Overexpression of an exotic thermotolerant β-glucosidase in trichoderma reesei and its significant increase in cellulolytic activity and saccharification of barley straw

Background: Trichoderma reesei is a widely used industrial strain for cellulase production, but its low yield of beta-glucosidase has prevented its industrial value. In the hydrolysis process of cellulolytic residues by T. reesei, a disaccharide known as cellobiose is produced and accumulates, which inhibits further cellulases production. This problem can be solved by adding beta-glucosidase, which hydrolyzes cellobiose to glucose for fermentation. It is, therefore, of high vvalue to construct T. reesei strains which can produce sufficient beta-glucosidase and other hydrolytic enzymes, especially when those enzymes are capable of tolerating extreme conditions such as high temperature and acidic or alkali pH. Results: We successfully engineered a thermostable beta-glucosidase gene from the fungus Periconia sp. into the genome of T. reesei QM9414 strain. The engineered T. reesei strain showed about 10.5-fold (23.9 IU/mg) higher beta-glucosidase activity compared to the parent strain (2.2 IU/mg) after 24 h of incubation. The transformants also showed very high total cellulase activity (about 39.0 FPU/mg) at 24 h of incubation whereas the parent strain almost did not show any total cellulase activity at 24 h of incubation. The recombinant beta-glucosidase showed to be thermotolerant and remains fully active after two-hour incubation at temperatures as high as 60 C. Additionally, it showed to be active at a wide pH range and maintains about 88% of its maximal activity after four-hour incubation at 25 C in a pH range from 3.0 to 9.0. Enzymatic hydrolysis assay using untreated, NaOH, or Organosolv pretreated barley straw as well as microcrystalline cellulose showed that the transformed T. reesei strains released more reducing sugars compared to the parental strain. Conclusions: The recombinant T. reesei overexpressing Periconia sp. beta-glucosidase in this study showed higher beta-glucosidase and total cellulase activities within a shorter incubation time (24 h) as well as higher hydrolysis activity using biomass residues. These features suggest that the transformants can be used for beta-glucosidase production as well as improving the biomass conversion using cellulases.