Improving Stress Tolerance in Industrial Saccharomyces cerevisiae Strains for Ethanol Production from Lignocellulosic Biomass

Abstract: The present work was aimed at developing industrial S. cerevisiae strains with improved tolerance to two types of stressors encountered during the fermentation of lignocellulosic biomass that affect ethanol yield and productivity, namely hydrolysate-derived inhibitors and high temperature, and at understanding the response of yeast and mechanisms of adaptation to such stressors. In one part of the study, key amino acid substitutions that were responsible for the acquired ability of a mutated yeast enzyme to convert HMF, one of the lignocellulosic inhibitors (LI), into a less inhibitory compound were identified in the active site of the enzyme. The specific properties of the mutant were investigated. In the second part of the thesis,
different strategies were applied to develop yeast strains with increased tolerance to combined stresses. In one approach, the effects of two targeted proteins that were previously shown to be involved in the response to oxidative stress in laboratory yeast strains were re-evaluated under process-mimicking conditions, i.e. using a robust industrial strain background and fermenting highly inhibitory spruce
hydrolysate. The beneficial effects on tolerance to LI were confirmed in the industrial strain, but they were shown to be strain-dependent and limited to the fermentation of 6-carbon sugars (C6); unexpected
negative interactions were also identified for one of the candidates in the fermentation of C5 sugars. The second approach focused on improving the tolerance to high temperature in the presence of LI. A strain with combined tolerance to both stressors was obtained by long-term adaptation. In contrast to its parental strain, the evolved strain was capable of growing and fermenting C6 in the presence of LI and at high temperature (39°C). Possible mechanisms behind the improved performance of this strain were investigated using genome-wide approaches. Significant differences were found in lipid composition,which correlated with changes at the genome level in different genes involved in lipid transport, synthesis,
and other steps of lipid metabolism, thereby indicating that alterations in membrane composition may be behind the improved combined tolerance. Overall, the work performed for this thesis resulted in the development of several strains with improved characteristics that are suitable for fermentation of LI. The
work has also contributed to a better understanding of the mechanisms of stress response in yeast.