Multilevel metabolic engineering of Shewanella oneidensis as a heme-tolerant host for efficient porphyrin production

Porphyrin-derived high-value chemicals are important for food, medicine, and fine chemicals manufacturing, yet the physiological complexity of tetrapyrrole metabolism constrains efficient bioproduction. Here, we systematically engineered the cytochrome-rich bacterium Shewanella oneidensis MR-1 for porphyrin production. A modular coproporphyrin III biosynthetic pathway was reconstructed and progressively optimized through hemB homolog selection, antioxidant defense engineering, attenuation of competing tetrapyrrole branches, genome-scale model-guided target mining, plasmid-backbone reassignment, and ribosome-binding site tuning. The resulting strain achieved a remarkable coproporphyrin III production of 1525.5 ± 37.3 mg L^-1 and was further raised to 1906.8 ± 39.2 mg L^-1 under 5-aminolevulinic acid supplementation condition. Integrated metabolomic analyses identified a riboflavin-associated redox response linked to improved production physiology, while extracellular product accumulation enabled efficient acetone-assisted crude recovery. The platform was further extended to protoporphyrin IX and heme production with titers of 612.7 ± 4.8 mg L^-1 and 107.4 ± 3.3 mg L^-1, respectively. This work demonstrates an integrated metabolic engineering strategy combining pathway reconstruction, physiological stress mitigation, model-guided target selection, and multilevel expression balancing for developing S. oneidensis as a porphyrin-producing cell factory.