| An economical and efficient technology for lignocellulose biomass conversion is the key to provide renewable resources and solve the energy crisis. Plants were used as bioreactors for cellulases production because they offer large-scale and low-cost heterologous enzyme products. Trangenic plant bodies with recombinant cellulases accumulated inside can further be "self degradiation", decreasing the cost of pretreatments. To explore the optimum expression methods. Two bacterial thermostable cellulases (E2 and E3) and a E3-E2 fusion form were expressed in tobacco, driven by a double 35S promoter and 5'TEV-UTL. The enzymes were targeted to the apoplast and cytosol via N-terminal signal peptides and C-terminal retention signal peptides, respectively, and all showed functional activities.Transgenic plants that expressed apoplast-localized E2 had the highest average activity, about 1.5 and 3 times higher than those expressed ER-localized and cytosolic E2, respectively. The recombinant E3 showed the lowest activity, while the E3-E2 enzyme exhibited nearly the same activity as the apoplast-localized E2 alone.Effect of subcellular compartment localization was due primarily to post-transcriptional modification, since mRNA abundances were similar by Real-Time PCR analysis despite the range of cellulase activities obtained. The recombinant E2 cellulases exhibited good thermostability around 60℃. As the temperature raised further to 70℃, the activities decreased fast. After storing for three days at -20℃and 28℃, the enzymes lost nearly 20% and 80% of activity, respectively, indicating the transgenic plants obtained in this research need expensive storage facilities. All transgenic plants exhibited normal growth compared to wild type, indicating the recombinant cellulases cause little damage to the hosts. The results suggested a potential application for heterologous expression of cellulases in plant for biomass conversion. |
