Discovery of Molecular Factors in the Non‐ CYP51A Route for Tebuconazole Resistance: An Adaptive Laboratory Evolution Research

Saccharomyces cerevisiae is a well‐established model organism for studying antifungal responses and resistance mechanisms. Its well‐characterized genetics and conserved cellular processes provide a useful framework for investigating responses to azole fungicides such as tebuconazole. The present work aimed to discover molecular factors underlying tebuconazole resistance in S. cerevisiae . For this purpose, genetically stable, tebuconazole‐resistant S. cerevisiae mutants were obtained by adaptive laboratory evolution (ALE) and were investigated at the physiological and genomic level to determine molecular factors responsible for the tebuconazole resistance. ALE analysis showed that tebuconazole‐resistant mutants could be developed without using any physical and chemical mutagens prior to the selection, demonstrating a potential mutagenic effect of tebuconazole. The resistance analyses showed that the mutants could still survive at tebuconazole levels up to 40 μg/mL (130 μM). The cross‐resistance and cell wall degradation analyses revealed that the selected three mutants exhibited resistance to cell membrane disruption (0.006% SDS), acid stress (30 mM acetic acid), lignocellulosic hydrolysate stress (8 mM vanillin), antifungal stress (100 μg/mL prochloraz), oxidative stress (1 mM H 2 O 2 ), and cell wall disruption (lyticase), compared to the wild‐type strain. Whole genome sequencing (WGS) analysis indicated that three mutants possessed 16 common nonsynonymous variations, and they were distributed into eight genes ( Q0182 , FLO9 , FIT1 , COS8 , YHL008C , HSP150, YMR057C , and YMR306C‐A ) independent of the CYP51A pathway. Biological process annotation analysis by STRING revealed that the mutant proteins and their interacting partners were associated with cell wall‐related functions (Hsp150p), iron metabolism (Fit1p), flocculation (Flo9p), and largely uncharacterized biological processes (Cos8p and Q0182). According to YEASTRACT analysis to determine the potential transcription factors responsible for tebuconazole resistance, Yap1p, Pdr3p, and Rpn4p were identified as common transcriptional regulators of the six mutated genes found in the three mutants, suggesting that these transcription factors may contribute to the development of tebuconazole resistance. The findings of this study suggested that FLO9 , FIT1 , COS8 , HSP150 , YHL008C , Q0182, YMR057C , and YMR306C‐A were novel genes whose genetic variations were associated with tebuconazole resistance through a non‐CYP51A (ERG11)‐mediated mechanism.