江大张荣珍教授团队：突破胆红素合成瓶颈，实现克级高效生物制造

Core Viewpoint - The research led by Professor Zhang Rongzhen's team has successfully developed an efficient in vitro enzyme-catalyzed system for high-level bilirubin biosynthesis, achieving a yield of 1.7 g/L and a conversion rate of 95.8%, laying a solid foundation for industrial production [2][4]. Group 1: Enzyme Optimization - The study clarified the classical two-enzyme catalytic pathway from heme to bilirubin and identified the most efficient enzyme combinations through systematic evolutionary analysis [4]. - The combination of rat HO-1 and its specific cytochrome P450 reductase (CPR) showed the highest efficiency in converting heme to biliverdin, while the rat BVR-A subtype performed best in reducing biliverdin to bilirubin [4]. - Structural analysis of key enzymes led to the development of soluble truncated variants RnCPRΔ54 and RnHO-1Δ22, which maintained substrate affinity and catalytic efficiency while improving solubility [4][5]. Group 2: Addressing Inhibitory Factors - Initial one-pot reactions showed a heme conversion rate of 83.9%, but bilirubin yield was only 48.1%, indicating material loss due to bottlenecks in the first step of heme oxygenase catalysis [5]. - The study identified Fe²⁺ as a negative factor affecting product stability, as it formed complexes with deprotonated biliverdin or bilirubin, leading to degradation [7]. - The researchers developed a dual strategy to stabilize the reaction, using HEDP to chelate Fe²⁺ and lowering the reaction pH to suppress deprotonation, which together increased bilirubin yield to 80.1% [7][8]. Group 3: Integrated Multi-Enzyme System - The research revealed carbon monoxide (CO) as another inhibitory factor, as it formed stable complexes with heme, obstructing substrate binding and enzyme activity [8]. - To eliminate all inhibitory factors, the team constructed a fully integrated multi-enzyme system, incorporating a CO dehydrogenase and a formate dehydrogenase for in situ NADPH regeneration [8]. - This integrated system successfully converted 3 mM of heme to 2.87 mM of bilirubin in a 1-liter reaction, achieving a yield of 95.8%, confirmed as the bioactive bilirubin IXα isomer [8].