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

Abstract

Background: Acetic 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.

Results: Haploid 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.

Conclusions: This 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.

Keywords: Acetic acid tolerance; Bioethanol production; Inbreeding; Polygenic analysis; Pooled-segregant whole-genome sequence analysis; QTL mapping; Saccharomyces cerevisiae.

Fig. 1

Fermentation profiles in the presence of different concentrations of acetic acid. CO₂ production was determined from the weight loss during fermentation and expressed as percentage of initial weight of the total culture medium. a Acetic acid sensitive diploid strain Ethanol Red. b Acetic acid sensitive Ethanol Red haploid segregant ER18. c Acetic acid-tolerant diploid strain JT22689. d Acetic acid-tolerant JT22689 haploid segregant 16D. Strains were inoculated in YPD medium with 2 % glucose at pH 4 and various concentrations of acetic acid: 0 % (), 0.4 % (v/v) (), 0.5 % (v/v) (), 0.6 % (v/v) (), 0.7 % (v/v) (), 0.8 % (v/v) (), 0.9 % (v/v) (), 1.0 % (v/v) (). Data points are the average of duplicate measurements; error bars represent the maximum deviation of the average

Fig. 2

QTL mapping of high acetic acid tolerance. The mapping was performed with pooled F1 segregants (green), pooled F7 segregants (red), and individual F7 segregants (black, second row). Pooled F1 and pooled F7 segregants (27 segregants for both pools) were subjected to sequence analysis with the Illumina platform at BGI. Individual F7 segregants were sequenced with the Illumina platform at EMBL. P values calculated using the individual sequencing data from F7 segregants were plotted against the respective chromosomal position (third row). p values <0.05 (indicated by dotted line) were considered statistically significant. Unselected pools consisting of 27 randomly selected segregants were also sequenced to eliminate linkage to inadvertently selected traits (bottom row, F1 segregants: green, F7 segregants: red)

Fig. 3

QTL mapping with selected SNPs in the individual segregants. Selected SNPs in QTL1 (a) and QTL2 (b) were scored with allele-specific PCR, the SNP variant frequency and the corresponding p values were calculated, and the p values were plotted over the length of the chromosomes, XIII for QTL1 (a) and XVI for QTL2 (b)

Fig. 4

Identification of HAA1 as the causative allele in QTL2 on Chr. XVI. a Fermentation profiles of the hemizygous diploid strains used in the RHA for HAA1. Fermentations were performed in YPD medium with 2 % glucose and supplemented with 0.7 % (v/v) acetic acid at pH 4.0. Three diploid hybrid strains were tested and compared: ER18 haa1Δ x 16D (); ER18 × 16D haa1Δ (); ER18 × 16D (). Data points are the average of duplicate measurements. Error bars represent the maximum deviation of the average. b. Fermentation profiles of the strains 16D (), ER18 (), and ER18-HAA1 () (ER18 in which the complete HAA1 gene with promoter, ORF and terminator, was replaced by the HAA1 allele from 16D) in YPD medium with 2 % glucose and supplemented with 0.6 % (v/v) acetic acid at pH 4.0

Fig. 5

QTL mapping of high acetic acid tolerance using individual F7 segregants. a For each of the individual F7 segregants, the parent-of-origin linkage information was determined by using a distance-based method followed by the segmentation of individual chromosomes. Genomic regions linked either to the genome of the superior parent (>0; green) or to that of the inferior parent (<0; red) were derived by averaging the parent-of-origin linkage information for the nine most tolerant segregants of the F7 pool. Horizontal lines are included to mark the threshold values of ±0.7 used for QTL identification (see “Methods” for details). b Detailed view of the identified QTL regions when different numbers of F7 strains (the 9, 18, or 27 strains with highest acetic acid tolerance) are considered. Inclusion of additional strains with less extreme acetic acid tolerance results in QTL regions becoming either less pronounced (QTL1, QTL3, QTL5), emerge when less tolerant strains are considered (QTL4), or are present when only the most tolerant strains are included in the analysis (QTL6)

Fig. 6

Fermentation profiles of the hemizygous diploid strains used in the RHA for GLO1 (a), CUP2 (b), DOT5 (c), and VMA7 (d). CO₂ production was determined from the weight loss during fermentation and expressed as percentage of initial weight of the total culture medium. Fermentations were performed in YPD [4 % glucose (w/v)] medium supplemented with 0.8 % (v/v) acetic acid at pH 4.0. Three diploid hybrid strains were tested and compared: ER18geneΔ × 16D (); ER18 × 16Dgene∆ (); ER18 × 16D (). Data points are the average of duplicate measurements. Error bars represent the maximum deviation of the average

Fig. 7

Fermentation profiles of the diploid strains GSE16-T18 and GSE16-T18-HAA1* (carrying the unique HAA1* mutation, changing G to A at position c.1517, of strain 16D in both HAA1 alleles). Semi-anaerobic static fermentations were performed in YP medium with 20 % glucose at pH 5.2 and varying concentrations of acetic acid. Strains: GSE16-T18 () and GSE16-T18-HAA1* (). a No acetic acid; b 1.0 % acetic acid; c 1.2 % acetic acid; d 1.4 % acetic acid; e 1.6 % acetic acid; f 2.0 % acetic acid