# US 10,501,727 B2 · Mutant enzymes

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- Number: US 10,501,727 B2
- Document ID: US10501727B2
- Status: Granted
- Kind: B2
- Application number: 15/802,321
- Filed: 2017-11-02
- Published: 2018-05-31
- Granted: 2019-12-10
- Earliest claimed: 2007-10-08
- Assignees: Isis Innovation Limited
- Applicants: Oxford University Innovation Limited
- Inventors: Luet Lok Wong; Stephen Graham Bell; Christopher Whitehouse
- Web page: https://patentgrep.com/patent/US10501727B2

## CPC

- C12N 9/0071: {acting on paired donors with incorporation of molecular oxygen (1.14)}
- C12P 7/16: Butanols
- C12P 7/22: aromatic
- C12P 7/26: Ketones
- C12P 7/40: containing a carboxyl group {including Peroxycarboxylic acids}
- C12Y 114/14001: Unspecific monooxygenase (1.14.14.1)
- Y02E 50/10: Biofuels, e.g. bio-diesel
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- 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)}
- C12Y: ENZYMES
- Y02E: REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION

## Abstract

This invention relates to mutant enzymes with enhanced properties and processes for oxidation of organic compound substrates using such enzymes.

## Claims

### Claim 1

A process for oxidising a substrate which is an organic compound, comprising the step of contacting said organic compound substrate with a mutant CYP102A (Cytochrome P450 family 102A sub-family member) enzyme, wherein said CYP102A enzyme comprises a fusion of a heme monooxygenase domain comprising a P450 fold to a reductase domain, and said mutant CYP102A enzyme comprises substitutions in the polypeptide chain of a wild-type CYP102A enzyme at positions corresponding to amino acid residue positions 307, 47 and 51 of SEQ ID NO:2, thereby enhancing monooxygenase activity and/or altering product selectivity of the mutant enzyme.

### Claim 2 (depends on 1)

The process according to claim 1, wherein said substrate is a short-chain alkane, or a substituted derivative thereof, or is an aromatic compound, or an alkylbenzene, or a substituted derivative thereof, wherein a substituted derivative is capable of being oxidised by the mutant CYP102A (Cytochrome P450 family 102A sub-family member) enzyme.

### Claim 3 (depends on 1)

The process according to claim 1, wherein said substrate is a halo aromatic compound or an acyclic or cyclic terpene or a terpenoid or a sesquiterpene or a damascone or ionone, or a cycloalkene, or a saturated fatty acid; or a substituted derivative thereof, wherein a substituted derivative is capable of being oxidised by the mutant CYP102A (Cytochrome P450 family 102A sub-family member) enzyme.

### Claim 4 (depends on 2)

The process according to claim 2, wherein said short chain alkane is pentane, 3-methylpentane, 2-methylbutane, butane, propane, ethane or methane; or wherein said alkylbenzene is propylbenzene, ethylbenzene, toluene, butylbenzene, t-butylbenzene, o-oxylene, m-xylene, cumene, p-cymene, or ethylanisole; or wherein the aromatic compound is naphthalene or fluorene.

### Claim 5 (depends on 3)

The process according to claim 3 wherein said acyclic or cyclic terpene is limonene or pinene; or wherein said sesquiterpene is valencene or caryophyllene; or wherein said ionone is β-ionone; or wherein said saturated fatty acid is lauric acid or decanoic acid.

### Claim 6 (depends on 1)

The process according to claim 1, wherein the organic compound substrate is oxidized in a cell which expresses said mutant enzyme.

### Claim 7 (depends on 1)

The process according to claim 1, wherein said mutant CYP102A enzyme additionally comprises substitutions at one or more amino acid residue positions in the polypeptide chain of the wild-type CYP102A enzyme corresponding to positions 82, 87, 171, 263, 267, 319, 328 and 401 of SEQ ID NO: 2.

### Claim 8 (depends on 1)

The process according to claim 1, wherein said mutant CYP102A enzyme comprises substitutions at amino acid residue positions in the polypeptide chain of the wild-type CYP102A enzyme corresponding to positions 47, 51 and 307 of SEQ ID NO:2.

### Claim 9 (depends on 8)

The process according to claim 8, wherein said mutant CYP102A enzyme comprises the group of mutations F87A/H171L/Q307H/N319Y or a corresponding group of mutations at amino acid residue positions in the polypeptide chain of the wild-type CYP102A enzyme corresponding to positions 87, 171, 307 and 319 of SEQ ID NO:2.

### Claim 10 (depends on 1)

The process according to claim 1, wherein said mutant CYP102A enzyme comprises one or more of the following mutations or groups of mutations: i) A330P; ii) F87A/A330P/E377A/D425N; iii) R47L/Y51F/A330P/I401P; iv) Q403P; v) R47L/Y51F/Q403P; or vi) R47L/Y51F/F87A/Q403P, or a corresponding mutation or group of mutations at amino acid residue position(s) in the polypeptide chain of the wild-type CYP102A enzyme corresponding to positions 47, 51, 87, 330, 377, 403,and 425 of SEQ ID NO: 2.

### Claim 11 (depends on 1)

The process according to claim 1, further comprising one of the following mutations or group of mutations: i) I401P; ii) R47L/Y51F/140IP; iii) F87A/I401P; or iv) R47L/Y51F/F87A/I401P, or a corresponding mutation or group of mutations at amino acid residue position(s) in the polypeptide chain of the wild-type CYP102A enzyme corresponding to positions 47, 51, 87, and 401 of SEQ ID NO: 2.

### Claim 12 (depends on 1)

The process according to claim 1, wherein the CYP102A enzyme is CYP102A2 or CYP102A3.

### Claim 13 (depends on 1)

The process of claim 1, wherein said mutant CYP102A enzyme further comprises substitution at one or more amino acid residue positions in the polypeptide chain of the wild-type CYP102A enzyme corresponding to positions 330, 401 and 403 of SEQ ID NO: 2.