[Directed evolution of amine dehydrogenase for the catalytic resolution of (1R,5S)-3-ethylbicyclo [3.2.0] hept-3-en-6-one, a chiral intermediate of mirogabalin].

(1R,5S)-3-ethylbicyclo[3.2.0]hept-3-en-6-one[(1R,5S)-1a] is the key chiral starting material for the synthesis of mirogabalin. Existing chemical and biocatalytic resolution processes both suffer from cumbersome post-processing procedures and high production costs. To achieve the efficient and green resolution of (1R,5S)-1a, the present study employed racemic ketone 1a as the substrate and identified LfAmDH(M0) as the template for directed evolution through enzyme library screening. The substrate binding site and the key amino acid residues around the substrate access channel were selected, and site-directed mutagenesis and iterative mutagenesis were performed in turn to construct a multi-round mutant library. Following library screening, the specific activities of the mutants were elevated to 1.3- to 5.9-fold that of the starting enzyme, demonstrating significantly optimized catalytic performance. The substrate spectrum characteristics of the starting enzyme M0 and the dominant mutants were investigated, revealing that the mutants exhibited appreciable catalytic activity and stereoselectivity toward aliphatic ketones, cyclic ketones, arylalkyl ketones and aromatic ketones. In a 50 mL reaction system, the optimal mutant L40T/L125R/V291A(M3) converted 150 mmol/L of substrate. After the reaction was completed, the product was subjected to extraction and acid washing, affording the chiral ketone with an ee value of 97.1% and a yield of 45.4%. This study successfully developed a novel amine dehydrogenase-catalyzed resolution approach for (1R,5S)-1a, which simplifies the post-processing workflow of the product and reduces the consumption of organic reagents, thereby providing robust technical support for the efficient industrial-scale biomanufacturing of (1R,5S)-1a.