STREPTOCOCCUS EQUISIMILIS HYALURONAN SYNTHASE GENE AND EXPRESSION THEREOF IN BACILLUS SUBTILIS

• US 20050287646A1
• Filed: 05/02/2005
• Published: 12/29/2005
• Est. Priority Date: 07/23/1997
• 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 7,029,880 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 19/04 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

Abstract

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.

Claims

1. A recombinant host cell, wherein the recombinant host cell is a Bacillus subtilis cell transformed with a recombinant vector comprising a purified nucleic acid segment having a coding region encoding enzymatically active hyaluronan synthase, wherein the coding region encodes a single protein that is a dual-action catalyst that utilizes UDP-GlcA and UDP-GlcNAc to synthesize HA.

2. The recombinant host cell of claim 1, wherein the purified nucleic acid segment encodes the Streptococcus equisimilis hyaluronan synthase of SEQ ID NO:

2.

3. The recombinant host cell of claim 1, wherein the purified nucleic acid segment comprises a nucleotide sequence in accordance with SEQ ID NO:

1.

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

8. The recombinant host cell of claim 1, wherein the purified nucleic acid segment encodes a Group A hyaluronan synthase.

9. 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 Bacillus subtilis promoter.

10. The recombinant host cell of claim 1, wherein the Bacillus subtilis cell is transformed with a recombinant vector comprising a purified nucleic acid segment having a coding region encoding enzymatically active UDP-glucose dehydrogenase.

11. The recombinant host cell of claim 1, wherein the purified nucleic acid segment encodes a Group C hyaluronan synthase.

12. The recombinant host cell of claim 1, herein the recombinant host cell has an enhanced production of at least one of UPD-GlcA and UDP-GlcNAc.

13. The recombinant host cell of claim 12, wherein the recombinant host cell further includes at least one mutated RNA polymerase promoter capable of expressing RNA polymerase in an amount greater than an endogenous RNA polymerase promoter.

14. The recombinant host cell of claim 12, wherein the recombinant host cell is transformed with a recombinant vector comprising a purified nucleic acid segment having a coding region encoding a UDP-sugar precursor biosynthesis pathway enzyme.

15. The recombinant host cell of claim 14, wherein the UDP-sugar precursor biosynthesis pathway enzyme is UDP-glucose dehydrogenase.

16. The recombinant host cell of claim 14, wherein the UDP-sugar precursor biosynthesis pathway enzyme is UDP-glucose pyrophosphorylase.

17. The recombinant host cell of claim 12, wherein the recombinant host cell further includes at least one additional messenger RNA stabilizing element than is found in a native Bacillus subtilis cell.

18. The recombinant host cell of claim 12,wherein the recombinant host cell further includes at least one less messenger RNA destabilizing element than is found in a native Bacillus subtilis cell.

19. The recombinant host cell of claim 12, wherein the recombinant host cell 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.

20. The recombinant host cell of claim 12, wherein the recombinant host cell further includes at least one mutated UDP-sugar precursor gene wherein the mutated UDP-sugar precursor gene increases a half-life of a transcribed messenger RNA.

21. The recombinant host cell of claim 12, wherein the recombinant host cell further includes at least one mutated UDP-sugar precursor gene encoding a messenger RNA having an increased translational efficiency.

22. The recombinant host cell of claim 21, wherein the mutated UDP-sugar precursor gene occurs in a ribosome binding site in the UDP-sugar precursor gene such that a ribosome has an increased binding affinity for the ribosome binding site.

5. A recombinant 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 subtilis strain, wherein the coding region encodes a single protein that is a dual-action catalyst that utilizes UDP-GlcA and UDP-GlcNAc to synthesize HA;

growing the Bacillus subtilis strain in a medium to secrete hyaluronic acid; and

recovering the secreted hyaluronic acid.

6. The method according to claim 5, wherein the step of recovering the hyaluronic acid comprises extracting the secreted hyaluronic acid from the medium.

7. The method according to claim 6, further comprising the step of purifying the extracted hyaluronic.

23. The method of claim 5, further comprising the step of introducing a purified nucleic acid segment having a coding region encoding enzymatically active UDP-glucose dehydrogenase in the Bacillus subtilis strain.

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

25. The method of claim 5 wherein, in the step of introducing a purified nucleic acid segment, the purified nucleic acid segment encodes a Group A hyaluronan synthase.

26. The method of claim 5 wherein, in the step of introducing a purified nucleic acid segment having a coding region encoding enzymatically active hyaluronan synthase into a Bacillus subtilis strain, the purified nucleic acid segment is introduced by at least one of transforming, transfecting, transducing, and electroporating.

27. The method of claim 5 wherein, in the step of introducing a purified nucleic acid segment, the purified nucleic acid segment encodes the enzymatically active hyaluronan synthase of SEQ ID NO:

2.

28. The method of claim 5 wherein, in the step of introducing a purified nucleic acid segment, the purified nucleic acid segment comprises a nucleotide sequence in accordance with SEQ ID NO:

1.

29. The method of claim 5 wherein, in the step of introducing a purified nucleic acid segment having a coding region encoding enzymatically active hyaluronan synthase into a Bacillus subtilis strain, the Bacillus subtilis strain has an enhanced production of at least one of UDP-GlcA and UDP-GlcNAc.

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

31. The method according to claim 29, wherein in the step of introducing a purified nucleic acid segment having a coding region encoding enzymatically active hyaluronan synthase into a Bacillus subtilis strain, the Bacillus subtilis 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.

32. The method according to claim 31, wherein the UDP-sugar precursor biosynthesis pathway is UDP-glucose dehydrogenase.

33. The method according to claim 31, wherein the UDP-sugar precursor biosynthesis pathway is UDP-glucose pyrophosphorylase.

34. The method according to claim 29, wherein in the step of introducing a purified nucleic acid segment having a coding region encoding enzymatically active hyaluronan synthase into a Bacillus subtilis strain, the Bacillus subtilis strain further includes at least one additional messenger RNA stabilizing element than is found in a native Bacillus subtilis strain.

35. The method according to claim 29, wherein in the step of introducing a purified nucleic acid segment having a coding region encoding enzymatically active hyaluronan synthase into a Bacillus subtilis strain, the Bacillus subtilis strain further includes at least one less messenger RNA destabilizing element than is found in a native Bacillus subtilis strain.

36. The method according to claim 29, wherein in the step of introducing a purified nucleic acid segment having a coding region encoding enzymatically active hyaluronan synthase into a Bacillus subtilis strain, the Bacillus subtilis 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.

37. The method according to claim 29, wherein in the step of introducing a purified nucleic acid segment having a coding region encoding enzymatically active hyaluronan synthase into a Bacillus subtilis strain, the Bacillus subtilis 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.

38. The method according to claim 29, wherein in the step of introducing a purified nucleic acid segment having a coding region encoding enzymatically active hyaluronan synthase into a Bacillus subtilis strain, the Bacillus subtilis strain further includes at least one mutated UDP-sugar precursor gene encoding a messenger RNA having an increased translational efficiency.

39. The method according to claim 38, 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.