Systemic engineering and global regulation enabling high-level bilirubin biosynthesis.

Area of Science:

• Biochemistry
• Synthetic Biology
• Enzyme Engineering

Background:

• Bilirubin biosynthesis is hindered by low yields and poorly understood bottlenecks.
• Existing methods struggle with heme conversion efficiency.

Purpose of the Study:

• To establish a highly efficient in vitro pathway for bilirubin synthesis from heme.
• To identify and overcome key limitations in heme catabolism pathways.

Main Methods:

• Enzyme screening and mechanistic analysis to identify degradation pathways.
• Computational chemistry (DFT) to understand iron-induced degradation mechanisms.
• Enzyme engineering and cofactor regeneration systems (e.g., formate dehydrogenase) for pathway optimization.

Main Results:

• Achieved a bilirubin titer of 1.7 g/L with 95.8% yield.
• Identified Fe²⁺-induced oxidative degradation of intermediates as a major bottleneck.
• Mitigated degradation via iron chelation and protonation modulation, improving yield to 80.1%.
• Resolved heme-CO inhibition by introducing carbon monoxide dehydrogenase, restoring heme oxygenase activity.
• Enabled efficient and scalable bilirubin synthesis with a 20-fold improvement.

Conclusions:

• Controlling inhibitory byproducts is critical for stabilizing heme-related pathways.
• The developed pathway provides a generalizable framework for synthetic biology applications.
• This work significantly advances the efficiency and scalability of bilirubin production.