Redirecting Metabolic Flux to Minimize By‐Product Formation Enables Efficient De Novo Biosynthesis of Chlorogenic Acid in Escherichia coli

ABSTRACT

Chlorogenic acid (CGA) is an important natural hydroxycinnamic acid ester with diverse biological activities, including antioxidant, antibacterial, and potential antitumor properties. However, efficient microbial titer of CGA remains challenging because of insufficient hydroxylation efficiency, by‐product accumulation, and limited host tolerance. Herein, an efficient and sustainable microbial platform for de novo CGA biosynthesis was constructed in Escherichia coli . Enhancement of the NAD(H) biosynthetic pathway increased intracellular reducing power and increased hydroxylation efficiency. A synergistic hydroxylation module consisting of the p ‐coumarate 3‐hydroxylase from Saccharothrix espanaensis ( Se C3H) and the flavin reductase from Pseudomonas aeruginosa ( Pa HpaC)was further introduced to promote the conversion of p ‐coumaric acid (pCA) to caffeic acid (CA) and redirect metabolic flux toward CGA biosynthesis. Rational engineering of 4‐coumarate‐CoA ligase from Arabidopsis thaliana ( At 4CL) increased substrate selectivity and decreased the formation of the major by‐product p ‐coumaroylquinic acid (pCQA). In addition, transcriptomic analysis identified lsrG , a gene associated with the AI‐2 quorum‐sensing system, as a beneficial target whose overexpression further enhanced CGA titer. The final engineered strain produced 7220.3 mg/L CGA in a 50 L fed‐batch fermentation. The strategies developed herein may provide an effective metabolic engineering framework for the sustainable biosynthesis of CGA and other aromatic natural products.