Combined strategy of transcription factor manipulation and β-glucosidase gene overexpression in Trichoderma reesei and its application in lignocellulose bioconversion.

The industrial application of Trichoderma reesei has been greatly limited by insufficient β-glucosidase activity in its cellulase system. In this study, a novel β-glucosidase expression cassette was constructed and integrated at the target site in T. reesei ZU-02, which achieved the overexpression of β-glucosidase gene and in situ disruption of the cellulase transcriptional repressor ACE1. The resulting transformants showed significant increase in both β-glucosidase activity (BGA) and filter paper activity (FPA). The BGA and FPA increased to 25.13 IU/mL and 20.06 FPU/mL, respectively, 167- and 2.45-fold higher than that of the host strain. Meanwhile, the obtained cellulase system exhibited improved ratio of BGA to FPA, leading to better synergistic effect between cellulase components. Furthermore, submerged fermentation of the transformant was established in 50 m fermenter yielding 112.2 IU/mL β-glucosidase and 89.76 FPU/mL total cellulase. The newly constructed T. reesei transformant achieved improved hydrolysis yield (90.6%) with reduced enzyme loading (15 FPU/g substrate).