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. An isolated Yarrowia lipolytica cell genetically engineered to produce proteins comprising GlcNAc2Man3GlcNAc2 N-glycans, wherein said Yarrowia lipolytica cell is genetically engineered to comprise the following alterations:
a) deficient in Outer Chain elongation (OCH1) activity;
b) deficient in Asparagine linked Glycosylation 3 (ALG3) activity;
c) recombinantly expresses an α-1,2-mannosidase inclusive of a targeting sequence to an intracellular compartment;
d) recombinantly expresses Asparagine Linked Glycosylation 6 (ALG6); and
e) recombinantly expresses a GlcNAc-transferase I and GlcNAc-transferase II,
wherein both transferases are inclusive of a targeting sequence to an intracellular compartment,
wherein as a result of said alterations, said Yarrowia lipolytica cell produces proteins comprising GlcNAc 2 Man 3 GlcNAc 2 N-glycans.
2. The Yarrowia lipolytica cell of claim 1 , wherein said genetically engineered Yarrowia lipolytica 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.
3. The Yarrowia lipolytica cell of claim 1 , said Yarrowia lipolytica 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 Yarrowia lipolytica cell produces said protein comprising said GlcNAc 2 Man 3 GlcNAc 2 N-glycans.
4. The Yarrowia lipolytica cell of claim 1 , wherein said GlcNAc-transferase I and GlcNAc-transferase II are targeted to the Golgi apparatus.
5. The Yarrowia lipolytica cell of claim 1 , wherein said α-1,2-mannosidase is targeted to the endoplasmic reticulum.
6. A pure culture comprising Yarrowia lipolytica cells, according to claim 1 .
7. The culture of claim 6 , said Yarrowia lipolytica 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 Yarrowia lipolytica cell produces said protein comprising said GlcNAc 2 Man 3 GlcNAc 2 N-glycans.
8. The culture of claim 6 , wherein said GlcNAc-transferase I and GlcNAc-transferase II are targeted to the Golgi apparatus.
9. The culture of claim 6 , wherein said α-1,2-mannosidase is targeted to the endoplasmic reticulum.