Engineering Escherichia coli for High-Yield Protoporphyrin IX Biosynthesis via Cytotoxicity Mitigation and Pathway...

Area of Science:

• Synthetic biology
• Metabolic engineering
• Biotechnology

Background:

• Porphyrins, like protoporphyrin IX (PP IX), are vital tetrapyrroles in biological systems.
• PP IX is a precursor for heme and chlorophyll with broad applications.
• Microbial PP IX production faces challenges in cellular toxicity and biosynthesis regulation.

Purpose of the Study:

• To engineer an Escherichia coli platform for efficient and scalable microbial production of PP IX.
• To overcome limitations of cellular toxicity and endogenous regulatory controls in PP IX biosynthesis.
• To establish a new paradigm for producing cytotoxic metabolites via spatiotemporal pathway governance.

Main Methods:

• Implemented synthetic biology strategies in E. coli for pathway optimization.
• Introduced a hyperactive 5-aminolevulinic acid synthase and rebalanced branch pathways using sRNA-based knockdown.
• Integrated the MacAB-TolC efflux system to mitigate intracellular PP IX accumulation.

Main Results:

• Achieved unprecedented PP IX titer of 3.90 g/L and productivity of 65.0 mg/L/h in a 5 L bioreactor.
• Reduced intracellular PP IX accumulation by 16% through the efflux system.
• Demonstrated metabolic plasticity by coproducing 0.24 g/L heme via dynamic pathway modifications.

Conclusions:

• Established an efficient synthetic biology platform for microbial PP IX production, overcoming toxicity and regulatory hurdles.
• The engineered E. coli chassis provides a versatile platform for next-generation porphyrin biomanufacturing.
• Developed a novel approach for cytotoxic metabolite synthesis through spatiotemporal pathway control, bypassing traditional trade-offs.