# US 10,370,648 B2 · Engineered imine reductases and methods for the reductive amination of ketone and amine compounds

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- Number: US 10,370,648 B2
- Document ID: US10370648B2
- Status: Granted
- Kind: B2
- Application number: 15/519,949
- Filed: 2015-11-24
- Published: 2017-12-14
- Granted: 2019-08-06
- Earliest claimed: 2014-11-25
- Assignees: Codexis, Inc.
- Applicants: Codexis, Inc.
- Inventors: Oscar Alvizo; Melissa Ann Mayo; Jeffrey C. Moore
- Web page: https://patentgrep.com/patent/US10370648B2

## CPC

- C12N 9/0028: {with NAD or NADP as acceptor (1.5.1)}
- C12P 13/001: {Amines; Imines}
- C07K 14/195: from bacteria
- 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)}
- 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 application 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 polypeptide comprising an amino acid sequence with at least 90% sequence identity to the reference sequence of SEQ ID NO:8, wherein the alanine at the position corresponding to amino acid 239 of SEQ ID NO:8 is substituted with another amino acid in the amino acid sequence of the engineered polypeptide, and wherein the engineered polypeptide has imine reductase activity.

### Claim 2

An engineered polypeptide comprising an amino acid sequence with at least 90% sequence identity to the reference sequence of SEQ IDNO:8, wherein the isoleucine at the position corresponding to amino acid 330 of SEQ ID NO:8 is substituted with another amino acid in the amino acid sequence of the engineered polypeptide, wherein the amino acid sequence of the engineered polypeptide has a valine, threonine, tryptophan, glutamic acid, or tyrosine at the position corresponding to amino acid 283 of SEQ ID NO:8, and wherein the engineered polypeptide has imine reductase activity.

### Claim 3 (depends on 2)

The engineered polypeptide of claim 2, wherein the amino acid sequence of the engineered polypeptide comprises at least one residue difference as compared to the reference sequence of SEQ ID NO: 4, wherein the at least one residue difference is at a residue selected from the group consisting of 198, 153, 167, 265, 262, 108, 234, 284, 282, 220, 272, 256, 267, 242, 281, 197, 277, 224, and 143, and wherein the residue number corresponds to the amino acid sequence of SEQ ID NO: 4.

### Claim 4 (depends on 2)

The engineered polypeptide of claim 2, wherein the amino acid sequence of the engineered polypeptide comprises at least one residue difference as compared to the reference sequence of SEQ ID NO: 6, wherein the at least one residue difference is at a residue selected from the group consisting of 262, 9, 259, 220, 267, 153, 279, 200, 224, 256, 137, 143, 260, 261, 154, 276, and 185, and wherein the residue number corresponds to the amino acid sequence of SEQ ID NO: 6.

### Claim 5 (depends on 2)

The engineered polypeptide of claim 2, wherein the amino acid sequence of the engineered polypeptide further comprises at least one residue difference as compared to the reference sequence of SEQ ID NO:8, wherein the at least one residue difference is at a residue selected from the group consisting of 12, 31, 37, 44, 46, 48, 55, 56, 65, 82, 93, 98, 108, 137, 138, 141, 142, 143, 153, 154, 155, 156, 159, 166, 168, 173, 177, 178, 184, 185, 195, 197, 199, 200, 201, 203, 205, 206, 210, 213, 215, 216, 218, 220, 221 223, 226, 234, 239, 242, 245, 251, 253, 256, 257, 259, 260, 261, 262, 263, 265, 266, 267, 268, 269, 273, 274, 277, 278, 279, 280, 281, 282, 284, 285, 289, 290, 291, 292, 293, 294, and 349, and wherein the residue number corresponds to the amino acid sequence of SEQ ID NO: 8.

### Claim 6 (depends on 2)

The engineered polypeptide of claim 2, wherein the imine reductase activity comprises converting the ketone substrate
and the amine substrate
to produce the amino product compound
under suitable reaction conditions.

### Claim 7 (depends on 6)

The engineered polypeptide of claim 6, wherein the imine reductase activity in converting the ketone and the amine substrate compound to the amine product compound under suitable reaction conditions is increased at least 2-fold as compared to the corresponding activity of an imine reductase polypeptide comprising the amino acid sequence of SEQ ID NO: 4, 6, 12, 30, 126, 173, 360, or 550.

### Claim 8 (depends on 1)

The engineered polypeptide of claim 1, wherein the amino acid sequence of the engineered polypeptide comprises the amino acid sequence of SEQ ID NO: 1188.