Independent metabolism of oligosaccharides is the keystone of synchronous utilization of cellulose and hemicellulose in Myceliophthora

Abstract

The effective utilization of cellulose and hemicellulose, the main components of plant biomass, is a key technical obstacle that needs to be overcome for the economic viability of lignocellulosic biorefineries. Here, we firstly demonstrated that the thermophilic cellulolytic fungus Myceliophthora thermophila can simultaneously utilize cellulose and hemicellulose, as evidenced by the independent uptake and intracellular metabolism of cellodextrin and xylodextrin. When plant biomass serviced as carbon source, we detected the cellodextrin and xylodextrin both in cells and in the culture medium, as well as high enzyme activities related to extracellular oligosaccharide formation and intracellular oligosaccharide hydrolysis. Sugar consumption assay revealed that in contrast to inhibitory effect of glucose on xylose and cellodextrin/xylodextrin consumption in mixed-carbon media, cellodextrin and xylodextrin were synchronously utilized in this fungus. Transcriptomic analysis also indicated simultaneous induction of the genes involved in cellodextrin and xylodextrin metabolic pathway, suggesting carbon catabolite repression (CCR) is triggered by extracellular glucose and can be eliminated by the intracellular hydrolysis and metabolism of oligosaccharides. The xylodextrin transporter MtCDT-2 was observed to preferentially transport xylobiose and tolerate high cellobiose concentrations, which helps to bypass the inhibition of xylobiose uptake. Furthermore, the expression of cellulase and hemicellulase genes was independently induced by their corresponding inducers, which enabled this strain to synchronously utilize cellulose and hemicellulose. Taken together, the data presented herein will further elucidate the degradation of plant biomass by fungi, with implications for the development of consolidated bioprocessing-based lignocellulosic biorefinery.

Keywords: Myceliophthora thermophila; carbon catabolite repression; cellulose and hemicellulose; fungal biotechnology; independent utilization.

Fig. 1.

Synchronous utilization of cellulose and hemicellulose in M. thermophila. A) Utilization of cellulose and hemicellulose by M. thermophila during cultivation on 2% corncob substrate. B) Residual sugar content in the medium when M. thermophila was cultured using G1 (0.5% glucose), X1 (0.5% xylose), or GX (0.5% glucose and 0.5% xylose) as the carbon sources. C) and D) Analysis of extracellular oligosaccharide content detected by HPAEC-PAD chromatograms in M. thermophila cultures using 2% corncob and AX (1% Avicel and 1% xylan) as the carbon sources, respectively. E) Residual sugar content in the medium when M. thermophila was cultured using 0.5% cellobiose (G2), 0.5% xylobiose (X2), or G2X2 (0.5% cellobiose and 0.5% xylobiose) as the carbon sources. F) The consumption rate of sugar when M. thermophila was cultured on G2, X2, or G2X2. G) Hydrolytic activity of M. thermophila culture filtrates. Culture and induction conditions are detailed in the Materials and methods section. For assays, treated culture filtrates (50 μg protein) were added to either 2% Avicel or 2% xylan, followed by incubation at 45 °C for 24 h. Subsequent analysis focused on monosaccharides and disaccharides, specifically targeting glucose and cellobiose for 2% Avicel, and xylose and xylobiose for 2% xylan. The values and error bars represent means and SD of independent triplicate experiments, respectively. *** represents significant difference at P < 0.001. ns, no statistical significance; plas, plasma membrane-bound; cyto, cytoplasm; extr, extracellular; G1, glucose; G2, cellobiose; G3, cellotriose; X1, xylose; X2, xylobiose; X3, xylotriose; X4, xylotetraose.

Fig. 2.

The intracellular activities of BGL and BXL play a dominant role in the hydrolysis of oligosaccharides. A) Intracellular oligosaccharide content detected by HPAEC-PAD chromatogram in M. thermophila cultured with 2% corncob or AX (1% Avicel and 1% xylan). B) Extracellular and intracellular activities of BGL and BXL in M. thermophila cultured with 2% Avicel, 2% xylan, or AX (1% Avicel and 1% xylan) as the carbon sources. The values and error bars represent means and SD of independent triplicate experiments, respectively. ns, no statistical significance. ** and *** represent significant difference at P < 0.01 and P < 0.001, respectively.

Fig. 3.

Uptake and intracellular hydrolysis of cellodextrin and xylodextrin derived from plant biomass. A) Hierarchical cluster analysis of the expression of BGL, BXL, and sugar transporter (STP) genes in M. thermophila after the 4 h induction with no carbon (NC), Avicel, xylan, or AX (Avicel and xylan). Intracellular B) and extracellular C) BXL activity in the M. thermophila wild-type (WT) strain and its mutant strains following 1 day of incubation in 2% xylan medium. D) Intracellular monosaccharide and oligosaccharide contents in WT, Δbxl1Δbxl2, and ΔMtcdt-2 strains grown on xylan for 2 days. E) Dry cell weight of the WT, Δbxl1Δbxl2, and ΔMtcdt-2 strains grown on xylan for 1 day and on Avicel for 3 days. F) Xylobiose consumption of the WT, Δbxl1Δbxl2, and ΔMtcdt-2 strains. G) Protein concentration and endo-xylanase activity in the supernatant of the WT, Δbxl1Δbxl2, and ΔMtcdt-2 strains after the 2 days culture on xylan. plas, plasma membrane-bound; cyto, cytoplasm; extr, extracellular. The values and error bars represent means and SD of independent triplicate experiments, respectively. ns, no statistical significance. *, **, and *** represent significant difference at P < 0.05, P < 0.01, and P < 0.001, respectively.

Fig. 4.

Intracellular hydrolysis and metabolism of cellodextrin and xylodextrin eliminates carbon catabolite repression. A) The relative expression levels of bxl1, bxl2, xr, xdh, and xks in M. thermophila on GX (glucose and xylose), GX2 (glucose and xylobiose), or G2X2 (cellobiose and xylobiose), compared with those under no carbon condition. B) The relative expression levels of oligosaccharide hydrolase and xylose catabolism genes on different carbon sources, compared to those under starvation condition. The relative expression levels of Mycth_96047 C) and Mtcdt-2 D) in M. thermophila grown on G1 (glucose), X1 (xylose), GX, G2 (cellobiose), X2 (xylobiose), G2X2, G2X (cellobiose and xylose), GX2, Avicel, xylan, and AX, compared to the starvation condition. E) Confocal fluorescence microscopy images of S. cerevisiae strain EBY.VW4000 cells harboring EGFP and MtCDT-2-EGFP. F) Sugar uptake rates of xylobiose (X2) and cellobiose (G2) in the strain EBY.VW4000 with MtCDT-2, When G2, X2, or a mixed sugar composition of G2 and X2 (in ratios of 1:1, 2:1, and 10:1) were used as the substrates. The values and error bars represent means and SD of independent triplicate experiments, respectively. ns, no statistical significance. *, **, and *** represent significant difference at P < 0.05, P < 0.01, and P < 0.001, respectively.

Fig. 5.

Independent induction of hemicellulolytic and cellulolytic enzyme-encoding genes in M. thermophila. A) Hierarchical cluster analysis of the expression of cellulase and hemicellulase genes in M. thermophila after the 4 h induction with NC (no carbon), G2 (cellobiose), X2 (xylobiose), or G2X2 (cellobiose and xylobiose). B) Hierarchical cluster analysis of the expression of cellulase and hemicellulase in M. thermophila after the 4 h induction with NC, Avicel, xylan, or AX (Avicel and xylan). C) Analysis of the correlation in cellulase and hemicellulase gene expression across different carbon sources. Spearman's rank correlation test was performed to analyze correlations; *P < 0.05. D) Venn diagram showing hemicellulase and cellulase genes with up-regulated expression in M. thermophila cultured on G2, X2, or G2X2, compared to the NC condition. E) Relative expression of xylan degradation-related genes under single sugar (X2 and X1) and mixed sugar (G2X2, GX2, and GX) conditions, compared to NC condition. X2, xylobiose; X1, xylose; G2X2, mixed cellobiose and xylobiose; GX2, mixed glucose and xylobiose; GX, mixed glucose and xylose. EGL, endoglucanase; EXGL, exoglucanase; CDH, cellobiose dehydrogenase; LPMO, lytic polysaccharide monooxygenase; BGL, β-glucosidase; XLN, xylanase; BXL, β-xylosidase.