Leucine

S. cerevisiae strains deleted for multiple genes were generated using PCR deletion cassettes and marker rescue strategies as described before (Darwiche et al., 2017 (link)). Strains were grown in a minimal defined medium (containing 0.67% yeast nitrogen base without amino acids (US Biological, Salem, MA, United States), 0.73 g/L of an amino acid/nucleobase mixture, and 2% glucose) at 30°C under shaking condition at 180 rpm. The amino acid/nucleobase mixture contained 1.0 g adenine, 1.0 g argenine, 1.0 g histidine, 3.0 g leucine, 11.5 g lysine, 1.0 g methionine, 15.0 g threonine, 1.0 g tryptophan, and 2.0 g uracil (all from AppliChem GmbH, Darmstadt, Germany). For fatty acid analyses, pre-cultures were grown overnight in either SC-Ura or SC-Leu medium. The next morning, the main cultures were inoculated at 0.1 OD_600nm and after 24 h of growth, 3 or 5 OD_600nm units of cells and the corresponding culture medium were collected.

Stock solutions of Casamino Acids (Difco Laboratories, Detroit, MI), alanine (Merck, Darmstadt, Germany), aspartate (Merck), glutamate (Merck), glycine (Sigma-Aldrich, Taufkirchen, Germany), leucine (AppliChem, Darmstadt, Germany), threonine (Merck), tyrosine (Merck), valine (Merck), ribose (Sigma-Aldrich), formate (Sigma-Aldrich), succinate (Sigma-Aldrich), and glucose (AppliChem) were prepared with anoxic sodium phosphate buffer (36 mM, pH 7 [pH was adjusted with NaOH]). Solutions were filter sterilized (0.22-μm pore size, cellulose-acetate membrane) into sterile anoxic 100-ml serum vials that were crimp sealed with sterile butyl-rubber stoppers (Glasgerätebau Ochs Laborfachhandel e.K., Bovenden, Germany [product number 102049]); the vials were then flushed 10 min with sterile argon (100%).