In silico approaches to increase selectivity of sugar transporters from Trichoderma reesei for xylose

Metabolic engineering of Saccharomyces cerevisiae for pentose utilization (e.g., xylose and arabinose) has been explored to improve fermentation performance in second-generation (2G) ethanol production. However, due to the intrinsic nonspecificity of sugar transporters, competition between sugars (e.g., xylose and glucose) occurs, leading to reduced pentose transport efficiency and lower ethanol productivity. This study aimed to develop and characterize sugar transporters with a lower affinity for glucose and maintaining the ability to transport xylose through genetic improvement of Trichoderma reesei transporters for heterologous expression in S. cerevisiae. Motifs and phosphorylation sites were predicted to screen for the phenotype of interest. Wild-type and mutant transporters were modeled using the Robetta online server, and molecular docking was performed using Autodock Vina. Based on these results, the transporters of interest were transformed into an S. cerevisiae strain for characterization. To confirm the predicted profile, a drop assay and aerobic fermentation were performed. In silico analysis showed 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-type transporter. The ORFS were sequenced and confirmed. The drop assay showed reproducibility between the docking predictions and the in vivo analysis. The Str3 (Tr62380) mutants indeed lost affinity for hexoses and maintained affinity for xylose, as predicted. Furthermore, Tr82309 naturally has high xylose selectivity. In the aerobic fermentation assay, only Str3(Tr62380)_WT showed high efficiency in capturing sugars from the medium. The mutations inserted into Str3(Tr62380) reduced its glucose transport capacity, which was interesting for the objectives of this study. Among the interesting mutations, phosphorylation mimetics demonstrated for the first time in vivo that this post-translational modification can modulate the affinity of sugar transporters for different substrates. Thus, we suggest phosphorylation sites as targets for engineering xylose transporters. Docking was an excellent tool for screening engineering targets for the transporters studied, and experimental validation is essential to confirm the viability and fermentation efficiency of the transporters.