Faculty of Bioresource Sciences, Akita Prefectural University, Akita City, Akita, 010-0195, Japan.
Faculty of Bioresource Sciences, Akita Prefectural University, Akita City, Akita, 010-0195, Japan.
Biochem Biophys Res Commun. 2024 Nov 12;733:150694. doi: 10.1016/j.bbrc.2024.150694. Epub 2024 Sep 12.
Terpenes, one of the secondary metabolites produced by plants, have diverse physiological functions. They are volatile compounds with physiological bioactivities (e.g., insect repellent, attracting enemies, and interacting with other plants). Terpenoids are also essential for flavor and aroma in plant-derived foods. In coffee, its aroma decides the value of coffee beans. Linalool, one of the volatile terpene compounds, is dominant in the coffee aroma. Coffee, with its good flavor and aroma, has high demand worldwide. Because terpenoids generally accumulate as glycosides in plant cells, glycosylation is catalyzed by UDP-glycosyltransferases (UGTs). Two linalyl-diglycosides have been identified: terpenoids reflected as necessary for coffee flavor. However, these UGTs and their action mechanisms are unknown in the Coffea genus. To obtain knowledge of terpene UGTs and elucidate the mechanism of terpene glycosylation in coffee, this study isolated terpene UGT genes and analyzed their functions. In silico screening based on the sequence of UGT85K11, which catalyzes terpene glycosylation from Camellia sinensis, was performed to obtain sequence information on five candidate UGT genes (CaUGT4, CaUGT5, CaUGT10, CaUGT15, and CaUGT20). These genes were isolated by reverse transcription-polymerase chain reaction, and the recombinant enzymes were produced with the Escherichia coli expression system. In functional analysis using radioisotopes, CaUGT4 showed critical activity against linalool, which had a higher affinity for its substrate than that of UGT85A84 from Osmanthus fragrans. Liquid chromatography-tandem mass spectrometry also revealed that CaUGT4 mainly produces linalyl glucoside. In this study, the first linalyl UGT was isolated from coffee. These findings can be used to elucidate the fundamental mechanism of the chemical defense in plants and apply aroma precursors for the plant-derived food industry in the future.
萜类化合物是植物产生的次生代谢物之一，具有多种生理功能。它们是具有生理生物活性的挥发性化合物（例如，驱虫、吸引天敌和与其他植物相互作用）。类萜化合物也是植物源性食品风味和香气的重要组成部分。在咖啡中，其香气决定了咖啡豆的价值。芳樟醇是挥发性萜类化合物之一，是咖啡香气的主要成分。具有良好风味和香气的咖啡在全球有很高的需求。由于萜类化合物通常在植物细胞中以糖苷的形式积累，因此糖苷化由 UDP-糖基转移酶（UGTs）催化。已经鉴定出两种芳樟醇二糖苷：萜类化合物被认为是咖啡风味所必需的。然而，这些 UGT 及其作用机制在咖啡属中尚不清楚。为了获得萜类 UGT 的知识并阐明咖啡中萜类化合物的糖苷化机制，本研究分离了萜类 UGT 基因并分析了它们的功能。基于催化 Camellia sinensis 中萜类化合物糖苷化的 UGT85K11 的序列，进行了基于计算机的筛选，以获得五个候选 UGT 基因（CaUGT4、CaUGT5、CaUGT10、CaUGT15 和 CaUGT20）的序列信息。通过反转录聚合酶链式反应分离这些基因，并使用大肠杆菌表达系统产生重组酶。在使用放射性同位素的功能分析中，CaUGT4 对芳樟醇表现出关键活性，其对底物的亲和力高于Osmanthus fragrans 的 UGT85A84。液相色谱-串联质谱分析也表明 CaUGT4 主要产生芳樟醇葡萄糖苷。在本研究中，首次从咖啡中分离出芳樟醇 UGT。这些发现可用于阐明植物化学防御的基本机制，并在未来应用香气前体于植物源性食品工业。
