Adaptive laboratory evolution and rational engineering enabled xylose utilisation and xylan conversion in natural...

Second-generation biofuels produced from renewable lignocellulosic biomass (LCB) are attractive alternatives to environmentally damaging, non-renewable fossil fuels. A key challenge in converting LCB to bioethanol is the incomplete utilisation of all available sugars. To address this, the hemicellulose fraction, consisting mainly of xylan, should be converted to the desired product alongside cellulose. This study aimed to develop natural isolate strains of Saccharomyces cerevisiae capable of xylose utilisation and xylan degradation. Strains YI13, YI59 and FIN1 were selected for potential industrial applications due to their high fermentation performance levels under environmental stress and enhanced ethanol production compared to laboratory strains. Xylose utilisation was achieved in these strains by introducing heterologous xylose isomerase (XI) and xylulokinase (XKS) gene cassettes and a xylose transporter (XTR), followed by adaptive laboratory evolution (ALE) in minimal xylose media. The evolved strains were further engineered for cell-associated xylosidase and secreted xylanase activities, yielding variants with strong enzyme activities, optimized xylose metabolism, and high ethanol production from both xylose and xylan. The final engineered version of YI13 showed the best xylose and xylan conversion, with maximum ethanol titres of ∼7.1 g/L from 20 g/L xylose and ∼4.7 g/L from 40 g/L xylan, among the highest ethanol titres from polymeric xylan by direct microbial conversion reported to date. The development of these S. cerevisiae strains provides a useful platform for future development of robust xylan-converting S. cerevisiae strains for large-scale ethanol production, although validation on real-world lignocellulosic feedstocks is still required.