title: Functional analysis and transcriptional regulation of two orthologs of ARO10, encoding broad-substrate-specificity 2-oxo-acid decarboxylases, in the brewing yeast Saccharomyces pastorianus CBS1483.
abstract: The hybrid genomes of Saccharomyces pastorianus consist of subgenomes similar to those of S. cerevisiae and S. eubayanus, and impact of the genome structure on flavour production and its regulation is poorly understood. This study focuses on ARO10, a 2-oxo-acid decarboxylase involved in production of higher alcohols. In S. pastorianus CBS1483, four ARO10 copies were identified, three resembled S. cerevisiae ARO10 and one S. eubayanus ARO10. Substrate specificities of lager strain (Lg)ScAro10 and LgSeubAro10 were compared by individually expressing them in a pdc1Δ-pdc5Δ-pdc6Δ-aro10Δ-thi3Δ S. cerevisiae strain. Both isoenzymes catalysed decarboxylation of the 2-oxo-acids derived from branched-chain, sulphur-containing amino acids and preferably phenylpyruvate. Expression of both alleles was induced by phenylalanine, however in contrast to the S. cerevisiae strain, the two genes were not induced by leucine. Additionally, LgSeubARO10 showed higher basal expression levels during growth with ammonia. ARO80, which encodes ARO10 transcriptional activator, is located on CHRIV and counts three Sc-like and one Seub-like copies. Deletion of LgSeubARO80 did not affect LgSeubARO10 phenylalanine induction, revealing 'trans' regulation across the subgenomes. ARO10 transcript levels showed a poor correlation with decarboxylase activities. These results provide insights into flavour formation in S. pastorianus and illustrate the complexity of functional characterization in aneuploid strains.
authors.affiliation: Industrial Microbiology Section, Department of Biotechnology, Delft University of Technology, Delft, The Netherlands.
authors.forename: Irina
authors.initials: I
authors.lastname: Bolat
authors.forename: Gabriele
authors.initials: G
authors.lastname: Romagnoli
authors.forename: Feibai
authors.initials: F
authors.lastname: Zhu
authors.forename: Jack T
authors.initials: JT
authors.lastname: Pronk
authors.forename: Jean-Marc
authors.initials: JM
authors.lastname: Daran
mesh_terms: D000596:Amino Acids / Q000378:metabolism; D002262:Carboxy-Lyases / Q000235:genetics* / Q000378:metabolism*; D017353:Gene Deletion; D015966:Gene Expression Regulation, Fungal; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D007651:Keto Acids / Q000378:metabolism*; D005810:Multigene Family; D012440:Saccharomyces / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D000596:Amino Acids; D007527:Isoenzymes; D007651:Keto Acids; D002262:Carboxy-Lyases
keywords: ARO10; Brewing; Saccharomyces pastorianus; TPP-dependent 2-oxo acid decarboxylase; functional characterization; hybrid genome