Abstract: In order to find a new drug resistance target to solve the problem of bacterial drug resistance, the PurB overexpression strain (BW25113-purB) of Escherichia coli adenylosuccinate lyase was constructed by using the pCA24N expression vector, and the killing effect of aminoglycoside antibiotics was compared with that of Escherichia coli carrying empty plasmid at the logarithmic phase (BW25113-pCA24N). Then, the ANOVA algorithm was used to analyze the difference of the test results, and the effect of PurB overexpression of Escherichia coli in the environment of aminoglycoside antibiotics was tested. The results showed that PurB overexpression could significantly attenuate the killing effect of gentamicin and kanamycin on Escherichia coli, proving that PurB overexpression affected the tolerance of Escherichia coli to aminoglycoside antibiotics. These results indicated that the adenylosuccinate lyase played an important role in the production process of drug-resistant Escherichia coli, suggesting the feasibility of this enzyme as a new pharmacological target for Escherichia coli.