Engineered β1-3-<i>N</i>-Acetylglucosaminyltransferase Facilitating the One-Pot Multienzyme Synthesis of Human Milk Oligosaccharides

β1-3-linked <i>N</i>-acetylglucosaminide is a prevalent carbohydrate motif found in oligosaccharides, polysaccharides, glycoproteins, and glycolipids. It is a crucial component of human milk oligosaccharides (HMOs). <i>Neisseria meningitidis</i> β1-3-<i>N</i>-acetylglucosaminyltransferase (NmLgtA) catalyzes the formation of a glycosidic bond and has the potential for use in synthesizing HMOs. However, this application is hindered by challenges such as low levels of enzyme expression, poor stability, and significant aggregation. Since there is no available crystal structure for NmLgtA, we used its AlphaFold 2 predicted structure to identify potential unfavorable factors. We then modified the enzyme by removing the 17 N-terminal amino acids and substituting nine specific residues. The engineered NmLgtA-Opti exhibited improved thermal stability, increased soluble protein expression, complete relief from aggregation, and enhanced catalysis while maintaining its catalytic specificity and substrate promiscuity. Furthermore, NmLgtA-Opti maximizes substrate utilization and can be employed in a sequential one-pot multienzyme platform for high-yield production of HMOs.