Helmholtz International Lab for Anti-Infectives, Shandong University-Helmholtz Institute of Biotechnology, State Key Laboratory of Microbial Technology, Shandong University, Qingdao, Shandong 266237, China.
Genomics, Biotechnology Center, Center for Molecular and Cellular Bioengineering, Technische Universität Dresden, Tatzberg 47-51, 01307 Dresden, Germany.
Nucleic Acids Res. 2022 Feb 22;50(3):e15. doi: 10.1093/nar/gkab1076.
Recombineering assisted multiplex genome editing generally uses single-stranded oligonucleotides for site directed mutational changes. It has proven highly efficient for functional screens and to optimize microbial cell factories. However, this approach is limited to relatively small mutational changes. Here, we addressed the challenges involved in the use of double-stranded DNA substrates for multiplex genome engineering. Recombineering is mediated by phage single-strand annealing proteins annealing ssDNAs into the replication fork. We apply this insight to facilitate the generation of ssDNA from the dsDNA substrate and to alter the speed of replication by elevating the available deoxynucleoside triphosphate (dNTP) levels. Intracellular dNTP concentration was elevated by ribonucleotide reductase overexpression or dNTP addition to establish double-stranded DNA Recombineering-assisted Multiplex Genome Engineering (dReaMGE), which enables rapid and flexible insertional and deletional mutagenesis at multiple sites on kilobase scales in diverse bacteria without the generation of double-strand breaks or disturbance of the mismatch repair system. dReaMGE can achieve combinatorial genome engineering works, for example, alterations to multiple biosynthetic pathways, multiple promoter or gene insertions, variations of transcriptional regulator combinations, within a few days. dReaMGE adds to the repertoire of bacterial genome engineering to facilitate discovery, functional genomics, strain optimization and directed evolution of microbial cell factories.
基于同源重组的多重基因组编辑通常使用单链寡核苷酸进行定点突变。该方法已被证明在功能筛选和优化微生物细胞工厂方面非常有效。然而，这种方法仅限于相对较小的突变改变。在这里，我们解决了使用双链 DNA 底物进行多重基因组工程所涉及的挑战。同源重组是由噬菌体单链退火蛋白将 ssDNA 退火到复制叉中介导的。我们利用这一见解来促进从 dsDNA 底物产生 ssDNA，并通过提高可用的脱氧核苷三磷酸 (dNTP) 水平来改变复制速度。通过过度表达核糖核苷酸还原酶或添加 dNTP 来提高细胞内 dNTP 浓度，建立双链 DNA 基于同源重组的多重基因组编辑（dReaMGE），从而能够在多种细菌中快速灵活地在千碱基尺度上进行插入和缺失突变，而不会产生双链断裂或干扰错配修复系统。dReaMGE 可以实现组合基因组工程工作，例如，在几天内改变多个生物合成途径、多个启动子或基因插入、转录调节因子组合的变化等。dReaMGE 增加了细菌基因组工程的手段，有助于发现、功能基因组学、菌株优化和微生物细胞工厂的定向进化。
