Insights into cell robustness against lignocellulosic inhibitors and insoluble solids in bioethanol production processes

Increasing yeast robustness against lignocellulosic-derived inhibitors and insoluble solids in bioethanol production is essential for the transition to a bio-based economy. This work evaluates the effect exerted by insoluble solids on yeast tolerance to inhibitory compounds, which is crucial in high gravity processes. Adaptive laboratory evolution (ALE) was applied on a xylose-fermenting Saccharomyces cerevisiae strain to simultaneously increase the tolerance to lignocellulosic inhibitors and insoluble solids. The evolved strain gave rise to a fivefold increase in bioethanol yield in fermentation experiments with high concentration of inhibitors and 10% (w/v) of water insoluble solids. This strain also produced 5% (P > 0.01) more ethanol than the parental in simultaneous saccharification and fermentation of steam-exploded wheat straw, mainly due to an increased xylose consumption. In response to the stress conditions (solids and inhibitors) imposed in ALE, cells induced the expression of genes related to cell wall integrity (SRL1, CWP2, WSC2 and WSC4) and general stress response (e.g., CDC5, DUN1, CTT1, GRE1), simultaneously repressing genes related to protein synthesis and iron transport and homeostasis (e.g., FTR1, ARN1, FRE1), ultimately leading to the improved phenotype. These results contribute towards understanding molecular mechanisms that cells might use to convert lignocellulosic substrates effectively.