Accelerating yeast adaptation: harnessing thermal stress for genomic diversification and thermotolerance in Yarrowia lipolytica.

Temperature sensitivity remains a significant bottleneck in the industrial application of the oleaginous yeast Yarrowia lipolytica. While adaptive evolution and metabolic engineering have produced thermotolerant variants, the impact of thermal stress on the genomic integrity of this non-conventional yeast remains poorly understood. Here, we employed a mutation accumulation framework coupled with whole-genome sequencing to characterize the mutational landscape of Y. lipolytica at 37°C. We observed a 29- and 68-fold increase in single-nucleotide variations and small insertions/deletions, respectively, compared to optimal growth conditions (30°C). Mechanistic analysis revealed that this mutagenicity is independent of error-prone translesion and nonhomologous end joining pathways; instead, thermal stress overwhelms the replicative fidelity of DNA polymerases and the mismatch repair system. Furthermore, long-read sequencing identified severe telomere instability and large-scale chromosomal rearrangements, including the frequent formation of circular chromosomes and terminal fusions. Lastly, functional genomics revealed YALI1_B03404g to be a key negative regulator of heat resistance. Its deletion substantially enhances thermotolerance, a phenotype potentially mediated through the modulation of yeast-to-hypha transitions. Collectively, our findings enhance the understanding of genomic evolution in non-conventional yeasts under thermal stress and offer a valuable framework for developing robust industrial strains.