Structure-Oriented Engineering of Amidase: Modification of Twisted Access Tunnel for Efficient Synthesis of 2-Chloronicotinic Acid

Amidases are robust biocatalysts for the industrial synthesis of carboxylic acids. Amidase-catalyzed hydrolysis of 2-chloronicotinamide is regarded as a promising route for 2-chloronicotinic acid production, which is an important building block for agrochemicals and pharmaceuticals. However, the o-substituted Cl of the pyridine ring caused a strong electronic effect and steric hindrance. Engineering access tunnels is one of the most efficient strategies to improve catalytic efficiency by adjusting the transfer efficiency of ligands. Herein, we engineered a twisted access tunnel of amidase and the mutant PaAmiM1A378V+V402L+L403V was achieved, which exhibited increased catalytic efficiency toward 2-chloronicotinamide. Tunnel analysis, molecular dynamics simulations, and quantum mechanical calculations revealed that the straightened tunnel significantly improved the transport efficiency and simultaneously facilitated substrate binding and nucleophilic attack. With the best mutant as a biocatalyst, 1330 mM product was accumulated within 2 h and a high space-time-yield of 2515 gproduct L–1 d–1 was achieved, which is the highest ever reported. These results lay the foundation for industrial production of 2-chloronicotinic acid and give important guidance for engineering amidases into versatile biocatalysts.