Proteome profiling of enriched membrane-associated proteins unraveled a novel sophorose and cello-oligosaccharide transporter in Trichoderma reesei

Abstract

Background: Trichoderma reesei is an organism extensively used in the bioethanol industry, owing to its capability to produce enzymes capable of breaking down holocellulose into simple sugars. The uptake of carbohydrates generated from cellulose breakdown is crucial to induce the signaling cascade that triggers cellulase production. However, the sugar transporters involved in this process in T. reesei remain poorly identified and characterized.

Results: To address this gap, this study used temporal membrane proteomics analysis to identify five known and nine putative sugar transporters that may be involved in cellulose degradation by T. reesei. Docking analysis pointed out potential ligands for the putative sugar transporter Tr44175. Further functional validation of this transporter was carried out in Saccharomyces cerevisiae. The results showed that Tr44175 transports a variety of sugar molecules, including cellobiose, cellotriose, cellotetraose, and sophorose.

Conclusion: This study has unveiled a transporter Tr44175 capable of transporting cellobiose, cellotriose, cellotetraose, and sophorose. Our study represents the first inventory of T. reesei sugar transportome once exposed to cellulose, offering promising potential targets for strain engineering in the context of bioethanol production.

Keywords: Cellulose; Membrane-associated proteome; Sugar transporters; Trichoderma reesei.

Fig. 1

Abundance of the T. reesei sugar transporters in presence of glycerol and cellulose. The heat map represents the abundance of the sugar transporters identified during the culture of T. reesei in presence of glycerol and cellulose. The R heatmap package was utilized to perform hierarchical clustering. The hierarchical clustering of protein abundance in all conditions was carried out using the complete linkage method and Euclidean distance with row-centered values. The protein IDs correspond to the sugar transporters identification according with JGI database

Fig. 2

Sugar transporters subjected to phylogenetic classification. The tree was inferred using 335 protein sequences from T. reesei, A. niger, A. nidulans and N. crassa containing the Pfam PF00083 domain. The sugar transporters characterized experimentally in the literature [42] are highlighted with bold font and the transporters identified in this study are highlighted in red and bold. Each clade potential substrates are determined based on the functions of known sugar transporter members within those specific clades. Three clades in red did not resemble experimentally characterized sugar transporters. Support values from 1000 resamples are illustrated as gray dots

Fig. 3

Expression analysis of Tr44175 gene in T. reesei. The expression of the Tr44175 gene was analyzed using T. reesei QM9414 strain grown in the presence of cellulose (A) or sophorose (B). The 2^–∆CT method [22] was used for calculating Tr44175 gene expression relative to the endogenous control gene (actin). Values are the means of three biological replicates. Data were analyzed using a 1-way ANOVA followed by a Bonferroni post hoc test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.001

Fig. 4

Prediction of membrane protein topology and docking analysis. A Analyses of membrane protein topology and insertion energy were performed in QMEANBrane. Interactions between Tr44175 and glucose (B), sophorose (C), cellobiose (D), cellotriose (E) and cellotetraose (F) analyzed using LigPlot + v2.2.8

Fig. 5

Tr4475 functional characterization. A Confocal microscopy shows that Tr44175::GFP localizes to the S. cerevisiae plasma membrane. Confocal microscopy pictures were taken in both differential interference contrast (DIC) and fluorescence modes and then merged (Tr44175::GFP). B Design of Sc_Tr44175_BGL1 cells with a vector expressing the transporter fused with GFP and a vector carrying a β-glucosidase encoding gene gh1-1 from N. crassa. C Design of Sc_Tr44175_BGL1B cells with a vector carrying the transporter fused with GFP and a vector expressing a β-glucosidase encoding gene An03g03740 (designated bgl1B) from A. niger. D Growth of S. cerevisiae strain Sc_Tr44175_BGL1in YNB supplemented with 5 g/L, 10 g/L, or 20 g/L cellobiose concentrations. E Growth of S. cerevisiae strain Sc_Tr44175_BGL1B in the presence of 8 mg/L, 16 mg/L, and 24 mg/L of sophorose concentrations. F Growth of S. cerevisiae strain Sc_Tr44175_BGL1 in YNB supplemented with 12 mg/L of cellotriose or 16 mg/L of cellotetraose concentrations. The control strain contains the pRH195d empty vector and the gene encoding β-glucosidase GH1-1 or BGL1B. Yeast cells, when inoculated on media containing glucose or cellobiose, were incubated at 30 °C for 120 h, while those on media containing cellotriose, cellotetraose, or sophorose were incubated for 192 h

Fig. 6

Growth and cellobiose consumption of S. cerevisiae strains expressing Tr44175. Growth of Sc_Tr44175_BGL1 (44175::GFP) and Sc_pRH195d_BGL1 (control), as determined by optical density at 600 nm (OD_600nm) in YNB supplemented with 5 g/L cellobiose (A); 10 g/L cellobiose (C) or 20 g/L cellobiose (E). Cellobiose consumption of the Sc_Tr44175_BGL1 (44175::GFP) and Sc_pRH195d_BGL1 (control) when incubated in YNB supplemented with cellobiose 5 g/L (B); 10 g/L cellobiose (D) or 20 g/L cellobiose (F). Data were analyzed using a 1-way ANOVA followed by a Tukey post hoc test. *P *P < 0.01; **P < 0.001; ***P < 0.001

Fig. 7

Inventory of T. reesei Sugar Transportome in the Presence of Cellulose. A hypothesized model of sugar transporters-mediated cellulase induction in T. reesei. The figure presents an overview of the potential transport activity and cellulose signaling by known sugar transporters and their suggested role in cellulase induction. Besides that, this scheme shows all the sugar transporters identified in this study, including the uncharacterized ones