Yeast nitrogen base with ammonium sulphate

The S.cerevisiae strain 1783 (MATa can1 his4 leu2-3,112 trp1-1 ura3-52) (National BioResource Project, Hiroshima, Japan) was used in this study. Yeast cells were cultured at 30 °C with shaking at 200 rpm in Yeast Peptone Dextrose (YPD) medium (10 g/L yeast extract, 20 g/L peptone, and 20 g/L dextrose) (HIMEDIA, Mumbai, India) for general growth. Synthetic complete (SC) medium consisting of 2% (w/v) glucose (HIMEDIA, Mumbai, India), 0.67% (w/v) yeast nitrogen base with ammonium sulphate (HIMEDIA, Mumbai, India), complete supplement mixture without tryptophan (HIMEDIA, Mumbai, India) and supplemented tryptophan (HIMEDIA, Mumbai, India) was used for ageing cultures. The concentrations of amino acids in SC medium are listed in Supplementary Table S1.

The S. cerevisiae strains used in this study were MLC30M (MATa trp1-1 leu2-3,112 his3-11,15 ura3-1 ade2-1 can1-100 yrs1::HIS3 yrr1::TRP1 pdr1::hisG pdr3::hisG) (NBRP, Japan)^{29 (link),30} and BY4741 (MATa leu2Δ0 ura3Δ0 his3-Δ1 met15Δ0) (NBRP, Japan)^{31 ,32} . The gene deletion mutants of BY4741 were purchased from Euroscarf, Germany. The deleted genes were replaced by kanMX4. Yeast cells were cultured at 30 °C in Yeast Peptone Dextrose (YPD) medium (10 g/L yeast extract, 20 g/L peptone, and 20 g/L dextrose) (HIMEDIA, India) for general growth and CLS screen of the mutants. Synthetic complete (SC) medium consisting of 2% glucose (HIMEDIA, India), 0.67% yeast nitrogen base with ammonium sulphate (HIMEDIA, India), and amino acids (HIMEDIA, India) at appropriate concentrations and lacked certain amino acids (uracil, leucine) were used for plasmid selection and protein expression assays. The concentrations of amino acids in SC medium are listed in Table 1. Yeast transformation was performed as described^{33 (link)}, and transformants were selected from synthetic complete minus uracil (SC-Ura) agar.Table 1

Final concentrations of amino acids in SC medium.

Amino acid	Final concentration in SC medium (mg/L)
L-Isoleucine	30
L-Valine	150
Adenine sulphate	20
L-Arginine	20
L-Lysine	30
L-Methionine	20
L-Phenylalanine	50
L-Threonine	200
L-Tyrosine	30
L-Histidine	24
L-Tryptophan	24
L-Leucine	36