QTL mapping approaches have been extensively used to unravel the genetic basis of complex traits in a wide range of organisms . In the yeast S. cerevisiae, QTL mapping approaches have facilitated the association between genetic variants and industrially relevant traits . In this scenario, wild type isolated strains present a higher degree of genetic and natural selection-driven diversity compared with domesticated laboratory ones, which facilitate their use to resolve the genetic basis of so-desired relevant traits. In this study, a bioethanol industrial strain - PE-2, isolated from Brazilian mills - was used to unravel potential genetic variants associated with low pH resistance on S. cerevisiae.
An initial evaluation of growth performance of JAY270 (PE-2 derivate) and other 40 S. cerevisiae strains cultivated at a low pH condition confirmed the superior phenotype of PE-2, especially when compared to the laboratory strain CEN.PK122. In fact, PE-2 persistence on the ethanol production process in Brazilian mills has already been associated with its resistance to the acid wash cell recycle step typically performed on Brazilian E1G production process [5, 42]. On the other hand, common laboratory strains such as CEN.PK122 are typically cultivated at standard conditions that include neutral pH and consequently do not undergo natural selection for this specific condition. Our initial result corroborates with the idea that the harsh conditions faced by yeast strains during bioethanol production produce tailored strains that can easily outcompete non-adapted ones. Thus, industrial isolated strains from the Brazilian bioethanol process, such as PE-2, SA-1, CAT-1, BG-1 can be a good source of genetic variability to explore the genetic basis of industrial relevant traits.
In order to investigate the genetic architecture of PE-2 acid tolerance, we first developed a high-throughput fluorescence-based approach to isolate a large number of yeast segregants. In comparison to other BSA approaches, such as X-QTL , our method allows the rapid generation of large mapping populations without extensive strain engineering. Despite the similarity to the fluorescence-based approach described by Treusch et al (2015) , our method has the advantage of decreasing the number of wavelength gates necessary for segregant isolation. It makes use of the eGFP and CyOFP1 fluorescent proteins, which are excited at wavelengths of 515/545 (green) and 655/695 (orange), respectively, via a single FITC filter. This is the first report of the use of CyOFP1 in a fluorescent-based approach for yeast cell separation.
The developed fluorescence-based approach was successfully applied to isolate a large pool of segregants for BSA-based QTL mapping. BSA is an efficient approach for detection of major QTLs associated with complex traits in yeast [44–52]. This approach relies on phenotyping a progeny from a cross and genotyping two subsets of these offspring presenting opposite phenotypes . The developed fluorescence-based approach was successfully applied to isolate a large pool of segregants (500.000) from a cross between the ACY503 (PE-2 derivative) and CEN.PK113-1A strains. The segregants were collected in crescent challenging conditions of low pH, which allowed the selection of a pool of 79 superior and inferior haploids - representing less than 0.01% of the total analyzed population.
By analyzing differential SNPs presence on both pools (ΔSNP-index), we were able to identify a major QTL located at the end of chromosome XIII. Although the QTL region encompasses a genomic window of approximately 150 Kb containing several genes, we focused our analysis on the 50 Kb window surrounding the detected QTL peak. We also applied a protein function analysis as well as non-synonymous mutation information in these genes, narrowing down the number of potential candidates to only 4. Through generation of engineered reciprocal hemizygotes, we were able to identify GAS1 as the causative allele on the major QTL at chromosome XIII. Furthermore, by interchanging the GAS1 alleles between ACY503 and CEN.PK113-1A, we remarkably increased CEN.PK113-1A tolerance to low pH condition. Both results indicate GAS1 as the major causal variant responsible for low pH tolerance on PE-2 strain.
GAS1 encodes a cell wall-bound 1,3-beta-glucanosyltransferase involved in the formation and maintenance of 1,3-beta-glucan, which is the major polysaccharide of the cell wall (see review on ). The Gas1p is a GPI-anchored glycoprotein of 125–130 kDa localized at the plasma membrane and is a member of the GH72 family of β-1,3-glucanosyltransferases that also include the Candida albicans pH-responsive proteins - CaPhr1p and CaPhr2p . The enzyme is characterized by an N-terminal catalytic domain of about 310 residues (D23–P332), known as the β-(1,3)-glucan transferase domain (GluTD), a cysteine-rich region containing a motif of eight cysteines (C370–C462) and a serine-rich region in which 28 serines are clustered in a region between residues S485 and S525 .
A comparison between ACY503 and CEN.PK113-1A alleles showed the presence of two distinct mutations at nucleotides positions 631 and 1314, non-synonymous and synonymous, respectively. Thus, the non-synonymous mutation at A211 amino acid residue is located at N-terminal catalytic domain and more precisely on α-helices domain between the two activity glutamates residues E161 and E262 necessary for GAS1 activity as b-(1,3)-glucanase and b-(1,3)-glucanosyltransferase .
A more comprehensive analysis of the presence of both SNPs in other S. cerevisiae strains revealed that the non-synonymous mutation is common on wild strains isolated from distinct sources - e.g. wineries, bioethanol industries and oak trees. On the other hand, this mutation is absent in laboratory domesticated strains such as S288C, CEN.PK and W303. Yeasts are known to be organisms with the capacity to survive and ferment in a more acidic environment - pH 4–5 [33, 58]. Acidification of the extracellular environment can be a consequence of natural processes occurring during fermentation or as a consequence of human interference during a biotechnological process . However, acidic environments are rare in controlled laboratory conditions, where yeast growth is typically carried out in standard conditions that include neutral and controlled pH. The lack of selective pressure in laboratory growth conditions may have contributed to the loss of beneficial genetic variants associated with low pH resistance on laboratory strains.
Regarding the synonymous mutation at nucleotide 1314, it is restricted to the Brazilian strains isolated from bioethanol mills. Although synonymous mutations are not expected to cause phenotypic changes, there is emerging evidence that it can affect translational efficiency, mRNA stability and also protein folding and function [59–61]. However, additional studies are necessary to associate this SNP to the low pH resistance phenotype on the Brazilian industrial isolated strains.
The Gas1p role on yeast low pH resistance may be related to activation of cell wall integrity (CWI) pathway. The CWI pathway is responsible for maintenance and function of the yeast cell wall and its mechanism is controlled by the regulatory cascade led by protein kinase . In summary, the stress sensor Mid2 mediates a response to acidic conditions that leads to activation of the Rlm1 transcription factor through phosphorylation of MAP kinase Slt2p/Mpk1p [63, 64]. Gas1Δ mutant strains show hypersensitivity to low pH and present higher levels of dually phosphorylated Slt2 which may help explain the connection between Gas1p and CWI pathway. Some studies have also demonstrated the existence of a synthetic interaction between Gas1p, Slt2p and Rlm1 . Collectively these results point that maintenance of cell wall structure is an important response to low pH stress. Corroborating with this idea, transcriptomic analysis of yeast cells growing under low pH showed that GAS1 and other genes related to cell wall biogenesis appear up-regulated as a response to the damages caused by strong inorganic acids such as sulphuric acid , a stress response that may be triggered by CWI pathway activation.
Recently, Ribeiro et. al (2021)  demonstrated that Gas1p may be also related to yeast cell wall response to stress caused by organic acid such as acetic acid. The presence of 60 mM of acetic acid (pH 4.0) in the medium up-regulates the transcription of β-1,3-glucanosyltransferase encoded by gene GAS1 that leads to an increased content of cell wall β-glucans. This correlation between the increased levels of GAS1’s transcripts and the content of glucan in the cell wall suggests that at least partially, the cell wall remodeling under acetic acid presence is due to the action of β-1,3-glucanosyltransferase encoded by GAS1. This remodeling is essential for preventing acetate (dissociated form of acetic acid due to the low pH) entry through passive diffusion into the cell.
The GAS1 was also identified as responsible for low pH resistance of the multiple-stress-tolerant yeast Issatchenkia orientalis (Pichia kudriavzevii) . Matsushika et al. (2016) have screened on S. cerevisiae a genomic DNA library of I. orientalis identifying loGAS1 as the allele responsible for low pH resistance and also demonstrating that expression of loGAS1 in S. cerevisiae (S288C) improved its ethanol fermentation ability at pH = 2. In a complementary study, the same group demonstrated that S. cerevisiae GAS1 (ScGAS1) expression is pH-dependent and increases in low pH conditions . Also, overexpression of ScGAS1 improved growth and ethanol production under acid stress conditions, although the stress tolerance was inferior to that of the IoGAS1-overexpressing strain. The DNA sequences of both genes - loGAS1 and ScGAS1, possess approximately 60% of similarity . Interestingly, by analyzing and comparing the amino acids profile from loGas1p with the Gas1p produced by the ACY503 and CEN.PK113-1A alleles, we found that the non-synonymous mutation present in the ACY503 allele result in the same amino acid (alanine) as at that position in the loGAS1 gene.
Finally, to build evidence that GAS1ACY503 may be the main responsible to PE-2 tolerance to acid wash treatment of the cell recycle process on Brazilian bioethanol mills, we analyzed the cell survival rate of the susceptible strain CEN.PK113-1A and its mutant CEN.PK113-1A GAS1ACY503 when submitted to a H2SO4 solution. The result showed that the strain harboring mutant GAS1 allele preserves up to 12% more viable cells after 2 and 3 hours of acid treatment. This result might be indicative of PE-2 strain tolerance to the acid wash treatment and its prevalence on fermentation vessels, as described by Basso et al. (2008) . Furthermore, this result also opens the possibility to use genetic engineering to develop more robust strains to be used on ethanol production (E1G and E2G) and also other biotechnological processes where cells experienced loss of cell viability or productivity due to the acidic environment.