Assessing the role of deep eutectic solvents in Yarrowia lipolytica inhibition

Yarrowia lipolytica has gained recognition as a microorganism with biological relevance and extensive biotechnological applications. Some of its features include a high enzyme secretion capacity and a high cell-density fermentation mode. Hexokinase (YlHxk) is a vital enzyme in Y. lipolytica growth since it catalyzes glucose metabolism through phosphorylation in the glycolytic pathway. Given the potential application of deep eutectic solvents (DES) as novel solvents in biotechnological processes, this study evaluated the influence of eighteen DES on the growth of Y. lipolytica. Furthermore, this work examined the effects of individual ions on the YlHxk enzyme by analyzing its enzymatic tunnel structure, molecule transport, and molecular docking. The results revealed a significant reduction in yeast growth in the presence of most DES compared to the control (medium without DES), with the exception of the [N₈₈₈₁]Cl: hexanoic acid (1:1) DES. The growth varied between 11.95 ± 0.60 and 0.68 ± 0.17 g dry cell weight L^-1. According to the enzymatic tunnel analysis, DES components associated with the lowest microbial growth values were transported through tunnel 1. On the other hand, DES components had their pathway facilitated through tunnel 2 ([N₈₈₈₁]⁺ and hexanoic acid) and showed growth values close to the control. Molecular docking analysis identified a similarity between all the ligands in this tunnel (including substrate and product), presenting binding interactions with the ASN273 amino acid of the YlHxk active site. Combining experimental results with computational tools provided promising insights at the molecular level, while also potentially reducing analysis costs and time, paving the way for similar approaches in broad biocatalytic reactions.