Hyaluronan synthase gene and uses thereof
View Patent ↗The present invention relates to a nucleic acid segment having a coding region segment encoding enzymatically active Streptococcus equisimilis hyaluronate synthase (seHAS), and to the use of this nucleic acid segment in the preparation of recombinant cells which produce hyaluronate synthase and its hyaluronic acid product. Hyaluronate is also known as hyaluronic acid or hyaluronan.
1. A recombinant method for producing hyaluronic acid, comprising the steps of:
providing a host cell having at least one expression construct comprising a hyaluronan synthase gene, wherein the hyaluronan synthase gene encodes a single protein that is a dual-action catalyst that utilizes UDP-GIcA and UDP-GIcNAc to synthesize HA; and
culturing the host cell under conditions appropriate for the production of hyaluronic acid.
2. The method of claim 1 , further comprising the step of separating the hyaluronic acid from the host cell.
3. The method 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 method 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, or an epimerase, capable of producing UDP-GIcNAc or UDP-GIcUA.
5. The method of claim 1 , wherein the at least one expression construct further comprises at least one biosynthetic pathway gene of a hyaluronic acid sugar precursor.
6. The method of claim 1 , wherein the at least one expression construct further includes at least one gene encoding an enzyme for synthesis of a hyaluronic acid sugar precursor.
7. The method of claim 6 , 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.
8. The method of claim 1 , wherein the host cell includes at least one integrated or plasmid-borne gene encoding an enzyme for synthesis of a hyaluronic acid sugar precursor.
9. The method of claim 1 , wherein nutrients utilized for a hyaluronic acid sugar precursor biosynthetic pathway are supplied to the host cell.
10. The method of claim 9 , wherein the nutrients supplying the hyaluronic acid sugar precursor biosynthetic pathway are fed to the host cell.
11. The method of claim 9 , wherein the hyaluronic acid sugar precursors are expressed in the host cell by endogenous genes thereof.
12. The method of claim 9 , wherein the hyaluronic acid sugar precursors enzymes are expressed in the host cell by an introduced construct including genes thereof.
13. The method of claim 1 , wherein the hyaluronan synthase gene is isolated from a streptococcal source.
14. The method of claim 1 , wherein the hyaluronan synthase gene is isolated from a microorganism.
15. The method of claim 1 wherein the hyaluronan synthase protein comprises a central intracellular domain, at least two transmembrane domains at an amino end thereof, and at least two membrane-associated domains at a carboxyl end thereof.
16. A recombinant method for producing hyaluronic acid, comprising the steps of:
providing a host cell having at least one expression construct comprising a hyaluronan synthase gene from a microorganism, wherein the hyaluronan synthase gene encodes a single protein that is a dual-action catalyst that utilizes UDP-GIcA and UDP-GIcNAc to synthesize HA; and
culturing the host cell under conditions appropriate for the production of hyaluronic acid.
17. The method of claim 16 , further comprising the step of separating the hyaluronic acid from the host cell.
18. The method of claim 16 , wherein the at least one expression construct further comprises at least one gene encoding an enzyme for synthesis of a hyaluronic acid sugar precursor.
19. The method of claim 16 , 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.
20. The method of claim 16 , wherein the at least one expression construct further comprises at least one biosynthetic pathway gene of a hyaluronic acid sugar precursor.
21. The method of claim 16 , wherein the at least one expression construct further includes at least one gene encoding an enzyme for synthesis of a hyaluronic acid sugar precursor.
22. The method of claim 21 , 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.
23. The method of claim 16 , wherein the host cell includes at least one integrated or plasmid-borne gene encoding an enzyme for synthesis of a hyaluronic acid sugar precursor.
24. The method of claim 16 , wherein nutrients utilized for a hyaluronic acid sugar precursor biosynthetic pathway are supplied to the host cell.
25. The method of claim 24 , wherein the nutrients supplying the hyaluronic acid sugar precursor biosynthetic pathway are fed to the host cell.
26. The method of claim 24 , wherein the hyaluronic acid sugar precursors are expressed in the host cell by endogenous genes thereof.
27. The method of claim 24 , wherein the hyaluronic acid sugar precursors enzymes are expressed in the host cell by an introduced construct including genes thereof.
28. The method of claim 16 , wherein the hyaluronan synthase gene is isolated from a streptococcal source.
29. The method of claim 16 wherein the hyaluronan synthase protein comprises a central intracellular domain, at least two transmembrane domains at an amino end thereof, and at least two membrane-associated domains at a carboxyl end thereof.
30. A recombinant method for producing hyaluronic acid, comprising the steps of:
providing a host cell having at least one expression construct comprising a hyaluronan synthase gene wherein the hyaluronan synthase gene encodes a single protein that is a dual-action catalyst that utilizes both sugar precursors to synthesize HA, and wherein the hyaluronan synthase protein comprises a central intracellular domain, at least two transmembrane domains at an amino end thereof, and at least two membrane-associated domains at a carboxyl end thereof; and
culturing the host cell under conditions appropriate for the production of hyaluronic acid.
31. The method of claim 30 , further comprising the step of separating the hyaluronic acid from the host cell.
32. The method of claim 30 , wherein the at least one expression construct further comprises at least one gene encoding an enzyme for synthesis of a hyaluronic acid sugar precursor.
33. The method of claim 30 , 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.
34. The method of claim 30 , wherein the at least one expression construct further comprises at least one biosynthetic pathway gene of a hyaluronic acid sugar precursor.
35. The method of claim 30 , wherein the at least one expression construct further includes at least one gene encoding an enzyme for synthesis of a hyaluronic acid sugar precursor.
36. The method of claim 35 , 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.
37. The method of claim 30 , wherein the host cell includes at least one integrated or plasmid-borne gene encoding an enzyme for synthesis of a hyaluronic acid sugar precursor.
38. The method of claim 30 , wherein nutrients utilized for a hyaluronic acid sugar precursor biosynthetic pathway are supplied to the host cell.
39. The method of claim 38 , wherein the nutrients supplying the hyaluronic acid sugar precursor biosynthetic pathway are fed to the host cell.
40. The method of claim 38 , wherein the hyaluronic acid sugar precursors are expressed in the host cell by endogenous genes thereof.
41. The method of claim 38 , wherein the hyaluronic acid sugar precursors enzymes are expressed in the host cell by an introduced construct including genes thereof.
42. The method of claim 30 , wherein the hyaluronan synthase gene is isolated from a streptococcal source.