Yeast Growth Optimization in Minimal Media

Single colonies were grown in 10 mL YPDA media (Takara) in 50 mL tubes for 24 h. Cultures were washed and inoculated in minimal media at starting optical densities (ODs) of 0.3 or 0.6 according to the experimental design. Minimal media contained 20 g/L glucose (Acros Organics) and 1.7 g/L yeast nitrogen base without amino acids or ammonium sulfate (BD Difco). A 60.5 mM nitrogen concentration in the media was obtained with 4 g/L ammonium sulfate (Acros Organics) or 1.82 g/L urea (Acros Organics). When required, media was buffered at a pH of 7 using 126 mM Na₂HPO₄ (Acros Organics) and 18 mM citric acid (Sigma‐Aldrich) (Prins & Billerbeck, 2021 (link)) and/or supplemented with 5 mM Phe (Sigma‐Aldrich) and/or 5 mM Glu (Sigma‐Aldrich). Cells were grown for 48 h at the required temperature and agitation speed in 50 mL mini‐bioreactor tubes (Corning) in an Innova 44 incubator (New Brunswick Scientific). At the end of the cultivation samples for OD measurements and pCA quantification were taken.