Establishment of a tobacco BY2 cell line devoid of plant-specific xylose and fucose as a platform for the production of biotherapeutic proteins

Plant Biotechnol J. 2017 Sep;15(9):1120-1129. doi: 10.1111/pbi.12702. Epub 2017 Mar 3.


Plant-produced glycoproteins contain N-linked glycans with plant-specific residues of β(1,2)-xylose and core α(1,3)-fucose, which do not exist in mammalian-derived proteins. Although our experience with two enzymes that are used for enzyme replacement therapy does not indicate that the plant sugar residues have deleterious effects, we made a conscious decision to eliminate these moieties from plant-expressed proteins. We knocked out the β(1,2)-xylosyltranferase (XylT) and the α(1,3)-fucosyltransferase (FucT) genes, using CRISPR/Cas9 genome editing, in Nicotiana tabacum L. cv Bright Yellow 2 (BY2) cell suspension. In total, we knocked out 14 loci. The knocked-out lines were stable, viable and exhibited a typical BY2 growing rate. Glycan analysis of the endogenous proteins of these lines exhibited N-linked glycans lacking β(1,2)-xylose and/or α(1,3)-fucose. The knocked-out lines were further transformed successfully with recombinant DNaseI. The expression level and the activity of the recombinant protein were similar to that of the protein produced in the wild-type BY2 cells. The recombinant DNaseI was shown to be totally free from any xylose and/or fucose residues. The glyco-engineered BY2 lines provide a valuable platform for producing potent biopharmaceutical products. Furthermore, these results demonstrate the power of the CRISPR/Cas9 technology for multiplex gene editing in BY2 cells.

Keywords: CRISPR/Cas9; DNaseI; bio-pharming; gene editing; glyco-engineering; plant glycans.

MeSH terms

  • Biological Therapy*
  • Cell Line
  • Clustered Regularly Interspaced Short Palindromic Repeats
  • Fucose / metabolism*
  • Fucosyltransferases / genetics
  • Fucosyltransferases / metabolism
  • Gene Editing
  • Genetic Vectors
  • Glycoproteins / genetics
  • Glycoproteins / metabolism*
  • Pentosyltransferases / genetics
  • Pentosyltransferases / metabolism
  • Plant Proteins / genetics
  • Plant Proteins / metabolism
  • Polysaccharides
  • Recombinant Proteins
  • Tobacco / genetics*
  • Tobacco / metabolism
  • Xylose / metabolism*


  • Glycoproteins
  • Plant Proteins
  • Polysaccharides
  • Recombinant Proteins
  • Fucose
  • Xylose
  • Fucosyltransferases
  • Pentosyltransferases
  • UDP xylose-protein xylosyltransferase