General

Plasmids were constructed using NEBuilder HiFi DNA assembly (New England Biolabs, Frankfurt am Main, Germany) and SLiCE cloning (Zhang et al., 2012). Plasmid and primer sequences are given in Supplementary Tables 1, 2, respectively. PCR amplification of DNA fragments was performed using high-fidelity polymerases: Phusion Polymerase (Thermo Fisher Scientific), Q5 DNA Polymerase (New England Biolabs, Frankfurt am Main, Germany), or PrimeSTAR GXL DNA Polymerase (Takara Bio, Saint-Germain-en-Laye, France) according to the manufacturer’s recommendations. All restriction enzymes were purchased from New England Biolabs (Frankfurt am Main, Germany). Amplified DNA fragments were gel-purified prior to further use. The primers were ordered from Biomers.net (Ulm, Germany). Escherichia coli ER2925, NEB 5α, or NEB 10β cells (New England Biolabs) were transformed with the plasmids. Strains were grown in Luria-Bertani medium containing an appropriate selection marker at 37°C (Ampicillin, 100 μg/mL or Kanamycin, 50 μg/mL). The plasmid constructs were confirmed by sequencing (Microsynth Seqlab, Goettingen, Germany).

Genetic transformation of the plasmids or linearized DNA fragments from S. cerevisiae was performed using the LiAc/SS carrier DNA/PEG method (Gietz and Schiestl, 2007). The adopted LiAc/SS carrier DNA/PEG method was used to transform P. pastoris with plasmids or linearized DNA fragments (Ito et al., 2018). The yeast strains were grown at 30°C in yeast extract peptone dextrose adenine (YPDA)-rich medium [1% (w/v) bacto yeast extract, 2% (w/v) peptone, 2% (w/v) glucose, 0.04% (w/v) adenine hemisulfate, and 2% (w/v) agar for solid medium] or in an appropriate synthetic complete (SC) media [0.67% (w/v) yeast nitrogen base with ammonium sulfate, 2% (w/v) glucose, and 2% (w/v) agar for solid medium] lacking amino acids to allow selection for transformed cells. When required, glucose was replaced by 2% (w/v) glucose. Single copy integration of each linearized plasmid into the GUT1 site of the P. pastoris genome was verified by colony PCR using primers GNPP001 and GNPP002.