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Lignocellulosic biomass is still considered a feasible source of bioethanol production. Saccharomyces cerevisiae can adapt to detoxify lignocellulose-derived inhibitors, including furfural. Tolerance of strain performance has been measured by the extent of the lag phase for cell proliferation following the furfural inhibitor challenge. The purpose of this work was to obtain a tolerant yeast strain against furfural through overexpression of YPR015C using the in vivo homologous recombination method. The physiological observation of the overexpressing yeast strain showed that it was more resistant to furfural than its parental strain. Fluorescence microscopy revealed improved enzyme reductase activity and accumulation of oxygen reactive species due to the harmful effects of furfural inhibitor in contrast to its parental strain. Comparative transcriptomic analysis revealed 79 genes potentially involved in amino acid biosynthesis, oxidative stress, cell wall response, heat shock protein, and mitochondrial-associated protein for the YPR015C overexpressing strain associated with stress responses to furfural at the late stage of lag phase growth. Both up- and down-regulated genes involved in diversified functional categories were accountable for tolerance in yeast to survive and adapt to the furfural stress in a time course study during the lag phase growth. This study enlarges our perceptions comprehensively about the physiological and molecular mechanisms implicated in the YPR015C overexpressing strain’s tolerance under furfural stress.

The yeast Saccharomyces cerevisiae able to tolerate lignocellulose-derived inhibitors like furfural. Yeast strain performance tolerance has been measured by the length of the lag phase for cell growth in response to the furfural inhibitor challenge. The aims of this work were to obtain RDS1 yeast tolerant strain against furfural through overexpression using a method of in vivo homologous recombination. Here, we report that the overexpressing RDS1 recovered more rapidly and displayed a lag phase at about 12 h than its parental strain. Overexpressing RDS 1 strain encodes a novel aldehyde reductase with catalytic function for reduction of furfural with NAD(P)H as the co-factor. It displayed the highest specific activity (24.8 U/mg) for furfural reduction using NADH as a cofactor. Fluorescence microscopy revealed improved accumulation of reactive oxygen species resistance to the damaging effects of inhibitor in contrast to the parental. Comparative transcriptomics revealed key genes potentially associated with stress responses to the furfural inhibitor, including specific and multiple functions involving defensive reduction–oxidation reaction process and cell wall response. A significant change in expression level of log2 (fold change >1) was displayed for RDS1 gene in the recombinant strain, which demonstrated that the introduction of RDS1 overexpression promoted the expression level. Such signature expressions differentiated tolerance phenotypes of RDS 1 from the innate stress response of its parental strain. Overexpression of the RDS1 gene involving diversified functional categories is accountable for stress tolerance in yeast S. cerevisiae to survive and adapt the furfural during the lag phase. [ABSTRACT FROM AUTHOR]

In industrial production, the excessive discharge of furfural can pose harm to soil microorganisms, aquatic animals and plants, as well as humans. Therefore, it is crucial to develop efficient and cost-effective methods for degrading furfural in the environment. Currently, the use of Saccharomyces cerevisiae for furfural degradation in water has shown effectiveness, but there is a need to explore improved efficiency and tolerance in S. cerevisiae for this purpose. In this study, we isolated and evolved highly efficient furfural degradation strains, namely YBA_08 and F60C. These strains exhibited remarkable capabilities, degrading 59% and 99% furfural in the YPD medium after 72 h of incubation, significantly higher than the 31% achieved by the model strain S288C. Through analysis of the efficient degradation mechanism in the evolutionary strain F60C, we discovered a 326% increase in the total amount of NADH and NADPH. This increase likely promotes faster furfural degradation through intracellular aldehyde reductases. Moreover, the decrease in NADPH content led to a 406% increase in glutathione content at the background level, which protects cells from damage caused by reactive oxygen species. Mutations and differential expression related to cell cycle and cell wall synthesis were observed, enabling cell survival in the presence of furfural and facilitating rapid furfural degradation and growth recovery. Based on these findings, it is speculated that strains YBA_08 and F60C have the potential to contribute to furfural degradation in water and the production of furfuryl alcohol, ethanol, and FDCA in biorefinery processes. [Display omitted] • Furfural induced damage of biological macromolecules and growth inhibition. • A 326% increase of NAD(P)H promoted the rapid degradation of furfural. • A 406% increase of glutathione in background level reduced oxidative stress. • DNA protection and extended lifespan ensured cell survival. [ABSTRACT FROM AUTHOR]