alpha-Ketoglutaric acid (alpha-KG) is a multifunctional dicarboxylic acid in the tricarboxylic acid (TCA) cycle, but microbial engineering for alpha-KG production is not economically efficient, due to the intrinsic inefficiency of its biosynthetic pathway. In this study, pathway engineering was used to improve pathway efficiency for alpha-KG production inEscherichia coli. First, the TCA cycle was rewired for alpha-KG production starting from pyruvate, and the engineered strainE. coliW3110 Delta 4-PCAI produced 15.66 g/L alpha-KG. Then, the rewired TCA cycle was optimized by designing various strengths of pyruvate carboxylase and isocitrate dehydrogenase expression cassettes, resulting in a large increase in alpha-KG production (24.66 g/L). Furthermore, acetyl coenzyme A (acetyl-CoA) availability was improved by overexpressing acetyl-CoA synthetase, leading to alpha-KG production up to 28.54 g/L. Finally, the engineered strainE. coliW3110 Delta 4-P((H))CAI((H))A was able to produce 32.20 g/L alpha-KG in a 5-L fed-batch bioreactor. This strategy described here paves the way to the development of an efficient pathway for microbial production of alpha-KG.