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The recently discovered wild yeast Wickerhamomyces sp. UFFS-CE-3.1.2 was analyzed through a high-throughput experimental design to improve ethanol yields in synthetic media with glucose, xylose, and cellobiose as carbon sources and acetic acid, furfural, formic acid, and NaCl as fermentation inhibitors. After Plackett–Burman (PB) and central composite design (CCD), the optimized condition was used in a fermentation kinetic analysis to compare this yeast's performance with an industrial Saccharomyces cerevisiae strain (JDY-01) genetically engineered to achieve a higher xylose fermentation capacity and fermentation inhibitors tolerance by overexpressing the genes XYL1, XYL2, XKS1, and TAL1. Our results show that furfural and NaCl had no significant effect on sugar consumption by UFFS-CE-3.1.2. Surprisingly, acetic acid negatively affected glucose but not xylose and cellobiose consumption. In contrast, the pH positively affected all the analyzed responses, indicating a cell's preference for alkaline environments. In the CCD, sugar concentration negatively affected the yields of ethanol, xylitol, and cellular biomass. Therefore, fermentation kinetics were carried out with the average concentrations of sugars and fermentation inhibitors and the highest tested pH value (8.0). Although UFFS-CE-3.1.2 fermented glucose efficiently, xylose and cellobiose were mainly used for cellular growth. Interestingly, the genetically engineered strain JDY-01 consumed ~ 30% more xylose and produced ~ 20% more ethanol. Also, while UFFS-CE-3.1.2 only consumed 32% of the acetic acid of the medium, JDY-01 consumed > 60% of it, reducing its toxic effects. Thus, the overexpressed genes played an essential role in the inhibitors' tolerance, and the applied engineering strategy may help improve 2G ethanol production. [ABSTRACT FROM AUTHOR]