pPM2-ZWF1

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	The pPM2 expression vectors overexpressing single P. pastoris genes ZWF1, SOL3, GND2, and RPE1 under the control of strong, constitutive glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter were described previously (Nocon et al. 2014).

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The pPM2 expression vectors overexpressing single P. pastoris genes ZWF1, SOL3, GND2, and RPE1 under the control of strong, constitutive glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter were described previously (Nocon et al. 2014).

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All P. pastoris strains used in this study, apart from the wild-type strain X-33 (Invitrogen), were based on the strain intracellularly producing human superoxide dismutase (Marx et al. 2009). The pPM2 expression vectors overexpressing single P. pastoris genes ZWF1, SOL3, GND2, and RPE1 under the control of strong, constitutive glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter were described previously (Nocon et al. 2014). For generation of double overexpression vectors, a second expression cassette, consisting of GAP promoter, gene, and terminator was inserted into vectors already containing one expression cassette using the restriction sites ApaI, MreI, and AgeI that generate overlapping ends. For this purpose, the expression cassettes of SOL3 and RPE1 were excised using MreI and AgeI and ligated into ZWF1 or GND1 expression vectors linearized by ApaI and MreI. The vectors were linearized in the genome integration locus (either 3′-region of AOX1 or the 5′-region of ENO1) and integrated into the genome of electrocompetent P. pastoris cells (Gasser et al. 2013). Hygromycin (HphMX) or Geneticin (KanMX) resistance were used as selection markers. Positive transformants were selected on YPD containing 500 μg/mL Zeocin and 500 μg/mL G418 or 200 μg/mL hygromycin. Additionally, to combine the first two PPP steps, a P. pastoris strain overexpressing SOL3 was transformed with the ZWF1 overexpression vector after recycling of the selection marker with Cre recombinase (Marx et al. 2008).

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