Abstract Pseudomonas putida vanillate- O-demethylase consisting of VanA and VanB was expressed in Escherichia coli strain K-12. Recombinant E. coli strain K-12 cells expressing VanAB efficiently converted vanillate into protocatechuate with glucose consumption. Mutant lacking either pgi or zwf showed higher or lower converting activity than the parental strain, respectively. Formaldehyde, which is the by-product of the demethylation, was converted into formate in the cellular reaction. Formate accumulation was blocked by gene disruption of the E. coli frmA that coded glutathione-dependent formaldehyde dehydrogenase.