Enzymatic Characterization and Engineering of Monomer Aspartokinase Mutant T379N/A380C/T65I/D173G from Corynebacterium pekinense

In the present study, four key residues in aspartokinase were selected for site-directed saturation mutagenesis, namely, Thr 379 and Ala 380 (the binding sites of the inhibitor Lys), Thr 65 (the binding site of the substrate Asp) and Asp 173 (the catalytically active site of both substrates and ATP binding site). The mutant T379N/A380C/T65I/D173G was successfully constructed using high-throughput screening. Compared with the wild type (WT) enzyme, the activity of the mutant was increased by 75.83 times. In addition, the characterization of kinetics and enzymatic properties showed that the K_m value of the mutant was reduced (1.34 versus 4.11 mmol/L) compared to the WT enzyme, indicating increased substrate affinity. The n value was reduced (1.07 versus 1.71), suggesting that the positive synergistic effect was weakened. Moreover, the optimal temperature, pH and half-life of the mutant were 30 ℃, 8.0 and 3.23 h compared to 25 ℃, 8.0 and 4.24 h for the WT enzyme, respectively. The inhibition effect of the inhibitors at 0.2–10 mmol/L on the mutant was weakened, especially for Lys and Lys + Thr. Through seamless cloning, it was transformed into Corynebacterium pekinense to construct an engineered bacterium. Compared with WT, the yields of lysine, threonine and methionine were increased by 83.05%, 29.36% and 30.77%, respectively. This work will provide a theoretical basis for optimizing the AK metabolic pathway and constructing strains capable of producing aspartate-family amino acids in high yield.