title: Characterisation of the broad substrate specificity 2-keto acid decarboxylase Aro10p of Saccharomyces kudriavzevii and its implication in aroma development.
abstract: BACKGROUND
  The yeast amino acid catabolism plays an important role in flavour generation since higher alcohols and acetate esters, amino acid catabolism end products, are key components of overall flavour and aroma in fermented products. Comparative studies have shown that other Saccharomyces species, such as S. kudriavzevii, differ during the production of aroma-active higher alcohols and their esters compared to S. cerevisiae.
  
  
  RESULTS
  In this study, we performed a comparative analysis of the enzymes involved in the amino acid catabolism of S. kudriavzevii with their potential to improve the flavour production capacity of S. cerevisiae. In silico screening, based on the severity of amino acid substitutions evaluated by Grantham matrix, revealed four candidates, of which S. kudriavzevii Aro10p (SkAro10p) had the highest score. The analysis of higher alcohols and esters produced by S. cerevisiae then revealed enhanced formation of isobutanol, isoamyl alcohol and their esters when endogenous ARO10 was replaced with ARO10 from S. kudriavzevii. Also, significant differences in the aroma profile were found in fermentations of synthetic wine must. Substrate specificities of SkAro10p were compared with those of S. cerevisiae Aro10p (ScAro10p) by their expression in a 2-keto acid decarboxylase-null S. cerevisiae strain. Unlike the cell extracts with expressed ScAro10p which showed greater activity for phenylpyruvate, which suggests this phenylalanine-derivative to be the preferred substrate, the decarboxylation activities measured in the cell extracts with SkAro10p ranged with all the tested substrates at the same level. The activities of SkAro10p towards substrates (except phenylpyruvate) were higher than of those for ScAro10p.
  
  
  CONCLUSIONS
  The results indicate that the amino acid variations observed between the orthologues decarboxylases encoded by SkARO10 and ScARO10 could be the reason for the distinct enzyme properties, which possibly lead to the enhanced production of several flavour compounds. The knowledge on the important enzyme involved in higher alcohols biosynthesis by S. kudriavzevii could be of scientific as well as of applied interest.
authors.affiliation: Food Biotechnology Department, Institute of Agrochemistry and Food Technology, (IATA-CSIC) Avda, Agustín Escardino, 7, Paterna, 46980, Valencia, Spain.
authors.forename: Jiri
authors.initials: J
authors.lastname: Stribny
authors.affiliation: Department of Biotechnology, Delft University of Technology, Delft, The Netherlands.|Kluyver Centre for Genomics of Industrial Fermentation, Delft, The Netherlands.
authors.forename: Gabriele
authors.initials: G
authors.lastname: Romagnoli
authors.affiliation: Food Biotechnology Department, Institute of Agrochemistry and Food Technology, (IATA-CSIC) Avda, Agustín Escardino, 7, Paterna, 46980, Valencia, Spain.
authors.forename: Roberto
authors.initials: R
authors.lastname: Pérez-Torrado
authors.affiliation: Department of Biotechnology, Delft University of Technology, Delft, The Netherlands.|Kluyver Centre for Genomics of Industrial Fermentation, Delft, The Netherlands.|Platform Green Synthetic Biology, Delft, The Netherlands.
authors.forename: Jean-Marc
authors.initials: JM
authors.lastname: Daran
authors.affiliation: Food Biotechnology Department, Institute of Agrochemistry and Food Technology, (IATA-CSIC) Avda, Agustín Escardino, 7, Paterna, 46980, Valencia, Spain. amquesi@uv.es.
authors.forename: Amparo
authors.initials: A
authors.lastname: Querol
mesh_terms: D000438:Alcohols / Q000378:metabolism; D002262:Carboxy-Lyases / Q000235:genetics* / Q000378:metabolism*; D003001:Cloning, Molecular; D003653:Decarboxylation / Q000235:genetics; D004952:Esters / Q000378:metabolism; D005285:Fermentation / Q000235:genetics; D005421:Flavoring Agents / Q000378:metabolism*; D015966:Gene Expression Regulation, Fungal; D012440:Saccharomyces / Q000201:enzymology / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D013379:Substrate Specificity; D014920:Wine
chemical_list: D000438:Alcohols; D004952:Esters; D005421:Flavoring Agents; D029701:Saccharomyces cerevisiae Proteins; D002262:Carboxy-Lyases
keywords: 2-keto acid decarboxylase; ARO10; Acetate esters; Amino acid catabolism; Grantham matrix; Higher alcohols; S. cerevisiae; Saccharomyces kudriavzevii