Whole-Cell Biosynthesis of Branched Human Milk Hexasaccharide Lacto-N-neohexaose

The inaccessibility of the enzyme responsible for β-1,6-GlcNAc branching has limited the production of branched human milk oligosaccharides (HMOs) using engineered strains. Herein, a recombinant Escherichia coli strain was metabolically engineered to serve as a whole-cell factory for the synthesis of lacto-N-neohexaose (LNnH), which represents the initial structure of branched HMOs, enabling access to diverse branched HMOs. Soluble expression of human β-1,6-N-acetylglucosaminyltransferase 2 (GCNT2) was achieved in E. coli SHuffle T7, providing a robust toolbox for the assembly of branched HMOs. Additionally, the genes encoding the LNnH biosynthesis pathway were well-designed, and the yield was increased from 1.04 to 21.19 mg/L. The production of LNnH was further increased to 173.21 mg/L in a 5 L bioreactor, representing a 166.5-fold increase over the starting engineered strain. This whole-cell biosynthetic strategy not only paves the way for accessing abundant branched HMOs but also enhances the field of HMO glycomics, opening new avenues for biological applications.