# US 11,441,165 B2 · Biosynthetic production of steviol glycosides rebaudioside J and rebaudioside N > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 11,441,165 B2 - Document ID: US11441165B2 - Status: Granted - Kind: B2 - Application number: 17/109,473 - Filed: 2020-12-02 - Published: 2021-06-24 - Granted: 2022-09-13 - Earliest claimed: 2018-03-12 - Assignees: Conagen Inc. - Applicants: Conagen Inc. - Inventors: Guohong Mao; Michael Batten; Phillip Hunt; Oliver (Xiaodan) Yu - Web page: https://patentgrep.com/patent/US11441165B2 ## CPC - C12P 19/56: having an oxygen atom of the saccharide radical directly bound to a condensed ring system having three or more carbocyclic rings, e.g. daunomycin, adriamycin - A23L 27/36: {Terpene glycosides} - C12N 9/1051: {Hexosyltransferases (2.4.1)} - C12N 9/1081: {transferring other glycosyl groups (2.4.99)} - C12N 15/8245: {involving modified carbohydrate or sugar alcohol metabolism, e.g. starch biosynthesis} - C12Y 204/01: Hexosyltransferases (2.4.1) - C12Y 204/01017: Glucuronosyltransferase (2.4.1.17) - C12Y 204/01013: Sucrose synthase (2.4.1.13) - C12Y 204/99: transferring other glycosyl groups (2.4.99) - C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)} - A23L: FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF (preservation thereof A23B) - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) - C12Y: ENZYMES ## Abstract The present disclosure relates to the production of steviol glycosides rebaudioside J and rebaudioside N through the use of rebaudioside A as a substrate and a biosynthetic pathway involving various 1,2 RhaT-rhamnosyltransferases. ## Claims ### Claim 1 A biosynthetic method of preparing rebaudioside N, the method comprising: reacting a steviol glycoside composition comprising rebaudioside A with a glucose donor moiety in the presence of a first recombinant polypeptide having glucosyltransferase activity to produce rebaudioside I, wherein said first recombinant polypeptide comprises (i) an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 7, SEQ ID NO: 11, or SEQ ID NO: 13, or (ii) an amino acid sequence having at least 97% sequence identity to SEQ ID NO: 15; and reacting said rebaudioside I with a rhamnose donor moiety in the presence of a second recombinant polypeptide having 1,2-rhamnosyltransferase activity to produce rebaudioside N; wherein said second recombinant polypeptide comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 3. ### Claim 2 (depends on 1) The method of claim 1, wherein said second recombinant polypeptide comprises the amino acid sequence of SEQ ID NO: 3. ### Claim 3 (depends on 1) The method of claim 1, wherein said first recombinant polypeptide comprises an amino acid sequence having at least 98% identity to SEQ ID NO: 7, SEQ ID NO: 11, SEQ ID NO: 13, or SEQ ID NO: 15. ### Claim 4 (depends on 3) The method of claim 3, wherein said first recombinant polypeptide comprises the amino acid sequence of SEQ ID NO: 7. ### Claim 5 (depends on 3) The method of claim 3, wherein said first recombinant polypeptide comprises the amino acid sequence of SEQ ID NO: 11. ### Claim 6 (depends on 3) The method of claim 3, wherein said first recombinant polypeptide comprises the amino acid sequence of SEQ ID NO: 13. ### Claim 7 (depends on 3) The method of claim 3, wherein said first recombinant polypeptide comprises the amino acid sequence of SEQ ID NO: 15. ### Claim 8 (depends on 1) The method of claim 1, wherein the rhamnose donor moiety is UDP-L-rhamnose. ### Claim 9 (depends on 1) The method of claim 1, wherein the glucose donor moiety is generated in situ. ### Claim 10 (depends on 1) The method of claim 1, comprising reacting said steviol glycoside composition comprising rebaudioside A with a glucose donor moiety in the presence of a third recombinant polypeptide having sucrose synthase activity. ### Claim 11 (depends on 1) The method of claim 1, comprising expressing said second recombinant polypeptide in a transformed cell. ### Claim 12 (depends on 11) The method of claim 11, wherein the transformed cell is selected from the group consisting of a yeast, a non-steviol glycoside producing plant, an alga, a fungus, and a bacterium. ### Claim 13 (depends on 12) The method of claim 12, wherein the transformed cell is a bacterium or yeast selected from the group consisting of Escherichia; Salmonella; Bacillus; Acinetobacter; Streptomyces; Corynebacterium; Methylosinus; Methylomonas; Rhodococcus; Pseudomonas; Rhodobacter; Synechocystis; Saccharomyces; Zygosaccharomyces; Kluyveromyces; Candida; Hansenula; Debaryomyces; Mucor; Pichia; Torulopsis; Aspergillus; Arthrobotlys; Brevibacteria; Microbacterium; Arthrobacter; Citrobacter; Klebsiella; Pantoea; and Clostridium. ### Claim 14 (depends on 13) The method of claim 13, comprising isolating said second recombinant polypeptide from the transformed cell and the step of reacting the rebaudioside I with the second recombinant polypeptide is performed in vitro. ### Claim 15 (depends on 11) The method of claim 11, wherein the step of reacting the rebaudioside I with the second recombinant polypeptide is performed in the transformed cell. ### Claim 16 (depends on 1) The method of claim 1, further comprising isolating rebaudioside N from the steviol glycoside composition. ### Claim 17 (depends on 1) The method of claim 1, further comprising purifying rebaudioside N to obtain a steviol glycoside composition enriched with rebaudioside N.