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
Department of Applied Biological Chemistry, Faculty of Agriculture, Meijo University, Aichi, 468-0073, Japan.
Food Research Center, Aichi Center for Industry and Science Technology, 2-1-1 Shimpukuji-cho, Nishi-ku, Nagoya, Aichi, 451-0083, Japan.
Appl Microbiol Biotechnol. 2016 Apr;100(7):3137-45. doi: 10.1007/s00253-015-7182-0. Epub 2015 Nov 28.
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.
2-羟基-4-甲基戊酸乙酯（亮氨酸乙酯）赋予日本清酒水果香味。米曲霉能从亮氨酸合成亮氨酸乙酯，随后酿酒酵母在清酒发酵过程中从亮氨酸乙酯生成2-羟基-4-甲基戊酸乙酯。在此，我们研究了米曲霉中参与亮氨酸乙酯合成的酶。鉴定了米曲霉中编码参与亮氨酸乙酯合成的酶的基因/cDNA，并在大肠杆菌和米曲霉宿主细胞中进行表达。纯化后的重组酶属于D-异构体特异性2-羟基酸脱氢酶家族，它以NADPH或NADH为依赖，将亮氨酸合成过程中可能的中间体4-甲基-2-氧代戊酸（MOA）还原为D-亮氨酸，且更偏好NADPH。因此，我们将这种新酶命名为MOA还原酶A（MorA）。此外，过表达morA的米曲霉菌株产生的亮氨酸乙酯比野生型菌株KBN8243多125倍。过表达MorA的菌株在清酒中产生的2-羟基-4-甲基戊酸乙酯比野生型菌株多6.3倍。这些发现表明，过表达morA的菌株有助于发酵出风味极佳的优质清酒。这是首次在真菌中鉴定出负责产生D-亮氨酸的MOA还原酶的研究。
