Yeast strains producing mammalian-like complex n-glycans

A method of making a Yarrowia lipolytica cell capable of producing proteins comprising GlcNAc2Man3GlcNAc2 N-glycans, said method comprising:

• a) providing a Yarrowia lipolytica cell genetically engineered to produce proteins comprising Man3GlcNAc2 N-glycans, wherein said cell is deficient in Outer CHain elongation (OCH1) activity and in Asparagine Linked Glycosylation 3 (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 an intracellular compartment, and wherein said cell further comprises a nucleic acid encoding Asparagine Linked Glycosylation 6 (ALG 6);
• 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, and a nucleic acid encoding a GlcNAc-transferase II, wherein said nucleic acid comprises a nucleotide sequence encoding a targeting sequence to target said encoded GlcNAc-transferase II to an intracellular compartment, wherein expression of said GlcNAc-transferase I and said GlcNAc-transferase II in said Yarrowia lipolytica cell produces proteins comprising GlcNAc2Man3GlcNAc2 N-glycans.

-32. (canceled)

-39. (canceled)

-43. (canceled)

A method of producing a target protein comprising GalGlcNAc2Man3GlcNAc2 or Gal2GlcNAc2Man3GlcNAc2 N-glycans, said method comprising:

• a) providing a Yarrowia lipolytica cell genetically engineered to be deficient in OCH1 activity and in ALG3 activity, comprising 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 an intracellular compartment, comprising a nucleic acid encoding ALG, 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 GalGlcNAc2Man3GlcNAc2 or Gal2GlcNAc2Man3GlcNAc2 N-glycans.

-47. (canceled)

-52. (canceled)

An isolated Yarrowia lipolytica cell genetically engineered to produce proteins comprising GlcNAc2Man3GlcNAc2 N-glycans, wherein said Yarrowia lipolytica cell is genetically engineered to be deficient in OCH1 activity and in ALG3 activity, 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 an intracellular compartment, comprises a nucleic acid encoding ALG6, 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 Yarrowia lipolytica cell produces proteins comprising GlcNAc2Man3GlcNAc2 N-glycans.

-55. (canceled)

-66. (canceled)

A method of producing a target protein comprising GlcNAc2Man3GlcNAc2 N-glycans, said method comprising:

• a) providing a Yarrowia lipolytica cell genetically engineered to be deficient in OCH1 activity and in ALG3 activity, comprising 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 an intracellular compartment, comprising a nucleic acid encoding ALG6, and comprising 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;
• b) introducing into said cell a nucleic acid encoding a target protein, wherein said cell produces said target protein comprising said GlcNAc2Man3GlcNAc2 N-glycans.

A substantially pure culture of Yarrowia lipolytica cells, a substantial number of which are genetically engineered to produce glycoproteins comprising GlcNAc2Man3GlcNAc2 N-glycans, wherein said cells are genetically engineered to be deficient in OCH1 activity and in ALG3 activity, comprise 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 an intracellular compartment, comprise a nucleic acid encoding ALG6, and comprise 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 cell produces proteins comprising GlcNAc2Man3GlcNAc2 N-glycans.