Engineering of isoleucine dioxygenase for efficient multi-enzymatic cascade synthesis of (2S, 3R,...

Fe(II)/α-ketoglutarate (α-KG)-dependent isoleucine dioxygenase (IDO) enables selective C-H hydroxylation to produce the antidiabetic agent (2S, 3R, 4S)-4-hydroxyisoleucine ((2S, 3R, 4S)-4-HIL), but its industrial application is limited by insufficient activity and stability and the high cost of α-KG. Here, the IDO from Bacillus thuringiensis was engineered using multiple strategies to generate a mutant MU3 (V146I/S105E/N104P) with increased substrate loading. The specific activity exhibited 15-fold higher (152.0 U/mg) with 15-fold improved catalytic efficiency (k_cat/K_m) for L-isoleucine hydroxylation. The thermostability was enhanced with a 10-fold decrease in thermal inactivation rate at 30 °C. Molecular dynamics simulation revealed that the communication alteration between the loops around the active site and the strengthened substrate binding contributed to the improvement of MU3. The MU3 mediated L-isoleucine hydroxylation was coupled with an optimized whole-cell module integrating L-glutamate oxidase and catalase for α-KG generation from low-cost L-glutamate sodium. By the stepwise whole-cell biocatalytic cascade system, both 500 mM and 750 mM of L-isoleucine could be completely converted into (2S, 3R, 4S)-4-HIL under scaled-up condition. The whole space-time-yield reached 196 g/L/d as the highest record up to date and the cost was decreased compared to the α-KG-supplemented approach. This work provides an efficient, cost-effective and scalable route for (2S, 3R, 4S)-4-HIL synthesis.