High-Yield Expression, Enhanced Stability, and Broad-Spectrum Substrate Catalysis of the Short-Chain Fungal Unspecify Peroxygenase from <i>Thermoascus thermophilus</i>

Unspecific peroxygenases (UPOs) show great industrial application potential, yet their heterologous expression efficiency remains unsatisfactory. Herein, a prokaryotic expression system for short-chain UPOs was constructed via multidimensional optimization. Efficient soluble expression of Thermoascus thermophilus TteUPO was first realized in Escherichia coli, with a yield of 28.4 mg/L, which was 1.67-fold higher than eukaryotic systems and reduced the culture period by 44 h. This platform was also applicable to three other fungal short-chain UPOs. Combined molecular dynamics simulations and experiments revealed that glycosylation did not affect TteUPO activity, while heme incorporation and protein folding dominated catalytic performance. The A167Y mutant obtained by semirational design exhibited improved thermal stability (Tm = 62.5 °C) and 48 h half-life. Moreover, its limonene epoxidation selectivity rose from 22.54 to 95.21% with maintained activity. TteUPO also performed efficiently on diverse terpenes with outstanding regioselectivity. This work supplies fundamental tools for large-scale UPO production and promotes its industrial utilization.