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 (<i>COMT</i>). In this study, we engineered <i>COMT</i> from <i>Arabidopsis thaliana</i> to match caffeic acid, and the mutant <i>COMT</i> ^{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 <i>Flavobacterium johnsoniae</i> (<i>FjTAL</i>), 4-hydroxyphenylacetate 3-hydroxylase from <i>Escherichia coli</i> (<i>EcHpaBC</i>), and mutant <i>COMT</i> ^{N129V-H313A-F174L}, and further increased tyrosine synthesis. Furthermore, we overexpressed two copies of <i>COMT</i> ^{N129V-H313A-F174L} and enhanced the supply of S-adenosyl-L-methionine (SAM) by expressed S-ribosylhomocysteine lyase (<i>luxS</i>) and 5'-methylthioadenosine/S-adenosylhomocysteine nucleosidase (<i>mtn</i>) 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, <i>COMT</i> 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.