A Rapid Lithium Ion‐Based Transformation Method for Kluyveromyces lactis

Kluyveromyces lactis is a budding yeast host useful for recombinant protein production; however, its transformation often relies on electroporation due to the perceived low efficiency of chemical methods, thereby limiting throughput. In this study, we developed a robust, small‐scale chemical transformation protocol (using 15 μL of competent cells) that achieves high efficiency, particularly with linear DNA. No significant differences in efficiency were observed between lithium chloride and lithium acetate, nor across concentrations ranging from 0.1 to 1.0 M, for making cells competent. The addition of dimethyl sulfoxide (DMSO) during heat shock enhanced colony counts, while ethanol inhibited the process. When PCR fragments containing the optimized pan‐genomic autonomous replication sequence (PanARS‐OPT) were used for transformation, the transformation efficiency was approximately 7‐fold higher than that of circular plasmids, yielding over 10 5 transformants/μg DNA. This high efficiency with linear DNA can be due to a potent non‐homologous end‐joining (NHEJ) activity in K. lactis . This rapid, “one‐tube” method eliminates the need for plasmid construction and specialized equipment, offering a tool for rapid and high‐throughput genetic engineering in K. lactis .