Tuning the Binding Affinity of Heme-Responsive Biosensor for Precise and Dynamic Pathway Regulation

Area of Science:

• Metabolic Engineering
• Synthetic Biology
• Biotechnology

Background:

• Achieving tunable control over artificial regulatory systems in metabolic engineering remains a significant challenge.
• Maintaining intracellular heme homeostasis is crucial for efficient metabolic pathway regulation.

Purpose of the Study:

• To design and construct a novel heme-responsive regulatory system for dynamic control of metabolic pathways.
• To engineer a tunable biosensor for precise regulation of gene expression based on intracellular heme levels.

Main Methods:

• Developed a heme-responsive system integrating the HrtR biosensor and CRISPR interference (CRISPRi).
• Employed semi-rational design with site-saturated mutagenesis of HrtR to create biosensors with varied sensitivity and thresholds.
• Validated HrtR's modified metabolite-binding affinity using heme titration and molecular dynamics simulations.
• Confirmed dynamic regulation by monitoring gene expression and intracellular heme concentration fluctuations.

Main Results:

• Engineered HrtR variants exhibited altered heme-binding affinities, confirmed by experimental and computational methods.
• The dynamic regulatory system effectively controlled gene expression and maintained heme homeostasis.
• Achieved a record 5.35 g/L yield of 5-aminolevulinic acid (ALA) in Escherichia coli batch fermentation.
• Demonstrated successful application in enhancing porphobilinogen (PBG) and porphyrins biosynthesis.

Conclusions:

• The developed heme-responsive regulatory system offers tunable dynamic control for metabolic engineering applications.
• This system significantly improves the production of valuable chemicals like ALA, PBG, and porphyrins.
• The platform holds broad applicability for optimizing various biological production processes.