Application of Hydroxyproline-O-Glycosylation Code for Enhanced Recombinant Protein Production In Plants and for Reconstruction of Plant Cell Wall for Improved Biomass Processability

Hydroxyproline-O-glycosylation involves post-translational hydroxylation of proline to hydroxyproline (Hyp) and subsequent glycosylation, a modification that is unique to plants and green algae. Our earlier work with synthetic genes encoding various Hyp-rich glycoproteins (HRGPs) expressed in plant cells elucidated a Hyp-O-glycosylation “code”, that is a peptide sequence directs the Hyp-O-glycosylation; specifically contiguous Hyp residues, as in X-Hyp-Hyp are sites of oligoarabinosylation; in contrast, clustered non-contiguous Hyp residues, as in X-Hyp-X-Hyp repeats are mainly sites of highly branched arabinogalactan polysaccharide addition. Where X is often Ser or Ala. These results demonstrated the feasibility of Hyp-O-based glycoprotein design in plants and triggered the following applications: 1) by introducing a Hyp-O-glycosylation tag to recombinant proteins expressed in tobacco BY-2 cells, we dramatically enhanced the production of secreted proteins up to 1500 fold; 2) using the same approach, we  significantly improved the yields  of recombinant proteins transiently expressed in Nicotiana benthamiana; 3) by engineering specific Hyp-O-based “designer” biopolymers into plants, we could reconstruct the plant cell walls for improved biomass processability.