HYALURONAN SYNTHASE GENE AND USES THEREOF

• US 20060216794A1
• Filed: 06/05/2006
• Published: 09/28/2006
• Est. Priority Date: 07/01/1994
• Status: Active Grant

• Current Assignee: Kshama Kumari, Paul Deangelis, Paul H. Weigel
• Original Assignee: Kshama Kumari, Paul Deangelis, Paul H. Weigel
• Inventors: Weigel, Paul H., Kumari, Kshama, DeAngelis, Paul
• Granted Patent: US 7,141,409 B2
• Time in Patent Office: Days
• US Class Current: 435/85
• CPC Class Codes: A61K 39/102 Pasteurellales, e.g. Actinobacillus, Pasteurella; Haemophilus
• CPC Class Codes: A61P 31/00 Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: C07H 21/04 with deoxyribosyl as saccharide radical
• CPC Class Codes: C08B 37/0072 Hyaluronic acid, i.e. HA or hyaluronan; Derivatives thereof, e.g. crosslinked hyaluronic acid (hylan) or hyaluronates
• CPC Class Codes: C12N 9/1051 Hexosyltransferases (2.4.1)
• CPC Class Codes: C12P 19/04 Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
• CPC Class Codes: C12P 19/26 Preparation of nitrogen-containing carbohydrates
• CPC Class Codes: Y10S 435/836 Bacillus licheniformis
• CPC Class Codes: Y10S 435/839 Bacillus subtilis
• CPC Class Codes: Y10S 435/848 Escherichia

Abstract

The present invention relates to a recombinant host cell that includes an expression construct having a hyaluronan synthase gene that encodes an enzymatically active hyaluronan synthase (HAS), and to the use of these recombinant cells that produce hyaluronan synthase and its hyaluronic acid product.

Claims

1. A recombinant host cell comprising at least one expression construct comprising a hyaluronan synthase gene, wherein the hyaluronan synthase gene encodes an enzymatically active hyaluronan synthase, wherein the enzymatically active hyaluronan synthase includes at least two amino acid motifs selected from the group consisting of:

2. The recombinant host cell of claim 1, wherein the recombinant host cell produces HA.

3. 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.

4. The recombinant host cell of claim 3, 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.

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

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

7. The recombinant host cell of claim 1, wherein hyaluronic acid sugar precursors are expressed in the recombinant host cell by endogenous genes thereof.

8. The recombinant host cell of claim 1, wherein the hyaluronan synthase gene is isolated from a prokaryotic source.

9. A recombinant host cell comprising at least one expression construct comprising a hyaluronan synthase gene, wherein the hyaluronan synthase gene encodes an enzymatically active hyaluronan synthase, wherein the enzymatically active hyaluronan synthase includes at least three amino acid motifs selected from the group consisting of:

10. The recombinant host cell of claim 9, wherein the recombinant host cell produces HA.

11. The recombinant host cell 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.

12. The recombinant host cell of claim 11, 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.

13. The recombinant host cell of claim 9, further comprising 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 9, wherein the recombinant host cell further 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 9, wherein hyaluronic acid sugar precursors are expressed in the recombinant host cell by endogenous genes thereof.

16. The recombinant host cell of claim 9, wherein the hyaluronan synthase gene is isolated from a prokaryotic source.

17. A recombinant host cell comprising at least one expression construct comprising a hyaluronan synthase gene, wherein the hyaluronan synthase gene encodes an enzymatically active hyaluronan synthase, wherein the enzymatically active hyaluronan synthase includes the amino acid motifs:

18. The recombinant host cell of claim 17, wherein the recombinant host cell produces HA.

19. 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.

20. The recombinant host cell of claim 19, 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.

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

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

23. The recombinant host cell of claim 17, wherein hyaluronic acid sugar precursors are expressed in the recombinant host cell by endogenous genes thereof.

24. The recombinant host cell of claim 17, wherein the hyaluronan synthase gene is isolated from a prokaryotic source.

25. A recombinant host cell comprising at least one expression construct comprising a hyaluronan synthase gene, wherein the hyaluronan synthase gene encodes an enzymatically active hyaluronan synthase, wherein the enzymatically active hyaluronan synthase includes at least two amino acid motifs selected from the group consisting of:

26. The recombinant host cell of claim 25, wherein the recombinant host cell produces HA.

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

28. The recombinant host cell of claim 27, 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.

29. The recombinant host cell of claim 25, further comprising 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 25, wherein the recombinant host cell further 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 25, wherein hyaluronic acid sugar precursors are expressed in the recombinant host cell by endogenous genes thereof.

32. The recombinant host cell of claim 25, wherein the hyaluronan synthase gene is isolated from a prokaryotic source.

33. A recombinant host cell comprising at least one expression construct comprising a hyaluronan synthase gene, wherein the hyaluronan synthase gene encodes an enzymatically active hyaluronan synthase, wherein the enzymatically active hyaluronan synthase includes at least three amino acid motifs selected from the group consisting of:

34. The recombinant host cell of claim 33, wherein the recombinant host cell produces HA.

35. 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.

36. The recombinant host cell of claim 35, 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.

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

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

39. The recombinant host cell of claim 33, wherein hyaluronic acid sugar precursors are expressed in the recombinant host cell by endogenous genes thereof.

40. The recombinant host cell of claim 33, wherein the hyaluronan synthase gene is isolated from a prokaryotic source.

41. A recombinant host cell comprising at least one expression construct comprising a hyaluronan synthase gene, wherein the hyaluronan synthase gene encodes an enzymatically active hyaluronan synthase, wherein the enzymatically active hyaluronan synthase includes the amino acid motifs:

42. The recombinant host cell of claim 41, wherein the recombinant host cell produces HA.

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

44. The recombinant host cell of claim 43, 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.

45. The recombinant host cell of claim 41, further comprising a second at least one expression construct having at least one gene encoding an enzyme for synthesis of a hyaluronic acid sugar precursor.

46. The recombinant host cell of claim 41, wherein the recombinant host cell further comprises at least one integrated or plasmid-borne gene encoding an enzyme for synthesis of a hyaluronic acid sugar precursor.

47. The recombinant host cell of claim 41, wherein hyaluronic acid sugar precursors are expressed in the recombinant host cell by endogenous genes thereof.

48. The recombinant host cell of claim 41, wherein the hyaluronan synthase gene is isolated from a prokaryotic source.