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

ABSTRACT Ferulic acid is a high‐value chemical synthesized in plants. The ferulic acid biosynthesis is still affected by the insufficient methylation activity of caffeic acid O‐methyltransferase ( COMT ). In this study, we engineered COMT from Arabidopsis thaliana to match caffeic acid, and the mutant COMT N129V‐H313A‐F174L showed 4.19‐fold enhanced catalytic efficiency for degrading caffeic acid. Then, we constructed the de novo synthesis pathway of ferulic acid by introducing tyrosine ammonia lyase from Flavobacterium johnsoniae ( FjTAL ), 4‐hydroxyphenylacetate 3‐hydroxylase from Escherichia coli ( EcHpaBC ), and mutant COMT N129V‐H313A‐F174L , and further increased tyrosine synthesis. Furthermore, we overexpressed two copies of COMT N129V‐H313A‐F174L and enhanced the supply of S‐adenosyl‐L‐methionine (SAM) by expressed S‐ribosylhomocysteine lyase ( luxS ) and 5′‐methylthioadenosine/S‐adenosylhomocysteine nucleosidase ( mtn ) to increase the production of ferulic acid. Finally, the production of ferulic acid reached 1260.37 mg/L in the shake‐flask fermentation and 4377 mg/L using a 50 L bioreactor by the engineered FA‐11. In conclusion, COMT enzyme engineering combined with global metabolic engineering effectively improved the production of ferulic acid and successfully obtained a fairly high level of ferulic acid production.