{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,23]],"date-time":"2026-10-23T20:05:13Z","timestamp":1792785913152,"version":"4.5.0"},"reference-count":37,"publisher":"Oxford University Press (OUP)","issue":"11","license":[{"start":{"date-parts":[[2026,8,19]],"date-time":"2026-08-19T00:00:00Z","timestamp":1787097600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"TKI Top Sector Agri & Food scheme for public-private partnerships"},{"name":"Japan Society for the Promotion of Science (JSPS) KAKENHI","award":["25H01430"],"award-info":[{"award-number":["25H01430"]}],"id":[{"id":"https:\/\/ror.org\/00hhkn466","id-type":"ROR","asserted-by":"crossref"}]},{"name":"Japan Society for the Promotion of Science (JSPS) KAKENHI","award":["25H01429"],"award-info":[{"award-number":["25H01429"]}],"id":[{"id":"https:\/\/ror.org\/00hhkn466","id-type":"ROR","asserted-by":"crossref"}]},{"name":"Human Glycome Atlas Project (HGA) from the Ministry of Education, Culture, Sports, Science and Technology","id":[{"id":"https:\/\/ror.org\/048rj2z13","id-type":"ROR","asserted-by":"crossref"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"published-print":{"date-parts":[[2026,9,29]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:p>The lactating mammary gland is uniquely adapted to synthesize unconjugated free glycans known as human milk oligosaccharides (HMOs). HMO biosynthesis is initiated by the formation of lactose, the common precursor from which structurally diverse neutral and acidic oligosaccharides are generated through the sequential action of Golgi-resident glycosyltransferases. Although microbial fermentation and chemical synthesis have enabled production of selected HMOs, human cell-based platforms remain largely unexplored despite providing the native glycosylation machinery required for HMO assembly. Consequently, the contributions of individual glycosyltransferases to HMO biosynthesis have remained difficult to define. Here, we describe the construction of HMO biosynthesis in human embryonic kidney (HEK293) cells through constitution of the lactose synthase complex and systematic engineering of downstream glycosyltransferases. This modular approach enables programmable production of structurally defined HMOs, including both simple sialylated oligosaccharides and more complex type-I and type-II structures, while providing a tractable platform for assigning glycosyltransferase functions within the HMO biosynthetic pathway. Together, this establishes mammalian glycoengineering as a versatile system for investigating HMO biosynthesis and for the tailored production of biologically relevant HMOs.<\/jats:p>","DOI":"10.1093\/glycob\/cwag066","type":"journal-article","created":{"date-parts":[[2026,8,15]],"date-time":"2026-08-15T12:48:02Z","timestamp":1786798082000},"source":"Crossref","is-referenced-by-count":0,"title":["Programmable HMO biosynthesis enabled by human cell-based glycoengineering"],"prefix":"10.1093","volume":"36","author":[{"given":"Stijn","family":"Kruf","sequence":"first","affiliation":[{"name":"Department of Biomolecular Chemistry, Institute for Molecules and Materials, Radboud University , Heyendaalseweg 135, 6525 AJ, Nijmegen ,","place":["the Netherlands"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Roy J B M","family":"Delahaije","sequence":"additional","affiliation":[{"name":"FrieslandCampina , Stationsplein 4, 3818 LE, Amersfoort ,","place":["the Netherlands"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0344-2408","authenticated-orcid":false,"given":"Morihisa","family":"Fujita","sequence":"additional","affiliation":[{"name":"Institute for Glyco-core Research (iGCORE), Gifu University , 1-1 Yanagido, Gifu 501-1193 ,","place":["Japan"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7785-2920","authenticated-orcid":false,"given":"Christian","family":"B\u00fcll","sequence":"additional","affiliation":[{"name":"Department of Biomolecular Chemistry, Institute for Molecules and Materials, Radboud University , Heyendaalseweg 135, 6525 AJ, Nijmegen ,","place":["the Netherlands"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"286","published-online":{"date-parts":[[2026,8,19]]},"reference":[{"key":"2026092912325177800_ref1","doi-asserted-by":"publisher","first-page":"71","DOI":"10.1016\/j.carres.2019.03.006","article-title":"Kilogram scale chemical synthesis of 2\u2032-fucosyllactose","volume":"476","author":"Agoston","year":"2019","journal-title":"Carbohydr Res"},{"issue":"9","key":"2026092912325177800_ref2","doi-asserted-by":"publisher","first-page":"1147","DOI":"10.1093\/glycob\/cws074","article-title":"Human milk oligosaccharides: every baby needs a sugar mama","volume":"22","author":"Bode","year":"2012","journal-title":"Glycobiology"},{"issue":"2","key":"2026092912325177800_ref3","doi-asserted-by":"publisher","first-page":"491","DOI":"10.1073\/pnas.59.2.491","article-title":"The role of alpha-lactalbumin and the a protein in lactose synthetase: a unique mechanism for the control of a biological reaction","volume":"59","author":"Brew","year":"1968","journal-title":"Proc Natl Acad Sci U S A"},{"issue":"1","key":"2026092912325177800_ref4","doi-asserted-by":"publisher","DOI":"10.1016\/j.xpro.2020.100017","article-title":"Cell-based glycan arrays-a practical guide to dissect the human Glycome","volume":"1","author":"Bull","year":"2020","journal-title":"STAR Protoc"},{"issue":"17","key":"2026092912325177800_ref5","doi-asserted-by":"crossref","DOI":"10.1073\/pnas.2026102118","article-title":"Probing the binding specificities of human Siglecs by cell-based glycan arrays","volume":"118","author":"Bull","year":"2021","journal-title":"Proc Natl Acad Sci U S A"},{"issue":"1","key":"2026092912325177800_ref6","doi-asserted-by":"publisher","DOI":"10.1080\/19490976.2023.2186115","article-title":"Functional effects of human milk oligosaccharides (HMOs)","volume":"15","author":"Dinleyici","year":"2023","journal-title":"Gut Microbes"},{"issue":"3","key":"2026092912325177800_ref7","doi-asserted-by":"publisher","first-page":"762","DOI":"10.1016\/S0021-9258(18)96903-6","article-title":"Resolution of a soluble lactose synthetase into two protein components and solubilization of microsomal lactose synthetase","volume":"241","author":"Ebner","year":"1966","journal-title":"J Biol Chem"},{"issue":"7","key":"2026092912325177800_ref8","doi-asserted-by":"publisher","DOI":"10.1016\/j.tifs.2023.104203","article-title":"Advances and challenges for obtaining human milk oligosaccharides: extraction from natural sources and synthesis by intentional design","volume":"141","author":"Gan","year":"2023","journal-title":"Trends Food Sci Technol"},{"issue":"1","key":"2026092912325177800_ref9","doi-asserted-by":"publisher","first-page":"46","DOI":"10.1038\/s41574-022-00742-y","article-title":"Hormonal regulation of mammary gland development and lactation","volume":"19","author":"Hannan","year":"2023","journal-title":"Nat Rev Endocrinol"},{"key":"2026092912325177800_ref10","doi-asserted-by":"publisher","first-page":"281","DOI":"10.1007\/978-1-0716-3670-1_24","article-title":"Recombinant production of glycoengineered mucins in HEK293-F cells","volume-title":"Methods Mol Biol","author":"Huang","year":"2024"},{"issue":"1","key":"2026092912325177800_ref11","doi-asserted-by":"publisher","first-page":"9668","DOI":"10.1038\/s41467-024-53738-9","article-title":"Mammalian cell-based production of glycans, glycopeptides and glycomodules","volume":"15","author":"Jaroentomeechai","year":"2024","journal-title":"Nat Commun"},{"issue":"1","key":"2026092912325177800_ref12","doi-asserted-by":"publisher","first-page":"2455","DOI":"10.1038\/s41467-022-29867-4","article-title":"Elucidating human milk oligosaccharide biosynthetic genes through network-based multi-omics integration","volume":"13","author":"Kellman","year":"2022","journal-title":"Nat Commun"},{"issue":"8","key":"2026092912325177800_ref13","doi-asserted-by":"publisher","first-page":"2336","DOI":"10.1016\/S0021-9258(19)45434-3","article-title":"The interaction of -lactalbumin and the a protein of lactose synthetase","volume":"247","author":"Klee","year":"1972","journal-title":"J Biol Chem"},{"issue":"8","key":"2026092912325177800_ref14","doi-asserted-by":"publisher","DOI":"10.1093\/glycob\/cwag048","article-title":"Biosynthesis of human milk oligosaccharides (HMOs) in glycoengineered human cells","volume":"36","author":"Kruf","year":"2026","journal-title":"Glycobiology"},{"issue":"49","key":"2026092912325177800_ref15","doi-asserted-by":"publisher","first-page":"27314","DOI":"10.1021\/acs.jafc.4c08703","article-title":"Efficient production of 3'-Sialyllactose using Escherichia coli","volume":"72","author":"Lv","year":"2024","journal-title":"J Agric Food Chem"},{"key":"2026092912325177800_ref16","doi-asserted-by":"publisher","DOI":"10.3389\/fnut.2023.1239349","article-title":"Human milk oligosaccharide composition and associations with growth: results from an observational study in the US","volume":"10","author":"Mainardi","year":"2023","journal-title":"Front Nutr"},{"issue":"1","key":"2026092912325177800_ref17","doi-asserted-by":"publisher","first-page":"10846","DOI":"10.1038\/s41598-022-14260-4","article-title":"In silico analysis of the human milk oligosaccharide glycome reveals key enzymes of their biosynthesis","volume":"12","author":"McDonald","year":"2022","journal-title":"Sci Rep"},{"key":"2026092912325177800_ref18","doi-asserted-by":"publisher","DOI":"10.1016\/j.carres.2024.109242","article-title":"Putting a cap on the glycome: dissecting human sialyltransferase functions","volume":"544","author":"Mohamed","year":"2024","journal-title":"Carbohydr Res"},{"issue":"2","key":"2026092912325177800_ref19","doi-asserted-by":"publisher","first-page":"394","DOI":"10.1016\/j.molcel.2019.05.017","article-title":"An atlas of human glycosylation pathways enables display of the human Glycome by gene engineered cells","volume":"75","author":"Narimatsu","year":"2019","journal-title":"Mol Cell"},{"key":"2026092912325177800_ref20","doi-asserted-by":"publisher","DOI":"10.1016\/j.jbc.2021.100448","article-title":"Genetic glycoengineering in mammalian cells","volume":"296","author":"Narimatsu","year":"2021","journal-title":"J Biol Chem"},{"key":"2026092912325177800_ref21","doi-asserted-by":"publisher","DOI":"10.1016\/j.ymben.2026.102494","article-title":"Reconstitution of human milk oligosaccharide biosynthesis in cultured mammalian cells","volume":"97","author":"Noda","year":"2026","journal-title":"Metab Eng"},{"issue":"9","key":"2026092912325177800_ref22","doi-asserted-by":"publisher","DOI":"10.1016\/j.isci.2025.113269","article-title":"Integrated 'omics analysis reveals human milk oligosaccharide biosynthesis programs in human lactocytes","volume":"28","author":"Nyquist","year":"2025","journal-title":"iScience"},{"key":"2026092912325177800_ref23","doi-asserted-by":"publisher","DOI":"10.1016\/j.cofs.2024.101154","article-title":"Recent advances in the microbial production of human milk oligosaccharides","volume":"57","author":"Pressley","year":"2024","journal-title":"Curr Opin Food Sci"},{"issue":"27","key":"2026092912325177800_ref24","doi-asserted-by":"publisher","first-page":"6954","DOI":"10.1073\/pnas.1701785114","article-title":"Synthesis of asymmetrical multiantennary human milk oligosaccharides","volume":"114","author":"Prudden","year":"2017","journal-title":"Proc Natl Acad Sci U S A"},{"issue":"1","key":"2026092912325177800_ref25","doi-asserted-by":"publisher","first-page":"205","DOI":"10.1006\/jmbi.2001.4757","article-title":"Crystal structure of lactose synthase reveals a large conformational change in its catalytic component, the beta1,4-galactosyltransferase-I","volume":"310","author":"Ramakrishnan","year":"2001","journal-title":"J Mol Biol"},{"key":"2026092912325177800_ref26","doi-asserted-by":"publisher","DOI":"10.3389\/fmolb.2025.1587602","article-title":"Glycosyltransferases: glycoengineers in human milk oligosaccharide synthesis and manufacturing","volume":"12","author":"Slater","year":"2025","journal-title":"Front Mol Biosci"},{"issue":"2","key":"2026092912325177800_ref27","doi-asserted-by":"publisher","first-page":"280","DOI":"10.1111\/jhn.12990","article-title":"Biology of human milk oligosaccharides: from basic science to clinical evidence","volume":"35","author":"Sprenger","year":"2022","journal-title":"J Hum Nutr Diet"},{"key":"2026092912325177800_ref28","doi-asserted-by":"publisher","first-page":"190","DOI":"10.3389\/fped.2018.00190","article-title":"Immunological effects of human milk oligosaccharides","volume":"6","author":"Triantis","year":"2018","journal-title":"Front Pediatr"},{"issue":"12","key":"2026092912325177800_ref29","doi-asserted-by":"publisher","first-page":"3382","DOI":"10.1016\/S0021-9258(18)93319-3","article-title":"The hormonal control of lactose synthetase in the developing mouse mammary gland","volume":"243","author":"Turkington","year":"1968","journal-title":"J Biol Chem"},{"key":"2026092912325177800_ref30","doi-asserted-by":"publisher","DOI":"10.1016\/j.bbadva.2024.100136","article-title":"Recent advances in the science of human milk oligosaccharides","volume":"7","author":"Urashima","year":"2025","journal-title":"BBA Adv"},{"issue":"12","key":"2026092912325177800_ref31","doi-asserted-by":"publisher","first-page":"1323","DOI":"10.1093\/glycob\/cwv091","article-title":"Symbol nomenclature for graphical representations of glycans","volume":"25","author":"Varki","year":"2015","journal-title":"Glycobiology"},{"issue":"14","key":"2026092912325177800_ref32","doi-asserted-by":"publisher","first-page":"5851","DOI":"10.1021\/acs.joc.6b00478","article-title":"Chemoenzymatic synthesis of a library of human milk oligosaccharides","volume":"81","author":"Xiao","year":"2016","journal-title":"J Org Chem"},{"issue":"1","key":"2026092912325177800_ref33","doi-asserted-by":"publisher","first-page":"38","DOI":"10.1186\/s12934-024-02312-5","article-title":"Efficient production of 2\u2032-fucosyllactose from fructose through metabolically engineered recombinant Escherichia coli","volume":"23","author":"You","year":"2024","journal-title":"Microb Cell Factories"},{"issue":"1","key":"2026092912325177800_ref34","doi-asserted-by":"publisher","first-page":"101","DOI":"10.1186\/s12934-018-0947-2","article-title":"Production of a human milk oligosaccharide 2\u2032-fucosyllactose by metabolically engineered Saccharomyces cerevisiae","volume":"17","author":"Yu","year":"2018","journal-title":"Microb Cell Factories"},{"issue":"11","key":"2026092912325177800_ref35","doi-asserted-by":"publisher","DOI":"10.3390\/molecules24112033","article-title":"Synthesis of human milk oligosaccharides: protein engineering strategies for improved enzymatic Transglycosylation","volume":"24","author":"Zeuner","year":"2019","journal-title":"Molecules"},{"issue":"5","key":"2026092912325177800_ref36","doi-asserted-by":"publisher","first-page":"787","DOI":"10.1016\/j.biotechadv.2019.04.011","article-title":"Microbial production of sialic acid and sialylated human milk oligosaccharides: advances and perspectives","volume":"37","author":"Zhang","year":"2019","journal-title":"Biotechnol Adv"},{"issue":"8","key":"2026092912325177800_ref37","doi-asserted-by":"publisher","first-page":"2083","DOI":"10.1080\/10408398.2020.1850413","article-title":"Recent advances on 2\u2032-fucosyllactose: physiological properties, applications, and production approaches","volume":"62","author":"Zhu","year":"2022","journal-title":"Crit Rev Food Sci Nutr"}],"container-title":["Glycobiology"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/academic.oup.com\/glycob\/advance-article-pdf\/doi\/10.1093\/glycob\/cwag066\/70709090\/cwag066.pdf","content-type":"application\/pdf","content-version":"am","intended-application":"syndication"},{"URL":"https:\/\/academic.oup.com\/glycob\/article-pdf\/36\/11\/cwag066\/70709090\/cwag066.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"syndication"},{"URL":"https:\/\/academic.oup.com\/glycob\/article-pdf\/36\/11\/cwag066\/70709090\/cwag066.pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,9,29]],"date-time":"2026-09-29T16:32:57Z","timestamp":1790699577000},"score":1,"resource":{"primary":{"URL":"https:\/\/academic.oup.com\/glycob\/article\/doi\/10.1093\/glycob\/cwag066\/8766054"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,8,19]]},"references-count":37,"journal-issue":{"issue":"11","published-print":{"date-parts":[[2026,9,29]]}},"URL":"https:\/\/doi.org\/10.1093\/glycob\/cwag066","relation":{},"ISSN":["1460-2423"],"issn-type":[{"value":"1460-2423","type":"electronic"}],"subject":[],"published-other":{"date-parts":[[2026,11]]},"published":{"date-parts":[[2026,8,19]]},"article-number":"cwag066"}}