YEAST STRAINS PRODUCING MAMMALIAN-LIKE COMPLEX N-GLYCANS
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.