Bacto yeast extract

Strains constructed in this study were generated using S. cerevisiae CEN.PK113-7D as a parent strain, except for strains FR013 and FR014 which were constructed in the CEN.PK113-9D background giving a possibility for using auxotrophic markers (31 ). Cultures were grown in complex medium (YEPD) comprised of 2% Difco™ phytone peptone (Becton–Dickinson, Franklin Lakes, NJ, USA), 1% Bacto™ yeast extract (BD) and either d-glucose (2%, Sigma Aldrich, St Louis, MO, USA) or galactose (2%, Sigma Aldrich) when induction of the GAL10 promoter was required. Selection was achieved using nourseothricin (NTC, 200 μg/ml, Jena Bioscience, Germany) or geneticin (G418, 200 μg/ml, Sigma Aldrich), when appropriate. Solid medium was prepared by addition of Difco™ granulated agar (BD) to the medium to a final concentration of 2% (w/v). Propagation of plasmids was performed using Escherichia coli NEB 10-beta cells (New England BioLabs, Ipswich, MA, USA). Bacterial cultures were prepared in 2*PY medium comprised of tryptone peptone (1.6%, BD), Bacto™ yeast extract (1.0%, BD) and NaCl (0.5%, Sigma Aldrich) and containing ampicillin (100 μg/ml, Sigma Aldrich) or neomycin (50 μg/ml, Sigma Aldrich).

All Saccharomycescerevisiae strains used and constructed in this study belong to the CEN.PK lineage (Entian and Kötter 2007 ; Nijkamp et al.2012 (link); Salazar et al.2017 (link)). Yeast cultures were grown on synthetic medium (SM, prepared and sterilised as described previously (Verduyn et al.1992 (link))) or YP medium (10 g L⁻¹ Bacto yeast extract (Becton Dickinson, Sparks, MD)), 20 g L⁻¹ Bacto peptone (Becton Dickinson); autoclaved at 121°C for 20 min. SM was supplemented with 1 mL L⁻¹ filter-sterilised vitamin solution (Verduyn et al.1992 (link)). Concentrated sterile d-glucose or d-xylose solutions (autoclaved at 110°C for 30 min) were added to SM and YP media to a concentration of 20 g L⁻¹, resulting in SMD or SMX and YPD or YPX, respectively. Shake-flask cultures were grown in 500-mL round-bottom flasks containing 100 mL medium and incubated in an Innova incubator (Brunswick Scientific, Edison, NJ) at 30°C and 200 rpm. Solid media contained 2% (w/v) Bacto agar (Becton Dickinson). Escherichia coli strains were grown in LB-ampicillin medium (10 g L⁻¹ Bacto tryptone, 5 g L⁻¹ Bacto yeast extract, 5 g L⁻¹ NaCl, 100 mg L⁻¹ ampicillin). For storage, sterile glycerol was added to stationary-phase cultures of yeast and E. coli to a final concentration of 30% (v/v), after which aliquots were stored at −80°C.