Xylose

For growth tests, a synthetic minimal medium (MM) (2 g L⁻¹ NaNO₃, 1 g L⁻¹ K₂HPO₄, 0.5 g L⁻¹ KCl, 0.5 g L⁻¹ MgSO₄·7H₂O, 0.01 g L⁻¹ FeSO₄·7H₂O) was prepared and supplemented with 1% (w/v) carbon source (glucose, sucrose, carboxymethyl cellulose (CMC), galacturonic acid (GA), polygalacturonic acid (PGA), xylose, xylan from beechwood (Carlroth, Karlsruhe, Germany), tween 80, olive oil, triolein, sodium acetate (NaAc)) and 1.5% agar (Oxoid, LP0011, Swedesboro, NJ, USA). pH was adjusted and buffered at 5 or 7 with a McIlvaine buffer. For inoculation, 7-mm diameter plugs with actively growing mycelium were used. The experiment was repeated independently three times (biological replicates), including three technical replicates (three plates) and the mean was calculated with 9 measures. The Petri dishes were incubated at 21 °C in the dark, and the growth diameter of the wild type, mutant, and complemented strains was measured to 4 dpi.

S. cerevisiae strains used in this study (Table I) were provided by DSM and may be made available for academic research under a strict Material Transfer Agreement with DSM (contact: paul.waal-de@dsm.com). The culture medium used was a defined mineral medium, with vitamins prepared as described by Verduyn et al. (1992). For storage of the strains, shake flask cultures were performed in the mineral medium supplemented with 2% maltose (Sigma–Aldrich, Rockville, MD) in case of the DS68616 and DS68625‐derivatives, or 2% xylose (Roth) in case of DS71054‐derivatives. With strains DS68625, DS68616, or DS71054, the Verduyn‐urea was supplemented with 0.02% histidine (Sigma–Aldrich) to complement for the auxotrophic marker. Cultures were maintained at 30°C in an orbital shaker until stationary growth phase was reached. After the addition of glycerol to 30% (v/v), samples were stored in 2 mL aliquots at −80°C.

S. cerevisiae strains used in this study (Table 1) were provided by DSM and may be made available for academic research under a strict Material Transfer Agreement with DSM (contact: paul.waal-de@dsm.com). The culture medium used was a defined mineral medium, with vitamins prepared as described by Verduyn et al. [40 (link)]. For storage of the strains, shake flask cultures were performed in the mineral medium supplemented with 2 % maltose (Sigma-Aldrich) in case of the DS68616/DS68625/DS68625-evo-derivatives, or 2 % xylose (Roth) in case of DS71054-derivatives. With strains DS68625, DS68616, or DS71054, the Verduyn-urea was supplemented with 0.02 % histidine (Sigma-Aldrich) to complement for the auxotrophic marker. Cultures were maintained at 30 °C in an orbital shaker until stationary growth phase was reached. After the addition of glycerol to 30 % (v/v), samples were stored in 2 ml aliquots at −80 °C.
Chemostat cultures were grown in a 3-L stirred tank bioreactor (Applikon, Schiedam, The Netherlands) filled with 500 ml of the mineral medium at a temperature of 30 °C, pH 4.5 and at a dilution rate of 0.01–0.05 h⁻¹. Stirring was performed at 400 rpm and the starting OD₆₀₀ was 1. Some diffusion of oxygen was allowed via the inflowing medium and the venting vessel on the reactor.