Engineering Xylose Fermentation in an Industrial Yeast: Continuous Cultivation as a Tool for Selecting Improved Strains

ABSTRACT Production of second-generation ethanol from lignocellulosic residues should be fueling the energy matrix in the near future. Lignocellulosic feedstock has received much attention as an alternative energy resource for biorefineries toward reducing the demand for fossil resources, contributing to a future sustainable bio-based economy. Fermentation of lignocellulosic hydrolysates poses many scientific and technological challenges as the drawback of Saccharomyces cerevisiae’s inability in fermenting pentose sugars (derived from hemicellulose). To overcome the inability of S. cerevisiae to ferment xylose and increase yeast robustness in the presence of inhibitory compound-containing media, the industrial S. cerevisiae strain SA-1 was engineered using CRISPR-Cas9 with the oxidoreductive xylose pathway from Scheffersomyces stipitis (encoded by XYL1, XYL2 , and XYL3 ). The engineered strain was then cultivated in a xylose-limited chemostat under increasing dilution rates (for 64 days) to improve its xylose consumption kinetics under aerobic conditions. The evolved strain (DPY06) and its parental strain (SA-1 XR/XDH) were evaluated under anaerobic conditions in complex media. DPY06 consumed xylose faster, exhibiting an increase of 70% in xylose consumption rate at 72h of cultivation compared to its parental strain, indicating that laboratory evolution improved xylose uptake of SA-1 XR/XDH. GRAPHICAL ABSTRACT