Methods of producing hyaluronic acid using a recombinant hyaluronan synthase gene

• US 20050202540A1
• Filed: 04/20/2005
• Published: 09/15/2005
• 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 7,060,466 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 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 method for producing hyaluronic acid, comprising the steps of:

providing a host cell having at least one expression construct comprising a hyaluronan synthase gene; and

culturing the host cell under conditions appropriate for the production of hyaluronic acid.

2. The method of claim 1, further comprising the step of separating the hyaluronic acid from the host cell.

3. The method of claim 1, wherein the at least one expression construct further comprises at least one gene encoding an enzyme for synthesis of a hyaluronic acid sugar precursor.

4. The method of claim 3, wherein the at least one gene encoding an enzyme for synthesis of a hyaluronic acid sugar precursor is selected from the group consisting of a pyrophosphorylase, a transferase, a mutase, a dehydrogenase, or an epimerase, capable of producing UDP-GlcNAc or UDP-GlcUA.

5. The method of claim 1, wherein the at least one expression construct further comprises at least one biosynthetic pathway gene of a hyaluronic acid sugar precursor.

6. The method of claim 1, wherein the at least one expression construct further includes at least one gene encoding an enzyme for synthesis of a hyaluronic acid sugar precursor.

7. The method of claim 1, further including a second at least one expression construct having at least one gene encoding an enzyme for synthesis of a hyaluronic acid sugar precursor.

8. The method of claim 1, wherein the host cell includes at least one integrated or plasmid-borne gene encoding an enzyme for synthesis of a hyaluronic acid sugar precursor.

9. The method of claim 1, wherein nutrients utilized for a hyaluronic acid sugar precursor biosynthetic pathway are supplied to the host cell.

10. The method of claim 9, wherein the nutrients supplying the hyaluronic acid sugar precursor biosynthetic pathway are fed to the host cell.

11. The method of claim 9, wherein the hyaluronic acid sugar precursors are expressed in the host cell by endogenous genes thereof.

12. The method of claim 9, wherein the hyaluronic acid sugar precursors enzymes are expressed in the host cell by an introduced construct including genes thereof.

13. The method of claim 1, wherein the hyaluronan synthase gene is isolated from a streptococcal source.

14. The method of claim 1, wherein the hyaluronan synthase gene is isolated from a microorganism.