Tryptophan

Yeast strains were pre-grown on YNB supplemented with 5 g/L of casamino acids (Difco), 1 g/L of tryptophan (Sigma) and 50 g/L of d-maltose for 24 h. Cells were then harvested by centrifugation, washed three times with sterile water and resuspended to an OD₆₀₀ of 10. Fermentation experiments were performed aerobically in 250 mL Erlenmeyer flasks using 70 mL of YNB supplemented with 5 g/L of casamino acids, 1 g/L of tryptophan (Sigma) and 10 g/L of xylose. For simultaneous glucose and xylose co- fermentation, 10 g/L of both sugars were used. The cells were incubated at 30 °C/200 rpm. Experiments were performed in triplicate and samples were collected to measure optical density and for HPLC analysis.

An IDO enzymatic activity was performed using a protocol adapted from previously described methods [53 (link), 54 (link)]. Murine samples used in the IDO activity assays were prepared in Dulbecco's Phosphate Buffered Saline (DPBS) (Fisher) containing protease inhibitors followed by sonication. 2X IDO reaction buffer (200 mg/ml catalase from bovine liver (Sigma), 800 mM L-tryptophan (Sigma), 100 mM DPBS, 40 mM L-ascorbic acid (Sigma), 20 mM methylene blue (Fisher), with and without tryptophan, was added to samples that were normalized for total protein by BCA assay. Samples were incubated at 37°C for 30 min and the reaction was stopped with 30% trichloroacetic acid (TCA) (Sigma). Samples were incubated again at 52°C for 30 min followed by centrifugation (5 min, 10,000 rpm, 4°C). Supernatants were used for spectrophotometric analyses by the addition of an equal amount of Ehrlich's Solution (Sigma). Samples were read at an absorbance of 480 nm and values were calculated based on a standard curve of L-Kynurenine (Sigma) from 0-30,000 mM. Final IDO activity values were determined by taking the difference between samples without tryptophan-containing IDO reaction buffer and those receiving tryptophan-containing IDO reaction buffer.