Genome-Wide A\n→ G and C → T Mutations\nInduced by Functional TadA Variants in Escherichia\ncoli

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