Patent ID: 8298811

Claim:
A uni- or multicellular fungal host cell which includes an α-1,2-mannosidase activity and a GlcNAc transferase I (GnT I) activity and is diminished or depleted in the activity of an initiating α-1,6-mannosyltransferase comprising a nucleic acid encoding a chimeric mannosidase enzyme comprising (a) a D. melanogaster mannosidase II catalytic domain fused to a cellular targeting signal peptide selected from the group consisting of Gls1-s, Mns1-s, Mns1-m, S.Sec-s, S.Sec-m, S.Sec-l, P.Sec-s, P.Sec-m, Mnn9-s, Van1-s, Van1-m, Van1-l, Anp1-s, Anp1-m, Anp1-l, Hoc1-s, Hoc1-m, Hoc 1-l , Mnn10-m, Mnn11-s, Mnt1-m, J3-m, Ktr1-s, Ktr2-s, Gnt1-s, Gnt1-m, Gnt1-l, Mnn2-s, Mnn2-m, Mnn2-1, Mnn5-m, Mnn1-s, Mnn1-m, Mnn1-l , Mnn6-s, and Mnn6-m or (b) a C. elegans mannosidase II catalytic domain fused to a cellular targeting signal peptide selected from the group consisting of Gls1-s, Mns1-s, Mns1-m, S.Sec-s, S.Sec-m, S.Sec-1, P.Sec-s, Van1-s, Van1-m, Van1-l, Anp1-s, Hoc1-m, Mnn10-s, Mnn10-m, Mnn10-1, Mnn11-s, Mnn11-m, Mnt1-s, Mnt1-m, Mnt1-l, D2-s, D2-m, D9-m, J3-m, Ktr2-s, Gnt1-s, Gnt1-m, Mnn2-s, Mnn2-m, Mnn2-l, Mnn5-s, Mnn5-m, Mnn1-s, Mnn1-m, and Mnn6-m, wherein said chimeric mannosidase in (a) or (b) is capable of hydrolyzing in vivo the Man α-1,3 and/or Man α-1,6 linkages of a GlcNAcMan5GlcNAc2 oligosaccharide substrate, whereby expression of said chimeric mannosidase produces one or more N-glycan structures on a recombinant glycoprotein expressed in said host cell wherein the N-glycan is characterized as having at least the oligosaccharide branch Manα1,3 (Manα1,6) Man β1,4-GlcNAc β1,4-GlcNAc-Asn.