Genome-wide A to G and C to 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 Cd2+ and kanamycin resistance, achieving an 8.0 mM Cd2+ and 200 g/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. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=98 SRC="FIGDIR/small/610230v1_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@176e445org.highwire.dtl.DTLVardef@848b7corg.highwire.dtl.DTLVardef@1e190b9org.highwire.dtl.DTLVardef@1e47b44_HPS_FORMAT_FIGEXP M_FIG C_FIG For TOC only