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

Abstract

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 trk1Δ was found to exhibit a much higher tolerance compared with the parental strain. Given that trk1Δ 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.

Keywords: Chemogenomic analysis; Formic acid tolerance; Formic acid toxicity; Haa1; Lignocellulosic hydrolysates; Trk1; Yeast robustness.

Fig. 1

Biological functions found to be enriched in the datasets of genes found to confer tolerance or susceptibility to formic acid. a Genes found to confer tolerance to formic acid, listed in Additional file 1, were clustered according their biological process GO assignments using the MIPS functional catalogue (http://mips.helmholtz-muenchen.de/proj/funcatDB), and functional categories were considered to be over-represented if p value ≤0.01. b Genes found to confer susceptibility to formic acid were manually clustered according to the information available in SGD (http://www.yeastgenome.org) into the indicated categories. The number of genes identified within each category is indicated in brackets

Fig. 2

The expression of HAA1 and of Haa1-regulated genes is required for S. cerevisiae adaptation and tolerance to formic acid. a Cells of the parental strain (squares) and deletion mutant haa1Δ (diamons) were grown in liquid MMB medium (pH 4.0) supplemented (closed symbols), or not (open symbols), with 30 mM of formic acid (upper panel) and growth was followed based on culture optical density at 600 nm (OD₆₀₀). Cell viability (CFU ml⁻¹) was also determined during cultivation (lower panel). b Growth curves of the parental BY4741 strain and of strains with the Haa1-target genes deleted were incubated in liquid MMB medium (pH 4.0) supplemented with 30 mM of formic acid. The growth curves indicated in panels (a) and (b) are representative of at least three independent experiments. c Comparison of the mRNA levels from HAA1 and selected Haa1-regulated genes assessed in wild-type cells incubated in basal medium (white bars), or exposed to formic acid (30 mM) for 30 min (diagonal stripes bars) and in haa1Δ cells in cultivated in those same conditions (grey and black bars, respectively). The bars represent the average values of at least three independent experiments, and the error bars the associated standard deviation

Fig. 3

TRK1 gene and genes encoding positive/negative regulators of Trk1 activity are determinants of susceptibility/tolerance to formic acid. a Wild-type (wt), trk1Δ and deletion mutants where the genes coding for regulators of Trk1 activity (HAL3, SAT4, HAL5 and PPZ1) were deleted were tested for their susceptibility to formic acid in solid MMB medium (pH 4.5) supplemented with the acid at the indicated concentrations. Resistance (R) and susceptibility (S) phenotypes. b Schematic representation of the positive and negative regulators of Trk1 activity. c Time-course accumulation of [¹⁴C]formic acid in non-adapted wild-type cells and trk1Δ cells cultivated in MM4 growth medium (at pH 4.0) after exposure to 30 mM of formic acid during a 45-min period. Values represent the average of at least three independent experiments and the error bars represent the associated standard deviation. The asterisks indicate a significant different (p value <0.05) between the accumulation of [¹⁴C]formic acid in wild-type and trk1Δ cells at that time point

Fig. 4

Effect of potassium concentration in tolerance to formic acid of yeast cells with different capacities of K⁺ uptake. Comparison of the susceptibility of wild-type (wt) BY4741, trk1Δ and trk2Δ deletion mutants to different concentrations of formic acid (0, 10, 20, 30, 40 and 50 mM) in ammonium phosphate medium supplemented with increasing concentrations of KCl (0.5, 2 and 20 mM). Serial dilutions (1:0, 1:3 and 1:15) of a water-diluted cellular suspension with an OD₆₀₀ of 0.05 were spotted onto agar plates and incubated during 5 days at 30 °C