Gene Co-expression Network Reveals Potential New Genes Related to Sugarcane Bagasse Degradation in Trichoderma reesei RUT-30

Abstract

The biomass-degrading fungus Trichoderma reesei has been considered a model for cellulose degradation, and it is the primary source of the industrial enzymatic cocktails used in second-generation (2G) ethanol production. However, although various studies and advances have been conducted to understand the cellulolytic system and the transcriptional regulation of T. reesei, the whole set of genes related to lignocellulose degradation has not been completely elucidated. In this study, we inferred a weighted gene co-expression network analysis based on the transcriptome dataset of the T. reesei RUT-C30 strain aiming to identify new target genes involved in sugarcane bagasse breakdown. In total, ~70% of all the differentially expressed genes were found in 28 highly connected gene modules. Several cellulases, sugar transporters, and hypothetical proteins coding genes upregulated in bagasse were grouped into the same modules. Among them, a single module contained the most representative core of cellulolytic enzymes (cellobiohydrolase, endoglucanase, β-glucosidase, and lytic polysaccharide monooxygenase). In addition, functional analysis using Gene Ontology (GO) revealed various classes of hydrolytic activity, cellulase activity, carbohydrate binding and cation:sugar symporter activity enriched in these modules. Several modules also showed GO enrichment for transcription factor activity, indicating the presence of transcriptional regulators along with the genes involved in cellulose breakdown and sugar transport as well as other genes encoding proteins with unknown functions. Highly connected genes (hubs) were also identified within each module, such as predicted transcription factors and genes encoding hypothetical proteins. In addition, various hubs contained at least one DNA binding site for the master activator Xyr1 according to our in silico analysis. The prediction of Xyr1 binding sites and the co-expression with genes encoding carbohydrate active enzymes and sugar transporters suggest a putative role of these hubs in bagasse cell wall deconstruction. Our results demonstrate a vast range of new promising targets that merit additional studies to improve the cellulolytic potential of T. reesei strains and to decrease the production costs of 2G ethanol.

Weighted Gene Co-expression Network Analysis Identifies Critical Genes for the Production of Cellulase and Xylanase in Penicillium oxalicum

“…Transcription of POX01960/ClrB was higher than that of POX05324/XlnR during the whole culture time on each carbon source ( Figure 12B), suggesting that POX01960/ClrB plays a much greater role in the regulation of cellulase and xylanaseencoding gene expression in P. oxalicum than POX05324/XlnR. This finding is in contrast to Xyr1 and XlnR in T. reesei (Mach-Aigner et al, 2008;Borin et al, 2018), which was confirmed through the previous genetic analysis (Li et al, 2015;Zhao et al, 2016).…”

“…They were divided into seven modules, MEivory, MEmidnightblue, MEorange, MEroyalblue, MEturquoise, MEgrey and MElightcyan1. WGCNA analysis of T. reesei grown on sugarcane bagasse identified 28 highly connected gene modules and one of these modules contained the most representative core of cellulolytic enzymes, with their regulators and transporters (Borin et al, 2018).…”

“…Most of them encoded proteins that were CAZymes, including the essential cellulases (CBH POX05587/Cel7A-2; EGs POX01166/Cel5B, POX05571/Cel7B, POX05570/Cel45A and POX01896/Cel5C; BGLs POX01646, POX03062, POX06079, and POX03641; Auxiliary activity POX08897/AA9), and their regulators such as POX01960/ClrB and POX02071/ClrB-2. It is not surprising that these cellulase genes and their regulatory genes in filamentous fungi including P. oxalicum, T. reesei, and Neurospora crassa, are clustered together because they are co-induced in response to most plant biomass substrates such as WB, sugarcane bagasse and Avicel (Craig et al, 2015;Yan et al, 2017;Borin et al, 2018). Under fungal growth conditions, in the presence of cellulose, cellulose is first digested into cellodextrins by constitutive levels of extracellular CBHs and EGs.…”

CRZ1 regulator and calcium cooperatively modulate holocellulases gene expression in Trichoderma reesei QM6a

“…Furthermore, we were able to observe that Ca 2+ seems to be critical for the expression of many holocellulases, Ca 2+ and sugar transporter coding genes more directly than the carbon source whose fungal growth was previously established. Albeit the effect of SCB on enzymes expression, Ca 2+ and sugar transporter proteins had already been reported in a T. reesei RUT-C30 gene co-expression network study by Borin et al [5], here we firstly described the seminal importance of Ca 2+ on the expression of sugar transporters in a CRZ1-dependent context in T. reesei QM6a strain, hypothesizing that under stress conditions T. reesei QM6a may increase the transcription of sugar transporters to keep metabolic homeostasis through Ca 2+ -CRZ1/Calcineurin pathway.…”

“…Despite the great potential of this fungus for high performance cellulase and hemicellulase production, the regulatory network controlling this system is a relevant field of study and not fully understood yet. Additionally, the comprehension of transcriptional networks which coordinate the transcription of holocellulases is one of the main focus to allow enzyme production in a viable at industrial scale [5].…”