# US 10,501,760 B2 · Methods for production of oxygenated terpenes

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- Number: US 10,501,760 B2
- Document ID: US10501760B2
- Status: Granted
- Kind: B2
- Application number: 15/505,022
- Filed: 2015-08-21
- Published: 2018-11-15
- Granted: 2019-12-10
- Earliest claimed: 2014-08-21
- Assignees: Givaudan SA; Manus Biosynthesis, Inc.
- Applicants: Givaudan SA; Manus Biosynthesis, Inc.
- Inventors: Ajikumar Parayil Kumaran; Chin Giaw Lim; Liwei Li; Souvik Ghosh; Christopher Pirie; Anthony Qualley; Geoff Marshall-Hill; Martin Preininger
- Web page: https://patentgrep.com/patent/US10501760B2

## CPC

- C12P 7/26: Ketones
- A01N 27/00: Biocides, pest repellants or attractants, or plant growth regulators containing hydrocarbons
- A23L 27/13: {from citrus fruits}
- C12N 9/0073: {with NADH or NADPH as one donor, and incorporation of one atom of oxygen 1.14.13}
- C12P 5/002: {cyclic (compounds containing at least three condensed carbocyclic rings C12P15/00)}
- C12Y 114/13078: Ent-kaurene oxidase (1.14.13.78)
- A23V 2002/00: Food compositions, function of food ingredients or processes for food or foodstuffs
- C07K 2319/03: containing a transmembrane segment
- 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)}
- A01N: PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF (preservation of food or foodstuff A23); BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES (preparations for medical, dental or toiletry purposes which kill or prevent the growth or proliferation of unwanted organisms A61K); PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- 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
- A23V: INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)

## Abstract

The present disclosure relates to methods for producing oxygenated terpenoids, and preparation of compositions and formulations thereof. Polynucleotides, derivative enzymes, and host cells for use in such methods are also provided.

## Claims

### Claim 1

A formulation, comprising:
nootkatone, wherein the nootkatone is present in the formulation in an amount ranging from about 55% to about 60% (w/w);
α-nootkatol, wherein the α-nootkatol is present in the formulation in an amount ranging from about 20% to about 25% (w/w); and
β-nootkatol, wherein the β-nootkatol is present in the formulation in an amount ranging from about 5% to about 10% (w/w).

### Claim 2 (depends on 1)

The formulation of claim 1, wherein at least one of the nootkatone, the α-nootkatol, and the β-nootkatol, is produced using a biosynthetic process.

### Claim 3 (depends on 1)

The formulation of claim 1, wherein the nootkatone, the α-nootkatol, and the β-nootkatol, are produced using at least one biosynthetic process.

### Claim 4 (depends on 1)

The formulation of claim 1, wherein the formulation is a natural flavor formulation.

### Claim 5 (depends on 1)

The formulation of claim 1, further comprising one or more non-sesquiterpene components or sesquiterpene components.

### Claim 6 (depends on 5)

The formulation of claim 5, wherein the one or more non-sesquiterpene components or sesquiterpene components is selected from Table 8A, products obtained from oxidation of valencene by the cytochrome P450 enzyme Stevia rebaudiana Kaurene Oxidase (SrKO).

### Claim 7 (depends on 1)

The formulation of claim 1, further comprising valencene.

### Claim 8 (depends on 7)

The formulation of claim 7, wherein the valencene is present in the formulation in an amount ranging from about 5% to about 10% (w/w).

### Claim 9 (depends on 7)

The formulation of claim 7, wherein at least one of the nootkatone, the α-nootkatol, the β-nootkatol, and the valencene is produced using a biosynthetic process.

### Claim 10 (depends on 7)

The formulation of claim 7, wherein the nootkatone, the α-nootkatol, the β-nootkatol, and the valencene are produced using at least one biosynthetic process.

### Claim 11 (depends on 8)

The formulation of claim 8, wherein the formulation is a natural flavor formulation.

### Claim 12 (depends on 8)

The formulation of claim 8, further comprising one or more non-sesquiterpene components or sesquiterpene components.

### Claim 13 (depends on 12)

The formulation of claim 12, wherein the one or more non-sesquiterpene components or sesquiterpene components is selected from products obtained from oxidation of valencene by the cytochrome P450 enzyme SrKO.