Hyaluronan synthases and methods of making and using same

• US 20060211104A1
• Filed: 05/31/2006
• Published: 09/21/2006
• Est. Priority Date: 12/03/2001
• Status: Abandoned Application

• Current Assignee: Kshama Kumari, Paul H. Weigel
• Original Assignee: Kshama Kumari, Paul H. Weigel
• Inventors: Weigel, Paul H., Kumari, Kshama
• Application Number:
  US11/444,093
• Publication Number:
  US 20060211104A1
• Time in Patent Office: Days
• US Class Current: 435/252.310
• 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 recombinant host cell comprising at least one expression construct comprising a hyaluronan synthase gene encoding a functionally active hyaluronan synthase such that the recombinant host cell is capable of producing hyaluronic acid, the functionally active hyaluronan synthase having an altered enzymatic activity as compared to a corresponding functionally active native hyaluronan synthase, 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.

2. The recombinant host cell of claim 1, wherein the corresponding functionally active native hyaluronan synthase is selected from the group consisting of spHAS, seHAS, suHAS and pmHAS.

3. The recombinant host cell of claim 1 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.

4. The recombinant host cell of claim 1, wherein the functionally active hyaluronan synthase has an amino acid sequence as set forth in at least one of SEQ ID NOS:

15-29.

5. The recombinant host cell of claim 1, wherein the functionally active hyaluronan synthase has an increased enzymatic activity.

6. The recombinant host cell of claim 1, wherein the functionally active hyaluronan synthase has a decreased enzymatic activity.

7. The recombinant host cell of claim 1, 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.

8. The recombinant host cell of claim 1, 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.

9. The recombinant host cell of claim 1, wherein the at least one expression construct further comprises at least one gene encoding an enzyme for synthesis of a hyaluronic acid sugar precursor.

10. The recombinant host cell of claim 9, 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, and an epimerase capable of producing UDP-GlcNAc or UDP-GlcUA.

11. The recombinant host cell of claim 1, wherein the at least one expression construct further comprises at least one biosynthetic pathway gene of a hyaluronic acid sugar precursor.

12. The recombinant host cell of claim 1, wherein the at least one expression construct further comprises at least one gene encoding an enzyme for synthesis of a hyaluronic acid sugar precursor.

13. The recombinant host cell of claim 1, wherein the recombinant host cell further comprises a second at least one expression construct having at least one gene encoding an enzyme for synthesis of a hyaluronic acid sugar precursor.

14. The recombinant host cell of claim 1, wherein the recombinant host cell comprises at least one integrated or plasmid-borne gene encoding an enzyme for synthesis of a hyaluronic acid sugar precursor.

15. The recombinant host cell of claim 1, wherein the recombinant host cell is a Bacillus cell.

16. The recombinant host cell of claim 15, wherein the recombinant host cell is a Bacillus subtilis cell or a Bacillus licheniformis cell.

17. A recombinant host cell comprising at least one expression construct comprising a hyaluronan synthase gene encoding a functionally active hyaluronan synthase such that the recombinant host cell is capable of producing hyaluronic acid, the functionally active hyaluronan synthase having at least one modified amino acid residue therein as compared to a corresponding functionally active native hyaluronan synthase, 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.

18. The recombinant host cell of claim 17, wherein the corresponding functionally active native hyaluronan synthase is selected from the group consisting of spHAS, seHAS, suHAS and pmHAS.

19. The recombinant host cell of claim 17 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.

20. The recombinant host cell of claim 17, wherein the functionally active hyaluronan synthase has an amino acid sequence as set forth in at least one of SEQ ID NOS:

15-29.

21. The recombinant host cell of claim 17, wherein the functionally active hyaluronan synthase has an increased enzymatic activity.

22. The recombinant host cell of claim 17, wherein the functionally active hyaluronan synthase has a decreased enzymatic activity.

23. The recombinant host cell of claim 17, 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.

24. The recombinant host cell of claim 17, 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.

25. The recombinant host cell of claim 17, wherein the at least one expression construct further comprises at least one gene encoding an enzyme for synthesis of a hyaluronic acid sugar precursor.

26. The recombinant host cell of claim 25, 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, and an epimerase capable of producing UDP-GlcNAc or UDP-GlcUA.

27. The recombinant host cell of claim 17, wherein the at least one expression construct further comprises at least one biosynthetic pathway gene of a hyaluronic acid sugar precursor.

28. The recombinant host cell of claim 17, wherein the at least one expression construct further comprises at least one gene encoding an enzyme for synthesis of a hyaluronic acid sugar precursor.

29. The recombinant host cell of claim 17, wherein the recombinant host cell further comprises a second at least one expression construct having at least one gene encoding an enzyme for synthesis of a hyaluronic acid sugar precursor.

30. The recombinant host cell of claim 17, wherein the recombinant host cell comprises at least one integrated or plasmid-borne gene encoding an enzyme for synthesis of a hyaluronic acid sugar precursor.

31. The recombinant host cell of claim 17, wherein the recombinant host cell is a Bacillus cell.

32. The recombinant host cell of claim 31, wherein the recombinant host cell is a Bacillus subtilis cell or a Bacillus licheniformis cell.

33. A recombinant host cell comprising at least one expression construct comprising a hyaluronan synthase gene encoding a functionally active hyaluronan synthase such that the recombinant host cell is capable of producing hyaluronic acid, the functionally active hyaluronan synthase having an amino acid sequence comprising at least one of SEQ ID NOS:

15-29.

34. The recombinant host cell of claim 33, wherein the functionally active hyaluronan synthase has an increased enzymatic activity.

35. The recombinant host cell of claim 33, wherein the functionally active hyaluronan synthase has a decreased enzymatic activity.

36. The recombinant host cell of claim 33, 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 recombinant host cell of claim 33, 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. The recombinant host cell of claim 33, wherein the at least one expression construct further comprises at least one gene encoding an enzyme for synthesis of a hyaluronic acid sugar precursor.

39. The recombinant host cell of claim 38, 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, and an epimerase capable of producing UDP-GlcNAc or UDP-GlcUA.

40. The recombinant host cell of claim 33, wherein the at least one expression construct further comprises at least one biosynthetic pathway gene of a hyaluronic acid sugar precursor.

41. The recombinant host cell of claim 33, wherein the at least one expression construct further comprises at least one gene encoding an enzyme for synthesis of a hyaluronic acid sugar precursor.

42. The recombinant host cell of claim 33, wherein the recombinant host cell further comprises a second at least one expression construct having at least one gene encoding an enzyme for synthesis of a hyaluronic acid sugar precursor.

43. The recombinant host cell of claim 33, wherein the recombinant host cell comprises at least one integrated or plasmid-borne gene encoding an enzyme for synthesis of a hyaluronic acid sugar precursor.

44. The recombinant host cell of claim 33, wherein the recombinant host cell is a Bacillus cell.

45. The recombinant host cell of claim 44, wherein the recombinant host cell is a Bacillus subtilis cell or a Bacillus licheniformis cell.