Engineered bacterial orthogonal DNA replication system for continuous evolution.
Nat Chem Biol
; 19(12): 1504-1512, 2023 Dec.
Article
en En
| MEDLINE
| ID: mdl-37443393
ABSTRACT
Continuous evolution can generate biomolecules for synthetic biology and enable evolutionary investigation. The orthogonal DNA replication system (OrthoRep) in yeast can efficiently mutate long DNA fragments in an easy-to-operate manner. However, such a system is lacking in bacteria. Therefore, we developed a bacterial orthogonal DNA replication system (BacORep) for continuous evolution. We achieved this by harnessing the temperate phage GIL16 DNA replication machinery in Bacillus thuringiensis with an engineered error-prone orthogonal DNA polymerase. BacORep introduces all 12 types of nucleotide substitution in 15-kilobase genes on orthogonally replicating linear plasmids with a 6,700-fold higher mutation rate than that of the host genome, the mutation rate of which is unchanged. Here we demonstrate the utility of BacORep-based continuous evolution by generating strong promoters applicable to model bacteria, Bacillus subtilis and Escherichia coli, and achieving a 7.4-fold methanol assimilation increase in B. thuringiensis. BacORep is a powerful tool for continuous evolution in prokaryotic cells.
Texto completo:
1
Colección:
01-internacional
Banco de datos:
MEDLINE
Asunto principal:
Saccharomyces cerevisiae
/
ADN Polimerasa Dirigida por ADN
Idioma:
En
Revista:
Nat Chem Biol
Asunto de la revista:
BIOLOGIA
/
QUIMICA
Año:
2023
Tipo del documento:
Article
País de afiliación:
China