| Reengineering plant cell walls with cell wall-modifying enzymes (CWMEs) represents a promising solution to overcome the recalcitrance for lignocellulosic biomass efficient conversion to liquid biofuels. This project investigated innovative application of hydroxyproline (Hyp)-O-glycosylation to engineer novel glycopeptide tags for heterologous CWMEs to maximize their hydrolytic functions in cell walls without disrupting composition and architecture. I addressed the hypothesis that a strategically designed glycopeptide tag fused to thermostable E1 endoglucanase and expressed in tobacco will facilitate the enzyme deposition into cell wall matrix to make the resulting biomass more subject to biochemical saccharification, while not adversely affecting the normal growth of transgenic plants. To address this hypothesis, the thermostable E1 endoglucanase (both the holoenzyme and catalytic domain) is engineered into tobacco plants as fusion with the (SP4)18 tag that consists of 18 tandem repeats of "Ser-Pro-Pro-Pro-Pro" motif and directs the Hyp-O-glycosylation with oligo-arabinosides. The results from this study show: (1) the (SP4)18 tag was extensively O-glycosylated, but this post-translational modification did not further enhance the secretion of the recombinant enzymes, and the enzyme activity was not negatively impacted by the (SP4)18 tag; (2) over-expression of recombinant enzymes influenced the phenotype of T0 plants, but the final yields of the plant biomass were not significantly impacted; (3) the created transgenic plants showed decreased recalcitrance to the enzymatic hydrolysis. This study partially supports the hypothesis and the feasibility of the HypGP engineering technology in plant cell wall reconstruction, which will contribute to the biomass and biofuel industry. |
