IP Library › Granted Patent US 12,378,585
Granted Patent B2
US 12,378,585 · App. 18/614,099 · Granted Aug 5, 2025

Ketoreductase polypeptides for the production of (R)-3-hydroxythiolane

Inventors: Jack S. Liang (South San Francisco, CA); Stephan Jenne (Foster City, CA); Emily Mundorff (Garden City, NY); Rama Voladri (Pleasanton, CA); James J. Lalonde (San Mateo, CA); Gjalt W. Huisman (Redwood City, CA)
Assignee: Codexis, Inc.
C12P17/00C12N9/0006C12P17/167C12Y101/01184Y02P20/52
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Quick Facts
Patent No.
US 12,378,585
App. No.
18/614,099
Granted
Aug 5, 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 chiral compounds.

Claims (135)

1. An engineered ketoreductase polypeptide comprising an amino acid sequence that is at least 90% identical to the reference sequence SEQ ID NO:4, and wherein said amino acid sequence comprises a substitution at position 223.

2. The engineered ketoreductase polypeptide of claim 1 , in which the ketoreductase polypeptide amino acid sequence further comprises one or more additional substitutions selected from:

the residue corresponding to X3 is asparagine, aspartic acid, or tyrosine;

the residue corresponding to X7 is glycine, histidine, serine or asparagine;

the residue corresponding to X11 is isoleucine or threonine;

the residue corresponding to X16 is threonine, alanine, valine, or glycine;

the residue corresponding to X19 is isoleucine or valine;

the residue corresponding to X23 is isoleucine or phenylalanine;

the residue corresponding to X41 is serine, alanine, or valine;

the residue corresponding to X45 is glutamic acid or glycine;

the residue corresponding to X49 is lysine or arginine;

the residue corresponding to X57 is isoleucine or valine;

the residue corresponding to X60 is phenylalanine, valine, or threonine;

the residue corresponding to X64 is alanine, serine, or threonine;

the residue corresponding to X72 is lysine or arginine, particularly arginine;

the residue corresponding to X82 is glycine or serine;

the residue corresponding to X94 is alanine, valine, threonine, serine, or arginine;

the residue corresponding to X95 is valine or alanine;

the residue corresponding to X96 is asparagine, serine, proline, alanine, or glutamic acid;

the residue corresponding to X97 is lysine, arginine or leucine;

the residue corresponding to X106 is glutamic acid or aspartic acid;

the residue corresponding to X108 is arginine or histidine;

the residue corresponding to X111 is leucine or methionine;

the residue corresponding to X117 is glycine or serine;

the residue corresponding to X126 is isoleucine or valine;

the residue corresponding to X127 is glutamine or arginine;

the residue corresponding to X145 is aspartic acid or serine;

the residue corresponding to X147 is phenylalanine, leucine or serine;

the residue corresponding to X152 is threonine, serine, or methionine;

the residue corresponding to X163 is valine or isoleucine;

the residue corresponding to X173 is aspartic acid or glycine;

the residue corresponding to X177 is lysine or arginine;

the residue corresponding to X192 is lysine, arginine or glutamic acid;

the residue corresponding to X194 is proline, glycine, aspartic acid, arginine, or leucine;

the residue corresponding to X198 is aspartic acid or glycine;

the residue corresponding to X200 is proline, glutamic acid, or lysine;

the residue corresponding to X206 is methionine or glutamine;

the residue corresponding to X208 is glutamine, histidine or arginine;

the residue corresponding to X210 is threonine or alanine;

the residue corresponding to X211 is lysine or glutamic acid;

the residue corresponding to X214 is methionine, valine or threonine, or serine;

the residue corresponding to X217 is isoleucine or phenylalanine; and

the residue corresponding to X226 is isoleucine or valine.

3. The engineered ketoreductase polypeptide of claim 1 , wherein the ketoreductase polypeptide amino acid sequence further comprises at least one additional substitution selected from:

the residue corresponding to X7 is a non-polar, polar, or constrained residue;

the residue corresponding to X41 is an aliphatic, non-polar, or polar residue;

the residue corresponding to X94 is a polar, basic, aliphatic, or non-polar residue;

the residue corresponding to X96 is a constrained, aliphatic, non-polar, acidic, or polar residue;

the residue corresponding to X108 is a basic, constrained, or aromatic residue;

the residue corresponding to X117 is a non-polar or polar residue;

the residue corresponding to X145 is a polar residue;

the residue corresponding to X147 is a non-polar or aliphatic residue;

the residue corresponding to X173 is an acidic or non-polar residue; and

the residue corresponding to X206 is a polar or non-polar residue.

4. The engineered ketoreductase polypeptide of claim 1 , in which the ketoreductase polypeptide amino acid sequence further comprises the following substitutions:

the residue corresponding to X7 is a non-polar, polar, or constrained residue;

the residue corresponding to X145 is a polar residue.

5. The engineered ketoreductase polypeptide of claim 1 , in which the ketoreductase polypeptide amino acid sequence further comprises the following substitutions:

the residue corresponding to X7 is a non-polar, polar, or constrained residue;

the residue corresponding to X117 is a non-polar or polar residue;

the residue corresponding to X145 is a polar residue;

the residue corresponding to X147 is a non-polar or aliphatic residue.

6. The engineered ketoreductase polypeptide of claim 1 , in which the ketoreductase polypeptide amino acid sequence further comprises at least one substitution selected from:

the residue corresponding to X7 is a non-polar, polar, or constrained residue;

the residue corresponding to X94 is a polar, basic, aliphatic, or non-polar residue;

the residue corresponding to X108 is a basic, constrained, or aromatic residue;

the residue corresponding to X117 is a non-polar or polar residue, particularly serine;

the residue corresponding to X147 is a non-polar or aliphatic residue.

7. The engineered ketoreductase polypeptide of claim 1 in which the ketoreductase polypeptide amino acid sequence comprises the following substitutions:

the residue corresponding to X7 is a non-polar, polar, or constrained residue;

the residue corresponding to X96 is a constrained, aliphatic, non-polar, acidic, or polar residue;

the residue corresponding to X108 is a basic, constrained, or aromatic residue;

the residue corresponding to X117 is a non-polar or polar residue, particularly serine; and

the residue corresponding to X147 is leucine.

8. The engineered ketoreductase polypeptide of claim 1 , in which the ketoreductase polypeptide amino acid sequence further comprises the following substitutions:

the residue corresponding to X7 is a non-polar, polar, or constrained residue;

the residue corresponding to X94 is a polar, basic, aliphatic, or non-polar residue;

the residue corresponding to X96 is a constrained, aliphatic, non-polar, acidic, or polar residue;

residue corresponding to X108 is a basic, constrained, or constrained residue;

the residue corresponding to X117 is a non-polar or polar residue, particularly serine.

9. The engineered ketoreductase polypeptide of claim 1 , in which the ketoreductase polypeptide amino acid sequence further comprises the following substitutions:

the residue corresponding to X7 is a non-polar, polar, or constrained residue;

the residue corresponding to X108 is a basic, constrained, or aromatic residue;

the residue corresponding to X117 is a non-polar or polar residue; and

the residue corresponding to X206 is a polar or non-polar residue.

10. The engineered ketoreductase polypeptide of claim 1 , in which the ketoreductase polypeptide amino acid sequence further comprises the following substitutions:

the residue corresponding to X7 is a non-polar, polar, or constrained residue;

the residue corresponding to X94 is a polar, basic, aliphatic, or non-polar residue;

the residue corresponding to X108 is a basic, constrained, or aromatic residue;

the residue corresponding to X117 is a non-polar or polar residue; and

the residue corresponding to X206 is a polar or non-polar residue.

11. The engineered ketoreductase polypeptide of claim 1 , in which the ketoreductase polypeptide amino acid sequence further comprises at least one of the following substitutions:

the residue corresponding to X7 is a non-polar, polar, or constrained residue;

the residue corresponding to X96 is a constrained, aliphatic, non-polar, acidic, or polar residue;

the residue corresponding to X108 is a basic, constrained, or aromatic residue;

the residue corresponding to X117 is a non-polar or polar residue; and

the residue corresponding to X206 is a polar or non-polar residue.

12. The engineered ketoreductase polypeptide of claim 1 , in which the ketoreductase polypeptide amino acid sequence further comprises at least one of the following substitutions:

the residue corresponding to X7 is a non-polar, polar, or constrained residue;

the residue corresponding to X94 is a polar, basic, aliphatic, or non-polar residue;

the residue corresponding to X96 is a constrained, aliphatic, non-polar, acidic, or polar residue;

the residue corresponding to X108 is a basic, constrained, or aromatic residue;

the residue corresponding to X117 is a non-polar or polar residue; and

the residue corresponding to X206 is a polar or non-polar residue.

13. The engineered ketoreductase polypeptide of claim 1 , in which the ketoreductase polypeptide amino acid sequence further comprises the following substitutions:

the residue corresponding to X117 is a non-polar or polar residue;

the residue corresponding to X173 is an acidic or non-polar residue; and

the residue corresponding to X206 is a polar or non-polar residue.

14. The engineered ketoreductase polypeptide of claim 1 , in which the ketoreductase polypeptide amino acid sequence further comprises the following substitutions:

the residue corresponding to X7 is a non-polar, polar, or constrained residue;

the residue corresponding to X117 is a non-polar or polar residue;

the residue corresponding to X173 is an acidic or non-polar residue; and

the residue corresponding to X206 is a polar or non-polar residue.

15. The engineered ketoreductase polypeptide of claim 1 , in which the ketoreductase polypeptide amino acid sequence further comprises the following substitutions:

the residue corresponding to X7 is a non-polar, polar, or constrained residue;

the residue corresponding to X94 is a polar, basic, aliphatic, or non-polar residue;

the residue corresponding to X117 is a non-polar or polar residue;

the residue corresponding to X173 is an acidic or non-polar residue; and

the residue corresponding to X206 is a polar or non-polar residue.

16. The engineered ketoreductase polypeptide of claim 1 , in which the ketoreductase polypeptide amino acid sequence further comprises at least one of the following substitutions:

the residue corresponding to X7 is a non-polar, polar, or constrained residue;

the residue corresponding to X96 is a constrained, aliphatic, non-polar, acidic, or polar residue;

the residue corresponding to X117 is a non-polar or polar residue;

the residue corresponding to X173 is an acidic or non-polar residue; and

the residue corresponding to X206 is a polar or non-polar residue.

17. The engineered ketoreductase polypeptide of claim 1 , in which the ketoreductase polypeptide amino acid sequence further comprises at least one of the following substitutions:

the residue corresponding to X7 is a non-polar, polar, or constrained residue;

the residue corresponding to X94 is a polar, basic, aliphatic, or non-polar residue;

the residue corresponding to X96 is a constrained, aliphatic, non-polar, acidic, or polar residue;

the residue corresponding to X117 is a non-polar or polar residue;

the residue corresponding to X173 is an acidic or non-polar residue;

the residue corresponding to X206 is a polar or non-polar residue.

18. The engineered ketoreductase polypeptide of claim 1 , wherein the percent stereomeric excess is at least 90%.

19. A method for stereoselectively reducing substrate 3-ketothiolane to product (R)-3-hydroxythiolane, which comprises contacting the 3-ketothiolane with the engineered ketoreductase polypeptide of claim 1 , under reaction conditions suitable for reducing or converting the 3-ketothiolane to the (R)-3-hydroxythiolane.

20. The method of claim 19 , wherein 3-ketothiolane is reduced to (R)-3-hydroxythiolane with at least 70% stereomeric excess.

Continuity (11)
Continuation 17813950 · Jul 21, 2022
Continuation 17080676 · Oct 26, 2020
Continuation 16457396 · Jun 28, 2019
Continuation 15902301 · Feb 22, 2018
Continuation 15184018 · Jun 16, 2016
Continuation 14597996 · Jan 15, 2015
Continuation 13525048 · Jun 15, 2012
Continuation 13110789 · May 18, 2011
Division 12197286 · Aug 24, 2008
Provisional Application 60957974 · Aug 24, 2007
Related Publication 20240229091A1 · Jul 11, 2024
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