Engineering Novel “Designer” Glycopeptides in Planta As a Molecular Carrier for Directing the Accumulation of Recombinant Proteins/Enzymes

This project is aimed at leveraging a plant-specific, O-glycosylation process called hydroxyproline (Hyp)-O-glycosylation for de novo design and engineering of Hyp-O-glycosylated peptides (HypGPs) in plant cells that function as a molecular carrier in facilitating extracellular secretion of conjoined heterologous proteins/enzyme. Hyp-O-glycosylation involves post-translational Pro to Hyp and subsequent O-glycosylation, a modification that is unique to higher plants and green algae. Strategically designed Pro-rich HypGP modules engineered in plants can direct extensive O-glycosylation on each Pro residues, with HypGPs comprised of contiguous Pro residues, as in extensin-like “Ser-Pro-Pro-Pro-Pro” motifs are glycosylated with oligo-arabinosides; HypGPs comprised of non-contiguous Pro residues, as clustered in arabinogalactan protein (AGP)-like “Ser-Hyp-Ser-Hyp…” motifs, are glycosylated with highly branched arabinogalactan (AG) polysaccharides. In plant cell culture, dramatically increased secreted protein yields up to 1500-fold were detected when heterologous proteins, including a reporter protein GFP and many therapeutic proteins such as interferon α2, human growth hormone, interleukin-12 and stem cell factor, were expressed with a designer HypGP tag. Engineering a thermostable endoglucanase from Acidothermus cellulolyticus in tobacco plants with a designer HypGP tag could facilitate the deposition and stabilization of the enzyme in the cell wall matrix, leading to increased biomass saccharification efficiency by 3. 5-fold compared to wild type plants. The growth and biomass accumulation of the transgenic plants was not significantly impacted.