Growth-Coupled Screening Enables Efficient Engineering of Key Enzymes for Chlorogenic Acid Biosynthesis

Area of Science:

• Biotechnology
• Metabolic Engineering
• Enzyme Engineering

Background:

• Chlorogenic acid (CGA) is a valuable phenolic compound with broad industrial applications.
• Current microbial production of CGA is limited by the low efficiency of the hydroxycinnamoyl-CoA quinate hydroxycinnamoyl transferase (HQT) enzyme.
• Overcoming enzymatic bottlenecks is crucial for high-yield CGA biosynthesis.

Purpose of the Study:

• To develop a novel selection system for improving HQT activity.
• To enhance chlorogenic acid (CGA) production in engineered Escherichia coli.
• To gain mechanistic insights into HQT enzyme function for rational engineering.

Main Methods:

• Developed a growth-coupled selection system leveraging HQT-mediated detoxification of caffeoyl-CoA.
• Employed iterative rounds of directed evolution to identify high-performance HQT variants.
• Performed structural analysis of the engineered HQT mutant.

Main Results:

• Identified a high-performance HQT variant that increased CGA production by 3.7-fold.
• Achieved a 1.8-fold improvement in the specific activity of the HQT enzyme.
• Structural analysis revealed the importance of the crossover loop in HQT activity modulation.

Conclusions:

• The developed selection system and HQT variant significantly enhance CGA biosynthesis.
• Understanding HQT structure-function relationships provides a framework for future enzyme engineering.
• This work addresses key enzymatic bottlenecks for improved microbial production of valuable compounds.