Trichoderma reesei CRE1-mediated Carbon Catabolite Repression in Re-sponse to Sophorose Through RNA Sequencing Analysis

Abstract

Carbon catabolite repression (CCR) mediated by CRE1 in Trichoderma reesei emerged as a mechanism by which the fungus could adapt to new environments. In the presence of readily available carbon sources such as glucose, the fungus activates this mechanism and inhibits the production of cellulolytic complex enzymes to avoid unnecessary energy expenditure. CCR has been well described for the growth of T. reesei in cellulose and glucose, however, little is known about this process when the carbon source is sophorose, one of the most potent inducers of cellulase production. Thus, we performed high-throughput RNA sequencing to better understand CCR during cellulase formation in the presence of sophorose, by comparing the mutant ∆cre1 with its parental strain, QM9414. Of the 9129 genes present in the genome of T. reesei, 184 were upregulated and 344 downregulated in the mutant strain ∆cre1 compared to QM9414. Genes belonging to the CAZy database, and those encoding transcription factors and transporters are among the gene classes that were repressed by CRE1 in the presence of sophorose; most were possible indirectly regulated by CRE1. We also observed that CRE1 activity is carbon-dependent. A recent study from our group showed that in cellulose, CRE1 repress different groups of genes when compared to sophorose. CCR differences between these carbon sources may be due to the release of cellodextrins in the cellulose polymer, resulting in different targets of CRE1 in both carbon sources. These results contribute to a better understanding of CRE1-mediated CCR in T. reesei when glucose comes from a potent inducer of cellulase production such as sophorose, which could prove useful in improving cellulase production by the biotechnology sector.

Keywords: CRE1; Carbon catabolite repression; RNA-seq; Sophorose; Trichoderma reesei.

Fig. (1)

Differentially expressed genes in the mutant ∆cre1 relative to the parental strain QM9414. The expression profile was calculated for conditions inlcuding sophorose. Genes that showed differential expression identified by DESeq package are shown in red. Among the 9129 T. reesei genes, 2368 were differentially expressed, using a p-value value ≤ 0.05 as threshold (A). Of these, 184 genes were upregulated and 344 genes were downregulated in the mutant ∆cre1 compared to QM9414 (B).

Fig. (2)

Enrichment analysis using Gene Ontology terms of the major genes upregulated in ∆cre1 compared to QM9414 during growth in sophorose. Functional categorization was performed using BayGO software [32] and the categories were considered significantly enriched when p ≤ 0.05.

Fig. (3)

CAZy genes upregulated in the mutant ∆cre1 compared to QM9414 in the presence of sophorose. Gene expression values are represented as Log₂ fold change.

Fig. (4)

Phylogenetic analysis of transporters upregulated in the mutant ∆cre1 during growth in sophorose. The analysis also includes other proteins involved with transport in different species. Highlights: five clusters formed by MFS permeases, maltose permeases, ABC transporters and sug-ar transporters. The tree was created using the Mega 4 program using the Maximum Parsimony method with 1000 bootstraps.

Fig. (5)

Comparison of the CRE1-mediated CCR mechanism under conditions of cellulose and sophorose. Initially, the action of cellulolytic enzymes in the cellulose polymer can release cellodextrins and sophorose (1). The enzymatic hydrolysis of these oligomers releases glucose (2) which sig-nals that the synthesis of new cellulolytic enzymes must be terminated by the fungus, since there is sufficient energy in the medium. By a mech-anism not yet elucidated, the fungus can differentiate glucose arising from cellodextrins from that derived from sophorose, and different genes are suppressed in each case. In cellulose, CRE1 acts primarily on genes involved in the early stages of cellulose deconstruction, whereas in sophorose this transcription factor acts by inhibiting a greater number of genes belonging to CAZy and membrane permeases, possibly includ-ing maltose permeases that could transport sophorose.