Fungal strains, media, and culture methods
The fungal strains used in this study are listed in Additional file 1. The S. cerevisiae strain EBY.VW4000  is a hexose-null mutant and it was used for the initial functional analysis of the Tr69957 transporter. The EBY.VW4000 and its mutants (EBY.VW4000 +pRH195m +pRH274 and Tr69957::GFP EBY.VW4000) were grown at 30 °C in broth or on solid yeast nitrogen base (YNB) medium (which contained 20 g/L agar, 7 g/L YNB without amino acids and supplemented with 0.05 g/L histidine, 0.1 g/L leucine, 0.1 g/L tryptophan, and 0.1 g/L uracil). Selection of transformants was done on the YNB medium lacking tryptophan and uracil. All the reagents were obtained from Sigma Aldrich (St. Louis, MO, USA). The yeast S. cerevisiae SC9721 strain (MATα his3-Δ200 URA3-52 leu2Δ1 lys2Δ202 trp1Δ63) (Fungal Genetic Stock Center—www.fgsc.net) was used to generate the deletion cassette by homologous recombination. This strain was grown at 30 °C in broth or on solid YNB medium (which contains 20 g/L agar, 7 g/L YNB without amino acids and supplemented with 0.05 g/L histidine, 0.1 g/L lysine, 0.1 g/L leucine, 0.1 g/L tryptophan, 0.1 g/L uridine, and 0.1 g/L uracil), along with 2% (w/v) glucose. The strain QM6a∆tmus53∆Pyr4  was used as a parental strain to construct the ∆69957 T. reesei mutant strain. This strain was obtained from the Institute of Chemical Engineering & Technical Biosciences of Vienna University of Technology, TU Vienna, Austria. The strain was maintained at 4 °C on MEX medium [3% (w/v) malt extract and 2% (w/v) agar–agar], which was supplemented with 5 mM uridine in the case of the pyr4 deletion strain. To perform gene expression assays, 106 cells/mL of each specific strain was inoculated into 25 mL of Mandels–Andreotti medium  (MAM) or Minimal Medium (MM) which contains 1% (w/v) of the selected sugar as the sole carbon source.
Conditions for maintenance of cultures
Trichoderma reesei strains QM6a∆tmus53∆Pyr4  and ∆69957 were grown in MEX medium at 28 °C for a period of 7 days. In all the experiments with SCB, the parental and Δ69957 mutant strains were initially grown in 1% (w/v) glycerol for 24 and 48 h, respectively, and then transferred to a medium containing SCB. For this purpose, 106 cells/mL of each specific strain was inoculated into 25 mL of MAM  containing 1% glycerol, and after 24 h (parental strain), the mycelium was transferred into 25 mL of MAM containing 1% of exploded sugarcane bagasse (ESB). Concerning the Δ69957 mutant strain, the mycelium was initially grown in 1% (w/v) glycerol for 48 h to achieve an initial mycelial biomass similar to the parental strain, and then, the mycelium was transferred into 25 mL of MAM containing 1% ESB. The ESB was prepared as described previously in the corresponding Ref. . Briefly, SCB in natura was treated with 14 kg/cm2 water steam, thoroughly washed with distilled water until the reducing sugars were not detected by dinitrosalicylic acid (DNS) , and dried at 40 °C for several days. SCB was kindly donated by Nardini Agroindustrial Ltd, Vista Alegre do Alto, São Paulo, Brazil.
The fungal cultures were incubated at 28 °C for 6, 24, and 48 h in an orbital shaker (200 rpm) for all the SCB experiments. All experiments were performed in three biological replicates. The resultant mycelia were collected by filtration, frozen in liquid nitrogen, and stored at − 80 °C for RNA extraction.
Construction of the ∆69957 mutant strain
The deletion of Tr69957 gene in T. reesei was performed as described previously by Schuster et al. . To construct the deletion cassette, the orotidine-5′-phosphate decarboxylase gene of Aspergillus niger (pyrG) was used as a selection marker. The flanking sequences of Tr69957 were obtained from the T. reesei genome database (http://genome.jgi.doe.gov/Trire2/Trire2.home.html). The marker gene and its respective 5′ and 3′ flanking sequences were amplified using the primers as described in Additional file 2. The primers were designed and analyzed with the help of OligoAnalyzer tool (https://eu.idtdna.com/calc/analyzer). To enable yeast-mediated recombination of the deletion cassette, we used the external 5′-UTR forward and 3′-UTR reverse primers containing cohesive ends with the vector pRS426 [19, 20] and the internal 5′-UTR reverse and 3′-UTR forward primers containing cohesive ends with the 5′ and 3′ sequence of the pyrG gene (Additional file 2). The 50-μL reaction mixture contained 1 U Platinum Taq DNA Polymerase High Fidelity (Thermo Scientific), 1 × High Fidelity PCR Buffer, 0.2 mM dNTPs, 0.3 µM forward and reverse primers, 1 µL T. reesei QM9414 and A. niger genomic DNA (150 ng/µL) as a template, and nuclease-free water. The PCR fragments were purified using a QIAquick PCR Purification Kit (Qiagen).
To perform yeast-mediated recombination, we used the yeast shuttle vector pRS426 (ampR lacZ URA3) [19, 20], which was digested with EcoRI and XhoI (Thermo Scientific), and purified with the QIAquick PCR Purification Kit (Qiagen). Yeast transformation was performed as described previously in the corresponding Ref. [21,22,23]. We prepared an overnight culture (200 rpm, 30 °C) of the yeast S. cerevisiae SC9721 strain (MATα his3-Δ200 URA3-52 leu2Δ1 lys2Δ202 trp1Δ63) obtained from Fungal Genetic Stock Center. Then, 1 mL of this overnight culture was added to 50 mL of fresh YPD medium (1% yeast extract, 2% peptone, 1% glucose: all of them were obtained from Sigma Aldrich) and incubated at 30 °C until the attainment of OD600 = 1. Thereafter, the cells were centrifuged and re-suspended in 100 mM lithium acetate for transformation. To this purpose, equimolar quantities of 5′ and 3′ flanking regions of pyrG gene and the digested pRS426 product were mixed and used for yeast transformation by the lithium acetate method . S. cerevisiae SC9721 transformants were selected for their ability to grow on YPD medium supplemented with lysine, histidine, leucine, and tryptophan, without uracil. S. cerevisiae genomic DNA was extracted using a previously described protocol [25, 26]. The deletion cassette of Tr69957 was PCR amplified using TaKaRa Ex Taq DNA Polymerase (Clontech) using Tr69957_pRS426_5 forward and Tr69957_pRS426_3 reverse primer (Additional file 2).
The selected transformants were analyzed by Southern hybridization using a previously described method , to demonstrate that the transformation cassettes had homologously integrated at the targeted T. reesei QM6aΔtmus53Δpyr4 loci (Additional file 3). To perform this analysis, 25 µg of total genomic DNA from the parental and mutant strains were digested with EcoRV (Fermentas) overnight, and then, this digested DNA was transferred onto GE Healthcare Amersham Hybond-N + membranes (GE). The probe was generated from the PCR-amplified fragment using Tr69957_pRS426_5fw and Tr69957_pyrG_5rv (Additional file 2) and labeled using a digoxigenin (DIG) DNA labeling kit (Roche, Mannheim, Germany) by following the manufacturer’s instructions. Labeling, hybridization, and immunological detection were performed using a non-radioactive labeling and immunological detection kit with CDP-Star as the chemiluminescent substrate (Roche, Mannheim, Germany), by following the methods that were previously described .
Measurement of dry weight
A total of 106 cells/mL of T. reesei parental or Δ69957 mutant strains were inoculated into 25 mL of MAM supplemented with 1% (w/v) glycerol. After 24 h, the parental strain mycelium was transferred into 25 mL of MAM containing 1% (w/v) xylose, cellobiose, or mannose. The mutant strain was grown for 48 h in MAM containing 1% (w/v) glycerol to achieve an initial mycelial biomass similar to the parental strain. After 6, 24, and 48 h, mycelia were harvested by filtration, dried by incubation at 70 °C for 3 h and subsequently weighed. The experiments were conducted in triplicates for each sample.
Determination of the extracellular sugar concentrations
The uptake of sugars was measured by HPLC analyses. A total of 106 cells/mL T. reesei parental and Δ69957 mutant strains were inoculated into 25 mL of MAM supplemented with 1% (w/v) glycerol. After 24 h, the parental strain mycelium was transferred into 25 mL of MAM containing 1% (w/v) or 30 mM of xylose, cellobiose, or mannose in the same culture, at 30 °C 200 rpm for 6, 24, and 48 h. The mutant strain was grown for 48 h in MAM containing 1% (w/v) glycerol to achieve an initial mycelial mass similar to that of the parental strain. S. cerevisiae Tr69957::GFP EBY.VW400 strain and EBY.VW4000 + pRH195m + pRH274 + pGH1 (control) were inoculated in 100 mL of YNB medium supplemented with 2% (w/v) maltose until they reached the exponential growth phase. Yeast cells were pelleted by centrifugation at 4000 rpm for 5 min, washed three times with 50-mL water, and re-suspended in water. Next, this cell suspension was inoculated (initial OD600 = 0.5) in 50-mL YNB-Trp-Ura-Leu medium without carbon source and then incubated at 30 °C for 3 h. Finally, this medium was supplemented with 1% (w/v) xylose, cellobiose, or mannose at 30 °C, 150 rpm for 144 h. At each timepoint, 2 mL of the culture were collected, centrifuged, and the supernatants were stored at − 80 °C. The amount of sugar in the supernatant at each timepoint was determined by ion chromatography (HPLC-Thermo Fisher-U3000), coupled with refractive index detector) Suplecogel C611 column (eluted with 5 mM H2SO4, at flow rate 0.5 mL/min and column temperature 60 °C) and Aminex HPX-87P column (eluted with H2O, at flow rate 0.5 mL/min and column temperature 85 °C).
Analysis of the effect of Tr69957 deletion on T. reesei growth in various carbon sources
To analyze the effect of the Tr69957 deletion on the growth of T. reesei in various carbon sources, phenotypic assays for the parental and mutant strains were performed by inoculating of 106 cells/mL on minimal media (MM) and potato dextrose agar (PDA) plates containing 1% of one of the following carbon sources: mannose, xylose, glucose, galactose, arabinose, raffinose, cellobiose, sophorose, lactose, xylitol, glycerol, fructose, or maltose . The experiment was performed in triplicates for each carbon source. The diameters of colonies were measured after 6 days.
Quantitative real-time PCR analysis
Trichoderma reesei mycelia were macerated and total RNA was extracted from each sample using TRIzol® RNA kit (Invitrogen Life Technologies, Carlsbad, CA, USA), according to the manufacturer’s instructions. RNA concentration was determined using a spectrophotometer at an optical density ratio of 260/280 nm, and RNA integrity was verified by electrophoresis in 1% agarose gels. To perform the expression analysis, initially, the total RNA (1 µg) from each sample was digested with DNase I (Fermentas) to remove genomic DNA. Then, cDNA synthesis was carried out using the RevertAid H Minus First Strand cDNA Synthesis kit (Waltham, Massachusetts, USA) according to the manufacturer’s instructions. Synthesized cDNA was diluted at 1:50 and used as a template for real-time PCR. Reactions were performed in the Bio-Rad CFX96™ using SsoFast™ EvaGreen® Supermix (Bio-Rad) for detection according to the manufacturer’s instructions. Each reaction mixture (10 µL) contained 5 µL SsoFast™ EvaGreen® Supermix (Bio-Rad), forward and reverse primers (500 nm each; Additional file 4), cDNA template, and nuclease-free water. PCR cycling conditions: 10 min at 95 °C (1 cycle), 10 s at 95 °C followed by 30 s at 60 °C (40 cycles), and a melting curve of 60–95 °C with an increment of 0.5 °C/10 s to check for primer dimers and nonspecific amplification. The transcript of the sar gene (encodes Sar1 GTPase from T. reesei) was used as an internal reference to normalize the amount of total RNA present in each reaction . To perform gene expression analysis in strains grown on SCB, cellobiose, mannose, and xylose, the expression levels of genes were calculated from the threshold cycle according to the 2−ΔCT method  relative to transcription levels of sar. The experiment was repeated three times for each sample. The results were analyzed using MeV v.4.6.1. Software and heat maps were constructed to assess the variety in the expression of genes among the strains under each indicated condition.
Construction of S. cerevisiae strain expressing T. reesei Tr69957
The construction of S. cerevisiae strain that expresses T. reesei Tr69957 was performed as described by dos Reis et al.  using the EBY.VW4000 yeast strain (Additional file 1). Briefly, the T. reesei Tr69957 ORF was PCR-amplified using cDNA obtained from the wild-type (WT) strain QM6A (Additional file 1) using specific primers 69957-F and 69957-R. To perform in vivo recombination, the plasmid pRH195 was linearized by double digestion with SpeI and SalI, originating the pRH195m (pRH195 without XKS1 gene). The GFP gene was PCR amplified from pCMC17apx plasmid using primers pRH195 GFP_CS_F-yeast and spacerGFP 5´R_pRH195-yeast. Furthermore, the linearized plasmid was co-transformed with the PCR-amplified sugar transporter and GFP fragments into the S. cerevisiae EBY.VW4000 strain, which already contained the pRH27412 and pGH1x plasmids. The pRH274 plasmid contains the xylulose kinase (XK), xylose reductase (XR), and xylose dehydrogenase (XDH) genes which encode enzymes of the xylose metabolic pathway while the pGH1 plasmid contains the β-glucosidase-encoding gene (gh1-1) from Neurospora crassa . The yeast transformation was performed using the lithium acetate method . Transformants were selected for tryptophan, leucine, and uridine prototrophy, and the yeast candidates were confirmed by PCR using the primers 69957-F and 69957-R. A list of all the primers used in this study can be accessed in Additional file 5.
Growth of S. cerevisiae strain on solid medium
Saccharomyces cerevisiae strains were inoculated in 50 mL YNB medium supplemented with 2% (w/v) maltose for 48 h at 30 °C, 200 rpm. Yeast cells were centrifuged at 4000 rpm for 5 min, washed three times with water, and re-suspended in water to a final concentration of 1.0 at OD600nm. A serial dilution of 1:10 of the yeast cells was done, and 5 μL of the cell suspensions were spotted on plates containing 1% (w/v) of specific carbon source. Plates were incubated at 30 °C for 120 h.
Saccharomyces cerevisiae EBY.VW400 strain containing the Tr69957 gene tagged with GFP was grown for 48 h in 0.5 mL of liquid YNB-Trp-Ura-Leu medium supplemented with 2% (w/v) maltose for 24 h at 30 °C in a well of 24-wells plate. Cells were washed with PBS and observed under a Carl Zeiss (Jena, Germany) microscope using the 100 × magnification oil immersion objective lens (EC Plan-Neofluar, NA 1.3) equipped with a 100-W HBO mercury lamp epifluorescence module. Phase contrast bright-field and fluorescent images were acquired with an AxioCam camera (Carl Zeiss), and images were processed using the AxioVision software version 3.1 and saved as TIFF files.
3-D structure prediction and molecular docking
The 3D model for Tr69957 was built using the I-TASSER online platform from the Yang Zhang’s Research Group (https://zhanglab.ccmb.med.umich.edu/I-TASSER/). To this purpose, an FASTA file containing the amino-acid sequence for the referred protein was obtained from the UniProt database (gene: TRIREDRAFT_69957) and submitted to the I-TASSER online server. The structure modeling approach used by this software is based on the sequence alignment to a protein template, which is identified by LOMETS, a method that selects the top ten alignments in a PDB library. The unaligned regions of the sequence are built through ab initio folding, by considering the lowest free energy states and minimized sterical clashes that are identified by SPICKER and TM-align, respectively, and the final models are built at an atomic level by REMO which optimizes the hydrogen-bonding profile. In addition, the I-TASSER online software also allows the prediction of protein biological functions, as it compares the designed models to different libraries of proteins with previously identified functions [34, 35].
The docking analysis was performed using the software iGEMDOCK v. 2.1, a Generic Evolutionary Method for molecular DOCKing, developed by Jinn-Moon Yang (http://gemdock.life.nctu.edu.tw/dock/igemdock.php). It computes the interaction between one or more ligands with the active site of the target protein through empirical scoring function and an evolutionary approach algorithm. It also provides post-analysis tools by k-means and hierarchical clustering methods from the docked poses and the protein–ligand properties, such as atom composition and energy function [36,37,38].
To perform the docking analysis, structure of all the ligands was downloaded from the PubChem database (https://www.ncbi.nlm.nih.gov/pccompound) in MOL2 format. [lactose (CID 6134), maltose (CID 6255), mannose (CID 18950), xylose (CID 644160), cellobiose (CID 10712), and fructose (CID 5984)]. The PDB file for the model was uploaded to the iGEMDOCK software along with all the ligands, and the docking was performed using standard settings, with a population size of 200 and a total of 70 generations. The post-docking parameters were analyzed and the best docked pose for each ligand was visualized by The PyMOL Molecular Graphics System, Version 184.108.40.206 Schrödinger, LLC (https://pymol.org/2/).