2020/12/18 by Lennard L. Bohlender, Juliana Parsons, Sebastian N. W. Hoernstein +9 · 1 citation
Biochemistry, Genetics and Molecular Biology · #Transgenic Plants and Applications #Glycosylation and Glycoproteins Research #Viral Infectious Diseases and Gene Expression in Insects
paper · pdf · doi:10.3389/fpls.2020.610032
openalex publication_date 2020/12/18 · openalex created_date 2025/10/10 · openalex updated_date 2026/07/23
Recombinantly produced proteins are indispensable tools for medical applications. Since the majority of them are glycoproteins, their N -glycosylation profiles are major determinants for their activity, structural properties and safety. For therapeutical applications, a glycosylation pattern adapted to product and treatment requirements is advantageous. Physcomitrium patens (Physcomitrella, moss) is able to perform highly homogeneous complex-type N -glycosylation. Additionally, it has been glyco-engineered to eliminate plant-specific sugar residues by knock-out of the β1,2-xylosyltransferase and α1,3-fucosyltransferase genes (Δxt/ft). Furthermore, Physcomitrella meets wide-ranging biopharmaceutical requirements such as GMP compliance, product safety, scalability and outstanding possibilities for precise genome engineering. However, all plants, in contrast to mammals, lack the capability to perform N -glycan sialylation. Since sialic acids are a common terminal modification on human N- glycans, the property to perform N -glycan sialylation is highly desired within the plant-based biopharmaceutical sector. In this study, we present the successful achievement of protein N -glycan sialylation in stably transformed Physcomitrella. The sialylation ability was achieved in a Δxt/ft moss line by stable expression of seven mammalian coding sequences combined with targeted organelle-specific localization of the encoded enzymes responsible for the generation of β1,4-galactosylated acceptor N -glycans as well as the synthesis, activation, transport and transfer of sialic acid. Production of free (Neu5Ac) and activated (CMP-Neu5Ac) sialic acid was proven. The glycosidic anchor for the attachment of terminal sialic acid was generated by the introduction of a chimeric human β1,4-galactosyltransferase gene under the simultaneous knock-out of the gene encoding the endogenous β1,3-galactosyltransferase. Functional complex-type N- glycan sialylation was confirmed via mass spectrometric analysis of a stably co-expressed recombinant human protein.