Uehara Kenji, Watanabe Jun, Mogi Yoshinobu, Tsukioka Yuichiro
Manufacturing Division, Yamasa Corporation, 2-10-1 Araoicho, Choshi, Chiba 288-0056, Japan.
Manufacturing Division, Yamasa Corporation, 2-10-1 Araoicho, Choshi, Chiba 288-0056, Japan.
J Biosci Bioeng. 2017 Mar;123(3):333-341. doi: 10.1016/j.jbiosc.2016.10.004. Epub 2016 Nov 16.
4-Hydroxy-2(or 5)-ethyl-5(or 2)-methyl-3(2H)-furanone (HEMF) is considered a key flavor compound in soy sauce. The compound has a caramel-like aroma and several important physiological activities, such as strong antioxidant activity. Here, we report the identification and characterization of an enzyme involved in the biosynthesis of HEMF in yeast. We fractionated yeast cell-free extract from Saccharomyces cerevisiae using column chromatography and partially purified a fraction with HEMF-forming activity. Peptide mass fingerprinting analysis showed that the partially purified fraction contains aldehyde reductase encoded by YNL134C. This reductase shares low sequence identity with enone oxidoreductase, which is responsible for the formation of 4-hydroxy-2,5-dimethyl-3(2H)-furanone (HDMF) and HEMF in plants. YNL134C was expressed heterologously in Escherichia coli, and the purified protein catalyzed the formation of HEMF from the mixture of Maillard reaction products, acetaldehydes, and NADPH. Multicopy expression in S. cerevisiae resulted in increased HEMF productivity, and gene knockout of YNL134C in S. cerevisiae resulted in decreased HEMF productivity. These data suggest that the translation product of YNL134C is the HEMF-producing enzyme in yeast. Detailed analyses of an intermediate in the enzymatic reaction mixture revealed that HEMF is synthesized from (2E)-2-ethylidene-4-hydroxy-5-methyl-3(2H)-furanone, which formed via Knoevenagel condensation between the acetaldehyde and 4-hydroxy-5-methyl-3(2H)-furanone derived from the Maillard reaction based on ribose and glycine, by YNL134Cp in an NADPH dependent manner. Overall, this study shed light on the molecular basis for the improvement of soy sauce flavor and the biotechnological production of HEMF.
4-羟基-2（或5）-乙基-5（或2）-甲基-3（2H）-呋喃酮（HEMF）被认为是酱油中的一种关键风味化合物。该化合物具有类似焦糖的香气以及多种重要的生理活性，如强大的抗氧化活性。在此，我们报告了酵母中参与HEMF生物合成的一种酶的鉴定与特性。我们使用柱色谱法对酿酒酵母的无细胞提取物进行分级分离，并部分纯化了具有HEMF形成活性的级分。肽质量指纹图谱分析表明，部分纯化的级分含有由YNL134C编码的醛还原酶。这种还原酶与烯酮氧化还原酶的序列同一性较低，烯酮氧化还原酶负责植物中4-羟基-2,5-二甲基-3（2H）-呋喃酮（HDMF）和HEMF的形成。YNL134C在大肠杆菌中进行了异源表达，纯化后的蛋白催化美拉德反应产物、乙醛和NADPH混合物形成HEMF。在酿酒酵母中多拷贝表达导致HEMF产量增加，而酿酒酵母中YNL134C的基因敲除导致HEMF产量降低。这些数据表明，YNL134C的翻译产物是酵母中产生HEMF的酶。对酶促反应混合物中的一种中间体进行的详细分析表明，HEMF由（2E）-2-亚乙基-4-羟基-5-甲基-3（2H）-呋喃酮合成，该中间体是基于核糖和甘氨酸的美拉德反应衍生的乙醛与4-羟基-5-甲基-3（2H）-呋喃酮之间通过克诺文纳格尔缩合形成的，由YNL134Cp以NADPH依赖的方式催化。总体而言，本研究揭示了改善酱油风味和HEMF生物技术生产的分子基础。
