DNA encoding hyaluronan synthase from Pasteurella multocida and methods of use

• US 7,604,973 B2
• Filed: 05/01/2007
• Issued: 10/20/2009
• Est. Priority Date: 04/02/1998
• Status: Expired due to Fees

• Current Assignee: The Board of Regents of the University of Colorado (University of Colorado System)
• Original Assignee: The Board of Regents of the University of Colorado (University of Colorado System)
• Inventors: DeAngelis, Paul
• Primary Examiner(s): Monshipouri; Maryam
• Application Number:
  US11/799,130
• Publication Number:
  US 20070298461A1
• Time in Patent Office: 903 Days
• Field of Search: 435/193, 435/320.1, 435/252.3, 435/6, 536/23.2
• US Class Current: 435/193
• CPC Class Codes: A61K 39/102 Pasteurellales, e.g. Actinobacillus, Pasteurella; Haemophilus
• CPC Class Codes: A61K 47/36 Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
• CPC Class Codes: A61L 24/08 Polysaccharides A61L24/043 takes precedence
• CPC Class Codes: A61L 29/085 Macromolecular materials
• CPC Class Codes: C08L 5/08 Chitin; Chondroitin sulfate; Hyaluronic acid; Derivatives thereof
• 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 Pasteurella multocida hyaluronate synthase (PmHAS), and to the use of this nucleic acid segment in the preparation of recombinant cells which produce hyaluronate synthase and its hyaluronic acid product.

Claims

1. 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 from Pasteurella into a host organism, wherein the enzymatically active hyaluronan synthase is a single protein that polymerizes UDP-GlcA and UDP-GlcNAc to form hyaluronic acid, and wherein the nucleic acid segment is selected from the group consisting of;

(a) the nucleic acid sequence as set forth in SEQ ID NO;

2;

(b) a nucleic acid segment encoding SEQ ID NO;

1; and

(c) a nucleic acid segment encoding SEQ ID NO;

17;

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

recovering the secreted hyaluronic acid.

2. The method according to claim 1, wherein the step of recovering the hyaluronic acid comprises extracting the secreted hyaluronic acid from the medium.

3. The method according to claim 2, further comprising the step of purifying the extracted hyaluronic acid.

16. The method of claim 1, wherein the host organism is a Bacillus strain.

4. 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 from Pasteurella into a host organism, wherein the enzymatically active hyaluronan synthase is a single protein that polymerizes UDP-GlcA and UDP-GlcNAc to form hyaluronic acid, and wherein a complement of the nucleic acid segment is capable of hybridizing to the nucleic acid sequence of SEQ ID NO;

2 under standard hybridization conditions comprising 1.2-1.8x HPB at 40-50°

C.;

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

recovering the secreted hyaluronic acid.

5. The method according to claim 4, wherein the step of recovering the hyaluronic acid comprises extracting the secreted hyaluronic acid from the medium.

6. The method according to claim 5, further comprising the step of purifying the extracted hyaluronic acid.

17. The method of claim 4, wherein the host organism is a Bacillus strain.

7. 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 from Pasteurella into a host organism, wherein the enzymatically active hyaluronan synthase is a single protein that polymerizes UDP-GlcA and UDP-GlcNAc to form hyaluronic acid, and wherein the nucleic acid segment is at least 85% identical to SEQ ID NO;

2;

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

recovering the secreted hyaluronic acid.

8. The method according to claim 7, wherein the step of recovering the hyaluronic acid comprises extracting the secreted hyaluronic acid from the medium.

9. The method according to claim 8, further comprising the step of purifying the extracted hyaluronic acid.

18. The method of claim 7, wherein the host organism is a Bacillus strain.

10. 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 from Pasteurella into a host organism, wherein the enzymatically active hyaluronan synthase is a single protein that polymerizes UDP-GlcA and UDP-GlcNAc to form hyaluronic acid, and wherein the enzymatically active hyaluronan synthase encoded by the purified nucleic acid segment is at least 89% identical to SEQ ID NO;

1;

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

recovering the secreted hyaluronic acid.

11. The method according to claim 10, wherein the step of recovering the hyaluronic acid comprises extracting the secreted hyaluronic acid from the medium.

12. The method according to claim 11, further comprising the step of purifying the extracted hyaluronic acid.

19. The method of claim 10, wherein the host organism is a Bacillus strain.

13. 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 from Pasteurella into a host organism, wherein the enzymatically active hyaluronan synthase is a single protein that polymerizes UDP-GlcA and UDP-GlcNAc to form hyaluronic acid, and wherein the hyaluronan synthase has SEQ ID NO;

18 and therein, and wherein a complement of the nucleic acid segment is capable of hybridizing to the nucleic acid sequence of SEQ ID NO;

2 under standard hybridization conditions comprising 1.2-1.8x HPB at 40-50°

C.;

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

recovering the secreted hyaluronic acid.

14. The method according to claim 13, wherein the step of recovering the hyaluronic acid comprises extracting the secreted hyaluronic acid from the medium.

15. The method according to claim 14, further comprising the step of purifying the extracted hyaluronic acid.

20. The method of claim 13, wherein the host organism is a Bacillus strain.

21. 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 enzymatically active hyaluronan synthase is a single protein that polymerizes UDP-GlcA and UDP-GlcNAc to form hyaluronic acid (HA), and 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 that is at least 85% identical to SEQ ID NO;

2; and

(B) a coding region wherein a complement thereof is capable of hybridizing to the nucleic acid sequence of SEQ ID NO;

2 under standard hybridization conditions comprising 1.2-1.8x HPB at 40-50°

C.

22. The recombinant host cell of claim 21, wherein the host cell produces hyaluronic acid.