Experimental evolution of Saccharomyces cerevisiae for caffeine tolerance alters multidrug resistance and target of rapamycin signaling pathways

Abstract

Caffeine is a natural compound that inhibits the major cellular signaling regulator target of rapamycin (TOR), leading to widespread effects including growth inhibition. Saccharomyces 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 showed 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.

Keywords: TOR signaling; caffeine; course-based research experience; experimental evolution; genome sequencing; multidrug resistance pathway; yeast.

Graphical Abstract

Fig. 1.

Evolution of caffeine tolerance. a) Growth curves of ancestors and evolved clones. Each curve represents 1 clone and is the average of 3 biological replicates. b) Doubling times for ancestors and evolved clones in a). Difference from ancestor by ANOVA with Tukey's honestly significant difference: *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Fig. 2.

Common mutations in caffeine-tolerant clones. a) Types of mutations in evolved clones with significantly increased caffeine tolerance. b) Genes with mutations in caffeine-tolerant evolved lineages and their prior support for connection to caffeine tolerance or TOR signaling. c) Doubling time of caffeine-tolerant clones grouped by mutations in MDR genes. Difference from ancestor by ANOVA with Tukey's honestly significant difference: ****P < 0.0001.

Fig. 3.

Gain-of-function mutations in MDR family genes contribute to cross-resistance to clotrimazole. a) Doubling time of WT (YMD5096) compared to clones with PDR1 and PDR3 mutations from caffeine (YMD4681 and YMD4684) or clotrimazole evolution experiments (YMD5097 and YMD5098). b) MDR transporters and their regulation by transcription factors based on Cui et al. (1998) and Kolaczkowska and Goffeau (1999), with effect on caffeine efflux from Tsujimoto et al. (2015). Dashed line arrows indicate basal activity; solid arrows indicate drug-induced activity. c) Reporter assay of PDR1 and PDR3 mutant strains expressing lacZ under the control of indicated promoters, grown 72 h on media containing X-gal. d) Growth in caffeine of CRISPR engineered strains with synonymous YRR1^T696= mutation, with or without YRR1^T699P mutation. e) Reporter assay of engineered YRR1 strains expressing lacZ under the control of indicated promoters, grown 72 h on media containing X-gal. Difference from wild type (WT) or synonymous by ANOVA with Tukey's honestly significant difference: **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Fig. 4.

Effect of deletions on caffeine and rapamycin tolerance. a) Doubling time and b) growth after 24 h in rapamycin for strains with indicated deletions. Difference from wild type (WT) by ANOVA with Tukey's honestly significant difference: *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Fig. 5.

Contributions of TOR effectors to caffeine tolerance. a) TOR signaling pathway (Huber et al. 2009; Numamoto et al. 2015; Ferrari et al. 2017; Ariño et al. 2019) annotated with mutations found in caffeine-tolerant clones in this study and prior literature support for effects of deletions on caffeine tolerance (Rempola et al. 2000; Brown et al. 2006; Reinke et al. 2006; Banuelos et al. 2010; Kapitzky et al. 2010; Hood-DeGrenier 2011). b) Doubling time for strains engineered to have synonymous TIP41^Q160=, with or without TIP41^S161X mutation. c) Growth after 24 h in 5 nM rapamycin for engineered strains. d) Doubling time for strains engineered to have SIT4 mutation; both have synonymous SIT4^L91= mutation. e) Doubling time for strains with SKY1 mutation; both have synonymous SKY1^T599= mutation. Difference from strain with only synonymous mutation by t-test: **P < 0.01 and ****P < 0.0001.