L-threonine, an essential amino acid, is widely used in the agricultural, pharmaceutical, and cosmetic industries. In the L-threonine fermentation process, the glyoxylate cycle functions as part of the feedback pathway. In this work, Escherichia coli THRD that can produce L-threonine were used as original strains. Red reconstruction technology was used to construct a strain lacking iclR (Escherichia coli THRD ΔiclR) and THRD P1 and THRD P2, in which aceBAK promoter in E. coli THRD was replaced by promoters of various strength.. Fluorescent quantitative real-time PCR analysis showed that the expression of the aceB gene in the different strains was upregulated by 1.89-, 2.11-, and 2.96-fold, respectively, compared with the original strain. The results from fermentation in shaken flasks showed that the L-threonine and sugar-acid conversion rate of E. coli THRD ΔiclR were 42.60±1.23 g/L and 32.77 g/g, respectively, which were 20.61% and 20.7% higher than those of the control strain THRD (35.32±1.07 g/L and 27.17 g/g). The L-threonine and sugar-acid conversion rate of E. coli THRD P1 were 36.50±1.42 g/L and 28.08 g/g, which increased by 3.34% and 3.39% from those obtained from the control strain, respectively. However, E. coli THRD P2 stopped growing after 8 h and resulted in L-threonine production of 8.31±1.31 g/L and a sugar-acid conversion rate of 20.78 g/, which were 76.47% and 23.52%, respectively, lower than those of the control strain THRD. In conclusion, moderate enhancement of the glyoxylate cycle benefited L-threonine accumulation, while excessive enhancement of glyoxylate cycle had a negative effect on the normal cell metabolism.