Human milk oligosaccharides: Overcoming production bottlenecks through novel large-scale strategies and applications

Breast milk comprises nutrient-dense bioactive constituents and a diversive bacterial population that encourages gut microbiota maturation, enhances immunological function, and aids in pathogen defense. Human milk oligosaccharides (HMOs), the third most abundant solid component, are key bioactive carbohydrates that shape intestinal microbiota, provide antibacterial effects, enhance the intestinal barrier, and regulate immune responses. For many years, research in this area has been constrained by the restricted availability of HMOs. Only human milk contains the majority of HMOs, and historically, it has been costly and time-consuming to isolate or synthesize them. This review examines novel approaches to mitigate production bottlenecks in HMOs, emphasizing chemoenzymatic synthesis, microbial engineering, and extraction from donor milk or dairy sources. It highlights progress in synthetic biology that improves biosynthetic pathways, increasing catalytic efficiency, stability, and diversity of intricate HMO structures. It evaluates the industrial scalability and technoeconomic feasibility of plant-based and microbial systems as economical and sustainable approaches for large-scale HMO production. • Structural constraints affecting the synthesis of complex human milk oligosaccharides. • Comparative features of biosynthetic, chemoenzymatic, and extraction-based HMO production routes. • Platform-dependent differences in achievable HMO structures and isomer control. • Analytical challenges in the characterization of complex and isomeric HMOs. • Technoeconomic evaluation supports industrial feasibility and future applications.