Wang, H.; Dong, Z.; Shi, J.; Chen, L.; Sun, T.; Zhang, W.

The fusion expression of DNA replication-related proteins with nucleotide deaminase enzymes promotes random mutations in bacterial genomes, thereby increasing genetic diversity. However, most previous studies have focused on cytosine deaminase, which only produces C to T mutations, significantly limiting the variety of mutation types. In this study, we developed a fusion expression system in Escherichia coli by combining DnaG (RNA primase) with adenine deaminase TadA-8e (DnaG-TadA), which rapidly introduced A to G mutations into the E. coli genome, resulting in a 664-fold increase in mutation rate. Additionally, we engineered a dual-functional TadA variant, TadAD, and fused it with DnaG. This construct introduced both C to T and A to G mutations into the 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 to adaptive laboratory evolution for Cd2+ and kanamycin resistance, achieving an 8.0 mM Cd2+ and 200 ug/mL kanamycin tolerance within just 17 days and 132 hours, respectively. Compared to standard evolution methods, the final tolerance levels increased by 320% and 266%, respectively. Our work offers a novel strategy for random mutagenesis in E. coli and potentially other species.