Heme biosensor-guided in vivo pathway optimization and directed evolution for efficient biosynthesis of heme

Area of Science:

• Metabolic Engineering
• Synthetic Biology
• Biotechnology

Background:

• Heme is crucial in medicine and food, but its biosynthesis is complex, involving multiple genes (hemBCDEFY) and enzymes like ferrochelatase (FECH).
• Current metabolic engineering approaches face challenges in optimizing gene expression, regulatory mechanisms, and enzymatic activity for efficient heme synthesis.

Purpose of the Study:

• To develop a heme biosensor-guided strategy for improving heme biosynthesis in Escherichia coli.
• To overcome limitations in heme production by optimizing the expression of hemBCDEFY genes and enhancing ferrochelatase activity.

Main Methods:

• Utilized a heme biosensor system employing the HrtR protein to link intracellular heme concentration with cell survival under selective conditions (tetracycline).
• Employed iterative evolution and saturation mutagenesis to screen for optimal ribosome binding site (RBS) variants of hemBCDEFY and high-activity FECH mutants.
• Applied fed-batch fermentation for large-scale production of heme using engineered E. coli strains.

Main Results:

• Achieved a protoporphyrin IX (PPIX) titer of 160.8 mg/L, the highest reported yield in shaken-flask fermentation, by optimizing hemBCDEFY expression.
• Identified a high-activity ferrochelatase (FECH) variant through directed evolution.
• Engineered strain SH20C produced 127.6 mg/L of heme via fed-batch fermentation using the optimized pathway and FECH variant.

Conclusions:

• Demonstrated the effectiveness of a heme biosensor-based strategy for sequential pathway optimization and in vivo directed evolution of key enzymes.
• Significantly improved heme biosynthesis efficiency, providing a robust platform for future metabolic engineering endeavors.
• Broadened the understanding of heme synthesis mechanisms and potential for industrial applications.