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

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