Glutaric acid is an important intermediate, which is widely used in chemical industry, agriculture, medicine and other fields. At present, there are some problems in the biosynthesis pathway of glutaric acid, such as long synthesis path, high consumption of cofactors and low yield. In order to develop an efficient method for the synthesis of glutaric acid, a new way to produce glutaric acid using glucose as substrate was constructed by combining enzyme engineering with metabolic engineering. Firstly, a novel catalytic pathway composed of lysine α-oxidase (LO), monoamine oxidase (MAO), α-ketoacid decarboxylase (KDC) and aldehyde dehydrogenase (ALDH) was designed by database mining. AB initio synthesis of glutaric acid was realized by introducing lysine producing strain E. coli CCTCC M2019435. In order to further improve the synthesis efficiency of this pathway, rational analysis and protein modification were carried out for the rate-limiting enzyme KpALDH of the pathway, and the catalytic efficiency of the enzyme was increased by 66.5 times. On this basis, the yield of glutaric acid was increased by 2.0 times through metabolic engineering to enhance the expression of rate-limiting enzyme KpALDH and block the by-product acetic acid metabolic branch. Finally, the glutaric acid fermentation conditions were optimized, the glutaric acid yield increased to 62.0 g/L at the end of fermentation, and the production intensity and yield reached 1.6 (g/L)/h and 0.3 g/g glucose, respectively.