Minicells derived from Escherichia coli Nissle 1917 for efficient phenylalanine degradation

Phenylketonuria is an inherited metabolic disorder characterized by impaired phenylalanine (Phe) catabolism and toxic Phe accumulation. Current therapies are limited by poor long-term adherence, high cost, and biosafety concerns associated with existing or emerging approaches. Here, a non-replicative yet metabolically active platform was developed using anucleate minicells derived from Escherichia coli Nissle 1917 (EcN). A dual phenylalanine degradation system was introduced to engineer these minicells into synthetic microreactors, designated PCB101. In vitro , PCB101 degraded 10 mM Phe within 1.5 h. In a murine hyperphenylalaninemia model, intravenous injection of PCB101 (10 9 cells/mL) reduced plasma Phe by 87.5%, and oral administration (10 10 cells/mL) achieved a 66% reduction in a dose-dependent manner. These results demonstrate that EcN-derived minicells provide a controllable and biosafe chassis for efficient Phe degradation both in vitro and in vivo , supporting their potential applicability in biotherapeutic strategies requiring stable dosing and prevention of microbial proliferation. IMPORTANCE Engineered microbial systems are widely applied to perform defined metabolic functions, yet most designs rely on viable and replicating cells, intrinsically coupling functional output to population expansion. This growth-dependent paradigm complicates dose stability and raises biosafety concerns in practical applications. This study demonstrates that anucleate minicells derived from Escherichia coli Nissle 1917 retain translational and metabolic activity despite lacking chromosomal DNA and replicative capacity. By incorporating a dual phenylalanine degradation pathway, the engineered minicells efficiently reduced phenylalanine both in vitro and in vivo. This study illustrates that metabolic function can be preserved independently of cell division, providing a controllable microbial platform for applications requiring stable functional output without population expansion. This work expands the conceptual framework for non-replicative microbial systems in applied microbiology.