The mulberry (Morus notabilis) is a medicinal and edible plant and contains diverse flavonoids and stilbenoids with significant medicinal benefits. The biosynthesis of these compounds has only been partially elucidated. In the present investigation, we identified and characterized two 4-coumarate: coenzyme A ligases (Mn4CL1 and Mn4CL2), two polyketide synthases (MnCHS and MnSTS), three chalcone reductases (MnCHR1–3), and two 2-oxoglutarate-dependent dioxygenases (MnFLS and MnF3H) involved in flavonoids and stilbenoid biosynthesis. MnCHS converts p-coumaroyl-CoA into naringenin and facilitates the novel conversion of 2,4-dihydroxycinnamoyl-CoA to steppogenin, which features hydroxyl groups at the 4′ and 6′ positions on the B ring. MnSTS could convert p-coumaroyl-CoA and 2,4-dihydroxycinnamoyl-CoA into resveratrol and oxyresveratrol, respectively. Furthermore, MnCHR1 was first identified in mulberry and collaborated with MnCHS to produce isoliquiritigenin and 2,4,2′,4′-tetrahydroxychalcone. A co-expression system of Mn4CL1, MnCHS, and MnCHR1 enabled the fermentation production of steppogenin and 2,4,2′,4′-tetrahydroxychalcone in engineered Escherichia coli. The in vitro enzymatic assays confirmed that MnFLS showed both FLS and F3H activities, whereas transgenic experiments revealed its predominant FLS function in vivo; MnF3H was confirmed as a bona fide F3H. These findings provide new insights into the flavonoids and stilbenoids biosynthesis pathway in mulberry and suggest a potential parallel pathway for 4′,6′-dihydroxylated flavonoids biosynthesis.

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