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^*

^*Corresponding author for this work

Institute of Molecular Biotechnology (6550)

Research output: Chapter in Book/Report/Conference proceeding › Chapter › peer-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 language	English
Title of host publication	Yeast Metabolic Engineering: Methods and Protocols
Editors	Valeria Mapelli, Maurizio Bettiga
Place of Publication	New York, U.S.A.
Publisher	Humana Press
Chapter	9
Pages	135-151
Number of pages	17
Edition	2nd
ISBN (Electronic)	978-1-0716-2399-2
ISBN (Print)	978-1-0716-2398-5
DOIs	https://doi.org/10.1007/978-1-0716-2399-2_9
Publication status	Published - 1 Jun 2022

Publication series

Name	Methods in Molecular Biology
Volume	2513
ISSN (Print)	1064-3745
ISSN (Electronic)	1940-6029

Keywords

Gene disruption
Homologous recombination
Knockout plasmids
Marker recycling
Pichia pastoris

ASJC Scopus subject areas

General Biochemistry,Genetics and Molecular Biology
Genetics
Molecular Biology

Fields of Expertise

Human- & Biotechnology

UN SDGs

This output contributes to the following UN Sustainable Development Goals (SDGs)

Access to Document

10.1007/978-1-0716-2399-2_9Licence: Unspecified

Cite this

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 (Eds.), Yeast Metabolic Engineering: Methods and Protocols (2nd ed., pp. 135-151). (Methods in Molecular Biology; Vol. 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. ed. / Valeria Mapelli; Maurizio Bettiga. 2nd. ed. New York, U.S.A.: Humana Press, 2022. p. 135-151 (Methods in Molecular Biology; Vol. 2513).

Research output: Chapter in Book/Report/Conference proceeding › Chapter › peer-review

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 (eds), Yeast Metabolic Engineering: Methods and Protocols. 2nd edn, Methods in Molecular Biology, vol. 2513, Humana Press, New York, U.S.A., pp. 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 -

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

Abstract

Publication series

Keywords

ASJC Scopus subject areas

Fields of Expertise

UN SDGs

Access to Document

Other files and links

Cite this