2025-08-13 ノースカロライナ州立大学(NCState)
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Illustration by Zidan Li. Created in BioRender. LI, Z. (2025) https://BioRender.com/c5d51o4.
<関連情報>
- https://news.ncsu.edu/2025/08/boosting-pdna-production/
- https://microbialcellfactories.biomedcentral.com/articles/10.1186/s12934-025-02821-x
E. coliによるpDNA生産の向上を目的とした誘導型ゲノムワイド変異導入法 Inducible genome-wide mutagenesis for improvement of pDNA production by E. coli
Zidan Li,George Sun,Ibrahim Al’Abri,Yihui Zhou & Nathan Crook
Microbial Cell Factories Published:13 August 2025
DOI:https://doi.org/10.1186/s12934-025-02821-x
Abstract
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.
