Hyaluronan synthase genes and expression thereof in bacillus hosts

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).

1 . An isolated and purified 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-GlcNAc (uridine diphosphate N-acetylglucosamine) to synthesize HA (hyaluronic acid), and wherein the isolated and purified nucleic acid segment comprises at least one of:
 (A) a nucleotide sequence in accordance with SEQ ID NO:11;   (B) a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:12;   (C) a nucleotide sequence that is at least 80% identical to the nucleotide sequence of SEQ ID NO:11; and   (D) a nucleotide sequence that hybridizes to a complement of the nucleotide sequence of SEQ ID NO:11 under conditions of 1.2×-1.8× High Phosphate Buffer (0.5 M NaCl, 5 nM EDTA, 0.1 M Na 2 PO 4 , pH 7.2) at 40° C.-50° C.   
     
     
         2 . The isolated and purified nucleic acid segment of  claim 1 , wherein the isolated and purified nucleic acid segment comprises (C). 
     
     
         3 . The isolated and purified nucleic acid segment of  claim 1 , wherein the isolated and purified nucleic acid segment comprises (D). 
     
     
         4 . A recombinant expression vector comprising the nucleic acid segment of  claim 1 . 
     
     
         5 . A recombinant host cell comprising the recombinant expression vector of  claim 4 . 
     
     
         6 . The recombinant host cell of  claim 5 , wherein the recombinant host cell is a  Bacillus  host cell. 
     
     
         7 . The recombinant host cell of  claim 6 , wherein the recombinant host cell is a  Bacillus subtilis  or  Bacillus licheniformis  cell. 
     
     
         8 . The recombinant host cell of  claim 5 , wherein the recombinant host cell produces hyaluronic acid. 
     
     
         9 . The recombinant host cell of  claim 5 , wherein 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. 
     
     
         10 . The recombinant host cell of  claim 5 , wherein the recombinant vector further comprises a purified nucleic acid segment having a coding region encoding enzymatically active UDP-glucose dehydrogenase. 
     
     
         11 . The recombinant host cell of  claim 5 , wherein the recombinant host cell has enhanced production of at least one of UDP-GlcUA and UDP-GlcNAc. 
     
     
         12 . The recombinant host cell of  claim 11 , 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. 
     
     
         13 . The recombinant host cell of  claim 12 , wherein the modification is a mutation or tandem promoter elements. 
     
     
         14 . The recombinant host cell of  claim 11 , 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.   
     
     
         15 . A recombinant method for producing hyaluronic acid, comprising the step of:
 culturing the recombinant host cell of  claim 5  under conditions appropriate for the expression of hyaluronan synthase from the at least one expression construct, whereby hyaluronic acid is produced.   
     
     
         16 . The method of  claim 15 , further comprising the step of separating the hyaluronic acid from the host cell. 
     
     
         17 . The method of  claim 15 , wherein the recombinant host cell is a  Bacillus  host cell. 
     
     
         18 . The method of  claim 17 , wherein the recombinant host cell is a  Bacillus subtilis  or  Bacillus licheniformis  cell.