Engineered bacterial orthogonal DNA replication system for continuous evolution.
Rongzhen TianRunzhi ZhaoHaoyu GuoKun YanChenyun WangCheng LuXueqin LvJianghua LiLong LiuGuocheng DuJian ChenYanfeng LiuPublished in: Nature chemical biology (2023)
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.
Keyphrases
- escherichia coli
- bacillus subtilis
- genome wide
- circulating tumor
- induced apoptosis
- single molecule
- cell free
- pseudomonas aeruginosa
- cell proliferation
- klebsiella pneumoniae
- cell death
- cell cycle arrest
- signaling pathway
- oxidative stress
- saccharomyces cerevisiae
- biofilm formation
- endoplasmic reticulum stress
- multidrug resistant
- genome wide analysis
- bioinformatics analysis