Insight into cryopreservation mechanism of natural deep eutectic solvents (NADESs) from the transcriptomics perspective

As a new cryoprotectant, natural deep eutectic solvents (NADESs) have been shown to achieve efficient and safe cryoprotection through excellent thermodynamic characteristics and strong recrystallisation inhibition ability. To further discover the cryoprotection mechanism of NADESs, the transcriptomics of yeast cells cryopreserved by different NADESs were examined. Notably, the PG cryoprotected yeast cells showed the highest viability (Q4: 81.39%) and the earliest regrowth peak (~ 6 h), indicating superior recovery performance. Transcriptomic analysis suggests that NADESs significantly modulate yeast cryotolerance. Differentially expressed genes (DEGs) were predominantly enriched in carbohydrate, amino acid, and fatty acid metabolism. Specifically, key genes involved in glycolysis, such as PCK1 , FBA1 , and ENO1 , were significantly regulated. Furthermore, the up-regulation of osmotic pressure-related genes ( STL1 , FPS1 ) in GF-cryopreserved yeast is consistent with observed osmotic changes. This suggests that proline-containing NADESs may help modulate osmotic pressure, thereby mitigating osmotic damage. Overall, this study supports a more comprehensive understanding of the cryopreservation mechanisms of emerging NADESs. Importantly, while these results support a role for NADES-induced shifts in glycolysis and osmoadaptation, the causality will be further validated in future work using HOG1 Δ, STL1 Δ, and FPS1 Δ strains, as well as pharmacologic MAPK inhibition. • Freezing negatively affects cell viability and cell proliferation. • NADES can prevent cell death and maintain cell proliferation ability. • NADES can activate the MAPK regulatory pathway. • The NADESs can significantly affect carbohydrate metabolism during freezing. • The presence of proline contributes to osmotic balance during freezing.