A novel NADH-dependent leucine dehydrogenase for multi-step cascade synthesis of L-phosphinothricin

L -Phosphinothricin ( L -PPT) is the effective constituent in racemic PPT (a high-efficiency and broad-spectrum herbicide), and the exploitation of green and sustainable synthesis route for L -PPT has always been the focus in pesticide industry. In recent years, “one-pot, two-step” enzyme-mediated cascade strategy is a mainstream pathway to obtain L -PPT. Herein, Rg DAAO and Bs LeuDH were applied to expand “one-pot, two-step” process. Notably, a NADH-dependent leucine dehydrogenase from Bacillus subtilis ( Bs LeuDH) was firstly characterized and attempted to generate L -PPT, achieving an excellent enantioselectivity (99.9% ee ). Meanwhile, a formate dehydrogenase from Pichia pastoris ( Pp FDH) was utilized to implement NADH cofactor regeneration and only CO 2 was by-product. Sufficient amount of the corresponding keto acid precursor PPO was obtained by oxidation of D -PPT relying on a D -amino acid oxidase from Rhodotorula gracilis ( Rg DAAO) with content conversion (46.1%). L -PPT was ultimately prepared from racemized PPT via oxidative deamination catalyzed by Rg DAAO and reductive amination catalyzed by Bs LeuDH, achieving 80.3% overall yield and > 99.9% ee value.