Glyco‐Engineering of Moss Lacking Plant‐Specific Sugar Residues

: The commercial production of complex pharmaceutical proteins from human origin in plants is currently limited through differences in protein N-glycosylation pattern between plants and humans. On the one hand, plant-specific α(1,3)-fucose and β(1,2)-xylose residues were shown to bear strong immunogenic potential. On the other hand, terminal β(1,4)-galactose, a sugar common on N-glycans of pharmaceutically relevant proteins, e.g., antibodies, is missing in plant N-glycan structures. For safe and flexible production of pharmaceutical proteins, the humanisation of plant protein N-glycosylation is essential. Here, we present an approach that combines avoidance of plant-specific and introduction of human glycan structures. Transgenic strains of the moss Physcomitrella patens were created in which the α(1,3)-fucosyltransferase and β(1,2)-xylosyltransferase genes were knocked out by targeted insertion of the human β(1,4)-galactosyltransferase coding sequence in both of the plant genes (knockin). The transgenics lacked α(1,3)-fucose and β(1,2)-xylose residues, whereas β(1,4)-galactose residues appeared on protein N-glycans. Despite these significant biochemical changes, the plants did not differ from wild type with regard to overall morphology under standard cultivation conditions. Furthermore, the glyco-engineered plants secreted a transiently expressed recombinant human protein, the vascular endothelial growth factor, in the same concentration as unmodified moss, indicating that the performed changes in glycosylation did not impair the secretory pathway of the moss. The combined knockout/knockin approach presented here, leads to a new generation of engineered moss and towards the safe and flexible production of correctly processed pharmaceutical proteins with humanised N-glycosylation profiles.