Leucine

Candida glabrata CBS138 (prototrophic), KUE100 (prototrophic) (Ueno et al., 2007 (link)) and L5U1 (cgura3Δ0; cgleu2Δ0) (Chen et al., 2007 (link)) strains were used in this study. C. glabrata cells were cultivated in rich YEPD medium, RPMI 1640 medium or BM minimal medium. YEPD medium contained per liter: 20 g D-(+)- glucose (Merk), 20 g bacterial-peptone (LioChem) and 10 g of yeast-extract (Difco); whereas RPMI 1640 medium (pH 4) contained 10.4 g RPMI 1640 (Sigma), 34.5 g MOPS (Sigma) and 18 g glucose (Merck) per liter. BM media contained 20 g/L of D-(+)-glucose (Merck), 1.7 g/L of Yeast-Nitrogen-Base, without amino acids or ammonium sulfate (Difco) and 2.65 g/L ammonium sulfate (Merck). L5U1 strain was grown in BM medium supplemented with 60 mg/L leucine (Sigma). Saccharomyces cerevisiae strain BY4741 (MATa, ura3Δ0, leu2Δ0, his3Δ1, met15Δ0) and the derived single deletion mutant BY4741_Δdtr1 were obtained from the Euroscarf collection and grown in BM medium supplemented with 20 mg/L methionine, 20 mg/L histidine, 60 mg/L leucine, and 20 mg/L uracil (all from Sigma). Solid media contained additionally 20 g/L of Bactoagar (Difco).

Carboxymethyl cellulose, birch wood xylan, 2,2′-azino-di-[3-ethylbenzothiazoline-6-sulphonic acid] (ABTS), dinitrosalicylic acid, sodium-potassium tartrate, bovine serum albumin (BSA), manganese sulphate, copper sulphate, tyrosine, leucine, cellobiose, avicel, xylose, tryptophan, aspartate, glutamate, hydrogen peroxide, and media components were products of Sigma-Aldrich (St Louis, MO, USA). Corn cob was purchased from a local market. The corn cob was sun-dried and powdered into fine particles which was utilized as carbon source in the basal media. Further processing on the powdered corn cob was carried out using standard sieve to an average size of 1 mm. All other chemicals used were of analytical grade.

The chemicals [2,2-diphenyl-1-picrylhydrazyl; 2′-azino-bis (3-ethylbenzothiazoline- 6-sulfonic acid); 2,4,6-Tris(2-pyridyl)-s-triazine], reagents (Folin–Ciocalteu reagent), standards: ferulic acid, ellagic acid, proline, tyrosine, glycine, lysine, histidine, leucine, aspartic acid, valine were obtained from Sigma Aldrich (St., MO, USA), Elisa kits for testosterone and estradiol from Abbexa (Cambridge, UK) and buffer components (chloroform, ethyl acetate, formic acid, ethanol, 1-butanol, 2-propanol, boric acid) were purchased from Avantor Performance Materials Poland SA (APM, Gliwice, Poland).

S. cerevisiae strains used in this study are listed in Table 1. Yeast cells were grown aerobically at 26 °C in an orbital shaker (at 140 rpm), with a ratio of flask volume:medium volume of 5:1. The growth medium used was synthetic complete (SC) medium, containing drop-out, 2% (w/v) glucose (ThermoFisher Scientific, Waltham, MA, USA), and 0.67% (w/v) yeast nitrogen base without amino acids (BD BioSciences, San Jose, CA, USA), supplemented with appropriate amino acids or nucleotides [0.008% (w/v) histidine (Sigma Aldrich, St. Louis, MO, USA), 0.038% (w/v) methionine (Sigma Aldrich, St. Louis, MO, USA), 0.04% (w/v) leucine (Sigma Aldrich, St. Louis, MO, USA), and 0.008% (w/v) uracil (Sigma Aldrich, St. Louis, MO, USA)]. Deletion of AFT1 in ncr1Δ cells was performed using a deletion fragment containing HIS3 and the flanking regions of AFT1. Deletion of YCK3 in wild-type cells was performed using a deletion fragment containing KanMX4 and the flanking regions of YCK3. Yeast cells were transformed using the lithium acetate/single-stranded carrier DNA/PEG method as described [96 (link)]. Cells were selected by growing in a medium lacking histidine or containing geneticin (300 µg mL⁻¹), and gene deletion was confirmed by standard PCR procedures.

Sulfuric acid, sodium hydroxide, acetonitrile (Ultra high performance liquid chromatography (UHPLC) grade), ethylenediaminetetraacetic acid (EDTA), sodium borate, and hydrochloric acid were obtained from Panreac (Castelar del Vallés, Barcelona, Spain). Sodium acetate (anhydrous), trimethylamine (TEA), phosphoric acid, ammonium molybdate, and methyl cellulose were purchased from Sigma-Aldrich (Darmstadt, Germany). Standard of amino acids (L-arginine (Arg), L-alanine (Ala), L-asparagine (Asn), L-aspartic acid (Asp), glycine (Gly), L-glutamic acid (Glu), L-glutamine (Gln), L-histidine (His), L-isoLeucine (Ileu), Leucine (Leu), L-Lysine (Lys), L-norvaline (Nor), L-phenylalanine (Phe), L-proline (Pro), L-serine (Ser), L-threonine (Thr), L-tryptophan (Trp), L-tyrosine (Tyr), and L-valine (Val)) were purchased from Sigma-Aldrich (Steinheim, Germany). All the other chemicals and reagents used were of analytical grade.