Experimental evolution of S. cerevisiae for caffeine tolerance alters multidrug resistance and TOR signaling pathways.

Caffeine is a natural compound that inhibits the major cellular signaling regulator TOR, leading to widespread effects including growth inhibition. S. cerevisiae yeast can adapt to tolerate high concentrations of caffeine in coffee and cacao fermentations and in experimental systems. While many factors affecting caffeine tolerance and TOR signaling have been identified, further characterization of their interactions and regulation remain to be studied. We used experimental evolution of S. cerevisiae to study the genetic contributions to caffeine tolerance in yeast, through a collaboration between high school students evolving yeast populations coupled with further research exploration in university labs. We identified multiple evolved yeast populations with mutations in PDR1 and PDR5, which contribute to multidrug resistance, and showed that gain-of-function mutations in multidrug resistance family transcription factors PDR1, PDR3, and YRR1 differentially contribute to caffeine tolerance. We also identified loss-of-function mutations in TOR effectors SIT4, SKY1, and TIP41, and show that these mutations contribute to caffeine tolerance. These findings support the importance of both the multidrug resistance family and TOR signaling in caffeine tolerance, and can inform future exploration of networks affected by caffeine and other TOR inhibitors in model systems and industrial applications.

yEvo: A modular eukaryotic genetics and evolution research experience for high school students.

The resources for carrying out and analyzing microbial evolution experiments have become more accessible, making it possible to expand these studies beyond the research laboratory and into the classroom. We developed five connected, standards-aligned yeast evolution laboratory modules, called "yEvo," for high school students. The modules enable students to take agency in answering open-ended research questions. In Module 1, students evolve baker's yeast to tolerate an antifungal drug, and in subsequent modules, investigate how evolved yeasts adapted to this stressful condition at both the phenotype and genotype levels. We used pre- and post-surveys from 72 students at two different schools and post-interviews with students and teachers to assess our program goals and guide module improvement over 3 years. We measured changes in student conceptions, confidence in scientific practices, and interest in STEM careers. Students who participated in yEvo showed improvements in understanding of activity-specific concepts and reported increased confidence in designing a valid biology experiment. Student experimental data replicated literature findings and has led to new insights into antifungal resistance. The modules and provided materials, alongside "proof of concept" evaluation metrics, will serve as a model for other university researchers and K - 16 classrooms interested in engaging in open-ended research questions using yeast as a model system.

Caffeine-tolerant mutations selected through an at-home yeast experimental evolution teaching lab.

yEvo is a curriculum for high school students centered around evolution experiments in S. cerevisiae . To adapt the curriculum for remote instruction, we created a new protocol to evolve non-engineered yeast in the presence of caffeine. Evolved strains had increased caffeine tolerance and distinct colony morphologies. Many possessed copy number variations, transposon insertions, and mutations affecting genes with known relationships to caffeine and TOR signaling - which is inhibited by caffeine - and in other genes not previously connected with caffeine. This demonstrates that our accessible, at-home protocol is sufficient to permit novel insights into caffeine tolerance.

Caffeine-tolerant mutations selected through an at-home yeast experimental evolution teaching lab.

yEvo is a curriculum for high school students centered around evolution experiments in S. cerevisiae . To adapt the curriculum for remote instruction, we created a new protocol to evolve non-GMO yeast in the presence of caffeine. Evolved strains had increased caffeine tolerance and distinct colony morphologies. Many possessed copy number variations, transposon insertions, and mutations affecting genes with known relationships to caffeine and TOR signaling - which is inhibited by caffeine - and in other genes not previously connected with caffeine. This demonstrates that our accessible, at-home protocol is sufficient to permit novel insights into caffeine tolerance.