Human milk oligosaccharides (HMOs), the third most abundant solid component in human milk after lactose and lipids, exert multiple physiological functions, including promoting infant gut health, immune development, and neurocognitive maturation. However, their natural sources are limited, and traditional chemical or enzymatic synthesis approaches suffer from complex procedures, high costs, and environmental burdens. In recent years, synthetic biology has provided a systematic solution for the efficient and sustainable production of HMOs. By employing Escherichia coli, Corynebacterium glutamicum, and Saccharomyces cerevisiae as chassis cells, researchers have achieved regeneration of sugar donors, optimization of glycosyltransferases, and modular reconstruction of metabolic pathways, resulting in fermentation titers of 141.27 g/L for 2′-fucosyllactose (2′-FL) and 56.8 g/L for 3′-sialyllactose (3′-SL), thereby accelerating industrial implementation. Meanwhile, the successful biosynthesis of structurally complex molecules such as lacto-N-fucopentaose I (LNFP I) marks the transition of HMOs research from laboratory exploration to engineering feasibility. This review summarizes the current advances and applications of synthetic biology in HMOs biosynthesis, with an emphasis on key technological bottlenecks, including insufficient donor recycling efficiency, poor enzyme stability, low host tolerance, and incomplete green manufacturing systems. Furthermore, it highlights future perspectives, suggesting that with the integration of AI-driven metabolic regulation, multi-omics analysis, and low-carbon biomanufacturing technologies, HMOs biosynthesis is poised to advance toward intelligent, precise, and sustainable production, offering new opportunities for the nutritional application of functional carbohydrates and innovation in the food industry.
