Insights into the Lignocellulose-Degrading Enzyme System of Humicola grisea var. thermoidea Based on Genome and Transcriptome Analysis

Abstract

Humicola grisea var. thermoidea is a thermophilic ascomycete and important enzyme producer that has an efficient enzymatic system with a broad spectrum of thermostable carbohydrate-active (CAZy) enzymes. These enzymes can be employed in lignocellulose biomass deconstruction and other industrial applications. In this work, the genome of H. grisea var. thermoidea was sequenced. The acquired sequence reads were assembled into a total length of 28.75 Mbp. Genome features correlate with what was expected for thermophilic Sordariomycetes. The transcriptomic data showed that sugarcane bagasse significantly upregulated genes related to primary metabolism and polysaccharide deconstruction, especially hydrolases, at both pH 5 and pH 8. However, a number of exclusive and shared genes between the pH values were found, especially at pH 8. H. grisea expresses an average of 211 CAZy enzymes (CAZymes), which are capable of acting in different substrates. The top upregulated genes at both pH values represent CAZyme-encoding genes from different classes, including acetylxylan esterase, endo-1,4-β-mannosidase, exoglucanase, and endoglucanase genes. For the first time, the arsenal that the thermophilic fungus H. grisea var. thermoidea possesses to degrade the lignocellulosic biomass is shown. Carbon source and pH are of pivotal importance in regulating gene expression in this organism, and alkaline pH is a key regulatory factor for sugarcane bagasse hydrolysis. This work paves the way for the genetic manipulation and robust biotechnological applications of this fungus. IMPORTANCE Most studies regarding the use of fungi as enzyme producers for biomass deconstruction have focused on mesophile species, whereas the potential of thermophiles has been evaluated less. This study revealed, through genome and transcriptome analyses, the genetic repertoire of the biotechnological relevant thermophile fungus Humicola grisea. Comparative genomics helped us to further understand the biology and biotechnological potential of H. grisea. The results demonstrate that this fungus possesses an arsenal of carbohydrate-active (CAZy) enzymes to degrade the lignocellulosic biomass. Indeed, it expresses more than 200 genes encoding CAZy enzymes when cultivated in sugarcane bagasse. Carbon source and pH are key factors for regulating the gene expression in this organism. This work shows, for the first time, the great potential of H. grisea as an enzyme producer and a gene donor for biotechnological applications and provides the base for the genetic manipulation and robust biotechnological applications of this fungus.

Keywords: CAZy enzymes; Humicola grisea; genome sequencing; pH regulation; sugarcane bagasse; transcriptome.

FIG 1

Genome features of 13 Ascomycetes genomes. (A) The RAxML maximum-likelihood phylogenetic tree using 200 single-copy genes shows the three orders Sordariales, Hypocreales, and Eurotiales. All nodes have maximum support value. Thermophile genomes are shown in red. (B) Genome size and percentage of GC of each genome. (C) Gene count and percentage of genes with the presence of at least one Pfam domain. (D) Distribution of CAZyme families among the genomes. CAZymes: auxiliary activities (AA), carbohydrate-binding module (CBM), carbohydrate esterases (CE), glycoside hydrolases (GH), glycosyl transferases (GT), and polysaccharide lyases (PL).

FIG 2

Distribution of gene expression of H. grisea grown in sugarcane bagasse normalized with growth on glucose as the sole carbon source. The two pH values, pH 5 (A) and pH 8 (B), exhibited differentially expressed genes (P value adjusted of <0.05) showed in red when normalized with glucose.

FIG 3

Differentially expressed genes at pH 5, pH 8, or both values. (A) Scatterplot of differentially expressed genes at pH 5 or pH 8. The colors denote genes expressed at pH 5 (red), pH 8 (green), and both conditions (blue). The Venn diagram shows the number of exclusive and shared genes between the pH values. (B) Fisher enrichment analysis of GO terms on each condition compared to the whole genome as background. No category exclusively enriched at pH 5 was found.

FIG 4

Expression of putative genes encoding glycoside hydrolases (GH) separated by the likely substrate. The asterisk (*) means the gene is potentially regulated by the PacC regulator.