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Objective The probiotic Escherichia coli Nissle 1917 (ECN) is engineered by synthetic biology to construct a tumor-targeting strain capable of colonizing the tumor tissue, converting glucose and metabolic waste ammonia in the tumor microenvironment into the photosensitizer precursor 5-aminolevulinic acid (5-ALA) and the immunomodulatory amino acid arginine, while synergizing with immune checkpoint inhibitors for enhanced antitumor efficacy. Methods The genes hemAM, hemL, and argA were co-expressed in ECN, and thyA was knocked out via the λ-Red homologous recombination system to improve the tumor-targeting specificity. Shake-flask fermentation experiments, UV spectrophotometry, and HPLC were employed to quantify 5-ALA and arginine production. The antitumor effects of the engineered ECN were systematically evaluated by in vitro cellular assays and a murine colorectal cancer model. Results The engineered strain achieved 5-ALA and arginine yields of (173.00±11.46) mg/L and (1.70±0.09) g/L, which represented 8.2-fold and 20-fold increases, respectively, over that of wild-type ECN (P<0.000 1). The deletion of thyA enabled selective proliferation of the strain in tumor cells (HCT116 and CT26), with a two-fold increase in OD600 compared with that in normal Vero cells (P<0.000 1), confirming enhanced tumor targeting. Both in vitro and in vivo experiments demonstrated sustained synthesis of 5-ALA and arginine in tumors. Compared with wild-type ECN, the engineered strain induced 2.7-fold and 1.9-fold increases in CD8+ and CD4+ T-cell infiltration (P<0.000 1), alongside 1.7-fold and 2.4-fold elevations in IL-6 and TNF-α secretion (P<0.000 1), respectively. The engineered strain combined with the anti-PD-L1 therapy achieved a tumor volume inhibition rate of 77.6% (P<0.000 1). Conclusion This study establishes a metabolically and immunologically dual-functional ECN platform that synergizes localized delivery of photodynamic therapy precursors, arginine-mediated immunometabolic reprogramming, and immune checkpoint blockade, providing a novel solution for the combined therapy against solid tumors. The engineered system offers a groundbreaking strategy for precise tumor microenvironment modulation, advancing the research on targeted cancer therapeutics.
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| 科 Family | 属数 Number of genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) | 属 Genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) |
|---|---|---|---|---|---|---|
| 鹅膏菌科Amanitaceae | 2 | 11 | 5.26 | 鹅膏菌属 Amanita | 10 | 4.78 |
| 小菇科 Mycenaceae | 2 | 12 | 5.74 | 丝盖伞属 Inocybe | 5 | 2.39 |
| 多孔菌科 Polyporaceae | 8 | 14 | 6.70 | 蜡蘑属 Laccaria | 5 | 2.39 |
| 红菇科 Russulaceae | 3 | 23 | 11.00 | 小皮伞属 Marasmius | 6 | 2.87 |
| 小菇属 Mycena | 11 | 5.26 | ||||
| 光柄菇属 Pluteus | 5 | 2.39 | ||||
| 红菇属 Russula | 17 | 8.13 | ||||
| 栓菌属 Trametes | 5 | 2.39 |
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