Polygenic analysis and targeted improvement of the complex trait of high acetic acid tolerance in the yeast Saccharomyces cerevisiae

Abstract

BackgroundAcetic acid is one of the major inhibitors in lignocellulose hydrolysates used for the production of second-generation bioethanol. Although several genes have been identified in laboratory yeast strains that are required for tolerance to acetic acid, the genetic basis of the high acetic acid tolerance naturally present in some Saccharomyces cerevisiae strains is unknown. Identification of its polygenic basis may allow improvement of acetic acid tolerance in yeast strains used for second-generation bioethanol production by precise genome editing, minimizing the risk of negatively affecting other industrially important properties of the yeast.ResultsHaploid segregants of a strain with unusually high acetic acid tolerance and a reference industrial strain were used as superior and inferior parent strain, respectively. After crossing of the parent strains, QTL mapping using the SNP variant frequency determined by pooled-segregant whole-genome sequence analysis revealed two major QTLs. All F1 segregants were then submitted to multiple rounds of random inbreeding and the superior F7 segregants were submitted to the same analysis, further refined by sequencing of individual segregants and bioinformatics analysis taking into account the relative acetic acid tolerance of the segregants. This resulted in disappearance in the QTL mapping with the F7 segregants of a major F1 QTL, in which we identified HAA1, a known regulator of high acetic acid tolerance, as a true causative allele. Novel genes determining high acetic acid tolerance, GLO1, DOT5, CUP2, and a previously identified component, VMA7, were identified as causative alleles in the second major F1 QTL and in three newly appearing F7 QTLs, respectively. The superior HAA1 allele contained a unique single point mutation that significantly improved acetic acid tolerance under industrially relevant conditions when inserted into an industrial yeast strain for second-generation bioethanol production.ConclusionsThis work reveals the polygenic basis of high acetic acid tolerance in S. cerevisiae in unprecedented detail. It also shows for the first time that a single strain can harbor different sets of causative genes able to establish the same polygenic trait. The superior alleles identified can be used successfully for improvement of acetic acid tolerance in industrial yeast strains.

Sphingolipid biosynthesis upregulation by TOR complex 2–Ypk1 signaling during yeast adaptive response to acetic acid stress

“…Also, phenotypic analysis of the S. cerevisiae genome-wide deletion collection identified a number of additional genes important for survival in response to acetic acid stress, including the protein kinase Ypk1 [20, 21]. The knowledge gathered so far on the understanding of molecular factors and cellular pathways involved in acetic acid tolerance has been used in the development of acetic acid-tolerant strains, either through the deletion or overexpression of single genes [22, 23], manipulation of genes that play a crucial role in the regulatory cascades that control acetic acid tolerance, such as manipulation of the Haa1-regulon [24–26], or the use of evolutionary engineering strategies to select strains that possess increased tolerance to the acid [27]. Manipulation of the composition of the growth medium in order to increase yeast tolerance to acetic acid has also been reported [20].…”

Adaptation to low pH and lignocellulosic inhibitors resulting in ethanolic fermentation and growth of Saccharomyces cerevisiae

“…As Meijnen et al (2016) found that tolerance towards acetic acid is a result of a polygenic response from yeast, evolutionary adaptation might be a suitable strategy to improve tolerance towards low pH and acetic acid with other lignocellulosic inhibitors since yeast might accrue beneficial properties under stress conditions over the time of evolution. Evolutionary engineering strategies have been successfully pursued to obtain yeast strains with enhanced tolerance against individual or combinations of several inhibitors in defined media (Dominik and Uwe 2008; Wright et al 2011) or in hydrolysates (Almario et al 2013; Hanqi et al 2014) with beneficial properties including better growth, improved viability, higher yield and ethanol productivity in comparison to the control strains.…”

The Zygosaccharomyces bailii transcription factor Haa1 is required for acetic acid and copper stress responses suggesting subfunctionalization of the ancestral bifunctional protein Haa1/Cup2

“…Our results and those reported by Keller et al [1] show that Haa1 is not involved in S. cerevisiae tolerance to copper. Although Cup2 was recently identified as a determinant of acetic acid tolerance in a quantitative trait loci analysis of pooled segregants that revealed the polygenic nature of high acetic acid tolerance in yeast, suggesting that in superior segregrants the CUP2 allele could take over the function of HAA1 [58], for S. cerevisiae S288c and under the experimental conditions used, we could not find evidences supporting the involvement of Cup2 in acetic acid tolerance. Our study points towards the evolution of both paralogues in a way that each one regulates a subset of the target genes all regulated by the single ancestral bifunctional transcription factor, given that ZbHaa1 activates Z. bailii genes homologous to both S. cerevisiae Haa1 and Cup2 target genes under acetic acid or copper-induced stresses.…”