Development of an aminotransferase‐driven biocatalytic cascade for deracemization of d,l‐phosphinothricin

Abstract 2‐oxo‐4‐[(hydroxy)(methyl)phosphinoyl]butyric acid (PPO) is the essential precursor keto acid for the asymmetric biosynthesis of herbicide l ‐phosphinothricin ( l ‐PPT). Developing a biocatalytic cascade for PPO production with high efficiency and low cost is highly desired. Herein, a d ‐amino acid aminotransferase from Bacillus sp. YM‐1 ( Ym DAAT) with high activity (48.95 U/mg) and affinity ( K m = 27.49 mM) toward d ‐PPT was evaluated. To circumvent the inhibition of by‐product d ‐glutamate ( d ‐Glu), an amino acceptor (α‐ketoglutarate) regeneration cascade was constructed as a recombinant Escherichia coli ( E. coli D), by coupling Ym d ‐AAT, d ‐aspartate oxidase from Thermomyces dupontii ( Td DDO) and catalase from Geobacillus sp. CHB1 . Moreover, the regulation of the ribosome binding site was employed to overcome the limiting step of expression toxic protein Td DDO in E. coli BL21(DE3). The aminotransferase‐driven whole‐cell biocatalytic cascade ( E. coli D) showed superior catalytic efficiency for the synthesis of PPO from d , l ‐phosphinothricin ( d , l ‐PPT). It revealed the production of PPO exhibited high space–time yield (2.59 g L −1 h −1 ) with complete conversion of d ‐PPT to PPO at high substrate concentration (600 mM d , l ‐PPT) in 1.5 L reaction system. This study first provides the synthesis of PPO from d , l ‐PPT employing an aminotransferase‐driven biocatalytic cascade.