Granted Patent B2

US 7,166,450 · App. 11/446,854 · Granted Jan 23, 2007

Assignee: The Board of Regents of the University of Oklahoma

Abstract

Claims (82)

1. A method of producing hyaluronic acid, comprising the steps of:

introducing a purified nucleic acid segment having a coding region encoding enzymatically active hyaluronan synthase into a Bacillus strain, wherein the coding region encodes an enzymatically active hyaluronan synthase that 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)(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 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 enzymatically active hyaluronan synthase; and

culturing the Bacillus strain under conditions appropriate for the production of hyaluronic acid.

2. The method of claim 1 , further comprising the step of recovering the hyaluronic acid.

3. The method of claim 1 , wherein the coding region encoding enzymatically active hyaluronan synthase is isolated from a prokaryotic source.

4. The method of claim 1 , wherein the coding region encoding enzymatically active hyaluronan synthase is isolated from a microbial source.

5. The method of claim 1 wherein, in the step of introducing a purified nucleic acid segment having a coding region encoding enzymatically active hyaluronan synthase into a Bacillus strain, the coding region encoding enzymatically active hyaluronan synthase of the purified nucleic acid segment is under control of a Bacillus promoter.

6. The method of claim 1 wherein, in the step of introducing a purified nucleic acid segment having a coding region encoding enzymatically active hyaluronan synthase into a Bacillus strain, the Bacillus strain has an enhanced production of at least one of UDP-GlCA and UDP-GlcNAc.

7. The method according to claim 6 , wherein in the step of introducing a purified nucleic acid segment having a coding region encoding enzymatically active hyaluronan synthase into a Bacillus strain, the Bacillus strain further includes at least one mutated RNA polymerase promoter having an increased promoter activity.

8. The method according to claim 6 , wherein in the step of introducing a purified nucleic acid segment having a coding region encoding enzymatically active hyaluronan synthase into a Bacillus strain, the Bacillus strain is transformed with a vector comprising a purified nucleic acid segment having a coding region encoding an endogenous UDP-sugar precursor biosynthesis pathway enzyme.

9. The method according to claim 8 , wherein the UDP-sugar precursor biosynthesis pathway enzyme is UDP-glucose dehydrogenase.

10. The method according to claim 8 , wherein the UDP-sugar precursor biosynthesis pathway enzyme is UDP-glucose pyrophosphorylase.

11. The method according to claim 6 , wherein in the step of introducing a purified nucleic acid segment having a coding region encoding enzymatically active hyaluronan synthase into a Bacillus strain, the Bacillus strain further includes at least one additional messenger RNA stabilizing element than is found in a native Bacillus strain.

12. The method according to claim 6 , wherein in the step of introducing a purified nucleic acid segment having a coding region encoding enzymatically active hyaluronan synthase into a Bacillus strain, the Bacillus strain further includes at least one less messenger RNA destabilizing element than is found in a native Bacillus strain.

13. The method according to claim 6 , wherein in the step of introducing a purified nucleic acid segment having a coding region encoding enzymatically active hyaluronan synthase into a Bacillus strain, the Bacillus strain further includes at least one nucleic acid segment having a coding region encoding a UDP-sugar precursor biosynthesis pathway enzyme having an activity greater than an endogenous UDP-sugar precursor biosynthesis pathway enzyme.

14. The method according to claim 6 , wherein in the step of introducing a purified nucleic acid segment having a coding region encoding enzymatically active hyaluronan synthase into a Bacillus strain, the Bacillus strain further includes at least one mutated UDP-sugar precursor gene wherein the mutation results in an increase of a half-life of a messenger RNA transcribed from the mutated UDP-sugar precursor gene.

15. The method according to claim 6 , wherein in the step of introducing a purified nucleic acid segment having a coding region encoding enzymatically active hyaluronan synthase into a Bacillus strain, the Bacillus strain further includes at least one mutated UDP-sugar precursor gene encoding a messenger RNA having an increased translational efficiency.

16. The method according to claim 15 , wherein the mutation to the UDP-sugar precursor gene occurs in a ribosome binding site in the UDP-sugar precursor gene such that a binding affinity of a ribosome for the ribosome binding site is increased.

17. The method according to claim 1 , wherein the Bacillus strain is a Bacillus subtilis strain.

18. The method according to claim 1 , wherein the hyaluronan synthase gene encodes an enzymatically active hyaluronan synthase that 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/CIT)(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 enzymatically active hyaluronan synthase.

19. A method of producing hyaluronic acid, comprising the steps of:

introducing a purified nucleic acid segment having a coding region encoding enzymatically active hyaluronan synthase into a Bacillus strain, wherein the coding region encodes an enzymatically active hyaluronan synthase that 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 enzymatically active hyaluronan synthase; and

culturing the Bacillus strain under conditions appropriate for the production of hyaluronic acid.

20. A method of producing hyaluronic acid, comprising the steps of:

introducing a purified nucleic acid segment having a coding region encoding enzymatically active hyaluronan synthase into a Bacillus strain, wherein the coding region encodes an enzymatically active hyaluronan synthase that 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 enzymatically active hyaluronan synthase; and

culturing the Bacillus strain under conditions appropriate for the production of hyaluronic acid.

21. The method of claim 20 , further comprising the step of recovering the hyaluronic acid.

22. The method of claim 20 , wherein the coding region encoding enzymatically active hyaluronan synthase is isolated from a prokaryotic source.

23. The method of claim 20 , wherein the coding region encoding enzymatically active hyaluronan synthase is isolated from a microbial source.

24. The method of claim 20 wherein, in the step of introducing a purified nucleic acid segment having a coding region encoding enzymatically active hyaluronan synthase into a Bacillus strain, the coding region encoding enzymatically active hyaluronan synthase of the purified nucleic acid segment is under control of a Bacillus promoter.

25. The method of claim 20 wherein, in the step of introducing a purified nucleic acid segment having a coding region encoding enzymatically active hyaluronan synthase into a Bacillus strain, the Bacillus strain has an enhanced production of at least one of UDP-GlCA and UDP-GlcNAc.

26. The method according to claim 25 , wherein in the step of introducing a purified nucleic acid segment having a coding region encoding enzymatically active hyaluronan synthase into a Bacillus strain, the Bacillus strain further includes at least one mutated RNA polymerase promoter having an increased promoter activity.

27. The method according to claim 25 , wherein in the step of introducing a purified nucleic acid segment having a coding region encoding enzymatically active hyaluronan synthase into a Bacillus strain, the Bacillus strain is transformed with a vector comprising a purified nucleic acid segment having a coding region encoding an endogenous UDP-sugar precursor biosynthesis pathway enzyme.

28. The method according to claim 27 , wherein the UDP-sugar precursor biosynthesis pathway enzyme is UDP-glucose dehydrogenase.

29. The method according to claim 27 , wherein the UDP-sugar precursor biosynthesis pathway enzyme is UDP-glucose pyrophosphorylase.

30. The method according to claim 25 , wherein in the step of introducing a purified nucleic acid segment having a coding region encoding enzymatically active hyaluronan synthase into a Bacillus strain, the Bacillus strain further includes at least one additional messenger RNA stabilizing element than is found in a native Bacillus strain.

31. The method according to claim 25 , wherein in the step of introducing a purified nucleic acid segment having a coding region encoding enzymatically active hyaluronan synthase into a Bacillus strain, the Bacillus strain further includes at least one less messenger RNA destabilizing element than is found in a native Bacillus strain.

32. The method according to claim 25 , wherein in the step of introducing a purified nucleic acid segment having a coding region encoding enzymatically active hyaluronan synthase into a Bacillus strain, the Bacillus strain further includes at least one nucleic acid segment having a coding region encoding a UDP-sugar precursor biosynthesis pathway enzyme having an activity greater than an endogenous UDP-sugar precursor biosynthesis pathway enzyme.

33. The method according to claim 25 , wherein in the step of introducing a purified nucleic acid segment having a coding region encoding enzymatically active hyaluronan synthase into a Bacillus strain, the Bacillus strain further includes at least one mutated UDP-sugar precursor gene wherein the mutation results in an increase of a half-life of a messenger RNA transcribed from the mutated UDP-sugar precursor gene.

34. The method according to claim 25 , wherein in the step of introducing a purified nucleic acid segment having a coding region encoding enzymatically active hyaluronan synthase into a Bacillus strain, the Bacillus strain further includes at least one mutated UDP-sugar precursor gene encoding a messenger RNA having an increased translational efficiency.

35. The method according to claim 34 , wherein the mutation to the UDP-sugar precursor gene occurs in a ribosome binding site in the UDP-sugar precursor gene such that a binding affinity of a ribosome for the ribosome binding site is increased.

36. The method according to claim 20 , wherein the Bacillus strain is a Bacillus subtilis strain.

37. The method according to claim 20 , wherein the hyaluronan synthase gene encodes an enzymatically active hyaluronan synthase that 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 enzymatically active hyaluronan synthase.

38. A method of producing hyaluronic acid, comprising the steps of:

introducing a purified nucleic acid segment having a coding region encoding enzymatically active hyaluronan synthase into a Bacillus strain, wherein the coding region encodes an enzymatically active hyaluronan synthase that 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 enzymatically active hyaluronan synthase; and

culturing the Bacillus strain under conditions appropriate for the production of hyaluronic acid.

Assignments (1)

Continuity (8)