Coupling continuous directed evolution with base editors identifies combinatorial antibiotic resistance

Antimicrobial resistance (AMR), mainly caused by the mutation of antibiotic resistance genes, poses a great threat to human health. Clinically, TEM-1 β-lactamase encoding bla_TEM-1 gene is the most frequently occurred antibiotic resistance gene. Identification, characterization, and monitor of high antibiotic resistance bla_TEM-1 mutants are crucial to AMR research and would provide the guidance for the development of next-generation antibiotics. To this end, we report here the adaption of the continuous directed evolution (CDE) along with base editor (BE) gene editing strategy to discover TEM-1 mutants. Firstly, TEM-1 mutants were identified in single-copy bacterial artificial chromosome vector via BE-mediated CDE. Then each mutant genotype was verified in high copy number pUC plasmid. Finally, combinatorial antibiotic resistance was observed when the TEM-1 phenotypes generated in CDE were combined with that of the reported TEM-1 variants. The Escherichia coli strain harboring the vector cloned with 10 TEM-1 mutations showed a minimum inhibitory concentration of 1280 μg/mL to antibiotic cefotaxime, which is the highest data reported thus far. The research highlights the application of gene editing methods to provide insight into the molecular basis of antibiotic resistance.