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 k_cat, K_m, and k_cat/K_m 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.

Keywords: CDH; LPMO; Phanerochaete chrysosporium; Trichoderma reesei; cellulase; electron donor.

Figure 1

Proposed reaction mechanisms of CDH as an electron donor of LPMO (Top) represents Type 1 or C1-LPMOs extracts H atom from position 1 and leads to lactone sugars formation. However, the Type 2 or C4-LPMOs break the H atom extracted from position 4 and lead to ketoaldoses formation. (Bottom) represents the electron donation from CDH which reduces Cu (II) of LPMO to Cu(I) and then binds to O₂. Copper superoxo intermediate results due to this internal transfer of electron and it can abstract the hydrogen ion from position 1 or 4 of the carbohydrate. Cu-bound hydroperoxide homolytic cleavage results due to a second electron from CDH. The substrate hydroxylation occurs when the substrate radical couples with copper oxo species (Cu−O•). The glycosidic bond is destabilized due to the addition of an oxygen atom and adjacent glucans are eliminated [8].

Figure 2

SDS-PAGE analysis of recombinant PcCDH protein. (A) SDS-PAGE analysis of PcCDH. M: molecular marker, 1: culture supernatant of recombinant PcCDH, 2: purified recombinant PcCDH, 3: repeat of 2. (B) Identification of the 80 kDa band in the PcCDH electrophoresis photograph; and (C) Identification of the 100 kDa band in the PcCDH electrophoresis photograph. The red peptides are identified using MS through molecular weight match.

Figure 3

In spectral analysis of PcCDH, the blue line represents oxidized while the red line represents reduced UV/Vis spectra of PcCDH. The inset shows the Magnified spectral range (500–600 nm) of heme b α- and β-peak.

Figure 4

Basic catalytic properties of PcCDH (A) Optimum pH of PcCDH. (B) Optimum reaction temperature and thermal stability of PcCDH at 40 °C and 50 °C. To analyze the effect of pH on enzyme activity, Britton-Robison buffer at pH 2.0–11.0 was used to adjust the pH. The reaction system contained 100 mM BR buffer, 500 μM cellobiose, 0.3 mM DCIP, 0.3 μM PcCDH. Error bars indicate the standard deviation (n = 3; independent experiments).

Figure 5

Substrate specificity of PcCDH. The catalytic activity of PcCDH on different substrates was analyzed through the Amplex red-horseradish method to measure the H₂O₂ production. The reaction mixture contained 100 mM phosphate buffer (pH 7.0), 50 μM Amplex Red, 2 μL of 40 U/mL HRP, 0.3 μM PcCDH and different substrates. Error bars indicate the standard deviation from three independent experiments.

Figure 6

PcCDH-LPMO lysis products. The blank bar shows the detection of PASC lysis products using LPMO and CDH. The black-colored bar in the graph shows the number of lysis products of PASC using LPMO, CDH, and cellobiose. Lysis products were assayed by the addition of extra cellobiose or cello-triose in the presence of PASC as substrate. Error bars indicate the standard deviation from three independent experiments.

Figure 7

Synergistic effect of PcCDH, LPMO, and cellulase. PASC degradation by cellulase alone or in different combinations with LPMO and PcCDH (cellulase, cellulase + LPMO, cellulase + PcCDH + cellobiose, and cellulase + LPMO + PcCDH + cellobiose) was measured through turbidimetry method. Initially, the effect of cellulase alone or in combinations had no obvious difference, however, with time cellulase alone or its combination (cellulase + LPMO + PcCDH) produced the best results at 12-24 h. Afterward, cellulase showed no further increment but its combination with LPMO and PcCDH still resulted in maximum degradation of PASC at 72 h. Error bars indicate the standard deviation (n = 3; independent experiments).

Figure 8

LPMO activity and its deactivation under various electron donors. PcCDH results in delayed autoxidative deactivation of LPMO and prolongs its enzymatic activity as compared to Asc when it is used alone. However, Asc when used alone or in combination with PcCDH increases the oxidative deactivation of LPMO.

Figure 9

LPMO and PcCDH product analysis through MALDI-TOF-MS (a) Products produced by LPMO when the substrate is PASC. (b) Product produced by LPMO when the substrate is PASC with cellobiose. (c) Magnified DP6 peaks of (b).