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

Abstract: Popular Abstract in English Oil resources are finite; and political and economic factors affecting oil production,together with the environmental damage associated with the increased use of oil by a rapidly growing population, have underscored the need for developing cleaner technologies based on sustainable resources. In this context, any lignocellulosic biomass that is not used in the food chain, such as woody crops and agricultural and forestry by-products, is an appealing raw material for the production of so-called “second-generation (2G) biofuels”, and more specifically 2G ethanol (as opposed to the “first-generation (1G) ethanol”, which is based on edible cellulosic biomass). Saccharomyces cerevisiae, commonly known as baker’s yeast (or even yeast) is the preferred microorganism for 1G ethanol production on a large scale, due to its capacity to convert the six-carbon (C6) sugars fast and efficiently. However due to the chemical properties of, and the wide sugar distribution in lignocellulosic biomass, there are specific challenges associated with 2G ethanol production in yeast. In particular, a pretreatment step is required to release all the sugar monomers from the complex lignocellulose chains and make them available for fermentation. During this step, inhibitory substances of different types are produced at the same time and their presence in the hydrolysate affects the fermentation performance of yeast. So the aim of the present work was to investigate the mechanisms of yeast tolerance to these inhibitors with or without other types of stressors encountered during the fermentation of lignocellulosic substrates, in order to develop efficient yeast biocatalysts for 2G ethanol production. In one part of the study, a mutated yeast enzyme responsible for the conversion of furaldehyde compounds, which are one group of lignocellulosic inhibitors (LI), into a less inhibitory compound, was studied and key mutated amino acids responsible for this conversion were identified. In the second part of the thesis, different strategies were used to develop yeast strains with increased tolerance to combined stresses. Two targeted proteins that were involved in the response to oxidative stress under laboratory conditions were re-evaluated under process-mimicking conditions. The beneficial effect on tolerance to LI was confirmed, although it was limited to the fermentation of C6 sugars, and unexpected negative interactions were identified for one candidate in the fermentation of C5 sugars. A second approach concentrated on obtaining a yeast strain with combined tolerance to LI and high temperature, as increased thermotolerance reduces production costs. Using long-term evolution under selective selection pressure, so-called “evolutionary engineering”, a strain capable of growing and fermenting in the presence of LI and at high temperature (39°C) was generated. Significant differences in the lipid composition of the evolved strain were found, which were confirmed by changes at the genome level in different genes involved in lipid transport, synthesis, and other steps of lipid metabolism, thereby implicating alterations in the composition of the yeast membrane as being responsible for combined tolerance. Overall, the work performed for this thesis resulted in the development of several strains with improved characteristics that were suitable for fermentation of LI. The work also contributed to a better understanding of the mechanisms of stress response in yeast.