A membrane-bound aromatic O -prenyltransferase catalyzes the last reaction step in citrus auraptene biosynthesis

Plants produce a variety of O -prenylated aromatics that exhibit biological activities beneficial to human health, and the presence of the O -prenyl moiety is often crucial to their functions. However, most aromatic O -prenylation genes remain unknown in plants. In this study, we report the molecular identification of an aromatic O -prenyltransferase (PT) involved in the biosynthesis of auraptene (7-geranyloxycoumarin), a citrus metabolite known for its preservative effect on human cognitive function. Based on in silico screening focusing on the membrane-bound PT family, CpPT4 was isolated as a candidate from grapefruit ( Citrus x paradisi ), an auraptene-rich species. Enzymatic characterization demonstrated that recombinant CpPT4 specifically catalyzes umbelliferone 7- O -geranyltransferase activity to form auraptene, which differs from the enzymatic functions of known O -PTs. This enzyme also catalyzed aromatic N -prenylation to produce a new-to-nature auraptene analog. Regarding organ- and organellar-specific localization, it is strongly suggested that CpPT4 functions in the outer pericarp plastids, where auraptene is expected be formed. Furthermore, we found that CpPT4 orthologs are widely distributed in citrus genomes. Intriguingly, mandarins and their descendant species possess dysfunctional orthologs, which is consistent with the low accumulation of auraptene and its downstream metabolites in these species. This study provides an example of the contribution of the UbiA superfamily to O -prenylated aromatic biosynthesis. Moreover, CpPT4 can be useful as a tool in the synthetic biology-based production of auraptene and its analogs, as well as a molecular marker in the breeding of auraptene-rich citrus varieties.