Boosting the cellulolytic oxidative and hydrolytic enzyme systems in Myceliophthora thermophila for the efficient enzymatic saccharification of lignocellulosic biomass.

The thermophilic fungus Myceliophthora thermophila serves as a vital platform for producing cellulolytic complex enzymes. However, their efficiency still requires enhancement to meet the cost-effective demands of lignocellulosic biomass conversion. Herein, secretome analysis revealed that the cellulolytic enzyme system of M. thermophila comprises the oxidative system consisting of lytic polysaccharide monooxygenases (LPMOs) and the hydrolytic system that includes endoglucanase, cellobiohydrolase, and β-glucosidase. Both in vitro supplementation and in vivo overexpression of MtLPMOs with C1 or C1/C4 oxidizing activity enhanced the enzymatic saccharification of Avicel using M. thermophila fermentation broth, resulting in a maximum increase of 485% in oxidized cello-oligosaccharides production. Furthermore, the simultaneous enhancement of LPMO and β-glucosidase expression in M. thermophila significantly improved cellulose depolymerization and lignocellulosic biomass degradation. Total production of native and oxidized cello-oligosaccharides from pretreated corncob residue increased from 5.55 to 0.27 mg/mL in the wild-type strain to 8.72 mg/mL and 0.61 mg/mL in the engineered strain. Taken together, these findings highlight the synergistic interaction between the oxidative and hydrolytic enzyme systems for efficient saccharification of lignocellulosic biomass, providing valuable insights for enhancing the performance of commercial cellulolytic enzyme products.