Heterologously Expressed Cellobiose Dehydrogenase Acts as Efficient Electron-Donor of Lytic Polysaccharide Monooxygenase for Cellulose Degradation in Trichoderma reesei

Abstract

The conversion of lignocellulosic biomass to second-generation biofuels through enzymes is achieved at a high cost. Filamentous fungi through a combination of oxidative enzymes can easily disintegrate the glycosidic bonds of cellulose. The combination of cellobiose dehydrogenase (CDH) with lytic polysaccharide monooxygenases (LPMOs) enhances cellulose degradation in many folds. CDH increases cellulose deconstruction via coupling the oxidation of cellobiose to the reductive activation of LPMOs by catalyzing the addition of oxygen to C-H bonds of the glycosidic linkages. Fungal LPMOs show different regio-selectivity (C1 or C4) and result in oxidized products through modifications at reducing as well as nonreducing ends of the respective glucan chain. T. reesei LPMOs have shown great potential for oxidative cleavage of cellobiose at C1 and C4 glucan bonds, therefore, the incorporation of heterologous CDH further increases its potential for biofuel production for industrial purposes at a reduced cost. We introduced CDH of Phanerochaete chrysosporium (PcCDH) in Trichoderma reesei (which originally lacked CDH). We purified CDH through affinity chromatography and analyzed its enzymatic activity, electron-donating ability to LPMO, and the synergistic effect of LPMO and CDH on cellulose deconstruction. The optimum temperature of the recombinant PcCDH was found to be 45 °C and the optimum pH of PcCDH was observed as 4.5. PcCDH has high cello-oligosaccharide kcat, Km, and kcat/Km values. The synergistic effect of LPMO and cellulase significantly improved the degradation efficiency of phosphoric acid swollen cellulose (PASC) when CDH was used as the electron donor. We also found that LPMO undergoes auto-oxidative inactivation, and when PcCDH is used an electron donor has the function of a C1-type LPMO electron donor without additional substrate increments. This work provides novel insights into finding stable electron donors for LPMOs and paves the way forward in discovering efficient CDHs for enhanced cellulose degradation.