttgcg cagaaggtgc aggttctcgc 360

ccggattggc tgattgtgga cacctttcat cactgggcgg cggcggcggc ggtggaaaac 420

aaagtgccgt gtgttatgct gctgctgggt gcagcaacgg tgatcgctgg tttcgcgcgt 480

ggtgttagcg aacatgcggc ggcggcggtg ggtaaagaac gtccggctgc ggaagccccg 540

agttttgaaa ccgaacgtcg caagctgatg accacgcaga atgcctccgg catgaccgtg 600

gcagaacgct atttcctgac gctgatgcgt agcgatctgg ttgccatccg ctcttgcgca 660

gaatgggaac cggaaagcgt ggcagcactg accacgctgg caggtaaacc ggtggttccg 720

ctgggtctgc tgccgccgag tccggaaggc ggtcgtggcg tttccaaaga agatgctgcg 780

gtccgttggc tggacgcaca gccggcaaag tcagtcgtgt acgtcgcact gggttcggaa 840

gtgccgctgc gtgcggaaca agttcacgaa ctggcactgg gcctggaact gagcggtgct 900

cgctttctgt gggcgctgcg taaaccgacc gatgcaccgg acgccgcagt gctgccgccg 960

ggtttcgaag aacgtacccg cggccgtggt ctggttgtca cgggttgggt gccgcagatt 1020

ggcgttctgg ctcatggtgc ggtggctgcg tttctgaccc actgtggctg gaactctacg 1080

atcgaaggcc tgctgttcgg tcatccgctg attatgctgc cgatcagctc tgatcagggt 1140

ccgaatgcgc gcctgatgga aggccgtaaa gtcggtatgc aagtgccgcg tgatgaatca 1200

gacggctcgt ttcgtcgcga agatgttgcc gcaaccgtcc gcgccgtggc agttgaagaa 1260

gacggtcgtc gcgtcttcac ggctaacgcg aaaaagatgc aagaaattgt ggccgatggc 1320

gcatgccacg aacgttgtat tgacggtttt atccagcaac tgcgcagtta caaggcgtga 1380

<210> 7

<211> 808

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic

<400> 7

Met Ala Asn Ala Glu Arg Met Ile Thr Arg Val His Ser Gln Arg Glu

1 5 10 15

Arg Leu Asn Glu Thr Leu Val Ser Glu Arg Asn Glu Val Leu Ala Leu

20 25 30

Leu Ser Arg Val Glu Ala Lys Gly Lys Gly Ile Leu Gln Gln Asn Gln

35 40 45

Ile Ile Ala Glu Phe Glu Ala Leu Pro Glu Gln Thr Arg Lys Lys Leu

50 55 60

Glu Gly Gly Pro Phe Phe Asp Leu Leu Lys Ser Thr Gln Glu Ala Ile

65 70 75 80

Val Leu Pro Pro Trp Val Ala Leu Ala Val Arg Pro Arg Pro Gly Val

85 90 95

Trp Glu Tyr Leu Arg Val Asn Leu His Ala Leu Val Val Glu Glu Leu

100 105 110

Gln Pro Ala Glu Phe Leu His Phe Lys Glu Glu Leu Val Asp Gly Val

115 120 125

Lys Asn Gly Asn Phe Thr Leu Glu Leu Asp Phe Glu Pro Phe Asn Ala

130 135 140

Ser Ile Pro Arg Pro Thr Leu His Lys Tyr Ile Gly Asn Gly Val Asp

145 150 155 160

Phe Leu Asn Arg His Leu Ser Ala Lys Leu Phe His Asp Lys Glu Ser

165 170 175

Leu Leu Pro Leu Leu Lys Phe Leu Arg Leu His Ser His Gln Gly Lys

180 185 190

Asn Leu Met Leu Ser Glu Lys Ile Gln Asn Leu Asn Thr Leu Gln His

195 200 205

Thr Leu Arg Lys Ala Glu Glu Tyr Leu Ala Glu Leu Lys Ser Glu Thr

210 215 220

Leu Tyr Glu Glu Phe Glu Ala Lys Phe Glu Glu Ile Gly Leu Glu Arg

225 230 235 240

Gly Trp Gly Asp Asn Ala Glu Arg Val Leu Asp Met Ile Arg Leu Leu

245 250 255

Leu Asp Leu Leu Glu Ala Pro Asp Pro Cys Thr Leu Glu Thr Phe Leu

260 265 270

Gly Arg Val Pro Met Val Phe Asn Val Val Ile Leu Ser Pro His Gly

275 280 285

Tyr Phe Ala Gln Asp Asn Val Leu Gly Tyr Pro Asp Thr Gly Gly Gln

290 295 300

Val Val Tyr Ile Leu Asp Gln Val Arg Ala Leu Glu Ile Glu Met Leu

305 310 315 320

Gln Arg Ile Lys Gln Gln Gly Leu Asn Ile Lys Pro Arg Ile Leu Ile

325 330 335

Leu Thr Arg Leu Leu Pro Asp Ala Val Gly Thr Thr Cys Gly Glu Arg

340 345 350

Leu Glu Arg Val Tyr Asp Ser Glu Tyr Cys Asp Ile Leu Arg Val Pro

355 360 365

Phe Arg Thr Glu Lys Gly Ile Val Arg Lys Trp Ile Ser Arg Phe Glu

370 375 380

Val Trp Pro Tyr Leu Glu Thr Tyr Thr Glu Asp Ala Ala Val Glu Leu

385 390 395 400

Ser Lys Glu Leu Asn Gly Lys Pro Asp Leu Ile Ile Gly Asn Tyr Ser

405 410 415

Asp Gly Asn Leu Val Ala Ser Leu Leu Ala His Lys Leu Gly Val Thr

420 425 430

Gln Cys Thr Ile Ala His Ala Leu Glu Lys Thr Lys Tyr Pro Asp Ser

435 440 445

Asp Ile Tyr Trp Lys Lys Leu Asp Asp Lys Tyr His Phe Ser Cys Gln

450 455 460

Phe Thr Ala Asp Ile Phe Ala Met Asn His Thr Asp Phe Ile Ile Thr

465 470 475 480

Ser Thr Phe Gln Glu Ile Ala Gly Ser Lys Glu Thr Val Gly Gln Tyr

485 490 495

Glu Ser His Thr Ala Phe Thr Leu Pro Gly Leu Tyr Arg Val Val His

500 505 510

Gly Ile Asp Val Phe Asp Pro Lys Phe Asn Ile Val Ser Pro Gly Ala

515 520 525

Asp Met Ser Ile Tyr Phe Pro Tyr Thr Glu Glu Lys Arg Arg Leu Thr

530 535 540

Lys Phe His Ser Glu Ile Glu Glu Leu Leu Tyr Ser Asp Val Glu Asn

545 550 555 560

Lys Glu His Leu Cys Val Leu Lys Asp Lys Lys Lys Pro Ile Leu Phe

565 570 575

Thr Met Ala Arg Leu Asp Arg Val Lys Asn Leu Ser Gly Leu Val Glu

580 585 590

Trp Tyr Gly Lys Asn Thr Arg Leu Arg Glu Leu Ala Asn Leu Val Val

595 600 605

Val Gly Gly Asp Arg Arg Lys Glu Ser Lys Asp Asn Glu Glu Lys Ala

610 615 620

Glu Met Lys Lys Met Tyr Asp Leu Ile Glu Glu Tyr Lys Leu Asn Gly

625 630 635 640

Gln Phe Arg Trp Ile Ser Ser Gln Met Asp Arg Val Arg Asn Gly Glu

645 650 655

Leu Tyr Arg Tyr Ile Cys Asp Thr Lys Gly Ala Phe Val Gln Pro Ala

660 665 670

Leu Tyr Glu Ala Phe Gly Leu Thr Val Val Glu Ala Met Thr Cys Gly

675 680 685

Leu Pro Thr Phe Ala Thr Cys Lys Gly Gly Pro Ala Glu Ile Ile Val

690 695 700

His Gly Lys Ser Gly Phe His Ile Asp Pro Tyr His Gly Asp Gln Ala

705 710 715 720

Ala Asp Thr Leu Ala Asp Phe Phe Thr Lys Cys Lys Glu Asp Pro Ser

725 730 735

His Trp Asp Glu Ile Ser Lys Gly Gly Leu Gln Arg Ile Glu Glu Lys

740 745 750

Tyr Thr Trp Gln Ile Tyr Ser Gln Arg Leu Leu Thr Leu Thr Gly Val

755 760 765

Tyr Gly Phe Trp Lys His Val Ser Asn Leu Asp Arg Leu Glu Ala Arg

770 775 780

Arg Tyr Leu Glu Met Phe Tyr Ala Leu Lys Tyr Arg Pro Leu Ala Gln

785 790 795 800

Ala Val Pro Leu Ala Gln Asp Asp

805

<210> 8

<211> 2427

<212> DNA

<213> Artificial sequence

<220>

<223> synthetic

<400> 8

atggcaaacg ctgaacgtat gataacgcgc gtccacagcc aacgtgagcg tttgaacgaa 60

acgcttgttt ctgagagaaa cgaagtcctt gccttgcttt ccagggttga agccaaaggt 120

aaaggtattt tacaacaaaa ccagatcatt gctgaattcg aagctttgcc tgaacaaacc 180

cggaagaaac ttgaaggtgg tcctttcttt gaccttctca aatccactca ggaagcaatt 240

gtgttgccac catgggttgc tctagctgtg aggccaaggc ctggtgtttg ggaatactta 300

cgagtcaatc tccatgctct tgtcgttgaa gaactccaac ctgctgagtt tcttcatttc 360

aaggaagaac tcgttgatgg agttaagaat ggtaatttca ctcttgagct tgatttcgag 420

ccattcaatg cgtctatccc tcgtccaaca ctccacaaat acattggaaa tggtgttgac 480

ttccttaacc gtcatttatc ggctaagctc ttccatgaca aggagagttt gcttccattg 540

cttaagttcc ttcgtcttca cagccaccag ggcaagaacc tgatgttgag cgagaagatt 600

cagaacctca acactctgca acacaccttg aggaaagcag aagagtatct agcagagctt 660

aagtccgaaa cactgtatga agagtttgag gccaagtttg aggagattgg tcttgagagg 720

ggatggggag acaatgcaga gcgtgtcctt gacatgatac gtcttctttt ggaccttctt 780

gaggcgcctg atccttgcac tcttgagact tttcttggaa gagtaccaat ggtgttcaac 840

gttgtgatcc tctctccaca tggttacttt gctcaggaca atgttcttgg ttaccctgac 900

actggtggac aggttgttta cattcttgat caagttcgtg ctctggagat agagatgctt 960

caacgtatta agcaacaagg actcaacatt aaaccaagga ttctcattct aactcgactt 1020

ctacctgatg cggtaggaac tacatgcggt gaacgtctcg agagagttta tgattctgag 1080

tactgtgata ttcttcgtgt gcccttcaga acagagaagg gtattgttcg caaatggatc 1140

tcaaggttcg aagtctggcc atatctagag acttacaccg aggatgctgc ggttgagcta 1200

tcgaaagaat tgaatggcaa gcctgacctt atcattggta actacagtga tggaaatctt 1260

gttgcttctt tattggctca caaacttggt gtcactcagt gtaccattgc tcatgctctt 1320

gagaaaacaa agtacccgga ttctgatatc tactggaaga agcttgacga caagtaccat 1380

ttctcatgcc agttcactgc ggatattttc gcaatgaacc acactgattt catcatcact 1440

agtactttcc aagaaattgc tggaagcaaa gaaactgttg ggcagtatga aagccacaca 1500

gcctttactc ttcccggatt gtatcgagtt gttcacggga ttgatgtgtt tgatcccaag 1560

ttcaacattg tctctcctgg tgctgatatg agcatctact tcccttacac agaggagaag 1620

cgtagattga ctaagttcca ctctgagatc gaggagctcc tctacagcga tgttgagaac 1680

aaagagcact tatgtgtgct caaggacaag aagaagccga ttctcttcac aatggctagg 1740

cttgatcgtg tcaagaactt gtcaggtctt gttgagtggt acgggaagaa cacccgcttg 1800

cgtgagctag ctaacttggt tgttgttgga ggagacagga ggaaagagtc aaaggacaat 1860

gaagagaaag cagagatgaa gaaaatgtat gatctcattg aggaatacaa gctaaacggt 1920

cagttcaggt ggatctcctc tcagatggac cgggtaagga acggtgagct gtaccggtac 1980

atctgtgaca ccaagggtgc ttttgtccaa cctgcattat atgaagcctt tgggttaact 2040

gttgtggagg ctatgacttg tggtttaccg actttcgcca cttgcaaagg tggtccagct 2100

gagatcattg tgcacggtaa atcgggtttc cacattgacc cttaccatgg tgatcaggct 2160

gctgatactc ttgctgattt cttcaccaag tgtaaggagg atccatctca ctgggatgag 2220

atctcaaaag gagggcttca gaggattgag gagaaataca cttggcaaat ctattcacag 2280

aggctcttga cattgactgg tgtgtatgga ttctggaagc atgtctcgaa ccttgaccgt 2340

cttgaggctc gccgttacct tgaaatgttc tatgcattga agtatcgccc attggctcag 2400

gctgttcctc ttgcacaaga tgattga 2427

<210> 9

<211> 1268

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic

<400> 9

Met Glu Asn Lys Thr Glu Thr Thr Val Arg Arg Arg Arg Arg Ile Ile

1 5 10 15

Leu Phe Pro Val Pro Phe Gln Gly His Ile Asn Pro Ile Leu Gln Leu

20 25 30

Ala Asn Val Leu Tyr Ser Lys Gly Phe Ser Ile Thr Ile Phe His Thr

35 40 45

Asn Phe Asn Lys Pro Lys Thr Ser Asn Tyr Pro His Phe Thr Phe Arg

50 55 60

Phe Ile Leu Asp Asn Asp Pro Gln Asp Glu Arg Ile Ser Asn Leu Pro

65 70 75 80

Thr His Gly Pro Leu Ala Gly Met Arg Ile Pro Ile Ile Asn Glu His

85 90 95

Gly Ala Asp Glu Leu Arg Arg Glu Leu Glu Leu Leu Met Leu Ala Ser

100 105 110

Glu Glu Asp Glu Glu Val Ser Cys Leu Ile Thr Asp Ala Leu Trp Tyr

115 120 125

Phe Ala Gln Ser Val Ala Asp Ser Leu Asn Leu Arg Arg Leu Val Leu

130 135 140

Met Thr Ser Ser Leu Phe Asn Phe His Ala His Val Ser Leu Pro Gln

145 150 155 160

Phe Asp Glu Leu Gly Tyr Leu Asp Pro Asp Asp Lys Thr Arg Leu Glu

165 170 175

Glu Gln Ala Ser Gly Phe Pro Met Leu Lys Val Lys Asp Ile Lys Ser

180 185 190

Ala Tyr Ser Asn Trp Gln Ile Leu Lys Glu Ile Leu Gly Lys Met Ile

195 200 205

Lys Gln Thr Lys Ala Ser Ser Gly Val Ile Trp Asn Ser Phe Lys Glu

210 215 220

Leu Glu Glu Ser Glu Leu Glu Thr Val Ile Arg Glu Ile Pro Ala Pro

225 230 235 240

Ser Phe Leu Ile Pro Leu Pro Lys His Leu Thr Ala Ser Ser Ser Ser

245 250 255

Leu Leu Asp His Asp Arg Thr Val Phe Gln Trp Leu Asp Gln Gln Pro

260 265 270

Pro Ser Ser Val Leu Tyr Val Ser Phe Gly Ser Thr Ser Glu Val Asp

275 280 285

Glu Lys Asp Phe Leu Glu Ile Ala Arg Gly Leu Val Asp Ser Lys Gln

290 295 300

Ser Phe Leu Trp Val Val Arg Pro Gly Phe Val Lys Gly Ser Thr Trp

305 310 315 320

Val Glu Pro Leu Pro Asp Gly Phe Leu Gly Glu Arg Gly Arg Ile Val

325 330 335

Lys Trp Val Pro Gln Gln Glu Val Leu Ala His Gly Ala Ile Gly Ala

340 345 350

Phe Trp Thr His Ser Gly Trp Asn Ser Thr Leu Glu Ser Val Cys Glu

355 360 365

Gly Val Pro Met Ile Phe Ser Asp Phe Gly Leu Asp Gln Pro Leu Asn

370 375 380

Ala Arg Tyr Met Ser Asp Val Leu Lys Val Gly Val Tyr Leu Glu Asn

385 390 395 400

Gly Trp Glu Arg Gly Glu Ile Ala Asn Ala Ile Arg Arg Val Met Val

405 410 415

Asp Glu Glu Gly Glu Tyr Ile Arg Gln Asn Ala Arg Val Leu Lys Gln

420 425 430

Lys Ala Asp Val Ser Leu Met Lys Gly Gly Ser Ser Tyr Glu Ser Leu

435 440 445

Glu Ser Leu Val Ser Tyr Ile Ser Ser Leu Gly Ser Gly Ala Asn Ala

450 455 460

Glu Arg Met Ile Thr Arg Val His Ser Gln Arg Glu Arg Leu Asn Glu

465 470 475 480

Thr Leu Val Ser Glu Arg Asn Glu Val Leu Ala Leu Leu Ser Arg Val

485 490 495

Glu Ala Lys Gly Lys Gly Ile Leu Gln Gln Asn Gln Ile Ile Ala Glu

500 505 510

Phe Glu Ala Leu Pro Glu Gln Thr Arg Lys Lys Leu Glu Gly Gly Pro

515 520 525

Phe Phe Asp Leu Leu Lys Ser Thr Gln Glu Ala Ile Val Leu Pro Pro

530 535 540

Trp Val Ala Leu Ala Val Arg Pro Arg Pro Gly Val Trp Glu Tyr Leu

545 550 555 560

Arg Val Asn Leu His Ala Leu Val Val Glu Glu Leu Gln Pro Ala Glu

565 570 575

Phe Leu His Phe Lys Glu Glu Leu Val Asp Gly Val Lys Asn Gly Asn

580 585 590

Phe Thr Leu Glu Leu Asp Phe Glu Pro Phe Asn Ala Ser Ile Pro Arg

595 600 605

Pro Thr Leu His Lys Tyr Ile Gly Asn Gly Val Asp Phe Leu Asn Arg

610 615 620

His Leu Ser Ala Lys Leu Phe His Asp Lys Glu Ser Leu Leu Pro Leu

625 630 635 640

Leu Lys Phe Leu Arg Leu His Ser His Gln Gly Lys Asn Leu Met Leu

645 650 655

Ser Glu Lys Ile Gln Asn Leu Asn Thr Leu Gln His Thr Leu Arg Lys

660 665 670

Ala Glu Glu Tyr Leu Ala Glu Leu Lys Ser Glu Thr Leu Tyr Glu Glu

675 680 685

Phe Glu Ala Lys Phe Glu Glu Ile Gly Leu Glu Arg Gly Trp Gly Asp

690 695 700

Asn Ala Glu Arg Val Leu Asp Met Ile Arg Leu Leu Leu Asp Leu Leu

705 710 715 720

Glu Ala Pro Asp Pro Cys Thr Leu Glu Thr Phe Leu Gly Arg Val Pro

725 730 735

Met Val Phe Asn Val Val Ile Leu Ser Pro His Gly Tyr Phe Ala Gln

740 745 750

Asp Asn Val Leu Gly Tyr Pro Asp Thr Gly Gly Gln Val Val Tyr Ile

755 760 765

Leu Asp Gln Val Arg Ala Leu Glu Ile Glu Met Leu Gln Arg Ile Lys

770 775 780

Gln Gln Gly Leu Asn Ile Lys Pro Arg Ile Leu Ile Leu Thr Arg Leu

785 790 795 800

Leu Pro Asp Ala Val Gly Thr Thr Cys Gly Glu Arg Leu Glu Arg Val

805 810 815

Tyr Asp Ser Glu Tyr Cys Asp Ile Leu Arg Val Pro Phe Arg Thr Glu

820 825 830

Lys Gly Ile Val Arg Lys Trp Ile Ser Arg Phe Glu Val Trp Pro Tyr

835 840 845

Leu Glu Thr Tyr Thr Glu Asp Ala Ala Val Glu Leu Ser Lys Glu Leu

850 855 860

Asn Gly Lys Pro Asp Leu Ile Ile Gly Asn Tyr Ser Asp Gly Asn Leu

865 870 875 880

Val Ala Ser Leu Leu Ala His Lys Leu Gly Val Thr Gln Cys Thr Ile

885 890 895

Ala His Ala Leu Glu Lys Thr Lys Tyr Pro Asp Ser Asp Ile Tyr Trp

900 905 910

Lys Lys Leu Asp Asp Lys Tyr His Phe Ser Cys Gln Phe Thr Ala Asp

915 920 925

Ile Phe Ala Met Asn His Thr Asp Phe Ile Ile Thr Ser Thr Phe Gln

930 935 940

Glu Ile Ala Gly Ser Lys Glu Thr Val Gly Gln Tyr Glu Ser His Thr

945 950 955 960

Ala Phe Thr Leu Pro Gly Leu Tyr Arg Val Val His Gly Ile Asp Val

965 970 975

Phe Asp Pro Lys Phe Asn Ile Val Ser Pro Gly Ala Asp Met Ser Ile

980 985 990

Tyr Phe Pro Tyr Thr Glu Glu Lys Arg Arg Leu Thr Lys Phe His Ser

995 1000 1005

Glu Ile Glu Glu Leu Leu Tyr Ser Asp Val Glu Asn Lys Glu His

1010 1015 1020

Leu Cys Val Leu Lys Asp Lys Lys Lys Pro Ile Leu Phe Thr Met

1025 1030 1035

Ala Arg Leu Asp Arg Val Lys Asn Leu Ser Gly Leu Val Glu Trp

1040 1045 1050

Tyr Gly Lys Asn Thr Arg Leu Arg Glu Leu Ala Asn Leu Val Val

1055 1060 1065

Val Gly Gly Asp Arg Arg Lys Glu Ser Lys Asp Asn Glu Glu Lys

1070 1075 1080

Ala Glu Met Lys Lys Met Tyr Asp Leu Ile Glu Glu Tyr Lys Leu

1085 1090 1095

Asn Gly Gln Phe Arg Trp Ile Ser Ser Gln Met Asp Arg Val Arg

1100 1105 1110

Asn Gly Glu Leu Tyr Arg Tyr Ile Cys Asp Thr Lys Gly Ala Phe

1115 1120 1125

Val Gln Pro Ala Leu Tyr Glu Ala Phe Gly Leu Thr Val Val Glu

1130 1135 1140

Ala Met Thr Cys Gly Leu Pro Thr Phe Ala Thr Cys Lys Gly Gly

1145 1150 1155

Pro Ala Glu Ile Ile Val His Gly Lys Ser Gly Phe His Ile Asp

1160 1165 1170

Pro Tyr His Gly Asp Gln Ala Ala Asp Thr Leu Ala Asp Phe Phe

1175 1180 1185

Thr Lys Cys Lys Glu Asp Pro Ser His Trp Asp Glu Ile Ser Lys

1190 1195 1200

Gly Gly Leu Gln Arg Ile Glu Glu Lys Tyr Thr Trp Gln Ile Tyr

1205 1210 1215

Ser Gln Arg Leu Leu Thr Leu Thr Gly Val Tyr Gly Phe Trp Lys

1220 1225 1230

His Val Ser Asn Leu Asp Arg Leu Glu Ala Arg Arg Tyr Leu Glu

1235 1240 1245

Met Phe Tyr Ala Leu Lys Tyr Arg Pro Leu Ala Gln Ala Val Pro

1250 1255 1260

Leu Ala Gln Asp Asp

1265

<210> 10

<211> 3807

<212> DNA

<213> Artificial sequence

<220>

<223> synthetic

<400> 10

atggagaata agacagaaac aaccgtaaga cggaggcgga ggattatctt gttccctgta 60

ccatttcagg gccatattaa tccgatcctc caattagcaa acgtcctcta ctccaaggga 120

ttttcaataa caatcttcca tactaacttt aacaagccta aaacgagtaa ttatcctcac 180

tttacattca ggttcattct agacaacgac cctcaggatg agcgtatctc aaatttacct 240

acgcatggcc ccttggcagg tatgcgaata ccaataatca atgagcatgg agccgatgaa 300

ctccgtcgcg agttagagct tctcatgctc gcaagtgagg aagacgagga agtttcgtgc 360

ctaataactg atgcgctttg gtacttcgcc caatcagtcg cagactcact gaatctacgc 420

cgtttggtcc ttatgacaag ttcattattc aactttcacg cacatgtatc actgccgcaa 480

tttgacgagt tgggttacct ggacccggat gacaaaacgc gattggagga acaagcgtcg 540

ggcttcccca tgctgaaagt caaagatatt aagagcgctt atagtaattg gcaaattctg 600

aaagaaattc tcggaaaaat gataaagcaa accaaagcgt cctctggagt aatctggaac 660

tccttcaagg agttagagga atctgaactt gaaacggtca tcagagaaat ccccgctccc 720

tcgttcttaa ttccactacc caagcacctt actgcaagta gcagttccct cctagatcat 780

gaccgaaccg tgtttcagtg gctggatcag caacccccgt cgtcagttct atatgtaagc 840

tttgggagta cttcggaagt ggatgaaaag gacttcttag agattgcgcg agggctcgtg 900

gatagcaaac agagcttcct gtgggtagtg agaccgggat tcgttaaggg ctcgacgtgg 960

gtcgagccgt tgccagatgg ttttctaggg gagagaggga gaatcgtgaa atgggttcca 1020

cagcaagagg ttttggctca cggagctata ggggcctttt ggacccactc tggttggaat 1080

tctactcttg aaagtgtctg tgaaggcgtt ccaatgatat tttctgattt tgggcttgac 1140

cagcctctaa acgctcgcta tatgtctgat gtgttgaagg ttggcgtgta cctggagaat 1200

ggttgggaaa ggggggaaat tgccaacgcc atacgccggg taatggtgga cgaggaaggt 1260

gagtacatac gtcagaacgc tcgggtttta aaacaaaaag cggacgtcag ccttatgaag 1320

ggaggtagct cctatgaatc cctagaatcc ttggtaagct atatatcttc gttaggttct 1380

ggtgcaaacg ctgaacgtat gataacgcgc gtccacagcc aacgtgagcg tttgaacgaa 1440

acgcttgttt ctgagagaaa cgaagtcctt gccttgcttt ccagggttga agccaaaggt 1500

aaaggtattt tacaacaaaa ccagatcatt gctgaattcg aagctttgcc tgaacaaacc 1560

cggaagaaac ttgaaggtgg tcctttcttt gaccttctca aatccactca ggaagcaatt 1620

gtgttgccac catgggttgc tctagctgtg aggccaaggc ctggtgtttg ggaatactta 1680

cgagtcaatc tccatgctct tgtcgttgaa gaactccaac ctgctgagtt tcttcatttc 1740

aaggaagaac tcgttgatgg agttaagaat ggtaatttca ctcttgagct tgatttcgag 1800

ccattcaatg cgtctatccc tcgtccaaca ctccacaaat acattggaaa tggtgttgac 1860

ttccttaacc gtcatttatc ggctaagctc ttccatgaca aggagagttt gcttccattg 1920

cttaagttcc ttcgtcttca cagccaccag ggcaagaacc tgatgttgag cgagaagatt 1980

cagaacctca acactctgca acacaccttg aggaaagcag aagagtatct agcagagctt 2040

aagtccgaaa cactgtatga agagtttgag gccaagtttg aggagattgg tcttgagagg 2100

ggatggggag acaatgcaga gcgtgtcctt gacatgatac gtcttctttt ggaccttctt 2160

gaggcgcctg atccttgcac tcttgagact tttcttggaa gagtaccaat ggtgttcaac 2220

gttgtgatcc tctctccaca tggttacttt gctcaggaca atgttcttgg ttaccctgac 2280

actggtggac aggttgttta cattcttgat caagttcgtg ctctggagat agagatgctt 2340

caacgtatta agcaacaagg actcaacatt aaaccaagga ttctcattct aactcgactt 2400

ctacctgatg cggtaggaac tacatgcggt gaacgtctcg agagagttta tgattctgag 2460

tactgtgata ttcttcgtgt gcccttcaga acagagaagg gtattgttcg caaatggatc 2520

tcaaggttcg aagtctggcc atatctagag acttacaccg aggatgctgc ggttgagcta 2580

tcgaaagaat tgaatggcaa gcctgacctt atcattggta actacagtga tggaaatctt 2640

gttgcttctt tattggctca caaacttggt gtcactcagt gtaccattgc tcatgctctt 2700

gagaaaacaa agtacccgga ttctgatatc tactggaaga agcttgacga caagtaccat 2760

ttctcatgcc agttcactgc ggatattttc gcaatgaacc acactgattt catcatcact 2820

agtactttcc aagaaattgc tggaagcaaa gaaactgttg ggcagtatga aagccacaca 2880

gcctttactc ttcccggatt gtatcgagtt gttcacggga ttgatgtgtt tgatcccaag 2940

ttcaacattg tctctcctgg tgctgatatg agcatctact tcccttacac agaggagaag 3000

cgtagattga ctaagttcca ctctgagatc gaggagctcc tctacagcga tgttgagaac 3060

aaagagcact tatgtgtgct caaggacaag aagaagccga ttctcttcac aatggctagg 3120

cttgatcgtg tcaagaactt gtcaggtctt gttgagtggt acgggaagaa cacccgcttg 3180

cgtgagctag ctaacttggt tgttgttgga ggagacagga ggaaagagtc aaaggacaat 3240

gaagagaaag cagagatgaa gaaaatgtat gatctcattg aggaatacaa gctaaacggt 3300

cagttcaggt ggatctcctc tcagatggac cgggtaagga acggtgagct gtaccggtac 3360

atctgtgaca ccaagggtgc ttttgtccaa cctgcattat atgaagcctt tgggttaact 3420

gttgtggagg ctatgacttg tggtttaccg actttcgcca cttgcaaagg tggtccagct 3480

gagatcattg tgcacggtaa atcgggtttc cacattgacc cttaccatgg tgatcaggct 3540

gctgatactc ttgctgattt cttcaccaag tgtaaggagg atccatctca ctgggatgag 3600

atctcaaaag gagggcttca gaggattgag gagaaataca cttggcaaat ctattcacag 3660

aggctcttga cattgactgg tgtgtatgga ttctggaagc atgtctcgaa ccttgaccgt 3720

cttgaggctc gccgttacct tgaaatgttc tatgcattga agtatcgccc attggctcag 3780

gctgttcctc ttgcacaaga tgattga 3807

<210> 11

<211> 1272

<212> PRT

<213> Artificial sequence

<220>

<223> synthetic

<400> 11

Met Asp Ser Gly Tyr Ser Ser Ser Tyr Ala Ala Ala Ala Gly Met His

1 5 10 15

Val Val Ile Cys Pro Trp Leu Ala Phe Gly His Leu Leu Pro Cys Leu

20 25 30

Asp Leu Ala Gln Arg Leu Ala Ser Arg Gly His Arg Val Ser Phe Val

35 40 45

Ser Thr Pro Arg Asn Ile Ser Arg Leu Pro Pro Val Arg Pro Ala Leu

50 55 60

Ala Pro Leu Val Ala Phe Val Ala Leu Pro Leu Pro Arg Val Glu Gly

65 70 75 80

Leu Pro Asp Gly Ala Glu Ser Thr Asn Asp Val Pro His Asp Arg Pro

85 90 95

Asp Met Val Glu Leu His Arg Arg Ala Phe Asp Gly Leu Ala Ala Pro

100 105 110

Phe Ser Glu Phe Leu Gly Thr Ala Cys Ala Asp Trp Val Ile Val Asp

115 120 125

Val Phe His His Trp Ala Ala Ala Ala Ala Leu Glu His Lys Val Pro

130 135 140

Cys Ala Met Met Leu Leu Gly Ser Ala His Met Ile Ala Ser Ile Ala

145 150 155 160

Asp Arg Arg Leu Glu Arg Ala Glu Thr Glu Ser Pro Ala Ala Ala Gly

165 170 175

Gln Gly Arg Pro Ala Ala Ala Pro Thr Phe Glu Val Ala Arg Met Lys

180 185 190

Leu Ile Arg Thr Lys Gly Ser Ser Gly Met Ser Leu Ala Glu Arg Phe

195 200 205

Ser Leu Thr Leu Ser Arg Ser Ser Leu Val Val Gly Arg Ser Cys Val

210 215 220

Glu Phe Glu Pro Glu Thr Val Pro Leu Leu Ser Thr Leu Arg Gly Lys

225 230 235 240

Pro Ile Thr Phe Leu Gly Leu Met Pro Pro Leu His Glu Gly Arg Arg

245 250 255

Glu Asp Gly Glu Asp Ala Thr Val Arg Trp Leu Asp Ala Gln Pro Ala

260 265 270

Lys Ser Val Val Tyr Val Ala Leu Gly Ser Glu Val Pro Leu Gly Val

275 280 285

Glu Lys Val His Glu Leu Ala Leu Gly Leu Glu Leu Ala Gly Thr Arg

290 295 300

Phe Leu Trp Ala Leu Arg Lys Pro Thr Gly Val Ser Asp Ala Asp Leu

305 310 315 320

Leu Pro Ala Gly Phe Glu Glu Arg Thr Arg Gly Arg Gly Val Val Ala

325 330 335

Thr Arg Trp Val Pro Gln Met Ser Ile Leu Ala His Ala Ala Val Gly

340 345 350

Ala Phe Leu Thr His Cys Gly Trp Asn Ser Thr Ile Glu Gly Leu Met

355 360 365

Phe Gly His Pro Leu Ile Met Leu Pro Ile Phe Gly Asp Gln Gly Pro

370 375 380

Asn Ala Arg Leu Ile Glu Ala Lys Asn Ala Gly Leu Gln Val Ala Arg

385 390 395 400

Asn Asp Gly Asp Gly Ser Phe Asp Arg Glu Gly Val Ala Ala Ala Ile

405 410 415

Arg Ala Val Ala Val Glu Glu Glu Ser Ser Lys Val Phe Gln Ala Lys

420 425 430

Ala Lys Lys Leu Gln Glu Ile Val Ala Asp Met Ala Cys His Glu Arg

435 440 445

Tyr Ile Asp Gly Phe Ile Gln Gln Leu Arg Ser Tyr Lys Asp Gly Ser

450 455 460

Gly Ala Asn Ala Glu Arg Met Ile Thr Arg Val His Ser Gln Arg Glu

465 470 475 480

Arg Leu Asn Glu Thr Leu Val Ser Glu Arg Asn Glu Val Leu Ala Leu

485 490 495

Leu Ser Arg Val Glu Ala Lys Gly Lys Gly Ile Leu Gln Gln Asn Gln

500 505 510

Ile Ile Ala Glu Phe Glu Ala Leu Pro Glu Gln Thr Arg Lys Lys Leu

515 520 525

Glu Gly Gly Pro Phe Phe Asp Leu Leu Lys Ser Thr Gln Glu Ala Ile

530 535 540

Val Leu Pro Pro Trp Val Ala Leu Ala Val Arg Pro Arg Pro Gly Val

545 550 555 560

Trp Glu Tyr Leu Arg Val Asn Leu His Ala Leu Val Val Glu Glu Leu

565 570 575

Gln Pro Ala Glu Phe Leu His Phe Lys Glu Glu Leu Val Asp Gly Val

580 585 590

Lys Asn Gly Asn Phe Thr Leu Glu Leu Asp Phe Glu Pro Phe Asn Ala

595 600 605

Ser Ile Pro Arg Pro Thr Leu His Lys Tyr Ile Gly Asn Gly Val Asp

610 615 620

Phe Leu Asn Arg His Leu Ser Ala Lys Leu Phe His Asp Lys Glu Ser

625 630 635 640

Leu Leu Pro Leu Leu Lys Phe Leu Arg Leu His Ser His Gln Gly Lys

645 650 655

Asn Leu Met Leu Ser Glu Lys Ile Gln Asn Leu Asn Thr Leu Gln His

660 665 670

Thr Leu Arg Lys Ala Glu Glu Tyr Leu Ala Glu Leu Lys Ser Glu Thr

675 680 685

Leu Tyr Glu Glu Phe Glu Ala Lys Phe Glu Glu Ile Gly Leu Glu Arg

690 695 700

Gly Trp Gly Asp Asn Ala Glu Arg Val Leu Asp Met Ile Arg Leu Leu

705 710 715 720

Leu Asp Leu Leu Glu Ala Pro Asp Pro Cys Thr Leu Glu Thr Phe Leu

725 730 735

Gly Arg Val Pro Met Val Phe Asn Val Val Ile Leu Ser Pro His Gly

740 745 750

Tyr Phe Ala Gln Asp Asn Val Leu Gly Tyr Pro Asp Thr Gly Gly Gln

755 760 765

Val Val Tyr Ile Leu Asp Gln Val Arg Ala Leu Glu Ile Glu Met Leu

770 775 780

Gln Arg Ile Lys Gln Gln Gly Leu Asn Ile Lys Pro Arg Ile Leu Ile

785 790 795 800

Leu Thr Arg Leu Leu Pro Asp Ala Val Gly Thr Thr Cys Gly Glu Arg

805 810 815

Leu Glu Arg Val Tyr Asp Ser Glu Tyr Cys Asp Ile Leu Arg Val Pro

820 825 830

Phe Arg Thr Glu Lys Gly Ile Val Arg Lys Trp Ile Ser Arg Phe Glu

835 840 845

Val Trp Pro Tyr Leu Glu Thr Tyr Thr Glu Asp Ala Ala Val Glu Leu

850 855 860

Ser Lys Glu Leu Asn Gly Lys Pro Asp Leu Ile Ile Gly Asn Tyr Ser

865 870 875 880

Asp Gly Asn Leu Val Ala Ser Leu Leu Ala His Lys Leu Gly Val Thr

885 890 895

Gln Cys Thr Ile Ala His Ala Leu Glu Lys Thr Lys Tyr Pro Asp Ser

900 905 910

Asp Ile Tyr Trp Lys Lys Leu Asp Asp Lys Tyr His Phe Ser Cys Gln

915 920 925

Phe Thr Ala Asp Ile Phe Ala Met Asn His Thr Asp Phe Ile Ile Thr

930 935 940

Ser Thr Phe Gln Glu Ile Ala Gly Ser Lys Glu Thr Val Gly Gln Tyr

945 950 955 960

Glu Ser His Thr Ala Phe Thr Leu Pro Gly Leu Tyr Arg Val Val His

965 970 975

Gly Ile Asp Val Phe Asp Pro Lys Phe Asn Ile Val Ser Pro Gly Ala

980 985 990

Asp Met Ser Ile Tyr Phe Pro Tyr Thr Glu Glu Lys Arg Arg Leu Thr

995 1000 1005

Lys Phe His Ser Glu Ile Glu Glu Leu Leu Tyr Ser Asp Val Glu

1010 1015 1020

Asn Lys Glu His Leu Cys Val Leu Lys Asp Lys Lys Lys Pro Ile

1025 1030 1035

Leu Phe Thr Met Ala Arg Leu Asp Arg Val Lys Asn Leu Ser Gly

1040 1045 1050

Leu Val Glu Trp Tyr Gly Lys Asn Thr Arg Leu Arg Glu Leu Ala

1055 1060 1065

Asn Leu Val Val Val Gly Gly Asp Arg Arg Lys Glu Ser Lys Asp

1070 1075 1080

Asn Glu Glu Lys Ala Glu Met Lys Lys Met Tyr Asp Leu Ile Glu

1085 1090 1095

Glu Tyr Lys Leu Asn Gly Gln Phe Arg Trp Ile Ser Ser Gln Met

1100 1105 1110

Asp Arg Val Arg Asn Gly Glu Leu Tyr Arg Tyr Ile Cys Asp Thr

1115 1120 1125

Lys Gly Ala Phe Val Gln Pro Ala Leu Tyr Glu Ala Phe Gly Leu

1130 1135 1140

Thr Val Val Glu Ala Met Thr Cys Gly Leu Pro Thr Phe Ala Thr

1145 1150 1155

Cys Lys Gly Gly Pro Ala Glu Ile Ile Val His Gly Lys Ser Gly

1160 1165 1170

Phe His Ile Asp Pro Tyr His Gly Asp Gln Ala Ala Asp Thr Leu

1175 1180 1185

Ala Asp Phe Phe Thr Lys Cys Lys Glu Asp Pro Ser His Trp Asp

1190 1195 1200

Glu Ile Ser Lys Gly Gly Leu Gln Arg Ile Glu Glu Lys Tyr Thr

1205 1210 1215

Trp Gln Ile Tyr Ser Gln Arg Leu Leu Thr Leu Thr Gly Val Tyr

1220 1225 1230

Gly Phe Trp Lys His Val Ser Asn Leu Asp Arg Leu Glu Ala Arg

1235 1240 1245

Arg Tyr Leu Glu Met Phe Tyr Ala Leu Lys Tyr Arg Pro Leu Ala

1250 1255 1260

Gln Ala Val Pro Leu Ala Gln Asp Asp

1265 1270

<210> 12

<211> 3819

<212> DNA

<213> Artificial sequence

<220>

<223> synthetic

<400> 12

atggattcgg gttactcttc ctcctatgcg gcggctgcgg gtatgcacgt tgttatctgt 60

ccgtggctgg cttttggtca cctgctgccg tgcctggatc tggcacagcg tctggcttca 120

cgcggccatc gtgtcagctt cgtgtctacc ccgcgcaata tttcgcgtct gccgccggtt 180

cgtccggcac tggctccgct ggttgcattt gtcgctctgc cgctgccgcg cgtggaaggt 240

ctgccggatg gtgcggaaag taccaacgac gtgccgcatg atcgcccgga catggttgaa 300

ctgcaccgtc gtgcattcga tggtctggca gcaccgtttt ccgaatttct gggtacggcg 360

tgcgccgatt gggtgatcgt tgacgtcttt catcactggg cggcggcggc ggcgctggaa 420

cataaagttc cgtgtgcaat gatgctgctg ggctcagctc acatgattgc gtcgatcgca 480

gaccgtcgcc tggaacgtgc agaaaccgaa agtccggctg cggccggcca gggtcgcccg 540

gcagctgcgc cgaccttcga agtggcccgc atgaaactga ttcgtacgaa aggcagctct 600

ggtatgagcc tggcagaacg ctttagtctg accctgtccc gtagttccct ggtggttggt 660

cgcagttgcg ttgaatttga accggaaacc gtcccgctgc tgtccacgct gcgtggtaaa 720

ccgatcacct ttctgggtct gatgccgccg ctgcatgaag gccgtcgcga agatggtgaa 780

gacgcaacgg tgcgttggct ggatgcacag ccggctaaaa gcgtcgtgta tgtcgccctg 840

ggctctgaag tgccgctggg tgtggaaaaa gttcacgaac tggcactggg cctggaactg 900

gctggcaccc gcttcctgtg ggcactgcgt aaaccgacgg gtgtgagcga tgcggacctg 960

ctgccggccg gttttgaaga acgtacccgc ggccgtggtg ttgtcgcaac gcgttgggtc 1020

ccgcaaatga gcattctggc gcatgccgca gtgggcgcct ttctgaccca ctgtggttgg 1080

aacagcacga tcgaaggcct gatgtttggt cacccgctga ttatgctgcc gatcttcggc 1140

gatcagggtc cgaacgcacg tctgattgaa gcgaaaaatg ccggcctgca agttgcgcgc 1200

aacgatggcg acggttcttt cgaccgtgag ggtgtggctg cggccattcg cgcagtggct 1260

gttgaagaag aatcatcgaa agtttttcag gcgaaagcca aaaaactgca agaaatcgtc 1320

gcggatatgg cctgccacga acgctacatt gatggtttca ttcagcaact gcgctcctac 1380

aaagacggtt ctggtgcaaa cgctgaacgt atgataacgc gcgtccacag ccaacgtgag 1440

cgtttgaacg aaacgcttgt ttctgagaga aacgaagtcc ttgccttgct ttccagggtt 1500

gaagccaaag gtaaaggtat tttacaacaa aaccagatca ttgctgaatt cgaagctttg 1560

cctgaacaaa cccggaagaa acttgaaggt ggtcctttct ttgaccttct caaatccact 1620

caggaagcaa ttgtgttgcc accatgggtt gctctagctg tgaggccaag gcctggtgtt 1680

tgggaatact tacgagtcaa tctccatgct cttgtcgttg aagaactcca acctgctgag 1740

tttcttcatt tcaaggaaga actcgttgat ggagttaaga atggtaattt cactcttgag 1800

cttgatttcg agccattcaa tgcgtctatc cctcgtccaa cactccacaa atacattgga 1860

aatggtgttg acttccttaa ccgtcattta tcggctaagc tcttccatga caaggagagt 1920

ttgcttccat tgcttaagtt ccttcgtctt cacagccacc agggcaagaa cctgatgttg 1980

agcgagaaga ttcagaacct caacactctg caacacacct tgaggaaagc agaagagtat 2040

ctagcagagc ttaagtccga aacactgtat gaagagtttg aggccaagtt tgaggagatt 2100

ggtcttgaga ggggatgggg agacaatgca gagcgtgtcc ttgacatgat acgtcttctt 2160

ttggaccttc ttgaggcgcc tgatccttgc actcttgaga cttttcttgg aagagtacca 2220

atggtgttca acgttgtgat cctctctcca catggttact ttgctcagga caatgttctt 2280

ggttaccctg acactggtgg acaggttgtt tacattcttg atcaagttcg tgctctggag 2340

atagagatgc ttcaacgtat taagcaacaa ggactcaaca ttaaaccaag gattctcatt 2400

ctaactcgac ttctacctga tgcggtagga actacatgcg gtgaacgtct cgagagagtt 2460

tatgattctg agtactgtga tattcttcgt gtgcccttca gaacagagaa gggtattgtt 2520

cgcaaatgga tctcaaggtt cgaagtctgg ccatatctag agacttacac cgaggatgct 2580

gcggttgagc tatcgaaaga attgaatggc aagcctgacc ttatcattgg taactacagt 2640

gatggaaatc ttgttgcttc tttattggct cacaaacttg gtgtcactca gtgtaccatt 2700

gctcatgctc ttgagaaaac aaagtacccg gattctgata tctactggaa gaagcttgac 2760

gacaagtacc atttctcatg ccagttcact gcggatattt tcgcaatgaa ccacactgat 2820

ttcatcatca ctagtacttt ccaagaaatt gctggaagca aagaaactgt tgggcagtat 2880

gaaagccaca cagcctttac tcttcccgga ttgtatcgag ttgttcacgg gattgatgtg 2940

tttgatccca agttcaacat tgtctctcct ggtgctgata tgagcatcta cttcccttac 3000

acagaggaga agcgtagatt gactaagttc cactctgaga tcgaggagct cctctacagc 3060

gatgttgaga acaaagagca cttatgtgtg ctcaaggaca agaagaagcc gattctcttc 3120

acaatggcta ggcttgatcg tgtcaagaac ttgtcaggtc ttgttgagtg gtacgggaag 3180

aacacccgct tgcgtgagct agctaacttg gttgttgttg gaggagacag gaggaaagag 3240

tcaaaggaca atgaagagaa agcagagatg aagaaaatgt atgatctcat tgaggaatac 3300

aagctaaacg gtcagttcag gtggatctcc tctcagatgg accgggtaag gaacggtgag 3360

ctgtaccggt acatctgtga caccaagggt gcttttgtcc aacctgcatt atatgaagcc 3420

tttgggttaa ctgttgtgga ggctatgact tgtggtttac cgactttcgc cacttgcaaa 3480

ggtggtccag ctgagatcat tgtgcacggt aaatcgggtt tccacattga cccttaccat 3540

ggtgatcagg ctgctgatac tcttgctgat ttcttcacca agtgtaagga ggatccatct 3600

cactgggatg agatctcaaa aggagggctt cagaggattg aggagaaata cacttggcaa 3660

atctattcac agaggctctt gacattgact ggtgtgtatg gattctggaa gcatgtctcg 3720

aaccttgacc gtcttgaggc tcgccgttac cttgaaatgt tctatgcatt gaagtatcgc 3780

ccattggctc aggctgttcc tcttgcacaa gatgattga 3819