KETOREDUCTASE POLYPEPTIDES AND USES THEREOF

• US 20090155863A1
• Filed: 09/29/2008
• Published: 06/18/2009
• Est. Priority Date: 09/28/2007
• Status: Active Grant

• Current Assignee: Codexis, Inc.
• Original Assignee: Codexis, Inc.
• Inventors: LIANG, JACK, LALONDE, JAMES, BORUP, BIRTHE, MITCHELL, VESNA, MUNDORFF, EMILY, HUISMAN, GJALT W.
• Granted Patent: US 8,088,610 B2
• Time in Patent Office: Days
• US Class Current: 435/122
• CPC Class Codes: C12N 9/0006 acting on CH-OH groups as donors (1.1)
• CPC Class Codes: C12P 17/12 containing a six-membered hetero ring

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

1. A ketoreductase polypeptide capable of converting the substrate 2-[3-[3-[2-(7-chloro-2-quinolinyl)ethenyl]phenyl]-3-oxopropyl]benzoic acid methyl ester to the product (S,E)-methyl 2-(3-(3-(2-(7-chloroquinolin-2-yl)vinyl)phenyl)-3-hydroxypropyl)benzoate at a rate that is at least 1.5 times the rate of the polypeptide of SEQ ID NO:

4.

2. The polypeptide of claim 1, which is capable of converting the substrate to the product with a percent stereomeric excess of at least about 95%.

3. The polypeptide of claim 1, which is capable of converting the substrate to the product with a percent stereomeric excess of at least about 99%.

4. The polypeptide of claim 1, which comprises an amino acid sequence that is at least about 85% identical to a reference sequence based on SEQ ID NO:

2, 4 or 114 having the following features;

residue corresponding to X190 is proline, and residue corresponding to X202 is tryptophan;

with the proviso that the ketoreductase polypeptide has an amino acid sequence in which the residue corresponding X190 is a constrained residue, and the residue corresponding to X202 is an aromatic residue.

5. The polypeptide of claim 1, which comprises an amino acid sequence that is at least about 85% identical to a reference sequence based on SEQ ID NO:

2, 4 or 114 having the following features;

residue corresponding to X190 is proline;

residue corresponding to X195 is arginine; and

the residue correspond X202 is a tryptophan;

with the proviso that the ketoreductase polypeptide has an amino acid sequence in which the residue corresponding X190 is a constrained residue, the residue corresponding to X195 is a basic, acidic, non-polar or polar residue; and

the residue corresponding to X202 is an aromatic residue.

6. The polypeptide of claim 4 which comprises an amino acid sequence in which the residue corresponding to X190 is proline, and the residue corresponding to X202 is tryptophan.

7. The polypeptide of claim 5 which comprises an amino acid sequence in which residue corresponding to X190 is proline, residue corresponding to X195 is arginine, and residue corresponding to X202 is tryptophan.

8. The polypeptide of claim 4 in which the ketoreductase amino acid sequence has additionally one or more of the following features:

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

residue corresponding to X17 is a non-polar or aliphatic residue;

residue corresponding to X25 is a polar or acidic residue;

residue corresponding to X27 is an aromatic residue;

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

residue corresponding to X53 is a polar, non-polar, or acidic residue;

residue corresponding to X57 is a non-polar or aliphatic residue;

residue corresponding to X64 is a polar, aliphatic, or non-polar residue;

residue corresponding to X66 is an acidic residue;

residue corresponding to X68 is a polar, acidic, aliphatic, or non-polar residue;

residue corresponding to X72 is a basic residue;

residue corresponding to X76 is an aliphatic, non-polar, or polar residue;

residue corresponding to X80 is an aliphatic, non-polar, or polar residue;

residue corresponding to X93 is a polar, aliphatic, or non-polar residue;

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

residue corresponding to X95 is an aliphatic, non-polar, or acidic residue;

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

residue corresponding to X97 is a basic, constrained or polar residue;

residue corresponding to X100 is a basic, acidic, polar, aliphatic, or non-polar residue;

residue corresponding to X108 is a basic or constrained residue;

residue corresponding to X111 is a non-polar or aliphatic residue;

residue corresponding to X139 is a non-polar or aliphatic residue;

residue corresponding to X145 is an acidic, aliphatic, or non-polar residue;

residue corresponding to X147 is an aromatic, aliphatic, or non-polar residue;

residue corresponding to X148 is a non-polar or aliphatic residue;

residue corresponding to X151 is a constrained, non-polar or aliphatic residue;

residue corresponding to X152 is a polar, aromatic, aliphatic, or non-polar residue;

residue corresponding to X163 is a non-polar or aliphatic residue;

residue corresponding to X165 is a polar, non-polar or aliphatic residue;

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

residue corresponding to X179 is an aromatic, basic, aliphatic, or non-polar residue;

residue corresponding to X194 is a constrained, polar, aliphatic, or non-polar residue;

residue corresponding to X196 is a non-polar or aliphatic residue;

residue corresponding to X197 is an acidic residue;

residue corresponding to X198 is an acidic or polar residue;

residue corresponding to X210 is a polar or basic residue;

residue corresponding to X211 is a basic, aliphatic, non-polar, acidic, or polar residue;

residue corresponding to X212 is a polar, aliphatic, or non-polar;

residue corresponding to X216 is a constrained or basic residue;

residue corresponding to X223 is a non-polar or aliphatic residue;

residue corresponding o X226 is a non-polar or polar residue;

residue corresponding to X233 is a polar or acidic;

residue corresponding to X235 is a polar or aromatic residue;

residue corresponding to X248 is a non-polar or aromatic residue;

residue corresponding to X249 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 the reference sequence.

9. The polypeptide of claim 4 in which the ketoreductase amino acid sequence has additionally one or more of the following features:

residue corresponding to X7 is histidine;

residue corresponding to X17 is methionine;

residue corresponding to X25 is aspartic acid;

residue corresponding to X27 is tyrosine;

residue corresponding to X41 is serine or alanine;

residue corresponding to X53 is aspartic acid;

residue corresponding to X57 is valine;

residue corresponding to X64 is valine;

residue corresponding to X66 is glutamic acid;

residue corresponding to X68 is valine;

residue corresponding to X72 is arginine;

residue corresponding to X76 is alanine;

residue corresponding to X80 is threonine or valine;

residue corresponding to X93 is serine;

residue corresponding to X94 is cysteine or glycine;

residue corresponding to X95 is leucine or glutamic acid;

residue corresponding to X96 is cysteine, asparagine, valine, leucine, isoleucine, or threonine;

residue corresponding to X97 histidine or serine;

residue corresponding to X100 is lysine;

residue corresponding to X108 is histidine;

residue corresponding to X111 is methionine;

residue corresponding to X139 is leucine;

residue corresponding to X145 is glutamine or leucine;

residue corresponding to X147 is leucine;

residue corresponding to X148 is valine;

residue corresponding to X151 is alanine;

residue corresponding to X152 is serine;

residue corresponding to X163 is isoleucine;

residue corresponding to X165 is asparagine;

residue corresponding to X173 is valine;

residue corresponding to X179 is phenylalanine, lysine, or leucine;

residue corresponding to X194 is glutamine, serine, or leucine;

residue corresponding to X196 is leucine;

residue corresponding to X197 is glutamic acid;

residue corresponding to X198 is glutamic acid or glutamine;

residue corresponding to X210 is arginine;

residue corresponding to X211 is valine, leucine, threonine, or isoleucine;

residue corresponding to X212 is serine or valine;

residue corresponding to X216 is arginine;

residue corresponding to X223 is valine;

residue corresponding to X226 is threonine;

residue corresponding to X233 is asparagine or aspartic acid;

residue corresponding to X235 is tryptophan;

residue corresponding to X248 is glycine or tryptophan;

residue corresponding to X249 is tryptophan; and

wherein optionally the amino acid sequence has one or more residue differences at other amino acid residue positions as compared to the reference sequence.

10. The polypeptide of claim 4 in which the ketoreductase amino acid sequence has additionally one or more or at least all of the following features:

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

residue corresponding to X96 is a cysteine, polar, aliphatic, or non-polar residue, and residue corresponding to X211 is a basic, aliphatic, non-polar, acidic, or polar residue; and

wherein optionally the amino acid sequence has one or more residue differences at other amino acid residue positions as compared to the reference sequence.

11. The polypeptide of claim 4 in which the ketoreductase amino acid sequence has additionally one or more or at least all of the following features:

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

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

residue corresponding to X147 is an aromatic, aliphatic, or non-polar residue; and

wherein optionally the amino acid sequence has one or more residue differences at other amino acid residue positions as compared to the reference sequence.

12. The polypeptide of claim 4 in which the ketoreductase amino acid sequence has additionally one or more or at least all of the following features:

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

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

residue corresponding to X147 is an aromatic, aliphatic, or non-polar residue;

residue corresponding to X211 is a basic, aliphatic, non-polar, acidic, or polar residue; and

wherein optionally the amino acid sequence has one or more residue differences at other amino acid residue positions as compared to the reference sequence.

13. The polypeptide of claim 4 in which the ketoreductase amino acid sequence has additionally one or more or at least all of the following features:

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

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

residue corresponding to X194 is a constrained, polar, aliphatic, or non-polar residue;

residue corresponding to X211 is a basic, aliphatic, non-polar, acidic, or polar residue; and

wherein optionally the amino acid sequence has one or more residue differences at other amino acid residue positions as compared to the reference sequence.

14. The polypeptide of claim 4 in which the ketoreductase amino acid sequence has additionally one or more or at least all of the following features:

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

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

residue corresponding to X147 is an aromatic, aliphatic, or non-polar residue;

residue corresponding to X194 is a constrained, polar, aliphatic, or non-polar residue;

residue corresponding to X211 is a basic, aliphatic, non-polar, acidic, or polar residue; and

wherein optionally the amino acid sequence has one or more residue differences at other amino acid residue positions as compared to the reference sequence.

15. The polypeptide of claim 4 in which the ketoreductase amino acid sequence has additionally one or more or at least all of the following features:

residue corresponding to X93 is a polar, aliphatic, or non-polar residue;

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

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

residue corresponding to X211 is a basic, aliphatic, non-polar, acidic, or polar residue; and

wherein optionally the amino acid sequence has one or more residue differences at other amino acid residue positions as compared to the reference sequence.

16. The polypeptide of claim 4 in which the ketoreductase amino acid sequence has additionally one or more or at least all of the following features:

residue corresponding to X93 is a polar, aliphatic, or non-polar residue;

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

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

residue corresponding to X100 is a basic, acidic, polar, aliphatic or non-polar residue;

residue corresponding to X147 is an aromatic, aliphatic, or non-polar residue;

residue corresponding to X152 is a polar, aromatic, aliphatic, or non-polar residue;

residue corresponding to X194 is a constrained, polar, aliphatic, or non-polar residue;

residue corresponding to X196 is a non-polar or aliphatic residue;

residue corresponding to X211 is a basic, aliphatic, non-polar, acidic, or polar residue; and

wherein optionally the amino acid sequence has one or more residue differences at other amino acid residue positions as compared to the reference sequence.

17. The polypeptide of claim 4 in which the ketoreductase amino acid sequence has additionally one or more or at least all of the following features:

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

residue corresponding to X66 is an acidic residue;

residue corresponding to X93 is a polar, aliphatic, or non-polar residue;

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

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

residue corresponding to X147 is an aromatic, aliphatic or non-polar residue;

residue corresponding to X194 is a constrained, polar, aliphatic, or non-polar residue;

residue corresponding to X196 is a non-polar or aliphatic residue;

residue corresponding to X198 is an acidic or polar residue;

residue corresponding to X211 is a basic, aliphatic, non-polar, acidic, or polar residue; and

wherein optionally the amino acid sequence has one or more residue differences at other amino acid residue positions as compared to the reference sequence.

18. The polypeptide of claim 4 in which the ketoreductase amino acid sequence has additionally one or more or at least all of the following features:

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

residue corresponding to X17 is a non-polar or aliphatic residue;

residue corresponding to X27 is an aromatic residue;

residue corresponding to X64 is polar, non-polar or aliphatic residue;

residue corresponding to X66 is an acidic residue;

residue corresponding to X80 is an aliphatic, non-polar or polar residue;

residue corresponding to X93 is a polar, aliphatic, or non-polar residue;

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

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

residue corresponding to X100 is a basic or constrained residue;

residue corresponding to X147 is an aromatic, aliphatic or non-polar residue;

residue corresponding to X194 is a constrained, polar, aliphatic, or non-polar residue;

residue corresponding to X196 is a non-polar or aliphatic residue;

residue corresponding to X198 is an acidic or polar residue;

residue corresponding to X211 is a basic, aliphatic, non-polar, acidic, or polar residue;

residue corresponding to X216 is a constrained or basic residue;

wherein optionally the amino acid sequence has one or more residue differences at other amino acid residue position positions as compared to the reference sequence.

19. The polypeptide of claim 4 in which the ketoreductase amino acid sequence has additionally all of the following features:

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

residue corresponding to X17 is a non-polar or aliphatic residue;

residue corresponding to X27 is an aromatic residue;

residue corresponding to X64 is polar, non-polar or aliphatic residue;

residue corresponding to X80 is an aliphatic, non-polar or polar residue;

residue corresponding to X93 is a polar, aliphatic, or non-polar residue;

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

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

residue corresponding to X100 is a basic or constrained residue;

residue corresponding to X147 is an aromatic, aliphatic or non-polar residue;

residue corresponding to X194 is a constrained, polar, aliphatic, or non-polar residue;

residue corresponding to X196 is a non-polar or aliphatic residue;

residue corresponding to X198 is an acidic or polar residue;

residue corresponding to X211 is a basic, aliphatic, non-polar, acidic, or polar residue;

residue corresponding to X216 is a constrained or basic residue;

wherein optionally the amino acid sequence has one or more residue differences at other amino acid residue positions as compared to the reference sequence.

20. The polypeptide of claim 4 in which the ketoreductase amino acid sequence has additionally all of the following features:

residue corresponding to X7 is histidine;

residue corresponding to X17 is methionine;

residue corresponding to X27 is an aromatic residue;

residue corresponding to X64 is tyrosine;

residue corresponding to X80 is threonine;

residue corresponding to X93 is serine;

residue corresponding to X94 is cysteine;

residue corresponding to X96 is valine, leucine, or isoleucine;

residue corresponding to X100 is lysine;

residue corresponding to X147 is leucine;

residue corresponding to X194 is glutamine;

residue corresponding to X196 is leucine;

residue corresponding to X198 is glutamic acid or glutamine;

residue corresponding to X211 is valine;

residue corresponding to X216 is arginine;

wherein optionally the amino acid sequence has one or more residue differences at other amino acid residue positions as compared to the reference sequence.

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

residue corresponding to X7 is histidine;

residue corresponding to X96 is threonine;

residue corresponding to X147 is leucine; and

residue corresponding to X196 is leucine.

22. The polypeptide of claim 21 in which the ketoreductase amino acid sequence has additionally the following features:

residue corresponding to X93 is serine;

residue corresponding to X94 is cysteine;

residue corresponding to X96 is leucine; and

residue corresponding to X211 is valine.

23. The polypeptide of claim 22 in which the ketoreductase amino acid sequence has additionally the following features:

residue corresponding to X17 is methionine;

residue corresponding to X27 is tyrosine;

residue corresponding to X64 is valine;

residue corresponding to X80 is threonine; and

residue corresponding to X100 is lysine.

24. The polypeptide of claim 22 in which the ketoreductase amino acid sequence has additionally the following features:

residue corresponding to X66 is glutamic acid;

residue corresponding to X96 is isoleucine;

residue corresponding to X194 is glutamine;

residue corresponding to X198 is glutamic acid; and

residue corresponding to X216 is arginine.

25. The polypeptide of claim 22 in which the ketoreductase amino acid sequence has additional the following features:

residue corresponding to X66 is glutamic acid;

residue corresponding to X96 is isoleucine;

residue corresponding to X194 is glutamine; and

residue corresponding to X198 is glutamic acid.

26. The polypeptide of claim 23 in which the ketoreductase amino acid sequence has additionally the following features:

residue corresponding to X194 is glutamine;

residue corresponding to X198 is glutamic acid; and

residue corresponding to X216 is arginine.

27. The polypeptide of claim 24 in which the ketoreductase amino acid sequence has additionally the following features:

residue corresponding to X17 is methionine;

residue corresponding to X27 is tyrosine;

residue corresponding to X64 is valine;

residue corresponding to X66 is aspartic acid;

residue corresponding to X80 is threonine;

residue corresponding to X96 is isoleucine or leucine; and

residue corresponding to X100 is lysine.

35. The polypeptide of claim 1, in which the ketoreductase comprises an amino acid sequence selected from SEQ ID NO:

6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 92, 94, 96, 98, 100, 102, 104, 106, 108, and 110.

42. The polypeptide of claim 1, which is capable of converting the substrate to the product at a rate that is at least 1000 times greater than the reference polypeptide.

43. The polypeptide of claim 42, which comprises an amino acid sequence corresponding to SEQ ID NO:

6, 8, 14, 16, and 20.

48. A polynucleotide encoding a polypeptide according to claim 1.

49. The polynucleotide of claim 48, which is selected from the sequence corresponding to SEQ ID NO:

5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, and 109.

50. An expression vector comprising the polynucleotide of claim 48 operably linked to control sequences suitable for directing expression in a host cell.

51. The expression vector of claim 50 in which the control sequence comprises a promoter.

53. A host cell comprising the expression vector of claim 50.

61. A method for reducing the substrate 2-[3-[3-[2-(7-chloro-2-quinolinyl)ethenyl]phenyl]-3-oxopropyl]benzoic acid methyl ester of formula (I) to the product (S,E)-methyl 2-(3-(3-(2-(7-chloroquinolin-2-yl)vinyl)phenyl)-3-hydroxypropyl)benzoate of formula (II), which comprises contacting the substrate of formula (I) with the ketoreductase polypeptide of claim 1 under reaction conditions suitable for reducing or converting the substrate to the product of formula (II).

62. The method of claim 61 in which the product is present at an stereomeric excess greater than about 99%.

63. The method of claim 61 in which at least about 95% of the substrate is reduced to the product in less than about 24 hours, wherein the concentration of the substrate is at least 100 g/L and the concentration of the polypeptide is less than about 5 g/L, and wherein the polypeptide comprises an amino acid sequence that corresponds to SEQ ID NO:

6, 8, 14, 16, and 20.

65. The method of claim 61 in which the ketoreductase is isolated and/or purified and the reduction reaction is carried out in the presence of a cofactor for the ketoreductase and optionally a regeneration system for the cofactor.

68. A method for reducing the substrate 2-[2-[3-[3-[2-(7-chloro-2-quinolinyl)ethenyl]phenyl]-3-oxopropyl]phenyl]-2-propanol of formula (III) to the product (S,E)-1-(3-(2-(7-chloroquinolin-2-yl)vinyl)phenyl)-3-(2-(2-hydroxypropan-2-yl)phenyl)propan-1-ol, which comprises contacting the substrate of formula (III) with the ketoreductase polypeptide of claim 1 under reaction conditions suitable for reducing the substrate to the product of formula (IV).

69. The method of claim 68, wherein the product is present at an stereomeric excess greater than about 99%.

70. The method of claim 68, wherein at least about 95% of the substrate is reduced to the product in less than about 24 hours, wherein the concentration of the substrate is at least 100 g/L and the concentration of the polypeptide is less than about 5 g/L, and wherein the polypeptide comprises an amino acid sequence that corresponds to SEQ ID NO:

6, 8, 14, 16, and 20.

72. The method of claim 68 in which the ketoreductase is isolated and/or purified and the reduction reaction is carried out in the presence of a cofactor for the ketoreductase and optionally a regeneration system for the cofactor.

75. A method for the synthesis of the compound of formula (XX):

77. A method for the synthesis of the compound of formula (XXI):

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