Glycosyltransferases for biosynthesis of oligosaccharides, and genes encoding them

Abstract

The present invention is directed to nucleic acids encoding glycosyltransferases, the proteins encoded thereby, and to methods for synthesizing oligosaccharides using the glycosyltransferases of the invention. In particular, the present application is directed to identification a glycosyltransferase locus of Neisseria gonorrhoeae containing five open reading frames for five different glycosyltransferases. The functionally active glycosyltransferases of the invention are characterized by catalyzing reactions such as adding Gal β1→4 to GlcNAc or Glc; adding GalNAc or GlcNAc β1→3 to Gal; and adding Gal α1→4 to Gal. The glycosyltransferases of the invention are particularly suited to the synthesis of the oligosaccharides Galβ1→4GlcNAcβ1→3Galβ1→4Glc (a mimic of lacto-N-neotetraose), GalNAcβ1→3Galβ1→4GlcNAcβ1→3Galβ1→4Glcβ1→4 (a mimic ganglioside), and Galβ1→4Galβ1→4Glcβ1→4Hep→R (a mimic of the saccharide portion of globo-glycolipids).

What is claimed is:  
     
         1 . A purified nucleic acid that is hybridizable under moderately stringent conditions to a nucleic acid having a nucleotide sequence corresponding to or complementary to the nucleotide sequence shown in FIG. 2 (SEQ ID NO:1).  
     
     
         2 . The nucleic acid of  claim 1  that is hybridizable under moderately stringent conditions to a nucleic acid having a nucleotide sequence corresponding to or complementary to a portion of the nucleotide sequence shown in FIG. 2 (SEQ ID NO: 1) that encodes a functionally active glycosyltransferase.  
     
     
         3 . The nucleic acid of  claim 2  that encodes a functionally active glycosyltransferase.  
     
     
         4 . The nucleic acid of  claim 1  that has a nucleotide sequence corresponding to or complementary to a portion of the nucleotide sequence shown in FIG. 2 (SEQ ID NO:1) that encodes a functionally active glycosyltransferase.  
     
     
         5 . The nucleic acid of  claim 4  that encodes a functionally active glycosyltransferase.  
     
     
         6 . The nucleic acid of  claim 1  that has a nucleotide sequence corresponding to or complementary to the nucleotide sequence shown in FIG. 2 (SEQ ID NO:1).  
     
     
         7 . The nucleic acid of  claim 3 , wherein the functionally active glycosyltransferase catalyzes a reaction selected from the group consisting of: 
 a) adding Gal β1→4 to GlcNAc or Glc;    b) adding GalNAc or GlcNAc β1→3 to Gal; and    c) adding Gal α1→4 to Gal.    
     
     
         8 . The nucleic acid of  claim 3  which encodes a glycosyltransferase having an amino acid sequence of SEQ ID NO:2.  
     
     
         9 . The nucleic acid of  claim 3  which encodes a glycosyltransferase having an amino acid sequence of SEQ ID NO:3.  
     
     
         10 . The nucleic acid of  claim 3  which encodes a glycosyltransferase having an amino acid sequence of SEQ ID NO:4.  
     
     
         11 . The nucleic acid of  claim 3  which encodes a glycosyltransferase having an amino acid sequence of SEQ ID NO:5.  
     
     
         12 . The nucleic acid of  claim 3  which encodes a glycosyltransferase having an amino acid sequence of SEQ ID NO:6.  
     
     
         13 . An expression vector comprising the nucleic acid of  claim 3  operatively associated with an expression control sequence.  
     
     
         14 . A recombinant host cell transformed with the expression vector of  claim 13 .  
     
     
         15 . A method for producing a glycosyltransferase comprising: 
 a) culturing the recombinant host cell of  claim 14  under conditions that allow expression of the glycosyltransferase; and    b) recovering the expressed glycosyltransferase.    
     
     
         16 . A glycosyltransferase having an amino acid sequence of SEQ ID NO:2, or a functionally active fragment thereof.  
     
     
         17 . A glycosyltransferase having an amino acid sequence of SEQ ID NO:3, or a functionally active fragment thereof.  
     
     
         18 . A glycosyltransferase having an amino acid sequence of SEQ ID NO:4, or a functionally active fragment thereof.  
     
     
         19 . A glycosyltransferase having an amino acid sequence of SEQ ID NO:5, or a functionally active fragment thereof.  
     
     
         20 . A glycosyltransferase having an amino acid sequence of SEQ ID NO:6, or a functionally active fragment thereof.  
     
     
         21 . A composition comprising a glycosyltransferase conjugated to a solid phase support, wherein the glycosyltransferase is selected from the group consist of: 
 a) a glycosyltransferase having an amino acid sequence of SEQ ID NO:2, or a functionally active fragment thereof;    b) a glycosyltransferase having an amino acid sequence of SEQ ID NO:3, or a functionally active fragment thereof;    c) a glycosyltransferase having an amino acid sequence of SEQ !ID NO:4, or a functionally active fragment thereof;    d) a glycosyltransferase having an amino acid sequence of SEQ ID NO:5, or a functionally active fragment thereof; and    e) a glycosyltransferase having an amino acid sequence of SEQ ID NO:6, or a functionally active fragment thereof.    
     
     
         22 . A method for adding GalNAc or GlcNAc β1→3 to Gal, comprising contacting a reaction mixture comprising an activated GalNAc or GlcNAc to an acceptor moiety comprising a Gal residue in the presence of the glycosyltransferase of  claim 16 .  
     
     
         23 . A method for adding Gal β1→4 to GlcNAc or Glc, comprising contacting a reaction mixture comprising an activated Gal to an acceptor moiety comprising a GlcNAc or Glc residue in the presence of the glycosyltransferase of  claim 17 .  
     
     
         24 . A method for adding Gal α1→4 to Gal, comprising contacting a reaction mixture comprising an activated Gal to an acceptor moiety comprising a Gal residue in the presence of the glycosyltransferase of  claim 18 .  
     
     
         25 . A method for adding GalNAc or GlcNAc β1→3 to Gal, comprising contacting a reaction mixture comprising an activated GalNAc or GlcNAc to an acceptor moiety comprising a Gal residue in the presence of the glycosyltransferase of  claim 19 .  
     
     
         26 . A method for adding Gal β1→4 to GlcNAc or Glc, comprising contacting a reaction mixture comprising an activated Gal to an acceptor moiety comprising a GlcNAc or Glc residue in the presence of the glycosyltransferase of  claim 20 .  
     
     
         27 . A method for preparing an oligosaccharide having the structure Galα1→4Galβ1→4Glc, which comprises sequentially performing the steps of: 
 a) contacting a reaction mixture comprising an activated Gal to an acceptor moiety comprising a Glc residue in the presence of a glycosyltransferase having an amino acid sequence of SEQ ID NO:6, or a functionally active fragment thereof; and  
 b) contacting a reaction mixture comprising an activated Gal to the acceptor moiety comprising Galβ1→4Glc in the presence of a glycosyltransferase having an amino acid sequence of SEQ ID NO:4, or a functionally active fragment thereof.  
 
     
     
         28 . A method for preparing an oligosaccharide having the structure Galβ1→4Glc, which comprises contacting a reaction mixture comprising an activated Gal to an acceptor moiety comprising a Glc residue in the presence of the glycosyltransferase of  claim 20 .  
     
     
         29 . A method for preparing an oligosaccharide having the structure GlcNAcβ1→3Galβ1→4Glc, which comprises contacting a reaction mixture comprising an activated GlcNAc to an acceptor moiety comprising a Galβ1→4Glc residue in the presence of the glycosyltransferase of  claim 16 .  
     
     
         30 . A method for preparing an oligosaccharide having the structure Galβ1→4GlcNAcβ1→3Galβ1→4Glc, which comprises contacting a reaction mixture comprising an activated Gal to an acceptor moiety comprising a GlcNAcβ1→3Galβ1→4Glc residue in the presence of the glycosyltransferase of  claim 17 .  
     
     
         31 . A method for preparing an oligosaccharide having the structure GlcNAcβ1→3Galβ1→4GlcNAcβ1→3Galβ1→4Glc, which comprises contacting a reaction mixture comprising an activated GalNAc to an acceptor moiety comprising a Galβ1→4GlcNAcβ1→3Galβ1→4Glc residue in the presence of the glycosyltransferase of  claim 19 .  
     
     
         32 . A method for preparing an oligosaccharide having the structure GalNAcβ1→3Galβ1→4GlcNAcβ1→3Galβ1→4Glc, which comprises sequentially performing the steps of: 
 a) contacting a reaction mixture comprising an activated Gal to an acceptor moiety comprising a Glc residue in the presence of a glycosyltransferase having an amino acid sequence of SEQ ID NO: 6, or a functionally active fragment thereof;  
 b) contacting a reaction mixture comprising an activated GlcNAc to the acceptor moiety comprising a Galβ1→4Glc residue in the presence of a glycosyltransferase having an amino acid sequence of SEQ ID NO:2, or a functionally active fragment thereof;  
 c) contacting a reaction mixture comprising an activated Gal to the acceptor moiety comprising a GlcNAcβ1→3Galβ1→4Glc residue in the presence of a glycosyltransferase having an amino acid of SEQ ID NO:3; and  
 d) contacting a reaction mixture comprising an activated GalNAc to the acceptor moiety comprising a Galβ1→4GlcNAcβ1→3Galβ1→4Glc residue in the presence of a glycosyltransferase having an amino acid sequenc of SEQ ID NO:5, or a functionally active fragment thereof.  
 
     
     
         33 . A method for preparing an oligosaccharide having the structure Galβ1→4GlcNAcβ1→3Galβ1→4Glc, which comprises sequentially performing the steps of: 
 a) contacting a reaction mixture comprising an activated Gal to an acceptor moiety comprising a Glc residue in the presence of a glycosyltransferase having an amino acid sequence of SEQ ID NO: 6, or a functionally active fragment thereof;  
 b) contacting a reaction mixture comprising an activated GlcNAc to the acceptor moiety comprising a Galβ1→4Glc residue in the presence of a glycosyltransferase having an amino acid sequence of SEQ ID NO:2, or a functionally active fragment thereof; and  
 c) contacting a reaction mixture comprising an activated Gal to the acceptor moiety comprising a GlcNAcβ1→3Galβ1→4Glc residue in the presence of a glycosyltransferase having an amino acid of SEQ ID NO:3.