Abstract

Chlorogenic acid (CGA) is a vital phenolic ester with extensive applications in the food, pharmaceutical, and cosmetic industries. Although numerous microbial cell factories have been developed for CGA biosynthesis, the low catalytic efficiency of the key enzyme hydroxycinnamoyl-CoA quinate hydroxycinnamoyl transferase (HQT) remains a major bottleneck for high-yield production. In this study, we developed a novel growth-coupled selection system based on HQT-mediated reduction of toxic caffeoyl-CoA accumulation, thereby relieving growth inhibition in engineered Escherichia coli. Through iterative rounds of directed evolution, we identified a high-performance HQT variant enhancing CGA production by 3.7-fold compared with the wild-type enzyme. The specific activity was improved by 1.8-fold. Structural analysis of the mutant revealed critical insights into the functional role of the crossover loop in modulating enzymatic activity, offering new perspectives for HQT rational engineering. This work provides both a mechanistic understanding and a practical framework for enhancing CGA biosynthesis by addressing key enzyme bottlenecks.