Construction of a grpE-based plasmid addiction system in Escherichia coli and its application in phloroglucinol biosynthesis.

This article focuses on the development and evaluation of a novel plasmid addiction system (PAS) based on the essential gene grpE in Escherichia coli BL21(DE3) to enhance plasmid stability and improve production of phloroglucinol (PG), a toxic chemical biosynthesized by engineered strains. The grpE-based PAS involves deleting the chromosomal grpE gene and complementing it with plasmid-borne grpE alleles, which selectively eliminate plasmid-free cells and thereby maintain plasmid presence without antibiotic selection. Implementation of this system in PG-producing strains resulted in significantly improved plasmid retention, increased tolerance to PG, and enhanced PG titers by 71.8% in shake-flask and 22.4% in fed-batch fermentations compared to controls. Transcriptomic analysis indicated that the improved performance was associated with upregulation of grpE, various chaperone genes, and the biotin ligase gene birA, suggesting enhanced protein processing and stress response contribute to the observed benefits. This grpE-based PAS represents a nonmetabolite-related strategy that may be broadly applicable for stable heterologous gene expression and microbial chemical production in E. coli.