Resurrection and characterization of ancestral xylose transporters enhance the capability of xylose uptake in the mixed sugar co-fermentation of Recombinant Saccharomyces cerevisiae.

Ancestral sequence reconstruction (ASR) offers a revolutionary approach to resurrect functional proteins, yet its potential in transporter engineering remains underexplored. Here, we pioneered the application of ASR to reconstructing ancestral xylose transporters, addressing the persistent challenge of glucose-mediated inhibition of xylose uptake in Saccharomyces cerevisiae during xylose co-fermentation. Through rigorous ASR analysis, we reconstructed ancestral xylose transporters (Xt) and selected two candidates—Xt3 (approximately 140 million years old) and Xt7 (approximately 40 million years old)—based on their phylogenetic positioning, degree of sequence divergence from extant homologs, and predicted structural integrity. Functional characterization demonstrated that both Xt3 and Xt7 significantly enhance xylose uptake efficiency and mitigate glucose-induced repression. In fermentation experiments with mixed sugars (40 g/L xylose and 40 g/L glucose) within 72 h, recombinant S. cerevisiae expressing Xt3 achieved 22.75 g/L xylose consumption, surpassing the benchmark N326FXltr1p (16.22 g/L) by 40.27% and outperforming Xt7 (21.36 g/L) by 6.51%, highlighting Xt3 as the most efficient transporter. Molecular docking suggested a potentially more favorable binding mode for xylose in the ancestral transporters (binding affinity: −3.68 kcal/mol for Xt3 vs. −3.15 kcal/mol for N326FXltr1p). Molecular dynamics simulations further demonstrated that the ancestral transporters formed complexes with xylose that exhibited faster convergence to a stable state and maintained significantly greater conformational stability throughout the simulation compared to the N326FXltr1p complex. These computational insights provide a plausible structural basis for their enhanced performance. This work contributes to the advancement of lignocellulosic biorefinery technology and provides a practical reference for resurrecting other valuable proteins using ASR’.