Glufosinate (2-amino-4-methyl-phosphino-butyric acid, PPT) is a non-selective herbicide, with advantages, including environmental friendliness, high efficiency, and a broad spectrum of activity. It is widely used to control stubborn weeds resistant to glyphosate and paraquat[1], [2]. PPT specifically inhibits the activity of glutamine synthetase, leading to ammonia accumulation and glutamine starvation, ultimately causing plant death [3]. Commercial PPT is a racemic mixture, containing both D-form and L-form, with only L-PPT exhibiting herbicidal activity[4]. Meanwhile, residual PPT degrades in soil, with the L-PPT exhibiting a higher degradation rate than D-PPT [2], [5]. Therefore, synthesizing optically pure L-PPT reduces the total amount of glufosinate released into the environment by half, which is of significant importance for improving atom utilization efficiency, reducing costs and alleviating environmental pressure [4], [6], [7].

In recent years, several biocatalytic methods have been developed as alternatives to traditional chemical approaches for producing optically pure L-PPT. The multi-enzyme cascade process involving D-amino acid oxidase (DAAO) and glutamate dehydrogenase (GDH) has emerged as the dominant route, exhibiting both strict stereoselectivity and high economic viability. DAAO first oxidatively deaminates the substrate D-PPT, producing 2-oxo-4-[(hydroxy)(methyl)phosphinyl] butyric acid (PPO). The reductive amination reaction is then catalyzed by GDH, yielding the final product, L-PPT. This process requires the addition of coenzymes NADH/NADPH [7], [8], [9], [10].

DAAO is a flavin adenine dinucleotide (FAD)-dependent enzyme that plays an important physiological role in biological cells [11]. Most reported DAAOs are derived from mammals, filamentous fungi, and yeasts. D-aspartate oxidase (DDO) is an enzyme that catalyzes the oxidative deamination of acidic D-amino acids, producing the corresponding α-keto acids and ammonia [12]. It is commonly used for the quantitative detection of acidic D-amino acids, particularly D-aspartate and D-glutamate [13]. Due to the high amino acid sequence identity between DDO and DAAO, they are considered to have originated from a common ancestral source [14]. As a structural analog of glutamate, phosphinothricin (PPT) can be stereoselectively oxidized by either DAAO or DDO. The most extensively studied include pkDAAO from porcine kidney[15], RgDAAO from Rhodotorula gracilis[16], and TvDAAO from Trigonopsis variabilis[17]. Due to its strict stereoselectivity and broad substrate specificity, DAAO is increasingly used in various biological transformation processes[18]. However, the low soluble expression and low activity of wild-type DAAO limit its industrial applications.

The heterologous expression of recombinant proteins in E. coli often results in non-functional insoluble inclusion bodies due to an imbalance between proper folding, aggregation, and degradation, presenting a major obstacle in the production of functional recombinant proteins[19]. Adding soluble fusion tags to the N- or C-terminus of the amino acid sequence of insoluble proteins is currently one of the most widely used and effective strategies[20]. These fusion proteins facilitate proper protein folding, thereby improving solubility and reducing the formation of inclusion bodies. The most widely used fusion tags include maltose-binding protein (MBP), N-utilization substance A (NusA), glutathione S-transferase (GST), thioredoxin (Trx), small ubiquitin-related modifier (SUMO), and Fh8[21]. Furthermore, peptide tags, which contain fewer amino acid residues and occupy a smaller fraction, generally do not alter the structure or function of the target protein when attached, thereby offering distinct advantages over fusion tags[22]. However, relying solely on fusion tags is not always effective, especially when the interaction between the fusion tag and the target protein negatively affects its structure and function[23]. Therefore, combining protein truncation strategies to progressively remove unnecessary regions has become another effective approach for optimizing expression[24]. The N-terminus is typically crucial for protein activation, recognition, and degradation. While C-terminal motifs are often linked to the initial/native state stability of protein folding[25]. By truncating specific segments, hydrophobic sequences or irrelevant domains that hinder protein folding are removed, thereby improving its solubility[26].

In recent years, the rapid development of protein engineering has enabled researchers to better understand the mechanisms of enzymes, enhance enzyme catalytic activity, stability, substrate specificity, and drive towards industrialization trends[27]. For example, Kazuyuki Yasukawa and colleagues conducted a structural analysis of pkDAAO from porcine kidney and developed novel variant through protein engineering that altered its substrate specificity, enabling its application in the production of more valuable chiral compounds[28]. Wang and colleagues performed a comprehensive computational design and variant library construction for protein stability engineering of DAAO from Rhodotorula taiwanensis, resulting in significant improvements in enzyme activity and thermal stability [29], [30].

Herein, we aimed to obtain DAAO with high catalytic activity toward D-PPT through various strategies. We enhanced the soluble expression of the protein by incorporating fusion tags and truncating its sequence. Furthermore, molecular docking and high-throughput screening were employed to perform saturation mutagenesis of the active site. The optimal variant efficiently catalyzed D-PPT, demonstrating its potential for industrial application.