TY - JOUR
T1 - Applying the auxin-based degron system for the inducible, reversible and complete protein degradation in Komagataella phaffii
AU - Lehmayer, Leonie
AU - Bernauer, Lukas
AU - Emmerstorfer-Augustin, Anita
N1 - Funding Information:
This work was supported by Erwin Schroedinger Fellowship J3787-B21 from the Austrian Science Fund (to A. E.-A.), and by BioTechMed-Graz Young Researcher Group project 'STEM' (to L. L, L. B., and A- E.-A.). This work was also aided, in part, by Astrid Radkohl and Franziska Gruber ( Graz University of Technology ), and Christoph Augustin ( Medical University of Graz ). Also, we want to thank Prof. Jeremy Thorner and Dr. Françoise Roelants for providing expression plasmids, as well as Prof. Harald Pichler and Prof. Anton Glieder for helpful discussions.
Funding Information:
This work was supported by Erwin Schroedinger Fellowship J3787-B21 from the Austrian Science Fund (to A. E.-A.), and by BioTechMed-Graz Young Researcher Group project 'STEM' (to L. L, L. B. and A- E.-A.). This work was also aided, in part, by Astrid Radkohl and Franziska Gruber (Graz University of Technology), and Christoph Augustin (Medical University of Graz). Also, we want to thank Prof. Jeremy Thorner and Dr. Françoise Roelants for providing expression plasmids, as well as Prof. Harald Pichler and Prof. Anton Glieder for helpful discussions. L. L. executed and analyzed experiments, and drafted the article, L. B. executed and analyzed experiments, and A. E.-A. designed, executed, and analyzed experiments, and drafted the article. The authors declare no competing interests.
Publisher Copyright:
© 2022 The Author(s)
PY - 2022/9/16
Y1 - 2022/9/16
N2 - The auxin-inducible degron (AID) system is a useful technique to rapidly deplete any protein of interest “on-demand.” In this study, we successfully established the AID system for the “biotech” yeast Komagataella phaffii. First, we tested different expression levels of TIR1 for auxin-induced degradation of the glycerol kinase Gut1. Moderate expression of TIR1 resulted in complete degradation of the target protein within several minutes. Second, we show that the absence of all three Wsc type sensors is detrimental to cell growth, which indicates that these are the dominant cell wall sensors this yeast. Third, down-regulation of Erg1, an essential enzyme of the ergosterol biosynthetic pathway, resulted in quick and efficient accumulation of squalene, a pharmaceutically relevant reagent. We conclude that AID is an extremely powerful tool that, for the first time, enables the analysis of gene essentiality and function in K. phaffii.
AB - The auxin-inducible degron (AID) system is a useful technique to rapidly deplete any protein of interest “on-demand.” In this study, we successfully established the AID system for the “biotech” yeast Komagataella phaffii. First, we tested different expression levels of TIR1 for auxin-induced degradation of the glycerol kinase Gut1. Moderate expression of TIR1 resulted in complete degradation of the target protein within several minutes. Second, we show that the absence of all three Wsc type sensors is detrimental to cell growth, which indicates that these are the dominant cell wall sensors this yeast. Third, down-regulation of Erg1, an essential enzyme of the ergosterol biosynthetic pathway, resulted in quick and efficient accumulation of squalene, a pharmaceutically relevant reagent. We conclude that AID is an extremely powerful tool that, for the first time, enables the analysis of gene essentiality and function in K. phaffii.
KW - microbiology
KW - molecular biology
UR - http://www.scopus.com/inward/record.url?scp=85136231012&partnerID=8YFLogxK
U2 - 10.1016/j.isci.2022.104888
DO - 10.1016/j.isci.2022.104888
M3 - Article
AN - SCOPUS:85136231012
SN - 2589-0042
VL - 25
JO - iScience
JF - iScience
IS - 9
M1 - 104888
ER -