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Alg3 of Saccharomyces cerevisiae catalyzes the mannosyl transfer from Man-P-Dol to Man5GlcNAc2-PP-Dol resulting in the formation of Man6GlcNAc2-PP-Dol, which is then further processed to the final precursor oligosaccharide Glc3Man9GlcNAc2 for N-glycosylation of proteins. Here, we identified the alg3 gene of the mushroom-forming fungus Schizophyllum commune by homology search. Its function was confirmed by the complementation of the Δalg3 strain of S. cerevisiae. Inactivation of alg3 in S. commune resulted in the production of predominantly Man3GlcNAc2 protein-linked N-glycans. No impact on growth nor a developmental phenotype due to the deletion was observed. This provides a first step toward engineering of a homogeneous, humanized N-glycosylation pattern for the production of therapeutic glycoproteins in mushrooms. [ABSTRACT FROM PUBLISHER]