Construction of Engineered Escherichia coli for Vanillin Production From Glucose: Pathway Module Optimization and Methyl-Donor Regulation.

Abstract

Vanillin is an important flavor compound widely used in the food, fragrance, and pharmaceutical industries. Current biotransformation processes from ferulic acid or eugenol are limited by high substrate cost and low carbon efficiency, motivating de novo biosynthesis from glucose. This study employed Escherichia coli as the chassis organism to establish a modular vanillin biosynthesis system based on the phenylpropanoid metabolic pathway. Heterologous expression of sam8, sam5, and comt established a biosynthetic module for the sequential conversion of l-tyrosine to p-coumaric acid, then to caffeic acid, and finally to ferulic acid. This module enabled the production of 15.86 mg/L ferulic acid from glucose. Two ferulic acid-to-vanillin modules were compared: a CoA-dependent deacetylation pathway (fcs/ech) and an oxidative decarboxylation pathway (fdc/cso2). With ferulic acid feeding, the deacetylation route produced 445.78 mg/L vanillin, far exceeding the 3.49 mg/L obtained via oxidative decarboxylation. When integrated with the upstream module, the deacetylation pathway enabled de novo vanillin production from glucose at 4.46 mg/L, whereas the oxidative decarboxylation route yielded only 0.46 mg/L, indicating better performance of the former under the tested conditions. Metabolite profiling indicated accumulation of caffeic acid and limited ferulic acid levels, identifying O-methylation and S-adenosyl-L-methionine (SAM) supply as major bottlenecks. Implementation of SAM regeneration modules revealed that mtn overexpression enhanced the vanillin titer by about 3-fold, to 12.36 mg/L, while luxS overexpression had a negligible effect. In summary, this study establishes a functional de novo phenylpropanoid pathway for vanillin in E. coli, underscores the critical role of terminal‑pathway selection, and demonstrates that SAM regeneration effectively improves vanillin production from glucose.