Transcriptome Profiling-Based Analysis of Carbohydrate-Active Enzymes in Aspergillus terreus Involved in Plant Biomass Degradation

Abstract

Given the global abundance of plant biomass residues, potential exists in biorefinery-based applications with lignocellulolytic fungi. Frequently isolated from agricultural cellulosic materials, Aspergillus terreus is a fungus efficient in secretion of commercial enzymes such as cellulases, xylanases and phytases. In the context of biomass saccharification, lignocellulolytic enzyme secretion was analyzed in a strain of A. terreus following liquid culture with sugarcane bagasse (SB) (1% w/v) and soybean hulls (SH) (1% w/v) as sole carbon source, in comparison to glucose (G) (1% w/v). Analysis of the fungal secretome revealed a maximum of 1.017 UI.mL^-1 xylanases after growth in minimal medium with SB, and 1.019 UI.mL^-1 after incubation with SH as carbon source. The fungal transcriptome was characterized on SB and SH, with gene expression examined in comparison to equivalent growth on G as carbon source. Over 8000 genes were identified, including numerous encoding enzymes and transcription factors involved in the degradation of the plant cell wall, with significant expression modulation according to carbon source. Eighty-nine carbohydrate-active enzyme (CAZyme)-encoding genes were identified following growth on SB, of which 77 were differentially expressed. These comprised 78% glycoside hydrolases, 8% carbohydrate esterases, 2.5% polysaccharide lyases, and 11.5% auxiliary activities. Analysis of the glycoside hydrolase family revealed significant up-regulation for genes encoding 25 different GH family proteins, with predominance for families GH3, 5, 7, 10, and 43. For SH, from a total of 91 CAZyme-encoding genes, 83 were also significantly up-regulated in comparison to G. These comprised 80% glycoside hydrolases, 7% carbohydrate esterases, 5% polysaccharide lyases, 7% auxiliary activities (AA), and 1% glycosyltransferases. Similarly, within the glycoside hydrolases, significant up-regulation was observed for genes encoding 26 different GH family proteins, with predominance again for families GH3, 5, 10, 31, and 43. A. terreus is a promising species for production of enzymes involved in the degradation of plant biomass. Given that this fungus is also able to produce thermophilic enzymes, this first global analysis of the transcriptome following cultivation on lignocellulosic carbon sources offers considerable potential for the application of candidate genes in biorefinery applications.

Keywords: Aspergillus terreus; biorefinery; carbohydrate-active enzymes; lignocellulosic biomass; transcriptome.

FIGURE 1

Scanning electron microscopy (SEM) analysis of the degradation of lignocellulosic carbon sources following growth of Aspergillus terreus BLU24 in liquid minimal medium supplemented with each carbon source. (A) Non-inoculated pre-treated sugarcane bagasse parenchyma in liquid minimal medium culture. (B) Inoculated pre-treated sugarcane bagasse in liquid minimal medium culture after 36 h incubation, 200X magnification. (C) Inoculated pre-treated sugarcane bagasse in liquid minimal medium culture after 48 h incubation, 450X magnification. (D) Non-inoculated pre-treated soybean hull parenchyma in liquid minimal medium culture. (E) Inoculated pre-treated soybean hull in liquid minimal medium culture after 36 h incubation, 200X magnification. (F) Inoculated pre-treated soybean hull in liquid minimal medium culture after 48 h incubation, 450X magnification. (G) A. terreus BLU24 conidiophore and conidiospore development, 2200X magnification. (H) A. terreus BLU24 conidiospores and hyphae on pre-treated sugarcane bagasse in liquid minimal medium culture after 48 h incubation, 2000X magnification. (I) A. terreus BLU24 conidiospores and hyphae on pre-treated soybean hull in liquid minimal medium culture after 48 h incubation, 2000X magnification.

FIGURE 2

Enzymatic activities of Aspergillus terreus BLU24 following growth in liquid minimal medium supplemented with sugarcane (1% w/v) (A), soybean hull (1% w/v) (B), or glucose (1% w/v) (C), as sole carbon source. Enzyme activities in response to xylan, carboxymethylcellulose, pectin, and filter paper substrate were determined through a DNS assay at pH 5.0.

FIGURE 3

Heatmap depiction of hierarchically clustered groups of Aspergillus terreus BLU24 genes according to gene expression modulation following growth on different carbon sources. Gene expression modulation was compared between the growth treatments SB36 and G36, SB48 and G48, SH36 and G36, and SH48 and G48. Statistically significant differentially expressed genes were considered if a log2 fold change (FC) was at least ≥2-fold and at a probability level of p ≤ 0.01. All FC values below –6 or above 6 were considered as minimum or maximum values, respectively.

FIGURE 4

Volcano plot analysis of differential gene expression in Aspergillus terreus BLU24 according to carbon source and incubation time period. (A) SB36 vs. G36; (B) SB48 vs. G48; (C) SH36 vs. G36; (D) SH48 vs. G48. Genes that were highly modulated, in relation to equivalent treatments with glucose as sole carbon source, appear further to the left and right sides of the plot, with highly significant changes in expression located higher on the plot. Yellow dots: CAZyme-encoding genes (all families); Red dots: genes encoding transcription factors; Green dots: genes encoding permeases; Blue dots: genes encoding oxidoreductases; Gray dots: genes of other function.

FIGURE 5

Barplot representation of genes related to cellulose, hemicellulose and pectin depolymerization in transcriptome data for Aspergillus terreus BLU24 following 36 and 48 hours growth on soybean hull (A) and sugarcane bagasse (B) as sole carbon source.

FIGURE 6

Venn diagram summary of differentially expressed CAZyme-encoding genes for Aspergillus terreus BLU24 among treatments SB36, SB48, SH36, and SH48. Differential gene expression was considered significant if relative expression in comparison to equivalent treatments with glucose as sole carbon source showed a log2 fold change (FC) of at least ≥2-fold, at a probability level of p ≤ 0.01. Overlapping regions of the diagram represent DEGs common to different growth treatments.

FIGURE 7

Representative model of conserved CAZyme families in Aspergillus terreus BLU24 involved in degradation of lignocellulosic biomasses sugarcane bagasse and soybean hull, based on genes with significant increased expression after growth on both carbon sources and at each evaluated time point, in comparison with equivalent growth on glucose.