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

Abstract

BackgroundTrichoderma reesei is a widely used industrial strain for cellulase production, but its low yield of β-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 β-glucosidase, which hydrolyzes cellobiose to glucose for fermentation. It is, therefore, of high vvalue to construct T. reesei strains which can produce sufficient β-glucosidase and other hydrolytic enzymes, especially when those enzymes are capable of tolerating extreme conditions such as high temperature and acidic or alkali pH.ResultsWe successfully engineered a thermostable β-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 β-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 β-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.ConclusionsThe recombinant T. reesei overexpressing Periconia sp. β-glucosidase in this study showed higher β-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 β-glucosidase production as well as improving the biomass conversion using cellulases.

Heterologous expression of cellobiohydrolases in filamentous fungi – An update on the current challenges, achievements and perspectives

“…Such strains with balanced homologous and heterologous enzyme activities, such as CBHs, EGs, and BGLs for on-site enzyme production, could eliminate the need for enzyme supplementation from other fungal fermentations [21,47]. Similar strategies would include either the heterologous expression of BGLs in efficient CBH producers [48][49][50][51] or the co-cultivation of compatible fungi producing high levels of each enzyme [21,[52][53][54]. These approaches could consolidate enzyme production into a single bioreactor, thus reducing the cost of the necessary equipment.…”

Microbial β-Glucosidases: Screening, Characterization, Cloning and Applications

“…Number of β-glucosidase genes from bacteria, yeast and fungi have been cloned and expressed in E. coli and eukaryotic systems such as Saccharomyces cerevisiae, Pichia pastoris, and T. reesei [168,180,181,182]. Cloning and expression has started with either 1) genomic DNA digestion, construction of genomic DNA library and then functional screening for β-glucosidase [183], or 2) construction of cDNA library and function-based screening of β-glucosidase [169,184].…”

“…After which the gel is incubated in buffered MUG solution and then visualized under UV light using UV transilluminator. Appearance of fluorescent bands indicates the liberation of methylumbelliferone due to enzyme activity [93,154,168,169].…”

Biochemical characterization and synergism of cellulolytic enzyme system from Chaetomium globosum on rice straw saccharification

“…T. reesei cellulase, a well-known cellulase in biomass industry was shown to lack β-glucosidase activity, resulting in strong inhibition of the cellulase by accumulated cellobiose in the reaction. This inhibition can be overcome by addition of external β-glucosidase enzymes from different microbial origins [40]. The synergistic action of T. reesei cellulase with a crude enzyme mixture from A. aculeatus containing various cell wall polysaccharide degrading enzymes with strong downstream cellulolytic and hemicellulolytic activities and a non-catalytic bacterial expansin, acting on physical loosening of the crystalline region of cellulose fibers, on saccharification of alkaline pretreated rice straw was reported [19].…”