Yeast strains producing mammalian-like complex n-glycans

Abstract

Described herein are methods and genetically engineered fungal cells useful for producing target molecules containing mammalian-like complex N-glycans or containing intermediates in a mammalian glycosylation pathway.

1 . A method of producing a fungal cell capable of producing proteins comprising GlcNAcMan 5 GlcNAc 2  N-glycans, said method comprising:
 a) providing a fungal cell genetically engineered to produce proteins comprising Man 5 GlcNAc 2  N-glycans;   b) introducing into said cell a nucleic acid encoding a GlcNAc-transferase I, wherein said nucleic acid comprises a nucleotide sequence encoding a targeting sequence to target the encoded GlcNAc-transferase I to an intracellular compartment, wherein expression of said GlcNAc-transferase I in said fungal cell produces proteins comprising GlcNAcMan 5 GlcNAc 2  N-glycans.   
     
     
         2 . The method of  claim 1 , said method further comprising introducing into said cell a nucleic acid encoding a target protein, wherein said cell produces said target protein modified to comprise said GlcNAcMan 5 GlcNAc 2  N-glycans. 
     
     
         3 . The method of  claim 2 , wherein said target protein binds to an Fc receptor. 
     
     
         4 . The method of  claim 3 , wherein said target protein is an antibody or fragment thereof. 
     
     
         5 . The method of  claim 2 , wherein said target protein is a therapeutic glycoprotein. 
     
     
         6 . The method of  claim 2 , wherein said target protein is Interferon-β, GM-CSF, Interferon γ, or erythropoietin. 
     
     
         7 . The method of  claim 1 , wherein said intracellular compartment is the Golgi apparatus. 
     
     
         8 . The method of  claim 1 , wherein said fungal cell genetically engineered to produce proteins comprising Man 5 GlcNAc 2  N-glycans is deficient in OCH1 activity and comprises a nucleic acid encoding an α-1,2-mannosidase, wherein said nucleic acid encoding said α-1,2-mannosidase comprises a nucleotide sequence encoding a targeting sequence to target the encoded α-1,2-mannosidase to the endoplasmic reticulum. 
     
     
         9 . The method of  claim 8 , wherein said targeting sequence is an HDEL sequence. 
     
     
         10 . The method of  claim 1 , wherein said fungal cell is  Yarrowia lipolytica  or  Arxula adeninivorans.    
     
     
         11 . The method of  claim 1 , said method further comprising introducing into said cell a nucleic acid encoding a mannosidase II, wherein said nucleic acid encoding said mannosidase II comprises a nucleotide sequence encoding a targeting sequence to target the encoded mannosidase II to the Golgi apparatus, wherein expression of said mannosidase II in said fungal cell produces proteins comprising GlcNAcMan 3 GlcNAc 2  N-glycans. 
     
     
         12 . The method of  claim 1  or  claim 11 , said method further comprising introducing into said cell a nucleic acid encoding a galactosyltransferase, wherein said nucleic acid encoding said galactosyltransferase comprises a nucleotide sequence encoding a targeting sequence to target the encoded galactosyltransferase to the Golgi apparatus, wherein expression of said galactosyltransferase in said fungal cell produces proteins comprising GalGlcNAcMan 5 GlcNAc 2  or GalGlcNAcMan 3 GlcNAc 2 N-glycans. 
     
     
         13 . The method of  claim 12 , said method further comprising introducing into said cell a nucleic acid encoding a target protein, wherein said cell produces said target protein modified to comprise said GalGlcNAcMan 5 GlcNAc 2  or GalGlcNAcMan 3 GlcNAc 2  N-glycans. 
     
     
         14 . The method of  claim 12 , wherein said galactosyltransferase is a fusion of a UDP-Glc-4-epimerase and the catalytic domain of a β-1,4-galactosyltransferase I. 
     
     
         15 . The method of  claim 13 , further comprising isolating said target protein modified to comprise said GalGlcNAcMan 5 GlcNAc 2  or GalGlcNAcMan 3 GlcNAc 2  N-glycans. 
     
     
         16 . A method of producing a target protein comprising GlcNAcMan 3 GlcNAc 2  N-glycans, said method comprising:
 a) providing a fungal cell genetically engineered to comprise a nucleic acid encoding a GlcNAc-transferase I, an α-1,2-mannosidase, and a mannosidase II, wherein said nucleic acid comprises nucleotide sequences encoding targeting sequences to target each encoded protein to an intracellular compartment, wherein said fungal cell is deficient in OCH1 activity; and   b) introducing into said cell a nucleic acid encoding a target protein, wherein said cell produces said target protein comprising said GlcNAcMan 3 GlcNAc 2  N-glycans.   
     
     
         17 . The method of  claim 16 , wherein said fungal cell is  Yarrowia lipolytica  or  Arxula adeninivorans.    
     
     
         18 . The method of  claim 16 , wherein said nucleic acid encoding said α-1,2-mannosidase comprises an endoplasmic reticulum targeting sequence to target the encoded α-1,2-mannosidase to the endoplasmic reticulum. 
     
     
         19 . The method of  claim 18 , wherein said targeting sequence is an HDEL sequence. 
     
     
         20 . The method of  claim 16 , wherein said nucleic acid encoding said GlcNAc-transferase I and said mannosidase II comprises nucleotide sequences encoding Golgi targeting sequences to target the encoded GlcNAc-transferase I and mannosidase II to the Golgi apparatus. 
     
     
         21 . The method of  claim 16 , wherein said target protein binds to an Fc receptor. 
     
     
         22 . The method of  claim 16 , wherein said target protein is an antibody or fragment thereof. 
     
     
         23 . The method of  claim 16 , wherein said target protein is a therapeutic glycoprotein. 
     
     
         24 . The method of  claim 16 , wherein said target protein is Interferon-β, GM-CSF, Interferon γ, or erythropoietin. 
     
     
         25 . The method of  claim 16 , further comprising introducing into said cell a nucleic acid encoding a galactosyltransferase, wherein said nucleic acid encoding said galactosyltransferase comprises a nucleotide sequence encoding a targeting sequence to target the encoded galactosyltransferase to the Golgi apparatus, wherein expression of said galactosyltransferase in said fungal cell produces said target protein modified to comprise GalGlcNAcMan 3 GlcNAc 2 N-glycans. 
     
     
         26 . The method of  claim 25 , further comprising isolating said target protein modified to comprise said GalGlcNAcMan 3 GlcNAc 2 N-glycans. 
     
     
         27 . A method of making a fungal cell capable of producing proteins comprising GlcNAcMan 3 GlcNAc 2 N-glycans, said method comprising:
 a) providing a fungal cell genetically engineered to produce proteins comprising Man 3 GlcNAc 2  N-glycans;   b) introducing into said cell a nucleic acid encoding a GlcNAc-transferase I, wherein said nucleic acid comprises a nucleotide sequence encoding a targeting sequence to target said encoded GlcNAc-transferase I to an intracellular compartment, wherein expression of said GlcNAc-transferase I in said fungal cell produces proteins comprising GlcNAcMan 3 GlcNAc 2  N-glycans.   
     
     
         28 . The method of  claim 27 , said method further comprising introducing into said cell a nucleic acid encoding a target protein, wherein said cell produces said target protein modified to comprise said GlcNAcMan 3 GlcNAc 2  N-glycans. 
     
     
         29 . The method of  claim 27 , wherein said target protein binds to an Fc receptor. 
     
     
         30 . The method of  claim 27 , wherein said target protein is an antibody or fragment thereof. 
     
     
         31 . The method of  claim 27 , wherein said target protein is a therapeutic glycoprotein. 
     
     
         32 . The method of  claim 27 , wherein said target protein is Interferon-β, GM-CSF, Interferon γ, or erythropoietin. 
     
     
         33 . The method of  claim 27 , wherein said intracellular compartment is the Golgi apparatus. 
     
     
         34 . The method of  claim 27 , wherein said fungal cell genetically engineered to produce proteins comprising Man 3 GlcNAc 2  N-glycans is deficient in ALG3 activity, and comprises a nucleic acid encoding an α-1,2-mannosidase, said nucleic acid comprising a nucleotide sequence encoding a targeting sequence to target the encoded α-1,2-mannosidase to the endoplasmic reticulum. 
     
     
         35 . The method of  claim 34 , wherein said fungal cell genetically engineered to produce proteins comprising Man 3 GlcNAc 2  N-glycans further is deficient in OCH1 activity. 
     
     
         36 . The method of  claim 34  or  35 , wherein said fungal cell genetically engineered to produce proteins comprising Man 3 GlcNAc 2  N-glycans further comprises a nucleic acid encoding an α-1,3-glucosyltransferase. 
     
     
         37 . The method of  claim 36 , wherein said α-1,3-glucosyltransferase is ALG6. 
     
     
         38 . The method of  claim 27 , wherein said fungal cell is  Yarrowia lipolytica  or  Arxula adeninivorans.    
     
     
         39 . The method of  claim 27 , said method further comprising introducing into said cell a nucleic acid encoding a GlcNAc-transferase II, wherein said nucleic acid encoding said GlcNAc-transferase II comprises a nucleotide sequence encoding a targeting sequence to target the encoded GlcNAc-transferase II to an intracellular compartment, wherein expression of said GlcNAc-transferase II in said fungal cell produces proteins comprising GlcNAc 2 Man 3 GlcNAc 2  N-glycans. 
     
     
         40 . The method of  claim 27  or  claim 39 , said method further comprising introducing into said cell a nucleic acid encoding a galactosyltransferase, wherein said nucleic acid encoding said galactosyltransferase comprises a nucleotide sequence encoding a targeting sequence to target the encoded galactosyltransferase to the Golgi apparatus, wherein expression of said galactosyltransferase in said fungal cell produces proteins comprising GalGlcNAcMan 3 GlcNAc 2  or Gal 2 GlcNAc 2 Man 3 GlcNAc 2  N-glycans. 
     
     
         41 . The method of  claim 40 , said method further comprising introducing into said cell a nucleic acid encoding a target protein, wherein said cell produces said target protein modified to comprise said GalGlcNAcMan 3 GlcNAc 2  or Gal 2 GlcNAc 2 Man 3 GlcNAc 2  N-glycans. 
     
     
         42 . The method of  claim 40 , wherein said galactosyltransferase is a fusion of a UDP-Glc-4-epimerase and catalytic domain of a β-1,4-galactosyltransferase I. 
     
     
         43 . The method of  claim 40  said method further comprising introducing into said cell a nucleic acid encoding the α and β subunits of a Glucosidase II, wherein expression of said α and β subunits of said Glucosidase II in said fungal cell produces proteins comprising GalGlcNAcMan 3 GlcNAc 2  or Gal 2 GlcNAc 2 Man 3 GlcNAc 2  N-glycans. 
     
     
         44 . A method of producing a target protein comprising Gal 2 GlcNAc 2 Man 3 GlcNAc 2  N-glycans, said method comprising:
 a) providing a fungal cell genetically engineered to be deficient in ALG3 activity and comprising a nucleic acid encoding a GlcNAc-transferase I, a GlcNAc-transferase II, and a galactosyltransferase, wherein said nucleic acid encoding said GlcNAc-transferase I, said GlcNAc-transferase II, and said galactosyltransferase comprises nucleotide sequences encoding targeting sequences to target each encoded protein to an intracellular compartment;   b) introducing into said cell a nucleic acid encoding a target protein, wherein said cell produces said target protein comprising said Gal 2 GlcNAc 2 Man 3 GlcNAc 2  N-glycans.   
     
     
         45 . The method of  claim 44 , wherein said fungal cell further is deficient in OCH1 activity. 
     
     
         46 . The method of  claim 44  or  45 , wherein said fungal cell further comprises a nucleic acid encoding an α-1,3-glucosyltransferase. 
     
     
         47 . The method of  claim 46 , wherein said nucleic acid encoding said α-1,3-glucosyltransferase is ALG6. 
     
     
         48 . The method of  claim 46 , wherein said fungal cell further comprises a nucleic acid encoding the α and β subunits of a Glucosidase II, wherein expression of said α and β subunits of said Glucosidase II in said fungal cell produces said target protein comprising said Gal 2 GlcNAc 2 Man 3 GlcNAc 2 N-glycans. 
     
     
         49 . An isolated fungal cell genetically engineered to produce proteins comprising GlcNAcMan 3 GlcNAc 2 N-glycans, wherein said fungal cell is deficient in OCH1 activity and comprises a nucleic acid encoding an α-1,2-mannosidase, a GlcNAc-transferase I, and a mannosidase II, wherein said nucleic acid encoding said α-1,2-mannosidase, said GlcNAc-transferase I, and said mannosidase II comprises nucleotide sequences encoding targeting sequences to target each encoded protein to an intracellular compartment, wherein expression of said α-1,2-mannosidase, said GlcNAc-transferase I, and said mannosidase II in said fungal cell produces proteins comprising GlcNAcMan 3 GlcNAc 2 N-glycans. 
     
     
         50 . The fungal cell of  claim 49 , wherein said genetically engineered fungal cell further comprises a nucleic acid encoding a target protein, wherein said cell produces said target protein modified to comprise said GlcNAcMan 3 GlcNAc 2 N-glycans. 
     
     
         51 . The fungal cell of  claim 49  or  claim 50 , wherein said genetically engineered fungal cell further comprises a nucleic acid encoding a GlcNAc-transferase II, wherein said nucleic acid encoding said GlcNAc-transferase II comprises a nucleotide sequence encoding a targeting sequence to target the encoded GlcNAc-transferase II to an intracellular compartment, wherein expression of said GlcNAc-transferase II in said fungal cell produces proteins comprising GlcNAc 2 Man 3 GlcNAc 2 N-glycans. 
     
     
         52 . The fungal cell of  claim 51 , said fungal cell further comprising a nucleic acid encoding a galactosyltransferase, wherein said nucleic acid encoding said galactosyltransferase comprises a nucleotide sequence encoding a targeting sequence to target the encoded galactosyltransferase to the Golgi apparatus, wherein expression of said galactosyltransferase in said fungal cell produces proteins comprising Gal 2 GlcNAc 2 Man 3 GlcNAc 2  N-glycans. 
     
     
         53 . An isolated fungal cell genetically engineered to produce proteins comprising GlcNAc 2 Man 3 GlcNAc 2  N-glycans, wherein said fungal cell is genetically engineered to be deficient in ALG3 activity and comprises a nucleic acid encoding a GlcNAc-transferase I and a GlcNAc-transferase II, wherein said nucleic acid encoding said GlcNAc-transferase I and said GlcNAc-transferase II comprises nucleotide sequences encoding targeting sequences to target each encoded protein to an intracellular compartment, wherein expression of said GlcNAc-transferase I, and said GlcNAc-transferase II in said fungal cell produces proteins comprising GlcNAc 2 Man 3 GlcNAc 2  N-glycans. 
     
     
         54 . The fungal cell of  claim 53 , wherein said genetically engineered fungal cell further is deficient in OCH1 activity. 
     
     
         55 . The fungal cell of  claim 53  or  claim 54 , wherein said genetically engineered fungal cell further comprises a nucleic acid encoding an α-1,3-glucosyltransferase. 
     
     
         56 . The fungal cell of  claim 53 , wherein said genetically engineered fungal cell further comprises a nucleic acid encoding a target protein, wherein said cell produces said target protein modified to comprise said GlcNAc 2 Man 3 GlcNAc 2  N-glycans. 
     
     
         57 . The fungal cell of  claim 53 , said fungal cell further comprising a nucleic acid encoding the α and β subunits of a Glucosidase II, wherein expression of said α and β subunits of said Glucosidase II in said fungal cell produces said protein comprising said GlcNAc 2 Man 3 GlcNAc 2  N-glycans. 
     
     
         58 . The fungal cell of  claim 57 , said fungal cell further comprising a nucleic acid encoding a galactosyltransferase, wherein said nucleic acid encoding said galactosyltransferase comprises a nucleotide sequence encoding a targeting sequence to target the encoded galactosyltransferase to the Golgi apparatus, wherein expression of said galactosyltransferase in said fungal cell produces proteins comprising Gal 2 GlcNAc 2 Man 3 GlcNAc 2  N-glycans. 
     
     
         59 . A substantially pure culture of  Yarrowia lipolytica  cells, a substantial number of which are genetically engineered to produce glycoproteins comprising Gal 2 GlcNac 2 Man 3 GlcNAc 2  N-glycans, wherein said cells are genetically engineered to be deficient in ALG3 activity and comprise a nucleic acid encoding a GlcNAc-transferase I, a GlcNAc-transferase II, and a galactosyltransferase, wherein said nucleic acid encoding said GlcNAc-transferase I, said GlcNAc-transferase II, and said galactosyltransferase comprises nucleotide sequences encoding targeting sequences to target each encoded protein to an intracellular compartment, wherein expression of said GlcNAc-transferase I, said GlcNAc-transferase II, and said galactosyltransferase in said cell produces proteins comprising Gal 2 GlcNAc 2 Man 3 GlcNAc 2  N-glycans. 
     
     
         60 . The substantially pure culture of  claim 59 , wherein said cells further are deficient in OCH1 activity. 
     
     
         61 . The substantially pure culture of  claim 59  or  claim 60 , wherein said cells further comprise a nucleic acid encoding an α-1,3-glucosyltransferase. 
     
     
         62 . The substantially pure culture of  claim 61 , wherein said cells further comprise a nucleic acid encoding the α and β subunits of a Glucosidase II, wherein expression of said α and β subunits of said Glucosidase II in said fungal cell produces said target protein comprising said Gal 2 GlcNAc 2 Man 3 GlcNAc 2  N-glycans. 
     
     
         63 . A substantially pure culture of  Yarrowia lipolytica  cells, a substantial number of which are genetically engineered to produce glycoproteins comprising Gal 2 GlcNAc 2 Man 3 GlcNAc 2  N-glycans, wherein said cells are genetically engineered to be deficient in OCH1 activity and comprise a nucleic acid encoding an α-1,2-mannosidase, a GlcNAc-transferase I, a mannosidase II, a GlcNAc-transferase II, and a galactosyltransferase, wherein said nucleic acid encoding said α-1,2-mannosidase, said GlcNAc-transferase I, said mannosidase II, said GlcNAc-transferase II, and said galactosyltransferase comprises nucleotide sequences encoding targeting sequences to target each encoded protein to an intracellular compartment, wherein expression of said α-1,2-mannosidase, said GlcNAc-transferase I, said mannosidase II, said GlcNAc-transferase II, and a galactosyltransferase in said cells produces proteins comprising Gal 2 GlcNAc 2 Man 3 GlcNAc 2 N-glycans. 
     
     
         64 . A composition comprising a glycoprotein, wherein at least 50% of the N-glycans on said glycoprotein are GlcNAc 2 Man 3 GlcNAc 2 N-glycans. 
     
     
         65 . The composition of  claim 64 , wherein at least 70% of the N-glycans on said glycoprotein are GlcNAc 2 Man 3 GlcNAc 2 N-glycans. 
     
     
         66 . The composition of  claim 64 , wherein at least 85% of the N-glycans on said glycoprotein are GlcNAc 2 Man 3 GlcNAc 2 N-glycans.