Bacteriocin peer selection for the production of antibiotic selection free biotherapeutic pDNA

Plasmid vectors are well established tools used to genetically engineer bacteria both in the laboratory and at industrial scale. The past few decades have seen a rising interest in the use of plasmid DNA (pDNA) for biotherapeutic applications. This interest is a strong driver for the development of technologies to increase pDNA production at biopharmaceutical scale in terms of decreasing production costs and meeting regulatory requirements. Although cell free technologies are emerging, pDNA vectors are still produced by fermentation in Escherichia coli strains. As plasmids are extra-chromosomic molecules there is a probability of losing a certain ratio within the E. coli population during the fermentation process leading to a decrease of DNA production efficiency. Maintaining pDNA in the population is thus a key element to reach efficient and robust production. Traditionally, antibiotic resistance genes and antibiotics have been used to generate a selective pressure to ensure pDNA stability in the microbial population during the production process. Nowadays, having an antibiotic resistance gene in the pDNA coding sequence represents a limitation both for safety and legal requirements and in terms of production yield. For this reason, we have developed a pDNA antibiotic-free bacteriocin-based selection system, based on the genes involved in the production, processing, secretion and immunity of the bacteriocin microcin V. Our approach is based on the peer pressure exerted by the bacteriocin and does not rely on the addition of any selective agent in the medium to limit population drift and ensure plasmid stability. This novel antibiotic-free approach may be applied to any pDNA vector in different E. coli strains and expands their potential applications in both animal and human health as delivery vectors for biotherapeutics.