7-O-Methyl aromadendrin (7-OMA) is an aglycone moiety of one of the important flavonoid-glycosides found in several plants, such asPopulus albaandEucalyptus maculata, with various medicinal applications. To produce such valuable natural flavonoids in large quantity, anEscherichia colicell factory has been developed to employ various plant biosynthetic pathways. Here, we report the generation of 7-OMA from its precursor,p-coumaric acid, inE. colifor the first time. Primarily, naringenin (NRN) (flavanone) synthesis was achieved by feedingp-coumaric acid and reconstructing the plant biosynthetic pathway by introducing the following structural genes: 4-coumarate–coenzyme A (CoA) ligase fromPetroselinum crispum, chalcone synthase fromPetunia hybrida, and chalcone isomerase fromMedicago sativa.In order to increase the availability of malonyl-CoA, a critical precursor of 7-OMA, genes for the acyl-CoA carboxylase α and β subunits (nfa9890andnfa9940), biotin ligase (nfa9950), and acetyl-CoA synthetase (nfa3550) fromNocardia farcinicawere also introduced. Thus, produced NRN was hydroxylated at position 3 by flavanone-3-hydroxylase fromArabidopsis thaliana, which was further methylated at position 7 to produce 7-OMA in the presence of 7-O-methyltransferase fromStreptomyces avermitilis. Dihydrokaempferol (DHK) (aromadendrin) and sakuranetin (SKN) were produced as intermediate products. Overexpression of the genes for flavanone biosynthesis and modification pathways, along with malonyl-CoA overproduction inE. coli, produced 2.7 mg/liter (8.9 μM) 7-OMA upon supplementation with 500 μMp-coumaric acid in 24 h, whereas the strain expressing only the flavanone modification enzymes yielded 30 mg/liter (99.2 μM) 7-OMA from 500 μM NRN in 24 h.