bisy e.U. 1 article published in JoVE Bioengineering Methanol Independent Expression by Pichia Pastoris Employing De-repression Technologies Jasmin Elgin Fischer1, Anna-Maria Hatzl2, Astrid Weninger2, Christian Schmid2, Anton Glieder1,2 1bisy e.U., 2Institute of Molecular Biotechnology, Technical University of Graz This method for the first time describes reliable experimental procedures for efficient inducible methanol-free recombinant protein production in Pichia pastoris (Komagataella phaffii), employing the carbon source regulated promoters PDCand PDF in small scale experiments while cultivation parameters can be monitored online, and a constant glycerol feed is applied.