Glutarate is a linear-chain dicarboxylic acid with wide applications in the production of polyesters and polyamides such as nylon-4,5 and nylon-5,5. Previous studies focused on the biological production of glutarate from lysine with low yields and titers. Here, we report on glutarate production by Escherichia coli using a five-step reverse adipate degradation pathway (RADP) identified in Thermobifida fusca By expressing the enzymes of RADP, the glutarate was detected by strain Bgl146 in shaken flasks. After fermentation optimization, the titer of glutarate by Bgl146 was increased to 4.7 ± 0.2 mM in shaken flasks. We further eliminated pathways for the major metabolites competing for carbon flux by CRISPR/Cas9 (ΔarcA, ΔldhA, ΔatoB, and ΔpflB). Moreover, the final strain Bgl4146 produced 36.5 ± 0.3 mM glutarate by fed-batch fermentation. These results constitute the highest glutarate titer reported in E. coliIMPORTANCE Glutarate is an important C5 linear-chain dicarboxylic acid, which is widely used in polyesters and polyamides such as nylon-4,5 and nylon-5,5 in the chemical industry. Glutarate is currently produced from the feedstocks derived from petroleum, specifically by oxidation of a mixture of cyclohexanone and cyclohexanol catalyzed by nitric acid. However, the chemical synthesis results in high pollution and dramatic greenhouse gas emission. Thus, the biological production of glutarate directly from the substrate is of great importance. Although there have been reports using Corynebacterium glutamicum to produce glutarate, it has serious limitations due to the limited lysine supply and long fermentation time. To solve this problem, a novel synthetic pathway was constructed in this study, and the highest glutarate titer was reported in Escherichia coli using a short fermentation time without lysine addition, making bio-based glutarate production much more feasible.