Methods for demannosylating phosphorylated N-glycans on a glycoprotein are described that use a mannosidase capable of hydrolyzing a terminal alpha-1,2 mannose linkage when the underlying mannose is phosphorylated.

Methods for demannosylating phosphorylated N-glycans on a glycoprotein are described that use a mannosidase capable of hydrolyzing a terminal alpha-1,2 mannose linkage when the underlying mannose is phosphorylated.

The present disclosure relates to compositions and methods useful for the production of recombinant glycoproteins in filamentous fungal cells, such as Trichoderma cells, wherein at least 90% (mol %), preferably at least 95% of the total neutral N-glycans of said produced recombinant glycoprotein are mammalian-like N-glycans. More specifically, the invention provides a filamentous fungal cell comprising i. one or more mutations that reduces or eliminates one or more endogenous protease activity compared to a parental filamentous fungal cell which does not have said mutation(s); ii.a polynucleotide encoding a heterologous catalytic subunit of oligosaccharyl transferase; iii. a recombinant polynucleotide for increasing 1, 2 mannosidase activity;and, iv. a recombinant polynucleotide encoding said heterologous glycoprotein.

Thermothelomyces heterothallica (formerly Myceliophthora thermophila) genetically modified to produce glycoproteins with N-glycans of mammalian proteins (particularly human, companion animal and other animal proteins) are provided, comprising deletion or disruption of the alg3 gene, expression of ER-targeted Mannosidase 1 (alpha-1.2-Mannosidase), and expression of ER-targeted Glucosidase 2 alpha-subunit. The Th. heterothallica may also further comprise heterologous GlcNAc transferase 1 (GNT1), GlcNAc transferase 2 (GNT2), STT3 subunit of a heterologous oligosaccharyltransferase and galactosyltransferase.

This disclosure relates to novel Pichia pastoris display systems, e.g., display systems featuring the Pichia pastoris strains (such as SuperMan5) with substantially homogeneous N-glycans displayed on cell surface proteins.