Ammonium sulphate

C. parapsilosis type strain CDC317 was batch cultured at 30 ºC in orbital agitation (250 rpm) in yeast extract–peptone–dextrose (YPD) for inoculating cultivation and in synthetic minimal media (SMM) for growth parameter determination. Media compositions were as follows: YPD: 20 g/L glucose (Merck, Darmstadt, Germany), 20 g/L peptone (Merck, Darmstadt, Germany), and 10 g/L yeast extract (Merck, Darmstadt, Germany); SMM: 20g/L glucose (Merck, Darmstadt, Germany), 2.7 g/L ammonium sulphate (Merck, Darmstadt, Germany), 0.05 g/L magnesium sulphate (Riddle-de-Haen), 2 g/L potassium dihydrogen phosphate (Panreac, Barcelona, Spain), 0.5 g/L calcium chloride (Merck, Darmstadt, Germany), and 100 µg/L biotin (Sigma).

A single colony of the recombinant strain SMB carrying the pFLDhα/L2 was inoculated in 10 mL YPD broth at 30 °C and 250 rpm overnight. Then, 100 μL of the culture were inoculated into 100 mL of MGAs [0.17% (w/v) YNB (Becton Dickinson, Franklin Lakes, NJ, USA), 0.5% (w/v) ammonium sulphate (Merck, GE), 1% (v/v) glycerol (Merck, DE), 4 × 10⁻⁵% (w/v) biotin (Sigma Aldrich, St. Louis, MO, USA)], and cultivated under the same condition until OD_600nm = 4. Then, the cells were harvested at 3000× g and room temperature (RT) for 5 min. The cell pellet was resuspended in 20 mL MMAs [same composition as MGAs with the exception that glycerol was replaced with 0.5% (v/v) methanol (Merck, DE)] and cultivation was continued for 5 days. methanol [0.5% (v/v)] was added every 24 h for induction. The strain SMB containing the empty vector (pFLDhα) was used as the control. Aliquots (15 mL) of the cultures were harvested at 8000× g and RT for 3 min every 24 h of post-induction. Finally, the supernatant was assayed colourimetrically using the previously described method at 70 °C. Recombinant strain SMB/pFLDhα/L2lipase (pFαS3) with the highest lipase expression was selected for further optimization.

Exophiala dermatitidis EXF-10123 strain (CBS 525.76; isolation source: human) was obtained from the Culture Collection Ex Infrastructural Centre Mycosmo, part of MRIC UL, Department of Biology, Biotechnical Faculty, University of Ljubljana, Ljubljana, Slovenia. Cultures were maintained on malt extract agar (MEA), incubated at 37 °C, for up to seven days. For the determination of morphological features, the fungus was cultured on MEA and oat meal agar (OA) and incubated for two weeks. For all other experiments, it was cultured on defined yeast nitrogen base (YNB) medium: 17% (w/v) YNB (Formedium, Hunstanton, UK), 0.5% (w/v) ammonium sulphate (Merck Millipore, Darmstadt, Germany), 2.0% (w/v) D-glucose in deionised water, pH 7.0. Fungal cell suspensions for the inoculation of media used for the determination of the assimilation of hydrocarbons and neurotransmitters were prepared by growing E. dermatitidis on MEA and preparing a suspension of 10⁵ cells/mL. In addition, S. cerevisiae Y00000 (S288c) wild type strain, provided by the Euroscarf collection (Scientific Research and Development GmbH, Oberursel, Germany), was cultured in the same media and at the same conditions as E. dermatitidis.