Synergistic Action of a Lytic Polysaccharide Monooxygenase and a Cellobiohydrolase from Penicillium funiculosum in Cellulose Saccharification under High-Level Substrate Loading

Abstract

Lytic polysaccharide monooxygenases (LPMOs) are crucial industrial enzymes required in the biorefinery industry as well as in the natural carbon cycle. These enzymes, known to catalyze the oxidative cleavage of glycosidic bonds, are produced by numerous bacterial and fungal species to assist in the degradation of cellulosic biomass. In this study, we annotated and performed structural analysis of an uncharacterized LPMO from Penicillium funiculosum (PfLPMO9) based on computational methods in an attempt to understand the behavior of this enzyme in biomass degradation. PfLPMO9 exhibited 75% and 36% sequence identities with LPMOs from Thermoascus aurantiacus (TaLPMO9A) and Lentinus similis (LsLPMO9A), respectively. Furthermore, multiple fungal genetic manipulation tools were employed to simultaneously overexpress LPMO and cellobiohydrolase I (CBH1) in a catabolite-derepressed strain of Penicillium funiculosum, PfMig1⁸⁸ (an engineered variant of P. funiculosum), to improve its saccharification performance toward acid-pretreated wheat straw (PWS) at 20% substrate loading. The resulting transformants showed improved LPMO and CBH1 expression at both the transcriptional and translational levels, with ∼200% and ∼66% increases in ascorbate-induced LPMO and Avicelase activities, respectively. While the secretome of PfMig⁸⁸ overexpressing LPMO or CBH1 increased the saccharification of PWS by 6% or 13%, respectively, over the secretome of PfMig1⁸⁸ at the same protein concentration, the simultaneous overexpression of these two genes led to a 20% increase in saccharification efficiency over that observed with PfMig1⁸⁸, which accounted for 82% saccharification of PWS under 20% substrate loading.IMPORTANCE The enzymatic hydrolysis of cellulosic biomass by cellulases continues to be a significant bottleneck in the development of second-generation biobased industries. While increasing efforts are being made to obtain indigenous cellulases for biomass hydrolysis, the high production cost of this enzyme remains a crucial challenge affecting its wide availability for the efficient utilization of cellulosic materials. This is because it is challenging to obtain an enzymatic cocktail with balanced activity from a single host. This report describes the annotation and structural analysis of an uncharacterized lytic polysaccharide monooxygenase (LPMO) gene in Penicillium funiculosum and its impact on biomass deconstruction upon overexpression in a catabolite-derepressed strain of P. funiculosum Cellobiohydrolase I (CBH1), which is the most important enzyme produced by many cellulolytic fungi for the saccharification of crystalline cellulose, was further overexpressed simultaneously with LPMO. The resulting secretome was analyzed for enhanced LPMO and exocellulase activities and the corresponding improvement in saccharification performance (by ∼20%) under high-level substrate loading using a minimal amount of protein.

Keywords: CBH1; LPMO; Penicillium funiculosum; PfMig188; cellobiohydrolase I; cellulase; fungi; fungus; lytic polysaccharide monooxygenase; saccharification.

FIG 1

Schematic representation of P. funiculosum LPMO (A) and phylogenetic tree of LPMO orthologs in fungi (B). Molecular phylogenetic analysis was performed using the maximum likelihood method and the Jones-Taylor-Thornton (JTT) matrix-based model. The tree with the highest log likelihood (−17,992.93) is shown. Initial trees for the heuristic search were obtained automatically by applying neighbor-joining and BIONJ (BIO neighbor-joining) algorithms to a matrix of pairwise distances estimated using the maximum composite likelihood (MCL) approach and then selecting the topology with a superior log-likelihood value. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The bootstrap support corresponding to the numbers on the tree branches was calculated per 1,000 bootstrap replicates (69). This analysis involved the catalytic domains of 29 LPMOs, most of which had been functionally characterized. Evolutionary analyses were conducted using MEGA X software.

FIG 2

Multiple-sequence alignment of PfLPMO9 with LPMOs in auxiliary activity family 9 (AA9) with known C-1/C-4-oxidizing activity. The proteins included are as follows: NCU07760 (UniProtKB accession no. Q7S111), PaLPMO9A (UniProtKB accession no. B2B629), TaLPMO9A (UniProtKB accession no. G3XAP7), FgLPMO9A (UniProtKB accession no. I1REU9), HjLPMO9B (UniProtKB accession no. O14405), GtLPMO9B (UniProtKB accession no. Q7SA19), and LsLPMO9A (UniProt accession no. A0A0S2GKZ1). Fully conserved residues are shown in white on a red background. Blue triangles indicate residues coordinating the copper ion at the active site. The positions of the four cysteine residues with potential disulfide bridge formation are indicated by green dashed lines. Blue frames indicate that more than 70% of the residues in the corresponding columns exhibit similar physicochemical properties (indicated as red residues on a white background). Black boxes indicate variable regions in the LPMO9 family with the corresponding names L2, L3, LC, and LS, which contribute to shaping the substrate-binding surface (70).

FIG 3

Structural model of PfLPMO9. (A) Best model of the catalytic domain of PfLPMO9, highlighting the three amino acids that make up the copper site. The loop regions that potentially contribute to functional variation among LPMOs, named L2, L3, LS, and LC, are marked with dark blue, light blue, green, and purple, respectively. (B) Ramachandran plot validation of the modeled structure evaluated by PROCHECK. An array of Phi (Φ) and Psi (Ψ) distributions of the nonglycine, nonproline residues is summarized on the plot.

FIG 4

Gluconic acid production in Avicel hydrolysates. The measured gluconic acid concentrations were produced in the absence and presence of 2 mM ascorbic acid (AA). Avicel hydrolysis was carried out with 7 FPU/g of the PfMig1⁸⁸ secretome. The differential gluconic acid [d(Gluconic acid)] concentration represents the actual concentration of gluconic acid produced during the enzymatic hydrolysis of Avicel in the presence of ascorbic acid. This was calculated by subtracting the background level of gluconic acid produced in the reaction mixture without ascorbic acid from the amount of gluconic acid produced in the presence of ascorbic acid.

FIG 5

Construction of the LPMO expression cassette and its overexpression in PfMig1⁸⁸. (A) Schematic diagram showing the assembly of the LPMO cassette from the P. funiculosum NCIM1228 genome. SP, signal peptide. (B) Transformants of pOAO1 after AMTM transformation in PfMig1⁸⁸. Transformants were selected on 100 μg/ml hygromycin. (C) Southern blotting of transformants confirmed by PCR. Lane M is the HindIII lambda DNA size marker used, lane C indicates the genomic DNA of the nontransformed parental strain, and lanes 1 to 5 represent the genomic DNA from transformants with LPMO cassette integration. (D) LPMO activity expressed as micromoles of H₂O₂ released per milliliter per minute and FPase activity in the fermentation broth of PfMig1⁸⁸ and five transformants.

FIG 6

Simultaneous overexpression of LPMO and CBH1 in PfMig1⁸⁸. (A) Schematic diagram showing the construction cassette for the dual overexpression of the LPMO and CBH1 genes from P. funiculosum NCIM1228. Pro, promoter; SP, signal peptide; Ter, terminator. (B) Transformants of pOAO5 after AMTM transformation in PfMig1⁸⁸. Transformants were selected on 100 μg/ml hygromycin. (C) Southern blotting of transformants confirmed by PCR. Lane M is the HindIII lambda DNA size marker used, lane C indicates the genomic DNA of the nontransformed parental strain, and lanes 1 to 5 represent the genomic DNA from transformants with LPMO/CBH1 cassette integration. (D) Enzymatic profile and H₂O₂ production of the overexpressed enzymes in the fermentation broth of PfMig1⁸⁸ and six transformants.

FIG 7

Determination of cellulase expression and activities in P. funiculosum NCIM1228, PfMig1⁸⁸, and all the engineered strains. (A) The transcriptional expression of LPMO, cellobiohydrolase, endoglucanase, β-glucosidase, and xylanase in NCIM1228, PfMig1⁸⁸, and all the engineered strains was measured by quantitative real-time PCR after growing the strains for 48 h in the presence of 4% Avicel. The expression levels were normalized to those in NCIM1228 and plotted. (B) CBH1 activity determined using Avicel as the substrate. (C) H₂O₂ production by PfLPMO expressed as micromoles of H₂O₂ released per milliliter determined using Amplex red. (D) β-Glucosidase activity determined using p-nitrophenyl-β-d-glucoside (pNPG). (E) Overall cellulase activity on filter paper. (F) Endoglucanase activity determined using CMC as the substrate. (G) Xylanase activity measured using beechwood xylan as the substrate. (H) Total secreted proteins in all the strains. The data are presented as the means from three independent experiments, and error bars express the standard deviations.

FIG 8

Time course of the saccharification of nitric acid-pretreated wheat straw by the secretomes of NCIM1228, PfMig1⁸⁸, and all the engineered strains under 20% solid loading and a protein concentration of 30 mg/g biomass. (A) Percent sugar release measured at 24-h intervals over the 96-h saccharification period; (B) total fermentable sugar obtained at the 72-h saccharification time point.