Engineered Antibiotic Marker-Free Probiotic Dual Expression Chassis

Probiotics present an attractive strategy for long-term delivery of therapeutic molecules in vivo as they may colonise as part of the human microbiota. Synthetic biology genetic circuits that facilitate the expression of these therapeutic molecules are usually stabilized in cells on plasmid by antibiotic resistance genes as selection markers. However, antibiotic resistance genes are not suitable for in vivo application as constant antibiotic selection pressure is required for plasmid retention. Moreover, the presence of antibiotic resistance genes introduces the risk of potential horizontal transfer of resistance to the native microbiome. In this study, we engineered an antibiotic marker-free dual expression system in a probiotic strain, Escsherichia coli Nissle. The essential genes involved in alanine and aspartate metabolisms were deleted from the bacterial genome to create a double auxotrophic E. coli strain. The auxotrophic phenotypes could be rescued by the balanced co-expression of the deleted genes on two separate plasmids to achieve plasmid selection and retention. To further enhance the dual plasmid system, a regulatory switch was implemented on the two co-expressed plasmids to ensure their stability. Our study demonstrates that the co-expressed plasmids could be maintained in the probiotic expression chassis without antibiotic selection. This dual expression system selected for auxotrophic complementation can be used for gastrointestinal delivery of therapeutic molecules in vivo with a high degree of biosafety.