Method for plasmid-based antibiotic-free fermentation.

backgroundAntibiotic-based plasmid selection and maintenance is a core tool in molecular biology; however, while convenient, this strategy has numerous drawbacks for biological manufacturing. Overuse of antibiotics and antibiotic resistance genes (ARG) contributes to the development of antimicrobial resistance, which is a growing threat to modern medicine. Antibiotics themselves are costly and therefore often omitted in fermentations, leading to plasmid loss and a corresponding loss in product yield. Furthermore, constitutive expression of a plasmid-encoded antibiotic resistance gene imposes a significant metabolic burden on the cells. For many fermentation products (e.g., in nutrition and medicine), the use of antibiotic resistance genes is subject to strict regulations and should be avoided. We present a method for plasmid selection and maintenance with stringent selection pressure that is independent of antibiotics and ARG. Furthermore, it can be used without any restrictions regarding culture medium and temperature.

resultsThe developed method involves modification of a bacterial strain such that an essential gene is expressed genomically under the control of an inducible promoter. A copy of the same essential gene with the endogenous promoter is supplied on a plasmid for selection. In the absence of the inducer for the genomic copy of the essential gene, cells rely on expression of the plasmid-encoded gene copy, leading to tight selection for plasmid maintenance. Induction of the genomic copy of the essential gene enables the engineered strain to be propagated in the absence of a plasmid. Here, we describe the genetic setup and demonstrate long-term, tight selection for plasmid maintenance with a variety of different plasmids and E. coli strains.

conclusionsThis method facilitates plasmid-based fermentations by eliminating the need for antibiotic selection and improving plasmid maintenance.