Yeast nitrogen base

Liquid-cultivated yeast were grown at a temperature of 30°C with a shaking frequency of 250 rpm. Auxotrophic strains were cultivated in YPD medium (10 g/L yeast extract [BD Biosciences: 288630], 20 g/L Bacto-peptone [BD Biosciences: 211830], 5 g/L agar [BD Biosciences: 214530]). Yeast were transformed as described previously (Gietz and Schiestl, 2007 (link)) with the pHLUM plasmid (Mülleder et al., 2016b (link)) and selected for growth on SD media with inositol 5 g/L ammonium sulfate (Sigma-Aldrich: A4418), 1.7 g/L Yeast Nitrogen base (BD Biosciences: 233530), 20 g/L D-(+)-glucose (Sigma-Aldrich: G8270), and 10 mg/L myo-inositol (Sigma-Aldrich: I5125). For growth shift experiments, yeast were cultivated in or shifted to SD proline media (5 g/L potassium sulfate [Sigma-Aldrich: P0772], 1.7 g/L Yeast Nitrogen base [BD Biosciences: 233530], 20 g/L D-(+)-glucose [Sigma-Aldrich: G8270], 750 mg/L proline [Sigma-Aldrich: P0380], and 10 mM potassium phthalate [pH 5, Sigma-Aldrich: 60360]).

The reference strain C. glabrata ATCC 2001 was used in this study (American Type Culture Collection, Manassas, VA, USA). Standard culture media were used, including YPD (Becton, Dickinson and Company, Franklin Lakes, NJ, USA): yeast extract (1%, w/v), peptone (2%, w/v), glucose (2%, w/v), agar (1.5%, w/v), and yeast nitrogen base (YNB) without amino acids (Becton, Dickinson and Company, USA): yeast nitrogen base (0.67%, w/v), ammonium sulfate (0.5%, w/v). Synthetic complete (SC) media were prepared with YNB without amino acids, supplemented with complete supplement mixture (0.2%, w/v) (Formedium, Hunstanton, UK), glucose (2%, w/v), and agar (2%, w/v). In addition, glucose was replaced with alternative carbon sources: acetate (2%, w/v), lactate (2%, v/v), ethanol (2%, v/v), glycerol (2%, v/v), or oleic acid (0.2%, w/v) (Sigma-Aldrich, St. Louis, MO, USA) as the sole carbon source in SC media [2 (link),51 (link)].

Saccharomyces cerevisiae strains were grown in SD minimal medium [0.67% Yeast Nitrogen Base (Becton, Dickinson & Company^®, Franklin Lakes, NJ, USA), 2% sucrose (Chempur^®, Piekary Śląskie, Poland)] supplemented with amino acids [5 mg/mL adenine, 2 mg/mL histidine, 6 mg/mL leucine, 4 mg/mL tryptophan, 2 mg/mL uracil (Sigma-Aldrich^®, St. Louis, MO, USA)], with addition of 1.5% agar (Becton, Dickinson & Company^®) for solid medium. For treatment with compounds, cells were grown to mid-logarithmic phase (OD600 0.4–0.6) at 30 °C. The yeast strains used in this study are listed in Table 1.
To prepare rho⁰ strain W303-1A yeast were grown at 30 °C at 200 RPM in YPD medium o mid-logarithmic phase (OD600 0.5–0.8), ethidium bromide was added to the medium to 30 μg/mL final concentration, and cells were incubated for 24 h at 30 °C 200 rpm. Following incubation, cells were plated on a YPD medium to obtain single colonies and incubated for 3 days at 30 °C. Obtained colonies were plated on a YPGly medium containing glycerol as a carbon source to identify respiratory deficient cells. To verify loss of mtDNA, strains displaying no growth on YPGly plates were grow cells to mid-log phase, DAPI (Sigma-Aldrich, St. Louis, MO, USA) was added to a final concentration of 5 μg/mL, cells were incubated at 30 °C 200 rpm for 30 min, washed with PBS, and observe under a fluorescent microscope.

Strains and plasmids used in this work are shown in Table 1. E. coli strains were grown in LB medium containing 30 μg/mL kanamycin. S.cerevisiae strains were prepared as previously described [19 (link)], and were grown in synthetic minimal (SD) medium containing 0.67% yeast nitrogen base (Becton-Dickinson), amino acid supplements (Sunrise), and 2% glucose. Expression in E. coli was induced by adding IPTG at OD₆₀₀ of 0.4.

“Hydrolysate 3” was either adjusted to pH 5.5 with KOH, then centrifuged and filter sterilized or detoxified by 24 h pervaporation as described previously [21 (link)]. Saccharomyces cerevisiae SA-1 was provided by the Yeast Biochemistry and Technology Laboratory, Biological Science Department, Luiz de Queiroz College of Agriculture, University of Sao Paulo, Brazil and was grown at 30 °C at 200 rpm in 10 mL of synthetic complete media (SC-80). SC-80 contains 80 g/L glucose, 2 g/L dropout mix (US Biological, Salem, MA, USA), 6.7 g/L yeast nitrogen base (Becton Dickinson, Franklin Lakes, NJ, USA), 19.5 g/L 2-ethanesulfonic acid (MES) buffer, and a small amount of KOH to adjust the pH to 5.5. After overnight growth, cells were harvested by centrifugation. Fermentation was performed in 25 mL Hungate bottles under anaerobic conditions. The fermentation broth contained either 25 % (v/v) of “hydrolysate 3” or pervaporation-detoxified “hydrolysate 3” (with added water to match the amount removed by pervaporation), with the addition of the components of SC-80 to match SC-80 levels, and harvested SA-1 yeast cells to obtain an initial OD600 of 0.3. The fermentation was performed at 34 °C and 200 rpm for 67 h.