Targeting of mitochondrial Saccharomyces cerevisiae Ilv5p to the cytosol and its effect on vicinal diketone formation in brewing.
Vicinal diketones (VDK) cause butter-like off-flavors in beer and are formed by a non-enzymatic oxidative decarboxylation of alpha-aceto-alpha-hydroxybutyrate and alpha-acetolactate, which are intermediates in isoleucine and valine biosynthesis taking place in the mitochondria. On the assumption that part of alpha-acetolactate can be formed also in the cytosol due to a mislocalization of the responsible acetohydroxyacid synthase encoded by ILV2 and ILV6, functional expression in the cytosol of acetohydroxyacid reductoisomerase (Ilv5p) was explored. Using the cytosolic Ilv5p, I aimed to metabolize the cytosolically formed alpha-aetolactate, thereby lowering the total VDK production. Among mutant Ilv5p enzymes with varying degrees of N-terminal truncation, one with a 46-residue deletion (Ilv5pDelta46) exhibited an unequivocal localization in the cytosol judged from microscopy of the Ilv5pDelta46-green fluorescent protein fusion protein and the inability of Ilv5pDelta46 to remedy the isoleucine/valine requirement of an ilv5Delta strain. When introduced into an industrial lager brewing strain, a robust expression of Ilv5pDelta46 was as effective as that of a wild-type Ilv5p in lowering the total VDK production in a 2-l scale fermentation trial. Unlike the case of the wild-type Ilv5p, an additional expression of Ilv5pDelta46 did not alter the quality of the resultant beer in terms of contents of aromatic compounds and organic acids.
Omura
Fumihiko
F
Suntory Research Center, 1-1-1 Wakayamadai, Shimamoto-cho, Mishima-gun, Osaka, 618-8503, Japan. Fumihiko_Omura@suntory.co.jp
D000429:Alcohol Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D001515:Beer / Q000382:microbiology*; D003600:Cytosol / Q000201:enzymology / Q000378:metabolism*; D004795:Enzyme Stability; D005285:Fermentation; D005816:Genetic Complementation Test; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D007532:Isoleucine / Q000378:metabolism; D007659:Ketones / Q000378:metabolism*; D008928:Mitochondria / Q000201:enzymology* / Q000378:metabolism; D024101:Mitochondrial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000201:enzymology* / Q000378:metabolism; D017384:Sequence Deletion; D014633:Valine / Q000378:metabolism
D007659:Ketones; D024101:Mitochondrial Proteins; D011993:Recombinant Fusion Proteins; D007532:Isoleucine; D049452:Green Fluorescent Proteins; D000429:Alcohol Oxidoreductases; C470636:Ilv5 protein, S cerevisiae; D014633:Valine