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Targeted C-to-T and A-to-G dual mutagenesis system for RhtA transporter in vivo evolution.
- Source :
-
Applied and environmental microbiology [Appl Environ Microbiol] 2023 Oct 31; Vol. 89 (10), pp. e0075223. Date of Electronic Publication: 2023 Sep 20. - Publication Year :
- 2023
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Abstract
- T7 RNA polymerase (T7RNAP) has been fused with cytosine or adenine deaminase individually, enabling in vivo 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 in vivo 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 <superscript>-3</superscript> and 1.20 × 10 <superscript>-2</superscript> , respectively, with 24 h culturing and distributed mutations evenly across the target gene. The T7-DualMuta system was used to in vivo 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 in vivo 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 in vivo mutagenesis was created. The mutagenesis frequency was 4.02 × 10 <superscript>7</superscript> fold higher than the spontaneous mutation, which was reported to be approximately 10 <superscript>-10</superscript> bases per nucleotide per generation. This mutant system can be utilized for various applications and research in Enzymology and Synthetic Biology studies.<br />Competing Interests: The authors declare no conflict of interest.
Details
- Language :
- English
- ISSN :
- 1098-5336
- Volume :
- 89
- Issue :
- 10
- Database :
- MEDLINE
- Journal :
- Applied and environmental microbiology
- Publication Type :
- Academic Journal
- Accession number :
- 37728922
- Full Text :
- https://doi.org/10.1128/aem.00752-23