5 fluoroorotic acid 5 foa

All chemicals were purchased from Sigma-Aldrich, unless otherwise stated. Phusion DNA polymerase, as well as all restriction endonucleases, T4 DNA ligase, shrimp alkaline phosphatase (rSAP) and Gibson Assembly cloning kit, were purchased from New England Biolabs. Zymolyase 20T was purchased from MP Biomedicals, 5-fluoroorotic acid (5-FOA) was purchased from Thermo Fisher Scientific. Primers for PCR amplification were purchased from Sigma-Aldrich (see ESI Table S3† for a list of oligonucleotides used in this study).

The wild-type (WT), monokaryotic D. squalens strain CBS464.89, derived from the WT dikaryon D. squalens FBCC312, was obtained from the FBCC-HAMBI culture collection (www.helsinki.fi/hambi/ accessed on 01 March 2018) and maintained at 28 °C on 2% (w/v) malt extract, 1.5% (w/v) agar (MEA) plates. The cultures for protoplast preparation were performed as previously described [33 (link)]. Transformants were selected on regeneration agar with 18–25 µg/mL geneticin (G-418, Roche, Mannheim, Germany) or 0.5–2 µg/mL carboxin (Sigma-Aldrich, St. Louis, MO, USA) or 1–2 mg/mL 5-fluoroorotic acid (5-FOA, Thermo Scientific, Vilnus, Lithuania) and 20 mM uridine (Molekula, Darlington, UK). The positive transformants were subcultured on MEA plates containing selective pressure and then maintained on MEA. For growth assays, mycelia-covered plugs (0.5 cm in diameter) from a freshly grown MEA plate were used to inoculate a low-nitrogen, asparagine-succinate (LN-AS, pH 4.5) medium with 1.5% (w/v) agar [35 (link)] and 25 mM of glucose, xylose, arabinose or galactose as a carbon source. Media of the uridine auxotrophic mutants were supplemented with 20 mM uridine. The strains used in this study are listed in Table 1.

Plasmid shuffling experiments were conducted following the protocol of Nathan et al.^{60 (link)}, using the ∆PCLDα S. cerevisiae strain from S. Lindquist’s laboratory deficient in genomic Hsp82 and Hsc82 containing a plasmid coding for WT Hsp82. The pKAT6 plasmid is constitutively expressed under the control of the glycerinaldehyde-3-phosphate dehydrogenase gene promotor (GPD promotor) and carries a URA selection marker for the selection of cells that have lost the WT Hsp82 plasmid in the medium supplemented with 5-Fluoroorotic Acid (5-FOA) (Thermo Fisher Scientific). The cells were transformed with either the empty vector p413 (negative control), the p413 vector, coding for the WT Hsp82 (positive control), and the p413 vector, coding for the Hsp82 R32A variant. Hsp82 is essential for yeast survival and the loss of the pKAT6 plasmid, due to 5-FOA-induced selection, inhibits yeast growth. Transformation of p413 vectors containing Hsp82 variants might restore yeast growth, depending on the characteristics of the Hsp82 variants.