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

Uri Hanania, Tami Ariel, Yoram Tekoah, Liat Fux, Maor Sheva, Yehuda Gubbay, Mara Weiss, Dina Oz, Yaniv Azulay, Albina Turbovski, Yehava Forster, Yoseph Shaaltiel

Research output: Contribution to journalArticlepeer-review

69 Scopus citations


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.

Original languageEnglish
Pages (from-to)1120-1129
Number of pages10
JournalPlant Biotechnology Journal
Issue number9
StatePublished - Sep 2017
Externally publishedYes

Bibliographical note

Publisher Copyright:
© 2017 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.


  • CRISPR/Cas9
  • DNaseI
  • bio-pharming
  • gene editing
  • glyco-engineering
  • plant glycans


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