Genomic and transcriptomic insights for the development of Saccharomyces cerevisiae strains tolerant to formic acid

Climate change has reached alarming levels in recent years, driven primarily by massive CO₂ emissions from economic systems and specific sectors such as transportation. Bioethanol emerges as a promising alternative to fossil fuels in the short and medium term. However, its production from lignocellulosic biomass, a highly promising substrate, faces significant challenges. The pre-treatment required for lignocellulosic materials generates inhibitors, such as weak acids, that impair the growth and fermentation of Saccharomyces cerevisiae, reducing the overall ethanol yield and decreasing the economical sustainability of the entire process. This study aimed to explore the response of natural S. cerevisiae strains to specific inhibitors associated with second-generation bioethanol production, focusing on weak acids such as formic and acetic acid. A hybrid approach combining biotechnology and bioinformatics was adopted to analyse a cluster of 20 yeast strains, including natural, industrial and oenological isolates with diverse phenotypic traits. Genomic analyses highlighted significant single nucleotide polymorphisms (SNPs) and copy number variations (CNVs) in key genes. Among the 20 strains studied, four natural S. cerevisiae – namely YI30, Fp89, Fp90, and CESPLG05 – displayed remarkable resistance to formic acid, one of the most inhibitory weak acids present in pre-treated lignocellulosic biomass. These strains shared two SNPs in GSH2 – a glutathione synthase –, a potential target for further engineering. To further understand formic acid resistance, transcriptional and metabolic responses of the most resistant strains (YI30 and CESPLG05) and a sensitive reference strain (DSM 70449) were investigated. RNA-seq analysis revealed adaptations in critical pathways, including glycolysis, the tricarboxylic acid (TCA) cycle, the pentose phosphate pathway (PPP), the glycerol pathway, and ergosterol biosynthesis. Resistance mechanisms involved the upregulation of SAT4, a positive regulator of TRK1 (a putative formic acid transporter), and FDH1, which detoxifies formic acid. Resistant strains also exhibited enhanced glycerol metabolism for osmotic stress adaptation and intracellular NAD+ regeneration, as evidenced by the upregulation of GPD2 and GPP2. Moreover, strains YI30 and CESPLG05 demonstrated superior ethanol production under stressful conditions, achieving ethanol yields of 93 and 89% of the theoretical maximum, respectively, in synthetic media containing 50 g/L glucose and 4.0 g/L formic acid. This work presents, for the first time, a hypothesis of innate resistance mechanisms to formic acid in natural isolates of S. cerevisiae, supported by phenotypic and transcriptional data. The findings provide valuable insights into the complexity and adaptive capacity of natural S. cerevisiae strains to environmental stresses. By identifying key genomic targets and leveraging their industrial fitness, this study lays the foundation for engineering robust yeast strains optimized for lignocellulosic bioethanol production and other biotechnological applications in challenging environments.