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

• US 7,977,078 B2
• Filed: 08/24/2008
• Issued: 07/12/2011
• Est. Priority Date: 08/24/2007
• Status: Active Grant

• Current Assignee: Codexis, Inc.
• Original Assignee: Codexis, Inc.
• Inventors: Voladri, Rama, Mundorff, Emily, Lalonde, James, Jenne, Stephane J., Liang, Jack, Huisman, Gjalt
• Primary Examiner(s): RAGHU, GANAPATHIRAM
• Application Number:
  US12/197,286
• Publication Number:
  US 20090093031A1
• Time in Patent Office: 1,052 Days
• Field of Search: 435/189, 435/190, 435/25, 435/117, 435/121, 435/135, 536/23.2
• US Class Current: 435/189
• CPC Class Codes: C12N 9/0006 acting on CH-OH groups as donors (1.1)
• CPC Class Codes: C12P 17/00 Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms C12P13/04 - C12P13/24 take precedence
• CPC Class Codes: C12P 17/167 Heterorings having sulfur atoms as ring heteroatoms, e.g. vitamin B1, thiamine nucleus and open chain analogs
• CPC Class Codes: C12Y 101/01184 Carbonyl reductase (NADPH) (1.1.1.184)
• CPC Class Codes: Y02P 20/52 using catalysts, e.g. selective catalysts

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

1. An engineered ketoreductase polypeptide capable of stereoselectively reducing a 3-ketothiolane to (R)-3-hydroxythiolane with a percent stereomeric excess of at least 70%, which comprises an amino acid sequence that is at least 90% identical to a reference sequence based on SEQ ID NO:

4 and which has at the residue corresponding to X145 a serine.

2. The polypeptide of claim 1 in which the ketoreductase polypeptide amino acid sequence has one or more features selected from the following:

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

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

residue corresponding to X11 is isoleucine or threonine;

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

residue corresponding to X19 is isoleucine or valine;

residue corresponding to X23 is isoleucine or phenylalanine;

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

residue corresponding to X45 is glutamic acid or glycine;

residue corresponding to X49 is lysine or arginine;

residue corresponding to X57 is isoleucine or valine;

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

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

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

residue corresponding to X82 is glycine or serine;

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

residue corresponding to X95 is valine or alanine;

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

residue corresponding to X97 is lysine, arginine or leucine;

residue corresponding to X106 is glutamic acid or aspartic acid;

residue corresponding to X108 is arginine or histidine;

residue corresponding to X111 is leucine or methionine;

residue corresponding to X117 is glycine or serine;

residue corresponding to X126 is isoleucine or valine;

residue corresponding to X127 is glutamine or arginine;

residue corresponding to X147 is phenylalanine, leucine or serine;

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

residue corresponding to X157 is asparagine, glutamine, threonine, serine, or aspartic acid;

residue corresponding to X163 is valine or isoleucine;

residue corresponding to X173 is aspartic acid or glycine;

residue corresponding to X177 is lysine or arginine;

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

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

residue corresponding to X198 is aspartic acid or glycine;

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

residue corresponding to X206 is methionine or glutamine;

residue corresponding to X208 is glutamine, histidine or arginine;

residue corresponding to X210 is threonine or alanine;

residue corresponding to X211 is lysine or glutamic acid;

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

residue corresponding to X217 is isoleucine or phenylalanine;

residue corresponding to X223 is isoleucine or valine;

residue corresponding to X226 is isoleucine or valine; and

wherein the amino acid sequence can optionally have one or more differences at other amino acid residues as compared to the reference sequence.

3. The polypeptide of claim 1 in which the ketoreductase polypeptide amino acid sequence has one or more or at least all of the features selected from the following:

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

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

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

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

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

residue corresponding to X157 is a polar or acidic residue;

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

residue corresponding to X206 is a polar or non-polar residue;

residue corresponding to X223 is an aliphatic or non-polar residue; and

wherein the amino acid sequence can optionally have one or more differences at other amino acid residues as compared to the reference sequence.

4. The polypeptide of claim 1 in which the ketoreductase polypeptide amino acid sequence has at least the following features;

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

residue corresponding to X157 is a polar or acidic residue; and

wherein the amino acid sequence can optionally have one or more differences at other amino acid residues as compared to the reference sequence.

5. The polypeptide of claim 1 in which the ketoreductase polypeptide amino acid sequence has at least the following features:

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

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

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

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

residue corresponding to X157 is a polar or acidic residue, particularly a threonine; and

wherein the amino acid sequence can optionally have one or more differences at other amino acid residues as compared to the reference sequence.

6. The polypeptide of claim 1 in which the ketoreductase polypeptide amino acid sequence has at least the following features:

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

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

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

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

residue corresponding to X157 is a polar or acidic residue, particularly a threonine; and

wherein the amino acid sequence can optionally have one or more differences at other amino acid residues as compared to the reference sequence.

7. The polypeptide of claim 1 in which the ketoreductase polypeptide amino acid sequence has at least the following features:

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

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

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;

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

residue corresponding to X157 is a polar or acidic residue, particularly a threonine; and

wherein the amino acid sequence can optionally have one or more differences at other amino acid residues as compared to the reference sequence.

8. The polypeptide of claim 1 in which the ketoreductase polypeptide amino acid sequence has at least the following features:

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

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

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

residue corresponding to X157 is a polar or acidic residue;

residue corresponding to X206 is a polar or non-polar residue; and

wherein the amino acid sequence can optionally have one or more differences at other amino acid residues as compared to the reference sequence.

9. The polypeptide of claim 1 in which the ketoreductase polypeptide amino acid sequence has at least the following features:

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

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

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

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

residue corresponding to X157 is a polar or acidic residue;

residue corresponding to X206 is a polar or non-polar residue; and

wherein the amino acid sequence can optionally have one or more differences at other amino acid residues as compared to the reference sequence.

10. The polypeptide of claim 1 in which the ketoreductase polypeptide amino acid sequence has at least the following features:

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

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

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

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

residue corresponding to X157 is a polar or acidic residue;

residue corresponding to X206 is a polar or non-polar residue; and

wherein the amino acid sequence can optionally have one or more differences at other amino acid residues as compared to the reference sequence.

11. The polypeptide of claim 1 in which the ketoreductase polypeptide amino acid sequence has at least the following features:

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

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

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

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

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

residue corresponding to X157 is a polar or acidic residue;

residue corresponding to X206 is a polar or non-polar residue; and

wherein the amino acid sequence can optionally have one or more differences at other amino acid residues as compared to the reference sequence.

12. The polypeptide of claim 1 in which the ketoreductase polypeptide amino acid sequence has at least the following features:

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

residue corresponding to X157 is a polar or acidic residue;

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

residue corresponding to X206 is a polar or non-polar residue; and

wherein the amino acid sequence can optionally have one or more differences at other amino acid residues as compared to the reference sequence.

13. The polypeptide of claim 1 in which the ketoreductase polypeptide amino acid sequence has at least the following features:

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

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

residue corresponding to X157 is a polar or acidic residue;

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

residue corresponding to X206 is a polar or non-polar residue; and

wherein the amino acid sequence can optionally have one or more differences at other amino acid residues as compared to the reference sequence.

14. The polypeptide of claim 1 in which the ketoreductase polypeptide amino acid sequence has at least the following features:

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

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

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

residue corresponding to X157 is a polar or acidic residue;

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

residue corresponding to X206 is a polar or non-polar residue; and

wherein the amino acid sequence can optionally have one or more differences at other amino acid residues as compared to the reference sequence.

15. The polypeptide of claim 1 in which the ketoreductase polypeptide amino acid sequence has at least the following features:

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

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

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

residue corresponding to X157 is a polar or acidic residue;

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

residue corresponding to X206 is a polar or non-polar residue; and

wherein the amino acid sequence can optionally have one or more differences at other amino acid residues as compared to the reference sequence.

16. The polypeptide of claim 1 in which the ketoreductase polypeptide amino acid sequence has at least the following features:

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

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

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

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

residue corresponding to X157 is a polar or acidic residue;

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

residue corresponding to X206 is a polar or non-polar residue; and

wherein the amino acid sequence can optionally have one or more differences at other amino acid residues as compared to the reference sequence.

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

18. The polypeptide of claim 17, which comprises an amino acid sequence corresponding to SEQ ID NO:

98.

19. The polypeptide of claim 1, wherein the percent stereomeric excess is at least 98%.

20. The polypeptide of claim 1, which is capable of converting 3-ketothiolane to (R)-3-hydroxythiolane at a rate that is improved over the wild-type polypeptide of SEQ ID NO:

4.

21. The polypeptide of claim 1, which is capable of of stereoselectively reducing 3-ketothiolane to (R)-3-hydroxythiolane with a percent stereomeric excess of at least 95%, which has improved activity and thermostability as compared to the wild-type polypeptide of SEQ ID NO:

4.

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

23. The method of claim 22, wherein 3-ketothiolane is reduced to (R)-3-hydroxythiolane with at least 70% stereomeric excess.

24. The method of claim 22, wherein 3-ketothiolane is reduced to (R)-3-hydroxythiolane with at least 90% stereomeric excess with the ketoreductase polypeptide.

25. The method of claim 22, wherein the 3-ketothiolase is reduced to (R)-3-hydroxythiolane with at least 98% stereomeric excess.

26. The method of claim 22, wherein the substrate is at least 100 g/L and at least 90% of the substrate is converted to product in less than 24 hrs, wherein the ketoreductase polypeptide is about 0.8-1.0 g/L.

27. The method of claim 22, wherein the substrate is at least 100 g/L and at least 90% of the substrate is converted to product in less than 20 hrs, wherein the ketoreductase polypeptide is about 0.8-1.0 g/L.

28. A composition comprising 3-ketothiolane and a ketoreductase of claim 1.

29. The polypeptide of claim 1 in which the ketoreductase polypeptide amino acid sequence has at least the following features:

residue corresponding to X72 is a basic residue;

residue corresponding to X117 is a polar residue;

residue corresponding to X157 is a polar residue;

residue corresponding to X223 is an aliphatic residue; and

wherein the amino acid sequence can optionally have one or more differences at other amino acid residues as compared to the reference sequence.

30. The polypeptide of claim 29 in which the ketoreductase polypeptide amino acid sequence has at least the following features:

residue corresponding to X72 is arginine;

residue corresponding to X117 is serine;

residue corresponding to X157 is threonine; and

residue corresponding to X223 is valine.

31. The polypeptide of claim 1 in which the ketoreductase polypeptide amino acid sequence has at least the following features:

;

residue corresponding to X7 is a polar residue;

residue corresponding to X108 is a constrained residue;

residue corresponding to X117 is a polar residue;

residue corresponding to X157 is a polar residue;

residue corresponding to X223 is an aliphatic residue; and

wherein the amino acid sequence can optionally have one or more differences at other amino acid residues as compared to the reference sequence.

32. The polypeptide of claim 31 in which the ketoreductase polypeptide amino acid sequence has at least the following features:

residue corresponding to X7 is serine;

residue corresponding to X108 is histidine;

residue corresponding to X117 is serine;

residue corresponding to X157 is threonine; and

residue corresponding to X223 is valine.

33. The polypeptide of claim 1 in which the ketoreductase polypeptide amino acid sequence has at least the following features:

residue corresponding to X7 is a polar residue;

residue corresponding to X49 is a basic residue;

residue corresponding to X111 is a non-polar residue;

residue corresponding to X117 is a polar residue;

residue corresponding to X157 is a polar residue;

residue corresponding to X173 is a non-polar residue;

residue corresponding to X223 is an aliphatic residue; and

wherein the amino acid sequence can optionally have one or more differences at other amino acid residues as compared to the reference sequence.

34. The polypeptide of claim 33 in which the ketoreductase polypeptide amino acid sequence has at least the following features:

residue corresponding to X7 is serine;

residue corresponding to X49 is arginine;

residue corresponding to X111 is methionine;

residue corresponding to X117 is serine;

residue corresponding to X157 is threonine;

residue corresponding to X173 is glycine; and

residue corresponding to X223 is valine.

35. The polypeptide of claim 1 in which the ketoreductase polypeptide amino acid sequence has at least the following features:

residue corresponding to X7 is a polar residue;

residue corresponding to X94 is a non-polar or polar residue;

residue corresponding to X96 is a constrained residue;

residue corresponding to X108 is a constrained residue;

residue corresponding to X117 is a polar residue;

residue corresponding to X157 is a polar residue;

residue corresponding to X206 is a polar residue;

residue corresponding to X223 is an aliphatic residue; and

wherein the amino acid sequence can optionally have one or more differences at other amino acid residues as compared to the reference sequence.

36. The polypeptide of claim 35 in which the ketoreductase polypeptide amino acid sequence has at least the following features:

residue corresponding to X7 is serine;

residue corresponding to X94 is threonine;

residue corresponding to X96 is proline;

residue corresponding to X108 is histidine;

residue corresponding to X117 is serine;

residue corresponding to X157 is threonine;

residue corresponding to X206 is glutamine; and

residue corresponding to X223 is valine.

37. In a method for the synthesis of an antibiotic of structural formula (III):

38. The polypeptide of claim 1, which comprises an amino acid sequence corresponding to SEQ ID NO:

98.

39. The method of claim 22, wherein the ketoreductase polypeptide is SEQ ID NO:

98.