Advanced Plant-Based Glycan Engineering

Abstract

With respect to biomanufacturing, glycosylation is one of the most addressed post-translational modifications, since it is well-known that the attachment of sugar residues efficiently affects protein homogeneity and functionality. Much effort has been taken into engineering various expression systems to control glycosylation and to generate molecules with targeted sugar profiles. Nevertheless, engineering of N- and O-linked glycans on well-established expression systems remains challenging. On the one side the glycosylation machinery in mammalian cells is hard to control due to its complexity. Most bacteria, on the other side, completely lack such glycan formations, and in general exhibit fundamental differences in their glycosylation abilities. Beyond that, plants generate complex N-glycans typical of higher eukaryotes, but simpler than those produced by mammals. Paradoxically, it seems that the limited glycosylation capacity of plant cells is an advantage for specific glycan manipulations. This review focuses on recent achievements in plant glycan engineering and provides a short outlook on how new developments (in synthetic biology) might have a positive impact.

Keywords: N- and O-linked glycans; glycan engineering; glyco proteins; glycosylation; plant-biotechnology; plants.

Figure 1

Schematic presentation of plant-based glycan engineering (A) N-linked glycans. Native: The three major complex glycoforms frequently detected in wild-type plants are 1: GnGnXF, 2: MMXF paucimannosidic structures, and 3: Lewis A structures. Simplified: Glycan simplification and homogeneity by knockout/down approaches (creating the “chassis”). 4: Man5 structure generated by GnTI knockout/down; 4′: single GlcNAc (GlycoDelete); 5: GnGn (common eukaryotic core). Augmentation of plant glycans by stable/transient overexpression of foreign glycosylation enzymes. 6: bisected (GnGnbi) structure; 7: branched ([GnGn][GnGn]) structure; 8: helminth LDN-F structure; 9: Lewis X structure; 10: AA (β1,4-galactosylated structures); 11: NaNa (α2,6-sialylated glycoforms); 12: polysialylated glycans. (B) O-linked glycans. 13: common native O-glycans (hydroxyproline, Hyp, decorated with arabinose residues); 14: PH4 knockout to prevent conversion of prolines to Hyp; 15: generation of mucin type O-glycans by the overexpression of human polypeptide GalNAc-transferase 2 (GalNAcT2) and Drosophila melanogaster core β1,3-galactosyltransferase (C1GalT1); 16: sialylation of O-glycans by overexpression of α2,6- and α2,3-sialyltransferases (ST6GalNAc, ST3Gal-I). ΔXF: knockout/down of α1,3-fucosyltransferase and core β1,2-xylosyltransferase; ΔHEXO: knockout of β-N-acetylhexosaminidases HEXO1 and HEXO3; Sia Pathway: recombinant expression of the sialic acid pathway as described by Castilho et al. (2010); PolyST: α2,8 polysialyltransferase II, IV; P4H: prolyl-4-hydroxylase. Glycan nomenclature: Consortium of functional glycomics.