USO09522929B2 (12) United States Patent (10) Patent No.: US 9,522,929 B2 Mao et al. (45) Date of Patent: Dec. 20, 2016 (54) NON-CALORIC SWEETENER WO 2013093880 A1 6, 2013 WO 2013093881 A1 6, 2013 WO 2013096.290 A1 6, 2013 (71) Applicant: Conagen Inc., St. Louis, MO (US) WO 2013O96663 A2 6, 2013 WO 2013110673 A1 8, 2013 (72) Inventors: Guohong Mao, St. Louis, MO (US); WO 2013176738 A1 11 2013 Venkata Sai Prakash Chaturvedula, Mission Viejo, CA (US); Oliver Yu, OTHER PUBLICATIONS Chesterfield, MO (US) Prakash et al. (2014) Natural Product Communication, vol. 9, No. (73) Assignee: Conagen Inc., Bedford, MA (US) 8, pp. 1135-1138.* Reeck et al., “Homology” in Proteins and Nucleic Acids: A Termi (*) Notice: Subject to any disclaimer, the term of this nology Muddle and a Way out of it: Cell. vol. 50:667, 1987. patent is extended or adjusted under 35 Bieniawska et al., Analysis of the Sucrose synthase gene family in U.S.C. 154(b) by 0 days. Arabidopsis, The Plant Journal (2007), 49: 810-828. Terasaka K et al. In Situ UDP-Glucose Regeneration Unravels Diverse Functions of Plant Secondary Product (21) Appl. No.: 14/269,435 Glycosyltransgerases. FEBS Letters. Nov. 15, 2012, vol. 586; pp. 4344-4350. (22) Filed: May 5, 2014 Masada, Set al. An Efficient Chemoenzymatic Production of Small Molecule Glucosides With In Situ UDP-Glucose Recycling. FEBS (65) Prior Publication Data Letters. May 8, 2007, vol. 581, pp. 2562-2566. US 2015/0315623 A1 Nov. 5, 2015 Prakash, I et al. Catalytic Hydrogenation of the Sweet Principles of Stevia Rebaudiana, Rebaudioside B, Rebaudioside C, and Int. C. Rebaudioside D and Sensory Evaluation of Their Reduced Deriva (51) tives. International Journal of Molecular Sciences. Nov. 16, 2012, CI2P 19/56 (2006.01) vol. 13, pp. 15126-15136. C7H 5/24 (2006.01) Prakash, I et al. Isolation and Characterization of a Novel C7H 5/256 (2006.01) Rebaudioside M Isomer From a Bioconversion Reaction of CI2N 9/10 (2006.01) Rebaudioside A and NMR Comparison Studies of Rebaudioside M (52) U.S. C. Isolated From Stevia Rebaudiana Bertoni and Stevia Rebaudiana CPC ............ C07H 15/24 (2013.01); C07H 15/256 Morita. Biomolecules. Mar. 21, 2014, vol. 2, pp. 374-389. Chaturvedula, VSP et al. NMR Spectral Analysis and Hyrdolysis (2013.01); C12N 9/10 (2013.01); CI2N Studies of Rebaudioside N. A Minor Steviol Glycoside of Stevia 9/1048 (2013.01); C12N 9/1051 (2013.01); Rebaudiana Bertoni. Food and Nutrition Science. Oct. 2013, vol. 4, CI2N 9/1062 (2013.01); CI2P 19/56 pp. 1004-1008. (2013.01); C12Y 204/01013 (2013.01); C12Y International Search Report and Written Opinion issued in PCT/ 204/01017 (2013.01) US2015/029163 issued Oct. 1, 2015. (58) Field of Classification Search None * cited by examiner See application file for complete search history. Primary Examiner — Nashaat Nashed (56) References Cited (74) Attorney, Agent, or Firm — Bryon V. Olsen; Wolf, Greenfield & Sacks, P.C. U.S. PATENT DOCUMENTS 2008. O148432 A1 6, 2008 Abad (57) ABSTRACT 2009,0183270 A1 7/2009 Adams et al. Disclosed is a steviol glycoside referred to as rebaudioside 2014/0357588 A1 12/2014 Markosyan et al. D3. Rebaudioside D3 has five B-D-glucosyl units connected to the aglycone Steviol. Also disclosed are methods for FOREIGN PATENT DOCUMENTS producing rebaudioside D3, a UDP-glycosyltransferase WO 0210210 A2 2, 2002 fusion enzyme, and methods for producing rebaudioside D WO 2011028671 A1 3, 2011 and rebaudioside E. WO 2012.125991 A2 9, 2012 WO 2013022989 A2 2, 2013 5 Claims, 10 Drawing Sheets U.S. Patent Dec. 20, 2016 Sheet 1 of 10 US 9,522,929 B2 -ry : Y--- y EUG1 FIG. 1A U.S. Patent US 9,522,929 B2 C OH U.S. Patent Dec. 20, 2016 Sheet 3 of 10 US 9,522,929 B2 UG 76G EUGT11 --> Rebaudioside D HO HO Y H O HO O o O O HO O vario OH HO O HO OH HO ES 17' HO ge 7.2-e O HO O F G 1 C Rebaudioside A U.S. Patent Dec. 20, 2016 Sheet 4 of 10 US 9,522,929 B2 kD 250 150 100 75 50 EUGT11 FIG. 2A KD 250 150 100 75 50 EUG11-AtSUS 1 FIG. 2B U.S. Patent US 9,522,929 B2 09 U.S. Patent Dec. 20, 2016 Sheet 6 of 10 US 9,522,929 B2 |09 |0|084 |00}, Q8|| |004. |084 |00}, U.S. Patent Dec. 20, 2016 Sheet 7 of 10 US 9,522,929 B2 .'sssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssarasaasaarsssssssssssssssssssssssssssssssssssssssssssssss x: Š3 -----S-S-S-S- SSSSSSSSS U.S. Patent Dec. 20, 2016 Sheet 8 of 10 US 9,522,929 B2 s& s y xxx U.S. Patent Dec. 20, 2016 Sheet 9 of 10 US 9,522,929 B2 sugar Hi OH HO Sugar I HOHO HO O O HO OH sugar V HO FIG. 7 U.S. Patent Dec. 20, 2016 Sheet 10 of 10 US 9,522,929 B2 %, US 9,522,929 B2 1. 2 NON-CALORIC SWEETENER are provided herein and are herein incorporated by refer ence. This Sequence Listing consists of SEQ ID NOs: 1-6. CROSS-REFERENCE TO RELATED BACKGROUND OF THE DISCLOSURE APPLICATIONS The present disclosure relates generally to natural Sweet This disclosure is a PCT Application entitled A Non eners. More particularly, the present disclosure relates to a Caloric Sweetener. This application claims priority to U.S. non-caloric Sweetener and methods for synthesizing the patent application Ser. No. 14/269.435, filed on May 5, non-caloric Sweetener. 2014, which is incorporated by reference herein in its Steviol glycosides are natural products isolated from entirety. 10 Stevia rebaudiana leaves. Steviol glycosides are widely used as high intensity, low-calorie Sweeteners and are signifi cantly Sweeter than Sucrose. Naturally occurring Steviol STATEMENT IN SUPPORT FOR FILING A glycosides share the same basic steviol structure, but differ SEQUENCE LISTING in the content of carbohydrate residues (e.g., glucose, rham 15 nose and xylose residues) at the C13 and C19 positions. A paper copy of the Sequence Listing and a computer Steviol glycosides with known structures include, Steviol, readable form of the Sequence Listing containing the file Stevioside, rebaudioside A, rebaudioside B, rebaudioside C, named “32559-11 ST25.txt, which is 32,670 bytes in size rebaudioside D, rebaudioside E, rebaudioside F and dulco (as measured in MICROSOFT WINDOWS(R) EXPLORER), side A (see e.g., Table 1). TABLE 1. Steviol glycosides. Molecular Molecular Name Structure Formula Weight Stevio C20HoO3 318 Stevioside C38H60018 804 US 9,522,929 B2 TABLE 1-continued Steviol glycosides. Molecular Molecular Name Structure Formula Weight Rebaudioside HO HO CAH70O23 966 A. HO HO Rebaudioside- HO C38H6OO18 804 B HO HO US 9,522,929 B2 TABLE 1-continued Steviol glycosides. Molecular Molecular Name Structure Formula Weight Rebaudioside CAH70O22 950 Rebaudioside CsoHsoC2s 1128 HO O HO HO OH US 9,522,929 B2 7 TABLE 1-continued Steviol glycosides. Molecular Molecular Name Structure Formula Weight Rebaudioside HO CAH70O23 966 E Rebaudioside C43H68O22 936 US 9,522,929 B2 TABLE 1-continued Steviol glycosides. Molecular Molecular Name Structure Formula Weight Rebaudioside CsoH80028 1128 D2 Ducloside C38H6OO17 788 A. On a dry weight basis, Stevioside, rebaudioside A, rebau- can vary from about 20% to more than 90% of the total dioside C, and dulcoside A, account for 9.1, 3.8, 0.6, and Steviol glycoside content, while the amount of rebaudioside 0.3% of the total weight of the steviol glycosides in the 60 B can be about 1-2%, the amount of rebaudioside C can be leaves, respectively, while the other steviol glucosides are about 7-15%, and the amount of rebaudioside D can be present in much lower amounts. Extracts from the Stevia about 2% of the total steviol glycosides. rebaudiana plant are commercially available, which typi Steviol glycosides differ from each other not only by cally contain Stevioside and rebaudioside A as primary molecular structure, but also by their taste properties. For compounds. The other Steviol glycosides typically are pres 65 example, different steviol glycosides have different degrees ent in the Stevia extract as minor components. For example, of Sweetness and after-taste. Stevioside, for example, is the amount of rebaudioside A in commercial preparations 100-150 times sweeter than sucrose, but has a bitter after US 9,522,929 B2 11 12 taste. Rebaudioside A and rebaudioside E, for example, are Method of Producing Rebaudioside D3 from Rebaudio 250-450 times sweeter than sucrose and have less of an side E. after-taste than Stevioside. Rebaudioside C is between 40-60 In another aspect, the present disclosure is directed to a times sweeter than sucrose. Dulcoside A is about 30 times method for synthesizing rebaudioside D3 from rebaudioside Sweeter than Sucrose. E. The method comprises preparing a reaction mixture The majority of steviol glycosides are formed by several glycosylation reactions of Steviol, which are typically cata comprising rebaudioside E; a substrate selected from the lyzed by the UDP-glycosyltransferases (UGTs) using uri group consisting of Sucrose, uridine diphosphate (UDP) and dine 5'-diphosphoglucose (UDP-glucose) as a donor of the uridine diphosphate-glucose (UDP-glucose); and a uridine Sugar moiety. UGTs in plants make up a very diverse group dipospho glycosyltransferase (UDP-glycosyltransferase) of enzymes that transfer a glucose residue from UDP 10 selected from the group consisting of a uridine diphospho glucose to steviol. For example, glycosylation of the C-3' of glycosyltransferase and a UDP-glycosyl transferase fusion the C-13-O-glucose of stevioside yields rebaudioside A; and enzyme comprising a uridine diphosphoglycosyltransferase glycosylation of the C-2 of the 19-O-glucose of the stevio domain coupled to a Sucrose synthase domain; and incubat side yields rebaudioside E.