Biosynthesis of vanillin glucoside in Escherichia coli

Vanillin is a widely used flavor compound in the food, cosmetic, and pharmaceutical industries. Although microbial biosynthesis offers a green alternative to conventional chemical synthesis, challenges such as unstable heterologous enzyme expression, insufficient methyl donor supply, and product cytotoxicity limit de novo production. Here, we engineered Escherichia coli to produce vanillyl alcohol, vanillin, and vanillin glucoside directly from glycerol. Genomic integration of the alcohol dehydrogenase ADH6 and multi-copy of the caffeic acid O-methyltransferase COMT gene eliminated the need for plasmid-based expression, yielding 1.09 g/L vanillyl alcohol in shake flasks. To overcome methylation bottlenecks, we reinforced the S-adenosyl-l-methionine (SAM) cycle by enhancing SAM biosynthesis, accelerating S-adenosyl-l-homocysteine (SAH) hydrolysis, and establishing methionine regeneration, which increased vanillyl alcohol production to 1.95 g/L. Subsequent deletion of four endogenous aldehyde-reduction genes together with removal of heterologous ADH6 enabled vanillin accumulation at 551.6 mg/L. Introduction of the glucosyltransferase UGT72E3/2 converted vanillin into vanillin glucoside, reaching 1.37 g/L in shake flasks and 2.12 g/L in a 3-L fed-batch fermenter. This work establishes a modular platform for efficient production of vanillin and its derivatives in E. coli.