Characterization of an α‐ketoglutarate‐dependent oxygenase involved in converting 2‐(2‐phenylethyl)chromones into 2‐styrylchromones in agarwood

2-Phenylethylchromones (PECs) and 2-styrylchromones (SCs) are the primary components responsible for the delightful fragrance and bioactivity of agarwood, a highly valuable aromatic resinous heartwood. PECs are derived from a common precursor with a diarylpentanoid skeleton (C₆-C₅-C₆). However, the biosynthesis of SCs remains unclear. In this study, based on the successful conversion of the PEC skeleton, rather than a dehydrogenated diarylpentanoid, into SCs by Aquilaria sinensis suspension cells, we demonstrated that double bond formation of the styryl group in SCs occurs after the creation of the PEC skeleton, not before this step from a dehydrogenated diarylpentanoid precursor. Through transcriptomic data mining, transient expression in Nicotiana benthamiana and A. sinensis suspension cells, we identified a new 2-oxoglutarate-dependent oxygenase (As2OG1) that plays a crucial role in the conversion of PECs into SCs. Further protein structure prediction and mutagenesis studies, combined with probing of the catalytic potential of As2OG1 using chemically synthesized hydroxylated intermediates, suggested that As2OG1 possibly uses diradical or carbocation intermediates, rather than hydroxylated intermediates, to install double bonds in SCs. The results not only provide insights into the molecular mechanism of agarwood formation but also facilitate the overproduction of pharmaceutically important SCs using metabolic engineering approaches.