A genome‐edited N. benthamiana line for industrial‐scale production of recombinant glycoproteins with targeted N‐glycosylation

• Published in: Biotechnology journal 2024-01, Vol.19 (1), p.e2300323-n/a
• Main Authors: Kogelmann, Benjamin, Melnik, Stanislav, Bogner, Michaela, Kallolimath, Somanath, Stöger, Eva, Sun, Lin, Strasser, Richard, D'Aoust, Marc‐André, Lavoie, Pierre‐Olivier, Saxena, Pooja, Gach, Johannes S., Steinkellner, Herta
• Format: Article
• Language: English
• Subjects: CRISPR/Cas9; fucose; Glycoproteins - genetics; Glycosylation; Humans; Nicotiana benthamiana; N‐glycan engineering; Plants, Genetically Modified - genetics; Plants, Genetically Modified - metabolism; Polysaccharides - chemistry; Rapid Communication; recombinant glycoproteins; Recombinant Proteins - metabolism; UDP Xylose-Protein Xylosyltransferase
• ISSN: 1860-6768; 1860-7314; 1860-7314
• DOI: 10.1002/biot.202300323

Control over glycosylation is an important quality parameter in recombinant protein production. Here, we demonstrate the generation of a marker‐free genome edited Nicotiana benthamiana N‐glycosylation mutant (NbXF‐KO) carrying inactivated β1,2‐xylosyltransferase and α1,3‐fucosyltransferase genes. The knockout of seven genes and their stable inheritance was confirmed by DNA sequencing. Mass spectrometric analyses showed the synthesis of N‐glycans devoid of plant‐specific β1,2‐xylose and core α 1,3‐fucose on endogenous proteins and a series of recombinantly expressed glycoproteins with different complexities. Further transient glycan engineering towards more diverse human‐type N‐glycans resulted in the production of recombinant proteins decorated with β1,4‐galactosylated and α2,6‐sialylated structures, respectively. Notably, a monoclonal antibody expressed in the NbXF‐KO displayed glycosylation‐dependent activities. Collectively, the engineered plants grow normally and are well suited for upscaling, thereby meeting industrial and regulatory requirements for the production of high‐quality therapeutic proteins. Graphical and Lay Summary Engineering glycosylation pathway in plants increases quality of recombinantly expressed proteins. A Nicotiana benthamiana line that lacks active xylosyltransferase genes was used to knock out core fucosyltransferase genes by multi‐target CRISPR/Cas9‐based gene editing. The engineered line synthesizes human‐type GnGn structures well suited for industrial‐scale production of proteins with homogeneous N‐glycans and serves as template for further glycan diversifications.