Metabolic Engineering of Human Milk Oligosaccharides

Human milk oligosaccharides (HMOs) constitute a structurally diverse group of complex carbohydrates that are the third most abundant solid component in breast milk. They play pivotal roles in infant health by functioning as prebiotics, anti-adhesive antimicrobials and modulators of immune development. Natural extraction yields are limited by low concentrations and high purification costs, driving the application of metabolic engineering to establish microbial cell factories capable of scalable HMO biosynthesis. Two principal approaches are employed: de novo synthesis, in which a host organism is engineered to convert simple sugars through integrated pathways for nucleotide-activated donor formation and glycosyltransferase-catalysed assembly; and the salvage pathway, which supplements exogenous monosaccharides such as fucose to generate specific HMOs. Key advances focus on enhancing precursor supply—especially GDP-l-fucose—optimising heterologous expression of glycosyltransferases, and balancing metabolic flux through chromosomal integration, promoter engineering and cofactor regeneration. Model hosts include Escherichia coli, Bacillus subtilis and Saccharomyces cerevisiae, each offering distinct advantages in safety status, tolerance to feedstocks and secretion capacity. Progress in enzyme engineering has improved regio- and stereoselectivity, enabling the synthesis of principal HMOs such as 2′-fucosyllactose and lacto-N-neotetraose. Ongoing efforts aim to refine bioprocess parameters and downstream recovery, paving the way for cost-effective production of HMOs as additives in infant formula, nutritional supplements and therapeutic applications.

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Metabolic Engineering of Human Milk Oligosaccharides publication trend

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Technical terms

Human Milk Oligosaccharides (HMOs): Complex unconjugated glycans found in human breast milk that promote infant health through prebiotic, immunomodulatory and antimicrobial functions.

2′-Fucosyllactose (2′-FL): A principal HMO consisting of lactose fucosylated at the 2′ position, noted for its ability to inhibit pathogen adhesion and support beneficial gut microbiota.

Metabolic engineering: The strategic modification of cellular pathways to optimise production of target compounds through gene insertion, deletion or regulation.

De novo synthesis pathway: A fully integrated biosynthetic route enabling host cells to generate nucleotide-activated sugar donors from simple carbon sources without external monosaccharide feeding.

Salvage pathway: A strategy that introduces exogenous monosaccharides into engineered hosts, which convert them into activated sugar donors for glycosyltransferase-mediated HMO assembly.

Fucosyltransferase: An enzyme that transfers fucose from GDP-l-fucose to acceptor molecules such as lactose or oligosaccharide backbones, establishing α-1,2 or α-1,3 linkages characteristic of fucosylated HMOs.

References

1. Nondigestible Functional Oligosaccharides: Enzymatic Production and Food Applications for Intestinal Health. Annual Review of Food Science and Technology (2023).
2. Multi-Path Optimization for Efficient Production of 2′-Fucosyllactose in an Engineered Escherichia coli C41 (DE3) Derivative. Frontiers in Bioengineering and Biotechnology (2020).
3. Engineered Bacillus subtilis for the de novo production of 2′-fucosyllactose. Microbial Cell Factories (2022).
4. Synthesis of Human Milk Oligosaccharides: Protein Engineering Strategies for Improved Enzymatic Transglycosylation. Molecules (2019).