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

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

*Korrespondierende/r Autor/-in für diese Arbeit

Publikation: Beitrag in Buch/Bericht/KonferenzbandBeitrag in Buch/BerichtBegutachtung

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.
Originalspracheenglisch
TitelYeast Metabolic Engineering: Methods and Protocols
Redakteure/-innenValeria Mapelli, Maurizio Bettiga
ErscheinungsortNew York, U.S.A.
Herausgeber (Verlag)Humana Press
Kapitel9
Seiten135-151
Seitenumfang17
Auflage2nd
ISBN (elektronisch)978-1-0716-2399-2
ISBN (Print)978-1-0716-2398-5
DOIs
PublikationsstatusVeröffentlicht - 1 Juni 2022

Publikationsreihe

NameMethods in Molecular Biology
Band2513
ISSN (Print)1064-3745
ISSN (elektronisch)1940-6029

ASJC Scopus subject areas

  • Allgemeine Biochemie, Genetik und Molekularbiologie
  • Genetik
  • Molekularbiologie

Fields of Expertise

  • Human- & Biotechnology

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