Gene isolation and enzymatic characterization of UDP-glycosyltransferases of terpene glucoside biosynthesis involved in linalyl-glycoside accumulation in Coffea arabica.

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.