BIOCATALYSTS AND METHODS FOR THE SYNTHESIS OF (S)-3-(1-AMINOETHYL)-PHENOL

• US 20130089898A1
• Filed: 06/16/2011
• Published: 04/11/2013
• Est. Priority Date: 06/17/2010
• Status: Active Grant

• Current Assignee: Codexis, Inc.
• Original Assignee: Codexis, Inc.
• Inventors: Gohel, Anupam, Oh, Seong Ho, Smith, Derek, Lalonde, James, Wong, Brian, Cabirol, Fabien
• Granted Patent: US 8,852,900 B2
• Time in Patent Office: Days
• US Class Current: 435/128
• CPC Class Codes: C07B 2200/07 Optical isomers
• CPC Class Codes: C07C 213/00 Preparation of compounds containing amino and hydroxy, amino and etherified hydroxy or amino and esterified hydroxy groups bound to the same carbon skeleton
• CPC Class Codes: C07C 213/08 by reactions not involving the formation of amino groups, hydroxy groups or etherified or esterified hydroxy groups
• CPC Class Codes: C07C 215/50 with amino groups and the six-membered aromatic ring, or the condensed ring system containing that ring, bound to the same carbon atom of the carbon chain
• CPC Class Codes: C07C 269/00 Preparation of derivatives of carbamic acid, i.e. compounds containing any of the groups , the nitrogen atom not being part of nitro or nitroso groups
• CPC Class Codes: C07C 271/44 to hydrogen atoms or to carbon atoms of unsubstituted hydrocarbon radicals
• CPC Class Codes: C07K 2319/02 containing a signal sequence
• CPC Class Codes: C07K 2319/21 containing a His-tag
• CPC Class Codes: C07K 2319/41 containing a Myc-tag
• CPC Class Codes: C12N 9/1096 transferring nitrogenous groups (2.6)
• CPC Class Codes: C12P 13/001 Amines; Imines
• CPC Class Codes: C12Y 206/01018 Beta-alanine-pyruvate transaminase (2.6.1.18)

Abstract

The present disclosure provides engineered transaminase polypeptides having improved properties as compared to naturally occurring transaminases including the ability of converting the substrate, 3′-hydroxyacetophenone to (S)-3-(1-aminoethyl)-phenol in enantiomeric excess and high percentage conversion. Also provided are polynucleotides encoding the engineered transaminases, host cells capable of expressing the engineered transaminases, and methods of using the engineered transaminases to synthesize (S)-3-(1-aminoethyl)-phenol and related compounds useful in the production of active pharmaceutical ingredients.

Claims

1. An engineered transaminase polypeptide capable of converting 3-hydroxyacetophenone (compound (1)) to (S)-3-(1-aminoethyl)-phenol (compound (2)),

2. The polypeptide of claim 1 in which the transaminase polypeptide has at least 1.2 times the activity of SEQ ID NO:

2 in the rate of conversion of 3-hydroxyacetophenone to (S)-3-(1-aminoethyl)-phenol under reaction conditions of 35 g/L of 3-hydroxyacetophenone, 1.5 M isopropylamine, 1 mM pyridoxal phosphate, 5% v/v DMSO, pH 7.5, and 45°

C.

3. The polypeptide of claim 1 in which the amino acid residue differences as compared to SEQ ID NO:

2 are selected from the following;

residue at position X18 is A;

residue at position X163 is F or H;

residue at position X235 is P;

residue at position X244 is T;

residue at position X323 is T;

residue at position X383 is V;

residue at position X424 is A; and

residue at position X427 is Y.

4. The polypeptide of claim 1 or 3 in which the transaminase polypeptide amino acid sequence has additional amino acid residue differences as compared to SEQ ID NO:

2 at one or more of the following positions;

X21, X31, X74;

X113, X122, X130, X133, X146, X147, X153, X164, X167, X168, X174, X233, X245, X286, X293, X315, X316, X318, X332, X375, X394, and X418.

5. The polypeptide of claim 4 in which the additional amino acid residue differences as compared to SEQ ID NO:

2 are selected from the following;

residue at position X21 is T;

residue at position X31 is M;

residue at position X74 is T;

residue at position X113 is V;

residue at position X122 is E;

residue at position X130 is L or M;

residue at position X133 is R;

residue at position X146 is L;

residue at position X147 is K;

residue at position X153 is S;

residue at position X164 is S or G;

residue at position X167 is K;

residue at position X168 is D or K;

residue at position X174 is E;

residue at position X233 is T or I;

residue at position X245 is T;

residue at position X286 is S;

residue at position X293 is S;

residue at position X312 is D;

residue at position X315 is G;

residue at position X316 is H;

residue at position X318 is D;

residue at position X332 is T;

residue at position X375 is V;

residue at position X394 is G; and

residue at position X418 is Q.

6. The polypeptide of claim 1 in which the polypeptide amino acid sequence comprises at least the following amino acid difference as compared to SEQ ID NO:

2;

residue at position X163 is F or H.

7. The polypeptide of claim 5 in which the polypeptide amino acid sequence comprises at least the following amino acid residue differences as compared to SEQ ID NO:

2;

residue at position X153 is S; and

residue at position X233 is I.

8. The polypeptide of claim 5 in which the polypeptide amino acid sequence comprises at least the following amino acid residue differences as compared to SEQ ID NO:

2;

residue at position X153 is S;

residue at position X163 is F or H; and

residue at position X233 is T or I.

9. The polypeptide of claim 5 in which the polypeptide amino acid sequence has at least the following amino acid residue differences as compared to SEQ ID NO:

2;

residue at position X153 is S;

residue at position X163 is F or H;

residue at position X233 is T or I;

residue at position X235 is P;

residue at position X424 is A; and

residue at position X427 is Y.

10. The polypeptide of claim 5 in which the polypeptide amino acid sequence has at least the following amino acid residue differences as compared to SEQ ID NO:

2;

residue at position X18 is A;

residue at position X21 is H;

residue at position X153 is S;

residue at position X163 is F or H;

residue at position X233 is T or I;

residue at position X235 is P;

residue at position X323 is T;

residue at position X383 is V;

residue at position X424 is A; and

residue at position X427 is Y.

11. The polypeptide of claim 5 in which the polypeptide amino acid sequence has at least the following amino acid residue differences as compared to SEQ ID NO:

2;

residue at position X18 is A;

residue at position X21 is H;

residue at position X147 is K;

residue at position X153 is S;

residue at position X163 is F or H;

residue at position X233 is T or I;

residue at position X235 is P;

residue at position X244 is T;

residue at position X323 is T;

residue at position X383 is V;

residue at position X424 is A; and

residue at position X427 is Y.

12. The polypeptide of claim 5 in which the polypeptide amino acid sequence has at least the following amino acid residue differences as compared to SEQ ID NO:

2;

residue at position X18 is A;

residue at position X21 is H;

residue at position X133 is R;

residue at position X146 is L;

residue at position X147 is K;

residue at position X153 is S;

residue at position X163 is F;

residue at position X233 is T or I;

residue at position X235 is P;

residue at position X244 is T;

residue at position X323 is T;

residue at position X383 is V;

residue at position X424 is A; and

residue at position X427 is Y.

13. The polypeptide of claim 5 in which the polypeptide amino acid sequence has at least the following amino acid residue differences as compared to SEQ ID NO:

2;

residue at position X18 is A;

residue at position X21 is H;

residue at position X31 is M;

residue at position X133 is R;

residue at position X146 is L;

residue at position X147 is K;

residue at position X153 is S;

residue at position X163 is F or H;

residue at position X233 is T or I;

residue at position X235 is P;

residue at position X244 is T;

residue at position X318 is D;

residue at position X323 is T;

residue at position X383 is V;

residue at position X424 is A; and

residue at position X427 is Y.

14. The polypeptide of any one of claims 1 to 13 in which the polypeptide amino acid sequence has additional amino acid residue differences as compared to SEQ ID NO:

2 at one or more of the following positions;

X9, X45, X86, X177, X211, X294, X324, and X391.

15. The polypeptide of claim 14 in which the additional amino acid residue differences as compared to SEQ ID NO:

2 at one or more of positions X9, X45, X86, X177, X211, X294, X324, and X391 is selected from the following;

residue at position X9 is T,

residue at position X45 is H,

residue at position X86 is Y,

residue at position X177 is L;

residue at position X211 is K,

residue at position X294 is V,

residue at position X324 is G, and

residue at position X391 is A.

16. The polypeptide of claim 14 in which the polypeptide amino acid sequence comprises at least the following amino acid residue differences as compared to SEQ ID NO:

2;

residue at position X153 is S;

residue at position X177 is L; and

residue at position X233 is T, or I.

17. The polypeptide of claim 1 in which the polypeptide amino acid sequence comprises a sequence selected from the group consisting of SEQ ID NO:

8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, and 44.

18. A polynucleotide encoding the polypeptide of any one of claims 1 to 17.

19. The polynucleotide of claim 18 in which the polynucleotide sequence comprises a sequence selected from the group consisting of SEQ ID NO:

7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, and 43.

20. An expression vector comprising the polynucleotide of claim 18 or 19.

21. The expression vector of claim 20 comprising a control sequence.

22. A host cell comprising the polynucleotide of claims 18 to 19 or the expression vector of any one of claims 20 to 21.

23. A method for preparing a polypeptide of any one of claims 1 to 17 comprising culturing a host cell of claim 22 and isolating the polypeptide from the cell.

24. A method for preparing (S)-3-(1-aminoethyl)-phenol (compound (2)) or an analog of compound (2) in enantiomeric excess,

25. The method of claim 24 in which the suitable reaction conditions comprise the amino donor at a concentration of at least about 0.5 M, at least about 1.0 M, at least about 2.5 M, at least about 5.0 M, at least about 7.5 M, at least about 10.0 M, or more.

26. The method of claim 24 in which the amino donor is selected from the group consisting of isopropylamine, putrescine, L-lysine, D-ornithine, L-ornithine, D-alanine, L-alanine, and mixtures thereof.

27. The method of claim 24 in which the amino donor is isopropylamine or isopropylamine acetate.

28. The method of claim 24 in which the amino donor is isopropylamine acetate.

29. The method of claim 27 which further comprises the removal of acetone formed from the isopropylamine.

30. The method of claim 24 in which the suitable reaction conditions comprise a compound (1) loading of at least about 20 g/L, at least about 25 g/L, at least about 30 g/L, at least about 40 g/L, at least about 50 g/L, at least about 75 g/L, at least about 100 g/L, at least about 150 g/L, at least about 200 g/L, at least about 250 g/L, or even greater.

31. The method of claim 24 in which the suitable reaction conditions comprise an engineered polypeptide concentration of about 0.1 to about 15 g/L, about 0.5 to about 10 g/L, about 1.0 to about 5 g/L, about 2 to about 5 g/L, about 15 g/L, about 10 g/L, about 5, g/L, about 3 g/L, about 2 g/L, about 1.5 g/L, about 1.0 g/L, about 0.75 g/L, or even lower concentration.

32. The method of claim 24 in which the suitable reaction conditions comprise a temperature of about 15°

C. to about 60°

C., about 20°

C. to about 55°

C., about 30°

C. to about 50°

C., about 40°

C. to about 60°

C., about 40°

C. to about 50°

C., about 45°

C. to about 55°

C., about 25°

C., about 30°

C., about 35°

C., about 40°

C., about 45°

C., about 50°

C., about 55°

C., or about 60°

C., or more.

33. The method of claim 24 in which the suitable reaction conditions comprise a pH of about 5.0 to about 11.0, a pH of about 6.0 to about 10.5, a pH of about 6.5 to about 10.0, a pH of about 7.0 to about 9.5, a pH of about 7.0 to about 9.0, a pH of about 7.0 to about 8.5, or a pH of about 7.2 to about 7.8, or about pH 7.5.

34. The method of claim 24 in which the suitable reaction conditions comprise a buffer solution of about 0.05 M TEA to about 0.25 M TEA, or about 0.1 M TEA.

35. The method of claim 24 in which the suitable reaction conditions comprise a substrate loading of compound (1) of at least about 20 g/L, 30 g/L, 40 g/L, 50 g/L, 60 g/L, 70 g/L, 100 g/L, or more, and wherein the method results in at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater conversion of compound (1) to compound (2) in about 48 h or less, in about 36 h or less, or in about 24 h or less.

36. The method of claim 35 in which the amino donor is isopropylamine or isopropylamine acetate at a concentration of at least about 0.5 M, at least about 1.0 M, at least about 2.5 M, at least about 5.0 M, at least about 7.5 M, at least about 10.0 M, or more.

37. The method of claim 35 in which the suitable reaction conditions further comprise an engineered polypeptide concentration of about 0.1 to about 15 g/L, about 0.5 to about 10 g/L, about 1.0 to about 5 g/L, about 2 to about 5 g/L.

38. The method of claim 24, wherein the method results in an enantiomeric excess of compound (2) of at least 97%, 98, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9%.

39. The method of claim 24 in which the suitable reaction conditions comprise addition of the polypeptide to a solution having an initial substrate loading of at least about 20 g/L, 30 g/L, or 40 g/L, followed by addition of further substrate to the solution at a rate of about 4 g/L/h until a final substrate loading of at least about 30 g/L, 40 g/L, 50 g/L, 60 g/L, 70 g/L, 100 g/L or more, is reached.

40. The method of claim 39 in which the further substrate added is in a solution comprising isopropylamine or isopropylamine acetate at a concentration of at least about 0.5 M, at least about 1.0 M, at least about 2.5 M, at least about 5.0 M, at least about 7.5 M, at least about 10.0 M.

41. The method of claim 24 in which the method comprises contacting compound (1) with the transaminase polypeptide.

42. The method of claim 24 in which the suitable reaction conditions comprise:

(1) substrate loading of about 25-200 g/L compound (1);

(2) polypeptide concentration of about 1.0-15 g/L;

(3) IPM concentration of about 0.1-10 M;

(4) PLP cofactor at a concentration of about 0.1-0.3 g/L;

(5) about pH 5.0-10.0; and

(6) temperature of about 30-60°

C.

43. The method of claim 24 in which the suitable reaction conditions comprise:

(1) substrate loading of about 25-75 g/L compound (1);

(2) polypeptide concentration of about 1.0-15 g/L;

(3) IPM concentration of about 0.5-2.5 M;

(4) PLP cofactor at a concentration of about 0.1-0.3 g/L;

(5) about pH 7.0-8.0; and

(6) temperature of about 35-45°

C.

44. The method of claim 24 in which the suitable reaction conditions comprise:

(1) substrate loading of about 25-75 g/L compound (1);

(2) polypeptide concentration of about 1.0-10 g/L;

(3) IPM concentration of about 1.0-2.0 M;

(4) PLP cofactor at a concentration of about 0.1-0.3 g/L;

(5) about pH 7.2-7.8; and

(6) temperature of about 35-45°

C.

45. The method of claim 24, wherein the analog of compound (1) is a compound of Formula I

46. The method of any one of claims 24 to 45 in which the method further comprises one or more steps selected from:

extraction of compound (2) or the analog of compound (2);

isolation of compound (2) or the analog of compound (2);

purification of compound (2) or the analog of compound (2); and

crystallization of compound (2) or the analog of compound (2).

47. The method of any one of claims 24 to 45 in which the method further comprises a step of dimethylating compound (2) or an analog of compound (2) thereby forming compound (3) or an analog of compound (3)

48. The method of claim 47 in which the demethylating step comprises contacting compound (2), or the analog of compound (2), with triethylamine, formic acid, and formaldehyde, under suitable reaction conditions.

49. The method of claim 48 in which the suitable reaction conditions comprise about 0.8 to about 1.6, about 1.0 to about 1.5, about 1.2 to about 1.4 equivalents, or about 1.3 equivalents of triethylamine.

50. The method of claim 48 in which the suitable reaction conditions comprise about 10 to about 18 equivalents, or 12 to 15 equivalents, or about 13.5 equivalents of formic acid.

51. The method of claim 48 in which the suitable reaction conditions comprise about 7 to about 9 equivalents, about 8 to about 9 equivalents, or about 9 equivalents of formaldehyde.

52. The method of claim 48 in which the suitable reaction conditions comprise at temperature of about 60°

C. to about 90°

C., about 70°

C. to about 80°

C., or about 75°

C.

53. The method of claim 48 in which the method results in at least about 95%, 96%, 97%, 98%, 99% or greater conversion of compound (2) to compound (3).

54. The method of any one of claims 47 to 49 in which the method further comprises one or more steps selected from:

extraction of compound (3) or the analog of compound (3);

isolation of compound (3) or the analog of compound (3);

purification of compound (3) or the analog of compound (3); and

crystallization of compound (3) or the analog of compound (3).

55. A process for the preparation of compound (4) in enantiomeric excess,

56. The process of claim 55 in which the step in the process comprises contacting compound (1) with the transaminase polypeptide.

57. The process of claim 55 in which the amino donor is isopropylamine.

58. The method of any one of claims 24 to 57 in which the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO:

8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, and 44.