Engineering Caffeic Acid O-Methyltransferase for Efficient De Novo Ferulic Acid Synthesis

Area of Science:

• Biotechnology
• Metabolic Engineering
• Enzyme Engineering

Background:

• Ferulic acid is a valuable plant-derived chemical.
• Insufficient methylation activity of caffeic acid O-methyltransferase (COMT) limits ferulic acid biosynthesis.
• Engineering COMT and optimizing metabolic pathways are crucial for enhanced production.

Purpose of the Study:

• To improve ferulic acid production through enzyme engineering of COMT and metabolic pathway construction.
• To enhance the catalytic efficiency of COMT for caffeic acid degradation.
• To establish a de novo synthesis pathway for ferulic acid in a microbial host.

Main Methods:

• Engineered *Arabidopsis thaliana* COMT to create a mutant (COMT^{N129V-H313A-F174L}) with enhanced catalytic efficiency.
• Constructed a de novo ferulic acid synthesis pathway using enzymes from *Flavobacterium johnsoniae* (FjTAL) and *Escherichia coli* (EcHpaBC).
• Optimized S-adenosyl-L-methionine (SAM) supply by expressing *luxS* and *mtn*, and overexpressed the engineered COMT.

Main Results:

• The mutant COMT^{N129V-H313A-F174L} exhibited a 4.19-fold increase in catalytic efficiency for caffeic acid.
• Engineered strain FA-11 achieved ferulic acid production of 1260.37 mg/L in shake-flask fermentation.
• Ferulic acid production reached 4377 mg/L in a 50 L bioreactor using the engineered FA-11 strain.

Conclusions:

• COMT enzyme engineering combined with global metabolic engineering effectively enhances ferulic acid production.
• The study successfully established a microbial strain for high-level ferulic acid production.
• This work provides a robust platform for the industrial biosynthesis of ferulic acid.