title: Functional characterization of three Coffea arabica L. monoterpene synthases: insights into the enzymatic machinery of coffee aroma.
abstract: The chemical composition of the coffee beverage is extremely complex, being made up of hundreds of volatile and non-volatile compounds, many of which are generated in the thermal reactions that occur during the roasting process. However, in the raw coffee bean there are also compounds that survive roasting and are therefore extracted into the beverage. Monoterpenes are an example of this category, as their presence has been reported in the coffee flower, fruit, seed, roasted bean and in the beverage aroma. The present work describes the isolation, heterologous expression and functional characterization of three Coffea arabica cDNAs coding for monoterpene synthases. RNA was purified from C. arabica (cv. Catuai Red) flowers, seeds and fruits at 4 successive ripening stages. Degenerate primers were designed on the most conserved regions of the monoterpene synthase gene family, and then used to isolate monoterpene synthase-like sequences from the cDNA libraries. After 5'- and 3'-RACE, the complete transcripts of 4 putative C. arabica monoterpene synthases (CofarTPS) were obtained. Gene expression in different tissues and developmental stages was analysed. After heterologous expression in Escherichia coli, enzyme activity and substrate specificity were evaluated in vitro by incubation of the recombinant proteins with geranyl pyrophosphate (GPP), geranylgeranyl pyrophosphate (GGPP) and farnesyl pyrophosphate (FPP), precursors respectively of mono-, di- and sesquiterpenes. The reaction products were characterized by HS-SPME GC-MS. CofarTPS1 was classified as a limonene synthase gene, while CofarTPS2 and 3 showed lower activity with the production of linalool and β-myrcene.
authors: illycaffè SpA, via Flavia 110, 34147 Trieste, Italy. lorenzo.delterra@illy.com
authors: Lorenzo
authors: L
authors: Del Terra
authors: Valentina
authors: V
authors: Lonzarich
authors: Elisa
authors: E
authors: Asquini
authors: Luciano
authors: L
authors: Navarini
authors: Giorgio
authors: G
authors: Graziosi
authors: Furio
authors: F
authors: Suggi Liverani
authors: Alberto
authors: A
authors: Pallavicini
mesh_terms: D000595:Amino Acid Sequence; D055162:Biocatalysis; D003001:Cloning, Molecular; D040503:Coffea / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D018506:Gene Expression Regulation, Plant; D019753:Intramolecular Lyases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D009812:Odorants / Q000032:analysis*
chemical_list: D018076:DNA, Complementary; D019753:Intramolecular Lyases; C040265:pinene cyclase I