Characterization and employment of novel α-ketoglutarate dependent oxygenases for efficient synthesis of antifungal alkyl citrates

Fungal alkyl citrates (ACs) exhibit potent antifungal activity, offering significant potential for drug development. Biosynthetic studies of sporothriolide and squalestatin S1 have shown that α-ketoglutarate dependent oxygenases (αKG OX), SpoG and MfR1/2, transform early-stage alkyl citrate precursors into diverse scaffolds through regio- and stereospecific oxidations. Here, we report two previously uncharacterized αKG OX, OryG and PaR5, which specifically hydroxylate the alkyl side chain and epoxidate the terminal alkene of alkyl itaconic acid 3, respectively. Furthermore, the catalytic mechanism and regioselectivity of PaR5 and OryG were investigated through molecular docking, site-directed mutagenesis, molecular dynamics simulation, and quantum mechanics calculation. In PaR5, the distance from the Fe(_IV)=O species to C-6 of alkyl itaconic acid 3 was greater than to C-13, which well explained the oxidation preference at the olefin position. On the contrary, the Fe(_IV)=O species are closer to C-6 than to C-13 in the OryG complex, which matches the consequence of alkyl side chain hydroxylation. By integrating Aspergillus oryzae biosynthesis, whole-cell biotransformation, and chemical workup, we developed an efficient synthetic strategy for the large-scale synthesis of antifungal sporothriolide 2 and novel epoxide 9 and lactone 10. Bioactivity assays demonstrated that sporothriolide 2 exhibits antifungal activity comparable to nystatin, while epoxide 9 and lactone 10 showed modest inhibition. Our study sheds light on the development and large-scale synthesis of novel alkyl citrates with promising antifungal activity.