Yeast nitrogen base

Standard yeast media and genetic procedures were previously described39 (link)40 (link). CAD medium consists of 0.67% yeast nitrogen base (Sigma), 2% glucose, 0.5% casamino acid (BD) and appropriate supplements (20 μg ml⁻¹ uracil and 40 μg ml⁻¹ tryptophan) as needed. CARaf and CAGal media are the same as CAD, except that they contain 2% raffinose or 2% galactose, respectively, in place of glucose. SRaf medium consists of 0.67% yeast nitrogen base and 2% raffinose with an appropriate yeast synthetic drop-out medium supplement. Buffer A for co-immunoprecipitation assays contains 50 mM Tris-HCl (pH 7.5), 15 mM EDTA, 15 mM EGTA, 2 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 1 mM benzamidine, 5 μg ml⁻¹ leupeptin, 50 mM NaF, 25 mM β-glycerophosphate and 150 mM NaCl. Buffer C2 for detergent-free membrane preparation contains 50 mM Tris-HCl (pH 7.2), 15 mM EDTA, 15 mM EGTA, 150 mM NaCl, 1 mM phenylmethylsulfonyl fluoride, 1 mM benzamidine and 5 μg ml⁻¹ leupeptin. Buffer X for crosslinking contains 50 mM Tris-HCl (pH 7.2) and 15 mM EDTA. Buffer Z for β-galactosidase assay contains 60 mM Na₂HPO₄, 40 mM NaH₂PO₄, 10 mM KCl and 1 mM MgSO₄, adjusted to pH 7.0. SDS loading buffer (1 × ) contains 50 mM Tris-HCl (pH 6.8), 2% SDS, 0.01% Bromophenol Blue, 10% glycerol and 700 mM 2-ME. For non-reducing conditions, 2-ME was omitted.

Aspergillus fumigatus (strain AF4215, MYA 1163; American Type Culture Collection) was grown on Sabouraud glucose agar plates (BD Bioscience, San Jose, CA, USA) for three days at 37 °C. Conidia were collected in Hanks’ balanced saline solution (Gibco) and filtered through a cell strainer. The estimation of the conidial number was performed microscopically in a Neubauer slide (LO–Laboroptik, Friedrichsdorf, Germany). Resting conidia were immediately used or were stored at 4 °C for a maximum of one week. A. fumigatus hyphae were prepared by seeding of resting conidia in flat-bottom cell culture plates (Nunc, Langenselbold, Germany) in Yeast Nitrogen Base (Sigma-Aldrich, Taufkirchen, Germany) medium for 17 h at 37 °C.

For E. coli selection, we used Lysogeny Broth (LB) with 100 mg/L ampicillin. For the selection of yeast strains, we used Yeast-Peptone-Dextrose (YPD) agar supplemented with 200 mg/L G418 for selection of Cas9 vector and 100 mg/L nourseothricin for selection of the gRNA vector. Synthetic Complete (SC) medium was prepared using 6.7 g/L Yeast Nitrogen Base without amino acids from Sigma-Aldrich, 1.92 g/L Synthetic Drop-out supplement without histidine from Sigma-Aldrich and 76 mg/L histidine. For ERG production, yeast strains were cultivated in SC medium with 20 or 40 g/L glucose or with 60 g/L EnPump substrate (and 0.6% enzyme reagent) as carbon source. Precultures for cultivation experiments were prepared by inoculating a single colony of a strain into 5 mL of the medium used in the cultivation experiment and incubating at 30°C and 250 rpm for 24 h. The cultivations were performed in 24-deep-well plates from EnzyScreen, 3 mL of medium was used per well and the starting OD₆₀₀ was 0.5. The plates were incubated at 30°C with 250 rpm agitation.

The A. fumigatus strain AF4215 (MYA 1163; American Type Culture Collection) was grown on Sabouraud glucose agar (BD Bioscience, San Jose, USA) at 37°C for 2-3 days. Conidia were harvested by gently washing the surface with PBS (Gibco) supplemented with 0.05% Tween-20 (Sigma-Aldrich, Taufkirchen, Germany). The number of the conidia was estimated in a Neubauer slide (LO-Laboroptik, Friedrichsdorf, Germany). Resting conidia were immediately used or stored at 4°C. For preparation of A. fumigatus hyphae, 1.25×10⁴ resting conidia were plated in 48-well flat-bottom cell culture plates (Nunc, Langenselbold, Germany) and incubated in 500 μL Yeast Nitrogen Base (Sigma-Aldrich) supplemented with (D)-Glucose (Sigma-Aldrich) at 37°C for 17 hours to allow formation of mycelium.

In the Bioscreen C system (Oy Growth Curves Ab Ltd., Finland), the growth of strains was tested. For tests YNB (Yeast Nitrogen Base, Sigma) medium supplemented with glycerol 5% (w/v) was used. The inoculation cultures were grown for 24 h in YPD medium. Consequently, the overnight cultures were centrifuged and the pellets were washed with sterile water. The strains were grown in 100-well plates in 200 μL of YNB medium. The OD₆₀₀ of the cells was standardized to 0.15. Each strain were grown in five repetitions at 28 °C under a constant agitation rate. Growth was monitored by measuring the optical density (OD) at 420–600 nm every 30 min for 48 h.

This study used Saccharomyces cerevisiae BY4741 as the wild type background strain, and deletion mutants from the Yeast Deletion Collection^{30 (link)}. YPD media; 10 g/L yeast extract, 20 g/L peptone, 20 g/L glucose. CSM media; 6.7 g/L yeast nitrogen base (Sigma Y0626), 790 mg/L Complete Supplement Mixture (Formedium DCS001), 20 g/L glucose.