Expressions Of Acidothermus Cellulolyticus Cellulases In Plants

Large-scale production of low-cost cellulose hydrolytic enzymes for lignocellulosic biomass conversion is desirable in terms of encomic benefits. Transgenic plants that overexpress recombinant cellulases offer an alternative as’bioreactors’to produce huge quantities of inexpensive and usable cellulase. In this study, we report the expressions of two thermostable Acidothermus cellulolyticus cellulases, endo-1,4-β-D-glucanase (El) and exoglucanase (Guxl) in tobacco and rice plants.The expression plasmids was separately introduced into tobacco and rice via Agrobacterium-mediated transformation technique. The presence of the recombinant cellulase genes within the transformed plants were confirmed by PCR. To evaluate of the heterologous cellulases expression level, the cellulase genes were targeted to various subcellular compartments via5’transit peptides. Transgenic tobacco plants that expressed apoplast-localized El showed the highest activity, which was nearly3times higher than cytoplasm-localized El, which the catalytic domain of exoglucanase Guxlcd showed the lowest level. The highest expression level in the transgenic rice was the primal El gene with a specific activity of14.3nmol MU mg-1total soluble protein min-1, about1.3and1.6times higher than Elcd and Guxlcd, respectively. The El-Guxlcd fusion enzyme shows insignificant enhancement in cellulase activity over El alone. And no noticeable increasement for the cellulase expression level with KOZAK-CALSP signal peptide was observed for transgenic tobacco plants. Thus, the PR-S sequence had observably effect on the transcription genes, indicating that the differences in the levels of cellulase activity were mainly affected by sub-cellular targeting.The recombinant El cellulases in transgenic rice exhibited good thermostability around70℃, and the optimum pH of recombinant enzymes were5.0, and great loss in cellulase activity occurred as the pH was increased to7. Above all, rice straw samples that were stored for a month lost about25%of the initial cellulase activity at room temperature. All transgenic plants exhibited normal growth compared to wild type, indicating the recombinant cellulases caused little damage to the hosts. The presented technique showed high reliability, which suggests its potential application in cellulosic ethanol applications.