CYP82G1 Enzyme Functions as Chromone Hydroxylase in the Biosynthesis of 6‐Hydroxy‐2‐(2‐Phenylethyl)chromone in <i>Aquilaria sinensis</i>

Aquilaria sinensis, a plant of the genus Aquilaria in the family Thymelaeaceae, can form resinous and fragrant agarwood in response to wounding or infection. The resin physically occludes xylem vessels and ray parenchyma, limiting pathogen mobility and resource loss. The quality of agarwood is often evaluated using highly oxygenated 2-(2-phenylethyl)chromones, such as agarotetrol, 2-[2-(4-methoxy)phenylethyl]chromone, 4'-methoxyagarotetrol and 6,7-dimethoxy-2-(2-phenylethyl)chromone. Among the derivatives of 2-(2-phenylethyl)chromone (PEC), the majority possess a hydroxyl or methoxy group at the C-6 position of the chromone, with subsequent substitutions occurring at the C-5, C-7 and/or C-8 positions. The hydroxylation at the C-6 position is a critical step in the biosynthesis of these 2-(2-phenylethyl)chromones (PECs). In this study, we investigated the gene expression differences and accumulation patterns of PECs in two-year-old wild A. sinensis and Qi-Nan A. sinensis, using transcriptomic and LC-MS/MS analysis. Based on these analyses, we identified 52 candidate genes encoding cytochrome P450 (CYPs). Further investigations using a yeast expression system and functional analysis in Nicotiana benthamiana and A. sinensis revealed that AsCYP82G1 could hydroxylate 2-(2-phenylethyl)chromone to form 6-hydroxy-2-(2-phenylethyl)chromone. Homology modelling and molecular docking demonstrated that the residues methionine (MET) and arginine (ARG) at positions 222 and 223 of the AsCYP82G1 protein are likely to play an important role in the catalysis of 2-(2-phenylethyl)chromone to 6-hydroxy-2-(2-phenylethyl)chromone. Finally, the reaction mechanism of AsCYP82G1 was studied by quantum mechanical calculation. Our research provides new insights into the molecular mechanism of PECs biosynthesis and lays the foundation for the breeding of high-quality agarwood varieties and large-scale production of PECs using synthetic biology.