Inducible complementation for antibiotic-free plasmid-based biomanufacturing in industrially relevant strains

Antibiotic-based plasmid maintenance is a core method in molecular biotechnology; however, reliance on antibiotics in biomanufacturing has significant drawbacks. Antibiotic use in novel industrial processes is being evaluated with increasing scrutiny due to rising levels of antimicrobial resistance (AMR). From an economic standpoint, antibiotic use is undesirable as supplementation on a manufacturing scale is prohibitively expensive and adds production costs, including purification steps to eliminate residual antibiotics and wastewater pre-treatment to prevent environmental contamination. Recently, we introduced a method for antibiotic-free plasmid selection and maintenance in Escherichia coli that retains the flexibility and ease of use enjoyed by antibiotic-based selection. This method uses inducible complementation of an essential gene. An essential gene in the production strain was replaced with an inducible copy so that in the absence of the inducer, a plasmid expressing the essential gene is required for growth. The ability to induce the genomic copy of the essential gene enables the engineered stain to be easily transformed with a complementing plasmid without requiring antibiotics, as non-supplemented media selects for plasmid uptake. Following the introduction of this method in an E. coli cloning strain, this study is designed to characterize and demonstrate the application of the method in various industrially relevant E. coli strains. Here, we modify a wide range of E. coli laboratory strains from the phylogenetic groups K-12, B and W as well as the probiotic strain Nissle1917, and demonstrate robust, antibiotic-free plasmid maintenance in all strain backgrounds in the absence of the inducer. We subsequently characterize growth rates and protein expression in the engineered strains, and find the modified strains performed equally well or better than their unmodified counterparts. Finally, we use this technology to scale up antibiotic-free protein expression to 10,000 L, demonstrating compatibility with large-scale production and downstream processing.