Plasmid-based gene knockout strategy with subsequent marker recycling in Pichia pastoris

Simon Kobalter, Astrid Radkohl, Helmut Schwab, Anita Emmerstorfer-Augustin, Harald Pichler*

*Corresponding author for this work

Research output: Chapter in Book/Report/Conference proceedingChapterpeer-review

Abstract

Gene knockout is a key technology in the development of cell factories and basic research alike. The methylotrophic yeast Pichia pastoris is typically employed as a producer of proteins and of fine chemicals, due to its ability to accumulate high cell densities in conjunction with a set of strong inducible promoters. However, protocols for genome engineering in this host are still cumbersome and time-consuming. Moreover, extensive genome engineering raises the need for a multitude of selection markers, which are limited in P. pastoris. In this chapter, we describe a fast and efficient method for gene disruption in P. pastoris that utilizes marker recycling to enable repetitive genome engineering cycles. A set of ready-to-use knockout vectors simplifies cloning procedures and facilitates quick knockout generation.
Original languageEnglish
Title of host publicationYeast Metabolic Engineering: Methods and Protocols
EditorsValeria Mapelli, Maurizio Bettiga
Place of PublicationNew York, U.S.A.
PublisherHumana Press
Chapter9
Pages135-151
Number of pages17
Edition2nd
ISBN (Electronic)978-1-0716-2399-2
ISBN (Print)978-1-0716-2398-5
DOIs
Publication statusPublished - 1 Jun 2022

Publication series

NameMethods in Molecular Biology
Volume2513
ISSN (Print)1064-3745
ISSN (Electronic)1940-6029

Keywords

  • Gene disruption
  • Homologous recombination
  • Knockout plasmids
  • Marker recycling
  • Pichia pastoris

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)
  • Genetics
  • Molecular Biology

Fields of Expertise

  • Human- & Biotechnology

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