Leveraging AlphaFold for Rational Redesign of Glycosyltransferases in Long-Chain Human Milk Oligosaccharide Biosynthesis

AlphaFold (AF) has transformed protein structure prediction and is increasingly enabling the rational redesign of glycosyltransferases (GT) involved in the biosynthesis of human milk oligosaccharides (HMOs). AF-derived structural insights deepen our understanding of GT structure-function relationships, supporting targeted engineering to enhance the catalytic efficiency, broaden substrate scope, and improve regiospecificity─key requirements for scalable microbial production of complex long-chain HMOs. Using lacto-N-fucopentaose I (LNFP I) synthesis as a case study, we illustrate how AF-guided modeling facilitates GT redesign for efficient HMO biosynthesis, while also discussing current limitations and challenges toward precision GT engineering.