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

Olive (Olea europaea L.) 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 flavor and quality of olive oil. 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 olive fruit mesocarp of six commercial olive cultivars with varying oleuropein content, during maturation and ripening. Using this data we discovered three polyphenol oxidases with oleuropein synthase (OS) activity, a novel oleoside-11-methyl ester glucosyl transferase (OMEGT) synthesizing a potential intermediate in the route, and a 7-epi-loganic acid O-methyltransferase (7eLAMT). Interestingly, using transcriptome assemblies of 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, allowed the discovery of two 2-oxoglutarate dependent dioxygenases (named 7eLAS) that synthesize 7-epi-loganic acid; in 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 big datasets for untargeted discovery of enzymes. Given the increasing availability of expression data from species across the plant kingdom, the methods used for gene discovery used in this work can be readily applied to other untraced pathways.