In silico rational design and functional analysis of novel glucose-insensitive sugar 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 consists of the expression of 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 that had lower affinity for glucose and maintained 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 phenotype of interest were transformed into 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 is not yet characterized, it was decided to proceed with the characterization of the wild transporter. The drop assay showed that there was reproducibility between the docking predictions and the in vivo analysis. The mutants of Str3 (Tr62380) did indeed lose their natural affinity for hexoses. In addition, Tr82309 naturally has high specificity for xylose. In the aerobic fermentation assay, only Str3 (Tr62380)_WT had high efficiency in the uptake of sugars from the medium; the mutations inserted in Str3 (Tr62380) reduced the ability to transport sugars, mainly glucose. Phosphorylation mimetics showed for the first time in vivo that this post-translational modification can modulate the affinity of sugar transporters for different substrates. Thus, we also present phosphorylation sites as a new target for engineering studies of sugar transporters. Docking was an excellent tool for screening the engineering targets of the transporters studied. However, experimental validation is indispensable.