Auxin-Induced Degradation of Target Proteins in Pichia pastoris.

The disruption of protein production is a major approach used for investigating protein function in a cell. In yeasts, this is mostly achieved by introducing an insertion or deletion ("indel") into a certain gene or by disrupting the whole reading frame. However, these methods have noteworthy limitations. For example, whole gene deletions may influence promoter or terminator regions of genes located up- and downstream of this particular gene; essential genes cannot simply be knocked out in order to study their function; and the overall absence of a protein can allow time for compensatory mechanisms that confound interpretation of results. To overcome these limitations, this chapter presents the application of the auxin-inducible degron (AID) technology for fast and efficient degradation of a target protein in Komagataella phaffii. The AID system is based on the formation of a chimeric SCF complex with the plant F-box protein TIR1, which, in the presence of auxin, leads to the polyubiquitination of the target sequence, the so-called AID tag. If the AID tag is fused to a protein of interest, this protein is immediately subjected to ubiquitin-mediated proteasome degradation upon incubation with auxin. In this work, the usability and efficiency of auxin-inducible degradation of Gut1, a well-studied glycerol kinase with an important role in glycerol metabolism, is demonstrated. Important parameters for experimental design for the use of this system in K. phaffii are being evaluated. The results prove that this toolkit can be expanded to all areas of K. phaffii-related research.