Efficient kinetic resolution of D,L‐phosphinothricin using an aminotransferase-mediated cascade

2-oxo-4-[(hydroxy)(methyl)phosphinoyl]butyric acid (PPO) is a valuable prochiral keto acid for the asymmetric synthesis of the important herbicide L-phosphinothricin (L-PPT). Developing an efficient biocatalytic pathway to meet the increasing demand for PPO is highly attractive. In this study, a robust D-amino acid aminotransferase from Cytobacillus firmus ( Cf DAAT) was screened, which exhibited excellent tolerability to D,L-PPT and showed remarkable specific activity and affinity toward pyruvate (61.9 U/mg, Km = 7.4   mM). A designed co-substrate regeneration system was then introduced to shift the reaction equilibrium forward by coupling with D-amino acid oxidase (DAAO) and catalase (CAT). An efficient whole-cell biocatalyst was obtained by fine-tuning RBS intensity to coordinate the co-expression of the three enzymes. Moreover, the optimum conditions to promote overall conversion efficiency were identified by response surface methodology. Consequently, with only a catalytic amount of pyruvate (3.33   mol%) added in a 1   L reaction system, 200   mM D-PPT (400   mM D,L-PPT) was completely converted in 10   h, resulting in a PPO productivity of 3.33   g⋅L −1 ⋅h −1 , and a notable conversion rate of >99% at 300   mM D-PPT in 16   h. It demonstrated a considerable catalytic level and provided a competitive route for industrial PPO production from D,L-PPT.