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Lignocellulose-derived biofuels present an attractive carbon-neutral alternative to fossil fuels amidst growing global energy and climate concerns. Bioethanol in particular, has been shown to be a viable additive to and / or replacement for petroleum in countries such as Brazil. The bioconversion of lignocellulose biomass to bioethanol is a developing technology with the yeast Saccharomyces cerevisiae playing a pivotal role in the fermentation-based processes. This yeast however, is challenged to ferment under harsh conditions, specifically, during exposure to lignocellulose-derived microbial inhibitors that are formed during pretreatment processes. This detrimentally affects biocatalyst performance, ultimately decreasing ethanol productivity and yield. To this end, S. cerevisiae strains are in development to increase the yeast microbial inhibitor resistance towards cost-effective cellulosic bioethanol production. This review discusses the current status of inhibitor resistance in S. cerevisiae strains and perspectives on the future of multi-tolerant phenotypes with a specific focus on existing and emerging strain development strategies designed to improve resistance phenotypes.

Natural Saccharomyces cerevisiae isolates from vineyards in the Western Cape, South Africa were evaluated for ethanol production in industrial conditions associated with the production of second-generation biofuels. The strains displayed high phenotypic diversity including the ability to grow at 45 °C and in the presence of 20% (v/v) ethanol, strain YI13. Strains HR4 and YI30 were inhibitor-tolerant under aerobic and oxygen-limited conditions, respectively. Spore-to-spore hybridization generated progeny that displayed heterosis, including increased ethanol productivity and improved growth in the presence of a synthetic inhibitor cocktail. Hybrid strains HR4/YI30#6 and V3/YI30#6 were able to grow at a high salt concentration (2 mol/L NaCl) with V3/YI30#6 also able to grow at a high temperature (45 °C). Strains HR4/YI30#1 and #3 were inhibitor-tolerant, with strain HR4/YI30#3 having similar productivity (0.36 ± 0.0036 g/L per h) as the superior parental strain, YI30 (0.35 ± 0.0058 g/L per h). This study indicates that natural S. cerevisiae strains display phenotypic variation and heterosis can be achieved through spore-to-spore hybridization. Several of the phenotypes (temperature-, osmo-, and inhibitor tolerance) displayed by both the natural strains and the generated progeny were at the maximum conditions reported for S. cerevisiae strains.

The second-generation (2G) fermentation environment for lignocellulose conversion presents unique challenges to the fermentative organism that do not necessarily exist in other industrial fermentations. While extreme osmotic, heat, and nutrient starvation stresses are observed in sugar-and starch-based fermentation environments, additional pre-treatment-derived inhibitor stress, potentially exacerbated by stresses such as pH and product tolerance, exist in the 2G environment. Furthermore, in a consolidated bioprocessing (CBP) context, the organism is also challenged to secrete enzymes that may themselves lead to unfolded protein response and other stresses. This review will discuss responses of the yeast Saccharomyces cerevisiae to 2G-specific stresses and stress modulation strategies that can be followed to improve yeasts for this application. We also explore published -omics data and discuss relevant rational engineering, reverse engineering, and adaptation strategies, with the view of identifying genes or alleles that will make positive contributions to the overall robustness of 2G industrial strains. Keypoints• Stress tolerance is a key driver to successful application of yeast strains in biorefineries.• A wealth of data regarding stress responses has been gained through omics studies.• Integration of this knowledge could inform engineering of fit for purpose strains.

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