Hyaluronan synthase genes and expression thereof in Bacillus hosts

• US 20060263858A1
• Filed: 06/26/2006
• Published: 11/23/2006
• Est. Priority Date: 07/01/1994
• Status: Active Grant

• Current Assignee: Kshama Kumari, Paul H. Weigel, Paul L. Deangelis
• Original Assignee: Kshama Kumari, Paul H. Weigel, Paul L. Deangelis
• Inventors: DeAngelis, Paul L., Weigel, Paul H., Kumari, Kshama
• Granted Patent: US 7,229,796 B2
• Time in Patent Office: Days
• US Class Current: 435/85
• 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 19/26 Preparation of nitrogen-containing carbohydrates

Abstract

The present invention relates to a recombinant Bacillus host cell containing a recombinant vector including a nucleic acid segment having a coding region segment encoding enzymatically active hyaluronan synthase (HAS). The recombinant Bacillus host cell is utilized in a method for producing hyaluronic acid (HA).

Claims

1. A recombinant host cell, wherein the recombinant host cell is a Bacillus cell comprising a recombinant vector comprising a purified nucleic acid segment having a coding region encoding enzymatically active hyaluronan synthase, wherein the coding region is under control of a promoter, and wherein the coding region encoding enzymatically active hyaluronan synthase is selected from the group consisting of:

(a) a coding region encoding enzymatically active hyaluronan synthase of SEQ ID NO;

16;

(b) a coding region encoding enzymatically active hyaluronan synthase of SEQ ID NO;

8;

(c) a nucleotide sequence in accordance with SEQ ID NO;

15;

(d) a nucleotide sequence in accordance with SEQ ID NO;

7;

(e) a coding region encoding enzymatically active hyaluronan synthase that is at least 70% identical to SEQ ID NO;

14; and

(f) a coding region encoding enzymatically active hyaluronan synthase, wherein the coding region is capable of hybridizing to a complementary sequence of SEQ ID NO;

13.

2. The recombinant host cell of claim 1, wherein the recombinant host cell is a Bacillus subtilis or a Bacillus licheniformis cell.

3. The recombinant host cell of claim 1, wherein the host cell produces hyaluronic acid.

4. The recombinant host cell of claim 1, wherein the coding region encoding enzymatically active hyaluronan synthase of the purified nucleic acid segment is under control of a Gram positive bacterial-compatible promoter.

5. The recombinant host cell of claim 4, wherein the promoter is a Bacillus compatible promoter.

6. The recombinant host cell of claim 1, wherein the Bacillus subtilis cell further comprises a recombinant vector comprising a purified nucleic acid segment having a coding region encoding enzymatically active UDP-GlcUA biosynthetic pathway enzyme, wherein the enzymatically active UDP-GlcUA biosynthetic pathway enzyme is selected from the group consisting of UDP-glucose dehydrogenase, UDP-glucose pyrophosphorylase, and combinations thereof.

7. The recombinant host cell of claim 1, wherein the recombinant vector further comprises a purified nucleic acid segment having a coding region encoding enzymatically active UDP-glucose dehydrogenase.

8. The recombinant host cell of claim 7, wherein the coding region encoding enzymatically active HAS and the coding region encoding enzymatically active UDP-glucose dehydrogenase are under the control of different promoters.

9. The recombinant host cell of claim 1, wherein the recombinant host cell has enhanced production of at least one of UDP-GlcUA and UDP-GlcNAc.

10. The recombinant host cell of claim 9, wherein the recombinant host cell further includes at least one modified RNA polymerase promoter wherein, when the modified RNA polymerase promoter is recognized by an RNA polymerase, the RNA polymerase is capable of expressing RNA in an amount greater than an endogenous RNA polymerase promoter.

11. The recombinant host cell of claim 10, wherein the modification is a mutation.

12. The recombinant host cell of claim 10, wherein the modification is tandem promoter elements.

13. The recombinant host cell of claim 9, wherein the recombinant host cell further includes at least one additional messenger RNA stabilizing element than is found in a native Bacillus cell.

14. The recombinant host cell of claim 9, wherein the recombinant host cell further includes at least one less messenger RNA destabilizing element than is found in a native Bacillus cell.

15. The recombinant host cell of claim 9, wherein the recombinant host cell further includes at least one nucleic acid segment having a coding region encoding a functionally active enzyme in a UDP-sugar precursor biosynthesis pathway such that the recombinant host cell has an activity greater than a native host cell expressing an endogenous UDP-sugar precursor biosynthesis pathway enzyme.

16. The recombinant host cell of claim 9, wherein the recombinant host cell further includes at least one mutated UDP-sugar precursor biosynthesis gene, wherein the mutated UDP-sugar precursor gene increases a half-life of a transcribed messenger RNA.

17. The recombinant host cell of claim 9, wherein the recombinant host cell further includes at least one mutated UDP-sugar precursor biosynthesis gene encoding a messenger RNA having an increased translational efficiency.

18. The recombinant host cell of claim 14, wherein the mutation in the UDP-sugar precursor biosynthesis gene occurs in a ribosome binding site in the UDP-sugar precursor biosynthesis gene such that a ribosome has an increased binding affinity for the ribosome binding site.

19. A method for 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 host wherein the coding region is under control of a promoter, and wherein the coding region encoding enzymatically active hyaluronan synthase is selected from the group consisting of;

(a) a coding region encoding enzymatically active hyaluronan synthase of SEQ ID NO;

16;

(b) a coding region encoding enzymatically active hyaluronan synthase of SEQ ID NO;

8;

(c) a nucleotide sequence in accordance with SEQ ID NO;

15;

(d) a nucleotide sequence in accordance with SEQ ID NO;

7;

(e) a coding region encoding enzymatically active hyaluronan synthase that is at least 70% identical to SEQ ID NO;

14; and

(f) a coding region encoding enzymatically active hyaluronan synthase, wherein the coding region is capable of hybridizing to a complementary sequence of SEQ ID NO;

13;

growing the host organism in a medium to secrete hyaluronic acid; and

recovering the secreted hyaluronic acid.

20. The method of claim 19, wherein the Bacillus host is Bacillus subtilis or Bacillus licheniformis.

21. The method according to claim 19, wherein the step of recovering the hyaluronic acid comprises extracting the secreted hyaluronic acid from the medium.

22. The method according to claim 21, further comprising the step of purifying the extracted hyaluronic acid.

23. The method of claim 19, further comprising the step of introducing a purified nucleic acid segment having a coding region encoding enzymatically active UDP-glucose dehydrogenase into the Bacillus host.

24. The method of claim 23, wherein the coding region encoding enzymatically active hyaluronan synthase and the coding region encoding enzymatically active UDP-glucose dehydrogenase are under the control of the same promoter.

25. The method of claim 23, wherein the coding region encoding enzymatically active hyaluronan synthase and the coding region encoding enzymatically active UDP-glucose dehydrogenase are under the control of different promoters.

26. The method of claim 19, wherein, in the step of introducing a purified nucleic acid segment having a coding region encoding enzymatically active hyaluronan synthase into a Bacillus host, the coding region encoding enzymatically active hyaluronan synthase of the purified nucleic acid segment is under control of a Gram positive bacterial-compatible promoter.

27. The method of claim 23, wherein the promoter is a Bacillus-compatible promoter.

28. The method of claim 19 wherein, in the step of introducing a purified nucleic acid segment having a coding region encoding enzymatically active hyaluronan synthase into a Bacillus host, the Bacillus host has an enhanced production of at least one of UDP-GlcUA and UDP-GlcNAc.

29. The method of claim 28 wherein, in the step of introducing a purified nucleic acid segment having a coding region encoding enzymatically active hyaluronan synthase into a Bacillus host, the Bacillus host further includes at least one modified RNA polymerase promoter having an increased promoter activity.

30. The method of claim 29, wherein the modification of the RNA polymerase promoter is a mutation.

31. The method of claim 29, wherein the modification of the RNA polymerase promoter is tandem promoter elements.

32. The method of claim 28 wherein, in the step of introducing a purified nucleic acid segment having a coding region encoding enzymatically active hyaluronan synthase into a Bacillus host, the Bacillus host further includes at least one additional messenger RNA stabilizing element than is found in a native Bacillus host.

33. The method of claim 28 wherein, in the step of introducing a purified nucleic acid segment having a coding region encoding enzymatically active hyaluronan synthase into a Bacillus host, the Bacillus host further includes at least one less messenger RNA destabilizing element than is found in a native Bacillus host.

34. The method of claim 28 wherein, in the step of introducing a purified nucleic acid segment having a coding region encoding enzymatically active hyaluronan synthase into a Bacillus host, the Bacillus host further includes at least one nucleic acid segment having a coding region encoding a UDP-sugar precursor biosynthesis pathway enzyme such that the recombinant host cell has an activity greater than a native host cell expressing an endogenous UDP-sugar precursor biosynthesis pathway enzyme.

35. The method of claim 28 wherein, in the step of introducing a purified nucleic acid segment having a coding region encoding enzymatically active hyaluronan synthase into a Bacillus host, the Bacillus host comprises at least one mutated endogenous UDP-sugar precursor biosynthesis gene wherein the mutation results in an increase of a half-life of a messenger RNA transcribed from the mutated UDP-sugar precursor gene.

36. The method of claim 19 wherein, in the step of introducing a purified nucleic acid segment having a coding region encoding enzymatically active hyaluronan synthase into a Bacillus host, the Bacillus host comprises at least one mutated UDP-sugar precursor biosynthesis gene encoding a messenger RNA having an increased translational efficiency.

37. The method according to claim 36, wherein the mutation in the UDP-sugar precursor biosynthesis gene occurs in a ribosome binding site in the UDP-sugar precursor biosynthesis gene such that a ribosome has an increased binding affinity for the ribosome binding site.