# US 10,308,966 B2 · Nucleic acids encoding engineered imine reductases

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- Number: US 10,308,966 B2
- Document ID: US10308966B2
- Status: Granted
- Kind: B2
- Application number: 16/195,480
- Filed: 2018-11-19
- Published: 2019-03-14
- Granted: 2019-06-04
- Earliest claimed: 2012-05-11
- Assignees: Codexis, Inc.
- Applicants: Codexis, Inc.
- Inventors: Haibin Chen; Steven J. Collier; Jovana Nazor; Joly Sukumaran; Derek Smith; Jeffrey C. Moore; Gregory Hughes; Jacob Janey; Gjalt W. Huisman; Scott J. Novick; Nicholas J. Agard; Oscar Alvizo; Gregory A. Cope; Wan Lin Yeo; Stefanie Ng Minor
- Web page: https://patentgrep.com/patent/US10308966B2

## CPC

- C12P 13/02: Amides, e.g. chloramphenicol {or polyamides; Imides or polyimides; Urethanes, i.e. compounds comprising N-C=O structural element or polyurethanes (peptides C12P21/00 or C07K)}
- C12N 9/0028: {with NAD or NADP as acceptor (1.5.1)}
- C12P 13/001: {Amines; Imines}
- C12P 13/04: Alpha- or beta- amino acids {(other amino acids C12P13/005)}
- C12P 13/06: Alanine; Leucine; Isoleucine; Serine; Homoserine
- C12P 17/10: Nitrogen as only ring hetero atom
- C12P 17/12: containing a six-membered hetero ring
- C12P 17/165: {Heterorings having nitrogen atoms as the only ring heteroatoms}
- C12P 17/185: {Heterocyclic compounds containing sulfur atoms as ring hetero atoms in the condensed system (cepam nucleus C12P35/00; penam nucleus C12P37/00)}
- C12P 17/188: {Heterocyclic compound containing in the condensed system at least one hetero ring having nitrogen atoms and oxygen atoms as the only ring heteroatoms (ergot-alcaloids C12P17/183)}
- C12Y 105/01: with NAD+ or NADP+ as acceptor (1.5.1)
- C12Y 105/01023: Tauropine dehydrogenase (1.5.1.23)
- C12Y 105/01024: N5-(Carboxyethyl)ornithine synthase (1.5.1.24)
- C12Y 105/01028: Opine dehydrogenase (1.5.1.28)
- Y02P 20/52: using catalysts, e.g. selective catalysts
- 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
- Y02P: CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS

## Abstract

The present disclosure provides engineered polypeptides having imine reductase activity, polynucleotides encoding the engineered imine reductases, host cells capable of expressing the engineered imine reductases, and methods of using these engineered polypeptides with a range of ketone and amine substrate compounds to prepare secondary and tertiary amine product compounds.

## Claims

### Claim 1

An engineered polynucleotide encoding an engineered polypeptide having imine reductase activity, wherein said polypeptide has at least 90% sequence identity to the polypeptide SEQ ID NO:2, and comprises a substitution at a position corresponding to position 293 of SEQ ID NO:2, wherein the amino acid at position 293 has been replaced with an aromatic, non-polar, aliphatic, basic, or polar residue.

### Claim 2 (depends on 1)

The engineered polynucleotide encoding the engineered polypeptide having imine reductase activity of claim 1, wherein said polypeptide has at least 90% sequence identity to the polypeptide of SEQ ID NO:2, wherein the amino acid at the position corresponding to position 293 of SEQ ID NO:2 has been replaced with leucine.

### Claim 3 (depends on 1)

The engineered polynucleotide encoding the engineered polypeptide of claim 1, wherein said polypeptide is capable of converting substrate compound (1a) pyruvate,
and substrate compound (2b) butylamine
to product compound (3b), N-2-(butylamino)propanoic acid,
under suitable reaction conditions.

### Claim 4 (depends on 1)

The engineered polynucleotide encoding the engineered polypeptide of claim 1, wherein said polypeptide is capable of converting substrate compound (1b) cyclohexanone,
and substrate compound (2a) L-norvaline
to product compound (3c), (S)-2-(cyclohexylamino)pentanoic acid,
under suitable reaction conditions.

### Claim 5 (depends on 1)

The engineered polynucleotide encoding the engineered polypeptide of claim 1, wherein said polypeptide is capable of converting substrate compound (1b) cyclohexanone,
and substrate compound (2b) butylamine
to product compound (3d), N-butylcyclohexanamine,
under suitable reaction conditions.

### Claim 6 (depends on 1)

The engineered polynucleotide encoding the engineered polypeptide of claim 1, wherein said polypeptide is capable of converting substrate compound (1i),
and substrate compound (2b)
to product compound (3n),
under suitable reaction conditions.

### Claim 7 (depends on 1)

The engineered polynucleotide encoding the engineered polypeptide of claim 1, wherein said polypeptide is capable of converting substrate compound (1j),
and substrate compound (2b)
to product compound (3o),
under suitable reaction conditions.

### Claim 8 (depends on 1)

A vector comprising the engineered polynucleotide of claim 1.

### Claim 9 (depends on 8)

The vector of claim 8, further comprising at least one control sequence.

### Claim 10 (depends on 8)

A host cell comprising the vector of claim 8.

### Claim 11 (depends on 9)

A host cell comprising the vector of claim 9.