IP Library › Granted Patent US 12,421,503
Granted Patent B2
US 12,421,503 · App. 18/472,540 · Granted Sep 23, 2025

Ketoreductase polypeptides for the production of azetidinone

Inventors: Onorato Campopiano (Hayward, CA); Emily Mundorff (Garden City, NY); Birthe Borup (Wesel, DE); Rama Voladri (Pleasanton, CA)
Assignee: Codexis, Inc.
C12N9/0006C12P17/10C12Y101/01184Y02P20/52
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Quick Facts
Patent No.
US 12,421,503
App. No.
18/472,540
Filed
Sep 22, 2023
Granted
Sep 23, 2025
Kind
B2
Examiner
LEE, JAE W
Art Unit
1656
USPC
435/348
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 (72)

1. An engineered ketoreductase polypeptide having ketoreductase activity,

wherein said ketoreductase polypeptide is at least 85% identical to SEQ ID NO:4, and

wherein the ketoreductase polypeptide comprises the following fetures:

the resiude at the position corresponding to position 153 is alanine or serine,

the residue at position corresponding to position 202 is valine or leucine, and

wherein the residue at the position corresponding to position 94 is threonine.

2. The engineered ketoreductase polypeptide of claim 1 , wherein said ketoreductase polypeptide further comprises at least one of the following features recited under [1] or [2]:

[1] the residue at the position corresponding to position 199 is a histidine, alanine, or asparagine; or

[2] the residue at the position corresponding to position 199 is an aliphatic, constrained or polar residue.

3. The engineered ketoreductase polypeptide of claim 1 , wherein the residue at the position corresponding to position 199 is alanine, histidine, or asparagine.

4. The engineered ketoreductase polypeptide of claim 1 , wherein the residue at the position corresponding to position 199 is an aliphatic, constrained or polar residue.

5. The engineered ketoreductase polypeptide of claim 1 , wherein the residue at the position corresponding to position 199 is alanine, histidine, or asparagine.

6. The engineered ketoreductase polypeptide of claim 1 , further comprising one or more of the following features:

the residue at the position corresponding to position 2 is a polar, non-polar, or aliphatic residue;

the residue at the position corresponding to position 4 is a basic residue or cysteine;

the residue at the position corresponding to position 11 is a non-polar, aliphatic, or aromatic residue;

the residue at the position corresponding to position 80 is a non-polar, aliphatic, or polar residue;

the residue at the position corresponding to position 86 is a non-polar, aliphatic, or polar residue;

the residue at the position corresponding to position 96 is a polar, aromatic, non-polar, or aliphatic residue;

the residue at the position corresponding to position 105 is a non-polar, aliphatic, basic or acidic residue;

the residue at the position corresponding to position 129 is a non-polar or polar residue;

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

the residue at the position corresponding to position 190 is an aromatic or constrained residue;

the residue at the position corresponding to position 195 is a non-polar or aliphatic residue;

the residue at the position corresponding to position 196 is a non-polar or aliphatic residue;

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

the residue at the position corresponding to position 248 is a non-polar or basic residue; and

the residue at the position corresponding to position 249 is an aromatic residue; and

wherein optionally the amino acid sequence has one or more residue differences at other amino acid residue positions as compared to SEQ ID NO:4.

7. The engineered ketoreductase polypeptide of claim 1 , further comprising one or more of the following features:

the residue at the position corresponding to position 2 is alanine;

the residue at the position corresponding to position 4 is cysteine;

the residue at the position corresponding to position 11 is phenylalanine;

the residue at the position corresponding to position 80 is threonine;

the residue at the position corresponding to position 86 is isoleucine;

the residue at the position corresponding to position 96 is valine or phenylalanine;

the residue at the position corresponding to position 105 is glycine;

the residue at the position corresponding to position 129 is threonine;

the residue at the position corresponding to position 147 is methionine or leucine;

the residue at the position corresponding to position 190 is histidine or proline;

the residue at the position corresponding to position 195 is valine;

the residue at the position corresponding to position 196 is leucine;

the residue at the position corresponding to position 206 is phenylalanine;

the residue at the position corresponding to position 248 is lysine, or arginine; and

the residue at the position corresponding to position 249 is tryptophan;

wherein optionally the amino acid sequence has one or more residue differences at other amino acid residue positions as compared to SEQ ID NO:4.

8. The engineered ketoreductase polypeptide of claim 1 , further comprising the feature that the residue corresponding to X147 is an aromatic, non-polar or aliphatic residue.

9. The engineered ketoreductase polypeptide of claim 1 , further comprising one or more of the following features:

the residue at the position corresponding to position 96 is a polar, aromatic, non-polar, or aliphatic;

the residue at the position corresponding to position 195 is a non-polar or aliphatic residue;

the residue at the position corresponding to position 196 is a non-polar or aliphatic residue;

the residue at the position corresponding to position 248 is a non-polar or basic residue; and

the residue at the position corresponding to position 249 is an aromatic residue.

10. The engineered ketoreductase polypeptide of claim 1 , further comprising one or more of the following features:

the residue at the position corresponding to position 2 is a polar, non-polar, or aliphatic residue;

the residue at the position corresponding to position 4 is a basic residue or cysteine;

the residue at the position corresponding to position 11 is a non-polar, aliphatic, or aromatic residue;

the residue at the position corresponding to position 80 is a non-polar, aliphatic, or polar residue;

the residue at the position corresponding to position 86 is a non-polar, aliphatic, or polar residue;

the residue at the position corresponding to position 105 is a non-polar, aliphatic, basic or acidic residue;

the residue at the position corresponding to position 129 is a non-polar or polar residue;

the residue at the position corresponding to position 190 is an aromatic or constrained residue; and

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

11. The engineered ketoreductase polypeptide of claim 1 , further comprising one or more of the following features: the residue at the position corresponding to position 147 is an aromatic, non-polar or aliphatic residue; and wherein optionally the amino acid sequence has one or more residue differences at other amino acid residue positions as compared to SEQ ID NO:4.

12. The engineered ketoreductase polypeptide of claim 1 , further comprising one or more of the following features: the residue at the position corresponding to position 96 is a polar, aromatic, non-polar, or aliphatic residue; the residue at the position corresponding to position 147 is an aromatic, non-polar or aliphatic residue; and wherein optionally the amino acid sequence has one or more residue differences at other amino acid residue positions as compared to SEQ ID NO:4.

13. The engineered ketoreductase polypeptide of claim 1 , further comprising one or more of the following features:

residue at the position corresponding to position 96 is a polar, aromatic, non-polar, or aliphatic residue;

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

residue at the position corresponding to position 195 is a non-polar or aliphatic residue;

residue at the position corresponding to position 196 is a non-polar or aliphatic residue; and

wherein optionally the amino acid sequence has one or more residue differences at other amino acid residue positions as compared to SEQ ID NO:4.

14. The engineered ketoreductase polypeptide of claim 1 , further comprising the feature that the residue at the position corresponding to position 147 is methionine or leucine.

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 (15)
Continuation 17357673 · Jun 24, 2021
Continuation 16790474 · Feb 13, 2020
Division 16404988 · May 7, 2019
Division 15631359 · Jun 23, 2017
Continuation 15408673 · Jan 18, 2017
Continuation 15272664 · Sep 22, 2016
Division 15175317 · Jun 7, 2016
Continuation 14822624 · Aug 10, 2015
Continuation 14658407 · Mar 16, 2015
Continuation 13925096 · Jun 24, 2013
Continuation 13569900 · Aug 8, 2012
Division 12977825 · Dec 23, 2010
Continuation 12243968 · Oct 1, 2008
Provisional Application 60976555 · Oct 1, 2007
Related Publication 20240141304A1 · May 2, 2024
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