Abstract: Possessing good biocompatibility, biodegradability, and environmental friendliness, polylactic acid (PLA) is one of the important biodegradable materials. In this study, a knockout experiment was designed based on genetic engineering technology for the three key genes ackA, iclR and arcA that affect the content of acetyl-CoA in metabolic flux, as well as for the minCD gene that affects cell division mode. The results show that the double-knockout strain BL21(DE3)ΔackAΔarcA/pETDuet-1_phac_pct/pACYCDuet-1_sulA_ldhA is of the modified strain with the highest yield, obtaining the final fermentation product yield 322.3 mg/L, the dry weight of cells 14.90 wt%, and the average molecular weight 26 352. The research achievement is beneficial to the further optimization and development of key technologies, providing theoretical and data reference for polylactic acid bio-manufacturing.