Plasmid-Based Gene Knockout Strategy with Subsequent Marker Recycling in Pichia pastoris.

Autor:	Kobalter S; Graz Institute of Molecular Biotechnology, University of Technology, Graz, Austria.; Austrian Centre of Industrial Biotechnology (ACIB), Graz, Austria., Radkohl A; Graz Institute of Molecular Biotechnology, University of Technology, Graz, Austria., Schwab H; Graz Institute of Molecular Biotechnology, University of Technology, Graz, Austria.; Austrian Centre of Industrial Biotechnology (ACIB), Graz, Austria., Emmerstorfer-Augustin A; Graz Institute of Molecular Biotechnology, University of Technology, Graz, Austria.; Austrian Centre of Industrial Biotechnology (ACIB), Graz, Austria., Pichler H; Graz Institute of Molecular Biotechnology, University of Technology, Graz, Austria. harald.pichler@tugraz.at.; Austrian Centre of Industrial Biotechnology (ACIB), Graz, Austria. harald.pichler@tugraz.at.
Jazyk:	angličtina
Zdroj:	Methods in molecular biology (Clifton, N.J.) [Methods Mol Biol] 2022; Vol. 2513, pp. 135-151.
DOI:	10.1007/978-1-0716-2399-2_9
Abstrakt:	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. (© 2022. Springer Science+Business Media, LLC, part of Springer Nature.)
Databáze:	MEDLINE
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