Plant secondary metabolites have been attracting people's attention for centuries, due to their potentials; however, their production is still difficult and costly. The rich diversity of microbes and microbial genome sequence data provide unprecedented gene resources that enable to develop efficient artificial pathways in microorganisms. Here, by mimicking a natural pathway of plants using microbial genes, a new metabolic route was developed in E. coli for the synthesis of vanillin, the most widely used flavoring agent. A series of factors were systematically investigated for raising production, including efficiency and suitability of genes, gene dosage, and culture media. The metabolically engineered strain produced 97.2 mg/L vanillin from l-tyrosine, 19.3 mg/L from glucose, 13.3 mg/L from xylose and 24.7 mg/L from glycerol. These results show that the metabolic route enables production of natural vanillin from low-cost substrates, suggesting that it is a good strategy to mimick natural pathways for artificial pathway design.