Genomic and transcriptomic analysis of the thermophilic lignocellulose-degrading fungus Thielavia terrestris LPH172

Abstract

Background: Biomass-degrading enzymes with improved activity and stability can increase substrate saccharification and make biorefineries economically feasible. Filamentous fungi are a rich source of carbohydrate-active enzymes (CAZymes) for biomass degradation. The newly isolated LPH172 strain of the thermophilic Ascomycete Thielavia terrestris has been shown to possess high xylanase and cellulase activities and tolerate low pH and high temperatures. Here, we aimed to illuminate the lignocellulose-degrading machinery and novel carbohydrate-active enzymes in LPH172 in detail.

Results: We sequenced and analyzed the 36.6-Mb genome and transcriptome of LPH172 during growth on glucose, cellulose, rice straw, and beechwood xylan. 10,128 predicted genes were found in total, which included 411 CAZy domains. Compared to other fungi, auxiliary activity (AA) domains were particularly enriched. A higher GC content was found in coding sequences compared to the overall genome, as well as a high GC3 content, which is hypothesized to contribute to thermophilicity. Primarily auxiliary activity (AA) family 9 lytic polysaccharide monooxygenase (LPMO) and glycoside hydrolase (GH) family 7 glucanase encoding genes were upregulated when LPH172 was cultivated on cellulosic substrates. Conventional hemicellulose encoding genes (GH10, GH11 and various CEs), as well as AA9 LPMOs, were upregulated when LPH172 was cultivated on xylan. The observed co-expression and co-upregulation of genes encoding AA9 LPMOs, other AA CAZymes, and (hemi)cellulases point to a complex and nuanced degradation strategy.

Conclusions: Our analysis of the genome and transcriptome of T. terrestris LPH172 elucidates the enzyme arsenal that the fungus uses to degrade lignocellulosic substrates. The study provides the basis for future characterization of potential new enzymes for industrial biomass saccharification.

Keywords: Biomass degradation; Carbohydrate active enzymes; Cellulose; Filamentous fungi; LPMO; Thermostable enzymes; Transcriptome; Xylan.

Fig. 1

Growth of T. terrestris LPH172 and other biomass-degrading filamentous fungi on different carbon sources. Seven different carbohydrate substrates at 2% (w/v) were used as sole carbon sources for growth on agar plates: monosaccharides (glucose, xylose), disaccharides (cellobiose), and polysaccharides (starch, Avicel, carboxymethyl cellulose—CMC, beechwood xylan). No carbon source was added in the control. The plates were incubated at 30 °C (S. commune, A. oryzae) or 50 °C (M. thermophila, M. cinnamomea, T. terrestris) for 2–7 days. Growth was evaluated visually and categorized from − (no growth) to +++ (very good growth), depending on the extent and density of the mycelium

Fig. 2

Relative numbers of CAZy domains from six CAZy families in various filamentous fungi. For each species, the numbers of predicted CAZyme domains were normalized to the total number of predicted CAZyme domains. GH, glycoside hydrolase; GT, glycoside transferase; AA, auxiliary activity; CE, carbohydrate esterase; PL, polysaccharide lyase; CBM, carbohydrate-binding module. Predictions were made with dbCAN2 (HMMER algorithm). CE10 family domains were excluded

Fig. 3

Number of auxiliary activity (AA) CAZyme family domains in different filamentous fungi. For each species, the number of predicted AA domains categorized into AA families 1–16 is shown. Predictions were made with dbCAN2 (HMMER algorithm)

Fig. 4

Combined expression and upregulation values of CAZyme-encoding genes during cultivation on four different substrates. Putative CAZyme-encoding genes involved in biomass degradation were analyzed for their expression levels (TPM, transcripts per million) from three biological replicates, as well as their differential expression (log2FC). The heatmap shows a combination of top forty most highly upregulated CAZyme-encoding genes on three substrates Avicel, rice straw (RS), beechwood xylan (BX) when compared to glucose (Glc). Shading ranges from low expression (light blue) to high expression (magenta). Log2 fold-change (log2FC) shows gene expression during cultivation on Avicel, RS, and BX compared to cultivation on glucose. Shading of upregulated genes (i.e., gene transcripts more abundant on Avicel, RS, and/or BX than on glucose) ranges from light yellow (low upregulation) to dark green (high upregulation). Downregulated genes or genes for which no differential expression was detected or where upregulation was not significant are indicated by blank cells. Only significantly upregulated genes are shown (p ≤ 0.05). All numbers were rounded to the nearest integer. The putative activities of the gene products are based on BLASTp predictions. CAZy domains were analyzed with dbCAN2. The presence of putative signal peptides (SP), predicted by SignalP 4.0, is indicated by a small s. Putative substrates of the CAZymes are: C, cellulose; Ch, chitin; GM, glucomannan; L, lignin; P, pectin; S, starch; X, xylan; XG, xyloglucan