Synthesising human milk oligosaccharide using biotechnology-driven enzymes

Abstract

Human milk oligosaccharides (HMOs) are a crucial ingredient in mother’s milk for infant health. These are a structurally diverse group of soluble bioactive glycans having antiviral and anti-bacterial properties and promote the growth of probiotics, particularly Bifidobacteria. They modulate the immune system and are important for infant’s brain development. Not all infants are fortunate enough to receive mother milk, and rely on industrially produced infant formulas which lack HMOs. Thus, synthesizing HMOs through different methods is becoming more appealing form an industrial perspective. Despite huge efforts to obtain predominant HMOs through chemical, chemo-enzymatic, enzymatic, and whole-cell biotransformation of microorganisms, a limited number of HMOs could be produced in sufficient quantities due to lack of canonical enzymes that can add different metabolic pathways together to produce several HMO. Particularly, lacto-N-neotetraose and 2′-fucosyllactose are being added to infant formula and are well-tolerated by infants. Amide other methods, enzymatic and whole-cell biotransformation of microorganisms are promising approaches, but it requires intervention of innovative methodologies of synthetic biology. This review highlights innovative approaches, such as using plant system, and employing synthetic biology including redesigning of enzyme structure for producing HMOs at the industrial level. An interesting approach for synthesizing whole milk components in mammary cell lines is also mentioned.

Keywords

Human milk oligosaccharides / Infant health / Enzymatic synthesis / Whole-cell biotransformation / Synthesis in plants / Biological Sciences / Biochemistry and Cell Biology

References

[1] SaumonneauA, et al. . Design of an alpha-L-transfucosidase for the synthesis of fucosylated HMOs. Glycobiology, 2016, 26: 261-269

[2] ZivkovicAM, GermanJB, LebrillaCB, MillsDA. Human milk glycobiome and its impact on the infant gastrointestinal microbiota. Proc Natl Acad Sci USA, 2011, 108Suppl 14653-4658.

[3] Jantscher-KrennE, von SchirndingL, TrotzmullerM, KofelerH, KurtovicU, FluhrH, MullerA, BagciS. Human milk oligosaccharides are present in amniotic fluid and show specific patterns dependent on gestational age. Nutrients, 2022.

[4] WiseA, RobertsonB, ChoudhuryB, RautavaS, IsolauriE, SalminenS, BodeL. Infants are exposed to human milk oligosaccharides already in utero. Front Pediatr, 2018, 6: 270.

[5] AagaardK, MaJ, AntonyKM, GanuR, PetrosinoJ, VersalovicJ. The placenta harbors a unique microbiome. Sci Translat Med, 2014, 6: 237ra265

[6] ColladoMC, RautavaS, AakkoJ, IsolauriE, SalminenS. Human gut colonisation may be initiated in utero by distinct microbial communities in the placenta and amniotic fluid. Sci Rep, 2016, 6: 23129.

[7] AckermanDL, DosterRS, WeitkampJH, AronoffDM, GaddyJA, TownsendSD. Human milk oligosaccharides exhibit antimicrobial and antibiofilm properties against Group B Streptococcus. ACS Infect Dis, 2017, 3: 595-605.

[8] BondueP, MilaniC, ArnouldE, VenturaM, DaubeG, LaPointeG, DelcenserieV. Bifidobacterium mongoliense genome seems particularly adapted to milk oligosaccharide digestion leading to production of antivirulent metabolites. BMC Microbiol, 2020, 20: 111.

[9] WuS, TaoN, GermanJB, GrimmR, LebrillaCB. Development of an annotated library of neutral human milk oligosaccharides. J Proteome Res, 2010, 9: 4138-4151.

[10] NewburgDS, TanritanirAC, ChakrabartiS. Lactodifucotetraose, a human milk oligosaccharide, attenuates platelet function and inflammatory cytokine release. J Thromb Thrombolysis, 2016, 42: 46-55.

[11] CarrLE, VirmaniMD, RosaF, MunblitD, MatazelKS, ElolimyAA, YeruvaL. Role of human milk bioactives on infants’ gut and immune health. Front Immunol, 2021, 12: 604080.

[12] SuligojT, VigsnaesLK, AbbeelePVD, ApostolouA, KaralisK, SavvaGM, McConnellB, JugeN. Effects of human milk oligosaccharides on the adult gut microbiota and barrier function. Nutrients, 2020.

[13] ZhuB, et al. . Immune modulation by Lacto-N-fucopentaose III in experimental autoimmune encephalomyelitis. Clin Immunol, 2012, 142: 351-361.

[14] MaessenSE, DerraikJGB, BiniaA, CutfieldWS. Perspective: human milk oligosaccharides: fuel for childhood obesity prevention?. Adv Nutr, 2020, 11: 35-40.

[15] GeorgiG, BartkeN, WiensF, StahlB. Functional glycans and glycoconjugates in human milk. Am J Clin Nutr, 2013, 98: 578S-585S.

[16] WangB, Brand-MillerJ. The role and potential of sialic acid in human nutrition. Eur J Clin Nutr, 2003, 57: 1351-1369.

[17] WangB, Brand-MillerJ, McVeaghP, PetoczP. Concentration and distribution of sialic acid in human milk and infant formulas. Am J Clin Nutr, 2001, 74: 510-515.

[18] LvY, et al. . Sialic acid in human milk and infant formulas in China: concentration, distribution and type. Brit J Nutrit, 2024, 13191-7.

[19] Al-KhafajiAH, JepsenSD, ChristensenKR, VigsnæsLK. The potential of human milk oligosaccharides to impact the microbiota-gut-brain axis through modulation of the gut microbiota. J Funct Foods, 2020, 74: 104176.

[20] VazquezE, BarrancoA, RamirezM, GruartA, Delgado-GarciaJM, JimenezML, BuckR, RuedaR. Dietary 2′-Fucosyllactose enhances operant conditioning and long-term potentiation via gut-brain communication through the vagus nerve in rodents. PLoS ONE, 2016, 11: e0166070.

[21] PessentheinerAR, et al. . The human milk oligosaccharide 3′sialyllactose reduces low-grade inflammation and atherosclerosis development in mice. JCI insight, 2024.

[22] KellmanBP, et al. . Elucidating human milk oligosaccharide biosynthetic genes through network-based multi-omics integration. Nat Commun, 2022, 13: 2455.

[23] TurckD, et al. . Safety of the extension of use of 2′-fucosyllactose (2′-FL) as a novel food pursuant to Regulation (EU) 2015/2283. EFSA J, 2023, 13: 4184

[24] SinghRP, NiharikaJ, KondepudiKK, BishnoiM, TingirikariJMR. Recent understanding of human milk oligosaccharides in establishing infant gut microbiome and roles in immune system. Food Res Int, 2022, 151: 110884.

[25] NinonuevoMR, et al. . A strategy for annotating the human milk glycome. J Agric Food Chem, 2006, 54: 7471-7480.

[26] KostopoulosI, et al. . Akkermansia muciniphila uses human milk oligosaccharides to thrive in the early life conditions in vitro. Sci Rep, 2020, 10: 14330.

[27] GarádiZ, DancsóA, PiskarevV, BéniS. From mother's milk to structural insights: 1H–15N NMR analysis of Lewis X antigen-bearing oligosaccharides isolated from human milk. Carbohyd Polym, 2025, 347: 122534.

[28] Plaza-DiazJ, FontanaL, GilA. Human milk oligosaccharides and immune system development. Nutrients, 2018, 10: 1038.

[29] ChenX. Human milk oligosaccharides (HMOS): structure, function, and enzyme-catalyzed synthesis. Adv Carbohydr Chem Biochem, 2015, 72: 113-190.

[30] PachecoAR, BarileD, UnderwoodMA, MillsDA. The impact of the milk glycobiome on the neonate gut microbiota. Annual Rev Anim Biosci, 2015, 3: 419-445.

[31] SmilowitzJT, LebrillaCB, MillsDA, GermanJB, FreemanSL. Breast milk oligosaccharides: structure-function relationships in the neonate. Annu Rev Nutr, 2014, 34: 143-169.

[32] XunY, et al. . Profile of twenty-three human milk oligosaccharides in han chinese mothers throughout postpartum 1 year. J Food Qual, 2022, 2022: 6230832.

[33] SeferovicMD, MohammadM, PaceRM, EngevikM, VersalovicJ, BodeL, HaymondM, AagaardKM. Maternal diet alters human milk oligosaccharide composition with implications for the milk metagenome. Sci Rep, 2020, 10: 22092.

[34] ThurlS, HenkerJ, SiegelM, TovarK, SawatzkiG. Detection of four human milk groups with respect to Lewis blood group dependent oligosaccharides. Glycoconj J, 1997, 14: 795-799.

[35] MarinoK, LaneJA, AbrahamsJL, StruweWB, HarveyDJ, MarottaM, HickeyRM, RuddPM. Method for milk oligosaccharide profiling by 2-aminobenzamide labeling and hydrophilic interaction chromatography. Glycobiology, 2011, 21: 1317-1330.

[36] FongB, MaK, McJarrowP. Quantification of bovine milk oligosaccharides using liquid chromatography-selected reaction monitoring-mass spectrometry. J Agric Food Chem, 2011, 59: 9788-9795.

[37] ChatziioannouAC, BenjaminsE, PellisL, HaandrikmanA, DijkhuizenL, van LeeuwenSS. Extraction and quantitative analysis of goat milk oligosaccharides: composition, variation, associations, and 2′-FL variability. J Agric Food Chem, 2021, 69: 7851-7862.

[38] SprengerN, OdenwaldH, KukkonenAK, KuitunenM, SavilahtiE, KunzC. FUT2-dependent breast milk oligosaccharides and allergy at 2 and 5 years of age in infants with high hereditary allergy risk. Eur J Nutr, 2017, 56: 1293-1301.

[39] BandaraMD, StineKJ, DemchenkoAV. The chemical synthesis of human milk oligosaccharides: Lacto-N-tetraose (Galbeta1–>3GlcNAcbeta1–>3Galbeta1–>4Glc). Carbohyd Res, 2019, 486: 107824.

[40] BandaraMD, StineKJ, DemchenkoAV. Chemical Synthesis of Human Milk Oligosaccharides: Lacto-N-hexaose Galbeta1–>3GlcNAcbeta1–>3 [Galbeta1–>4GlcNAcbeta1–>6] Galbeta1–>4Glc. J Org Chem, 2019, 84: 16192-16198.

[41] BandaraMD, StineKJ, DemchenkoAV. Chemical synthesis of human milk oligosaccharides: lacto-N-neohexaose (Galbeta1 –> 4GlcNAcbeta1–>)(2) 3,6Galbeta1 –> 4Glc. Org Biomol Chem, 2020, 18: 1747-1753.

[42] Fernandez-MayoralasA, Martin-LomasM. Synthesis of 3- and 2′-fucosyl-lactose and 3,2′-difucosyl-lactose from partially benzylated lactose derivatives. Carbohyd Res, 1986, 154: 93-101.

[43] Arboe JennumC, Hauch FengerT, BruunLM, MadsenR. One-pot glycosylations in the synthesis of human milk oligosaccharides. european journal of organic chemistry. Eur J Org Chem, 2014, 2014: 3232-3241.

[44] LoveKR, SeebergerPH. Solution syntheses of protected type II Lewis blood group oligosaccharides: study for automated synthesis. J Org Chem, 2005, 70: 3168-3177.

[45] BoonsGJ, DemchenkoAV. Recent advances in o-sialylation. Chem Rev, 2000, 100: 4539-4566.

[46] EspositoD, HurevichM, CastagnerB, WangCC, SeebergerPH. Automated synthesis of sialylated oligosaccharides. Beilstein J Org Chem, 2012, 8: 1601-1609.

[47] HsuCH, HungSC, WuCY, WongCH. Toward automated oligosaccharide synthesis. Angew Chem Int Ed Engl, 2011, 50: 11872-11923.

[48] SinghY, EscopyS, ShadrickM, BandaraMD, StineKJ, DemchenkoAV. Chemical synthesis of human milk oligosaccharides: para-Lacto-N-hexaose and para-Lacto-N-neohexaose. Chemistry, 2023, 29: e202302288.

[49] XiaoZ, et al. . Chemoenzymatic synthesis of a library of human milk oligosaccharides. J Org Chem, 2016, 81: 5851-5865.

[50] WuY, SunY, PeiC, PengX, LiuX, QianEW, DuY, LiJJ. Automated chemoenzymatic modular synthesis of human milk oligosaccharides on a digital microfluidic platform. RSC Adv, 2024, 14: 17397-17405.

[51] ZhengJ, XuH, FangJ, ZhangX. Enzymatic and chemoenzymatic synthesis of human milk oligosaccharides and derivatives. Carbohydr Polym, 2022, 291: 119564.

[52] FlitschSLCarreiraEM, YamamotoH. 7.21 Enzymatic Carbohydrate Synthesis. Comprehensive chirality, 2012AmsterdamElsevier454-464.

[53] ZeunerB, TezeD, MuschiolJ, MeyerAS. Synthesis of human milk oligosaccharides: protein engineering strategies for improved enzymatic transglycosylation. Molecules, 2019, 24: 2033.

[54] MurataT, UsuiT. Enzymatic synthesis of oligosaccharides and neoglycoconjugates. Biosci Biotechnol Biochem, 2006, 70: 1049-1059.

[55] WeijersCA, FranssenMC, VisserGM. Glycosyltransferase-catalyzed synthesis of bioactive oligosaccharides. Biotechnol Adv, 2008, 26: 436-456.

[56] PeruginoG, TrinconeA, RossiM, MoracciM. Oligosaccharide synthesis by glycosynthases. Trends Biotechnol, 2004, 22: 31-37.

[57] SinghRP, et al. . Utilization of dietary mixed-linkage b-glucans by the Firmicute Blautia producta. J Biol Chem, 2023, 299: 104806.

[58] LiA, BenkouloucheM, LadevezeS, DurandJ, CiociG, LavilleE, Potocki-VeroneseG. Discovery and biotechnological exploitation of glycoside-phosphorylases. Int J Mol Sci, 2022, 23: 3043.

[59] PuchartV. Glycoside phosphorylases: structure, catalytic properties and biotechnological potential. Biotechnol Adv, 2015, 33: 261-276.

[60] O'NeillEC, PergolizziG, StevensonCEM, LawsonDM, NepogodievSA, FieldRA. Cellodextrin phosphorylase from Ruminiclostridium thermocellum: X-ray crystal structure and substrate specificity analysis. Carbohyd Res, 2017, 451: 118-132.

[61] SinghRP, PergolizziG, NepogodievSA, de AndradeP, KuhaudomlarpS, FieldRA. Preparative and kinetic analysis of beta-1,4- and beta-1,3-glucan phosphorylases informs access to human milk oligosaccharide fragments and analogues thereof. Chembiochem : Euro J Chem Biol, 2020, 21: 1043-1049.

[62] NishimotoM. Large scale production of lacto-N-biose I, a building block of type I human milk oligosaccharides, using sugar phosphorylases. Biosci Biotechnol Biochem, 2020, 84: 17-24.

[63] MuschiolJ, MeyerAS. A chemo-enzymatic approach for the synthesis of human milk oligosaccharide backbone structures. Z Naturforsch C J Biosci, 2019, 74: 85-89.

[64] LiY, et al. . Enzymatic modular synthesis of asymmetrically branched human milk oligosaccharides. Carbohydr Polym, 2024, 333: 121908.

[65] PruddenAR, et al. . Synthesis of asymmetrical multiantennary human milk oligosaccharides. Proc Natl Acad Sci USA, 2017, 114: 6954-6959.

[66] HeinzlerR, FischöderT, EllingL, FranzrebM. Toward automated enzymatic glycan synthesis in a compartmented flow microreactor system. Adv Synth Catal, 2019, 361: 4506-4516.

[67] FarhadihosseinabadiB, et al. . The in vivo effect of Lacto-N-neotetraose (LNnT) on the expression of type 2 immune response involved genes in the wound healing process. Sci Rep, 2020, 10: 997.

[68] NishimotoM, KitaokaM. Practical preparation of lacto-N-biose I, a candidate for the bifidus factor in human milk. Biosci Biotechnol Biochem, 2007, 71: 2101-2104.

[69] YuH, et al. . Highly efficient chemoenzymatic synthesis of β1-3-linked galactosides. Chem Commun (Camb), 2010, 46: 7507-7509.

[70] VetereA, MiletichM, BoscoM, PaolettiS. Regiospecific glycosidase-assisted synthesis of lacto-N-biose I (Galbeta1-3GlcNAc) and 3'-sialyl-lacto-N-biose I (NeuAcalpha2-3Galbeta1-3GlcNAc). Eur J Biochem, 2000, 267: 942-949.

[71] BayónC, CortésÁ, BerenguerJ, HernáizMJ. Highly efficient enzymatic synthesis of Galβ-(1→3)-GalNAc and Galβ-(1→3)-GlcNAc in ionic liquids. Tetrahedron, 2013, 69: 4973-4978.

[72] LiY, et al. . Donor substrate promiscuity of bacterial β1-3-N-acetylglucosaminyltransferases and acceptor substrate flexibility of β1-4-galactosyltransferases. Bioorg Med Chem, 2016, 24: 1696-1705.

[73] YuH, et al. . Synthetic disialyl hexasaccharides protect neonatal rats from necrotizing enterocolitis. Angew Chem Int Ed Engl, 2014, 53: 6687-6691.

[74] LiuYH, WangL, HuangP, JiangZQ, YanQJ, YangSQ. Efficient sequential synthesis of lacto-N-triose II and lacto-N-neotetraose by a novel beta-N-acetylhexosaminidase from Tyzzerella nexilis. Food Chem, 2020, 332: 127438.

[75] LiuY, MaJ, ShiR, LiT, YanQ, JiangZ, YangS. Biochemical characterization of a β-N-acetylhexosaminidase from Catenibacterium mitsuokai suitable for the synthesis of lacto-N-triose II. Process Biochem, 2021, 102: 360-368.

[76] NekvasilovaP, HovorkovaM, MeszarosZ, PetraskovaL, PelantovaH, KrenV, SlamovaK, BojarovaP. Engineered glycosidases for the synthesis of analogs of human milk oligosaccharides. Int J Mol Sci, 2022, 23: 4106.

[77] LiuXW, XiaC, LiL, GuanWY, PettitN, ZhangHC, ChenM, WangPG. Characterization and synthetic application of a novel beta1,3-galactosyltransferase from Escherichia coli O55:H7. Bioorg Med Chem, 2009, 17: 4910-4915.

[78] McArthurJB, YuH, ChenX. A bacterial β1–3-galactosyltransferase enables multigram-scale synthesis of human milk lacto-N-tetraose (LNT) and its fucosides. ACS Catal, 2019, 91210721-10726.

[79] MiyazakiT, SatoT, FurukawaK, AjisakaK. Enzymatic synthesis of lacto-N-difucohexaose I which binds to Helicobacter pylori. Methods Enzymol, 2010, 480: 511-524.

[80] ZhuY, LuoG, LiZ, ZhangP, ZhangW, MuW. Efficient biosynthesis of lacto-N-neotetraose by a novel beta-1,4-galactosyltransferase from Aggregatibacter actinomycetemcomitans NUM4039. Enzyme Microb Technol, 2022, 153: 109912.

[81] SakanakaM, et al. . Evolutionary adaptation in fucosyllactose uptake systems supports bifidobacteria-infant symbiosis. Sci Adv, 2019.

[82] AlbermannC, PiepersbergW, WehmeierUF. Synthesis of the milk oligosaccharide 2′-fucosyllactose using recombinant bacterial enzymes. Carbohyd Res, 2001, 334: 97-103.

[83] EngelsL, EllingL. WbgL: a novel bacterial α1,2-fucosyltransferase for the synthesis of 2′-fucosyllactose. Glycobiology, 2014, 24: 170-178.

[84] ZhouW, JiangH, LiangX, QiuY, WangL, MaoX. Discovery and characterization of a novel α-l-fucosidase from the marine-derived Flavobacterium algicola and its application in 2′-fucosyllactose production. Food Chem, 2022, 369: 130942.

[85] LezykM, JersC, KjaerulffL, GotfredsenCH, MikkelsenMD, MikkelsenJD. Novel alpha-L-fucosidases from a soil metagenome for production of fucosylated human milk oligosaccharides. PLoS ONE, 2016, 11: e0147438.

[86] Escamilla-LozanoY, Guzmán-RodríguezF, Alatorre-SantamaríaS, García-GaribayM, Gómez-RuizL, Rodríguez-SerranoG, Cruz-GuerreroA. Synthesis of fucosyl-oligosaccharides using α-L-fucosidase from Lactobacillus rhamnosus GG. Molecules, 2019, 24132402.

[87] ShiR, MaJ, YanQ, YangS, FanZ, JiangZ. Biochemical characterization of a novel alpha-L-fucosidase from Pedobacter sp. and its application in synthesis of 3′-fucosyllactose and 2′-fucosyllactose. Appl Microbiol Biotechnol, 2020, 104: 5813-5826.

[88] LiL, et al. . Efficient chemoenzymatic synthesis of novel galacto-N-biose derivatives and their sialylated forms. Chem Commun, 2015, 515110310-10313.

[89] BaiJ, et al. . Biochemical characterization of Helicobacter pylori α1-3-fucosyltransferase and its application in the synthesis of fucosylated human milk oligosaccharides. Carbohydr Res, 2019, 480: 1-6.

[90] HolckJ, et al. . Enzyme catalysed production of sialylated human milk oligosaccharides and galactooligosaccharides by Trypanosoma cruzi trans-sialidase. New Biotechnol, 2014, 31: 156-165.

[91] LiZ, NiZ, ChenX, WangG, WuJ, YaoJ. Multi-enzymatic cascade one-pot biosynthesis of 3′-sialyllactose using engineered Escherichiacoli. Molecules, 2020, 25: 3567.

[92] GuoL, ChenX, XuL, XiaoM, LuL. Enzymatic synthesis of 6′-sialyllactose, a dominant sialylated human milk oligosaccharide, by a novel exo-alpha-sialidase from bacteroides fragilis NCTC9343. Appl Environ Microbiol, 2018.

[93] LiuJJ, et al. . Biosynthesis of a functional human milk oligosaccharide, 2′-Fucosyllactose, and l-Fucose using engineered Saccharomycescerevisiae. ACS Synth Biol, 2018, 7: 2529-2536.

[94] LiL, KimSA, HeoJE, KimTJ, SeoJH, HanNS. One-pot synthesis of GDP-l-fucose by a four-enzyme cascade expressed in Lactococcus lactis. J Biotechnol, 2017, 264: 1-7.

[95] LiZ, RinasU. Recombinant protein production associated growth inhibition results mainly from transcription and not from translation. Microb Cell Fact, 2020, 19: 1-11.

[96] TaoM, YangL, ZhaoC, ZhangW, ZhuY, MuW. Efficient biosynthesis of Lacto-N-Biose I, a building block of type i human milk oligosaccharides, by a metabolically engineered Escherichiacoli. J Agric Food Chem, 2024, 72: 5860-5866.

[97] PriemB, GilbertM, WakarchukWW, HeyraudA, SamainE. A new fermentation process allows large-scale production of human milk oligosaccharides by metabolically engineered bacteria. Glycobiology, 2002, 12: 235-240.

[98] BaumgartnerF, ConradJ, SprengerGA, AlbermannC. Synthesis of the human milk oligosaccharide lacto-N-tetraose in metabolically engineered, plasmid-free E. coli. Chembiochem: Euro J Chem Biol, 2014, 15: 1896-1900.

[99] BaumgartnerF, JurzitzaL, ConradJ, BeifussU, SprengerGA, AlbermannC. Synthesis of fucosylated lacto-N-tetraose using whole-cell biotransformation. Bioorg Med Chem, 2015, 23: 6799-6806.

[100] HuD, WuH, ZhuY, ZhangW, MuW. Engineering Escherichia coli for highly efficient production of lacto-N-triose II from N-acetylglucosamine, the monomer of chitin. Biotechnol Biofuels, 2021, 14: 198.

[101] LiuY, YanQ, MaJ, YangS, LiT, JiangZ. Production of lacto-N-triose II and lacto-N-neotetraose from chitin by a novel β-N-acetylhexosaminidase expressed in Pichiapastoris. ACS Sustain Chem Eng, 2020, 8: 15466-15474.

[102] ZhuY, WanL, MengJ, LuoG, ChenG, WuH, ZhangW, MuW. Metabolic engineering of Escherichia coli for lacto-N-triose II production with high productivity. J Agric Food Chem, 2021, 69: 3702-3711.

[103] ZhuY, LiZ, LuoG, WuH, ZhangW, MuW. Metabolic engineering of escherichia coli for efficient biosynthesis of Lacto-N-tetraose Using a Novel β-1,3-Galactosyltransferase from Pseudogulbenkiania ferrooxidans. J Agric Food Chem, 2021, 69: 11342-11349.

[104] LiZ, ZhuY, ZhangP, ZhangW, MuW. Pathway optimization and Uridine 5′-Triphosphate regeneration for enhancing Lacto-N-Tetraose biosynthesis in engineered Escherichiacoli. J Agric Food Chem, 2022, 70: 7727-7735.

[105] HuM, LiM, MiaoM, ZhangT. Engineering Escherichiacoli for the High-Titer Biosynthesis of Lacto-N-tetraose. J Agric Food Chem, 2022, 70: 8704-8712.

[106] DongX, LiN, LiuZ, LvX, LiJ, DuG, WangM, LiuL. Modular pathway engineering of key precursor supply pathways for lacto-N-neotetraose production in Bacillus subtilis. Biotechnol Biofuels, 2019, 12: 212.

[107] DongX, et al. . CRISPRi-guided multiplexed fine-tuning of metabolic flux for enhanced Lacto-N-neotetraose Production in Bacillussubtilis. J Agric Food Chem, 2020, 68: 2477-2484.

[108] ZhangP, ZhuY, LiZ, ZhangW, GuangC, MuW. Designing a highly efficient biosynthetic route for Lacto-N-Neotetraose production in Escherichiacoli. J Agric Food Chem, 2022, 70: 9961-9968.

[109] ZhangM, et al. . High-level production of Lacto-N-neotetraose in Escherichiacoli by stepwise optimization of the biosynthetic pathway. J Agric Food Chem, 2023, 71: 16212-16220.

[110] TaoM, YangL, ZhaoC, ZhaoM, ZhangW, ZhuY, MuW. Implementation of a quorum-sensing system for highly efficient biosynthesis of lacto-n-neotetraose in engineered Escherichiacoli MG1655. J Agric Food Chem, 2024, 72: 7179-7186.

[111] LiaoY, et al. . High-yield synthesis of Lacto-N-Neotetraose from glycerol and glucose in engineered Escherichiacoli. J Agric Food Chem, 2024, 72: 5325-5338.

[112] JungSM, ChinYW, LeeYG, SeoJH. Enhanced production of 2'-fucosyllactose from fucose by elimination of rhamnose isomerase and arabinose isomerase in engineered Escherichiacoli. Biotechnol Bioeng, 2019, 116: 2412-2417.

[113] ChinYW, KimJY, KimJH, JungSM, SeoJH. Improved production of 2′-fucosyllactose in engineered Escherichiacoli by expressing putative alpha-1,2-fucosyltransferase, WcfB from Bacteroidesfragilis. J Biotechnol, 2017, 257: 192-198.

[114] NiZ, LiZ, WuJ, GeY, LiaoY, YuanL, ChenX, YaoJ. Multi-Path Optimization for efficient production of 2′-Fucosyllactose in an engineered Escherichiacoli C41 (DE3) Derivative. Front Bioeng Biotechnol, 2020, 8: 611900.

[115] YuS, LiuJJ, YunEJ, KwakS, KimKH, JinYS. Production of a human milk oligosaccharide 2′-fucosyllactose by metabolically engineered Saccharomycescerevisiae. Microb Cell Fact, 2018, 17: 101.

[116] DengJ, et al. . Engineering the substrate transport and cofactor regeneration systems for enhancing 2′-Fucosyllactose synthesis in Bacillussubtilis. ACS Synth Biol, 2019, 8: 2418-2427.

[117] Seo J.-H, Young-Wook C, Hae-Yong J (2018) Method of producing 2′-fucosyllactose using Corynebacterium glutamicumed. Google Patents

[118] ChenR, ZhuY, WangH, LiuY, MengJ, ChenY, MuW. Engineering Escherichiacoli MG1655 for Highly efficient biosynthesis of 2′-Fucosyllactose by De Novo GDP-Fucose pathway. J Agric Food Chem, 2023, 71: 14678-14686.

[119] DuZ, ZhuY, LuZ, ChenR, HuangZ, ChenY, GuangC, MuW. Combinatorial optimization strategies for 3-Fucosyllactose Hyperproduction in Escherichiacoli. J Agric Food Chem, 2024, 72: 14191-14198.

[120] ZhangA, SunL, BaiY, YuH, McArthurJB, ChenX, AtsumiS. Microbial production of human milk oligosaccharide lactodifucotetraose. Metab Eng, 2021, 66: 12-20.

[121] ZhuY, et al. . De Novo biosynthesis of Difucosyllactose by artificial pathway construction and alpha1,3/4-Fucosyltransferase rational design in Escherichiacoli. J Agricult Food Chem, 2024, 72: 9247

[122] LuM, MoslehI, AbbaspourradA. Engineered microbial routes for human milk oligosaccharides synthesis. ACS Synth Biol, 2021, 10: 923-938.

[123] HuM, LiM, LiC, ZhangT. Biosynthesis of Lacto-N-fucopentaose I in Escherichiacoli by metabolic pathway rational design. Carbohydr Polym, 2022, 297: 120017.

[124] FierfortN, SamainE. Genetic engineering of Escherichiacoli for the economical production of sialylated oligosaccharides. J Biotechnol, 2008, 134: 261-265.

[125] ZhangJ, ZhuY, ZhangW, MuW. Efficient production of a functional human milk Oligosaccharide 3′-Sialyllactose in genetically engineered Escherichiacoli. ACS Synth Biol, 2022, 11: 2837-2845.

[126] LiC, LiM, HuM, MiaoM, ZhangT. Metabolic engineering of Escherichiacoli for High-Titer Biosynthesis of 3′-Sialyllactose. J Agric Food Chem, 2024, 72: 5379-5390.

[127] LvX, ChenX, LiuY, YuanL, WuJ, YaoJ. Efficient production of 3′-Sialyllactose using Escherichiacoli. J Agricult Food Chem, 2024.

[128] DrouillardS, MineT, KajiwaraH, YamamotoT, SamainE. Efficient synthesis of 6′-sialyllactose, 6,6′-disialyllactose, and 6′-KDO-lactose by metabolically engineered E. coli expressing a multifunctional sialyltransferase from the Photobacterium sp. JT-ISH-224. Carbohyd Res, 2010, 345: 1394-1399.

[129] LiC, LiM, HuM, GaoW, MiaoM, ZhangT. Engineering Escherichiacoli for the efficient biosynthesis of 6′-sialyllactose. Food Biosci, 2023, 55: 103040.

[130] BarnumCR, et al. . Engineered plants provide a photosynthetic platform for the production of diverse human milk oligosaccharides. Nature food, 2024, 5: 480-490.

[131] LundemoP, KarlssonEN, AdlercreutzP. Eliminating hydrolytic activity without affecting the transglycosylation of a GH1 β-glucosidase. Appl Microbiol Biotechnol, 2017, 101: 1121-1131.

[132] ZeunerB, MarleneV, HolckJ, MuschioJ, MeyerAS. Loop engineering of an alpha-1,3/4-l-fucosidase for improved synthesis of human milk oligosaccharides. Enzyme Microb Technol, 2018, 115: 37-44.

[133] JamekSB, MuschiolJ, HolckJ, ZeunerB, BuskPK, MikkelsenJD, MeyerAS. Loop protein engineering for improved transglycosylation Activity of a β-N-Acetylhexosaminidase. ChemBioChem, 2018, 19: 1858-1865.

[134] JersC, et al. . Rational design of a new Trypanosoma rangeli trans-sialidase for efficient sialylation of glycans. PLoS ONE, 2014, 9: e83902.

[135] NyffeneggerC, et al. . Backbone structures in human milk oligosaccharides: trans-glycosylation by metagenomic β-N-acetylhexosaminidases. Appl Microbiol Biotechnol, 2015, 99: 7997-8009.

[136] ParisG, RatierL, AmayaMF, NguyenT, AlzariPM, FraschAC. A sialidase mutant displaying trans-sialidase activity. J Mol Biol, 2005, 345: 923-934.

[137] Jimenez-PerezC, Guzman-RodriguezF, Cruz-GuerreroAE, Alatorre-SantamariaS. The dual role of fucosidases: tool or target. Biologia (Bratisl), 2023.

[138] SugiyamaY, et al. . Introduction of H-antigens into oligosaccharides and sugar chains of glycoproteins using highly efficient 1,2-α-l-fucosynthase. Glycobiology, 2016, 26: 1235-1247

[139] WadaJ, et al. . 1,2-alpha-l-Fucosynthase: a glycosynthase derived from an inverting alpha-glycosidase with an unusual reaction mechanism. FEBS Lett, 2008, 582: 3739-3743.

[140] SakuramaH, et al. . 1,3–1,4-α-L-fucosynthase that specifically introduces Lewis a/x antigens into type-1/2 chains. J Biol Chem, 2012, 287: 16709-16719.

[141] SugiartoG, LauK, LiY, KhedriZ, YuH, LeDT, ChenX. Decreasing the sialidase activity of multifunctional Pasteurella multocida alpha2-3-sialyltransferase 1 (PmST1) by site-directed mutagenesis. Mol BioSyst, 2011, 7: 3021-3027.

[142] SugiartoG, et al. . A sialyltransferase mutant with decreased donor hydrolysis and reduced sialidase activities for directly sialylating LewisX. ACS Chem Biol, 2012, 7: 1232-1240.

[143] MichalakM, et al. . Biocatalytic production of 3′-sialyllactose by use of a modified sialidase with superior trans-sialidase activity. Process Biochem, 2014, 49: 265-270.

[144] ChoiYH, ParkBS, SeoJH, KimBG. Biosynthesis of the human milk oligosaccharide 3-fucosyllactose in metabolically engineered Escherichia coli via the salvage pathway through increasing GTP synthesis and beta-galactosidase modification. Biotechnol Bioeng, 2019, 116: 3324-3332.

[145] TaoM, YangL, ZhaoC, HuangZ, ZhaoM, ZhangW, ZhuY, MuW. Rational modification of Neisseria meningitidis β1,3-N-acetylglucosaminyltransferase for lacto-N-neotetraose synthesis with reduced long-chain derivatives. Carbohyd Polym, 2024, 345: 122543.

[146] MészárosZ, NekvasilováP, BojarováP, KřenV, SlámováK. Advanced glycosidases as ingenious biosynthetic instruments. Biotechnol Adv, 2021, 49: 107733.

[147] RuzicL, BolivarJM, NidetzkyB. Glycosynthase reaction meets the flow: Continuous synthesis of lacto-N-triose II by engineered beta-hexosaminidase immobilized on solid support. Biotechnol Bioeng, 2020, 117: 1597-1602.

[148] HayesMR, PietruszkaJ. Synthesis of glycosides by glycosynthases. Molecules, 2017, 22: 1434.

[149] KapešováJ, et al. . Transglycosidase activity of glycosynthase-type mutants of a fungal GH20 β-N-acetylhexosaminidase. Int J Biol Macromol, 2020, 161: 1206-1215.

[150] SchmölzerK, WeingartenM, BaldeniusK, NidetzkyB. Glycosynthase principle transformed into biocatalytic process technology: Lacto-N-triose II production with engineered exo-hexosaminidase. ACS Catal, 2019, 9: 5503-5514.

[151] MiyazakiT, SatoT, FurukawaK, AjisakaKFukudaM. Chapter Twenty-Four—Enzymatic synthesis of Lacto-N-Difucohexaose I which binds to Helicobacterpylori. Methods in enzymology, 2010Academic Press511-524

[152] McArthurJB, YuH, ChenX. A Bacterial β1–3-Galactosyltransferase enables multigram-scale synthesis of human Milk Lacto-N-tetraose (LNT) and Its fucosides. ACS Catal, 2019, 9: 10721.

[153] LiuY, YanQ, MaJ, YangS, LiT, JiangZ. Production of Lacto-N-triose II and Lacto-N-neotetraose from Chitin by a Novel β-N-Acetylhexosaminidase Expressed in Pichia pastoris. ACS Sustainable Chem Eng, 2020, 8: 15466.

[154] ZhaoC, et al. . The one-pot multienzyme (OPME) synthesis of human blood group H antigens and a human milk oligosaccharide (HMOS) with highly active Thermosynechococcus elongatus α1–2-fucosyltransferase. Chem Commun, 2016, 52: 3899-3902.

[155] BaumgärtnerF, ConradJ, SprengerGA, AlbermannC. Synthesis of the human milk Oligosaccharide Lacto-N-Tetraose in metabolically engineered. Plasmid-Free E coli, 2014, 15: 1896-1900

[156] ChinYW, SeoN, KimJH, SeoJH. Metabolic engineering of Escherichiacoli to produce 2′-fucosyllactose via salvage pathway of guanosine 5′-diphosphate (GDP)-l-fucose. Biotechnol Bioeng, 2016, 113: 2443-2452.

[157] HuangD, YangK, LiuJ, XuY, WangY, WangR, LiuB, FengL. Metabolic engineering of Escherichiacoli for the production of 2′-fucosyllactose and 3-fucosyllactose through modular pathway enhancement. Metab Eng, 2017, 41: 23-38.

[158] SeydametovaE, et al. . Development of a quantitative assay for 2 -fucosyllactose via one-pot reaction with alpha1,2-fucosidase and l-fucose dehydrogenase. Anal Biochem, 2019, 582: 113358.

[159] ChenQ, WuH, JiM, XieY, LiS, LiY, ShiJ, SunJ. Engineering a colanic acid biosynthesis pathway in E. coli for manufacturing 2′-fucosyllactose. Process Biochem, 2020, 94: 79-85.

[160] HollandsK, et al. . Engineering two species of yeast as cell factories for 2′-fucosyllactose. Metab Eng, 2019, 52: 232-242.

[161] LiM, LiC, LuoY, HuM, LiuZ, ZhangT. Multi-level metabolic engineering of Escherichiacoli for high-titer biosynthesis of 2′-fucosyllactose and 3-fucosyllactose. Microb Biotechnol, 2022, 15: 2970-2981.

[162] EndoT, KoizumiS, TabataK, OzakiA. Large-scale production of CMP-NeuAc and sialylated oligosaccharides through bacterial coupling. Appl Microbiol Biotechnol, 2000, 53: 257-261.

Funding

Department of Biotechnology, Ministry of Science and Technology, India(BT/PR32876/PFN/20/1471/2020)

RIGHTS & PERMISSIONS

Jiangnan University