Granted Patent B2

US 7,141,409 · App. 11/446,871 · Granted Nov 28, 2006

Assignee: The Board of Regents of the University of Oklahoma

Abstract

Claims (84)

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:

(i) GKR(E/H)(V/C/A)(M/L/Q)(Y/A)(T/W)(A/G)F(X) 3-7 (A/S/D)(W/V/A)(DIN)(V/A/Y)(F/V/I)(L/Q/V)(T/V/L)(V/C/I) DSDTX(L/I)(D/E/Y)(P/E/K)X(A/S/C)XXE (SEQ ID NO:11);

(ii) (L/M)(S/T/V)(D/S/A)XRY(W/Y/D)XAF(G/N/C) (V/I)ER(A/S)(C/A)QSX(F/T)X(N/C/T)(V/I)(L/Q/S)(C/V) (I/V/C)(S/G)GPL(G/S)XY(K/R) (SEQ ID NO:12);

(iii) (D/E)X(W/Y)(X) 2 QXF(L/M)G(X) 2 (C/V)(S/T) XGDDR(H/R/C)LTN (SEQ ID NO:13); and

(iv) (E/D)(T/S/V)P(X) 5 (W/Y/F)(L/I)XQQ(T/N) RW(S/T/N)KS(Y/W/F)(F/C)RE (SEQ ID NO:14),

with X being selected from the group consisting of any amino acid; and

wherein the at least two amino acid motifs are found in the relative order of (i), (ii), (iii), and (iv) from an amino terminus to a carboxyl terminus of the recombinantly produced, enzymatically active hyaluronan synthase.

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:

(i) GKR(E/H)(V/C/A)(M/L/Q)(Y/A)(T/W)(A/G)F(X) 3-7 (A/S/D)(W/V/A)(D/N)(V/A/Y)(F/V/I)(L/Q/V)(T/V/L)(V/C/I) DSDTX(L/I)(D/E/Y)(P/E/K)X(A/S/C)XXE (SEQ ID NO:11);

(ii) (L/M)(S/T/V)(D/S/A)XRY(W/Y/D)XAF(G/N/C) (V/I)ER(A/S)(C/A)QSX(F/T)X(N/C/T)(V/I)(L/Q/S)(C/V) (I/V/C)(S/G)GPL(G/S)XY(K/R) (SEQ ID NO:12);

(iii) (D/E)X(W/Y)(X) 2 QXF(L/M)G(X) 2 (C/V)(S/T) XGDDR(H/R/C)LTN (SEQ ID NO:13); and

(iv) (E/D)(T/S/V)P(X) 5 (W/Y/F)(L/I)XQQ(T/N) RW(S/T/N)KS(Y/W/F)(F/C)RE (SEQ ID NO:14),

with X being selected from the group consisting of any amino acid; and

wherein the at least three amino acid motifs are found in the relative order of (i), (ii), (iii), and (iv) from an amino terminus to a carboxyl terminus of the recombinantly produced, enzymatically active hyaluronan synthase.

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:

(i) GKR(E/H)(V/C/A)(M/L/Q)(Y/A)(T/W)(A/G)F(X) 3-7 (A/S/D)(W/V/A)(D/N)(V/A/Y)(F/V/I)(L/Q/V)(T/V/L)(V/C/I) DSDTX(L/I)(D/E/Y)(P/E/K)X(A/S/C)XXE (SEQ ID NO:11);

(ii) (L/M)(S/T/V)(D/S/A)XRY(W/Y/D)XAF(G/N/C) (V/I)ER(A/S)(C/A)QSX(F/T)X(N/C/T)(V/I)(L/Q/S)(C/V) (I/V/C)(S/G)GPL(G/S)XY(K/R) (SEQ ID NO:12);

(iii) (D/E)X(W/Y)(X) 2 QXF(L/M)G(X) 2 (C/V)(S/T) XGDDR(H/R/C)LTN (SEQ ID NO:13); and

(iv) (E/D)(T/S/V)P(X) 5 (W/Y/F)(L/I)XQQ(T/N) RW(S/T/N)KS(Y/W/F)(F/C)RE (SEQ ID NO:14),

with X being selected from the group consisting of any amino acid; and

wherein the amino acid motifs are found in the relative order of (i), (ii), (iii), and (iv) from an amino terminus to a carboxyl terminus of the recombinantly produced, enzymatically active hyaluronan synthase.

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:

(i) GKR(X) 6 F(X) 11-15 DSDT(X) 8 E (SEQ ID NO:15);

(ii) RY(X) 2 AF(X) 2 ER(X) 2 QS(X) 9 GPL(X) 2 Y (SEQ ID NO:16);

(iii) QXFXG(X) 5 GDDRXLTN (SEQ ID NO:17); and

(iv) P(X) 8 QQXRWXKS(X) 2 RE (SEQ ID NO:18),

with X being selected from the group consisting of any amino acid; and

wherein the at least two amino acid motifs are found in the relative order of (i), (ii), (iii), and (iv) from an amino terminus to a carboxyl terminus of the recombinantly produced, enzymatically active hyaluronan synthase.

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:

(i) GKR(X) 6 F(X) 11-15 DSDT(X) 8 E (SEQ ID NO:15);

(ii) RY(X) 2 AF(X) 2 ER(X) 2 QS(X) 9 GPL(X) 2 Y (SEQ ID NO:16);

(iii) QXFXG(X) 5 GDDRXLTN (SEQ ID NO:17); and

(iv) P(X) 8 QQXRWXKS(X) 2 RE (SEQ ID NO:18),

with X being selected from the group consisting of any amino acid; and

wherein the at least three amino acid motifs are found in the relative order of (i), (ii), (iii), and (iv) from an amino terminus to a carboxyl terminus of the recombinantly produced, enzymatically active hyaluronan synthase.

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:

(i) GKR(X) 6 F(X) 11-15 DSDT(X) 8 E (SEQ ID NO:15);

(ii) RY(X) 2 AF(X) 2 ER(X) 2 QS(X) 9 GPL(X) 2 Y (SEQ ID NO:16);

(iii) QXFXG(X) 5 GDDRXLTN (SEQ ID NO:17); and

(iv) P(X) 8 QQXRWXKS(X) 2 RE (SEQ ID NO:18),

with X being selected from the group consisting of any amino acid; and

wherein the amino acid motifs are found in the relative order of (i), (ii), (iii), and (iv) from an amino terminus to a carboxyl terminus of the recombinantly produced, enzymatically active hyaluronan synthase.

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.

Assignments (2)

Continuity (8)