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Engineered bacterial orthogonal DNA replication system for continuous evolution
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DOI：10.1038/s41589-023-01387-2.png)
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Abstract
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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.
Keywords:
PRIMER-TERMINUS STABILIZATION
DIRECTED EVOLUTION
BACILLUS-CEREUS
3-5 EXONUCLEASE
PROTEIN
BINDING
MUTAGENESIS
POLYMERASE
DESIGN
GIL01
Journal
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13.7
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4.8K
Citations:
3.3W
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