ENGINEERED PHENYLALANINE AMMONIA LYASE ENZYMES

20240425841

18/690104

September 08, 2022

Aspects of the disclosure relate to phenylalanine ammonia lyase (PAL) enzymes, including engineered PAL enzymes, and their use in catalyzing chemical reactions. [chemical expression included]

Ginkgo Bioworks, Inc. (Boston, MA, US), Synlogic Operating Company, Inc. (Cambridge, MA, US)

1. A host cell that comprises a heterologous polynucleotide encoding a phenylalanine ammonia lyase (PAL) enzyme, wherein the PAL comprises a sequence that is at least 90% identical to any one of SEQ ID NOs: 1, 5, 7, 9, 11, 13 or 15, and wherein the host cell is not a plant cell.

2. The host cell of claim 1, wherein the PAL comprises the sequence of any one of SEQ ID NOs: 1, 5, 7, 9, 11, 13 or 15.

3. The host cell of claim 1 or 2, wherein the heterologous polynucleotide is at least 90% identical to any one of SEQ ID NOs: 2, 6, 8, 10, 12, 14 or 16.

4. The host cell of claim 3, wherein the heterologous polynucleotide comprises the sequence of any one of SEQ ID NOs: 2, 6, 8, 10, 12, 14 or 16.

5. The host cell of any one of claims 1-4, wherein the host cell is a bacterial cell, an archaebacterial cell, a fungal cell, a yeast cell, an animal cell, a mammalian cell, or a human cell.

6. The host cell of claim 5, wherein the host cell is a bacterial cell.

7. The host cell of claim 6, wherein the bacterial cell is an Escherichia coli (E. coli) cell.

8. The host cell of claim 6, wherein the bacterial cell is a Bacillus cell.

9. The host cell of any one of claims 1-5, wherein the host cell is a filamentous fungi cell or a yeast cell.

10. The host cell of claim 7, wherein the E. coli cell is an E. coli Nissle 1917 cell.

11. The host cell of any one of claims 1-10, wherein the PAL comprises one or more amino acid substitutions, additions, and/or deletions relative to the sequence of any one of SEQ ID NOs: 1, 5, 7, 9, 11, 13 or 15.

12. A host cell that comprises a heterologous polynucleotide encoding a phenylalanine ammonia lyase (PAL) enzyme, wherein the PAL comprises an amino acid substitution at one or more of the following amino acid residues relative to SEQ ID NO: 3: I77, F84, M87, A88, V90, A91, Q96, S98, L104, V105, W106, T110, Y158, I165, V172, S175, L214, L219, T243, D253, T345, L364, A394, L406, L407, K413, N415, Q422, I423, C424, F450, K522 and Y529.

13. The host cell of claim 12, wherein the PAL comprises one or more of the following amino acid substitutions relative to SEQ ID NO: 3: I77V, F84Y, M87L, A88S, V90T, A91V, Q96E, S98A, V105F, W106Q, T110S, Y158H, Y158V, 1165V, V172A, S175G, S175T, L214Q, L219I, T243L, D253A, T345S, L364H, A394M, L406V, L407A, L407C, L407S, L407T, K413G, K413S, N415H, Q422H, I423L, C424T, F450A, F450G, F450H, N453A, N453S, K522P and Y529L.

14. The host cell of claim 12 or 13, wherein the PAL further comprises an amino acid substitution at one or more of the following amino acid residues relative to SEQ ID NO: 3: LA, T102, L104, L108, M222, G218 and N453.

15. The host cell of claim 14, wherein the PAL comprises one or more of the following amino acid substitutions relative to SEQ ID NO: 3: L4P, T102E, T102K, T102R, T102S, L104A, L104M, L108A, L108H, L108Q, L108T, L108V, M222I, M222L, M222N, M222T, M222V, G218A, G218S, N453A and N453S.

16. The host cell of any one of claims 12-15, wherein the PAL comprises an amino acid substitution at the following amino acid residues relative to SEQ ID NO: 3: (i) L104 and L219; (ii) L4 and G218; (iii) T102 and G218; (iv) L104, L219 and M222; (v) T102 and G218; (vi) T102 and M222; (vii) L4, L104 and G218; (viii) G218, L104 and L219; (ix) L104, L219 and N453; or (x) G218, L4 and M222.

17. The host cell of claim 16, wherein the PAL comprises any of the following relative to SEQ ID NO: 3: (i) L104M and L219I; (ii) L4P and G218S; (iii) T102K and G218A; (iv) L104M, L219I and M222L; (v) T102S and G218A; (vi) T102K and M222L; (vii) L4P, L104M and G218S; (viii) G218A, L104M and L219I; (ix) L104M, L219I and N453S; or (x) G218S, L4P and M222L.

18. The host cell of claim 17, wherein the PAL comprises the following amino acid substitutions relative to SEQ ID NO: 3: L104M and L219I.

19. The host cell of claim 17, wherein the PAL comprises the following amino acid substitution relative to SEQ ID NO: 3: L4P and G218S.

20. A host cell that comprises a heterologous polynucleotide encoding a phenylalanine ammonia lyase (PAL) enzyme, wherein the PAL comprises an amino acid substitution at one or more of the following amino acid residues relative to SEQ ID NO: 17: T40, M111, N258, N275, C288, M402, S499 and I502.

21. The host cell of claim 20, wherein the PAL comprises one or more of the following amino acid substitutions relative to SEQ ID NO: 17: T40I, M111V, N258I, N258R, N275S, C288S, M402L, S499P and I502V.

22. The host cell of claim 20 or 21, wherein the PAL further comprises an amino acid substitution at one or more of the following amino acid residues relative to SEQ ID NO: 17: S92, H133, 1167, A263, L432, A433 and V470.

23. The host cell of claim 22, wherein the PAL further comprises one or more of the following amino acid substitutions relative to SEQ ID NO: 17: S92G, H133F, H133M, I167K, A263T, L432I, A433S and V470A.

24. The host cell of any one of claims 20-23, wherein the PAL comprises an amino acid substitution at the following amino acid residues relative to SEQ ID NO: 17: (i) C288, T40, 1502 and S499; (ii) S92, L432, V460, H133 and N258; (iii) S92, L432, V470, N258 and T40; (iv) S92, L432, V470 and T40; (v) N258, T40, 1502 and S499; (vi) S92, V470, M111 and T40; (vii) S92, L432, V470, N258 and T40; (viii) S92, V470, A263 and T40; or (ix) S92, L432, M111 and T40.

25. The host cell of claim 24, wherein the PAL comprises any one of the following relative to SEQ ID NO: 17: (i) C288S, T40I, I502V and S499P; (ii) S92G, L432I, V460A, H133F and N258I; (iii) S92G, L432I, V470A, N258I and T40I; (iv) S92G, L432I, V470A and T40I; (v) N258I, T40I, I502V and S499P; (vi) S92G, V470A, M111V and T40I; (vii) S92G, L432I, V470A, N258R and T40I; (viii) S92G, V470A, A263T and T40I; or (ix) S92G, L432I, M111V and T40I.

26. The host cell of any one of claims 20-25, wherein the PAL comprises the amino acid substitution N258R relative to SEQ ID NO: 17.

27. The host cell of claim 25, wherein the PAL comprises the following amino acid substitutions relative to SEQ ID NO: 17: T40I, C288S, S499P and I502V.

28. The host cell of any one of claims 12-27, wherein the host cell is a bacterial cell.

29. The host cell of claim 28, wherein the bacterial cell is an Escherichia coli (E. coli) cell.

30. The host cell of claim 28, wherein the bacterial cell is a Bacillus cell.

31. The host cell of any one of claims 12-27, wherein the host cell is a filamentous fungi cell or a yeast cell.

32. The host cell of claim 29, wherein the E. coli cell is an E. coli Nissle 1917 cell.

33. The host cell of any one of claims 1-32, wherein the PAL is able to convert phenylalanine to trans-cinnamic acid.

34. A method of treating or protecting against a metabolic disorder associated with excess phenylalanine, comprising administering to a subject in need thereof a therapeutically effective amount of a phenylalanine ammonia lyase (PAL) enzyme or a polynucleotide encoding a PAL enzyme, wherein the PAL comprises a sequence that is at least 90% identical to any one of SEQ ID NOs: 1, 5, 7, 9, 11, 13 or 15.

35. A method of treating or protecting against a metabolic disorder associated with excess phenylalanine, comprising administering to a subject in need thereof a cell comprising a heterologous polynucleotide encoding a therapeutically effective amount of a phenylalanine ammonia lyase (PAL) enzyme, wherein the PAL comprises a sequence that is at least 90% identical to any one of SEQ ID NOs: 1, 5, 7, 9, 11, 13 or 15.

36. The method of claim 35, wherein the cell is a human cell, an animal cell, a yeast cell, or a bacterial cell.

37. A method of converting phenylalanine to trans-cinnamic acid, comprising contacting phenylalanine with a phenylalanine ammonia lyase (PAL) enzyme, wherein the PAL comprises a sequence that is at least 90% identical to any one of SEQ ID NOs: 1, 5, 7, 9, 11, 13 or 15.

38. The method of claim 37, wherein the PAL is encoded by a polynucleotide comprising a sequence that is at least 90% identical to any one of SEQ ID NO: 2, 6, 8, 10, 12, 14 or 16.

39. The method of any one of claims 35-38, wherein the PAL comprises one or more amino acid substitutions relative to the sequence of any one of SEQ ID NOs: 1, 5, 7, 9, 11, 13 or 15.

40. A method of treating or protecting against a metabolic disorder associated with excess phenylalanine, comprising administering to a subject in need thereof a therapeutically effective amount of a phenylalanine ammonia lyase (PAL) enzyme or a polynucleotide encoding a PAL enzyme, wherein the PAL comprises an amino acid substitution at one or more of the following amino acid residues relative to SEQ ID NO: 3: I77, F84, M87, A88, V90, A91, Q96, S98, L104, V105, W106, T110, Y158, I165, V172, S175, L214, L219, T243, D253, T345, L364, A394, LA06, L407, K413, N415, Q422, I423, C424, F450, K522 and Y529.

41. A method of treating or protecting against a metabolic disorder associated with excess phenylalanine, comprising administering to a subject in need thereof a cell comprising a heterologous polynucleotide encoding a therapeutically effective amount of a phenylalanine ammonia lyase (PAL) enzyme, wherein the PAL comprises an amino acid substitution at one or more of the following amino acid residues relative to SEQ ID NO: 3: I77, F84, M87, A88, V90, A91, Q96, S98, L104, V105, W106, T110, Y158, I165, V172, S175, L214, L219, T243, D253, T345, L364, A394, L406, L407, K413, N415, Q422, I423, C424, F450, K522 and Y529.

42. The method of claim 41, wherein the cell is a human cell, an animal cell, a yeast cell, or a bacterial cell.

43. A method of converting phenylalanine to trans-cinnamic acid, comprising contacting phenylalanine with a phenylalanine ammonia lyase (PAL) enzyme, wherein the PAL comprises an amino acid substitution at one or more of the following amino acid residues relative to SEQ ID NO: 3: I77, F84, M87, A88, V90, A91, Q96, S98, L104, V105, W106, T110, Y158, I165, V172, S175, L214, L219, T243, D253, T345, L364, A394, L406, L407, K413, N415, Q422, 1423, C424, F450, K522 and Y529.

44. The method of any one of claims 40-43, wherein the PAL comprises one or more of the following amino acid substitutions relative to SEQ ID NO: 3: I77V, F84Y, M87L, A88S, V90T, A91V, Q96E, S98A, V105F, W106Q, T110S, Y158H, Y158V, 1165V, V172A, S175G, S175T, L214Q, L219I, T243L, D253A, T345S, L364H, A394M, L406V, L407A, L407C, L407S, L407T, K413G, K413S, N415H, Q422H, I423L, C424T, F450A, F450G, F450H, N453A, N453S, K522P and Y529L.

45. The method of any one of claims 40-44, wherein the PAL further comprises an amino acid substitution at one or more of the following amino acid residues relative to SEQ ID NO: 3: L4, T102, L104, L108, M222, G218 and N453.

46. The method of claim 45, wherein the PAL comprises one or more of the following amino acid substitutions relative to SEQ ID NO: 3: L4P, T102E, T102K, T102R, T102S, L104A, L104M, L108A, L108H, L108Q, L108T, L108V, M222I, M222L, M222N, M222T, M222V, G218A, G218S, N453A and N453S.

47. The method of any one of claims 40-46, wherein the PAL comprises an amino acid substitution at the following amino acid residues relative to SEQ ID NO: 3: (i) L104 and L219; (ii) L4 and G218; (iii) T102 and G218; (iv) L104, L219 and M222; (v) T102 and G218; (vi) T102 and M222; (vii) L4, L104 and G218; (viii) G218, L104 and L219; (ix) L104, L219 and N453; or (x) G218, L4 and M222.

48. The method of claim 47, wherein the PAL comprises any of the following relative to SEQ ID NO: 3: (i) L104M and L219I; (ii) L4P and G218S; (iii) T102K and G218A; (iv) L104M, L219I and M222L; (v) T102S and G218A; (vi) T102K and M222L; (vii) L4P, L104M and G218S; (viii) G218A, L104M and L219I; (ix) L104M, L219I and N453S; or (x) G218S, L4P and M222L.

49. The method of claim 48, wherein the PAL comprises the following amino acid substitutions relative to SEQ ID NO: 3: L104M and L219I.

50. The method of claim 48, wherein the PAL comprises the following amino acid substitution relative to SEQ ID NO: 3: L4P and G218S.

51. A method of treating or protecting against a metabolic disorder associated with excess phenylalanine, comprising administering to a subject in need thereof a therapeutically effective amount of a phenylalanine ammonia lyase (PAL) enzyme or a polynucleotide encoding a PAL enzyme, wherein the PAL comprises an amino acid substitution at one or more of the following amino acid residues relative to SEQ ID NO: 17: T40, M111, N258, N275, C288, M402, S499 and I502.

52. The method of claim 51, wherein the PAL further comprises an amino acid substitution at one or more of the following amino acid residues relative to SEQ ID NO: 17: S92, H133, 1167, A263, L432, A433 and V470.

53. A method of treating or protecting against a metabolic disorder associated with excess phenylalanine, comprising administering to a subject in need thereof a cell comprising a heterologous polynucleotide encoding a therapeutically effective amount of a phenylalanine ammonia lyase (PAL) enzyme, wherein the PAL comprises an amino acid substitution at one or more of the following amino acid residues relative to SEQ ID NO: 17: T40, M111, N258, N275, C288, M402, S499 and I502.

54. The method of claim 53, wherein the PAL further comprises an amino acid substitution at one or more of the following amino acid residues relative to SEQ ID NO: 17: S92, H133, 1167, A263, L432, A433 and V470.

55. The method of claim 53 or 54, wherein the cell is a human cell, an animal cell, a yeast cell, or a bacterial cell.

56. A method of converting phenylalanine to trans-cinnamic acid, comprising contacting phenylalanine with a phenylalanine ammonia lyase (PAL) enzyme, wherein the PAL comprises an amino acid substitution at one or more of the following amino acid residues relative to SEQ ID NO: 17: T40, M111, N258, N275, C288, M402, S499 and I502.

57. The method of claim 56, wherein the PAL further comprises an amino acid substitution at one or more of the following amino acid residues relative to SEQ ID NO: 17: S92, H133, 1167, A263, L432, A433 and V470.

58. The method of any one of claims 51-57, wherein the PAL comprises one or more of the following amino acid substitutions relative to SEQ ID NO: 17: T40I, M111V, N258I, N258R, N275S, C288S, M402L, S499P and I502V.

59. The method of claim 58, wherein the PAL further comprises one or more of the following amino acid substitutions relative to SEQ ID NO: 17: S92G, H133F, H133M, I167K, A263T, L432I, A433S and V470A.

60. The method of any one of claims 51-59, wherein the PAL comprises an amino acid substitution at the following amino acid residues relative to SEQ ID NO: 17: (i) C288, T40, 1502 and $499; (ii) S92, L432, V460, H133 and N258; (iii) S92, L432, V470, N258 and T40; (iv) S92, L432, V470 and T40; (v) N258, T40, 1502 and S499; (vi) S92, V470, M111 and T40; (vii) S92, L432, V470, N258 and T40; (viii) S92, V470, A263 and T40; or (ix) S92, L432, M111 and T40.

61. The method of claim 60, wherein the PAL comprises any one of the following relative to SEQ ID NO: 17: (i) C288S, T40I, I502V and S499P; (ii) S92G, L432I, V460A, H133F and N258I; (iii) S92G, L432I, V470A, N258I and T40I; (iv) S92G, L432I, V470A and T40I; (v) N258I, T40I, I502V and S499P; (vi) S92G, V470A, M111V and T40I; (vii) S92G, L432I, V470A, N258R and T40I; (viii) S92G, V470A, A263T and T40I; or (ix) S92G, L432I, M111V and T40I.

62. The method of any one of claims 51-61, wherein the PAL comprises the amino acid substitution N258R relative to SEQ ID NO: 17.

63. The method of claim 61, wherein the PAL comprises the following amino acid substitutions relative to SEQ ID NO: 17: T40I, C288S, S499P and I502V.

64. The method of any one of claims 34-63, wherein the method is a method of protecting a subject against phenylketonuria or hyperphenylalaninemia.

65. The method of any one of claims 34-63, wherein the method is a method of treating a subject that has phenylketonuria or hyperphenylalaninemia.

66. A phenylalanine ammonia lyase (PAL) enzyme, wherein the PAL comprises an amino acid substitution at one or more of the following amino acid residues relative to SEQ ID NO: 3: I77, F84, M87, A88, V90, A91, Q96, S98, L104, V105, W106, T110, Y158, I165, V172, S175, L214, L219, T243, D253, T345, L364, A394, L406, L407, K413, N415, Q422, I423, C424, F450, K522 and Y529.

67. The PAL enzyme of claim 66, wherein the PAL comprises one or more of the following amino acid substitutions relative to SEQ ID NO: 3: I77V, F84Y, M87L, A88S, V90T, A91V, Q96E, S98A, V105F, W106Q, T110S, Y158H, Y158V, 1165V, V172A, S175G, S175T, L214Q, L219I, T243L, D253A, T345S, L364H, A394M, L406V, L407A, L407C, L407S, L407T, K413G, K413S, N415H, Q422H, I423L, C424T, F450A, F450G, F450H, N453A, N453S, K522P and Y529L.

68. The PAL enzyme of claim 66 or 67, wherein the PAL further comprises an amino acid substitution at one or more of the following amino acid residues relative to SEQ ID NO: 3: L4, T102, L104, L108, M222, G218 and N453.

69. The PAL enzyme of claim 68, wherein the PAL comprises one or more of the following amino acid substitutions relative to SEQ ID NO: 3: L4P, T102E, T102K, T102R, T102S, L104A, L104M, L108A, L108H, L108Q, L108T, L108V, M222I, M222L, M222N, M222T, M222V, G218A, G218S, N453A and N453S.

70. The PAL enzyme of any one of claims 66-69, wherein the PAL comprises an amino acid substitution at the following amino acid residues relative to SEQ ID NO: 3: (i) L104 and L219; (ii) L4 and G218; (iii) T102 and G218; (iv) L104, L219 and M222; (v) T102 and G218; (vi) T102 and M222; (vii) L4, L104 and G218; (viii) G218, L104 and L219; (ix) L104, L219 and N453; or (x) G218, L4 and M222.

71. The PAL enzyme of claim 70, wherein the PAL comprises any of the following relative to SEQ ID NO: 3: (i) L104M and L219I; (ii) L4P and G218S; (iii) T102K and G218A; (iv) L104M, L219I and M222L; (v) T102S and G218A; (vi) T102K and M222L; (vii) L4P, L104M and G218S; (viii) G218A, L104M and L219I; (ix) L104M, L219I and N453S; or (x) G218S, L4P and M222L.

72. The PAL enzyme of claim 71, wherein the PAL comprises the following amino acid substitutions relative to SEQ ID NO: 3: L104M and L219I.

73. The PAL enzyme of claim 71, wherein the PAL comprises the following amino acid substitution relative to SEQ ID NO: 3: L4P and G218S.

74. A phenylalanine ammonia lyase (PAL) enzyme, wherein the PAL comprises an amino acid substitution at one or more of the following amino acid residues relative to SEQ ID NO: 17: T40, M111, N258, N275, C288, M402, S499 and I502.

75. The PAL enzyme of claim 74, wherein the PAL comprises one or more of the following amino acid substitutions relative to SEQ ID NO: 17: T40I, M111V, N258I, N258R, N275S, C288S, M402L, S499P and I502V.

76. The PAL enzyme of claim 74 or 75, wherein the PAL enzyme further comprises an amino acid substitution at one or more of the following amino acid residues relative to SEQ ID NO: 17: S92, H133, 1167, A263, L432, A433 and V470.

77. The PAL enzyme of claim 76, wherein the PAL enzyme further comprises one or more of the following amino acid substitutions relative to SEQ ID NO: 17: S92G, H133F, H133M, I167K, A263T, L432I, A433S and V470A.

78. The PAL enzyme of any one of claims 74-77, wherein the PAL comprises an amino acid substitution at the following amino acid residues relative to SEQ ID NO: 17: (i) C288, T40, 1502 and S499; (ii) S92, L432, V460, H133 and N258; (iii) S92, L432, V470, N258 and T40; (iv) S92, L432, V470 and T40; (v) N258, T40, 1502 and $499; (vi) S92, V470, M111 and T40; (vii) S92, L432, V470, N258 and T40; (viii) S92, V470, A263 and T40; or (ix) S92, L432, M111 and T40.

79. The PAL enzyme of claim 78, wherein the PAL comprises any one of the following relative to SEQ ID NO: 17: (i) C288S, T40I, I502V and S499P; (ii) S92G, L432I, V460A, H133F and N258I; (iii) S92G, L432I, V470A, N258I and T40I; (iv) S92G, L432I, V470A and T40I; (v) N258I, T40L, I502V and S499P; (vi) S92G, V470A, M111V and T40I; (vii) S92G, L432I, V470A, N258R and T40I; (viii) S92G, V470A, A263T and T40I; or (ix) S92G, L432I, M111V and T40I.

80. The PAL enzyme of any one of claims 74-79, wherein the PAL comprises the amino acid substitution N258R relative to SEQ ID NO: 17.

81. The PAL enzyme of claim 79, wherein the PAL comprises the following amino acid substitutions relative to SEQ ID NO: 17: T40I, C288S, S499P and I502V.

12; 61; 12; 12

edspap.20240425841