Mentor: Andrew Jones, Ph.D.<

Catechol-O-methyltransferases (COMTs) catalyze regioselective O-methylation of phenolic substrates that are valuable biocatalysts to produce functionalized aromatic compounds. In this study, four COMTs originating from diverse biological sources – LwOMT10 (Lophophora williamsii), AtOMT (Arabidopsis thaliana), sCOMT (Homo sapiens), and MtbOMT (Mycobacterium tuberculosis) – were evaluated for enzymatic activity and regiospecificity toward cinnamic acid derived substrates from the lignin pathway. Each enzyme was expressed in Escherichia coli BL21(DE3*) and screened against caffeic acid, ferulic acid, isoferulic acid, and 3,4-dimethoxycinnamic acid. Product formation and regioselectivity were quantified using high-performance liquid chromatography-mass spectrometry (HPLC-MS). Based on screening performance, selected COMTs were integrated into engineered biosynthetic pathways that enabled de novo production of 3,4-dimethoxycinnamic acid from L-tyrosine. Multigene operons incorporating tyrosine ammonia-lyase (RgTAL), hydroxyphenyl acetate monooxygenase (EcHpaBC) in either wild type or mutant variants were assembled with sCOMT and AtOMT using plasmid construction. Recombinant strains were fermented under inducible conditions, and metabolites were analyzed using HPLC-MS to assess pathway efficiency and methylation. Comparative analysis discovered distinct substrate preference and regiospecific methylation profiles among the four COMTs, with sCOMT and AtOMT demonstrating the strongest catalytic activity and effective conversion to demethylated products. The engineered strains were successful in achieving de novo biosynthesis of 3,4-dimethoxycinnamic acid directly from tyrosine, which confirms functional pathway integration and enzyme compatibility. The results highlight the importance of COMT selection for pathway optimization and demonstrate a versatile microbial platform for sustainable biosynthesis of high-value aromatic compounds.

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