Engineering 4-Vinylcatechol Biosynthesis in Escherichia coli Through Host-Enzyme Combination

De novo biosynthesis of 4-vinylcatechol (VCAT), a valuable bio-based monomer and potent antioxidant, has emerged as a crucial objective due to its profound industrial and pharmacological significance. However, its biosynthesis is frequently hindered by limited precursor availability. In this study, we developed a highly efficient, dual-plasmid platform utilizing engineered Escherichia coli cell factories to synthesize VCAT directly from glucose without precursor feeding. To establish optimal production cells, we evaluated combinations of different host strains and phenolic acid decarboxylase (PAD) sources. Our results indicate that BL21(DE3) is less suitable for efficient de novo VCAT biosynthesis under the conditions tested. In contrast, the rpoA14(DE3), featuring a reprogrammed shikimate pathway, provided a more favorable host for VCAT production and enabled endogenous tyrosine supply, thereby eliminating the need for exogenous tyrosine supplementation required in BL21(DE3). To maximize this enhanced intracellular flux, we comparatively evaluated padC genes from different Bacillus subtilis strains. Comparative analysis demonstrated that the rpoA14(DE3)-10A5 exhibited significantly superior production titers, markedly outperforming both its 168-PAD counterpart and the standard BL21(DE3) expression hosts. Cultivated in single-step, aqueous fermentation at mild conditions, this optimal host-enzyme combination achieved de novo accumulation of 200 mg/L VCAT, promising scalable biosynthesis.