## 2 Materials and Methods
### Activity Screening and Quantitative Enzyme Assays  
_T.reg2_ transformants were inoculated on YPD liquid media at 30 \({}^{\circ}\)C overnight. Cultures were then spotted on carboxymethylcellulose (CMC, Sigma Aldrich, St. Louis, MI, USA) solid media containing: 1% (_w/v_) carboxymethyl cellulose (CMC), 3 g/L Yeast nitrogen base with amino acids, 20 g/L glucose; 5 g/L ammonium sulphate and 20 g/L agar and incubated at 30 \({}^{\circ}\)C for 24 h. Following incubation, the plate was washed with water and stained with 0.1% Congo Red for 30 min, followed by washing with 1.2 M NaCl\({}_{2}\). For quantitative assays, transformed strains were cultivated in triplicate in 10 mL YPD media and cultivated at 30 \({}^{\circ}\)C for 48-72 h on an orbital shaker at 180 rpm for all assays. The samples were taken at 48- and 72-h cultivation times, and OD\({}_{600}\) readings were used to calculate the dry cell weight (DCW) for the strains [27]. Cells were removed by centrifugation, and the supernatant was used for the determination of the endoglucanase (EG) and cellobiohydrolase (CBH) activity, while the total culture was used for \(\beta\)-glucosidase (BGL) activity determination.  
The dinitrosalicylic acid (DNS) EG assay was performed, as previously described by [PERSON] et al. [28], using 1% CMC as substrate. A standard curve was set using D-glucose at concentrations of 0.5-10 g/L. All spectrophotometric readings for the enzymatic assays were taken at 540 nm on a FLUOstar Omega Microplate Reader (BMG LABTECH, Ortenberg, Germany), and media blanks were included. The CBH activity of transformants was measured on the soluble fluorescent substrate 4-methylumbelliferyl-\(\beta\)-D-lactoside (MULac; Sigma) using the method previously described by [PERSON] et al. [19] with a reaction time of 30 min at 37 \({}^{\circ}\)C and compared to a 4-methylumbelliferone (MU) standard curve set between 0.63 \(\upmu\)M and 20 \(\upmu\)M. The liberation of MU was detected by fluorescence measurement (excitation wavelength = 355 nm, emission wavelength = 460 nm). To evaluate the BGL activity of the recombinant strains, assays were carried out using _p_-nitrophenyl-\(\beta\)-D-glucopyranoside (pNPG; Sigma) as substrate, at 50 \({}^{\circ}\)C with a reaction time of 5 min as was previously described [29] and compared to a standard curve set between 0.075 and 1.25 mM pNP after centrifugation to remove cells. Assays were performed in biological and technical triplicates, and values were given as averages of these repeats with standard deviation indicated. For all enzyme assays, one unit was defined as the amount of enzyme required to produce one \(\upmu\)mol of reducing sugar or equivalent in one minute under the assay conditions.  
For the cellulose conversion assay, a substrate mixture containing 2% (_w/v_) Avicel PH101 (Sigma Aldrich), 50 mM sodium acetate (pH 5.0) and 0.02% (_w/v_) sodium azide was made and continuously stirred to ensure homogeneity. In a 96-deep-well plate, a 1:1 mixture of the substrate mix and yeast culture supernatant was added to a final volume of 600 \(\upmu\)L, with assays performed in triplicate. A sample was taken at 0 h as a baseline measurement for background sugar. The reaction was performed at 35 \({}^{\circ}\)C, shaking at 1000 rpm in a Heidolph Titramax 1000 microplate shaker/incubator (Heidolph, Schwabach, Germany) and sampled at 24 and 48 h. The amount of glucose in the supernatant was determined by performing a modified DNS assay method with absorbance determined at an OD of 540 nm, as previously described [29]. Using 72-h data, significant differences between activities attained were investigated using two-tailed \(t\)-tests, assuming unequal variance. A \(p\)-value lower than 0.05 was deemed significant.