title: Improvement of yeast tolerance to acetic acid through Haa1 transcription factor engineering: towards the underlying mechanisms.
abstract: BACKGROUND
  Besides being a major regulator of the response to acetic acid in Saccharomyces cerevisiae, the transcription factor Haa1 is an important determinant of the tolerance to this acid. The engineering of Haa1 either by overexpression or mutagenesis has therefore been considered to be a promising avenue towards the construction of more robust strains with improved acetic acid tolerance.
  
  
  RESULTS
  By applying the concept of global transcription machinery engineering to the regulon-specific transcription factor Haa1, a mutant allele containing two point mutations could be selected that resulted in a significantly higher acetic acid tolerance as compared to the wild-type allele. The level of improvement obtained was comparable to the level obtained by overexpression of HAA1, which was achieved by introduction of a second copy of the native HAA1 gene. Dissection of the contribution of the two point mutations to the phenotype showed that the major improvement was caused by an amino acid exchange at position 135 (serine to phenylalanine). In order to further study the mechanisms underlying the tolerance phenotype, Haa1 translocation and transcriptional activation of Haa1 target genes was compared between Haa1 mutant, overproduction and wild-type strains. While the rapid Haa1 translocation from the cytosol to the nucleus in response to acetic acid was not affected in the Haa1S135Fmutant strain, the levels of transcriptional activation of four selected Haa1-target genes by acetic acid were significantly higher in cells of the mutant strain as compared to cells of the wild-type strain. Interestingly, the time-course of transcriptional activation in response to acetic acid was comparable for the mutant and wild-type strain whereas the maximum mRNA levels obtained correlate with each strain's tolerance level.
  
  
  CONCLUSIONS
  Our data confirms that engineering of the regulon-specific transcription factor Haa1 allows the improvement of acetic acid tolerance in S. cerevisiae. It was also shown that the beneficial S135F mutation identified in the current work did not lead to an increase of HAA1 transcript level, suggesting that an altered protein structure of the Haa1S135Fmutant protein led to an increased recruitment of the transcription machinery to Haa1 target genes.
authors.affiliation: Department of Life Sciences and Chemistry, Jacobs University Bremen gGmbH, Campus Ring 1, 28759, Bremen, Germany.
authors.forename: Steve
authors.initials: S
authors.lastname: Swinnen
authors.affiliation: Department of Bioengineering, Institute for Bioengineering and Biosciences, Instituto Superior Técnico, Universidade de Lisboa, 1049-001, Lisbon, Portugal.
authors.forename: Sílvia F
authors.initials: SF
authors.lastname: Henriques
authors.affiliation: Department of Life Sciences and Chemistry, Jacobs University Bremen gGmbH, Campus Ring 1, 28759, Bremen, Germany.
authors.forename: Ranjan
authors.initials: R
authors.lastname: Shrestha
authors.affiliation: Department of Life Sciences and Chemistry, Jacobs University Bremen gGmbH, Campus Ring 1, 28759, Bremen, Germany.
authors.forename: Ping-Wei
authors.initials: PW
authors.lastname: Ho
authors.affiliation: Department of Bioengineering, Institute for Bioengineering and Biosciences, Instituto Superior Técnico, Universidade de Lisboa, 1049-001, Lisbon, Portugal. isacorreia@tecnico.ulisboa.pt.
authors.forename: Isabel
authors.initials: I
authors.lastname: Sá-Correia
authors.affiliation: Department of Life Sciences and Chemistry, Jacobs University Bremen gGmbH, Campus Ring 1, 28759, Bremen, Germany. e.nevoigt@jacobs-university.de.
authors.forename: Elke
authors.identifier: http://orcid.org/0000-0001-7947-3000
authors.initials: E
authors.lastname: Nevoigt
mesh_terms: D019342:Acetic Acid / Q000494:pharmacology*; D015870:Gene Expression; D017354:Point Mutation; D011401:Promoter Regions, Genetic; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D029701:Saccharomyces cerevisiae Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism*; D014157:Transcription Factors / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism*
chemical_list: C439059:Haa1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D019342:Acetic Acid
keywords: Acetic acid tolerance; Haa1; Response and adaptation to acetic acid; Saccharomyces cerevisiae; Transcription factor engineering