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 (PpGluDH) exhibited relatively high catalytic activity and favorable soluble expression. This enzyme was purified to homogeneity for further characterization. The specific activity of PpGluDH 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, PpGluDH 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 PpGluDH I170M is a promising biocatalyst for highly enantioselective synthesis of L-phosphinothricin by reductive amination.

Glutamate dehydrogenases (GluDHs) are promising biocatalysts for the synthesis of chiral aamino acids by asymmetric reductive amination of a-keto acids. However, their strict substrate specificity limits their applications. To address this problem, we developed a molecular engineering method for GluDHs that enhances the asymmetric reductive amination of bulky a-keto acids. Based on rational design, a "cave" located in the active site pocket of PpGluDH (GluDH from Pseudomonas putida), which plays an essential role in substrate recognition, was tailored to facilitate the accepting of bulky substrates. Two mutants (A167G and V378A) were discovered to have significantly enhanced catalytic activity toward 2-oxo-4-[(hydroxy)(methyl)phosphinyl]butyric acid (PPO) and several other bulky substrates. This molecular engineering method was then applied to ten other GluDHs from different sources and with different properties. All engineered GluDHs acquired substantial improvements in PPO-oriented catalytic activity. The most efficient mutant of NADP + (nicotinamide adenine dinucleotide phosphate)-specific GluDHs showed up to 1820-fold increased activity and the specific activity reached 111.02 U/mg-protein. The NAD + (nicotinamide adenine dinucleotide)-specific GluDHs, which have no detectable wild type activity toward PPO, acquired a considerable level of activity (1.90-29.48 U/mg-protein). In batch production of L-phosphinothricin, these "cave-tailored" GluDHs exhibited markedly improved catalytic efficiencies compared with their wild types and ee values of > 99%. The space-time yields (STY) varied from 818.16 to 1482.96 g · L À1 · d À1 , suggesting potential practical applications of these mutants.

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Sour rot, caused by Geotrichum citri-aurantii, is a major postharvest disease in citrus and results in significant economic losses. The genus Beauveria is recognized as a promising source of biocontrol agents for agricultural applications. Herein, we established a targeted strategy by integrating genomics and metabolomics to accelerate the discovery of new cyclopeptides from antagonistic metabolites produced by the marine-derived fungus Beauveria felina SYSU-MS7908. As a result, we isolated and characterized seven cyclopeptides, including six new molecules, isaridins I-N (1−6). Their chemical structures and conformational analysis were extensively elucidated using spectroscopic techniques (NMR, HRMS, and MS'MS data), modified Mosher's and Marfey's methods, and single-crystal X-ray diffraction. Notably, isaridin K (3) contains a peptide backbone with an N-methyl-2aminobutyric acid residue rarely found in natural cyclopeptides. Bioassays showed that compound 2 could significantly inhibit the mycelial growth of G. citri-aurantii by destroying the cell membrane. These findings provide an effective strategy for searching for new fungal peptides for potential agrochemical fungicides and also pave the way for further exploration of applications in agriculture, food, and medicine.