Now, Rongzhen Zhang and colleagues report a strategy that achieves gram-scale bilirubin production through an in vitro cascade that effectively mitigates the impact of interfering side products. In biological systems, bilirubin (BR) is produced through a two-step process from heme. First, heme oxygenase catalyses its oxidative cleavage, generating biliverdin (BV). Next, biliverdin is converted into BR by biliverdin reductase, an enzyme that requires NADPH as a cofactor (pictured). During the first step, Fe2+ ions and carbon monoxide are generated as stoichiometric by-products of heme oxygenase activity. The researchers showed that Fe2+ forms complexes with deprotonated biliverdin and bilirubin, narrowing the HOMO–LUMO gap, which increases their reactivity and ultimately promotes oxidative breakdown. To suppress this unwanted degradation, metal coordination was suppressed through etidronic acid chelation combined with pH control. Moreover, computational simulations showed that the other side product CO acts as a stronger ligand than O2. This results in a heme–CO complex that is not compatible with the catalytic pocket of heme oxygenase and prevents necessary O2 activation for catalysis. This CO poisoning of the heme oxygenase active site was overcome by integrating an O2-tolerant carbon monoxide dehydrogenase, which continuously oxidizes CO to CO2. Coupled with formate dehydrogenase for NADPH recycling, these strategies enabled an enzyme cascade that achieves near-quantitative conversion of heme to BR at litre scale with a titer of 1,678.4 mg l−1.
By moving the emphasis away from enzyme-focused optimization to system-level control of inhibitory intermediates, this work demonstrates how mechanistic insight and process design can converge to unlock leaps in performance. For the catalysis community the following lesson can be drawn from this study: sometimes, the key to high performance lies not in the catalyst itself, but in balancing the different chemistries.
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