Production of glycoproteins with mammalian-like N-glycans in filamentous fungi

A method for producing a Trichoderma or Myceliophthora filamentous fungal cell which produces a human antibody comprising mammalian-like neutral N-glycans, the method comprising:

• i. introducing one or more mutations into the Trichoderma or Myceliophthora filamentous fungal cell that reduces or eliminates one or more endogenous protease activity compared to a parental Trichoderma or Myceliophthora filamentous fungal cell which does not have said mutation(s) and wherein endogenous protease is selected from the group consisting of aspartic proteases, trypsin-like serine proteases, subtilisin proteases, glutamic proteases, aminopeptidase proteases, sep proteases, and sedolisin proteases;
• ii. introducing a polynucleotide encoding a heterologous catalytic subunit of oligosaccharyl transferase into the Trichoderma or Myceliophthora filamentous fungal cell;
• iii. introducing a mutation in the gene encoding dolichyl-P-Man:Man(5)GlcNAc(2)-PPdolichyl mannosyltransferase (ALG3) that eliminates the corresponding ALG3 expression compared to the level of expression of ALG3 gene in a parental Trichoderma or Myceliophthora cell which does not have such mutation,
• iv. recombinant polynucleotide for expression of at least 2 distinct genes encoding a-glucosidase II, where the α-glucosidase II is selected from the group consisting of an, α recombinant polynucleotide encoding a Trichoderma α-glucosidase II, a recombinant polynucleotide encoding an Aspergillus α-glucosidase II catalytic domain, a recombinant polynucleotide encoding a microalgae α-glucosidase II catalytic domain, and a recombinant polynucleotide encoding a Trypanosoma α-glucosidase II catalytic domain,
• v. introducing a polynucleotide encoding an N-acetylglucosaminyltransferase I (GnTI) catalytic domain into the Trichoderma or Myceliophthora filamentous fungal cell,
• vi. introducing a polynucleotide encoding an N-acetylglucosaminyltransferase II catalytic domain into the Trichoderma or Myceliophthora filamentous fungal cell,
• vii. optionally, introducing a recombinant polynucleotide encoding a α1,6 fucosyltransferase activity and a recombinant polynucleotide encoding a GDP fucose synthesizing activity,
• viii. introducing a recombinant polynucleotide for increasing α1,2 mannosidase activity into the Trichoderma or Myceliophthora filamentous fungal cell;
• and,
• ix. introducing a recombinant polynucleotide encoding said human antibody into the Trichoderma or Myceliophthora filamentous fungal cell;
• wherein at least 90% (mol %), or at least 95% (mol %), of the total neutral N-glycans of said human antibody are Man3, GIcNAcMan3, G0, G1, G2, FG0, FG1 and/or FG2 glycoform, and less than 10% of the total neutral N-glycans of said human antibody are Hex6 glycoform, wherein Hex6 has structure of FIG. 1.