Genome-wide A→G and C→T Mutations Induced by Functional TadA Variants in Escherichia coli

The fusion expression of DNA replication-related proteins with nucleotide deaminase enzymes promotes random mutations in bacterial genomes, thereby increasing genetic diversity among population. Most previous studies have focused on cytosine deaminase, which produces only C→T mutations, significantly limiting the variety of mutation types. In this study, we developed a fusion expression system by combining DnaG (RNA primase) with adenine deaminase TadA-8e (DnaG-TadA) in Escherichia coli , which is capable of rapidly introducing A→G mutations into the E. coli genome, resulting in a 664-fold increase in terms of mutation rate. Additionally, we engineered a dual-functional TadA variant, TadAD, and then fused it with DnaG. This construct introduced both C→T and A→G mutations into the E. coli genome, with the mutation rate further increased by 370-fold upon co-expression with an uracil glycosylase inhibitor (DnaG-TadAD-UGI). We applied DnaG-TadA and DnaG-TadAD-UGI systems to the adaptive laboratory evolution for Cd ²⁺ and kanamycin resistance, achieving an 8.0 mM Cd ²⁺ and 200 μg/mL kanamycin tolerance within just 17 days and 132 hours, respectively. Compared to conventional evolution methods, the final tolerance levels were increased by 320% and 266%, respectively. Our work offers a novel strategy for random mutagenesis in E. coli and potentially other prokaryotic species.