(499f) Dynamic Regulation of 4-Hydroxycoumarin Synthesis in Engineered Escherichia coli

4-Hydroxycoumarin (4-HC) is an important pharmacophore for manufacturing of oral anticoagulants. The de novo biosynthetic pathway of 4-HC has been established by condensation of salicoyl-CoA and malonyl-CoA in Escherichia coli. However, the imbalance between the co-precursors, salicylate and malonyl-CoA, would hinder the production of 4-HC. To address the imbalance, we recruited an engineered MarR-based salicylate biosensor system to dynamically control the 4-HC pathway. In this dynamic control system, the buildup of salicylate will activate the downstream 4-HC pathway under control of the engineered PmarO promoter and repress the major PEP consumption pathway via salicylate-inducible CRISPRi, redirecting the carbon flux to 4-HC production. The pyruvate released from the 4-HC production will be taken up for TCA cycle and malonyl-CoA biosynthesis. Further CRISPRi knockdown of the fatty acid pathway increased the availability of malonyl-CoA for 4-HC production. Compared with the parental strain, the engineered strain with the dynamic control system significantly increased 4-HC production from glycerol. This study demonstrated the over-production of 4-HC by integrating a bifunctional dynamic control system, which could serve as a new strategy for intelligent metabolic rewiring and balance in biosynthetic pathways.