Novel 4-methyl-2-oxopentanoate reductase involved in synthesis of the Japanese sake flavor, ethyl leucate.
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.
['Shimizu|Motoyuki|M|', 'Yamamoto|Tatsuya|T|', 'Okabe|Natsumi|N|', 'Sakai|Kiyota|K|', 'Koide|Emiri|E|', 'Miyachi|Yuta|Y|', 'Kurimoto|Maki|M|', 'Mochizuki|Mai|M|', 'Yoshino-Yasuda|Shoko|S|', 'Mitsui|Shun|S|', 'Ito|Akitoshi|A|', 'Murano|Hirotatsu|H|', 'Takaya|Naoki|N|', 'Kato|Masashi|M|']
D000429:Alcohol Oxidoreductases D000434:Alcoholic Beverages D001236:Aspergillus oryzae D003001:Cloning, Molecular D004926:Escherichia coli D005285:Fermentation D005421:Flavoring Agents D019649:Food Industry D005656:Fungal Proteins D015870:Gene Expression D007218:Industrial Microbiology D007700:Kinetics D011994:Recombinant Proteins D012441:Saccharomyces cerevisiae D013379:Substrate Specificity D014631:Valerates
Japanese sake flavor Ethyl leucate Leucate MOA reductase 4-Methyl-2-oxopentanate (MOA) Glyoxylate/hydroxypyruvate reductase