Inducer-free cellulase production system based on the constitutive expression of mutated XYR1 and ACE3 in the...

Trichoderma reesei is a widely used host for producing cellulase and hemicellulase cocktails for lignocellulosic biomass degradation. Here, we report a genetic modification strategy for industrial T. reesei that enables enzyme production using simple glucose without inducers, such as cellulose, lactose and sophorose. Previously, the mutated XYR1^V821F or XYR1^A824V was known to induce xylanase and cellulase using only glucose as a carbon source, but its enzyme composition was biased toward xylanases, and its performance was insufficient to degrade lignocellulose efficiently. Therefore, we examined combinations of mutated XYR1^V821F and constitutively expressed CRT1, BGLR, VIB1, ACE2, or ACE3, known as cellulase regulators and essential factors for cellulase expression to the T. reesei E1AB1 strain that has been highly mutagenized for improving enzyme productivity and expressing a ß-glucosidase for high enzyme performance. The results showed that expression of ACE3 to the mutated XYR1^V821F expressing strain promoted cellulase expression. Furthermore, co-expression of these two transcription factors also resulted in increased productivity, with enzyme productivity 1.5-fold higher than with the conventional single expression of mutated XYR1^V821F. Additionally, that productivity was 5.5-fold higher compared to productivity with an enhanced single expression of ACE3. Moreover, although the DNA-binding domain of ACE3 had been considered essential for inducer-free cellulase production, we found that ACE3 with a partially truncated DNA-binding domain was more effective in cellulase production when co-expressed with a mutated XYR1^V821F. This study demonstrates that co-expression of the two transcription factors, the mutated XYR1^V821F or XYR1^A824V and ACE3, resulted in optimized enzyme composition and increased productivity.