Mechanistic Multi-Enzyme Engineering for High-Yield Bilirubin Biosynthesis

Bilirubin biosynthesis has long been limited by low yields and unclear bottlenecks. Here, we report a fully in vitro pathway that coverts heme to bilirubin with the highest reported titer. Through systematically screening and engineering, we identified two hidden challenges: Fe²⁺ causes intermediate degradation, and CO inhibits heme oxygenase activity. Initial yields stalled at 48.1% due to Fe²⁺-induced biliverdin and bilirubin breakdown. We revealed that Fe²⁺ interacts with deprotonated biliverdin and bilirubin, triggering oxidative ring-opening degradation via O₂-mediated radical mechanism. DFT calculations showed Fe²⁺-ligand complexes reduce the HOMO-LUMO gap, enhancing their elector transfer susceptibility. Competitive chelation of Fe ²⁺ and protonation-modulation boosted yield to 80.1%. Furthermore, heme-CO complexes block O ₂ -activation for accessing heme oxygenase. Introducing carbon monoxide dehydrogenase for CO removal and formate dehydrogenase for NADPH-recycling enabled efficient bilirubin synthesis of 1.7 g/L and 95.8% yield—a 20-fold improvement. Our work shows byproducts control is the key to stabilize heme-related pathways and as an advanced tool in synthetic biology.