Lignin valorization allows the generation of a number of value-added products such as _cis,cis_-muconic acid (ccMA), which is widely used for the synthesis of chemicals for the production of biodegradable plastic materials. In the present work, we reported the first multi-enzymatic, one-pot bioconversion process of vanillin into ccMA. In details, we used four sequential reactions catalyzed by xanthine oxidase, _O_-demethylase LigM (and the tetrahydrofolate-regeneration enzyme methyl transferase MetE), decarboxylase AroY (based on the use of _E. coli_ transformed cells) and catechol 1,2-dioxygenase CatA. The optimized lab-scale procedure allowed to reach, for the first time, the conversion of 5 mM vanillin into ccMA in ∼ 30 h with a 90% yield: this achievement represents an improvement in terms of yields and time when compared to the use of a whole-cell system. This multi-enzymatic system represents a sustainable alternative for the production of a high value added product from a renewable resource.