Monday, May 5, 2008
6-16

Methods of Increasing β-glucosidase Expression in Trichoderma reesei for Improved Hydrolysis of Pretreated Corn Stover

Sandy Merino and Suchindra Maiyuran. Artificial Evolution, Novozymes, 1445 Drew Ave., Davis, CA 95618

Trichoderma reesei produces two cellobiohydrolases (CBH I and CBH II), five endoglucanases (EGI-V), and two β-glucosidases (BG).  Cellulose is hydrolyzed to cellobiose, a water-soluble beta-1,4-linked dimer of glucose, through synergistic action of cellobiohydrolases and endoglucanases.  Cellobiose is then hydrolyzed to glucose by β-glucosidases.  Wildtype T. reesei secretes levels of β-glucosidase insufficient to hydrolyze all the  cellobiose under conditions of high substrate concentration, resulting in product inhibition of CBH and EG enzymes and a reduced enzymatic hydrolysis of cellulose.  One method of economically alleviating this inhibition is by increasing the expression levels of β-glucosidase in T. reesei through molecular approaches. Initially the BG from Aspergillus oryzae was recombinantly expressed in T. reesei, but expression levels were still insufficient.  The native signal sequence from the A. oryzae BG was replaced with a recombinant signal sequence from a Humicola insolens protein that is highly expressed in T. reesei.  This swap effectively increased BG expression and resulted in a 2-fold improvement in the conversion of cellulose to glucose at the substrate levels tested.  Further improvement in BG expression levels were obtained by creating a fusion protein comprised of an endoglucanase catalytic domain fused at the N-terminus of BG.  This fusion resulted in additional expression and further improvement of the cellulase activity in PCS hydrolysis assays.

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