Development of a hardened industrial strain of S. cerevisiae for bioethanol production from sugarcane bagasse hydrolysates

Sugarcane bagasse is a priority source of industrially available lignocellulose for producing residue-based fuels using microbes such as Saccharomyces cerevisiae. This process requires pre-treatment (such as dilute acid or steam explosion) of the lignocellulosic material, which often releases the monomeric sugars (glucose and xylose) and selected microbial inhibitors. A key bottleneck, however, remains the limited xylose ability and toxicity of the released microbial inhibitors that negatively affect the fermentation ability of the yeast. Hence, this study engineered the industrial, xylose-utilizing Saccharomyces cerevisiae CelluX^TM4 strain for improved resistance to pre-treatment-derived microbial inhibitors by overexpressing various genes associated with inhibitor resistance phenotypes. Combinations of six homologous genes were overexpressed through 3 rounds of genomic integrations, resulting in the C4TP1 and C4TP3 groups of transformants. These were screened in 50% w/w sugarcane hydrolysate fermentations under industrially relevant conditions for bioethanol production. Key findings show that the highest bioethanol titers were from C4TP1 and C4TP3 transformants, ranging from 1.8–35.2 g/L, which far outperformed the 2.1–3.2 g/L achieved by the CelluX^TM4 industrial strain. Moreover, the TFA3.3 and TFA1.4 transformants achieved 39.4 and 40.1 g/L bioethanol titers, respectively. Thus, the overexpressed genes contributed to the improved tolerance to inhibitors, resulting in a step-change improvement in fermentation performance.