Abstract
BackgroundHigh acetic acid tolerance is of major importance in industrial yeast strains used for second-generation bioethanol production, because of the high acetic acid content of lignocellulose hydrolysates. It is also important in first-generation starch hydrolysates and in sourdoughs containing significant acetic acid levels. We have previously identified snf4E269* as a causative allele in strain MS164 obtained after whole-genome (WG) transformation and selection for improved acetic acid tolerance.ResultsWe have now performed polygenic analysis with the same WG transformant MS164 to identify novel causative alleles interacting with snf4E269* to further enhance acetic acid tolerance, from a range of 0.8–1.2% acetic acid at pH 4.7, to previously unmatched levels for Saccharomyces cerevisiae. For that purpose, we crossed the WG transformant with strain 16D, a previously identified strain displaying very high acetic acid tolerance. Quantitative trait locus (QTL) mapping with pooled-segregant whole-genome sequence analysis identified four major and two minor QTLs. In addition to confirmation of snf4E269* in QTL1, we identified six other genes linked to very high acetic acid tolerance, TRT2, MET4, IRA2 and RTG1 and a combination of MSH2 and HAL9, some of which have never been connected previously to acetic acid tolerance. Several of these genes appear to be wild-type alleles that complement defective alleles present in the other parent strain.ConclusionsThe presence of several novel causative genes highlights the distinct genetic basis and the strong genetic background dependency of very high acetic acid tolerance. Our results suggest that elimination of inferior mutant alleles might be equally important for reaching very high acetic acid tolerance as introduction of rare superior alleles. The superior alleles of MET4 and RTG1 might be useful for further improvement of acetic acid tolerance in specific industrial yeast strains.
Highlights
High acetic acid tolerance is of major importance in industrial yeast strains used for second-generation bioethanol production, because of the high acetic acid content of lignocellulose hydrolysates
In previous work, we have identified snf4E269* as a causative allele in strain MS164 obtained after whole-genome (WG) transformation and selection for improved acetic acid tolerance (Stojiljkovic et al, submitted for publication)
These results show that a causative allele generated during whole-genome transformation (WGT) can be identified by Quantitative trait locus (QTL) mapping and reciprocal hemizygosity analysis (RHA) analysis with mixed genetic backgrounds obtained by crossing with an unrelated strain
Summary
High acetic acid tolerance is of major importance in industrial yeast strains used for second-generation bioethanol production, because of the high acetic acid content of lignocellulose hydrolysates. It is important in first-generation starch hydrolysates and in sourdoughs containing significant acetic acid levels. In first-generation bioethanol production with hydrolysates of starch derived from food crops or molasses, significant levels of acetic acid can be present. It is produced by contaminating bacteria and accumulates due to water recycling practices [9]. Tolerance to acetic acid is important in baker’s yeast used in sourdoughs where acetic acid is produced in the preceding bacterial fermentation [10]
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