Novel 4-methyl-2-oxopentanoate reductase involved in synthesis of the Japanese sake flavor, ethyl leucate

• Published in: Applied microbiology and biotechnology 2016-04, Vol.100 (7), p.3137-3145
• Main Authors: Shimizu, Motoyuki, Yamamoto, Tatsuya, Okabe, Natsumi, Sakai, Kiyota, Koide, Emiri, Miyachi, Yuta, Kurimoto, Maki, Mochizuki, Mai, Yoshino-Yasuda, Shoko, Mitsui, Shun, Ito, Akitoshi, Murano, Hirotatsu, Takaya, Naoki, Kato, Masashi
• Format: Article
• Language: English
• Subjects: Agricultural biotechnology; Alcohol; Alcohol Oxidoreductases - chemistry; Alcohol Oxidoreductases - genetics; Alcohol Oxidoreductases - metabolism; Alcoholic beverages; Alcoholic Beverages - analysis; Aspergillus; Aspergillus oryzae; Aspergillus oryzae - chemistry; Aspergillus oryzae - enzymology; Aspergillus oryzae - genetics; Beverages; Biomedical and Life Sciences; Biosynthesis; Biotechnologically Relevant Enzymes and Proteins; Biotechnology; Chemical properties; Cloning, Molecular; complementary DNA; Composition; Dehydrogenases; E coli; Enzymes; Escherichia coli; Escherichia coli - genetics; Escherichia coli - metabolism; Ethyl compounds; Fermentation; flavor; Flavoring Agents - chemistry; Flavoring Agents - metabolism; Food Industry; Fungal Proteins - chemistry; Fungal Proteins - genetics; Fungal Proteins - metabolism; Fungi; Gene Expression; genes; Identification and classification; Industrial Microbiology; Kinetics; leucine; Life Sciences; Microbial Genetics and Genomics; Microbiology; NADP (coenzyme); Observations; Plasmids; Proteins; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Rice; Saccharomyces cerevisiae; Saccharomyces cerevisiae - chemistry; Saccharomyces cerevisiae - enzymology; Saccharomyces cerevisiae - genetics; sake; Sake (Beverage); Studies; Substrate Specificity; Valerates - chemistry; Valerates - metabolism; Wines; Yeast; Yeasts
• ISSN: 0175-7598; 1432-0614
• DOI: 10.1007/s00253-015-7182-0

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.