Hyaluronan synthases and methods of making and using same

• US 20030235893A1
• Filed: 12/03/2002
• Published: 12/25/2003
• Est. Priority Date: 10/26/1998
• Status: Active Grant

• Current Assignee: The Board of Regents of the University of Colorado (University of Colorado System)
• Original Assignee: The Board of Regents of the University of Colorado (University of Colorado System)
• Inventors: Weigel, Paul H., Kumari, Kshama
• Granted Patent: US 7,094,581 B2
• Time in Patent Office: Days
• US Class Current: 435/101
• CPC Class Codes: C12N 9/1051 Hexosyltransferases (2.4.1)
• CPC Class Codes: C12P 19/26 Preparation of nitrogen-containing carbohydrates

Abstract

A functionally active hyaluronan synthase having at least one modified amino acid residue therein as compared to a corresponding functionally active native hyaluronan synthase such that the functionally active hyaluronan synthase has an altered enzymatic activity as compared to the corresponding functionally active native hyaluronan synthase is disclosed. Methods of producing hyaluronic acid utilizing a recombinant host cell having an expression construct encoding the functionally active hyaluronan synthase with altered enzymatic activity are also disclosed.

Claims

1. A functionally active hyaluronan synthase having at least one modified amino acid residue therein as compared to a corresponding functionally active native hyaluronan synthase.

2. The functionally active hyaluronan synthase of claim 1, wherein the functionally active hyaluronan synthase having at least one modified amino acid residue therein is selected from the group consisting of spHAS, seHAS, suHAS and pmHAS.

3. The functionally active hyaluronan synthase of claim 1, wherein the at least one modified amino acid residue is a cysteine.

4. The functionally active hyaluronan synthase of claim 3, wherein the cysteine is modified to an alanine or a serine residue.

5. The functionally active hyaluronan synthase of claim 1, wherein the functionally active hyaluronan synthase having at least one modified amino acid residue therein has an amino acid sequence essentially as set forth in at least one of SEQ ID NOs:

15-92.

6. A functionally active hyaluronan synthase having an altered enzymatic activity as compared to a corresponding functionally active native hyaluronan synthase.

7. The functionally active hyaluronan synthase having an altered enzymatic activity of claim 6, wherein the functionally active hyaluronan synthase having an altered enzyme activity is selected from the group consisting of spHAS, seHAS, suHAS and pmHAS.

8. The functionally active hyaluronan synthase having an altered enzymatic activity of claim 7, wherein the functionally active hyaluronan synthase having an altered enzymatic activity has at least one modified amino acid residue therein as compared to a corresponding functionally active native hyaluronan synthase.

9. The functionally active hyaluronan synthase having an altered enzymatic activity of claim 8, wherein the at least one modified amino acid residue is a cysteine.

10. The functionally active hyaluronan synthase having an altered enzymatic activity of claim 9, wherein the cysteine is modified to an alanine or a serine residue.

11. The functionally active hyaluronan synthase having an altered enzymatic activity of claim 10, wherein the functionally active hyaluronan synthase having at least one modified amino acid residue therein has an amino acid sequence essentially as set forth in at least one of SEQ ID NOs:

15-92.

12. A functionally active hyaluronan synthase having at least one modified amino acid residue therein as compared to a corresponding functionally active native hyaluronan synthase, wherein the corresponding functionally active native hyaluronan synthase has an amino acid sequence essentially as set forth in at least one of SEQ ID NOs:

2, 4, 6, 8, 10, 12 and 14.

13. The functionally active hyaluronan synthase of claim 12, wherein the at least one modified amino acid residue is a cysteine.

14. The functionally active hyaluronan synthase of claim 13, wherein the cysteine is modified to an alanine or a serine residue.

15. The functionally active hyaluronan synthase of claim 12, wherein the functionally active hyaluronan synthase having at least one modified amino acid residue therein has an amino acid sequence essentially as set forth in at least one of SEQ ID NOs:

15-92.

16. A functionally active hyaluronan synthase having an altered enzymatic activity as compared to a corresponding functionally active native hyaluronan synthase, wherein the corresponding functionally active native hyaluronan synthase has an amino acid sequence essentially as set forth in at least one of SEQ ID NOs:

2, 4, 6, 8, 10, 12 and 14.

17. The functionally active hyaluronan synthase having an altered enzymatic activity of claim 16, wherein the functionally active hyaluronan synthase having an altered enzymatic activity has at least one modified amino acid residue therein as compared to the corresponding functionally active native hyaluronan synthase.

18. The functionally active hyaluronan synthase having an altered enzymatic activity of claim 17, wherein the at least one modified amino acid residue is a cysteine.

19. The functionally active hyaluronan synthase having an altered enzymatic activity of claim 18, wherein the cysteine is modified to an alanine or a serine residue.

20. The functionally active hyaluronan synthase having an altered enzymatic activity of claim 16, wherein the functionally active hyaluronan synthase having at least one modified amino acid residue therein has an amino acid sequence essentially as set forth in at least one of SEQ ID NOs:

15-92.

21. A host cell having a functionally active hyaluronan synthase having an altered enzymatic activity as compared to a corresponding functionally active native hyaluronan synthase incorporated therein such that the host cell is capable of producing hyaluronan.

22. The host cell of claim 21 wherein the corresponding functionally active native hyaluronan synthase has an amino acid sequence essentially as set forth in at least one of SEQ ID NOs:

2, 4, 6, 8, 10, 12 and 14.

23. The host cell of claim 21, wherein the functionally active hyaluronan synthase having an altered enzyme activity is selected from the group consisting of spHAS, seHAS, suHAS and pmHAS.

24. The host cell of claim 21, wherein the functionally active hyaluronan synthase having an altered enzymatic activity has at least one modified amino acid residue therein as compared to a corresponding functionally active native hyaluronan synthase.

25. The host cell of claim 24, wherein the at least one modified amino acid residue is a cysteine.

26. The host cell of claim 25, wherein the cysteine is modified to an alanine or a serine residue.

27. The host cell of claim 21, wherein the functionally active hyaluronan synthase having at least one modified amino acid residue therein has an amino acid sequence essentially as set forth in at least one of SEQ ID NOs:

15-92.

28. A functionally active hyaluronan synthase having an amino acid sequence comprising at least one of SEQ ID NOs:

15-92.

29. A functionally active hyaluronan synthase having an amino acid sequence essentially set forth in at least one of SEQ ID NOs:

15-92.

30. A method of providing a functionally active hyaluronan synthase having an altered enzymatic activity as compared to a corresponding functionally active native hyaluronan synthase, comprising the steps of:

providing a hyaluronan synthase; and

modifying at least one amino acid residue of the hyaluronan synthase to provide a functionally active hyaluronan synthase having an altered enzymatic activity.

31. The method of claim 30 wherein, in the step of providing a hyaluronan synthase, the hyaluronan synthase has an amino acid sequence essentially as set forth in at least one of SEQ ID NOs:

2, 4, 6, 8, 10, 12 and 14.

32. The method of claim 30, wherein in the step of providing a hyaluronan synthase, the hyaluronan synthase is selected from the group consisting of spHAS, seHAS, suHAS and pmHAS.

33. The method of claim 30, wherein in the step of modifying at least one amino acid residue of the hyaluronan synthase to provide a functionally active hyaluronan synthase having an altered enzymatic activity, the at least one modified amino acid residue is a cysteine.

34. The method of claim 33, wherein the cysteine is modified to an alanine or a serine residue.

35. The method of claim 30 wherein, in the step of modifying at least one amino acid residue of the hyaluronan synthase, the functionally active hyaluronan synthase having an altered enzymatic activity has an amino acid sequence essentially as set forth in at least one of SEQ ID NOs:

15-92.

36. The method of claim 30 wherein, in the step of modifying at least one amino acid residue of the hyaluronan synthase to provide a functionally active hyaluronan synthase having an altered enzymatic activity, the functionally active hyaluronan synthase has an increased enzymatic activity.

37. The method of claim 30 wherein, in the step of modifying at least one amino acid residue of the hyaluronan synthase to provide a functionally active hyaluronan synthase having an altered enzymatic activity, the functionally active hyaluronan synthase has a decreased enzymatic activity.

38. The method of claim 30 wherein, in the step of modifying at least one amino acid residue of the hyaluronan synthase to provide a functionally active hyaluronan synthase having an altered enzymatic activity, the functionally active hyaluronan synthase produces hyaluronic acid having an average molecular mass that is less than an average molecular mass of hyaluronic acid produced by a corresponding functionally active native hyaluronan synthase.

39. The method of claim 30 wherein, in the step of modifying at least one amino acid residue of the hyaluronan synthase to provide a functionally active hyaluronan synthase having an altered enzymatic activity, the functionally active hyaluronan synthase produces hyaluronic acid having an average molecular mass that is greater than an average molecular mass of hyaluronic acid produced by a corresponding functionally active native hyaluronan synthase.

40. A method for producing hyaluronic acid, comprising the steps of:

providing a host cell having at least one expression construct comprising a hyaluronan synthase gene encoding a functionally active hyaluronan synthase incorporated therein such that the host cell is capable of producing hyaluronan, wherein the functionally active hyaluronan synthase has an altered enzymatic activity as compared to a corresponding functionally active native hyaluronan synthase; and

culturing the host cell under conditions appropriate for the production of hyaluronic acid.

41. The method of claim 40 wherein, in the step of providing a host cell, the corresponding functionally active native hyaluronan synthase has an amino acid sequence essentially as set forth in at least one of SEQ ID NOs:

2, 4, 6, 8, 10, 12 and 14.

42. The method of claim 40 wherein, in the step of providing a host cell, the functionally active hyaluronan synthase having an altered enzymatic activity has at least one modified amino acid residue therein as compared to a corresponding functionally active native hyaluronan synthase.

43. The method of claim 42, wherein the at least one modified amino acid residue is a cysteine.

44. The method of claim 43, wherein the cysteine is modified to an alanine or a serine residue.

45. The method of claim 40 wherein, in the step of providing a host cell, the functionally active hyaluronan synthase having an altered enzymatic activity has an amino acid sequence essentially as set forth in at least one of SEQ ID NOs:

15-92.

46. The method of claim 40, further comprising the step of separating the hyaluronic acid from the host cell.

47. The method of claim 40, wherein the at least one expression construct further comprises at least one gene encoding an enzyme for synthesis of a hyaluronic acid sugar precursor.

48. The method of claim 47, wherein the at least one gene encoding an enzyme for synthesis of a hyaluronic acid sugar precursor is selected from the group consisting of a pyrophosphorylase, a transferase, a mutase, a dehydrogenase, or an epimerase, capable of producing UDP-GIcNAc or UDP-GIcUA.

49. The method of claim 40, wherein the at least one expression construct further comprises at least one biosynthetic pathway gene of a hyaluronic acid sugar precursor.

50. The method of claim 40, wherein the at least one expression construct further includes at least one gene encoding an enzyme for synthesis of a hyaluronic acid sugar precursor.

51. The method of claim 40, further including a second at least one expression construct having at least one gene encoding an enzyme for synthesis of a hyaluronic acid sugar precursor.

52. The method of claim 40, wherein the host cell includes at least one integrated or plasmid-borne gene encoding an enzyme for synthesis of a hyaluronic acid sugar precursor.

53. The method of claim 40, wherein nutrients utilized for a hyaluronic acid sugar precursor biosynthetic pathway are supplied to the host cell.

54. The method of claim 53, wherein the nutrients supplying the hyaluronic acid sugar precursor biosynthetic pathway are fed to the host cell.

55. The method of claim 53, wherein the hyaluronic acid sugar precursors are expressed in the host cell by endogenous genes thereof.

56. The method of claim 53, wherein the hyaluronic acid sugar precursors enzymes are expressed in the host cell by an introduced construct including genes thereof.

57. The method of claim 40 wherein, in the step of providing a host cell, the functionally active hyaluronan synthase has an increased enzymatic activity.

58. The method of claim 40 wherein, in the step of providing a host cell, the functionally active hyaluronan synthase has a decreased enzymatic activity.

59. The method of claim 40 wherein, in the step of providing a host cell, the functionally active hyaluronan synthase produces hyaluronic acid having an average molecular mass that is less than an average molecular mass of hyaluronic acid produced by a corresponding functionally active native hyaluronan synthase.

60. The method of claim 40 wherein, in the step of providing a host cell, the functionally active hyaluronan synthase produces hyaluronic acid having an average molecular mass that is greater than an average molecular mass of hyaluronic acid produced by a corresponding functionally active native hyaluronan synthase.