Production of sialylated n-glycans in lower eukaryotes

Abstract

The present invention relates to eukaryotic host cells which have been modified to produce sialylated glycoproteins by the heterologous expression of a set of glycosyltransferases, including sialyltransferase and/or trans-sialidase, to become host-strains for the production of mammalian, e.g., human therapeutic glycoproteins. Novel eukaryotic host cells expressing a CMP-sialic acid biosynthetic pathway for the production of sialylated glycoproteins are also provided. The invention provides nucleic acid molecules and combinatorial libraries which can be used to successfully target and express mammalian enzymatic activities (such as those involved in sialylation) to intracellular compartments in a eukaryotic host cell. The process provides an engineered host cell which can be used to express and target any desirable gene(s) involved in glycosylation.

1 . A method for producing a recombinant sialylated glycoprotein in a  Pichia pastoris  host cell, the host cell selected or engineered to produce glycoproteins comprising a Gal (1-4) GlcNAc (1-4) Man 3 GlcNAc 2  glycoform, the method comprising the step of transforming into the host cell:
 a. a nucleic acid encoding an enzyme having sialyltransferase activity, wherein the nucleic acid encodes a silayltransferase enzyme comprising the amino acid sequence of SEQ ID NO:104 or a catalytic domain thereof;   b. a nucleic acid encoding a CMP-sialic acid transporter; and   c. one or more nucleic acids encoding a CMP-sialic acid pathway consisting of: a bifunctional UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase, an N-acetylneuraminate-9-phosphate synthase, and a CMP-sialic acid synthase;   
       wherein, upon passage of the recombinant glycoprotein through the secretory pathway of the host cell, a recombinant glycoprotein comprising a NANA (1-4) Gal (1-4) GlcNAc (1-4) Man 3 GlcNA 2  glycoform is produced. 
     
     
         2 - 11 . (canceled) 
     
     
         12 . The method of  claim 1 , wherein said host cell comprises a cellular pool of CMP-sialic acid. 
     
     
         13 - 15 . (canceled) 
     
     
         16 . The method of  claim 1 , wherein the method further comprises culturing said host cell in the presence of a sialic acid donor or a precursor of a sialic acid donor. 
     
     
         17 . The method of  claim 1 , wherein the method further comprises the step of introducing into the host cell one or more additional nucleic acids encoding one or more enzymes selected from the group consisting of glycosyltransferases, glycosidases and sugar transporters. 
     
     
         18 - 34 . (canceled) 
     
     
         35 . The method of  claim 1 , wherein the host cell is transformed with:
 a. a nucleic acid encoding a mouse CMP-sialic acid transporter; and   b. one or more nucleic acids encoding a: human a bifunctional UDP-N_acetylglucosamine-2-epimerase/N-acetylmannosamine kinase, a human N-acetylneuraminate-9-phosphate synthase, and a human CMP-sialic acid synthase;