Engineering of an Aldehyde Dehydrogenase from <i>Neurospora crassa</i> for Crocetin Production

Microbial synthesis represents a promising strategy for crocetin production but suffers from a low yield. The limited catalytic efficiency of aldehyde dehydrogenase (ALDH) highlighted it as a critical target for the microbial synthesis of crocetin. In this study, the NcALDH from <i>Neurospora crassa</i> was biochemically characterized and its catalytic activity was significantly improved through structure-guided enzyme engineering. NcALDH showed optimal activity toward crocetin dialdehyde at 30 °C and pH 8.0. Then, the G118W mutant with enhanced catalytic activity was obtained by redesigning the substrate-binding pocket of NcALDH. Furthermore, the mechanism behind the enhanced activity of the G118W mutant was elucidated through molecular dynamics simulations. Finally, 77.86 μM crocetin was achieved by using the recombinant strain harboring G118W as a whole-cell biocatalyst, marking a 113% increase compared with the titer achieved by the control strain. This study establishes a strong foundation for high-efficiency microbial crocetin synthesis.