Engineering <i>Escherichia coli</i> for High-Yield Protoporphyrin IX Biosynthesis via Cytotoxicity Mitigation and Pathway Optimization

Porphyrins are essential tetrapyrroles that play critical roles in biological electron-transfer and light-harvesting systems. As the universal precursor of heme and chlorophyll, protoporphyrin IX (PP IX) has transformative potential for fields as diverse as biomedicine, materials, food, and agriculture. However, large-scale microbial PP IX production is subject to challenges regarding cellular toxicity and regulation of tetrapyrrole biosynthesis. Herein, we report a synthetic-biology-driven <i>Escherichia coli</i> platform enabled by spatially resolved pathway optimization and cytotoxicity mitigation. By introducing a hyperactive 5-aminolevulinic acid synthase and rebalancing branch pathways via sRNA-based knockdown, we decoupled the PP IX synthesis from endogenous regulatory constraints. Integration of the MacAB-TolC efflux system reduced intracellular PP IX accumulation by 16%, synergistically enhancing extracellular productivity. PP IX titer values of 3.90 g/L and 65.0 mg/L/h productivity were achieved in a 5 L bioreactor, the highest ever reported. The engineered chassis exhibits metabolic plasticity, coproducing 0.24 g/L heme through dynamic pathway modifications. This work establishes a new paradigm for cytotoxic metabolite synthesis through spatiotemporal pathway governance, circumventing classical toxicity-productivity trade-offs. Our work establishes an efficient platform for microbial PP IX production. Furthermore, the engineered chassis developed here enables versatile applications in next-generation porphyrin biomanufacturing.