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

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.