Experimental evolution of Saccharomyces cerevisiae for caffeine tolerance alters multidrug resistance and target of rapamycin signaling pathways
Authors: Geck Renee C , Moresi Naomi G , Anderson Leah M ,   yEvo Students, Addepalli Isabel , Aggarwal Deepti , Agnihotri Prisha , Ali Ahlaam A , Amorosi Clara J , Anand Abhinav , Atukuri Ashna , Awi Thang , Basrai Insiya , Bathala Hitha , Bhide Sarang , Cantor Benjamin B , Cervantes Jocelyn , Chakraborty Tridib , Champlin James , Chbihi Ameen , Chen Felicia , Chenfang Hayley , Choi Reagan , Chokka Sebastian , D'Souza Julian , Dandu Vivek , Das Arkesh , Dawoud Margrette , Dong Victoria , Dutta Riya , Edoff Graeme , Fan Cecelia , Foo Rena , Glanville Liam T , Golat Cristian , Grewal Suhavi , Guan Faye , Gurav Aarya , Gupta Aranav , Gupta Krish , Gupta Siya , Hameed Osman , Hede-Sakhardande Rhea , Hossain Nushaba , Ibrahim Youssef , Isaac Jemi , Jalf Udayvir , Jasti Medha , Jazvin Amar , Johnny Okichy , Kamat Vidhi , Kandula Venya , Katuri Lekhana , Kebede Keabe E , Khuperkar Om , Kim Emily , Konduru Rishi , Kyaw Salimah , Lee Daniel , Syun Lin Tian , Luo Karen , Magsoosi Jwan , Mahmood Mlahat , Marzan Ronald Brent F , Masud Noyonima , Mathew Jessica , Miciuda Ava , Morey Trevor , Nair Anagha , Natarajan Naveen , Abdul Nazeer Aahil , Noa Usoatua Levei P , Pagadala Shashank , Paik Hamin , Palomino John , Parikh Kush , Phadke Naisha , Phan Michelle V , Pingree Britta , Podhuturi Neal , Prasad Arya , Puri Sonia , Rajkumar Sanjini , Ramanan Ananya , Russell Elliot M , Saad Zachary L , Sabalsa Gaytan Magdalena , Salazar Francis L , Sanil Anjali , Shah Neespruha , Sharba Mustafa , Sharma Prihensha , Showman Sophia , Showman Soyeon , Shyn Heejin , Singh Aryan , Sovani Saakshi , Srugaram Shreya , Stroia Rachel , Sunilkumar Sanjana , Suthy Nihil , Syed Asma , Thavamani Ruthesh , Upadhye Nitya , Varghese Rebecca , Wang Annie , Wang Cynthia , Wang Roger , Watson Miya A , Wei Theresa , Woody Myra L , Yao Nancy , Yee Tyler , Cindy Yeh Chiann-Ling , Yoon Jungbin , Zhou Jiaying , Zhu Tianhui , Fredstrom Noah , Pennington Sandra , Counihan Scarlett , Burris Owen , Jimenez Garcia Marisol , Godin Dennis , Brewer Rebecca , Renz Timothy R , Taylor Matthew Bryce , Dunham Maitreya J 
Published: July 2024

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.