Difco yeast nitrogen base without amino acids and ammonium sulfate

A95 (CBS 123872) was used as recipient (WT) for genetic manipulation. A95 and derivatives (Tables S2 and S3) were cultivated in malt extract broth/agar (MEB/MEA)^{24 (link)} at 25 °C. Osmotically stabilised media (MEBS, MEAS) contained 10.8% (w/v) sucrose^{34 (link)}. Transformants were incubated on MEA containing 24 µg ml⁻¹ hygromycin B (HYG) (Duchefa Biochemie) and/or 5 µg ml⁻¹ nourseothricin (NAT) (Carl Roth GmbH). Basal synthetic media for growth assays were SD [2% (w/v) glucose, 0.67% (w/v) Difco Yeast Nitrogen Base without Amino Acids (BD Biosciences), 2% (w/v) agar] and SD/NO₃ [2% (w/v) glucose, 0.17% (w/v) Difco Yeast Nitrogen Base without Amino Acids and Ammonium Sulfate (BD Biosciences), 0.3% (w/v) NaNO₃, 2% (w/v) agar], which were supplemented with 50 mg l⁻¹ uracil (URA), 100 mg l⁻¹ adenine (ADE), 1 g l⁻¹ 5-fluoroorotic acid (5-FOA), 48 g l⁻¹ KClO₃ and/or 0.5 g l⁻¹ yeast extract (YE). For inoculation of growth assays, one inoculation loop of cells were taken from surface-grown colonies, transferred to 0.6 ml phosphate-buffered saline (PBS) containing 6–8 glass beads (3–5 mm), and dispersed using a TissueLyser (Retsch) (2 × 20 s at 40 Hz). Dispersed cells were transferred to fresh tubes, cell titers were determined using a Thoma cell counting chamber and adjusted with PBS to 5 × 10⁵ cells ml⁻¹. Solid media were inoculated with 10 µl droplets containing 5 × 10³ cells.