Creation of bioactive non‐natural flavonoids via combinatorial prenylation–glycosylation cascades

Impact statement Flavonoids have significant medicinal potential; however, the reliance on plant extraction for their production poses a substantial barrier to their practical application. Innovative approaches like enzymatic cascade catalysis and microbial synthesis present promising avenues for the efficient production of these structurally complex compounds. In this study, two non‐natural flavonoids (compounds 7 and 8 ) were synthesized, with compound 7 displaying antifungal properties. This was achieved through the cascade enzymatic catalysis of naringenin, a key intermediate in flavonoid biosynthesis, using glycosyl‐ and prenyltransferases. Additionally, metabolic engineering was used to bolster the supply of the dimethylallyl pyrophosphate precursor via the isopentenol utilization pathway in Escherichia coli , facilitating the de novo creation of non‐natural flavonoids. Consequently, the titers of compounds 7 and 8 reached 47.4 and 6.6 mg/l, respectively. This research highlights the potential of modular enzyme assembly in generating bioactive flavonoid derivatives and establishes a sustainable platform for the discovery of non‐natural flavonoids.