Engineered bacterial orthogonal DNA replication system for continuous evolution.
Journal
Nature chemical biology
ISSN: 1552-4469
Titre abrégé: Nat Chem Biol
Pays: United States
ID NLM: 101231976
Informations de publication
Date de publication:
Dec 2023
Dec 2023
Historique:
received:
09
07
2022
accepted:
16
06
2023
medline:
27
11
2023
pubmed:
14
7
2023
entrez:
14
7
2023
Statut:
ppublish
Résumé
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.
Identifiants
pubmed: 37443393
doi: 10.1038/s41589-023-01387-2
pii: 10.1038/s41589-023-01387-2
doi:
Substances chimiques
DNA, Bacterial
0
DNA-Directed DNA Polymerase
EC 2.7.7.7
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
1504-1512Subventions
Organisme : National Natural Science Foundation of China (National Science Foundation of China)
ID : 32172349
Organisme : Natural Science Foundation of Jiangsu Province (Jiangsu Provincial Natural Science Foundation)
ID : BK20202002, BK20200085
Informations de copyright
© 2023. The Author(s), under exclusive licence to Springer Nature America, Inc.
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