Hyaluronan synthase gene and uses thereof

• US 20070087416A1
• Filed: 11/14/2006
• Published: 04/19/2007
• Est. Priority Date: 07/01/1994
• Status: Active Grant

• Current Assignee: Kshama Kumari, Paul Deangelis, Paul Weigel
• Original Assignee: Kshama Kumari, Paul Deangelis, Paul Weigel
• Inventors: Kumari, Kshama, DeAngelis, Paul, Weigel, Paul
• Granted Patent: US 7,504,246 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 recombinantly produced, enzymatically active hyaluronan synthase, wherein the recombinantly produced, enzymatically active hyaluronan synthase is a single protein that is a dual-action catalyst that utilizes UDP-GlcA and UDP-GlcNAc to synthesize HA. The recombinantly produced, enzymatically active hyaluronan synthase may be produced from a hyaluronan synthase gene isolated from a microbial source, such as a streptococcal source.

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 expression of hyaluronan synthase from the at least one expression construct and for 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 3, 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.

6. 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.

7. 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.

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

9. The method of claim 8, wherein the nutrients supplying the hyaluronic acid sugar precursor biosynthetic pathway are fed to the host cell.

10. The method of claim 8, wherein the hyaluronic acid sugar precursors are expressed in the host cell by endogenous genes thereof.

11. The method of claim 8, wherein the hyaluronic acid sugar precursors enzymes are expressed in the host cell by an introduced construct including genes thereof.

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

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

14. The method of claim 1 wherein the hyaluronan synthase protein a central intracellular domain, at least two transmembrane domains at an amino end thereof, and at least two membrane-associated domains at a carboxyl end thereof.

15. The method of claim 1 wherein the hyaluronan synthase gene comprises the nucleotide sequence of SEQ ID NO:

1.

16. The method of claim 1 wherein the hyaluronan synthase comprises the amino acid sequence of SEQ ID NO:

2.

17. The method of claim 1 wherein the hyaluronan synthase gene is at least 70% identical to the nucleotide sequence of SEQ ID NO:

1.

18. The method of claim 1 wherein the hyaluronan synthase gene hybridizes to a complement of the nucleotide sequence of SEQ ID NO:

1 under conditions of 1.2-1.8×

HPB at 40-50°

C.