Abstract
BACKGROUND: 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 also important in first-generation starch hydrolysates and in sourdoughs containing significant acetic acid levels. We have previously identified snf4 (E269*) as a causative allele in strain MS164 obtained after whole-genome (WG) transformation and selection for improved acetic acid tolerance. RESULTS: We have now performed polygenic analysis with the same WG transformant MS164 to identify novel causative alleles interacting with snf4 (E269*) 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 snf4 (E269*) 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. CONCLUSIONS: The 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.