Efficient Production of Vanillyl Alcohol in Escherichia coli via Protein and Pathway Engineering.

Vanillyl alcohol (VNA) is a high-value compound used in the pharmaceutical industry and other fields. Its microbial de novo biosynthesis is severely limited by the low catalytic efficiency of O-methyltransferase. Here, we constructed a V314I mutant of caffeate O-methyltransferase via structure-guided protein engineering, which showed a 95% increase in catalytic efficiency toward 3,4-dihydroxybenzyl alcohol in Escherichia coli. Molecular simulations revealed that the mutation narrows the active-site cavity, constraining the smaller substrate into a catalytically competent orientation. To alleviate growth inhibition caused by 3,4-dihydroxybenzaldehyde accumulation, alcohol dehydrogenase gene ADH6 was introduced into the host. Moreover, S-Adenosylmethionine supply was enhanced by genomic integration of luxS-mtn and methionine supplementation. Finally, a VNA titer of 6.21 g/L was achieved in a 3 L fermenter from glycerol. This work establishes a VNA biomanufacturing platform and demonstrates an integrative strategy for the microbial production of methylated aromatic natural products, combining enzyme active-site spatial engineering and pathway engineering.