Targeted knockouts of Physcomitrella lacking plant‐specific immunogenic N‐glycans

Using plants as production factories for therapeutic proteins requires modification of their N‐glycosylation pattern because of the immunogenicity of plant‐specific sugar residues. In an attempt towards such humanization, we disrupted the genes for α1,3‐fucosyltransferase and β1,2‐xylosyltransferase in Physcomitrella patens by homologous recombination. The single Δfuc‐t and Δxyl‐t plants, as well as the double knockout, lacked transcripts of the corresponding genes, but did not differ from the wild‐type moss in morphology, growth, development, and ability to secrete a recombinant protein, the human vascular endothelial growth factor VEGF₁₂₁, into the culture medium. N‐Glycan analysis, however, revealed the absence of 1,3‐fucosyl and/or 1,2‐xylosyl residues, respectively. Therefore, the modifications described here represent the key step towards the generation of moss lines suitable for the production of plant‐made glycosylated biopharmaceuticals with nonallergenic N‐glycans.