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.
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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: Mapelli, V., Bettiga, M. (eds) Yeast Metabolic Engineering. Methods in Molecular Biology, vol 2513. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2399-2_9
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DOI: https://doi.org/10.1007/978-1-0716-2399-2_9
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