Comparative transcriptomic and co-expression analyses enable the discovery of key enzymes responsible for oleuropein biosynthesis in olive (Olea europaea)

Olive (Olea europaea) is one of the most important crop trees, with olive oil being a key ingredient of the Mediterranean diet. Oleuropein, an oleoside-type secoiridoid, is the major determinant of olive oil flavor and quality. Iridoid biosynthesis has been elucidated in Catharanthus roseus, which produces secologanin-type secoiridoids, but iridoid biosynthesis in other species remains unresolved. In this work, we sequenced RNA from the fruit mesocarp of six commercial olive cultivars with various oleuropein contents during maturation and ripening. Using these data, we discovered three polyphenol oxidases with oleuropein synthase (OS) activity, a novel oleoside-11-methyl ester glucosyltransferase (OMEGT) that synthesizes a potential intermediate in the pathway, and a 7-epi-loganic acid O-methyltransferase (7eLAMT). Interestingly, the use of transcriptome assemblies for 15 plant species from three iridoid-producing plant orders (Lamiales, Gentianales, and Cornales) for orthogroup inference, and integration of two tissue expression panels from Jasminum sambac and Fraxinus excelsior, enabled the discovery of two 2-oxoglutarate-dependent dioxygenases (named 7eLAS) that synthesize 7-epi-loganic acid; by contrast, C. roseus 7-deoxy-loganic acid hydroxylase (7DLH), a known bottleneck in MIA production, is a cytochrome P450. This comparative co-expression method, which combines guilt-by-association and comparative transcriptomics approaches, can successfully leverage large datasets for untargeted discovery of enzymes. Given the increasing availability of expression data from species across the plant kingdom, the methods for gene discovery used in the present work can be readily applied to other untraced pathways.