An Orthogonal T7 Replisome for Continuous Hypermutation and Accelerated Evolution in E. coli.
bioRxiv
; 2024 Jul 26.
Article
en En
| MEDLINE
| ID: mdl-39211285
ABSTRACT
Systems that perform continuous hypermutation of designated genes without compromising the integrity of the host genome can dramatically accelerate the evolution of new or enhanced protein functions. We describe an orthogonal DNA replication system in E. coli based on the controlled expression of the replisome of bacteriophage T7. The system replicates circular plasmids that enable high transformation efficiencies and seamless integration into standard molecular biology workflows. Engineering of T7 DNA polymerase yielded variant proteins with mutation rates of 1.7 × 10 -5 substitutions per base in vivo - 100,000-fold above the genomic mutation rate. Continuous evolution using the mutagenic T7 replisome was demonstrated by expanding the substrate scope of TEM-1 ß-lactamase and increase activity 1,000-fold against clinically relevant monobactam and cephalosporin antibiotics in less than one week.
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1
Colección:
01-internacional
Base de datos:
MEDLINE
Idioma:
En
Revista:
BioRxiv
Año:
2024
Tipo del documento:
Article
Pais de publicación:
Estados Unidos