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Here, we report an engineered \u03b12,3-sialyltransferase variant, R60V, with enhanced catalytic performance toward the 3-fucosyllactose (3-FL) substrate. R60V exhibited a 3.6-fold enhancement in transglycosylation activity and a nearly 5-fold lower apparent Km for 3-FL. Its half-life at 40 \u00b0C extended from 42.55 to 89.82 h. Mechanistic analyses revealed that R60V alleviated steric hindrance for 3-FL binding, promoted productive interactions with the catalytic residue, and balanced local flexibility with global stability. Furthermore, a one-pot cascade system comprising R60V and a CMP-sialic acid synthetase was established, enabling in situ sialyl donor generation and affording 9.31 mM 3\u2032S3FL in 93.13% yield within 3 h under optimized conditions. This study provides a practical biocatalytic cascade platform for efficient 3\u2032S3FL production.<\/jats:p>","DOI":"10.1021\/acs.jafc.6c10399","type":"journal-article","created":{"date-parts":[[2026,9,24]],"date-time":"2026-09-24T21:02:19Z","timestamp":1790283739000},"page":"31044-31059","source":"Crossref","is-referenced-by-count":0,"title":["Multifaceted Cross-Rational\nModification of the \u03b12,3-Sialyltransferase\nMsST for High-Efficiency Production of 3\u2032-Sialyl-3-fucosyllactose\nvia One-Pot Cascade Reaction"],"prefix":"10.1021","volume":"74","author":[{"given":"Yifan","family":"Cao","sequence":"first","affiliation":[{"name":"Ocean University of China , , ,","place":["Qingdao, PR China, 266404"]},{"name":"Qingdao Key Laboratory of Green Manufacturing of Marine Bioproducts , , ,","place":["Qingdao, PR China, 266071"]},{"name":"China National Light Industry , , ,","place":["Qingdao, PR China, 266404"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Rui","family":"Chen","sequence":"additional","affiliation":[{"name":"Ocean University of China , , ,","place":["Qingdao, PR China, 266404"]},{"name":"Qingdao Key Laboratory of Green Manufacturing of Marine Bioproducts , , ,","place":["Qingdao, PR China, 266071"]},{"name":"China National Light Industry , , ,","place":["Qingdao, PR China, 266404"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tongyan","family":"Yin","sequence":"additional","affiliation":[{"name":"Ocean University of China , , ,","place":["Qingdao, PR China, 266404"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zhuoning","family":"Cao","sequence":"additional","affiliation":[{"name":"Ocean University of China , , ,","place":["Qingdao, PR China, 266404"]},{"name":"Qingdao Key Laboratory of Green Manufacturing of Marine Bioproducts , , ,","place":["Qingdao, PR China, 266071"]},{"name":"China National Light Industry , , ,","place":["Qingdao, PR China, 266404"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4774-6873","authenticated-orcid":true,"given":"Jianan","family":"Sun","sequence":"additional","affiliation":[{"name":"Ocean University of China , , ,","place":["Qingdao, PR China, 266404"]},{"name":"Qingdao Key Laboratory of Green Manufacturing of Marine Bioproducts , , ,","place":["Qingdao, PR China, 266071"]},{"name":"China National Light Industry , , ,","place":["Qingdao, PR China, 266404"]},{"name":"Ocean University of China , , ,","place":["Sanya, China, 572025"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2939-2514","authenticated-orcid":true,"given":"Hong","family":"Jiang","sequence":"additional","affiliation":[{"name":"Ocean University of China , , ,","place":["Qingdao, PR China, 266404"]},{"name":"Qingdao Key Laboratory of Green Manufacturing of Marine Bioproducts , , ,","place":["Qingdao, PR China, 266071"]},{"name":"China National Light Industry , , ,","place":["Qingdao, PR China, 266404"]},{"name":"Ocean University of China , , ,","place":["Sanya, China, 572025"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6315-1338","authenticated-orcid":true,"given":"Xiangzhao","family":"Mao","sequence":"additional","affiliation":[{"name":"Ocean University of China , , ,","place":["Qingdao, PR China, 266404"]},{"name":"Qingdao Marine Science and Technology Center , , ,","place":["Qingdao, PR China, 266237"]},{"name":"Qingdao Key Laboratory of Green Manufacturing of Marine Bioproducts , , ,","place":["Qingdao, PR China, 266071"]},{"name":"China National Light Industry , , ,","place":["Qingdao, PR China, 266404"]},{"name":"Ocean University of China , , ,","place":["Sanya, China, 572025"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"316","published-online":{"date-parts":[[2026,9,24]]},"reference":[{"key":"2026100711074794400_cit1","doi-asserted-by":"publisher","first-page":"374","DOI":"10.1016\/j.tifs.2021.09.021","article-title":"How Far Is It from Infant Formula to Human Milk? A Look at the Human\nMilk Oligosaccharides","volume":"118","author":"Li","year":"2021","journal-title":"Trends Food Sci. Technol."},{"issue":"1","key":"2026100711074794400_cit2","doi-asserted-by":"publisher","first-page":"5010","DOI":"10.1038\/s41467-018-07476-4","article-title":"Human Milk\nOligosaccharides, Milk Microbiome and Infant Gut Microbiome Modulate\nNeonatal Rotavirus Infection","volume":"9","author":"Ramani","year":"2018","journal-title":"Nat. Commun."},{"key":"2026100711074794400_cit3","doi-asserted-by":"publisher","first-page":"118738","DOI":"10.1016\/j.carbpol.2021.118738","article-title":"Human Milk Oligosaccharides\nand Infant Gut Microbiota: Molecular Structures, Utilization Strategies\nand Immune Function","volume":"276","author":"Zhang","year":"2022","journal-title":"Carbohydr. Polym."},{"issue":"7","key":"2026100711074794400_cit4","doi-asserted-by":"publisher","first-page":"2061","DOI":"10.1021\/acs.jafc.2c00475","article-title":"Human Milk\nOligosaccharides: The\nNew Gold Standard for Premium Infant Formula","volume":"70","author":"Zhu","year":"2022","journal-title":"J. Agric. Food Chem."},{"issue":"1","key":"2026100711074794400_cit5","doi-asserted-by":"publisher","first-page":"181","DOI":"10.1080\/10408398.2020.1813683","article-title":"Human Milk Oligosaccharides\nas Bioactive Compounds in Infant Formula: Recent Advances and Trends\nin Synthetic Methods","volume":"62","author":"Pe\u0301rez-Escalante","year":"2022","journal-title":"Crit. Rev. Food Sci. Nutr."},{"key":"2026100711074794400_cit6","doi-asserted-by":"publisher","first-page":"12","DOI":"10.3389\/fimmu.2021.680911","article-title":"Human Milk Oligosaccharides: Their Effects on the Host\nand Their Potential as Therapeutic Agents","volume":"12","author":"Rousseaux","year":"2021","journal-title":"Front.\nImmunol."},{"key":"2026100711074794400_cit7","doi-asserted-by":"publisher","first-page":"108058","DOI":"10.1016\/j.biotechadv.2022.108058","article-title":"Recent Progress on\nHealth Effects and Biosynthesis of Two Key Sialylated Human Milk Oligosaccharides,\n3\u2032-Sialyllactose and 6\u2032-Sialyllactose","volume":"62","author":"Zhu","year":"2023","journal-title":"Biotechnol. Adv."},{"issue":"5","key":"2026100711074794400_cit8","doi-asserted-by":"publisher","first-page":"667","DOI":"10.1016\/j.biotechadv.2019.03.014","article-title":"Enzymatic\nand Cell Factory Approaches to the Production of Human Milk Oligosaccharides","volume":"37","author":"Faijes","year":"2019","journal-title":"Biotechnol. Adv."},{"issue":"43","key":"2026100711074794400_cit9","doi-asserted-by":"publisher","first-page":"15908","DOI":"10.1021\/acs.jafc.3c04412","article-title":"Human Milk\nOligosaccharides: A Critical Review on Structure, Preparation,\nTheir Potential as a Food Bioactive Component, and Future Perspectives","volume":"71","author":"Zhu","year":"2023","journal-title":"J. Agric. Food Chem."},{"issue":"43","key":"2026100711074794400_cit10","doi-asserted-by":"publisher","first-page":"13996","DOI":"10.1021\/acs.jafc.2c04499","article-title":"Improved Glycoqueuing\nStrategy Reveals Novel \u03912,3-Linked Di-\/Tri-Sialylated Oligosaccharide\nIsomers in Human Milk","volume":"70","author":"Jin","year":"2022","journal-title":"J. Agric. Food Chem."},{"issue":"2","key":"2026100711074794400_cit11","doi-asserted-by":"publisher","first-page":"261","DOI":"10.1016\/0008-6215(89)85070-0","article-title":"Isolation of Monosialylated Oligosaccharides\nfrom Human Milk and Structural Analysis of Three New Compounds","volume":"191","author":"Gro\u0308nberg","year":"1989","journal-title":"Carbohydr. Res."},{"issue":"1","key":"2026100711074794400_cit12","doi-asserted-by":"publisher","first-page":"52","DOI":"10.3168\/jds.S0022-0302(03)73583-8","article-title":"Sialyloligosaccharides\nin Human and Bovine Milk and in Infant Formulas: Variations with the\nProgression of Lactation","volume":"86","author":"Marti\u0301n-Sosa","year":"2003","journal-title":"J. Dairy Sci."},{"key":"2026100711074794400_cit13","doi-asserted-by":"publisher","first-page":"1","DOI":"10.1016\/j.idairyj.2018.07.015","article-title":"Lactational Changes in the Human Milk Oligosaccharide\nConcentration in Chinese and Malaysian Mothers\u2019 Milk","volume":"87","author":"Ma","year":"2018","journal-title":"Int. Dairy J."},{"issue":"3","key":"2026100711074794400_cit14","doi-asserted-by":"publisher","first-page":"383S","DOI":"10.3945\/an.111.001404","article-title":"Structure-Function Relationships\nof Human Milk Oligosaccharides","volume":"3","author":"Bode","year":"2012","journal-title":"Adv. Nutr."},{"key":"2026100711074794400_cit15","doi-asserted-by":"publisher","first-page":"113","DOI":"10.1016\/bs.accb.2015.08.002","article-title":"Human Milk\nOligosaccharides (HMOS): Structure, Function, and Enzyme-Catalyzed\nSynthesis","volume":"72","author":"Chen","year":"2015","journal-title":"Adv. Carbohydr. Chem. Biochem."},{"issue":"6","key":"2026100711074794400_cit16","doi-asserted-by":"publisher","first-page":"1402","DOI":"10.1160\/TH04-01-0055","article-title":"Inhibition of Monocyte,\nLymphocyte, and Neutrophil\nAdhesion to Endothelial Cells by Human Milk Oligosaccharides","volume":"92","author":"Bode","year":"2004","journal-title":"Thromb. Haemost."},{"issue":"4","key":"2026100711074794400_cit17","doi-asserted-by":"publisher","first-page":"820","DOI":"10.1189\/jlb.0304198","article-title":"Human Milk\nOligosaccharides Reduce Platelet-Neutrophil Complex Formation Leading\nto a Decrease in Neutrophil Beta 2 Integrin Expression","volume":"76","author":"Bode","year":"2004","journal-title":"J. Leukocyte Biol."},{"issue":"8","key":"2026100711074794400_cit18","doi-asserted-by":"publisher","DOI":"10.1111\/cmi.13340","article-title":"Burkholderia Cenocepacia\nBCAM2418-Induced\nAntibody Inhibits Bacterial Adhesion, Confers Protection to Infection\nand Enables Identification of Host Glycans as Adhesin Targets","volume":"23","author":"Pimenta","year":"2021","journal-title":"Cell. Microbiol."},{"issue":"2","key":"2026100711074794400_cit19","doi-asserted-by":"publisher","first-page":"254","DOI":"10.1021\/acsinfecdis.0c00807","article-title":"Prospecting\nHuman Milk Oligosaccharides\nas a Defense Against Viral Infections","volume":"7","author":"Moore","year":"2021","journal-title":"ACS Infect.\nDis."},{"issue":"27","key":"2026100711074794400_cit20","doi-asserted-by":"publisher","first-page":"6954","DOI":"10.1073\/pnas.1701785114","article-title":"Synthesis of Asymmetrical\nMultiantennary Human Milk Oligosaccharides","volume":"114","author":"Prudden","year":"2017","journal-title":"Proc. Natl. Acad. Sci. U. S. A."},{"key":"2026100711074794400_cit21","unstructured":"Pedersen, M.; Papadakis, M.; Becker, P.; Samain, E.; Peltier-Pain, P.; Kampmann, K. B.; Johanson, T.; Champion, E.; Dekany, G..  Oligosaccharide Production. WO 2,022,013,143 A1, 2022."},{"key":"2026100711074794400_cit22","unstructured":"Merighi, M.; Mccoy, J. M.; Heidtman, M. I..  Biosynthesis of Human\nMilk Oligosaccharides in Engineered Bacteria. WO 2,012,112,777\nA2, 2012."},{"key":"2026100711074794400_cit23","doi-asserted-by":"publisher","first-page":"107613","DOI":"10.1016\/j.biotechadv.2020.107613","article-title":"Bacterial Sialyltransferases\nand Their Use in Biocatalytic Cascades for Sialo-Oligosaccharide Production","volume":"44","author":"Schelch","year":"2020","journal-title":"Biotechnol. Adv."},{"key":"2026100711074794400_cit24","doi-asserted-by":"publisher","first-page":"119564","DOI":"10.1016\/j.carbpol.2022.119564","article-title":"Enzymatic and Chemoenzymatic Synthesis\nof Human Milk Oligosaccharides and Derivatives","volume":"291","author":"Zheng","year":"2022","journal-title":"Carbohydr. Polym."},{"issue":"5","key":"2026100711074794400_cit25","doi-asserted-by":"publisher","first-page":"2234","DOI":"10.1021\/acs.jafc.2c08436","article-title":"Biosynthesis of Human\nMilk Oligosaccharides: Enzyme Cascade and Metabolic Engineering Approaches","volume":"71","author":"Meng","year":"2023","journal-title":"J. Agric. Food Chem."},{"issue":"33","key":"2026100711074794400_cit26","doi-asserted-by":"publisher","first-page":"12390","DOI":"10.1021\/acs.jafc.3c02895","article-title":"An Overview\nof Sugar Nucleotide-Dependent Glycosyltransferases for Human Milk\nOligosaccharide Synthesis","volume":"71","author":"Zhao","year":"2023","journal-title":"J. Agric. Food Chem."},{"issue":"2","key":"2026100711074794400_cit27","doi-asserted-by":"publisher","first-page":"1178","DOI":"10.1021\/acs.jafc.3c07604","article-title":"Rational Design\nof an \u03b1-1,3-Fucosyltransferase for the Biosynthesis\nof 3-Fucosyllactose in Bacillus Subtilis ATCC 6051a via De Novo GDP-l-Fucose\nPathway","volume":"72","author":"Xie","year":"2024","journal-title":"J. Agric. Food Chem."},{"issue":"7","key":"2026100711074794400_cit28","doi-asserted-by":"publisher","first-page":"1232","DOI":"10.1021\/cb300125k","article-title":"A Sialyltransferase\nMutant with Decreased Donor Hydrolysis and Reduced Sialidase Activities\nfor Directly Sialylating Lewisx","volume":"7","author":"Sugiarto","year":"2012","journal-title":"ACS Chem. Biol."},{"issue":"153","key":"2026100711074794400_cit29","doi-asserted-by":"publisher","first-page":"11","DOI":"10.1016\/j.procbio.2025.03.002","article-title":"Improving 3\u2032-Sialyllactose\nBiosynthesis in Escherichia Coli by Engineering Neisseria Meningitidis 406Y \u03b12,3-Sialyltransferase","volume":"153","author":"Lin","year":"2025","journal-title":"Process Biochem."},{"issue":"3","key":"2026100711074794400_cit30","doi-asserted-by":"publisher","first-page":"387","DOI":"10.1093\/glycob\/cwq172","article-title":"Cloning and Characterization of a\nViral \u03912\u22123-Sialyltransferase (vST3Gal-I) for the Synthesis\nof Sialyl Lewisx","volume":"21","author":"Sugiarto","year":"2011","journal-title":"Glycobiology"},{"issue":"1","key":"2026100711074794400_cit31","doi-asserted-by":"publisher","first-page":"2455","DOI":"10.1038\/s41467-022-29867-4","article-title":"Elucidating Human\nMilk Oligosaccharide Biosynthetic Genes through Network-Based Multi-Omics\nIntegration","volume":"13","author":"Kellman","year":"2022","journal-title":"Nat. Commun."},{"issue":"11","key":"2026100711074794400_cit32","doi-asserted-by":"publisher","first-page":"3021","DOI":"10.1039\/C1MB05182B","article-title":"Decreasing\nthe Sialidase Activity of Multifunctional Pasteurella\nMultocida \u03912\u22123-Sialyltransferase 1 (PmST1) by Site-Directed\nMutagenesis","volume":"7","author":"Sugiarto","year":"2011","journal-title":"Mol. BioSyst."},{"issue":"2","key":"2026100711074794400_cit33","doi-asserted-by":"publisher","first-page":"159","DOI":"10.1093\/glycob\/cwt092","article-title":"Protein\nEngineering\nof \u03912,3\/2,6-Sialyltransferase to Improve the Yield and Productivity\nof in Vitro Sialyllactose Synthesis","volume":"24","author":"Choi","year":"2014","journal-title":"Glycobiology"},{"issue":"33","key":"2026100711074794400_cit34","doi-asserted-by":"publisher","first-page":"20547","DOI":"10.1021\/acs.jafc.5c03673","article-title":"An Overview of Industrial Enzyme\nEngineering to Achieve a pH Adaptability-Activity Trade-off: Current\nStatus and Future Perspectives","volume":"73","author":"Hu","year":"2025","journal-title":"J. Agric. Food\nChem."},{"issue":"33","key":"2026100711074794400_cit35","doi-asserted-by":"publisher","first-page":"20600","DOI":"10.1021\/acs.jafc.5c05979","article-title":"Computer-Aided Techniques in the Engineering of Enzyme\nBinding Pockets:\nNew Perspectives and Frontiers","volume":"73","author":"Xie","year":"2025","journal-title":"J. Agric. Food\nChem."},{"issue":"1","key":"2026100711074794400_cit36","doi-asserted-by":"publisher","first-page":"211","DOI":"10.1021\/acs.jafc.5c13923","article-title":"Leveraging\nAlphaFold for Rational Redesign of Glycosyltransferases in Long-Chain\nHuman Milk Oligosaccharide Biosynthesis","volume":"74","author":"Luo","year":"2026","journal-title":"J.\nAgric. Food Chem."},{"key":"2026100711074794400_cit37","doi-asserted-by":"publisher","first-page":"132081","DOI":"10.1016\/j.ijbiomac.2024.132081","article-title":"Highly Efficient Biosynthesis of\n3\u2032-Sialyllactose in Engineered\nEscherichia Coli","volume":"269","author":"Zhang","year":"2024","journal-title":"Int. J. Biol. Macromol."},{"issue":"W1","key":"2026100711074794400_cit38","doi-asserted-by":"publisher","first-page":"W132","DOI":"10.1093\/nar\/gkaf399","article-title":"Caver Web 2.0: Analysis of Tunnels\nand Ligand Transport\nin Dynamic Ensembles of Proteins","volume":"53","author":"Marques","year":"2025","journal-title":"Nucleic Acids\nRes."},{"issue":"50","key":"2026100711074794400_cit39","doi-asserted-by":"publisher","first-page":"17618","DOI":"10.1021\/ja0561690","article-title":"A Multifunctional\nPasteurella Multocida Sialyltransferase: A Powerful Tool for the Synthesis\nof Sialoside Libraries","volume":"127","author":"Yu","year":"2005","journal-title":"J. Am. Chem. Soc."},{"key":"2026100711074794400_cit40","article-title":"Chai-1: Decoding the Molecular Interactions of Life","author":"Discovery Team","year":"2024","journal-title":"bioRxiv"},{"issue":"12","key":"2026100711074794400_cit41","doi-asserted-by":"publisher","first-page":"355","DOI":"10.1007\/s00894-019-4232-z","article-title":"Toward a Better Numerical Simulation\nFramework","volume":"25","author":"Rakhshani","year":"2019","journal-title":"J. Mol. Model"},{"key":"2026100711074794400_cit42","doi-asserted-by":"publisher","first-page":"116","DOI":"10.1016\/j.jbiotec.2015.09.031","article-title":"A Semi-Multifunctional\nSialyltransferase from Bibersteinia Trehalosi and\nIts Comparison to the Pasteurella Multocida ST1Mutants","volume":"216","author":"Talafova\u0301","year":"2015","journal-title":"J. Biotechnol."},{"issue":"2","key":"2026100711074794400_cit43","doi-asserted-by":"publisher","first-page":"340","DOI":"10.1016\/j.bbrc.2007.10.201","article-title":"Conserved Amino Acid\nSequences in\nthe Bacterial Sialyltransferases Belonging to Glycosyltransferase\nFamily 80","volume":"365","author":"Yamamoto","year":"2008","journal-title":"Biochem. Biophys. Res. Commun."},{"issue":"21","key":"2026100711074794400_cit44","doi-asserted-by":"publisher","first-page":"6288","DOI":"10.1021\/bi700346w","article-title":"Crystal\nStructures of Pasteurella Multocida Sialyltransferase Complexes with\nAcceptor and Donor Analogues Reveal Substrate Binding Sites and Catalytic\nMechanism","volume":"46","author":"Ni","year":"2007","journal-title":"Biochemistry"},{"issue":"1","key":"2026100711074794400_cit45","doi-asserted-by":"publisher","first-page":"158","DOI":"10.1021\/acscatal.2c04735","article-title":"Reshaping Substrate-Binding\nPocket of Leucine Dehydrogenase\nfor Bidirectionally Accessing Structurally Diverse Substrates","volume":"13","author":"Wu","year":"2023","journal-title":"ACS Catal."},{"issue":"10","key":"2026100711074794400_cit46","doi-asserted-by":"publisher","DOI":"10.1126\/sciadv.aaw8451","article-title":"Directed Evolution\nof an \u03911,3-Fucosyltransferase Using a Single-Cell Ultrahigh-Throughput\nScreening Method","volume":"5","author":"Tan","year":"2019","journal-title":"Sci. Adv."},{"issue":"20","key":"2026100711074794400_cit47","doi-asserted-by":"publisher","first-page":"12800","DOI":"10.1021\/acscatal.2c02258","article-title":"Flexibility\nRegulation of Loops Surrounding the Tunnel Entrance in Cytochrome\nP450 Enhanced Substrate Access Substantially","volume":"12","author":"Li","year":"2022","journal-title":"ACS Catal."},{"key":"2026100711074794400_cit48","doi-asserted-by":"publisher","first-page":"134","DOI":"10.1016\/j.aca.2020.05.051","article-title":"Recent Advances in the Mass Spectrometric\nAnalysis of Glycosphingolipidome\n\u2212 a Review","volume":"1132","author":"Barrientos","year":"2020","journal-title":"Anal. Chim. Acta"},{"key":"2026100711074794400_cit49","doi-asserted-by":"publisher","first-page":"7537","DOI":"10.1021\/acscatal.6c00035","article-title":"Computer-Aided\nDirected Evolution Achieves Balanced\nActivity, Thermal Stability, and Selectivity in Stereoselective Carbonyl\nReductase","volume":"16","author":"Gu","year":"2026","journal-title":"ACS Catal."},{"issue":"24","key":"2026100711074794400_cit50","doi-asserted-by":"publisher","first-page":"15245","DOI":"10.1021\/acs.jafc.5c04205","article-title":"Rational Design of\nAlginate Lyase\nAlgL3199 for Thermal Stability and Specific Activity Enhancement","volume":"73","author":"Chen","year":"2025","journal-title":"J. Agric. 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