*3.8. Heterologous Expression of PsaPOX in Komagataella pfa*ffi*i*

The gene of PsaPOX was amplified with a C-terminal 6x His tag using the primers PsaPOX\_fw 5 -AAAAAGAATTCatgactacacctgcaccacccctcgacctcaacaa-3 and PsaPOX\_rev 5 -atatatGCGGCCGC tcaGTGGTGATGGTGATGATGggtagagatcggagcctgggcctg-3 (underlined are the EcoRI and NotI restriction sites, respectively; lower cases represent parts of the coding *PsaPOX*). In addition, it was inserted in frame with the *Saccharomyces cerevisiae* α-factor secretion signal sequence into the *K. pfa*ffi*i* pPIC9 expression vector (Invitrogen, Karlsruhe, Germany). The resulting expression construct pPIC-PsaPOX-His was transformed into *E. coli* TOP10 for vector propagation, isolated (NucleoSpin, Macherey-Nagel, Düren, Germany), linearized with PmeI, and used for transformation of *K. pfa*ffi*i* GS115 according to a standard protocol [48]. The linearized empty vector was transformed in the same way and served as negative control. Forty-eight transformants were tested for peroxidase activity after selection according to their ability to grow on histidine-deficient agar plates in 96-well plates for 120 h, as described elsewhere [49]. Gene expression was induced by daily addition of 1% (*v*/*v*) methanol.
