The objective of this study was to identify and exploit a robust biocatalyst that can be applied in reductive amination for enantioselective synthesis of the competitive herbicide L-phosphinothricin. Applying a genome mining-based library construction strategy, eight NADPH-specific glutamate dehydrogenases (GluDHs) were identified for reductively aminating 2-oxo-4-[(hydroxy)(methyl)phosphinoyl]butyric acid (PPO) to L-phosphinothricin. Among them, the glutamate dehydrogenase cloned from Pseudomonas putida ( Pp GluDH) exhibited relatively high catalytic activity and favorable soluble expression. This enzyme was purified to homogeneity for further characterization. The specific activity of Pp GluDH was 296.1 U/g-protein, which is significantly higher than the reported value for a GluDH. To the best of our knowledge, there has not been any report on protein engineering of GluDH for PPO-oriented activity. Taking full advantage of the available information and the diverse characteristics of the enzymes in the enzyme library, Pp GluDH was engineered by site-directed mutation based on multiple sequence alignment. The mutant I170M, which had 2.1-fold enhanced activity, was successfully produced. When the I170M mutant was applied in the batch production of L-phosphinothricin, it showed markedly improved catalytic efficiency compared with the wild type enzyme. The conversion reached 99% (0.1 M PPO) with an L-phosphinothricin productivity of 1.35 g/h·L, which far surpassed the previously reported level. These results show that Pp GluDH I170M is a promising biocatalyst for highly enantioselective synthesis of L-phosphinothricin by reductive amination.
Chiral amino acids are extensively applied in the pharmaceutical, food, cosmetic, and agricultural industries. As a representative example, l -phosphinothricin ( l -PPT, a chiral non-natural amino acid) is a broad-spectrum herbicide. An NAD(H)-driven biocatalytic system for the asymmetric synthesis of chiral amino acids (focused on l -PPT) with high efficiency and low cost is highly desired. The key for the development of such biocatalytic system is to obtain an NADH-dependent biocatalyst with high catalytic performance toward l -PPT pro-ketone PPO. Herein, an engineered glutamate dehydrogenase from Lysinibacillus composti ( Lc GluDH) with desired activity was obtained by gene mining and protein engineering. In silico analyses suggested that the volume of substrate-binding pocket was substantially enlarged from 330.5 Å 3 to 409.6 Å 3 . The stability of Lc GluDH was increased ( T m value increased from 47.3 °C to 65.3 °C) by introducing positively charged amino acids or aromatic amino acids at position 375. The desired biocatalytic system was constructed by coupling the engineered Lc GluDH and an NAD + -dependent FDH. Through this biocatalytic system, the batch production of l -PPT exhibited high space-time yield (207.3 g ⋅ L −1 ⋅ day −1 ) with strict stereoselectivity ( ee of l -PPT>99%). Furthermore, eight other chiral amino acids were synthesised by the developed NAD(H)-driven biocatalytic system with high ee values.
D-amino acid oxidase (DAAO)-catalyzed selective oxidative deamination is a very promising process for synthesizing l -amino acids including l -phosphinothricin ( l -PPT, a high-efficiency and broad-spectrum herbicide). However, the wild-type DAAO's low activity toward unnatural substrates like d -phosphinothricin ( d -PPT) hampers its application. Herein, a DAAO from Caenorhabditis elegans ( Ce DAAO) was screened and engineered to improve the catalytic potential on d -PPT. First, we designed a novel growth selection system, taking into account the intricate relationship between the growth of Escherichia coli ( E. coli ) and the catalytic mechanism of DAAO. The developed system was used for high-throughput screening of gene libraries, resulting in the discovery of a variant (M6) with significantly increased catalytic activity against d -PPT. The variant displays different catalytic properties on substrates with varying hydrophobicity and hydrophilicity. Analysis using Alphafold2 modeling and molecular dynamic simulations showed that the reason for the enhanced activity was the substrate-binding pocket with enlarged size and suitable charge distribution. Further QM/MM calculations revealed that the crucial factor for enhancing activity lies in reducing the initial energy barrier of the reductive half reaction. Finally, a comprehensive binding-model index to predict the enhanced activity of DAAO toward d -PPT, and an enzymatic deracemization approach was developed, enabling the efficient synthesis of l -PPT with remarkable efficiency.
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