Integrative Analysis of Selected Metabolites and the Fungal Transcriptome during the Developmental Cycle of Ganoderma lucidum Strain G0119 Correlates Lignocellulose Degradation with Carbohydrate and Triterpenoid Metabolism

Abstract

To systemically understand the biosynthetic pathways of bioactive substances, including triterpenoids and polysaccharides, in Ganoderma lucidum, the correlation between substrate degradation and carbohydrate and triterpenoid metabolism during growth was analyzed by combining changes in metabolite content and changes in related enzyme expression in G. lucidum over 5 growth phases. Changes in low-polarity triterpenoid content were correlated with changes in glucose and mannitol contents in fruiting bodies. Additionally, changes in medium-polarity triterpenoid content were correlated with changes in the lignocellulose content of the substrate and with the glucose, trehalose, and mannitol contents of fruiting bodies. Weighted gene coexpression network analysis (WGCNA) indicated that changes in trehalose and polyol contents were related to carbohydrate catabolism and polysaccharide synthesis. Changes in triterpenoid content were related to expression of the carbohydrate catabolic enzymes laccase, cellulase, hemicellulase, and polysaccharide synthase and to the expression of several cytochrome P450 monooxygenases (CYPs). It was concluded that the products of cellulose and hemicellulose degradation participate in polyol, trehalose, and polysaccharide synthesis during initial fruiting body formation. These carbohydrates accumulate in the early phase of fruiting body formation and are utilized when the fruiting bodies mature and a large number of spores are ejected. An increase in carbohydrate metabolism provides additional precursors for the synthesis of triterpenoids. IMPORTANCE Most studies of G. lucidum have focused on its medicinal function and on the mechanism of its activity, whereas the physiological metabolism and synthesis of bioactive substances during the growth of this species have been less studied. Therefore, theoretical guidance for cultivation methods to increase the production of bioactive compounds remains lacking. This study integrated changes in the lignocellulose, carbohydrate, and triterpenoid contents of G. lucidum with enzyme expression from transcriptomics data using WGCNA. The findings helped us better understand the connections between substrate utilization and the synthesis of polysaccharides and triterpenoids during the cultivation cycle of G. lucidum. The results of WGCNA suggest that the synthesis of triterpenoids can be enhanced not only through regulating the expression of enzymes in the triterpenoid pathway, but also through regulating carbohydrate metabolism and substrate degradation. This study provides a potential approach and identifies enzymes that can be targeted to regulate lignocellulose degradation and accelerate the accumulation of bioactive substances by regulating substrate degradation in G. lucidum.

Keywords: Ganoderma lucidum; carbohydrate; lignocellulose; triterpenoid.

FIG 1

Morphology of G. lucidum G0119 at 5 growth phases (P1 to P5). P1, fully grown mycelium (1 month); P2, primordium (2 months); P3, young fruiting bodies (2.5 months); P4, mature fruiting bodies (3.5 months); P5, fully ejected spores (4.5 months).

FIG 2

Pearson correlation coefficients for changes in the lignocellulose, carbohydrate, and triterpenoid contents of G. lucidum during five phases of growth. Red dots indicate positive correlations, and blue dots indicate negative correlations. White outlines indicate regions of positive and negative correlation. Abbreviations: U, upper layer of the substrate; M, middle layer of the substrate; L, lower layer of the substrate; P, pileus; S, stipe; B, base; Lig, lignin; Cel, cellulose; Hemi, hemicellulose; Glc, glucose; Ara, arabinitol; Man, mannitol; Tre, trehalose; Pol, polysaccharide; GaC2, ganoderic acid C2; GraB, ganoderenic acid B; GaB, ganoderic acid B; GaA, ganoderic acid A; GaD, ganoderic acid D; GaF, ganoderic acid F; Gmt, ganodermanontriol; GaDM, ganoderic acid DM; GaS, ganoderic acid S; GaT, ganoderic acid T; GlB, ganoderiol B.

FIG 3

Changes in the expression of lignocellulolytic enzymes in G. lucidum during the 5 phases of its growth (P1 to P5). Values that differ significantly (fold change greater than 2 and P < 0.05) are marked with black asterisks. Areas outlined in green indicate that the expression of lignin-modifying enzymes and hemicellulase decreased by more than 2-fold from P1 to P2. Areas outlined in red indicate that the expression of cellulase was upregulated by more than 2-fold from P2 to P3. From P4 to P5, most cellulases, several lignin-modifying enzymes, and hemicellulases were upregulated by more than 2-fold.

FIG 4

Changes in the expression of genes involved in carbohydrate catabolism in G. lucidum during the 5 phases of its growth (P1 to P5). Values that differ significantly (fold change greater than 2 and P < 0.05) are marked with black asterisks. Areas outlined in red indicate that the expression levels of EMP, HMP, and TCA pathway enzymes, as well as those of arabinitol, mannitol, trehalose, and polysaccharide metabolic enzymes, increased by more than 2-fold from P1 to P2 and from P2 to P3. Areas outlined in green indicate that the expression levels of EMP, HMP, and TCA pathway enzymes, as well as those of arabinitol, mannitol, trehalose, and polysaccharide metabolic enzymes, decreased by more than 2-fold from P4 to P5.

FIG 5

Changes in the expression of genes involved in triterpenoid and sterol metabolism in G. lucidum during the 5 phases of its growth (P1 to P5). Values that differ significantly (fold change greater than 2 and P < 0.05) are marked with black asterisks. Areas outlined in red indicate that the expression levels of ACAT, HMGS, HMGR, CYP505, MVD, IDI, SE, and LS peaked at P2 and P5.

FIG 6

WGCNA changes in gene expression and content of lignocellulose, carbohydrates, and triterpenoids in G. lucidum. (A) Dendrogram of modules indicated with different colors. (B) Module-trait relationships between content and gene expression changes. Areas outlined in black indicate metabolites that were most positively and negatively correlated with genes in the corresponding modules. Correlation with P values lower than 0.05 are indicated by black asterisks. Abbreviations: U, upper layer of the substrate; M, middle layer of the substrate; L, lower layer of the substrate; P pileus; S, stipe; B, base; Lig, lignin; Cel, cellulose; Hemi, hemicellulose; Glc, glucose; Ara, arabinitol; Man, mannitol; Tre, trehalose; Pol, polysaccharide; GaC2, ganoderic acid C2; GraB, ganoderenic acid B; GaB, ganoderic acid B; GaA, ganoderic acid A; GaD, ganoderic acid D; GaF, ganoderic acid F; Gmt, ganodermanontriol; GaDM, ganoderic acid DM; GaS, ganoderic acid S; GaT, ganoderic acid T; GlB, ganoderiol B. (C) Hub genes and changes in their expression in each module are correlated with content changes.

FIG 7

Potential correlation and changes in lignocellulose, carbohydrate, and triterpenoid levels during the growth of G. lucidum. In phases P1 and P2, lignocellulose is degraded into glucose and xylose, participates in carbohydrate catabolism, and is used to synthesize other carbohydrates, triggering the metabolism of triterpenoids. During P2 and P3, carbohydrate metabolism increases, and trehalose, glycogen, and polyol accumulate at high levels as the degradation of cellulose and hemicellulose increases. These changes provide more precursors for triterpenoid synthesis and increase the levels of medium-polarity triterpenoids. During P4 and P5, the production of many spores increases energy costs and generates a requirement for structural carbohydrate. Carbohydrate storage switches from anabolism to catabolism and, together with cellulose degradation, aids spore production. High levels of carbohydrate catabolism supply more precursors and increase the low-polarity triterpenoid content.