Docking-guided and phosphomimetic rational engineering of glucose-insensitive xylose transporters from Trichoderma reesei

The engineering of Saccharomyces cerevisiae for the use of xylose is fundamental to improving fermentation performance in the production of second-generation ethanol (2G) via pentose fermentation. For this, one of the main strategies involves expressing heterologous xylose transporters to ensure efficient uptake of this sugar. However, due to the intrinsic non-specificity of sugar transporters, competition occurs between sugars (e.g., xylose and glucose), leading to reduced pentose transport efficiency and lower ethanol productivity. This study aimed to develop and characterize sugar transporters with lower affinity for glucose, while maintaining the ability to transport xylose, through genetic improvement of Trichoderma reesei transporters for heterologous expression in S. cerevisiae. To this end, alignments were made to find motifs described as important for xylose transport, and phosphorylation sites were predicted to achieve the objective. Based on these predictions, the transporters were modeled and docked with glucose and xylose. The transporters with the desired phenotype were expressed in S. cerevisiae strains for characterization. Drop assays and aerobic fermentation trials were performed to confirm the predicted profile. In silico analysis shows that two mutations in Str3 (Tr62380) exhibited a promising phenotype. For Tr82309, which has not yet been characterized, it was decided to proceed with characterizing the wild transporter. Drop assays revealed qualitative differences in growth phenotypes among the tested transporters. Docking was used strictly as a qualitative, hypothesis-generating tool to prioritize mutations for experimental testing and was not intended to predict transport kinetics or substrate affinity. The mutants of Str3 (Tr62380) did indeed lose their natural affinity for hexoses. Additionally, Tr82309 showed limited activity in liquid assays but supported growth at higher xylose concentrations in solid medium, suggesting condition-dependent functionality rather than confirmed substrate specificity. In the aerobic fermentation assay, only Str3 (Tr62380)_WT exhibited high efficiency in the uptake of sugars from the medium; mutations inserted in Str3 (Tr62380) reduced the ability to transport sugars, primarily glucose. Phosphorylation mimetics provided in vivo evidence that post-translational modification can influence the substrate utilization profile of sugar transporters. Docking served as a qualitative screening step to guide the selection of mutations for experimental validation. We also present phosphorylation sites as a new target for engineering studies of sugar transporters. However, experimental validation is indispensable. In summary, our findings provide a rational framework for the engineering of glucose-insensitive xylose transporters, enabling more efficient co-fermentation in biorefineries.