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. [Display omitted] • A "one-pot, two-step" cascade process catalyzed by Rg DAAO and Bs LeuDH was explored to prepare L -phosphinothricin (L -PPT) starting from racemic PPT. • A novel NADH-dependent leucine dehydrogenase (Bs LeuDH) coupled with Pp FDH was applied to the synthesis of L -PPT for the first time. • The cascade yielded 80.3% L -PPT with 99.9% ee in a 12 h bioconversion of 0.1 M rac-PPT. [ABSTRACT FROM AUTHOR]