For the efficient industrial production of glutarate, an important C5 platform chemical that is widely used in the chemical and pharmaceutical industries, a five-enzyme cascade pathway was designed and reconstructed in vitro to synthesize glutarate from l-lysine. Then, the imbalanced enzyme expression levels of l-lysine decarboxylase from Escherichia coli (EcCA), putrescine aminotransferase (KpcPA) and gamma-aminovaleraldehyde dehydrogenase (KpcPD) from Klebsiella pneumoniae, and the poor catalytic efficiency of KpcPA were identified as the rate-limiting bottlenecks. To this end, ribosome binding site regulation was employed to coordinate the enzyme molar ratio of EcCA:KpcPA:KpcPD at approximately 4:8:7 (the optimum ratio obtained in vitro), and volume scanning and hydrophobicity scanning were applied to increase KpcPA activity toward cadaverine from 15.89 +/- 0.52 to 75.87 +/- 1.51 U center dot mg(-1). Furthermore, the extracellular accumulation of 5-aminovalerate (5AVA) was considerably reduced by overexpressing gabP encoding the 5AVA importer. Combining these strategies into the engineered strain Glu-02, 77.62 g/L glutarate, the highest titer by E. coli to date, was produced from 100 g/L l-lysine in 42 h, with a yield and productivity of 0.78 g/g l-lysine and 1.85 g/L/h, respectively, at a 5-L scale. The results presented here provide a novel and potential enzymatic process at industrial-scale to produce glutarate from cheaper amino acids.