High-Throughput Transformation of Chemical Competent Pichia pastoris Cells Using Lithium Acetate/Chloride and Carrier...

One of the most commonly used Komagataella phaffii transformation protocols is electroporation. Electroporation, i.e., applying an electric field to cells with the consequence that the cell membrane becomes permeable, is considered a fast and efficient method for introducing recombinant DNA into K. phaffii cells. The transformation method provides, depending on the quality of the electrocompetent cells, transformation efficiency between 2.1 × 10⁴ and 1.1 × 10⁵ CFU/μg of linear DNA (Kumar, Yeast 36(6):399-410, 2019). However, electroporation as a transformation method is hardly adaptable to a 96-well format and thus inconvenient for performing several transformations in parallel.In this chapter, we describe protocols to produce chemically competent K. phaffii cells using lithium acetate/chloride and the subsequent transformation of the cells supported by the use of carrier DNA. Although this transformation procedure is more laborious, time-consuming, and potentially suffers from a lower transformation efficiency (1 × 10² and 1 × 10³ CFU/μg DNA) than the conventional electroporation method, the lithium acetate/chloride transformation protocol is beneficial when very high transformation rates such as in the case of library generation are not essential.