title: Construction of self-cloning bottom-fermenting yeast with low vicinal diketone production by the homo-integration of ILV5.
abstract: The vicinal diketones (VDK), such as diacetyl and 2,3-pentandione, impart an unpleasant butter-like flavour to beer. Typically, these are required to be reduced below the flavour thresholds during the maturation (lagering) stages of the brewing process. To shorten beer maturation time, we constructed a self-cloning, bottom-fermenting yeast with low VDK production by integrating ILV5, a gene encoding a protein that metabolizes α-acetolactate and α-aceto-α-hydroxybutyrate (precursors of VDK). A DNA fragment containing Saccharomyces cerevisiae-type ILV5 was inserted upstream of S. cerevisiae-type ILV2 in bottom-fermenting yeast to construct self-cloning strains with an increased copy number of ILV5. Via transformation, ILV2 was replaced with the sulfometuron methyl (SM) resistance gene SMR1B, which differs by a single nucleotide, to create SM-resistant transformants. The wort fermentation test, using the SC-ILV5-homo inserted transformant, confirmed a consecutive reduction in VDK and a shortening period during which VDK was reduced to within the threshold. The concentrations of ethyl acetate, isoamyl acetate, isoamyl alcohol, 1-propanol, isobutyl alcohol and active isoamyl alcohol (flavour components) were not changed when compared with the parent strain. We successfully constructed self-cloning brewer's yeast in which SC-ILV5 was homo-inserted. Using the transformed yeast, the concentration of VDK in fermenting wort was reduced, whereas the concentrations of flavour components were not affected. This genetically stable, low VDK-producing, self-cloning bottom-fermenting yeast would contribute to the shortening of beer maturation time without affecting important flavour components produced by brewer's yeast.
authors.affiliation: Research Laboratories for Brewing, Asahi Breweries Ltd, Ibaraki, Japan. kusunoki@asahibeer.co.jp
authors.forename: K
authors.initials: K
authors.lastname: Kusunoki
authors.forename: T
authors.initials: T
authors.lastname: Ogata
mesh_terms: D000429:Alcohol Oxidoreductases / Q000235:genetics* / Q000378:metabolism; D001515:Beer / Q000382:microbiology / Q000592:standards; D003001:Cloning, Molecular; D003931:Diacetyl / Q000378:metabolism*; D005285:Fermentation; D006885:Hydroxybutyrates / Q000378:metabolism; D007773:Lactates / Q000378:metabolism; D024101:Mitochondrial Proteins / Q000235:genetics* / Q000378:metabolism; D030781:Organisms, Genetically Modified; D010422:Pentanones / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics* / Q000502:physiology; D013997:Time Factors; D014170:Transformation, Genetic
chemical_list: D006885:Hydroxybutyrates; D007773:Lactates; D024101:Mitochondrial Proteins; D010422:Pentanones; C027745:alpha-aceto-alpha-hydroxybutyrate; C006359:alpha-acetolactate; D000429:Alcohol Oxidoreductases; C470636:Ilv5 protein, S cerevisiae; D003931:Diacetyl; C013186:2,3-pentanedione