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<p>Bilirubin biosynthesis has long been constrained by low yields and poorly understood bottlenecks. Here, we report a fully in vitro pathway that converts heme to bilirubin with the titer of 1.7 g/L and 95.8%. Systematically, enzyme screening and mechanistic analysis reveal the hidden challenge: Fe²⁺-induced oxidative degradation of intermediates. We show that Fe²⁺ coordinates with deprotonated intermediates to trigger oxidative ring-opening degradation via O₂-mediated radical mechanism, as supported by DFT calculations indicating reduced HOMO-LUMO gap in Fe²⁺-ligand complexes. The degradation is mitigated through competitive iron chelation and protonation state modulation, improving yield to 80.1%. Furthermore, we have resolved heme-CO complexes blocking O₂ activation at heme oxygenase by introducing a carbon monoxide dehydrogenase to remove CO and restore enzyme activity. Coupled with NADPH-recycling via formate dehydrogenase, these interventions enable efficient, scalable bilirubin synthesis with a 20-fold improvement. Our work shows controlling inhibitory byproducts is critical for stabilizing heme-related pathways and as a generalizable framework for synthetic biology.</p>

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Systemic engineering and global regulation enabling high-level bilirubin biosynthesis

  • 期刊论文

  • 出版模式：

    开放获取

  • 发表日期：

    2025年12月23日

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  • Zhentao Jiang,
  • Jingxin Rao,
  • Caokai Zhu,
  • Yamiao Li,
  • Qiang Zhu,
  • Mingyue Zheng,
  • Wenchi Zhang,
  • Rongzhen Zhang

摘要

Bilirubin biosynthesis has long been constrained by low yields and poorly understood bottlenecks. Here, we report a fully in vitro pathway that converts heme to bilirubin with the titer of 1.7 g/L and 95.8%. Systematically, enzyme screening and mechanistic analysis reveal the hidden challenge: Fe²⁺-induced oxidative degradation of intermediates. We show that Fe²⁺ coordinates with deprotonated intermediates to trigger oxidative ring-opening degradation via O₂-mediated radical mechanism, as supported by DFT calculations indicating reduced HOMO-LUMO gap in Fe²⁺-ligand complexes. The degradation is mitigated through competitive iron chelation and protonation state modulation, improving yield to 80.1%. Furthermore, we have resolved heme-CO complexes blocking O₂ activation at heme oxygenase by introducing a carbon monoxide dehydrogenase to remove CO and restore enzyme activity. Coupled with NADPH-recycling via formate dehydrogenase, these interventions enable efficient, scalable bilirubin synthesis with a 20-fold improvement. Our work shows controlling inhibitory byproducts is critical for stabilizing heme-related pathways and as a generalizable framework for synthetic biology.