Rational Design of N-Acetylglucosamine-2-epimerase and N-Acetylneuraminic Lyase for Efficient N-Acetylneuraminic Acid Biosynthesis.

N-Acetylneuraminic acid (NeuAc) performs a variety of biological activities where it is used as a nutraceutical and pharmaceutical intermediate. N-Acetylglucosamine-2-epimerase (AGE) and N-acetylneuraminic lyase (NAL) are the most widely used key enzymes in the industrial production of NeuAc through whole-cell catalytic synthesis. However, both AGE and NAL catalyze reversible reactions, and the equilibrium of these two reactions lies between substrates and products, resulting in a lower conversion rate of NeuAc. In this study, affinity analysis based on the dynamic docking (ADD) strategy was used to rationally design the AGE and NAL to improve enzymes properties. The variant AGE^A172S/C118A showed a 2.19-fold improvement in the catalytic rate. Then, we combinatorially expressed the variant of AGE and NAL in two plasmids for whole cell catalytic synthesis. NeuAc production was 35% higher with the combination of AGE^A172S/C118A and NAL^F252M compared with the wild type. When substrate GlcNAc/Pyruvate was 3:8 and AGE^A172S/C118A and NAL^F252M expressed strains were 1:0.6, the molar conversion rate was 62%. Thus, our modification of AGE and NAL, the key enzymes in producing NeuAc, gave a better AGE variant AGE^A172S/C118A, which could produce 128 g/L NeuAc when using low substrate concentration (0.6 M GlcNAc).