Li Zidan, Sun George, Al'Abri Ibrahim, Zhou Yihui, Crook Nathan
Department of Chemical and Biomolecular Engineering, North Carolina State University, Raleigh, NC, USA.
Bioinformatics Research Center, North Carolina State University, Raleigh, NC, USA.
Microb Cell Fact. 2025 Aug 13;24(1):183. doi: 10.1186/s12934-025-02821-x.
Plasmid DNA (pDNA) is a cost-driving reagent for the production of gene therapies and DNA vaccines. Improving pDNA production in the most common production host (E. coli) has faced obstacles arising from the complex network of genes responsible for pDNA synthesis, with the specific enzyme(s) limiting pDNA yield remaining unidentified. To address this challenge, we employed an inducible genome-wide mutagenesis strategy, combined with fluorescent screening, to isolate E. coli NEB 5α strains with enhanced pDNA production. Following selection, we successfully isolated an E. coli strain (M3) with elevated plasmid copy numbers (PCNs) across multiple origin types. Specifically, we observed a 5.93-fold increase in PCN for the GFP reporter plasmid, a 1.93-fold increase for the gWiz DNA vaccine plasmid, and an 8.7-fold increase for the pAAV-CAGG-eGFP plasmid, all of which contain pUC origins. In addition, plasmids with p15A and pSC101 origins showed 1.44-fold and 1.68-fold increases in PCN, respectively. Whole-genome sequencing of the adapted strain M3 identified 85 mutations, including one in recG, which encodes an ATP-dependent DNA helicase. Replacement of the mutant recG with its wild-type counterpart in the mutant strain resulted in a 63% reduction in PCN, but the recG mutation alone was insufficient to increase PCN in the wild-type strain. These findings suggest that the recG mutation plays a synergistic role with other genomic mutations to drive PCN increases. Taken together, this study presents the development of a pDNA hyperaccumulating E. coli strain with promising applications in industrial and therapeutic pDNA production, while also offering important insights into key genes involved in pDNA production.
质粒DNA（pDNA）是基因治疗和DNA疫苗生产中成本高昂的试剂。在最常用的生产宿主（大肠杆菌）中提高pDNA产量面临着诸多障碍，这些障碍源于负责pDNA合成的复杂基因网络，而限制pDNA产量的特定酶仍未确定。为应对这一挑战，我们采用了诱导型全基因组诱变策略，并结合荧光筛选，以分离出pDNA产量提高的大肠杆菌NEB 5α菌株。经过筛选，我们成功分离出一株大肠杆菌菌株（M3），该菌株在多种复制起点类型中质粒拷贝数（PCN）均有所提高。具体而言，我们观察到，对于含有pUC复制起点的绿色荧光蛋白（GFP）报告质粒，其PCN增加了5.93倍；对于gWiz DNA疫苗质粒，增加了1.93倍；对于pAAV-CAGG-eGFP质粒，增加了8.7倍。此外，含有p15A和pSC101复制起点的质粒PCN分别增加了1.44倍和1.68倍。对适应性菌株M3进行全基因组测序，鉴定出85个突变，其中一个位于recG基因，该基因编码一种ATP依赖性DNA解旋酶。在突变菌株中将突变的recG基因替换为野生型对应基因后，PCN降低了63%，但仅recG突变不足以使野生型菌株的PCN增加。这些发现表明，recG突变与其他基因组突变发挥协同作用，推动PCN增加。综上所述，本研究展示了一种pDNA超积累大肠杆菌菌株的开发，该菌株在工业和治疗性pDNA生产中具有广阔应用前景，同时也为参与pDNA生产的关键基因提供了重要见解。
