title: Evolutionary engineering of a wine yeast strain revealed a key role of inositol and mannoprotein metabolism during low-temperature fermentation.
abstract: BACKGROUND
  Wine produced at low temperature is often considered to improve sensory qualities. However, there are certain drawbacks to low temperature fermentations: e.g. low growth rate, long lag phase, and sluggish or stuck fermentations. Selection and development of new Saccharomyces cerevisiae strains well adapted at low temperature is interesting for future biotechnological applications. This study aimed to select and develop wine yeast strains that well adapt to ferment at low temperature through evolutionary engineering, and to decipher the process underlying the obtained phenotypes.
  
  
  RESULTS
  We used a pool of 27 commercial yeast strains and set up batch serial dilution experiments to mimic wine fermentation conditions at 12 °C. Evolutionary engineering was accomplished by using the natural yeast mutation rate and mutagenesis procedures. One strain (P5) outcompeted the others under both experimental conditions and was able to impose after 200 generations. The evolved strains showed improved growth and low-temperature fermentation performance compared to the ancestral strain. This improvement was acquired only under inositol limitation. The transcriptomic comparison between the evolved and parental strains showed the greatest up-regulation in four mannoprotein coding genes, which belong to the DAN/TIR family (DAN1, TIR1, TIR4 and TIR3). Genome sequencing of the evolved strain revealed the presence of a SNP in the GAA1 gene and the construction of a site-directed mutant (GAA1 (Thr108)) in a derivative haploid of the ancestral strain resulted in improved fermentation performance. GAA1 encodes a GPI transamidase complex subunit that adds GPI, which is required for inositol synthesis, to newly synthesized proteins, including mannoproteins.
  
  
  CONCLUSIONS
  In this study we demonstrate the importance of inositol and mannoproteins in yeast adaptation at low temperature and the central role of the GAA1 gene by linking both metabolisms.
authors.affiliation: Departamento de Biotecnología de los alimentos, Instituto de Agroquímica y Tecnología de los Alimentos (CSIC), Avda. Agustín Escardino, 7, E-46980, Paterna, Valencia, Spain.
authors.forename: María
authors.initials: M
authors.lastname: López-Malo
authors.affiliation: Departamento de Biotecnología de los alimentos, Instituto de Agroquímica y Tecnología de los Alimentos (CSIC), Avda. Agustín Escardino, 7, E-46980, Paterna, Valencia, Spain.
authors.forename: Estéfani
authors.initials: E
authors.lastname: García-Rios
authors.affiliation: Departamento de Biotecnología de los alimentos, Instituto de Agroquímica y Tecnología de los Alimentos (CSIC), Avda. Agustín Escardino, 7, E-46980, Paterna, Valencia, Spain.
authors.forename: Bruno
authors.initials: B
authors.lastname: Melgar
authors.affiliation: Department of Genome Sciences, University of Washington, Seattle, WA, 98195, USA.
authors.forename: Monica R
authors.initials: MR
authors.lastname: Sanchez
authors.affiliation: Department of Genome Sciences, University of Washington, Seattle, WA, 98195, USA.
authors.forename: Maitreya J
authors.initials: MJ
authors.lastname: Dunham
authors.affiliation: Departamento de Biotecnología de los alimentos, Instituto de Agroquímica y Tecnología de los Alimentos (CSIC), Avda. Agustín Escardino, 7, E-46980, Paterna, Valencia, Spain. guillamon@iata.csic.es.
authors.forename: José Manuel
authors.initials: JM
authors.lastname: Guillamón
mesh_terms: D019143:Evolution, Molecular; D005285:Fermentation; D007294:Inositol / Q000235:genetics / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000235:genetics*; D060847:Metabolic Engineering; D013696:Temperature; D014920:Wine; D015003:Yeasts / Q000235:genetics / Q000378:metabolism
chemical_list: D008562:Membrane Glycoproteins; C025363:mannoproteins; D007294:Inositol