QTL mapping of a Brazilian bioethanol strain links the cell wall protein-encoding gene GAS1 to low pH tolerance in S. cerevisiae

Abstract

Background Saccharomyces cerevisiae is largely applied in many biotechnological processes, from traditional food and beverage industries to modern biofuel and biochemicals factories. During the fermentation process, yeast cells are usually challenged in different harsh conditions, which often impact productivity. Regarding bioethanol production, cell exposure to acidic environments is related to productivity loss on both first- and second-generation ethanol. In this scenario, indigenous strains traditionally used in fermentation stand out as a source of complex genetic architecture, mainly due to their highly robust background—including low pH tolerance. Results In this work, we pioneer the use of QTL mapping to uncover the genetic basis that confers to the industrial strain Pedra-2 (PE-2) acidic tolerance during growth at low pH. First, we developed a fluorescence-based high-throughput approach to collect a large number of haploid cells using flow cytometry. Then, we were able to apply a bulk segregant analysis to solve the genetic basis of low pH resistance in PE-2, which uncovered a region in chromosome X as the major QTL associated with the evaluated phenotype. A reciprocal hemizygosity analysis revealed the allele GAS1, encoding a β-1,3-glucanosyltransferase, as the casual variant in this region. The GAS1 sequence alignment of distinct S. cerevisiae strains pointed out a non-synonymous mutation (A631G) prevalence in wild-type isolates, which is absent in laboratory strains. We further showcase that GAS1 allele swap between PE-2 and a low pH-susceptible strain can improve cell viability on the latter of up to 12% after a sulfuric acid wash process. Conclusion This work revealed GAS1 as one of the main causative genes associated with tolerance to growth at low pH in PE-2. We also showcase how GAS1PE-2 can improve acid resistance of a susceptible strain, suggesting that these findings can be a powerful foundation for the development of more robust and acid-tolerant strains. Our results collectively show the importance of tailored industrial isolated strains in discovering the genetic architecture of relevant traits and its implications over productivity.

Crosstalk between Yeast Cell Plasma Membrane Ergosterol Content and Cell Wall Stiffness under Acetic Acid Stress Involving Pdr18

“…Interestingly, plasma membrane lipid composition, in particular concerning the content in ergosterol and complex sphingolipids, determines the proper raft association of Gas1 and sorting of the plasma membrane [74], ultimately influencing its activity. Interestingly, a recent QTL mapping to uncover the genetic basis that confers to the bioethanol industrial strain Pedra-2 (PE-2) tolerance during growth at low pH, identified a non-synonymous mutation (A631G) in GAS1, prevalent in wild-type isolates and absent in laboratory strains [75]. The chitin content, assessed by the quantification of calcofluor white fluorescence intensity, also increased during acetic-acid-induced latency, with a time-course pattern similar to the β-glucan profile.…”

The cell wall and the response and tolerance to stresses of biotechnological relevance in yeasts

“…Many of the genes found to be upregulated at low pH are related to cell wall biogenesis, including FKS1 (β1,3-glucan synthase), GAS1 (β-1,3-glucanosyltransferase involved in cell wall remodeling-elongation of (1 → 3)-β- D -glucan chains and branching), CHS1 (chitin synthase), NCW2 (GPI-protein involved in chitin-glucan assembly), KRE6 (glucosyl hydrolase required for β1,6-glucan synthesis) and MNN9 (mannosyltransferase subunit involved in wall protein mannosylation) ( De Melo et al, 2010 ; De Lucena et al, 2012 ; Figure 3 ). Interestingly, using QTL mapping to uncover the genetic basis of a bioethanol industrial strain Pedra-2 (PE-2) tolerance, a prevalent non-synonymous mutation (A631G) in GAS1 was identified during growth at low pH induced by sulfuric acid exposure, reinforcing the idea of the relevant role of this GPI-protein in yeast tolerance in acidic environments ( Coradini et al, 2021 ). Together with the up-regulation of genes related with 1,3-β-glucan synthesis, elongation, and anchoring, low pH stress leads to increased cell wall resistance to compounds with β1,3-glucanase activity and to the establishment of more alkali-sensitive linkages between CWPs and the β1,3-glucan network ( Kapteyn et al, 2001 ; De Melo et al, 2010 ; De Lucena et al, 2012 , 2015 ; Lucena et al, 2020 ).…”