Glycosyltransferases in human milk oligosaccharide synthesis: structural mechanisms and rational design

Human milk oligosaccharides (HMOs) play a pivotal role in infant health through their multifunctional bioactive properties. Recent advances in synthetic biology have revolutionized microbial platforms for HMO biosynthesis, with glycosyltransferases (GTs) emerging as indispensable biocatalytic tools that drive enzymatic lactose glycosylation to generate diversified oligosaccharides. This review systematically analyzes GT structural biology, elucidating conserved domains and catalytic mechanisms through crystallographic studies. We summarize contemporary optimization strategies for enhancing GT functionality, including solubility enhancement, catalytic efficiency improvement, and substrate specificity engineering via structure-guided rational design. Emerging deep learning algorithms demonstrate transformative potential in GT modifications and de novo design, providing innovative solutions to overcome bottlenecks in industrial-scale HMO synthesis. These approaches establish a framework for the precision engineering of carbohydrate-active enzymes.