Targeted C-to-T and A-to-G dual mutagenesis system for RhtA transporter evolution.

T7 RNA polymerase (T7RNAP) has been fused with cytosine or adenine deaminase individually, enabling C-to-T or A-to-G transitions on DNA sequence downstream of T7 promoter, and greatly accelerated directed protein evolution. However, its base conversion type is limited. In this study, we created a dual-functional system for simultaneous C-to-T and A-to-G mutagenesis, called T7-DualMuta, by fusing T7RNAP with both cytidine deaminase (PmCDA1) and a highly active adenine deaminase (TadA-8e). The C-to-T and A-to-G mutagenesis frequencies of T7-DualMuta were 4.02 × 10 and 1.20 × 10, respectively, with 24 h culturing and distributed mutations evenly across the target gene. The T7-DualMuta system was used to directed evolution of L-homoserine transporter RhtA, resulting in efficient variants that carried the four types of base conversions by T7-DualMuta. The evolved variants greatly increased the host growth rates at L-homoserine concentrations of 8 g/L, which was not previously achieved, and demonstrated the great evolution capacity. The novel molecular device T7-DualMuta efficiently provides both C/G-to-T/A and A/T-to-G/C mutagenesis on target regions, making it useful for various applications and research in Enzymology and Synthetic Biology studies. It also represents an important expansion of the base editing toolbox.ImportanceA T7-DualMuta system for simultaneous C-to-T and A-to-G mutagenesis was created. The mutagenesis frequency was 4.02 × 10 fold higher than the spontaneous mutation, which was reported to be approximately 10 bases per nucleotide per generation. This mutant system can be utilized for various applications and research in Enzymology and Synthetic Biology studies.