Efficient Biosynthesis of Chlorogenic Acid in Escherichia coli by Optimization of Precursors Metabolic Flow and Reduction of an Unknown Byproduct

Chlorogenic acid (CGA) is a natural phenylpropanoid compound with antioxidant properties that has received increasing attention for its anticancer potential. This study developed an efficient and sustainable microbial platform for the production of chlorogenic acid. Herein, the supply of precursors was first optimized by fine-tuning the expression level of aroD, encoding 3-dehydroquinate dehydratase, and ydiB, encoding shikimate dehydrogenase. For a significant unknown byproduct during the synthesis of CGA, successive purification, analysis, and verification were conducted. The key enzyme hydroxycinnamoyl CoA: quinic acid transferase from Nicotiana tabacum (NtHQT) was shown for the first time to catalyze the condensation of levodopa ( l -DOPA) and quinic acid to produce the byproduct. To reduce the production of this byproduct, the introduction of HpaBC from Pseudomonas aeruginosa to achieve a metabolic shunt of l -DOPA, and the catalytic pocket of NtHQT had been modified to alter its substrate selectivity. Finally, the CGA titer reached 4988.7 mg/L in a 5-L fermenter via optimization of the inoculation ratio and induction time. This study provides an environmentally friendly method for the industrial production of chlorogenic acid and a valuable reference for the identification and elimination of byproducts.