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 - 56 . (canceled) 
     
     
         57 . A method for producing CMP-sialic acid in a yeast host cell comprising:
 (a) transforming into the yeast host cell one or more nucleic acid molecules encoding the enzymes of a sialylation pathway consisting of  E. coli  NeuC,  E. coli  NeuB, and a CMP-Sialic acid synthase; and   (b) growing the yeast host cell under conditions effective for producing the CMP-sialic acid.   
     
     
         58 . The method of  claim 57 , wherein the yeast host cell is a  Pichia  sp.,  Pichia pastoris, Pichia finlandica, Pichia trehalophila, Pichia koclamae, Pichia membranaefaciens, Pichia opuntiae, Pichia thermotolerans, Pichia salictaria, Pichia guercuum, Pichia pijperi, Pichia stiptis, Pichia methanolica; Saccharomyces  sp.,  Saccharomyces cerevisiae, Hansenula polymorpha, Kluyveromyces  sp.,  Kluyveromyces lactis , or  Candida albicans.    
     
     
         59 . The method of  claim 57 , wherein the yeast host cell is  Pichia pastoris.    
     
     
         60 . A yeast host cell capable of producing CMP-sialic acid comprising:
 a sialylation pathway consisting of  E. coli  NeuC,  E. coli  NeuB, and a CMP-Sialic acid synthase, wherein the yeast host cell is capable of producing the CMP-sialic acid.   
     
     
         61 . The yeast host cell of  claim 60 , wherein the yeast host cell is a  Pichia  sp.,  Pichia pastoris, Pichia finlandica, Pichia trehalophila, Pichia koclamae, Pichia membranaefaciens, Pichia opuntiae, Pichia thermotolerans, Pichia salictaria, Pichia guercuum, Pichia pijperi, Pichia stiptis, Pichia methanolica; Saccharomyces  sp.,  Saccharomyces cerevisiae, Hansenula polymorpha, Kluyveromyces  sp.,  Kluyveromyces lactis , or  Candida albicans.    
     
     
         62 . The yeast host cell of  claim 60 , wherein the yeast host cell is  Pichia pastoris.    
     
     
         63 . A method for producing CMP-sialic acid comprising:
 (a) providing a yeast host cell comprising a CMP-sialic acid biosynthetic pathway consisting of  E. coli  NeuC,  E. coli  NeuB, and a CMP-Sialic acid synthase; and   (b) growing the yeast host cell under conditions effective for producing the CMP-sialic acid.   
     
     
         64 . The method of  claim 63 , wherein the yeast host cell is a  Pichia  sp.,  Pichia pastoris, Pichia finlandica, Pichia trehalophila, Pichia koclamae, Pichia membranaefaciens, Pichia opuntiae, Pichia thermotolerans, Pichia salictaria, Pichia guercuum, Pichia pijperi, Pichia stiptis, Pichia methanolica; Saccharomyces  sp.,  Saccharomyces cerevisiae, Hansenula polymorpha, Kluyveromyces  sp.,  Kluyveromyces lactis , or  Candida albicans.    
     
     
         65 . The method of  claim 63 , wherein the yeast host cell is  Pichia pastoris.