Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, China.
Science Center for Future Foods, Jiangnan University, Wuxi, China.
Nat Chem Biol. 2023 Dec;19(12):1504-1512. doi: 10.1038/s41589-023-01387-2. Epub 2023 Jul 13.
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.
连续进化可以为合成生物学生成生物分子，并能够进行进化研究。酵母中的正交 DNA 复制系统（OrthoRep）可以有效地以易于操作的方式突变长 DNA 片段。然而，在细菌中缺乏这样的系统。因此，我们开发了一种用于连续进化的细菌正交 DNA 复制系统（BacORep）。我们通过利用苏云金芽孢杆菌中的温和噬菌体 GIL16 DNA 复制机制，结合经过工程改造的易错正交 DNA 聚合酶来实现这一目标。BacORep 在正交复制的线性质粒上引入了所有 12 种核苷酸取代，在 15kb 基因中，突变率比宿主基因组高 6700 倍，而宿主基因组的突变率保持不变。在这里，我们通过生成适用于模型细菌枯草芽孢杆菌和大肠杆菌的强启动子，以及在苏云金芽孢杆菌中实现甲醇同化率提高 7.4 倍，证明了基于 BacORep 的连续进化的实用性。BacORep 是原核细胞连续进化的有力工具。
