Lignocellulose-based biorefineries have been gaining increasing attention to substitute current petroleum-based refineries. Biomass processing requires a pretreatment step to break lignocellulosic biomass recalcitrant structure, which results in the release of a broad range of microbial inhibitors, mainly weak acids, furans, and phenolic compounds. Saccharomyces cerevisiae is the most commonly used organism for ethanol production; however, it can be severely distressed by these lignocellulosederived inhibitors, in addition to other challenging conditions, such as pentose sugar utilization and the high temperatures required for an efficient simultaneous saccharification and fermentation step. Therefore, a better understanding of the yeast response and adaptation towards the presence of these multiple stresses is of crucial importance to design strategies to improve yeast robustness and bioconversion capacity from lignocellulosic biomass. This review includes an overview of the main inhibitors derived from diverse raw material resultants from different biomass pretreatments, and describes the main mechanisms of yeast response to their presence, as well as to the presence of stresses imposed by xylose utilization and high-temperature conditions, with a special emphasis on the synergistic effect of multiple inhibitors/stressors. Furthermore, successful cases of tolerance improvement of S. cerevisiae are highlighted, in particular those associated with other process-related physiologically relevant conditions. Decoding the overall yeast response mechanisms will pave the way for the integrated development of sustainable yeast cell-based biorefineries.

In this work, four robust yeast chassis isolated from industrial environments were engineered with the same xylose metabolic pathway. The recombinant strains were physiologically characterized in synthetic xylose and xylose-glucose medium, on non-detoxified hemicellulosic hydrolysates of fast-growing hardwoods (Eucalyptus and Paulownia) and agricultural residues (corn cob and wheat straw) and on Eucalyptus hydrolysate at different temperatures. Results show that the co-consumption of xylose-glucose was dependent on the yeast background. Moreover, heterogeneous results were obtained among different hydrolysates and temperatures for each individual strain pointing to the importance of designing from the very beginning a tailor-made yeast considering the specific raw material and process.

Background: Xylose isomerase (XI) and xylose reductase/xylitol dehydrogenase (XR/XDH) pathways have been extensively used to confer xylose assimilation capacity to Saccharomyces cerevisiae and tackle one of the major bottlenecks in the attainment of economically viable lignocellulosic ethanol production. Nevertheless, there is a lack of studies comparing the efficiency of those pathways both separately and combined. In this work, the XI and/or XR/ XDH pathways were introduced into two robust industrial S. cerevisiae strains, evaluated in synthetic media and corn cob hemicellulosic hydrolysate and the results were correlated with the differential enzyme activities found in the xylose-pathway engineered strains. Results: The sole expression of XI was found to increase the fermentative capacity of both strains in synthetic media at 30 °C and 40 °C: decreasing xylitol accumulation and improving xylose consumption and ethanol production. Similar results were observed in fermentations of detoxified hydrolysate. However, in the presence of lignocellulosicderived inhibitors, a positive synergistic effect resulted from the expression of both XI and XR/XDH, possibly caused by a cofactor equilibrium between the XDH and furan detoxifying enzymes, increasing the ethanol yield by more than 38%. Conclusions: This study clearly shows an advantage of using the XI from Clostridium phytofermentans to attain high ethanol productivities and yields from xylose. Furthermore, and for the first time, the simultaneous utilization of XR/ XDH and XI pathways was compared to the single expression of XR/XDH or XI and was found to improve ethanol production from non-detoxified hemicellulosic hydrolysates. These results extend the knowledge regarding S. cerevisiae xylose assimilation metabolism and pave the way for the construction of more efficient strains for use in lignocellulosic industrial processes.

In this work, the industrial Saccharomyces cerevisiae PE-2 strain, presenting innate capacity for xylitol accumulation, was engineered for xylitol production by overexpression of the endogenous GRE3 gene and expression of different xylose reductases from Pichia stipitis. The best-performing GRE3-overexpressing strain was capable to produce 148.5 g/L of xylitol from high xylose-containing media, with a 0.95 g/g yield, and maintained close to maximum theoretical yields (0.89 g/g) when tested in non-detoxified corn cob hydrolysates. Furthermore, a successful integrated strategy was developed for the production of xylitol from whole slurry corn cob in a presaccharification and simultaneous saccharification and fermentation process (15% solid loading and 36 FPU) reaching xylitol yield of 0.93 g/g and a productivity of 0.54 g/L·h. This novel approach results in an intensified valorization of lignocellulosic biomass for xylitol production in a fully integrated process and represents an advance towards a circular economy.

Acetic acid tolerance and xylose consumption are desirable traits for yeast strains used in industrial biotechnological processes. In this work, overexpression of a weak acid stress transcriptional activator encoded by the gene HAA1 and a phosphoribosyl pyrophosphate synthetase encoded by PRS3 in a recombinant industrial Saccharomyces cerevisiae strain containing a xylose metabolic pathway was evaluated in the presence of acetic acid in xylose- or glucose-containing media. HAA1 or PRS3 overexpression resulted in superior yeast growth and higher sugar consumption capacities in the presence of 4 g/L acetic acid, and a positive synergistic effect resulted from the simultaneous overexpression of both genes. Overexpressing these genes also improved yeast adaptation to a non-detoxified hardwood hydrolysate with a high acetic acid content. Furthermore, the overexpression of HAA1 and/or PRS3 was found to increase the robustness of yeast cell wall when challenged with acetic acid stress, suggesting the involvement of the modulation of the cell wall integrity pathway. This study clearly shows HAA1 and/or, for the first time, PRS3 overexpression to play an important role in the improvement of industrial yeast tolerance towards acetic acid. The results expand the molecular toolbox and add to the current understanding of the mechanisms involved in higher acetic acid tolerance, paving the way for the further development of more efficient industrial processes.

The biorefinery concept, consisting in using renewable biomass with economical and energy goals, appeared in response to the ongoing exhaustion of fossil reserves. Bioethanol is the most prominent biofuel and has been considered one of the top chemicals to be obtained from biomass. Saccharomyces cerevisiae, the preferred microorganism for ethanol production, has been the target of extensive genetic modifications to improve the production of this alcohol from renewable biomasses. Additionally, S. cerevisiae strains from harsh industrial environments have been exploited due to their robust traits and improved fermentative capacity. Nevertheless, there is still not an optimized strain capable of turning second generation bioprocesses economically viable. Considering this, and aiming to facilitate and guide the future development of effective S. cerevisiae strains, this work reviews genetic engineering strategies envisioning improvements in 2 nd generation bioethanol production, with special focus in process-related traits, xylose consumption, and consolidated bioprocessing. Altogether, the genetic toolbox described proves S. cerevisiae to be a key microorganism for the establishment of a bioeconomy, not only for the production of lignocellulosic bioethanol, but also having potential as a cell factory platform for overall valorization of renewable biomasses.

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PRS3, RPB4 and ZWF1 were previously identified as key genes for yeast tolerance to lignocellulose-derived inhibitors. To better understand their contribution to yeast resistance to the multiple stresses occurring during lignocellulosic hydrolysate fermentations, we overexpressed these genes in two industrial Saccharomyces cerevisiae strains, CCUG53310 and PE-2, and evaluated their impact on the fermentation of Eucalyptus globulus wood and corn cob hydrolysates. PRS3 overexpression improved the fermentation rate (up to 32%) and productivity (up to 48%) in different hydrolysates. ZWF1 and RPB4 overexpression did not improve the fermentation performance, but their increased expression in the presence of acetic acid, furfural and hydroxymethylfurfural was found to contribute to yeast adaptation to these inhibitors. This study expands our understanding about the molecular mechanisms involved in industrial yeast tolerance to the stresses occurring during lignocellulosic bioethanol production and highlights the importance of selecting appropriate strain backgrounds/hydrolysates combinations when addressing further improvement of these processes.