It runs in the family: Discovery of enzymes in the oleuropein pathway in Olive (Olea europaea) by comparative transcriptomics

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, integrating transcriptomics data from 15 plant species from three iridoid-producing plant orders (Lamiales, Gentianales, and Cornales), and tissue expression panels from Jasminum sambac and Fraxinus excelsior, we discovered 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. Key FindingsO_LIExpression of genes involved in iridoid biosynthesis, from the early MEP pathway to the last step of oleuropein biosynthesis, decreases during olive fruit maturation. C_LIO_LIWe discovered an oxoglutarate dependent dioxygenase, 7-epi-loganic acid synthase (7eLAS), catalyzing the stereoselective oxidation of 7-deoxy-loganic acid to 7-epi-loganic acid, in a reaction analogous to C. roseus 7-deoxy-loganic acid hydroxylase (7DLH), a cytochrome p450. C_LIO_LIWe report a 7-epi-loganic acid O-methyltransferase (7eLAMT) orthologous to Catharanthus roseus loganic acid O-methyltransferase and found a novel oleoside-11-methyl ester glucosyl transferase (OMEGT) synthesizing 7-{beta}-1-D-glucopyranosyl-oleoside-11-methyl ester, a potential intermediate in the oleuropein biosynthesis route. C_LIO_LIWe discovered three olive polyphenol oxidases that have oleuropein synthase (OS) activity, catalyzing the conversion of ligstroside to oleuropein. C_LI