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.
Highlights
Acetic acid is one of the major inhibitors in lignocellulose hydrolysates used for the production of sec‐ ond-generation bioethanol
We show that the increased recombination frequency in the F7 segregants results in reduced quantitative trait loci (QTL) size, facilitating the identification of causative genes, but unexpectedly in appearance of new QTLs and disappearance of previously validated QTLs, compared to QTL mapping with F1 segregants
The fermentation performance of this strain is severely affected by acetic acid, a weak organic acid present in high quantities in lignocellulose hydrolysates and other industrial fermentation media
Summary
Acetic acid is one of the major inhibitors in lignocellulose hydrolysates used for the production of sec‐ ond-generation bioethanol. Multiple attempts to rationally engineer increased acetic acid tolerance in yeast have met with limited success, possibly because a high number of genes appears to be involved in the response to acetic acid stress and in establishing high intrinsic acetic acid tolerance [8,9,10,11,12,13] Random approaches, such as evolutionary adaptation, have resulted in strains with improved acetic acid tolerance [14, 15], but this method of focused selection on a single trait often leads to loss of other important properties in industrial yeast strains. The genetic mapping of quantitative trait loci (QTL) is hampered by genetic heterogeneity, variable phenotypic contributions of each QTL, epistasis and gene-environment interactions [18] These limitations have stimulated the development of novel technologies to simultaneously identify genomic loci that are involved in complex traits. Phenotypes like high-temperature tolerance, efficient sporulation, and chemical resistance have been genetically unraveled [17, 19, 20]
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