QTL Mapping of a Brazilian Bioethanol Strain Unravels the Cell Wall Protein-Encoding Gene GAS1 as a Major Contributor to Low Ph Tolerance in S. Cerevisiae

Abstract

BACKGROUNDSaccharomyces 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. RESULTSIn this work, we pioneer the use of QTL mapping to uncover the genetic basis that endow industrial strain Pedra-2 (PE-2) with outstanding acid resistance. 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 XIII as the major QTL associated with the evaluated phenotype. A reciprocal hemizygosity analysis revealed allele GAS1, encoding a β-1,3-glucanosyltransferase, as the major contributor to this phenotype. The GAS1 sequence alignment of 48 S. cerevisiae strains pointed out a non-synonymous mutation (T211A) 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.CONCLUSIONThis work revealed GAS1 as the major causative gene associated with low pH resistance in PE-2, harboring a non-synonymous mutation persistent in industrial strains. 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 for the industrial production of economically-relevant goods. Our results collectively show the importance of tailored industrial isolated strains in the discovery of the genetic architecture of relevant traits and its implications over productivity.

Genetic mapping of a bioethanol yeast strain reveals new targets for aldehyde- and thermotolerance

“…QTL mapping usually reveals a major causative gene to a given phenotype in yeast (Feng et al 2018;Coradini et al 2021), but it was not the case for this study.…”

“…After overnight incubation at 30ºC, single colonies were isolated, and their mating type was PCR-checked. The confirmed F 1 diploid FMY097/BY4742 was transformed with plasmid pMF002 for further haploid collection, as described by (Coradini et al 2021). Sporulation was performed on conical glass tubes containing 2 mL of 1%KAc medium for 5-7 days.…”

“…Because the QTL approach to uncover the genetic basis of aldehyde Initially, we crossed the strains FMY097 and BY4742, resulting in the heterozygous F 1 hybrid FMY097/BY4742. The resulting diploid was transformed with plasmid pMF002 expressing different gene reporters with specific mating-type promoters, and further sporulated, as described by Coradini et al (Coradini et al 2021). Following the asci lysis, haploid collection was performed using flow cytometry.…”

Unveiling genetic anchors in Saccharomyces cerevisiae: QTL mapping identifies IRA2 as a key player in ethanol tolerance and beyond

“…In this regard, quantitative trait loci (QTL) mapping emerges as an efficient technique for uncovering gene mutations that confer higher tolerances, as well as identifying novel phenotype-associated genes (Deutschbauer and Davis, 2005). QTL mapping of industrially relevant traits in industrial strains has proven highly promising, as these strains represent a valuable resource for understanding and exploring intricate metabolism and uncovering gene mutations that confer tolerance, such as resistance to acidic pH, thermotolerance, and aldehydes (Coradini et al, 2021; de Mello et al, 2022a; Ho et al, 2021; Swinnen et al, 2012; Wang et al, 2019).…”