Establishing microbial co-cultures for 3-hydroxybenzoic acid biosynthesis on glycerol.

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 designed E. coli-E. coli co-culture systems were established for converting glycerol to 3-hydroxybenzoic acid (3HB). Specifically, the 3HB pathway was modularized and accommodated by two metabolically engineered E. coli strains. 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 the E. coli mono-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.