Method for providing hyaluronic acid

Abstract

Disclosed are DNA segments encoding hyaluronic acid synthase which are employed to construct recombinant cells useful in the production of hyaluronate synthase or hyaluronic acid (HA) the recombinant DNA segment identified in FIG. 5. In preferred aspects, chromosomal DNA from Streptococcus equisimilis is partially digested with EcoRI and the resultant fragments are ligated to form recombinant vectors. These vectors are useful in the transformation of host cells such as E. coli and or Streptococcal hosts. Resultant transformants are screened by the novel screening assays to identify colonies which have incorporated HA synthase DNA in a form that is being actively transcribed into the corresponding HA synthase enzyme. These colonies may be selected and employed in the production of the enzyme itself or its product, HA. The recombinant DNA segment identified in FIG. 5 is then inserted into a recombinant Streptococcal host for the production of hyaluronic acid ( HA ).

What is claimed is:  
     
       1. A DNA segment isolated from a eukaryotic or prokaryotic source comprising a sequence encoding hyaluronate synthase  method for providing hyaluronic acid employing a recombinant DNA segment, comprising the steps of: 
       ( i )  providing recombinant Streptococcal host, wherein the recombinant Streptococcal host contains a plasmid comprising the DNA segment identified in  FIG. 5 inserted therein;  
       ( ii )  culturing the recombinant Streptococcal host under conditions appropriate for the production of hyaluronic acid; and    
       ( iii )  separating the hyaluronic acid from the recombinant Streptococcal host.   
     
     
       2. The DNA segment of claim  1  wherein the encoded hyaluronate synthase comprises Streptococcal hyaluronate synthase. 
     
     
       3. A recombinant vector which incorporates a recombinant insert comprising a DNA segment in accordance with claim  1 . 
     
     
       4. The recombinant vector of claim  3 , further identified as a shuttle vector capable of replication in both a Streptococcal and E. coli host. 
     
     
       5. A recombinant host comprising the DNA segment of claim  1  or the vector of claim  3 . 
     
     
       6. The recombinant host of claim  5 , capable of expressing the DNA segment to produce hyaluronate synthase. 
     
     
       7. The recombinant host of claim  5 , further defined as a Streptococcal or an E. coli host. 
     
     
       8. The recombinant host of claim  5 , capable of expressing the hyaluronate synthase and producing hyaluronic acid. 
     
     
       9. The recombinant host of claim  5 , further defined as a hyaluronidase negative host. 
     
     
       10. A method of providing hyaluronic acid comprising: 
       (a) providing a recombinant host bearing a recombinant DNA segment encoding a hyaluronate synthase enzyme and capable of expressing the enzyme;  
       (b) culturing the recombinant host in a medium under conditions appropriate for the production of hyaluronate synthase or hyaluronic acid; and  
       (c) separating the hyaluronate synthase or hyaluronic acid from the recombinant host.