The efficient conversion of lignocellulosic sugars into bioethanol is constrained by the inability of Saccharomyces cerevisiae to metabolize xylose and by its preference for glucose when both sugars are available. Although recombinant strains have been developed to improve xylose utilization, further optimization is needed to achieve robust co-fermentation performance. In this study, three parental strains were used: a wild-type S. cerevisiae strain (GF16), a genetically engineered S. cerevisiae strain capable of metabolizing xylose (TMB3001), and a reference strain of Scheffersomyces stipitis (ATCC 58376). From these, we obtained an evolved S. cerevisiae strain (F2C7A) through a combination of UV mutagenesis, protoplast fusion, and adaptive laboratory evolution. In synthetic medium containing only xylose, F2C7A consumed 87.9% of the sugar after 72 h, compared with only 52.3% by its parental hybrid strain, although, its biomass yield was lower (0.20 g/g vs. 0.35 g/g). Under mixed-sugar conditions, F2C7A consumed all available glucose and 33% of xylose within 48 h, producing ethanol at 0.45 g/g yield with minimal xylitol accumulation. In culture medium containing only xylose, it reached a biomass yield of 0.86 g/g and a xylitol yield of 0.11 g/g. Transcriptomic analysis revealed strong induction of XYL1, XYL2, tricarboxylic acid cycle genes, and oxidative phosphorylation components under xylose, consistent with a respiratory phenotype. Mixed-sugar cultures displayed a respirofermentative profile and reduced xylitol formation, suggesting improved redox balance in the presence of glucose. Several nonspecific sugar transporters (HXT8, HGT1, STL1) were overexpressed under xylose, indicating potential compensatory uptake mechanisms. Changes in nitrogen metabolism included upregulation of GLT1 and repression of GDH1, suggesting a shift toward NADH-dependent glutamate synthesis. These findings demonstrate that combining classical and evolutionary strategies can enhance xylose metabolism in S. cerevisiae, providing a foundation for further improvement of strains intended for lignocellulosic bioethanol production.

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Journal Article

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López-deÁvila LM, Monsalve-Fonnegra ZI, Rodríguez-Cabal HA

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