Genome-wide search for candidate genes for yeast robustness improvement against formic acid reveals novel susceptibility (Trk1 and positive regulators) and resistance (Haa1-regulon) determinants

Background: Formic acid is an inhibitory compound present in lignocellulosic hydrolysates. Understanding the complex molecular mechanisms underlying Saccharomyces cerevisiae tolerance to this weak acid at the system level is instrumental to guide synthetic pathway engineering for robustness improvement of industrial strains envisaging their use in lignocellulosic biorefineries. Results: This study was performed to identify, at a genome-wide scale, genes whose expression confers protection or susceptibility to formic acid, based on the screening of a haploid deletion mutant collection to search for these phenotypes in the presence of 60, 70 and 80 mM of this acid, at pH 4.5. This chemogenomic analysis allowed the identification of 172 determinants of tolerance and 41 determinants of susceptibility to formic acid. Clustering of genes required for maximal tolerance to this weak acid, based on their biological function, indicates an enrichment of those involved in intracellular trafficking and protein synthesis, cell wall and cytoskeleton organization, carbohydrate metabolism, lipid, amino acid and vitamin metabolism, response to stress, chromatin remodelling, transcription and internal pH homeostasis. Among these genes is HAA1 encoding the main transcriptional regulator of yeast transcriptome reprograming in response to acetic acid and genes of the Haa1-regulon; all demonstrated determinants of acetic acid tolerance. Among the genes that when deleted lead to increased tolerance to formic acid, TRK1, encoding the high-affinity potassium transporter and a determinant of resistance to acetic acid, was surprisingly found. Consistently, genes encoding positive regulators of Trk1 activity were also identified as formic acid susceptibility determinants, while a negative regulator confers protection. At a saturating K+ concentration of 20 mM, the deletion mutant trk1 Delta was found to exhibit a much higher tolerance compared with the parental strain. Given that trk1 Delta accumulates lower levels of radiolabelled formic acid, compared to the parental strain, it is hypothesized that Trk1 facilitates formic acid uptake into the yeast cell. Conclusions: The list of genes resulting from this study shows a few marked differences from the list of genes conferring protection to acetic acid and provides potentially valuable information to guide improvement programmes for the development of more robust strains against formic acid.