# US 10,119,154 B2 · Anthocyanins with specific properties > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 10,119,154 B2 - Document ID: US10119154B2 - Status: Granted - Kind: B2 - Application number: 14/654,291 - Filed: 2013-12-23 - Published: 2015-12-03 - Granted: 2018-11-06 - Earliest claimed: 2012-12-21 - Assignees: Evolva SA - Applicants: Evolva SA - Inventors: Jorgen Hansen; Patricia Hoyle; Michael Naesby; Joseph M. Sheridan; Jonathan R. Heal - Web page: https://patentgrep.com/patent/US10119154B2 ## CPC - C12P 17/06: containing a six-membered hetero ring, e.g. fluorescein - C12N 9/1025: {Acyltransferases (2.3)} - C12N 9/1048: {Glycosyltransferases (2.4)} - C12N 15/1079: {Screening libraries by altering the phenotype or phenotypic trait of the host (reporter assays C12N15/1086)} - C12N 15/52: Genes encoding for enzymes or proenzymes - C12P 19/44: Preparation of O-glycosides, e.g. glucosides {(polysaccharides and not substituted disaccharides C12P19/04, C12P19/12)} - C12Y 203/00: Acyltransferases (2.3) - C12Y 204/00: Glycosyltransferases (2.4) - 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)} - 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 provides methods for identifying anthocyanins with improved stability, color, or hue using a screening method, and producing anthocyanins with improved stability, color, or hue in, for example, host cells comprising one or more heterologous glycosyltransferase nucleic acid molecules and one or more heterologous acyltransferase nucleic acid molecules. ## Claims ### Claim 1 A method of identifying modified anthocyanins with improved stability, color, or hue using a screening method comprising the steps of: (a) providing one or more microbial host cells capable of producing at least one anthocyanidin, wherein said host cells comprise one or more heterologous glycosyltransferase (GT) nucleic acid molecules and one or more heterologous acyltransferase (AT) nucleic acid molecules; (b) cultivating said host cells under conditions allowing growth of said cells and production of at least one anthocyanin thereby; (c) screening said host cells and/or culture medium for modified anthocyanins with improved stability, color, or hue; and (d) identifying modified anthocyanins with improved stability, color, or hue. ### Claim 2 A method of identifying modified anthocyanins with improved stability, color, or hue using a screening method comprising the steps of: (a) providing one or more microbial host cells capable of producing at least one anthocyanidin, wherein said host cells comprise one or more heterologous glycosyltransferase (GT) nucleic acid molecules, one or more heterologous acyltransferase (AT) nucleic acid molecules, or both one or more heterologous GT nucleic acid molecules and one or more heterologous AT nucleic acid molecules; (b) cultivating said host cells under conditions allowing growth of said cells and production of at least one anthocyanin thereby; (c) screening said host cells and/or culture medium for modified anthocyanins with improved stability, color, or hue; wherein the screening comprises: (i) isolating one or more anthocyanins from said host cells and/or from culture medium of the host cells; (ii) exposing the one or more anthocyanins to steadily increasing concentrations of a reagent to effect a change in the color intensity of the one or more anthocyanins; (iii) comparing the concentration where a loss of color intensity occurs with a control, wherein the ability to retain color intensity at higher concentrations of exposure to the reagent than the control indicates improved anthocyanin stability and (d) identifying modified anthocyanins with improved stability, color, or hue. ### Claim 3 (depends on 2) The method according to claim 2, wherein the reagent is a base, light, heat, and/or oxygen. ### Claim 4 (depends on 2) The method according to claim 2, wherein the reagent is a base. ### Claim 5 (depends on 2) The method according to claim 2, wherein the reagent comprises lithium hydroxide, sodium hydroxide, potassium hydroxide, and rubidium hydroxide. ### Claim 6 (depends on 5) The method according to claim 5, wherein the reagent further comprises sodium hydroxide. ### Claim 7 A method of identifying a host cell capable of producing a modified anthocyanin with a predetermined property, said method comprising the steps of: (a) providing a composition of microbial host cells, wherein each host cell is capable of producing at least one anthocyanidin, and wherein each host cell comprises one or more heterologous glycosyltransferase (GT) nucleic acid molecules and one or more heterologous acyltransferase (AT) AT nucleic acid molecules; (b) cultivating said host cells under conditions allowing growth of said host cells and production of at least one anthocyanin thereby; (c) screening the composition of host cells, for host cells producing a modified anthocyanin with the predetermined property; and (d) selecting said host cell; wherein a host cell capable of producing a modified anthocyanin with a predetermined property is identified. ### Claim 8 A method of producing modified anthocyanins with improved stability, color, or hue, said method comprising the steps of: (a) providing two or more microbial host cells capable of producing at least one anthocyanidin, wherein each host cell comprises one or more heterologous GT nucleic acid molecules and one or more heterologous AT nucleic acid molecules; (b) cultivating said host cells under conditions allowing growth of said cells and production of modified anthocyanins thereby; and (c) isolating one or more modified anthocyanins from the host cells and/or from the culture medium. ### Claim 9 (depends on 1) The method of claim 1, wherein said one or more host cells are capable of producing an anthocyanidin of the formula I wherein R1 is selected from the group consisting of H, —OH and OCH3; and R2 is selected from the group consisting of —H and —OH; and R3 is selected from the group consisting of —H, —OH and —OCH3; and R4 is selected from the group consisting of —H and —OH; and R5 is selected from the group consisting of —OH and —OCH3; and R6 is selected from the group consisting of —H and —OH; and R7 is selected from the group consisting of —OH and —OCH3. ### Claim 10 (depends on 1) The method of claim 1, wherein the anthocyanidin is selected from the group consisting of aurantinidin, cyanidin, delphinidin, europinidin, luteolinidin, pelargonidin, malvidin, peonidin, petunidin and rosinidin. ### Claim 11 (depends on 1) The method of claim 1, wherein the one or more host cells comprise one or more heterologous enzyme nucleic acid molecules, and wherein each heterologous enzyme nucleic acid molecule encodes an enzyme of the aurantinidin, cyanidin, delphinidin, europinidin, luteolinidin, pelargonidin, malvidin, peonidin, petunidin or rosinidin biosynthesis pathway. ### Claim 12 (depends on 11) The method according to claim 11, wherein the host cells comprise one or more heterologous enzyme nucleic acid molecules each encoding an enzyme of the cyanidin biosynthesis pathway. ### Claim 13 (depends on 12) The method according to claim 12, wherein the host cell comprises one or more of the following heterologous enzyme nucleic acid molecules encoding the polynucleotides comprising CHS, CHI, F3H, F3′H, F3′5′H, DFR, ANS (LDOX), FLS, LAR, and ANR. ### Claim 14 (depends on 1) The method of claim 1, wherein the one or more host cells are capable of producing one or more flavanones. ### Claim 15 (depends on 14) The method according to claim 14, wherein the one or more host cells comprise one or more heterologous enzyme nucleic acid molecules each encoding an enzyme of the naringenin biosynthesis pathway. ### Claim 16 (depends on 14) The method according to claim 14, wherein the one or more host cells comprise one or more of the following heterologous enzyme nucleic acid molecules: (a) a heterologous enzyme nucleic acid molecule encoding naringenin-chalcone synthase; or (b) a heterologous enzyme nucleic acid molecule encoding chalcone isomerase. ### Claim 17 (depends on 14) The method according to claim 14, wherein the one or more host cells comprise one or more heterologous enzyme nucleic acid molecules each encoding an enzyme of the coumaoryl-CoA biosynthesis pathway. ### Claim 18 (depends on 1) The method according to claim 1, wherein the one or more host cells comprise one or more of the following heterologous enzyme nucleic acid molecules: (a) a heterologous enzyme nucleic acid encoding phenylalanine ammonia lyase (PAL); (b) a heterologous enzyme nucleic acid encoding cinnamate 4-monooxygenase; or (c) a heterologous enzyme nucleic acid encoding 4-coumarate-CoA ligase. ### Claim 19 (depends on 1) The method according to claim 1, wherein at least 10 or more host cells each comprise different combinations of heterologous GT nucleic acid molecules and/or heterologous AT nucleic acid molecules. ### Claim 20 (depends on 1) The method according to claim 1, wherein at least 10 host cells comprise at least 10 different heterologous GT nucleic acid molecules each encoding a different glycosyltransferase.