Engineering of halide methyltransferases for synthesis of SAE and its application in biosynthesis of ethyl vanillin

Halide methyltransferase (HMT)-catalyzed synthesis of alkyl donors from S -adenosyl- L -homocysteine (SAH) and haloalkanes is a promising method for biocatalytic alkylation. However, the reported halide methyltransferases show limited catalytic capacity towards unnatural substrates such as ethyl iodide, which limits their application in biocatalytic ethylation. In this work, we performed structure-based directed evolution starting from the wild-type Aspergillus clavatus halide methyltransferase ( Ac HMT). The best variant, Ac HMT M3 , was identified with an activity of up to 421.5 mU mg –1 towards ethyl iodide, 38.7-fold higher than that of the wild type Ac HMT. Subsequently, molecular dynamics simulations provide structural insights into how mutations improve the catalytic activity towards iodoethane. Finally, Ac HMT M3 was utilized in one-pot cascade with the reported O -methyltransferase Rn COMT+Y200L to synthesize ethyl vanillin from 3,4-dihydroxybenzaldehyde (79% conversion). Our work demonstrated the potential of the engineered HMT in further biocatalytic alkylation.