Controlled production of cellulases in plants for biomass conversion. Annual report, March 11, 1997--March 14, 1998

The goal of this project is to facilitate conversion of plant biomass to usable energy by developing transgenic plants that express genes for microbial cellulases, which can be activated after harvest of the plants. In particular, the feasibility of targeting an endoglucanase and a cellobiohydrolase to the plant apoplast (cell wall milieu) is to be determined. To avoid detrimental effects of cellulose expression in plants, enzymes with high temperature optima were chosen; the genes for these enzymes are from thermophilic organisms that can use cellulose as a sole energy source. During the past year (year 2 of the grant), efforts have been focused on testing expression of endoglucanase E{sub 1}, from Acidothermus cellulolyticus, in the apoplast of both tobacco suspension cells and Arabidopsis thaliana plants. Using the plasmids constructed during the first year, transgenic cells and plants that contain the gene for the E{sub 1} catalytic domain fused to a signal peptide sequence were obtained. This gene was constructed so that the fusion protein will be secreted into the apoplast. The enzyme is made in large quantities and is secreted into the apoplast. More importantly, it is enzymatically active when placed under optimal reaction conditions (high temperature). Moreover, the plant cells and intact plants exhibit no obvious problems with growth and development under laboratory conditions. Work has also continued to improve binary vectors for Agrobacterium-mediated transformation, to determine activity of E{sub 1} at various temperatures, and to investigate the activity of the 35S Cauliflower Mosaic Virus promoter in E. coli. 9 figs