Hyaluronan synthase genes and expression thereof in Bacillus hosts

• US 8,735,102 B2
• Filed: 07/05/2011
• Issued: 05/27/2014
• Est. Priority Date: 07/01/1994
• 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 L., Weigel, Paul H., Kumari, Kshama
• Primary Examiner(s): KIM, ALEXANDER D
• Application Number:
  US13/176,344
• Publication Number:
  US 20110287487A1
• Time in Patent Office: 1,057 Days
• Field of Search: None
• US Class Current: 435/84
• 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 method for producing hyaluronic acid (HA), the method comprising the steps of:

obtaining a recombinant host cell capable of producing HA, the recombinant host cell containing a recombinant expression vector comprising a nucleic acid segment encoding an enzymatically active hyaluronan synthase, wherein the enzymatically active hyaluronan synthase is a single protein that is a dual-action catalyst that utilizes UDP-GlcA (uridine diphosphate glucuronic acid) and UDP-GIcNAc (uridine diphosphate N-acetylglucosamine) to synthesize HA, and wherein the nucleic acid segment comprises at least one of;

(A) the nucleotide sequence of nucleotides 23-1276 of SEQ ID NO;

11;

(B) a nucleotide sequence encoding the amino acid sequence of SEQ ID NO;

12; and

(C) a nucleotide sequence that is at least 90% identical to nucleotides 23-1276 of the nucleotide sequence of SEQ ID NO;

11;

culturing the recombinant host cell under conditions appropriate for the expression of hyaluronan synthase from the at least one expression construct, whereby hyaluronic acid is produced.

2. The method of claim 1 wherein, in the step of obtaining a recombinant host cell, the recombinant host cell is a Bacillus host cell.

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

4. The method of claim 1 wherein, in the step of obtaining a recombinant host cell, the recombinant host cell further comprises a recombinant vector comprising a purified nucleic acid segment having a coding region encoding an 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.

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

6. The method of claim 1 wherein, in the step of obtaining a recombinant host cell, the recombinant host cell has enhanced production of at least one of UDP-GlcUA and UDP-GlcNAc.

7. The method of claim 6, 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.

8. The method of claim 7, wherein the modification is at least one of a mutation and tandem promoter elements.

9. The method of claim 6, wherein the recombinant host cell further includes at least one of:

(A) at least one additional messenger RNA stabilizing element than is found in a native host cell;

(B) at least one less messenger RNA destabilizing element than is found in a native host cell;

(C) 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;

(D) 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;

(E) at least one mutated UDP-sugar precursor biosynthesis gene encoding a messenger RNA having an increased translational efficiency; and

(F) at least one mutated UDP-sugar precursor biosynthesis gene encoding a messenger RNA having an increased translational efficiency, 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.

10. The method of claim 9, further comprising the step of separating the hyaluronic acid from the recombinant host cell.

11. A recombinant method for producing hyaluronic acid (HA), the method comprising the steps of:

obtaining a recombinant host cell capable of producing HA, the recombinant host cell containing a recombinant expression vector comprising a nucleic acid segment encoding an enzymatically active hyaluronan synthase, wherein the enzymatically active hyaluronan synthase is a single protein that is a dual-action catalyst that utilizes UDP-GlcA (uridine diphosphate glucuronic acid) and UDP-GIcNAc (uridine diphosphate N-acetylglucosamine) to synthesize HA, and wherein the nucleic acid segment is at least 90% identical to nucleotides 23-1276 of the nucleotide sequence of SEQ ID NO;

11; and

culturing the recombinant host cell under conditions appropriate for the expression of hyaluronan synthase from the expression vector, whereby hyaluronic acid is produced.

12. The method of claim 11, further comprising the step of separating the hyaluronic acid from the recombinant host cell.

13. The method of claim 11 wherein, in the step of obtaining a recombinant host cell, the recombinant host cell is a Bacillus host cell.

14. The method of claim 13, wherein the recombinant host cell is a Bacillus subtilis or Bacillus licheniformis cell.

15. The method of claim 11 wherein, in the step of obtaining a recombinant host cell, the recombinant host cell further comprises a recombinant vector comprising a purified nucleic acid segment having a coding region encoding an 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.

16. The method of claim 11 wherein, in the step of obtaining a recombinant host cell, the recombinant vector further comprises a purified nucleic acid segment having a coding region encoding enzymatically active UDP-glucose dehydrogenase.

17. The method of claim 11 wherein, in the step of obtaining a recombinant host cell, the recombinant host cell has enhanced production of at least one of UDP-GlcUA and UDP-GlcNAc.

18. The method of claim 17, 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.

19. The method of claim 18, wherein the modification is at least one of a mutation and tandem promoter elements.

20. The method of claim 17, wherein the recombinant host cell further includes at least one of:

(A) at least one additional messenger RNA stabilizing element than is found in a native host cell;

(B) at least one less messenger RNA destabilizing element than is found in a native host cell;

(C) 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;

(D) 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;

(E) at least one mutated UDP-sugar precursor biosynthesis gene encoding a messenger RNA having an increased translational efficiency; and

(F) at least one mutated UDP-sugar precursor biosynthesis gene encoding a messenger RNA having an increased translational efficiency, 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.