Presentation Information
[2BRBP-12-KL]Engineering natural Saccharomyces cerevisiae isolates for consolidated bioprocessing of lignocellulosic biomass
○Riaan den Haan1,2, Letitia Minnaar2, Francois Kruger2, Kentaro Inokuma3, Tomohisa Hasunuma3 (1. Stellenbosch University (South Africa), 2. University of the Western Cape (South Africa), 3. Kobe University (Japan))
Keywords:
Saccharomyces cerevisiae,consolidated bioprocessing,lignocellulosic biomass,xylanase,cellulase
Background: The transition from fossil fuels to second-generation biofuels is hindered by the inefficient conversion of lignocellulosic biomass (LCB), particularly the incomplete utilization of hemicellulose and cellulose fractions. While Saccharomyces cerevisiae is a preferred industrial chassis, standard laboratory strains often lack the robustness and metabolic pathways required for efficient consolidated bioprocessing (CBP). This study utilized natural S. cerevisiae isolates (YI13, YI59, and FIN1), selected for their superior fermentation performance under environmental stress. To enable xylose and xylan utilization, strains were engineered with heterologous xylose isomerase (XI), xylulokinase (XKS), and a xylose transporter (XTR), followed by adaptive laboratory evolution (ALE). These evolved strains were further modified to express cell-associated xylosidase and secreted xylanase activity. In parallel, different cellulase expression strategies, comparing cell-tethered versus free enzyme systems, were evaluated under process-relevant conditions.
Results: The engineered strain YI13 demonstrated notable hemicellulose conversion, achieving maximum ethanol titres of ~7.1 g/L from 20 g/L xylose and ~4.7 g/L from 40 g/L xylan. In mixed-sugar fermentations, ethanol production reached 17.5 g/L from 20 g/L glucose and 20 g/L xylose (0.45 g/g yield). Regarding cellulose utilization, the cell-tethered enzyme system significantly outperformed free enzymes, enhancing both protein titres and strain robustness. Specifically, strain YI59_V2 produced ~10 g/L of ethanol from crystalline cellulose—approximately 88% of the theoretical yield—without exogenous enzyme supplementation.
Conclusion: By combining rational engineering with ALE in robust natural isolates, this research successfully developed strains capable of direct microbial conversion of both xylan and cellulose. These results highlight the importance of expression design in improving secretory capacity and stress tolerance, marking a significant step toward the industrial deployment of cost-effective, one-step cellulosic ethanol production.
Results: The engineered strain YI13 demonstrated notable hemicellulose conversion, achieving maximum ethanol titres of ~7.1 g/L from 20 g/L xylose and ~4.7 g/L from 40 g/L xylan. In mixed-sugar fermentations, ethanol production reached 17.5 g/L from 20 g/L glucose and 20 g/L xylose (0.45 g/g yield). Regarding cellulose utilization, the cell-tethered enzyme system significantly outperformed free enzymes, enhancing both protein titres and strain robustness. Specifically, strain YI59_V2 produced ~10 g/L of ethanol from crystalline cellulose—approximately 88% of the theoretical yield—without exogenous enzyme supplementation.
Conclusion: By combining rational engineering with ALE in robust natural isolates, this research successfully developed strains capable of direct microbial conversion of both xylan and cellulose. These results highlight the importance of expression design in improving secretory capacity and stress tolerance, marking a significant step toward the industrial deployment of cost-effective, one-step cellulosic ethanol production.
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