title: 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.
authors.affiliation: Laboratory of Molecular Cell Biology, Institute of Botany and Microbiology, KU Leuven, Leuven-Heverlee, Belgium ; Department of Molecular Microbiology, VIB, Kasteelpark Arenberg 31, Flanders, 3001 Leuven-Heverlee, Belgium.
authors.forename: Jean-Paul
authors.initials: JP
authors.lastname: Meijnen
authors.affiliation: Laboratory of Molecular Cell Biology, Institute of Botany and Microbiology, KU Leuven, Leuven-Heverlee, Belgium ; Department of Molecular Microbiology, VIB, Kasteelpark Arenberg 31, Flanders, 3001 Leuven-Heverlee, Belgium.
authors.forename: Paola
authors.initials: P
authors.lastname: Randazzo
authors.affiliation: Laboratory of Molecular Cell Biology, Institute of Botany and Microbiology, KU Leuven, Leuven-Heverlee, Belgium ; Department of Molecular Microbiology, VIB, Kasteelpark Arenberg 31, Flanders, 3001 Leuven-Heverlee, Belgium.
authors.forename: María R
authors.initials: MR
authors.lastname: Foulquié-Moreno
authors.affiliation: CBS, Fungal Biodiversity Centre (CBS-KNAW), Utrecht, The Netherlands.
authors.forename: Joost
authors.initials: J
authors.lastname: van den Brink
authors.affiliation: Laboratory of Molecular Cell Biology, Institute of Botany and Microbiology, KU Leuven, Leuven-Heverlee, Belgium ; Department of Molecular Microbiology, VIB, Kasteelpark Arenberg 31, Flanders, 3001 Leuven-Heverlee, Belgium.
authors.forename: Paul
authors.initials: P
authors.lastname: Vandecruys
authors.affiliation: Laboratory of Molecular Cell Biology, Institute of Botany and Microbiology, KU Leuven, Leuven-Heverlee, Belgium ; Department of Molecular Microbiology, VIB, Kasteelpark Arenberg 31, Flanders, 3001 Leuven-Heverlee, Belgium.
authors.forename: Marija
authors.initials: M
authors.lastname: Stojiljkovic
authors.affiliation: Laboratory of Molecular Cell Biology, Institute of Botany and Microbiology, KU Leuven, Leuven-Heverlee, Belgium ; Department of Molecular Microbiology, VIB, Kasteelpark Arenberg 31, Flanders, 3001 Leuven-Heverlee, Belgium.
authors.forename: Françoise
authors.initials: F
authors.lastname: Dumortier
authors.affiliation: Department of Biology, Biotechnical Faculty, University of Ljubljana, Večna pot 111, 1000 Ljubljana, Slovenia.
authors.forename: Polona
authors.initials: P
authors.lastname: Zalar
authors.affiliation: CBS, Fungal Biodiversity Centre (CBS-KNAW), Utrecht, The Netherlands.
authors.forename: Teun
authors.initials: T
authors.lastname: Boekhout
authors.affiliation: Department of Biology, Biotechnical Faculty, University of Ljubljana, Večna pot 111, 1000 Ljubljana, Slovenia ; Centre of Excellence for Integrated Approaches in Chemistry and Biology of Proteins, Jamova 39, 1000 Ljubljana, Slovenia.
authors.forename: Nina
authors.initials: N
authors.lastname: Gunde-Cimerman
authors.affiliation: Genialis d.o.o., Ulica Zore Majcnove 4, 1000 Ljubljana, Slovenia.
authors.forename: Janez
authors.initials: J
authors.lastname: Kokošar
authors.affiliation: Genialis d.o.o., Ulica Zore Majcnove 4, 1000 Ljubljana, Slovenia ; Faculty of Computer and Information Science, University of Ljubljana, Večna pot 113, Ljubljana, Slovenia.
authors.forename: Miha
authors.initials: M
authors.lastname: Štajdohar
authors.affiliation: Faculty of Computer and Information Science, University of Ljubljana, Večna pot 113, Ljubljana, Slovenia.
authors.forename: Tomaž
authors.initials: T
authors.lastname: Curk
authors.affiliation: Department of Molecular and Biomedical Sciences, Jožef Stefan Institute, Jamova 39, Ljubljana, Slovenia.
authors.forename: Uroš
authors.initials: U
authors.lastname: Petrovič
authors.affiliation: Laboratory of Molecular Cell Biology, Institute of Botany and Microbiology, KU Leuven, Leuven-Heverlee, Belgium ; Department of Molecular Microbiology, VIB, Kasteelpark Arenberg 31, Flanders, 3001 Leuven-Heverlee, Belgium.
authors.forename: Johan M
authors.initials: JM
authors.lastname: Thevelein
keywords: Acetic acid tolerance; Bioethanol production; Inbreeding; Polygenic analysis; Pooled-segregant whole-genome sequence analysis; QTL mapping; Saccharomyces cerevisiae