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Abstract Converting renewable feedstocks to aromatic compounds using engineered microbes offers a robust approach for sustainable, environment‐friendly, and cost‐effective production of these value‐added products without the reliance on petroleum. In this study, rationally designedE. coli–E. colico‐culture systems were established for converting glycerol to 3‐hydroxybenzoic acid (3HB). Specifically, the 3HB pathway was modularized and accommodated by two metabolically engineeredE. colistrains. The co‐culture biosynthesis was optimized by using different cultivation temperatures, varying the inoculum ratio between the co‐culture strains, recruitment of a key pathway intermediate transporter, strengthening the critical pathway enzyme expression, and adjusting the timing for inducing pathway gene expression. Compared with theE. colimono‐culture, the optimized co‐culture showed 5.3‐fold improvement for 3HB biosynthesis. This study demonstrated the applicability of modular co‐culture engineering for addressing the challenges of aromatic compound biosynthesis. 

Abstract Modular co‐culture engineering is an emerging approach for biosynthesis of complex natural products. In this study, microbial co‐cultures composed of two and threeEscherichia colistrains, respectively, are constructed for de novo biosynthesis of flavonoid acacetin, a value‐added natural compound possessing numerous demonstrated biological activities, from simple carbon substrate glucose. To this end, the heterologous biosynthetic pathway is divided into different modules, each of which is accommodated in a dedicatedE. colistrain for functional expression. After the optimization of the inoculation ratio between the constituent strains, the engineered co‐cultures show a 4.83‐fold improvement in production comparing to the mono‐culture controls. Importantly, cultivation of the three‐strain co‐culture in shake flasks result in the production of 20.3 mg L⁻¹acacetin after 48 h. To the authors' knowledge, this is the first report on acacetin de novo biosynthesis in a heterologous microbial host. The results of this work confirm the effectiveness of modular co‐culture engineering for complex flavonoid biosynthesis.