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

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

doi:10.1007/978-1-0716-2399-2_9

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

Institut für Molekulare Biotechnologie (6550)

Publikation: Beitrag in Buch/Bericht/Konferenzband › Beitrag in Buch/Bericht › Begutachtung

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.

Originalsprache	englisch
Titel	Yeast Metabolic Engineering: Methods and Protocols
Redakteure/-innen	Valeria Mapelli, Maurizio Bettiga
Erscheinungsort	New York, U.S.A.
Herausgeber (Verlag)	Humana Press
Kapitel	9
Seiten	135-151
Seitenumfang	17
Auflage	2nd
ISBN (elektronisch)	978-1-0716-2399-2
ISBN (Print)	978-1-0716-2398-5
DOIs	https://doi.org/10.1007/978-1-0716-2399-2_9
Publikationsstatus	Veröffentlicht - 1 Juni 2022

Publikationsreihe

Name	Methods in Molecular Biology
Band	2513
ISSN (Print)	1064-3745
ISSN (elektronisch)	1940-6029

ASJC Scopus subject areas

Biochemie, Genetik und Molekularbiologie (insg.)
Genetik
Molekularbiologie

Fields of Expertise

Human- & Biotechnology

UN SDGs

Dieser Output leistet einen Beitrag zu folgendem(n) Ziel(en) für nachhaltige Entwicklung

Zugriff auf Dokument

10.1007/978-1-0716-2399-2_9Lizenz: Nicht definiert

Andere Dateien und Links

Verknüpfung zur Publikation in Scopus

Dieses zitieren

Kobalter, S., Radkohl, A., Schwab, H., Emmerstorfer-Augustin, A., & Pichler, H. (2022). Plasmid-based gene knockout strategy with subsequent marker recycling in Pichia pastoris. in V. Mapelli, & M. Bettiga (Hrsg.), Yeast Metabolic Engineering: Methods and Protocols (2nd Aufl., S. 135-151). (Methods in Molecular Biology; Band 2513). Humana Press. https://doi.org/10.1007/978-1-0716-2399-2_9

Plasmid-based gene knockout strategy with subsequent marker recycling in Pichia pastoris. / Kobalter, Simon; Radkohl, Astrid; Schwab, Helmut et al.
Yeast Metabolic Engineering: Methods and Protocols. Hrsg. / Valeria Mapelli; Maurizio Bettiga. 2nd. Aufl. New York, U.S.A.: Humana Press, 2022. S. 135-151 (Methods in Molecular Biology; Band 2513).

Publikation: Beitrag in Buch/Bericht/Konferenzband › Beitrag in Buch/Bericht › Begutachtung

Kobalter, S, Radkohl, A, Schwab, H, Emmerstorfer-Augustin, A & Pichler, H 2022, Plasmid-based gene knockout strategy with subsequent marker recycling in Pichia pastoris. in V Mapelli & M Bettiga (Hrsg.), Yeast Metabolic Engineering: Methods and Protocols. 2nd Aufl., Methods in Molecular Biology, Bd. 2513, Humana Press, New York, U.S.A., S. 135-151. https://doi.org/10.1007/978-1-0716-2399-2_9

@inbook{5b7a3ca220eb4d1887fc7f419f418fd7,

title = "Plasmid-based gene knockout strategy with subsequent marker recycling in Pichia pastoris",

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.",

keywords = "Gene disruption, Homologous recombination, Knockout plasmids, Marker recycling, Pichia pastoris",

author = "Simon Kobalter and Astrid Radkohl and Helmut Schwab and Anita Emmerstorfer-Augustin and Harald Pichler",

year = "2022",

month = jun,

day = "1",

doi = "10.1007/978-1-0716-2399-2_9",

language = "English",

isbn = "978-1-0716-2398-5",

series = "Methods in Molecular Biology",

publisher = "Humana Press",

pages = "135--151",

editor = "Valeria Mapelli and Maurizio Bettiga",

booktitle = "Yeast Metabolic Engineering: Methods and Protocols",

address = "United States",

edition = "2nd",

}

TY - CHAP

T1 - Plasmid-based gene knockout strategy with subsequent marker recycling in Pichia pastoris

AU - Kobalter, Simon

AU - Radkohl, Astrid

AU - Schwab, Helmut

AU - Emmerstorfer-Augustin, Anita

AU - Pichler, Harald

PY - 2022/6/1

Y1 - 2022/6/1

N2 - 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.

AB - 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.

KW - Gene disruption

KW - Homologous recombination

KW - Knockout plasmids

KW - Marker recycling

KW - Pichia pastoris

UR - http://www.scopus.com/inward/record.url?scp=85133216434&partnerID=8YFLogxK

U2 - 10.1007/978-1-0716-2399-2_9

DO - 10.1007/978-1-0716-2399-2_9

M3 - Chapter

SN - 978-1-0716-2398-5

T3 - Methods in Molecular Biology

SP - 135

EP - 151

BT - Yeast Metabolic Engineering: Methods and Protocols

A2 - Mapelli, Valeria

A2 - Bettiga, Maurizio

PB - Humana Press

CY - New York, U.S.A.

ER -