Elimination of calnexin overcomes the secretion bottleneck of Trichoderma reesei β-glucosidase Cel3B which boosts...

The industrial filamentous fungus Trichoderma reesei has a prolific capability in secreting a large quantity of cellulolytic enzymes. The low production of β-glucosidase and presence of only one detectable β-glucosidase Cel3A in T. reesei cellulase cocktail lead to inefficiency in biomass degradation. To date, it is not clear that how the secretion of another β-glucosidase Cel3B, with remarkable catalytic properties, is hindered in T. reesei, which hampers rational strain engineering to enhance β-glucosidase production. Herein, we showed that elimination of the endoplasmic reticulum (ER) chaperone calnexin hardly affected the production of the main cellobiohydrolase or endoglucanase, but specifically promoted the secretion of Cel3B. Analyses of Cel3B expression with a constitutive promoter revealed that Cel3B was restricted from being secreted to extracellular space, while elimination of calnexin significantly increased its secretion level, with extracellular activity increased by ∼40 fold on Avicel cellulose. Notably, further analyses demonstrated that Cel3B protein secreted in the absence of calnexin exhibited a ∼37 % increase in specific activity as compared to that expressed in the parental strain. Moreover, the Cel3B protein had a superior performance in promoting pretreated corncob saccharification compared to Cel3A. These results unveil the regulatory role of calnexin in the coordinated secretion of cellulolytic enzymes in T. reesei, and provide insights into improvement of cellulose saccharification efficiency.