[A multi-enzyme cascade process for the preparation of L-phosphinothricin]

L-phosphinothricin (L-PPT) is an efficient broad-spectrum herbicide. To realize the multi-enzyme catalytic preparation of L-PPT, we constructed an engineered strain Escherichia coli YM-1 for efficient expression of D-amino acid transaminase, which could catalyze the generation of the intermediate 2-oxo-4-[(hydroxymethylphosphonyl)] butyric acid (PPO) from D-phosphinothricin (D-PPT). In addition, E. coli pLS was constructed to co-express glutamate dehydrogenase and glucose dehydrogenase, which not only catalyzed the generation of L-PPT from PPO but also regenerated the coenzyme nicotinamide adenine dinucleotide phosphate (NADPH). A fed-batch fermentation process was then established for E. coli YM-1 and pLS, and the apparent activities of D-amino acid transaminase and glutamate dehydrogenase were increased by 22.68% and 100.82%, respectively, compared with those in shake flasks. The process parameters were optimized for the catalytic preparation of L-PPT by whole-cell cascade of E. coli YM-1 and pLS with D, L-PPT as the substrate. After reaction for 8 h, 91.36% conversion of D-PPT was achieved, and the enantiomeric excess of L-PPT reached 90.22%. The findings underpin the industrial production of L-PPT.