Cultivation of E. coli, P. pastoris, and S. cerevisiae

Escherichia coli KRX (Promega, USA) was used for plasmid construction and propagation work. KRX was cultivated in LB (Lysogeny Broth) medium supplemented with 100 µg/mL ampicillin. For experiments involving P. pastoris CBS 7435 (ΔHIS4), obtained from ACIB (Austrian Center of Industrial Biotechnology, Austria) as well as the wild type CBS 7435 (CECT 11047 at Spanish Type Culture Collection, Spain), were used. Saccharomyces cerevisiae wild type strain LBG H620 was provided by the Institute for Agricultural Bacteriology and Fermentation Biology, ETH Zurich, Switzerland. Yeast shake flask cultivations were carried out in BMD (Buffered Minimal Dextrose) [49 ] or YPD (Yeast Peptone Dextrose) medium, supplemented with 4 mg/L l-histidine when necessary. Experiments in 96-deep-well plates with 2.4 mL total volume (Eppendorf, Germany) used BMD, BMM2 (Buffered Minimal Methanol) and BMM10 as previously described by Weis et al. [30 (link)] and Hartner et al. [31 (link)]. In brief, BMD is used for the growth phase while BMM2 and BMM10 induce expression of the target gene by maintaining a 0.5 % (v/v) methanol content in the culture medium. The 96-deep-well plates contained up to 500 µL of culture media, were sealed with sterile Breathseal film (Greiner, Germany) and were shaken at 340 rpm at 28 °C.