Cellulose is an important industrial raw material and the most abundant renewable natural resource produced in the biosphere. In order to harness cellulose for biological energy applications, a reduction in cellulase manufacturing cost, an improvement in enzyme activity, and an increase in fermentable sugar yields are all vital design parameters. Here, we demonstrate that cellobiohydrolases (i.e. CBHI and CBHII) from the filamentous fungi, Trichoderma reesei, can not only be expressed in E. coli, but can be transported to the periplasm and anchored to the outer membrane by fusing them to signal peptides and cell surface display proteins (i.e. alkaline phosphatase, Lpp-OmpA, etc.), respectively. Expression of these fungal cellulases in the periplasm will allow for correct disulfide bond formation in order to subsequently purify and perform in vitro activity assays on soluble cellulose substrates. Cellulose cannot be transported into the cell; therefore one of the goals is to assay the cellobiohydrolases in vivo by displaying them on the bacterial cell surface so that the enzymes release reducing sugars from the chain ends of the biological polymer. Upon the demonstration of active, cell-anchored cellulases, we will endeavor to engineer these crucial enzymes for improved activity.

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