Engineering Minicell Factories: RBS‑Mediated Optimization of Protein Expression and Minicell Yield in E. coli Nissle 1917

Background Bacterial minicells represent promising platforms for targeted drug delivery and enzymatic bioreactors. However, the relationship between recombinant protein expression and minicell production remains unclear, and strategies to simultaneously optimize both minicell yield and the quality of the expressed protein remain to be developed. Results An engineered minicell‑producing strain (ΔEcN) was constructed from Escherichia coli Nissle 1917. To precisely modulate protein expression, the native ribosome‑binding site (RBS) upstream of enhanced green fluorescent protein (eGFP) in the plasmid pUC19‑eGFP was replaced with two RBS variants (OmpA‑RBS and J61100‑RBS) of different strengths. The resulting plasmids were individually transformed into ΔEcN. In this strain, RBS engineering generated a gradient of eGFP expression levels (Lac-eGFP > J61100-eGFP > OmpA-eGFP). Higher eGFP expression was correlated with a significant reduction in minicell yield, suggesting a metabolic burden. Notably, while higher expression of eGFP in whole cells was associated with lower solubility, the solubilities of Lac-eGFP and J61100-eGFP were markedly greater within purified minicells than in parental bacteria. Despite the observed differences in minicell yield, viability remained consistent across all groups, and the hierarchical expression pattern imposed by the RBS variants was faithfully preserved in the minicells. Conclusions​ This study demonstrated that RBS strength can serve as a critical determinant for balancing the recombinant protein load and minicell yield. Furthermore, the finding that minicells provide a superior environment for the soluble accumulation of proteins of interest highlights their advantages as a production platform. In summary, this study establishes a rational technology platform for designing minicell-based systems, where RBS optimization can be used to concurrently maximize functional protein encapsulation and nanoparticle output for therapeutic and biocatalytic applications.