Lois Abbott - Senior Instructor, Retired

<p>Plant biomass consists primarily of cellulose, the most abundant organic compound on earth. Biomass is potentially a source of ethanol or hydrogen fuel, provided that the cellulose therein can be converted efficiently into monosaccharides, cellulase, which is needed for the enzymatic conversion of cellulose to glucose, is prohibitively expensive, thus hampering efforts to exploit an abundant source of renewable energy. We are addressing this problem by developing transgenic plants as bioreactors to produce large amounts of enzyme that can be activated after harvest to provide an inexpensive feedstock for fuel production. Enzymatic degradation of cellulose involves the concerted action of two classes of cellulase: an endoglucanase, which cleaves internal cellulosic linkages, and an exoglucanase, which releases oligosaccharides from free ends of the polymer. Many microorganisms, including thermophiles, secrete such enzymes, and the cloned genes are available. To avoid detrimental effects of expressing cellulase genes in plants, we are using microbial cellulases with high temperature optima. We have successfully generated transgenic tobacco cells and Arabidopsis thaliana plants that produce the catalytic domain of a thermostable (Topt=81 deg C) endo-1-4-Beta-D-glucanase, E1, from the eubacterium, Acidothermus cellulolyticus. Recombinant E1 catalytic domain was targeted to the ER by the signal peptide and secreted into the apoplast via the default pathway. Secretion of the enzyme did not detectably affect the growth rate of transgenic BY-2 cells, although the protein was enzymatically active at elevated temperatures. Similarly, transgenic plants exhibited no abnormal phenotypes between independent immunochemical and activity-based assays indicates that the enzyme accumulated to concentrations up to 26% of the total soluble protein in leaves of primary A. thaliana transformants. The amount of functional endoglucanase produced illustrates that plants can accumulate very large quantities of enzyme for commercial biomass conversion. We are developing strategies to sequester cellulases in cellular compartments and evaluating the effectiveness of a variety of enzymes to degrade cellulosic substracts in vitro under optimal enzyme reaction conditions.</p>