Target of rapamycin (TOR) signaling is a nutrient-sensitive pathway that plays a role in cell growth and aging. Caffeine is a factor that inhibits TOR signaling and growth, compromising overall cellular fitness. The budding yeast S. cerevisiae is an ideal organism to study TOR signaling in relation to caffeine tolerance as there are many resources available to study yeast genetics, and yeast shares many basic biological properties with other eukaryotic cells. Additionally, yeast allows us to study TOR signaling using experimental evolution, where under a defined selective pressure - here, caffeine - we observe what rare beneficial mutations arise and increase in frequency. While many inputs to the TOR signaling pathway are known, others are yet to be identified. Furthermore, how other pathways compensate for TOR signaling inhibition is not completely understood. Our goal is to identify factors involved in TOR signaling by growing yeast in inhibitory concentrations of caffeine to select for better growing mutants with increased resistance. We will then sequence the resistant strains’ genomes and study the resultant mutations to determine how they connect to TOR signaling and caffeine tolerance. We evolved yeast for 5-10 weeks in increased doses of caffeine, and sequenced clones from the evolved populations. Our yeast evolved increased caffeine tolerance, and mutations arose in drug-response pathways including in the Pdr1 transcription factor. We also observed mutations in processes regulated by TOR, such as nutrient sensing. With further genome sequencing of more evolved populations we aim to identify novel mutations and factors involved in caffeine tolerance and TOR signaling. Ultimately, these findings increase our understanding of how caffeine impacts TOR signaling and how other cellular processes are regulated by TOR signaling. More broadly, this research can aid in the continued development of how cell signaling pathways are related to nutrient response, aging, and growth.

The genetic makeup of beer-brewing yeast plays an essential role in determining the flavor profile during production of beer. With functional copies of genes on the MAL locus, beer yeasts, namely Saccharomyces cerevisiae, can utilize maltose and maltotriose as their carbon source. The three genes in this locus are responsible for the regulation (MALx3), transport across membrane (MALx1), and breakdown of sugars (MALx2) in brewers’ wort. If a strain lacks a functional copy of any of these three genes, it cannot digest these alternative sugars. Alternatively, some strains have more than one MAL locus, but which loci are functional, remains unknown. Apart from previous studies that have investigated a handful of MAL alleles, the function of the genes in these duplicated loci (or paralogs) cannot be determined based on sequences alone. To address this problem, I experimentally tested the function of alleles from 1,011 natural isolates. I focused on MALx3 because the reference strain lacks a functional MALx3 allele, preventing its growth in maltose. Therefore, introduction of any functional MALx3 alleles should permit growth in maltose. To test this, I cloned ~250 MALx3 alleles from three different loci (MAL1, MAL3, MAL7) and transformed the reference strain to generate three yeast libraries. The library with the MALx3 gene of the MAL3 locus, or MAL33, successfully grew to saturation after ~2.5 days of incubation in 2% maltose, showing this approach can be used to determine the function of MAL33 alleles. Looking forward, I will use DNA barcodes to track the growth of alleles in maltose to pinpoint which are functional. With the MAL loci serving as a great candidate for understanding paralog differences, by identifying the functional paralogs, we can better understand the evolutionary history of MAL genes and what role these loci play in the brewery and across all natural isolates.