Adaptive Laboratory Evolution of Pichia pastoris by Serial Batch Cultivation.

Adaptive laboratory evolution (ALE) is a research tool widely applied for the improvement and investigation of microorganisms. Multiple yeast species have been effectively targeted with this method, and Komagataella phaffii specifically is now well-suited for ALE experimentation due to advancements in its characterization and genetic manipulation. Here is presented a simple and generalizable protocol for the adaptation of K. phaffii towards growth on minimal media with methanol as the sole carbon source, along with suggestions on how this protocol may be adapted for other purposes. We give detailed steps for the repeated subculturing as well as recommended pre-experimental testing and post-experimental analysis. We also include notes and tips from hands-on experience with this methodology as applied specifically to K. phaffii.