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 . 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 is a dual-action catalyst that utilizes UDP-GlcA and UDP-GlcNAc to synthesize 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 encoding enzymatically active hyaluronan synthase that is at least 80% identical to SEQ ID NO:10; and    (B) a coding region that is capable of hybridizing to a complementary sequence of SEQ ID NO:9 under hybridization conditions of 1.2-1.8× High Phosphate Buffer (HPB) at 40-50° C.    
     
     
         2 . The recombinant host cell of  claim 1 , wherein the recombinant host cell is a  Bacillus subtilis  or a  Bacillus licheniformis  cell.  
     
     
         3 . The recombinant host cell of  claim 1 , wherein the host cell produces hyaluronic acid.  
     
     
         4 . The recombinant host cell of  claim 1 , wherein the coding region encoding enzymatically active hyaluronan synthase of the purified nucleic acid segment is under control of a Gram positive bacterial-compatible promoter.  
     
     
         5 . The recombinant host cell of  claim 1 , wherein the  Bacillus subtilis  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.  
     
     
         6 . The recombinant host cell of  claim 1 , wherein the recombinant vector further comprises a purified nucleic acid segment having a coding region encoding enzymatically active UDP-glucose dehydrogenase.  
     
     
         7 . The recombinant host cell of  claim 1 , wherein the recombinant host cell has enhanced production of at least one of UDP-GlcUA and UDP-GlcNAc.  
     
     
         8 . The recombinant host cell of  claim 7 , 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.  
     
     
         9 . The recombinant host cell of  claim 8 , wherein the modification is a mutation or tandem promoter elements.  
     
     
         10 . The recombinant host cell of  claim 7 , 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  Bacillus  cell;    (B) at least one less messenger RNA destabilizing element than is found in a native  Bacillus  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.    
     
     
         11 . 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 into a  Bacillus  host wherein the coding region is under control of a promoter, and wherein the enzymatically active hyaluronan synthase is a single protein that is a dual-action catalyst that utilizes UDP-GlcA and UDP-GlcNAc to synthesize HA, and wherein the coding region encoding enzymatically active hyaluronan synthase is selected from the group consisting of:    (A) a coding region encoding enzymatically active hyaluronan synthase that is at least 80% identical to SEQ ID NO:10; and    (B) a coding region that is capable of hybridizing to a complementary sequence of SEQ ID NO:9 under hybridization conditions of 1.2-1.8× High Phosphate Buffer (HPB) at 40-50° C.;    growing the host organism in a medium to secrete hyaluronic acid; and    recovering the secreted hyaluronic acid.    
     
     
         12 . The method of  claim 11 , wherein the  Bacillus  host is  Bacillus subtilis  or  Bacillus licheniformis.    
     
     
         13 . The method according to  claim 11 , wherein the step of recovering the hyaluronic acid comprises extracting the secreted hyaluronic acid from the medium.  
     
     
         14 . The method according to  claim 13 , further comprising the step of purifying the extracted hyaluronic acid.  
     
     
         15 . The method of  claim 11 , further comprising the step of introducing a purified nucleic acid segment having a coding region encoding enzymatically active UDP-glucose dehydrogenase into the  Bacillus  host.  
     
     
         16 . The method of  claim 11 , wherein, in the step of introducing a purified nucleic acid segment having a coding region encoding enzymatically active hyaluronan synthase into a  Bacillus  host, the coding region encoding enzymatically active hyaluronan synthase of the purified nucleic acid segment is under control of a Gram positive bacterial-compatible promoter.  
     
     
         17 . The method of  claim 11  wherein, in the step of introducing a purified nucleic acid segment having a coding region encoding enzymatically active hyaluronan synthase into a  Bacillus  host, the  Bacillus  host has an enhanced production of at least one of UDP-GlcUA and UDP-GlcNAc.  
     
     
         18 . The method of  claim 17  wherein, in the step of introducing a purified nucleic acid segment having a coding region encoding enzymatically active hyaluronan synthase into a  Bacillus  host, the  Bacillus  host further includes at least one modified RNA polymerase promoter having an increased promoter activity.  
     
     
         19 . The method of  claim 18 , wherein the modification of the RNA polymerase promoter is a mutation or tandem promoter elements.  
     
     
         20 . The method of  claim 17  wherein, in the step of introducing a purified nucleic acid segment having a coding region encoding enzymatically active hyaluronan synthase into a  Bacillus  host, the  Bacillus  host further includes at least one of: 
 (A) at least one additional messenger RNA stabilizing element than is found in a native  Bacillus  cell;    (B) at least one less messenger RNA destabilizing element than is found in a native  Bacillus  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.