Unraveling the different roles of enone oxidoreductases (FaEOs) in strawberry to improve aroma quality during cold storage

The unique aroma of strawberry ( Fragaria × ananassa ) is dominated by the accumulation of the key volatile compound, 4-hydroxy-2,5-dimethyl-3(2 H )-furanone (HDMF). Strawberry enone oxidoreductase (FaEO) emerges as a critical enzyme in catalyzing 4-hydroxy-5-methyl-2-methylene-3(2 H )-furanone (HMMF) to form HDMF. Here, 10 FaEO genes were isolated from strawberry, and the contributions of these genes to HDMF synthesis were analyzed. The sequence, docking and phylogenetic analysis revealed that these FaEOs could be classified into two distinct monophyletic groups, with three of them exhibiting a notable absence of the conserved G-X-G-X-X-G motif. During the ripening process, the expression of seven intact FaEO s surged, thereby enhancing HDMF accumulation, whereas cold storage conditions led to a marked reduction. In vivo enzymatic activity assays demonstrated that overexpressing FaEO s in both tobacco leaves and transgenic low-aroma strawberries significantly elevated HDMF levels within the plant, even though no discernible catalytic differences were observed among the seven enzymes in this context. Heterologous functional surveys of the seven FaEOs, using the Escherichia coli and the Yarrowia lipolytica expression systems, revealed that FaEO1 was more efficient than the others in catalyzing HDMF formation. Furthermore, considering predominant contribution of FaEO1 -transformant of Y. lipolytica in HDMF biosynthesis, this biotechnological strategy was successfully applied in restoring the aroma of diverse strawberry cultivars following prolonged cold storage, thereby offering a promising avenue for enhancing the postharvest quality and marketability of strawberries. • Different roles of FaEOs contribute to the strawberry aroma in vitro and in vivo . • FaEO1 -transformants of Y. lipolytica could restore the aroma of strawberry.