# US 10,246,687 B2 · Ketoreductase polypeptides

> Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt

- Number: US 10,246,687 B2
- Document ID: US10246687B2
- Status: Granted
- Kind: B2
- Application number: 16/031,177
- Filed: 2018-07-10
- Published: 2018-11-01
- Granted: 2019-04-02
- Earliest claimed: 2008-08-29
- Assignees: Codexis, Inc.
- Applicants: Codexis, Inc.
- Inventors: Emily Mundorff; Erik Jan De Vries
- Web page: https://patentgrep.com/patent/US10246687B2

## CPC

- C12N 9/0006: {acting on CH-OH groups as donors (1.1)}
- C12P 7/02: containing a hydroxy group
- C12P 17/10: Nitrogen as only ring hetero atom
- C12P 17/14: Nitrogen or oxygen as hetero atom and at least one other diverse hetero ring atom in the same ring
- C12P 41/002: {by oxidation/reduction reactions}
- C12Y 101/01184: Carbonyl reductase (NADPH) (1.1.1.184)
- C12N 15/00: Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor (mutants or genetically engineered microorganisms, per se C12N1/00, C12N5/00, C12N7/00; new plants per se A01H; plant reproduction by tissue culture techniques A01H4/00; new animals per se A01K67/00; use of medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases, gene therapy A61K48/00)
- C12N 15/75: for Bacillus
- Y02P 20/52: using catalysts, e.g. selective catalysts
- 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
- Y02P: CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS

## Abstract

The present disclosure provides engineered ketoreductase enzymes having improved properties as compared to a naturally occurring wild-type ketoreductase enzyme including the capability of reducing 5-((4S)-2-oxo-4-phenyl (1,3-oxazolidin-3-yl))-1-(4-fluorophenyl) pentane-1,5-dione to (4S)-3-[(5S)-5-(4-fluorophenyl)-5-hydroxypentanoyl]-4-phenyl-1,3-oxazolidin-2-one. Also provided are polynucleotides encoding the engineered ketoreductase enzymes, host cells capable of expressing the engineered ketoreductase enzymes, and methods of using the engineered ketoreductase enzymes to synthesize the intermediate (4S)-3-[(5S)-5-(4-fluorophenyl)-5-hydroxypentanoyl]-4-phenyl-1,3-oxazolidin-2-one in a process for making Ezetimibe.

## Claims

### Claim 1

An engineered polypeptide having ketoreductase activity, wherein said engineered polypeptide comprises a region with an amino acid sequence having at least 90% sequence identity to residues 90 to 211 of SEQ ID NO: 130, comprising a substitution at position X190, and further comprising one or more of the following features selected from:
residue corresponding to X94 is asparagine, glycine, serine, or a polar residue;
residue corresponding to X95 is an aliphatic residue;
residue corresponding to X96 is glutamine, asparagine, or threonine;
residue corresponding to X101 is an acidic, non-polar, or a polar residue;
residue corresponding to X105 is an acidic or non-polar residue;
residue corresponding to X108 is a hydrophilic, polar or constrained residue;
residue corresponding to X111 is a non-polar or aliphatic residue;
residue corresponding to X112 is an acidic or polar residue;
residue corresponding to X113 is a non-polar or aliphatic residue;
residue corresponding to X117 is a non-polar or a polar residue;
residue corresponding to X127 is a basic residue;
residue corresponding to X147 is a non-polar, aliphatic, aromatic, or hydrophobic residue;
residue corresponding to X152 is a non-polar, basic, or hydrophilic residue;
residue corresponding to X157 is a polar residue;
residue corresponding to X163 is a non-polar or aliphatic residue;
residue corresponding to X176 is a non-polar or aliphatic residue;
residue corresponding to X194 is a constrained, basic, or polar residue;
residue corresponding to X197 is a hydrophilic, acidic, basic, aliphatic or a non-polar residue;
residue corresponding to X198 is an acidic, basic, hydrophilic, or non-polar residue;
residue corresponding to X199 is an acidic, aliphatic, or non-polar residue;
residue corresponding to X200 is an acidic or constrained residue;
residue corresponding to X202 is a non-polar residue;
residue corresponding to X206 is a non-polar, aromatic, or hydrophobic residue;
residue corresponding to X211 is a basic residue, and
wherein the amino acid sequence can optionally have one or more differences at other amino acid residues in the domain as compared to the reference sequence.

### Claim 2

An engineered polypeptide having ketoreductase activity, wherein said engineered polypeptide comprises a region with an amino acid sequence having at least 90% sequence identity to residues-90 to 211 of SEQ ID NO: 130, comprising a substitution at position X190, and further comprising one or more of the following features selected from:
residue corresponding to X94 is asparagine, glycine, or serine;
residue corresponding to X95 is leucine or methionine;
residue corresponding to X96 is glutamine, asparagine, or threonine;
residue corresponding to X101 is glycine or asparagine;
residue corresponding to X105 is glycine;
residue corresponding to X108 is histidine or serine;
residue corresponding to X112 is aspartic acid;
residue corresponding to X113 is alanine;
residue corresponding to X117 is serine;
residue corresponding to X127 is arginine;
residue corresponding to X147 is leucine;
residue corresponding to X152 is methionine or lysine;
residue corresponding to X157 is threonine;
residue corresponding to X163 is isoleucine;
residue corresponding to X176 is valine;
residue corresponding to X194 is arginine or glutamine;
residue corresponding to X197 is valine or glutamic acid;
residue corresponding to X198 is glycine, glutamic acid, or lysine;
residue corresponding to X199 is aspartic acid;
residue corresponding to X200 is proline;
residue corresponding to X202 is glycine;
residue corresponding to X206 is a glycine;
residue corresponding to X211 is a arginine or lysine; and
wherein the amino acid sequence can optionally have one or more differences at other amino acid residues in the domain as compared to the reference sequence.