Hyaluronan synthases and methods of making and using same

• US 7,094,581 B2
• Filed: 12/03/2002
• Issued: 08/22/2006
• Est. Priority Date: 10/26/1998
• Status: Expired due to Fees

• 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
• Primary Examiner(s): Wax, Robert A.
• Application Number:
  US10/309,560
• Publication Number:
  US 20030235893A1
• Time in Patent Office: 1,358 Days
• Field of Search: None
• US Class Current: 435/84
• 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 an amino acid sequence comprising at least one of SEQ ID NOS:

15–

29.

2. A functionally active hyaluronan synthase having an amino acid sequence as set forth in at least one of SEQ ID NOS:

15–

29.

3. 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 as set forth in SEQ ID NO;

2; and

modifying at least one cysteine residue of the hyaluronan synthase to an alanine or serine residue to provide a functionally active hyaluronan synthase having an altered enzymatic activity.

4. The method of claim 3 wherein, in the step of modifying at least one cysteine residue of the hyaluronan synthase, the functionally active hyaluronan synthase having an altered enzymatic activity has an amino acid sequence as set forth in at least one of SEQ ID NOS:

15–

29.

5. The method of claim 3 wherein, in the step of modifying at least one cysteine 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.

6. The method of claim 3 wherein, in the step of modifying at least one cysteine 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.

7. The method of claim 3 wherein, in the step of modifying at least one cysteine 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.

8. The method of claim 3 wherein, in the step of modifying at least one cysteine 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.

9. 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, wherein the corresponding functionally active native hyaluronan synthase is seHAS, and wherein the functionally active hyaluronan synthase has at least one modified amino acid residue therein as compared to a corresponding functionally active native hyaluronan synthase, and wherein the at least one modified amino acid residue is a cysteine modified to an alanine or a serine residue; and

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

10. The method of claim 9 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 SEQ ID NO:

2.

11. The method of claim 9 wherein, in the step of providing a host cell, the functionally active hyaluronan synthase having an altered enzymatic activity has an amino acid sequence as set forth in at least one of SEQ ID NOS:

15–

29.

12. The method of claim 9, further comprising the step of separating the hyaluronic acid from the host cell.

13. The method of claim 9, wherein the at least one expression construct further comprises at least one gene encoding an enzyme for synthesis of a hyaluronic acid sugar precursor.

14. The method of claim 13, 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.

15. The method of claim 9, wherein the at least one expression construct further comprises at least one biosynthetic pathway gene of a hyaluronic acid sugar precursor.

16. The method of claim 9, wherein the at least one expression construct further includes at least one gene encoding an enzyme for synthesis of a hyaluronic acid sugar precursor.

17. The method of claim 9, 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.

18. The method of claim 9, wherein the host cell includes at least one integrated or plasmid-borne gene encoding an enzyme for synthesis of a hyaluronic acid sugar precursor.

19. The method of claim 9, wherein nutrients utilized for a hyaluronic acid sugar precursor biosynthetic pathway are supplied to the host cell.

20. The method of claim 19, wherein the nutrients supplying the hyaluronic acid sugar precursor biosynthetic pathway are fed to the host cell.

21. The method of claim 19, wherein the hyaluronic acid sugar precursors are expressed in the host cell by endogenous genes thereof.

22. The method of claim 19, wherein the hyaluronic acid sugar precursors enzymes are expressed in the host cell by an introduced construct including genes thereof.

23. The method of claim 9 wherein, in the step of providing a host cell, the functionally active hyaluronan synthase has an increased enzymatic activity.

24. The method of claim 9 wherein, in the step of providing a host cell, the functionally active hyaluronan synthase has a decreased enzymatic activity.

25. The method of claim 9 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.

26. The method of claim 9 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.

27. 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 is seHAS, and wherein the at least one modified amino acid residue is a cysteine modified to an alanine or a serine residue, and wherein the functionally active hyaluronan synthase has an amino acid residue as set forth in at least one of SEQ ID NOS:

15–

29.

28. The functionally active hyaluronan synthase of claim 27, wherein the functionally active hyaluronan synthase has an increased enzymatic activity.

29. The functionally active hyaluronan synthase of claim 27, wherein the functionally active hyaluronan synthase has a decreased enzymatic activity.

30. The functionally active hyaluronan synthase of claim 27, wherein 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.

31. The functionally active hyaluronan synthase of claim 27, wherein 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.

32. 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 is seHAS, and wherein the functionally active hyaluronan synthase has at least one modified amino acid residue therein as compared to a corresponding functionally active native hyaluronan synthase, and wherein the at least one modified amino acid residue is a cysteine modified to an alanine or a serine residue.

33. The functionally active hyaluronan synthase of claim 32, wherein the functionally active hyaluronan synthase having at least one modified amino acid residue therein has an amino acid sequence as set forth in at least one of SEQ ID NOS:

15–

29.

34. The functionally active hyaluronan synthase of claim 32, wherein the functionally active hyaluronan synthase has an increased enzymatic activity.

35. The functionally active hyaluronan synthase of claim 32, wherein the functionally active hyaluronan synthase has a decreased enzymatic activity.

36. The functionally active hyaluronan synthase of claim 32, wherein 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.

37. The functionally active hyaluronan synthase of claim 32, wherein 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.

38. 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 as set forth in SEQ ID NO:

2, wherein the at least one modified amino acid residue is a cysteine, and wherein the cysteine is modified to an alanine or a serine residue.

39. The functionally active hyaluronan synthase of claim 38, wherein the functionally active hyaluronan synthase has an amino acid sequence as set forth in at least one of SEQ ID NOS:

15–

29.

40. The functionally active hyaluronan synthase of claim 38, wherein the functionally active hyaluronan synthase has an increased enzymatic activity.

41. The functionally active hyaluronan synthase of claim 38, wherein the functionally active hyaluronan synthase has a decreased enzymatic activity.

42. The functionally active hyaluronan synthase of claim 38, wherein 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.

43. The functionally active hyaluronan synthase of claim 38, wherein 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.

44. 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 as set forth in SEQ ID NO:

2, wherein the functionally active hyaluronan synthase has at least one modified amino acid residue therein as compared to a corresponding functionally active native hyaluronan synthase, and wherein the at least one modified amino acid residue is a cysteine modified to an alanine or a serine residue.

45. The functionally active hyaluronan synthase of claim 44, wherein the functionally active hyaluronan synthase has an amino acid sequence as set forth in at least one of SEQ ID NOS:

15–

29.

46. The functionally active hyaluronan synthase of claim 44, wherein the functionally active hyaluronan synthase has an increased enzymatic activity.

47. The functionally active hyaluronan synthase of claim 44, wherein the functionally active hyaluronan synthase has a decreased enzymatic activity.

48. The functionally active hyaluronan synthase of claim 44, wherein 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.

49. The functionally active hyaluronan synthase of claim 44, wherein 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.