Establishing an Artificial Pathway for de Novo Biosynthesis of Vanillyl Alcohol in Escherichia coli

Vanillyl alcohol is a phenolic alcohol and is used as a flavoring agent in foods and beverages. In this paper, we propose a novel artificial pathway for microbial production of vanillyl alcohol from simple carbon sources. The pathway extends from 4-hydroxybenzoic acid (4-HBA), and needs only three heterologous enzymes, p-hydroxybenzoate hydroxylase (PobA), carboxylic acid reductase (CAR) and caffeate O-methyltransferase (COMT). First, we examined the promiscuous activity of COMT toward 3,4-dihydroxybenzyl alcohol and found a k_cat value of 0.097 s^-1. Meanwhile, 499.36 mg/L vanillyl alcohol was produced by COMT in vivo catalysis when fed with 1000 mg/L 3,4-dihydroxybenzyl alcohol. In the following experiment, de novo biosynthesis of vanillyl alcohol was carried out and 240.69 mg/L vanillyl alcohol was produced via modular optimization of pathway genes. This work was to date the first achievement for microbial production of vanillyl alcohol. Additionally, the present study demonstrates the application of enzyme promiscuity of COMT in the design of an artificial pathway for the production of high-value methylated aromatic compounds.