High-Efficiency Transformation of Integrative Libraries Under Zeocin Selection in Pichia pastoris

High-efficiency transformation is often a major bottleneck in the generation of large and diverse libraries. This is especially the case for the yeast Komagataella phaffii, where integration in the genome and selection via Zeocin are often desired but negatively affect the transformation efficiency.This chapter describes an improved transformation protocol for integrative plasmids in K. phaffii using Zeocin selection that achieves a high efficiency (10⁵ integrative clones per μg DNA) in a time-saving manner. Additionally, we show that by fine-tuning Zeocin concentration, the number of multi-insert transformants can be controlled. This method should be valuable for the construction of large-pooled libraries in K. phaffii and can be applied in multiple fields of study.