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Gene-targeted Random Mutagenesis to Select Heterochromatin-destabilizing Proteasome Mutants in Fission Yeast
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Pichia pastoris Random Mutagenesis and Screening.

Christine Winkler1, Joan Lin-Cereghino2, Geoff P Lin-Cereghino3

  • 1Institute of Molecular Biotechnology, Graz University of Technology, Graz, Austria.

Methods in Molecular Biology (Clifton, N.J.)
|October 1, 2025
PubMed
Summary
This summary is machine-generated.

Random mutagenesis in Komagataella phaffii enhances protein secretion. This method identifies novel genes that improve the production of recombinant proteins like horseradish peroxidase and beta-galactosidase.

Keywords:
Genome walkingInsertional mutagenesisKomagataella phaffiiPichia pastorisProtein secretionRestriction enzyme-mediated integration (REMI)

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Area of Science:

  • Biotechnology
  • Molecular Biology
  • Yeast Genetics

Background:

  • Random mutagenesis is crucial for understanding gene function and molecular pathways.
  • Komagataella phaffii is a methylotrophic yeast utilized for recombinant protein production.

Purpose of the Study:

  • To establish protocols for random mutagenesis in Komagataella phaffii.
  • To screen for mutants with enhanced recombinant protein secretion.
  • To identify novel genes involved in improving protein secretion.

Main Methods:

  • Random mutagenesis via non-homologous DNA cassette insertion in K. phaffii.
  • Identification of mutagenized loci using cassette-specific primers.
  • Screening for enhanced secretion of horseradish peroxidase (HRP) and β-galactosidase.

Main Results:

  • Developed and validated two protocols for random mutagenesis in K. phaffii.
  • Successfully enhanced the secretion of HRP and β-galactosidase.
  • Identified previously unknown genes that positively impact recombinant protein secretion.

Conclusions:

  • The developed random mutagenesis system is effective for gene discovery in K. phaffii.
  • This methodology holds significant potential for optimizing recombinant protein production in yeast.
  • The identified genes offer new targets for further research in protein secretion enhancement.