(620bn) Engineering Designer Glycopeptides As a Molecular Carrier for Cell Wall-Modifying Enzymes Expressed in Planta

Efficient conversion of lignocellulosic biomass to biofuels and biobased products has been hampered by recalcitrance of the plant cell wall to biochemical deconstruction. This project aims to leverage hydroxyproline (Hyp)-O-glycosylation – a process unique to plant cell wall glycoproteins – as an innovative technology for de novo design and engineering of novel “designer glycopeptide” (DGPs) that can facilitate plant cell wall reconstruction. This strategically designs Pro/Hyp-rich repetitive peptides, such as tandem repeats of “Ser-Pro” and “Ser-Pro-Pro-Pro-Pro” motifs as the DGP peptide backbone, which direct extensive Hyp-O-glycosylation in plants with arabinogalactan polysaccharides and oligoarabinosides, respectively. Such Hyp-O-glycosylated DGPs are proposed to be able to function as a molecular carrier for in planta-expressed cell wall-modifying (CWM) enzymes, targeting the enzymes to the cell wall matrix and stabilize the enzymes. Two types of  DGP design, (SP)₃₂ comprised of 32 tandem repeats of “Ser-Pro” motif and (SP4)₁₈ comprised of 18 tandem repeats of “Ser-Pro-Pro-Pro-Pro” motif, was each engineered into tobacco plants as fusion with a reporter protein, enhanced green fluorescence protein (EGFP). The (SP4)₁₈ tag showed a better effectiveness on improving the accumulation of the tagged EGFP in cell wall matrix than the (SP)₃₂ tag. A thermostable CWM enzyme, E1 endoglucanase from Acidothermus cellulolyticus, was then engineered into tobacco as a fusion with the (SP4)₁₈ tag. The enzyme activity and subcellular localization of the enzyme, as well as phenotype and enzymatic digestibility of the plants were investigated. These DGP tags are also being engineered into the bioenergy crop−switchgrass to study their function as a molecular carrier in a monocot plant.