HYALURONAN SYNTHASE GENE AND USES THEREOF

• US 20060216793A1
• 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 7,166,450 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 method of producing hyaluronic acid that includes introducing a purified nucleic acid segment having a coding region encoding enzymatically active hyaluronan synthase into a Bacillus strain, and culturing the Bacillus strain under conditions appropriate for the production of hyaluronic acid.

Claims

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(SEQ ID NO;

11)(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;

(ii)(L/M)(S/T/V)(D/S/A)XRY(W/Y/D)XAF(G/N/C)(SEQ ID NO;

12)(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);

(iii)(D/E)X(W/Y)(X)₂QXF(L/M)G(X)₂(C/V)(S/T)(SEQ ID NO;

13)XGDDR(H/R/C)LTN;

and (iv)(E/D)(T/S/V)P(X)₅(W/Y/F)(L/I)XQQ(T/N)(SEQ ID NO;

14)RW(S/T/N)KS(Y/W/F)(F/C)RE,

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 hyaluronan synthase gene is isolated from a prokaryotic source.

4. The method of claim 1, wherein the hyaluronan synthase gene 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 is UDP-glucose dehydrogenase.

10. The method according to claim 8, wherein the UDP-sugar precursor biosynthesis pathway 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:

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(SEQ ID NO;

11)(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;

(ii)(L/M)(S/T/V)(D/S/A)XRY(W/Y/D)XAF(G/N/C)(SEQ ID NO;

12)(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);

(iii)(D/E)X(W/Y)(X)₂QXF(L/M)G(X)₂(C/V)(S/T)(SEQ ID NO;

13)XGDDR(H/R/C)LTN;

and (iv)(E/D)(T/S/V)P(X)₅(W/Y/F)(L/I)XQQ(T/N)(SEQ ID NO;

14)RW(S/T/N)KS(Y/W/F)(F/C)RE,

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.

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)₆F(X)_11-15DSDT(X)₈E;

(SEQ ID NO;

15) (ii)RY(X)₂AF(X)₂ER(X)₂QS(X)₉GPL(SEQ ID NO;

16)(X)₂Y;

(iii)QXFXG(X)₅GDDRXLTN;

(SEQ ID NO;

17)and (iv)P(X)₈QQXRWXKS(X)₂RE,(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 hyaluronan synthase gene is isolated from a prokaryotic source.

23. The method of claim 20, wherein the hyaluronan synthase gene 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 is UDP-glucose dehydrogenase.

29. The method according to claim 27, wherein the UDP-sugar precursor biosynthesis pathway 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:

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)₆F(X)_11-15DSDT(X)₈E;

(SEQ ID NO;

15) (ii)RY(X)₂AF(X)₂ER(X)₂QS(X)₉GPL(SEQ ID NO;

16)(X)₂Y;

(iii)QXFXG(X)₅GDDRXLTN;

(SEQ ID NO;

17)and (iv)P(X)₈QQXRWXKS(X)₂RE,(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.