Rongzhen Zhang

Substrate inhibition limits the industrial use of tryptophan hydroxylase (TPH), the key catalyst for 5-hydroxytryptophan (5-HTP) production, by causing tunnel congestion and substrate trapping at high concentrations. We developed a mechanism-guided strategy to overcome this. The crystal structure of the Y235S (MS) variant revealed a 243% expansion of the substrate channel, reducing tunnel congestion and increasing activity 2.38-fold, though substrate affinity decreased. Mechanistic analysis showed loop II acts as a molecular gate controlling cofactor-substrate binding. Its rational stabilization in variant MS4 enhanced loop stability and optimized substrate orientation, increasing catalytic efficiency by over 150% compared to MS and specific activity by 285% compared to wild-type. This approach proved generalizable across TPH orthologs. Combined with a tetrahydrobiopterin regeneration system, MS4 broke through the substrate concentration limitation, achieving >5-fold higher whole-cell 5-HTP production (16.37 mM in 4 h). This work establishes a general framework for relieving tunnel congestion and substrate trapping through integrated structural, computational, and loop engineering.

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.

L-Threonine aldolase (LTA) is pivotal for β-hydroxy-α-amino acids (β-HAAs) synthesis but is limited by poor thermostability and activity-stability trade-off. Here, we applied interfacial engineering to Faecalimicrobium dakarense LTA, targeting nonconserved residues at subunit interfaces. The resulting variant M3 (L17F/V129R/L206W) exhibited 1.23-fold higher specific activity and significantly enhanced thermostability (T₅₀⁶⁰ + 20.5 °C; T_m + 13.1 °C). In a 1 L system, M3 produced the agrochemical precursor L-threo-4-methylsulfonyl-phenylserine in 2.49-fold higher yield than wild-type. Scaling up to 10 L, M3 achieved 95.7% conversion and 84.9% diastereomeric excess (de), with a high space-time yield of 10.5 g·L^-1·h^-1. Molecular simulations revealed tighter subunit packing, expanded hydrogen-bonding networks, and enhanced rigidity, collectively improving tetramer stability and catalytic performance. This work presents a generalizable interfacial engineering strategy to decouple activity-stability trade-offs in multimeric enzymes, advancing industrial β-HAAs synthesis.