title: Novel 4-methyl-2-oxopentanoate reductase involved in synthesis of the Japanese sake flavor, ethyl leucate.
abstract: Ethyl-2-hydroxy-4-methylpentanoate (ethyl leucate) contributes to a fruity flavor in Japanese sake. The mold Aspergillus oryzae synthesizes leucate from leucine and then the yeast Saccharomyces cerevisiae produces ethyl leucate from leucate during sake fermentation. Here, we investigated the enzyme involved in leucate synthesis by A. oryzae. The A. oryzae gene/cDNA encoding the enzyme involved in leucate synthesis was identified and expressed in E. coli and A. oryzae host cells. The purified recombinant enzyme belonged to a D-isomer-specific 2-hydroxyacid dehydrogenase family and it NADPH- or NADH-dependently reduced 4-methyl-2-oxopentanate (MOA), a possible intermediate in leucine synthesis, to D-leucate with a preference for NADPH. Thus, we designated this novel enzyme as MOA reductase A (MorA). Furthermore, an A. oryzae strain overexpressing morA produced 125-fold more leucate than the wild-type strain KBN8243. The strain overexpressing MorA produced 6.3-fold more ethyl leucate in the sake than the wild-type strain. These findings suggest that the strain overexpressing morA would help to ferment high-quality sake with an excellent flavor. This is the first study to identify the MOA reductase responsible for producing D-leucate in fungi.
authors.affiliation: Department of Applied Biological Chemistry, Faculty of Agriculture, Meijo University, Aichi, 468-0073, Japan. moshimi@meijo-u.ac.jp.
authors.forename: Motoyuki
authors.initials: M
authors.lastname: Shimizu
authors.affiliation: Department of Applied Biological Chemistry, Faculty of Agriculture, Meijo University, Aichi, 468-0073, Japan.
authors.forename: Tatsuya
authors.initials: T
authors.lastname: Yamamoto
authors.affiliation: Department of Applied Biological Chemistry, Faculty of Agriculture, Meijo University, Aichi, 468-0073, Japan.
authors.forename: Natsumi
authors.initials: N
authors.lastname: Okabe
authors.affiliation: Department of Applied Biological Chemistry, Faculty of Agriculture, Meijo University, Aichi, 468-0073, Japan.
authors.forename: Kiyota
authors.initials: K
authors.lastname: Sakai
authors.affiliation: Department of Applied Biological Chemistry, Faculty of Agriculture, Meijo University, Aichi, 468-0073, Japan.
authors.forename: Emiri
authors.initials: E
authors.lastname: Koide
authors.affiliation: Department of Applied Biological Chemistry, Faculty of Agriculture, Meijo University, Aichi, 468-0073, Japan.
authors.forename: Yuta
authors.initials: Y
authors.lastname: Miyachi
authors.affiliation: Department of Applied Biological Chemistry, Faculty of Agriculture, Meijo University, Aichi, 468-0073, Japan.
authors.forename: Maki
authors.initials: M
authors.lastname: Kurimoto
authors.affiliation: Department of Applied Biological Chemistry, Faculty of Agriculture, Meijo University, Aichi, 468-0073, Japan.
authors.forename: Mai
authors.initials: M
authors.lastname: Mochizuki
authors.affiliation: Food Research Center, Aichi Center for Industry and Science Technology, 2-1-1 Shimpukuji-cho, Nishi-ku, Nagoya, Aichi, 451-0083, Japan.
authors.forename: Shoko
authors.initials: S
authors.lastname: Yoshino-Yasuda
authors.affiliation: Food Research Center, Aichi Center for Industry and Science Technology, 2-1-1 Shimpukuji-cho, Nishi-ku, Nagoya, Aichi, 451-0083, Japan.
authors.forename: Shun
authors.initials: S
authors.lastname: Mitsui
authors.affiliation: Food Research Center, Aichi Center for Industry and Science Technology, 2-1-1 Shimpukuji-cho, Nishi-ku, Nagoya, Aichi, 451-0083, Japan.
authors.forename: Akitoshi
authors.initials: A
authors.lastname: Ito
authors.affiliation: Department of Applied Biological Chemistry, Faculty of Agriculture, Meijo University, Aichi, 468-0073, Japan.
authors.forename: Hirotatsu
authors.initials: H
authors.lastname: Murano
authors.affiliation: Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki, 305-8572, Japan.
authors.forename: Naoki
authors.initials: N
authors.lastname: Takaya
authors.affiliation: Department of Applied Biological Chemistry, Faculty of Agriculture, Meijo University, Aichi, 468-0073, Japan.
authors.forename: Masashi
authors.initials: M
authors.lastname: Kato
mesh_terms: D000429:Alcohol Oxidoreductases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000434:Alcoholic Beverages / Q000032:analysis*; D001236:Aspergillus oryzae / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005285:Fermentation; D005421:Flavoring Agents / Q000737:chemistry / Q000378:metabolism*; D019649:Food Industry; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D007218:Industrial Microbiology; D007700:Kinetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity; D014631:Valerates / Q000737:chemistry / Q000378:metabolism*
chemical_list: D005421:Flavoring Agents; D005656:Fungal Proteins; D011994:Recombinant Proteins; D014631:Valerates; C517080:ethyl 2-hydroxy-4-methylpentanoate; D000429:Alcohol Oxidoreductases; C083779:2-hydroxyacid dehydrogenase
keywords: 4-Methyl-2-oxopentanate (MOA); Ethyl leucate; Glyoxylate/hydroxypyruvate reductase; Japanese sake flavor; Leucate; MOA reductase