Combinatorial dna library for producing modified n-glycans in lower eukaryotes

Abstract

The present invention relates to eukaryotic host cells having modified oligosaccharides which may be modified further by heterologous expression of a set of glycosyltransferases, sugar transporters and mannosidases to become host-strains for the production of mammalian, e.g., human therapeutic glycoproteins. 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 glycosylation 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. Host cells with modified oligosaccharides are created or selected. N-glycans made in the engineered host cells have a Man 5 GlcNAc 2 core structure which may then be modified further by heterologous expression of one or more enzymes, e.g., glycosyltransferases, sugar transporters and mannosidases, to yield human-like glycoproteins. For the production of therapeutic proteins, this method may be adapted to engineer cell lines in which any desired glycosylation structure may be obtained.

What is claimed is: 
     
         1 . A method for producing a human-like glycoprotein in a non-human eukaryotic host cell that does not display a 1,6 mannosyltransferase activity with respect to the N-glycan on a glycoprotein, the method comprising the step of introducing into the host cell one or more enzymes for production of a Man 5 GlcNAc 2  carbohydrate structure, wherein Man 5 GlcNAc 2  is produced within the host cell at a yield of at least 30 mole percent. 
     
     
         2 .- 4 . (canceled) 
     
     
         5 . The method of  claim 1 , wherein at least one of the enzymes is targeted to a subcellular location in the host cell where the enzyme will have optimal activity. 
     
     
         6 . The method of  claim 5 , wherein the enzyme is targeted by means of a chimeric protein comprising a cellular targeting signal peptide not normally associated with the enzyme. 
     
     
         7 . (canceled) 
     
     
         8 . The method of  claim 1 , wherein at least one of the enzymes is selected from the group consisting of mannosidases, glycosyltransferases and glycosidases. 
     
     
         9 . The method of  claim 6 , wherein the enzyme is a mannosidase predominantly localized in the Golgi apparatus or the endoplasmic reticulum. 
     
     
         10 .- 11 . (canceled) 
     
     
         12 . The method of  claim 1 , wherein the glycoprotein comprises one or more sugars selected from the group consisting of GlcNAc, galactose, sialic acid, and fucose. 
     
     
         13 . The method of  claim 1 , wherein the glycoprotein comprises at least one oligosaccharide branch comprising the structure NeuNAc-Gal-GlcNAc-Man. 
     
     
         14 . (canceled) 
     
     
         15 . The method of  claim 1 , wherein the host is a lower eukaryotic cell. 
     
     
         16 . The method of  claim 15 , wherein the host cell is selected from the group consisting of  Pichia pastoris, Pichia finlandica, Pichia trehalophila, Pichia koclamae, Pichia membranaefaciens, Pichia opuntiae, Pichia thermotolerans, Pichia salictaria, Pichia guercuum, Pichia pijperi, Pichia stiptis, Pichia methanolica, Pichia  sp.,  Saccharomyces cerevisiae, Saccharomyces  sp.,  Hansenula polymorpha, Kluyveromyces  sp.,  Kluyveromyces lactis, Candida albicans, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, Chrysosporium lucknowense, Fusarium  sp.,  Fusarium gramineum, Fusarium venenatum  and  Neurospora crassa.    
     
     
         17 . The method of  claim 1 , wherein the host is deficient in the activity of one or more enzymes selected from the group consisting of mannosyltransferases and phosphomannosyltransferases. 
     
     
         18 . The method of  claim 17 , wherein the host does not express an enzyme selected from the group consisting of 1,6 mannosyltransferase; 1,3 mannosyltransferase; and 1,2 mannosyltransferase. 
     
     
         19 . The method of  claim 1 , wherein the host is an och1 mutant of  P. pastoris.    
     
     
         20 . The method of  claim 1 , wherein the host expresses GnTI and UDP-GlcNAc transporter activities. 
     
     
         21 . The method of  claim 1 , wherein the host expresses a UDP- or GDP-specific diphosphatase activity. 
     
     
         22 . The method of  claim 1 , further comprising the step of isolating the glycoprotein from the host. 
     
     
         23 .- 24 . (canceled) 
     
     
         25 . The method of  claim 1 , further comprising the step of introducing into the host a nucleic acid molecule encoding one or more mannosidase activities involved in the production of Man 5 GlcNAc 2  from Man 8 GlcNAc 2  or Man 9 GlcNAc 2 . 
     
     
         26 . The method of  claim 25 , wherein at least one of the encoded mannosidase activities has a pH optimum within about 1.4 pH units of the average pH optimum of other representative enzymes in the organelle in which the mannosidase activity is localized, or has optimal activity at a pH of between about 5.1 and about 8.0. 
     
     
         27 . The method of  claim 26 , wherein the mannosidase has optimal activity at a pH of between about 5.5 and about 7.5. 
     
     
         28 . The method of  claim 26 , wherein the mannosidase activity is an α-1,2-mannosidase derived from mouse, human,  Lepidoptera, Aspergillus nidulans , or  Bacillus  sp.,  C. elegans, D. melanogaster, P. citrinum  or  X. laevis.    
     
     
         29 .- 36 . (canceled) 
     
     
         37 . A nucleic acid library comprising at least two different genetic constructs, wherein at least one genetic construct comprises a nucleic acid fragment encoding a glycosylation enzyme ligated in-frame with a nucleic acid fragment encoding a cellular targeting signal peptide which it is not normally associated with. 
     
     
         38 . A DNA library of fusion constructs comprising:
 (a) at least two nucleotide sequences encoding a cellular targeting signal peptide and at least one nucleotide sequence encoding a catalytic domain region selected from the group consisting of mannosidases, glycosyltransferases and glycosidases; or   (b) at least one nucleotide sequence encoding a cellular targeting signal peptide and at least two nucleotide sequences encoding a catalytic domain region selected from the group consisting of mannosidases, glycosyltransferases and glycosidases;   wherein at least one nucleotide sequence encoding a catalytic domain region is ligated in-frame to a nucleotide sequence encoding a cellular targeting signal peptide.   
     
     
         39 .- 62 . (canceled) 
     
     
         63 . A host cell produced by the method of  claim 1 ,  24  or  55 . 
     
     
         64 . A human-like glycoprotein produced by the method of  claim 1 ,  24  or  55 . 
     
     
         65 .- 76 . (canceled)