Genetic Improvement of a Wild-Type Saccharomyces cerevisiae Strain for Enhanced Xylitol Production
Xylitol is a sugar alcohol of interest in the food, pharmaceutical, and healthcare industries due to its applications as a food sweetener and sugar substitute. A wild-type Saccharomyces cerevisiae yeast strain designated 202-3 was isolated from a Colombian distillery located near sugarcane fields. This diploid 202-3 strain showed non-common, modest but noticeable xylose consumption and xylitol production in lignocellulosic hydrolysates. To enhance its natural xylose consumption ability, the strain was genetically engineered and submitted to adaptive laboratory evolution (ALE). Firstly, it was considered the deletion of the GAL80 gene to enable continuous expression of GAL genes, enhancing the uptake and assimilation of xylose. While the deletion of one copy of GAL80 (strain 202-3/∆) showed improved xylose consumption and xylitol production, better results were obtained when both copies of GAL80 were silenced (strain 202-3/∆∆). Subsequently, ALE experiments were conducted for these three strains in rich medium containing 20 g/L xylose. While the parental 202-3 strain consumed 2.46 g/L xylose and produced 0.42 g/L xylitol, the evolved 202-3/∆∆/ALE strain was able to consume 5.61 g/L xylose and produced 4.87 g/L xylitol, with a xylitol yield of 0.87 g xylitol/g xylose, and also the highest xylitol volumetric productivity (0.034 g xylitol/L/h) among the strains. Thus, our engineered and evolutionary experiments allowed a significant improvement in terms of xylose consumption, xylitol production and xylitol yield.