PCB-101

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.

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.

Bovine mastitis is one of the diseases that affect cattle and has a considerable detrimental impact on both the economics and animal welfare internationally. S. aureus is among the most relevant causative agents of this disease. Due to developing bacterial resistance and public health regulations, the number of antibiotics to treat mastitis is restricted. Endolysins, cell-wall-degrading enzymes of bacteriophage origin, are emerging as a promising alternative to traditional antibiotics. Moreover, combination therapy is frequently utilized to combat drug-resistant bacterial pathogens. In this study, we evaluated the therapeutic efficacy of the recombinant endolysin S.AgEndo1332 in combination with the antibiotic gentamicin. The synergistic activity of this combination was assessed in vitro using checkerboard and time-kill assays, and in vivo using a mouse mastitis model induced by mastitogenic S. aureus. S.AgEndo1332, a homolog of GBS phage B30 lysin gene, was custom synthesized, expressed by prokaryotic expression system, and purified under native purification protocol. S.AgEndo1332 showed strong antimicrobial activity against Streptococcus agalactiae (MIC 25 µg/mL) and S. aureus (MIC 50 µg/mL) strains in Tris-HCl buffer system at pH 7.5. The lytic activity of S.AgEndo1332 wasretained at -80 °C for 12 weeks. The combination of S.AgEndo1332 (12.5 µg/mL) with gentamicin (0.0594 µg/mL) was showing synergistic activity by checkerboard assay with FIC index 0.375. The dosage for the combination treatment (10X concentration, S.AgEndo1332: 125 µg/mL and gentamicin: 0.594 µg/mL) used for the in vivo trial was determined based on cell viability by the MTT assay (> 93% cell viability), time kill kinetics study (30 min) and lytic activity in milk (within 30 min). In S. aureus induced-mouse mastitis model, combination therapy significantly reduced the intramammary bacterial load and result was further supported by consistent findings of expression of pro and anti-inflammatory cytokine genes (TNF-α, IL-6, IFN-γ, and IL-10), and histopathological analysis of the mammary gland tissue section. Overall, our findings highlight the potential of combining endolysin with antibiotics as a promising alternative treatment for S. aureus-induced bovine mastitis.