IP Library › Granted Patent US 12,415,990
Granted Patent B2
US 12,415,990 · App. 17/932,420 · Granted Sep 16, 2025

Ketoreductase polypeptides for the reduction of acetophenones

Inventors: Jack Liang (South San Francisco, CA); Stephane J. Jenne (Foster City, CA); Emily Mundorff (Garden City, NY); Charlene Ching (San Jose, CA); John M. Gruber (Mountain View, CA); Anke Krebber (Palo Alto, CA); Gjalt W. Huisman (Redwood City, CA)
Assignee: Codexis, Inc.
C12N9/0006C12P7/22C12Y101/01184C12Y101/01Y02E50/10Y02P20/52
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Quick Facts
Patent No.
US 12,415,990
App. No.
17/932,420
Granted
Sep 16, 2025
Kind
B2
Abstract

The present disclosure provides engineered ketoreductase enzymes having improved properties as compared to a naturally occurring wild-type ketoreductase enzyme. 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 a variety of chiral compounds.

Claims (26)

1. A recombinant ketoreductase polypeptide capable of stereoselectively reducing acetophenone to (S)-1-phenethanol, which comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 4 and comprises a substitution at the position corresponding to position 7 of the polypeptide of SEQ ID NO: 4.

2. The recombinant ketoreductase polypeptide of claim 1 , wherein the residue at the position corresponding to position 7 of the polypeptide of SEQ ID NO: 4 is an aromatic, non-polar, polar, histidine, proline, or basic residue.

3. The recombinant ketoreductase polypeptide of claim 1 , wherein said amino acid sequence of said engineered ketoreductase polypeptide further comprises at least one substitution at a position corresponding to a position in SEQ ID NO: 4, selected from the following substitutions:

the residue at the position corresponding to position 16 is a polar residue;

the residue at the position corresponding to position 43 is a nonpolar or polar residue;

the residue at the position corresponding to position 60 is an aromatic or non-polar, or aliphatic residue;

the residue at the position corresponding to position 94 is a cysteine, non-polar or an aliphatic residue;

the residue at the position corresponding to position 95 is a non-polar or aliphatic residue;

the residue at the position corresponding to position 96 is a polar or acidic residue;

the residue at the position corresponding to position 97 is a polar, non-polar, aliphatic, or basic residue;

the residue at the position corresponding to position 120 is an aromatic, non-polar or aliphatic residue;

the residue at the position corresponding to position 125 is a polar or non-polar residue;

the residue at the position corresponding to position 142 is a polar residue, serine or asparagine;

the residue at the position corresponding to position 147 is an aromatic, polar, non-polar, or aliphatic residue;

the residue at the position corresponding to position 149 is a non-polar or aromatic residue;

the residue at the position corresponding to position 150 is a constrained histidine, proline, or acidic residue;

the residue at the position corresponding to position 152 is a non-polar or polar residue;

the residue at the position corresponding to position 196 is an aliphatic, non-polar, or aromatic residue;

the residue at the position corresponding to position 202 is an aliphatic, aromatic, or a non-polar residue;

the residue at the position corresponding to position 205 is a basic, nonpolar or aliphatic residue; and

the residue at the position corresponding to position 206 is non-polar or aromatic residue.

4. The recombinant ketoreductase polypeptide of claim 1 , wherein the polypeptide is further capable of stereoselectively reducing the substrate 2′,6′-dichloro-3′-fluoroacetophenone to the product (S)-1-(2,6-dichloro-3-fluorophenyl) ethanol with a percent stereometric excess of at least 99%.

5. The recombinant ketoreductase polypeptide of claim 1 , wherein the polypeptide is further capable of reducing the substrate to the product at a rate greater than the rate capable by the ketoreductase polypeptide having the sequence of SEQ ID NO: 6 under the same conditions.

6. The recombinant ketoreductase polypeptide of claim 1 , wherein the polypeptide is further capable of reducing the substrate 2′,6′-dichloro-3′-fluoroacetophenone to the product (S)-1-(2,6-dichloro-3-fluorophenyl) ethanol at a rate that is at least 450% greater than the rate capable by the ketoreductase polypeptide having the sequence of SEQ ID NO: 6 under the same conditions.

7. The recombinant ketoreductase polypeptide of claim 1 , wherein the polypeptide is further capable of reducing the substrate 2′,6′-dichloro-3′-fluoroacetophenone to the product (S)-1-(2,6-dichloro-3-fluorophenyl) ethanol at a rate that is at least 1500% greater than the rate capable by the ketoreductase polypeptide having the sequence of SEQ ID NO: 6 under the same conditions.

8. The recombinant ketoreductase polypeptide of claim 1 , wherein the amino acid sequence of said recombinant ketoreductase polypeptide further comprises a proline at the position corresponding to position 190 of SEQ ID NO: 119.

Assignments (1)
SECURITY INTEREST Recorded Feb 15, 2024
From: CODEXIS, INC.
To: INNOVATUS LIFE SCIENCES LENDING FUND I, LP, AS COLLATERAL AGENT
Reel/Frame 066600/0650 →
Continuity (12)
Continuation 17149463 · Jan 14, 2021
Continuation 16253972 · Jan 22, 2019
Continuation 16126761 · Sep 10, 2018
Continuation 15915927 · Mar 8, 2018
Continuation 15840381 · Dec 13, 2017
Continuation 15353165 · Nov 16, 2016
Division 14501416 · Sep 30, 2014
Continuation 13970284 · Aug 19, 2013
Continuation 13682600 · Nov 20, 2012
Division 12210195 · Sep 13, 2008
Provisional Application 60972058 · Sep 13, 2007
Related Publication 20230257721A1 · Aug 17, 2023
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