Researchers have developed a new cell-based approach to produce several sugars naturally found in human breastmilk. Human milk oligosaccharides (HMOs) are complex sugars in human milk that can act as prebiotics and influence the infant gut environment. They can promote the growth of beneficial gut bacteria and may help limit the attachment or growth of certain pathogens.
More than 200 HMO structures have been identified, but only a limited number are currently available for use in infant formula. Now, researchers developed a cultured mammalian cell system capable of producing several structurally distinct HMOs. The results were published in Metabolic Engineering on 20 June .
“HMOs are important bioactive sugars in human milk, but many of them have complex structures that are difficult to produce or study. We wanted to understand whether the biosynthetic pathway for HMO could be reconstructed in cultured mammalian cells, and whether the types of HMOs produced could be controlled by changing glycosylation pathways,” said Professor Morihisa Fujita, a researcher at Gifu University in Gifu, Japan.
The first step researchers needed to solve was understanding why HEK293 cells did not produce detectable HMOs under standard culture conditions. They mapped the biosynthetic pathway of HMO production using a prediction tool called GlycoMaple and found that two components are essential: β1,4-galactosyltransferase (B4GALT1) and α-lactalbumin (LALBA). While HEK293 cells have plenty of B4GALT1, they did not express any LALBA. When the HEK293 cells were manipulated to express LALBA, they began producing several HMOs.
“Ordinary cultured mammalian cells can be converted into HMO-producing cells by introducing α-lactalbumin (LALBA). Moreover, by modifying glycosyltransferase expression, we can change the composition of HMOs produced by the cells. This provides a new cell-based platform to study how diverse HMOs are made and to design specific HMO profiles,” said Fujita.
Researchers continued to refine the process, eventually producing a variety of structurally diverse HMOs. These findings may support future efforts to produce a broader range of HMOs for research and potential use in infant nutrition. It will also help improve understanding of the enzyme combinations needed to produce HMOs and how those enzymes are organized.
The engineered cells also produced sialylated HMOs including LSTa and DSLNT. DSLNT is of particular interest because previous studies suggest that it may have protective effects against necrotizing enterocolitis, a deadly gastrointestinal illness that affects premature infants. Once these HMOs were produced by the engineered cells, it was confirmed that the cell-derived HMOs retained at least one expected biological activity. In particular, they the growth of an important gut bacterium for infants called B. infantis.
At almost the same time, an independent study by Büll and colleagues reported the production of HMOs in glycoengineered human cells (Kruf et al. (2026) Glycobiology ). Together, the two studies highlight the potential of engineered mammalian cells as platforms for investigating and controlling HMO biosynthesis.
Looking ahead, researchers hope to produce more HMOs through this system. “The next step is to improve the productivity of this system and expand the range of HMOs that can be produced by engineering additional glycosylation pathways. Our long-term goal is to establish a flexible platform for studying HMO biosynthesis and producing a wider range of HMOs for research and potential future applications in infant nutrition,” said Fujita.
Other contributors include Fuki Noda, Aika Ohno, Hiroko Ichihashi, Kazuki Nakajima, and Yasuhiko Kizuka of Gifu University; Aruto Nakajima, Takane Katayama, and Toshihiko Katoh of Kyoto University.
The Japan Society for the Promotion of Science (JSPS) KAKENHI Grants, the joint research program of the J-GlycoNet cooperative network, the Human Glycome Atlas Project from the Ministry of Education, Culture, Sports, Science, and Technology (MEXT), and a grant from the Takeda Science Foundation supported this research.
Metabolic Engineering
Experimental study
Cells
Reconstitution of Human Milk Oligosaccharide Biosynthesis in Cultured Mammalian Cells
20-Jun-2026