Genome-Wide A → G and C → T Mutations Induced by Functional TadA Variants in .

The fusion expression of deoxyribonucleic acid (DNA) replication-related proteins with nucleotide deaminase enzymes promotes random mutations in bacterial genomes, thereby increasing genetic diversity among the 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 , which is capable of rapidly introducing A → G mutations into the genome, resulting in a 664-fold increase in terms of mutation rate. Additionally, we tested a dual-functional TadA variant, TadAD, and then fused it with DnaG. This construct introduced both C → T and A → G mutations into the genome, with the mutation rate increased by 370-fold upon coexpression with a uracil glycosylase inhibitor (DnaG-TadAD-UGI). We applied DnaG-TadA and DnaG-TadAD-UGI systems to the adaptive laboratory evolution for Cd and kanamycin resistance, achieving an 8.0 mM Cd and 200 μg/mL kanamycin tolerance within just 17 days and 132 h, 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 and potentially other prokaryotic species.