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. 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. Our goal is to increase the expression level of β -glucosidase was increased in order to alleviate product inhibition. Initially the BG from Aspergillus oryzae was recombinantly expressed in T. reesei, but expression levels were low. In order to improve expression 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. The swap in signal sequences 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. The fusion protein resulted in additional BG expression and further improvement of the cellulase activity in PCS hydrolysis assays.
Full conference title:
9th EUROPEAN CONFERENCE ON FUNGAL GENETICS
- ECFG 9th (2008)