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

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

Methods in molecular biology (Clifton, N.J.) 2022

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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.

Citation

Simon Kobalter, Astrid Radkohl, Helmut Schwab, Anita Emmerstorfer-Augustin, Harald Pichler. Plasmid-Based Gene Knockout Strategy with Subsequent Marker Recycling in Pichia pastoris. Methods in molecular biology (Clifton, N.J.). 2022;2513:135-151