Adaptive Evolution Identifies MHY1 ‐Mediated Transcriptional Reprogramming for Polyphenol Tolerance in Yarrowia lipolytica

Polyphenols are bioactive compounds used in food, beverages, cosmetics, and medicine. Despite their expanding use, the mechanisms of polyphenol toxicity in microbial polyphenol producers and the adaptive strategies that confer tolerance, remain poorly characterized. Here, we applied adaptive laboratory evolution to evolve tolerance to three structurally distinct polyphenols, curcumin, naringenin, and resveratrol in Yarrowia lipolytica. Whole-genome resequencing of tolerant strains revealed recurrent loss-of-function mutations in the morphogenetic transcription factor MHY1 (YALI1_B28150g) and large segmental duplications on chromosome E, indicating multiple evolutionary solutions. Notably, resveratrol-evolved strains showed strong tolerance in the absence of MHY1 mutations, instead relying on chromosome-scale copy-number variation, highlighting compound-specific adaptive routes. Functional reconstruction demonstrated that MHY1 inactivation is sufficient to confer high-level tolerance, as both gene deletion (ΔMHY1) and independent point mutations (F240L, C153*) reproduce the tolerant phenotype across polyphenols. Transcriptomic analysis showed that MHY1 inactivation is associated with a distinct regulatory state characterized by reduced transcriptional representation of stress-associated pathways and relative stabilization of core metabolic functions. Together, these results identify MHY1 loss-of-function as a dominant and transferable regulatory mechanism for polyphenol tolerance, while also revealing alternative genome-level adaptations selected under severe chemical stress.