Research progress and development trends in the biosynthesis of neutral core human milk oligosaccharides

LIU Dan; WANG Jianyu; JIANG Zhengqiang

doi:10.12211/2096-8280.2025-083

您当前的位置：

首页 >

文章列表页 >

Research progress and development trends in the biosynthesis of neutral core human milk oligosaccharides

Invited Review | 更新时间：2025-11-12

- Research progress and development trends in the biosynthesis of neutral core human milk oligosaccharides
- Synthetic Biology Journal Vol. 6, Issue 5, Pages: 1126-1144(2025)
- 作者机构：
  
  1.中国农业大学食品科学与营养工程学院，北京 100083
  2.中原食品实验室，河南漯河 462300
  3.中国农业大学工学院，北京 100083
- 作者简介：
- 基金信息：
- DOI：10.12211/2096-8280.2025-083
  CLC： Q815
- Received：04 August 2025，
  
  Revised：2025-09-16，
  
  Published：31 October 2025
- 稿件说明：
移动端阅览
刘丹，王建宇，江正强. 中性核心母乳寡糖生物合成的研究进展和发展趋势［J］. 合成生物学， 2025， 6（5）： 1126-1144

LIU Dan， WANG Jianyu， JIANG Zhengqiang. Research progress and development trends in the biosynthesis of neutral core human milk oligosaccharides［J］. Synthetic Biology Journal， 2025， 6（5）： 1126-1144
刘丹，王建宇，江正强. 中性核心母乳寡糖生物合成的研究进展和发展趋势［J］. 合成生物学， 2025， 6（5）： 1126-1144 DOI： 10.12211/2096-8280.2025-083.

LIU Dan， WANG Jianyu， JIANG Zhengqiang. Research progress and development trends in the biosynthesis of neutral core human milk oligosaccharides［J］. Synthetic Biology Journal， 2025， 6（5）： 1126-1144 DOI： 10.12211/2096-8280.2025-083.

摘要

母乳寡糖（human milk oligosaccharide，HMO）是母乳中重要的功能和营养成分。其中，中性核心母乳寡糖（neutral core human milk oligosaccharide，ncHMO）主要包括乳糖-

-三糖（LNT Ⅱ）、乳糖-

-新四糖（LNnT）和乳糖-

-四糖（LNT），在婴幼儿健康发育过程中发挥着不可替代的生理作用。近年来，中性核心母乳寡糖的生物合成技术快速发展，其工业化生产从可能走向现实。本文综述了中性核心母乳寡糖的酶法和微生物细胞法合成研究现状，介绍了糖基转移酶和糖苷酶在酶法合成中性核心母乳寡糖的应用，讨论了糖基转移酶的催化特性、底盘细胞的种类及改造等关键因素对微生物细胞法合成中性核心母乳寡糖产量的影响，进一步对比了两种方法生物合成中性核心母乳寡糖的优缺点。目前，中性核心母乳寡糖的生物合成存在酶催化效率低、底盘细胞选择与适配性差、副产物多及内毒素污染等问题，需通过理性设计酶元件、优化安全底盘、动态调控代谢网络及强化发酵纯化工艺等策略协同攻关，有望实现母乳寡糖的低成本、高效绿色生产，为开发更具营养价值的婴幼儿健康食品提供核心支撑。

Abstract

Human milk oligosaccharides (HMOs) are essentially functional and nutritional components found in human milk. They can be primarily classified into fucosylated

neutral core

and sialylated HMOs. Lacto-

-triose Ⅱ (LNT Ⅱ)

lacto-

-neotetraose (LNnT)

and lacto-

-tetraose (LNT) are common neutral core human milk oligosaccharides (ncHMOs)

which can be extended to form longer-chain HMOs and play important roles in intestinal health. In recent years

the biosynthesis of ncHMOs has developed rapidly

and industrial-scale production is from theoretical possibility to practical reality. The synthesis approaches for ncHMOs include chemical synthesis

enzymatic synthesis

and microbial cell synthesis. As the rapid development in biotechnology

enzymatic and microbial cell synthesis have emerged as prominent methods in ncHMOs bios

ynthesis. Enzymatic synthesis is highly efficient

regioselective

and stereoselective. Currently

glycosyltransferases and glycoside hydrolases represent the two major types of enzymes used for biosynthesizing ncHMOs. Glycosidase-based enzymatic synthesis has demonstrated high conversion rates for LNT Ⅱ and LNnT production. However

the enzymatic synthesis of LNT is less efficient and requires further improvement. Notably

the production of LNnT and LNT typically relies on LNT Ⅱ as a key precursor

requiring a multi-step synthetic strategy. Microbial cell synthesis employs metabolic engineering to construct continuously synthetic pathways in microbial cells such as

Escherichia coli

and

Bacillus subtilis

. Knocking out genes in competitive pathway

optimizing genes expression

regenerating cofactors have significantly enhanced the yields of ncHMOs. The biosynthesis of ncHMOs faces several critical challenges

including the low activity and poor substrate specificity of key glycosyltransferases

such as β-1

-acetylglucosaminyltransferase and β-1

3-galactosyltransferase. Additionally

the transporters of LNT Ⅱ and LNnT are not clear in microbial cell. Furthermore

the yields of LNT Ⅱ should be substantially improved for industrial-scale production. Thus

it is important to overcome the interconnected limitations in enzyme engineering (particularly glycosyltransferase specificity and activity)

microbial cell modification (focusing on metabolic compatibility and pathway design)

and bioprocess optimization (through rational pathway redesign)

via

an integrated synthetic biology and fermentation engineering approach in the future. These strategies are essential for achieving efficient

cost-effective biosynthesis of ncHMO at industrial scale.

关键词

Keywords

references

史然 , 江正强 . 2′-岩藻糖基乳糖的酶法合成研究进展和展望 [J ] . 合成生物学 , 2020 , 1 ( 4 ): 481 - 494 .

SHI R , JIANG Z Q . Enzymatic synthesis of 2′-fucosyllactose: advances and perspectives [J ] . Synthetic Biology Journal , 2020 , 1 ( 4 ): 481 - 494 .

SOUSA Y R F , MEDEIROS L B , PINTADO M M E , et al . Goat milk oligosaccharides: composition, analytical methods and bioactive and nutritional properties [J ] . Trends in Food Science & Technology , 2019 , 92 : 152 - 161 .

URRUTIA-BACA V H , ÁLVAREZ-BUYLLA J R , GUEIMONDE M , et al . Comparative study of the oligosaccharide profile in goat, bovine, sheep, and human milk whey [J ] . Food Chemistry , 2025 , 463 : 141123 .

马心悦 , 黄纯翠 , 赵耀 , 等 . 人乳寡糖的结构及其分离分析 [J ] . 生物化学与生物物理进展 , 2023 , 50 ( 12 ): 2869 - 2878 .

MA X Y , HUANG C C , ZHAO Y , et al . Structure and separation analysis of human milk oligosaccharides [J ] . Progress in Biochemistry and Biophysics , 2023 , 50 ( 12 ): 2869 - 2878 .

BENSIMON J , LU X N . Human milk oligosaccharides produced by synthetic biology [J ] . Journal of Agriculture and Food Research , 2024 , 18 : 101361 .

BODE L . Human milk oligosaccharides: next-generation functions and questions [J ] . Nestle Nutrition Institute Workshop Series , 2019 , 90 : 191 - 201 .

LI Y Y , DU G C , CHEN J , et al . Glycosyltransferases in human milk oligosaccharide synthesis: structural mechanisms and rational design [J ] . Current Opinion in Biotechnology , 2025 , 93 : 103315 .

ZHU Y Y , YANG L H , ZHAO C H , et al . Microbial synthesis of lacto- N -fucopentaose Ⅰ with high titer and purity by screening of specific glycosyltransferase and elimination of residual lacto- N -triose Ⅱ and lacto- N -tetraose [J ] . Journal of Agricultural and Food Chemistry , 2024 , 72 ( 8 ): 4317 - 4324 .

WANG L , ZHU Y Y , ZHAO C H , et al . Engineering Escherichia coli for highly efficient biosynthesis of lacto- N -difucohexaose Ⅱ through de novo GDP-L-fucose pathway [J ] . Journal of Agricultural and Food Chemistry , 2024 , 72 ( 18 ): 10469 - 10476 .

PEI C X , PENG X L , WU Y R , et al . Characterization and application of active human α2,6-sialyltransferases ST6GalNAc Ⅴ and ST6GalNAc Ⅵ recombined in Escherichia coli [J ] . Enzyme and Microbial Technology , 2024 , 177 : 110426 .

SUGITA T , SAMPEI S , KOKETSU K . Efficient production of lacto- N -fucopentaose Ⅲ in engineered Escherichia coli using α1,3-fucosyltransferase from Parabacteroides goldsteinii [J ] . Journal of Biotechnology , 2023 , 361 : 110 - 118 .

孟佳炜 , 朱莺莺 , 罗国聪 , 等 . 乳酰- N -新四糖的生理功能、生物合成及其衍生化研究进展 [J ] . 中国食品学报 , 2022 , 22 ( 3 ): 320 - 328 .

MENG J W , ZHU Y Y , LUO G C , et al . Recent advances on physiological function, biosynthesis, and derivatization of lacto- N -neotetraose [J ] . Journal of Chinese Institute of Food Science and Technology , 2022 , 22 ( 3 ): 320 - 328 .

LI C C , LI M L , GAO W , et al . Biosynthesis of sialyllacto- N -tetraose c in engineered Escherichia coli [J ] . Journal of Agricultural and Food Chemistry , 2024 , 72 ( 46 ): 25836 - 25846 .

SPRENGER G A , BAUMGÄRTNER F , ALBERMANN C . Production of human milk oligosaccharides by enzymatic and whole-cell microbial biotransformations [J ] . Journal of Biotechnology , 2017 , 258 : 79 - 91 .

GALEOTTI F , COPPA G V , ZAMPINI L , et al . Capillary electrophoresis separation of human milk neutral and acidic oligosaccharides derivatized with 2-aminoacridone [J ] . ELECTROPHORESIS , 2014 , 35 ( 6 ): 811 - 818 .

TONON K M , MIRANDA A , ABRÃO A C F V , et al . Validation and application of a method for the simultaneous absolute quantification of 16 neutral and acidic human milk oligosaccharides by graphitized carbon liquid chromatography-electrospray ionization-mass spectrometry [J ] . Food Chemistry , 2019 , 274 : 691 - 697 .

BYCH K , MIKŠ M H , JOHANSON T , et al . Production of HMOs using microbial hosts: from cell engineering to large scale production [J ] . Current Opinion in Biotechnology , 2019 , 56 : 130 - 137 .

SCHENK S , BODE L , JENSEN S R , et al . Systemic availability of human milk oligosaccharides in infants and adults: a narrative review [J ] . Advances in Nutrition , 2025 , 16 ( 9 ): 100488 .

PALUR D S K , PRESSLEY S R , ATSUMI S . Microbial production of human milk oligosaccharides [J ] . Molecules , 2023 , 28 ( 3 ): 1491 .

GE H D , ZHU W X , ZHANG J , et al . Human milk microbiota and oligosaccharides in colostrum and mature milk: comparison and correlation [J ] . Frontiers in Nutrition , 2024 , 11 : 1512700 .

KONG C L , CHENG L H , KRENNING G , et al . Human milk oligosaccharides mediate the crosstalk between intestinal epithelial caco-2 cells and Lactobacillus plantarum WCFS1 in an in vitro model with intestinal peristaltic shear force [J ] . The Journal of Nutrition , 2020 , 150 ( 8 ): 2077 - 2088 .

LIAO J Q , WANG M H , LI H Y , et al . Human milk oligosaccharide LNnT promotes intestinal epithelial growth and maturation during the early life of infant mice [J ] . Journal of Agricultural and Food Chemistry , 2025 , 73 ( 11 ): 6678 - 6690 .

LI M L , LU H , XUE Y L , et al . An in vitro colonic fermentation study of the effects of human milk oligosaccharides on gut microbiota and short-chain fatty acid production in infants aged 0-6 months [J ] . Foods , 2024 , 13 ( 6 ): 921 .

CHEN Y L , LUO G X , SONG F B , et al . Truncated rotavirus VP4 proteins induce stronger protective immunity compared to P2-VP8 in animal models [J ] . Antiviral Research , 2025 , 238 : 106156 .

SUN X M , LI D D , QI J X , et al . Glycan binding specificity and mechanism of human and porcine P [6]/P [19] rotavirus VP8*s [J ] . Journal of Virology , 2018 , 92 ( 14 ): e00538-18 .

EL-HAWIET A , KITOVA E N , KLASSEN J S . Recognition of human milk oligosaccharides by bacterial exotoxins [J ] . Glycobiology , 2015 , 25 ( 8 ): 845 - 854 .

THOMAS P G , CARTER M R , ATOCHINA O , et al . Maturation of dendritic cell 2 phenotype by a helminth glycan uses a Toll-like receptor 4-dependent mechanism [J ] . Journal of Immunology , 2003 , 171 ( 11 ): 5837 - 5841 .

IDÄNPÄÄN-HEIKKILÄ I , SIMON P M , ZOPF D , et al . Oligosaccharides interfere with the establishment and progression of experimental pneumococcal pneumonia [J ] . The Journal of Infectious Diseases , 1997 , 176 ( 3 ): 704 - 712 .

OTTINO-GONZÁLEZ J , ADISE S , MACHLE C J , et al . Consumption of different combinations of human milk oligosaccharides in the first 6 months of infancy is positively associated with early cognition at 2 years of age in a longitudinal cohort of Latino children [J ] . The American Journal of Clinical Nutrition , 2024 , 120 ( 3 ): 593 - 601 .

CHEETHAM N W H , DUBE V E . Preparation of lacto- N -neotetraose from human milk by high-performance liquid chromatography [J ] . Journal of Chromatography A , 1983 , 262 : 426 - 430 .

ALY M R E , IBRAHIM E I , EL ASHRY E H , et al . Synthesis of lacto- N -neotetraose and lacto- N -tetraose using the dimethylmaleoyl group as amino protective group [J ] . Carbohydrate Research , 1999 , 316 ( 1-4 ): 121 - 132 .

BANDARA M D , STINE K J , DEMCHENKO A V . The chemical synthesis of human milk oligosaccharides: lacto- N -neotetraose (Galβ1→4GlcNAcβ1→3Galβ1→4Glc) [J ] . Carbohydrate Research , 2019 , 483 : 107743 .

BANDARA M D , STINE K J , DEMCHENKO A V . The chemical synthesis of human milk oligosaccharides: lacto- N -tetraose (Galβ1→3GlcNAcβ1→3Galβ1→4Glc) [J ] . Carbohydrate Research , 2019 , 486 : 107824 .

刘丹 , 孙柳 , 赵春华 , 等 . 母乳低聚糖LNnT和LNT的研究进展及法规市场情况概述 [J ] . 中国食品添加剂 , 2024 , 35 ( 8 ): 230 - 240 .

LIU D , SUN L , ZHAO C H , et al . Research progress and regulatory market overview of breast milk oligosaccharides LNnT and LNT [J ] . China Food Additives , 2024 , 35 ( 8 ): 230 - 240 .

EFSA Panel on Nutrition , Novel Foods and Food Allergens (NDA) , TURCK D , CASTENMILLER J , et al . Safety of lacto- N -neotetraose (LNnT) produced by derivative strains of E . coli BL21 as a novel food pursuant to Regulation (EU) 2015/2283 [J ] . EFSA Journal European Food Safety Authority , 2020 , 18 ( 11 ): e06305 .

EFSA Panel on Nutrition , Novel Foods and Food Allergens (NDA) , TURCK D , BOHN T , et al . Safety of lacto- N -tetraose (LNT) produced by derivative strains of Escherichia coli BL21 (DE3) as a Novel Food pursuant to Regulation (EU) 2015/2283 [J ] . EFSA Journal , 2022 , 20 ( 5 ): e07242 .

CHEN C C , ZHANG Y , XUE M Y , et al . Sequential one-pot multienzyme (OPME) synthesis of lacto- N -neotetraose and its sialyl and fucosyl derivatives [J ] . Chemical Communications , 2015 , 51 ( 36 ): 7689 - 7692 .

JENNINGS M P , HOOD D W , PEAK I R A , et al . Molecular analysis of a locus for the biosynthesis and phase-variable expression of the lacto- N -neotetraose terminal lipopolysaccharide structure in Neisseria meningitidis [J ] . Molecular Microbiology , 1995 , 18 ( 4 ): 729 - 740 .

PENG W J , PRANSKEVICH J , NYCHOLAT C , et al . Helicobacter pylori β1,3- N -acetylglucosaminyltransferase for versatile synthesis of type 1 and type 2 poly-LacNAcs on N -linked, O -linked and I-antigen glycans [J ] . Glycobiology , 2012 , 22 ( 11 ): 1453 - 1464 .

ZEUNER B , NYFFENEGGER C , MIKKELSEN J D , et al . Thermostable β-galactosidases for the synthesis of human milk oligosaccharides [J ] . New Biotechnology , 2016 , 33 ( 3 ): 355 - 360 .

MATSUO I , KIM S , YAMAMOTO Y , et al . Cloning and overexpression of β- N -acetylglucosaminidase encoding gene nagA from Aspergillus oryzae and enzyme-catalyzed synthesis of human milk oligosaccharide [J ] . Bioscience, Biotechnology, and Biochemistry , 2003 , 67 ( 3 ): 646 - 650 .

NYFFENEGGER C , NORDVANG R T , ZEUNER B , et al . Backbone structures in human milk oligosaccharides: trans-glycosylation by metagenomic β- N -acetylhexosaminidases [J ] . Applied Microbiology and Biotechnology , 2015 , 99 ( 19 ): 7997 - 8009 .

TEZE D , ZHAO J , WIEMANN M , et al . Rational enzyme design without structural knowledge: a sequence-based approach for efficient generation of transglycosylases [J ] . Chemistry-A European Journal , 2021 , 27 ( 40 ): 10323 - 10334 .

CHEN X D , JIN L , JIANG X K , et al . Converting a β- N -acetylhexosaminidase into two trans-β- N -acetylhexosaminidases by domain-targeted mutagenesis [J ] . Applied Microbiology and Biotechnology , 2020 , 104 ( 2 ): 661 - 673 .

SCHMÖLZER K , WEINGARTEN M , BALDENIUS K , et al . Glycosynthase principle transformed into biocatalytic process technology: lacto- N -triose Ⅱ production with engineered exo -hexosaminidase [J ] . ACS Catalysis , 2019 , 9 ( 6 ): 5503 - 5514 .

LIU Y H , MA J W , SHI R , et al . Biochemical characterization of a β- N -acetylhexosaminidase from Catenibacterium mitsuokai suitable for the synthesis of lacto- N -triose Ⅱ [J ] . Process Biochemistry , 2021 , 102 : 360 - 368 .

LI C Q , CAO Z N , JIANG H , et al . Characterization of a GH20 β- N -acetylhexosaminidase from Flavobacterium algicola suitable to synthesize lacto- N -triose Ⅱ [J ] . Journal of Agricultural and Food Chemistry , 2024 , 72 ( 9 ): 4849 - 4857 .

LIU Y H , YAN Q J , MA J W , et al . Production of lacto- N -triose Ⅱ and lacto- N -neotetraose from chitin by a novel β- N -acetylhexosaminidase expressed in Pichia pastoris [J ] . ACS Sustainable Chemistry & Engineering , 2020 , 8 ( 41 ): 15466 - 15474 .

LIU Y H , YAN Q J , MA J W , et al . Directed evolution of a β- N -acetylhexosaminidase from Haloferula sp. for lacto- N -triose Ⅱ and lacto- N -neotetraose synthesis from chitin [J ] . Enzyme and Microbial Technology , 2023 , 164 : 110177 .

JAMEK S B , MUSCHIOL J , HOLCK J , et al . Loop protein engineering for improved transglycosylation activity of a β- N -acetylhexosaminidase [J ] . ChemBioChem , 2018 , 19 ( 17 ): 1858 - 1865 .

CAO Z N , LI C Q , JIANG H , et al . Molecular modification of a GH84 β- N -acetylglucosaminidase from Streptomyces violascens for synthesis of lacto- N -triose Ⅱ using whey powder and chitin-derived N -acetyl chitobiose [J ] . Food Chemistry , 2025 , 474 : 143046 .

LIU Y H , WANG L , HUANG P , et al . Efficient sequential synthesis of lacto- N -triose Ⅱ and lacto- N -neotetraose by a novel β- N -acetylhexosaminidase from Tyzzerella nexilis [J ] . Food Chemistry , 2020 , 332 : 127438 .

ABDUL MANAS N H , MD ILLIAS R , MAHADI N M . Strategy in manipulating transglycosylation activity of glycosyl hydrolase for oligosaccharide production [J ] . Critical Reviews in Biotechnology , 2018 , 38 ( 2 ): 272 - 293 .

WAKARCHUK W , MARTIN A , JENNINGS M P , et al . Functional relationships of the genetic locus encoding the glycosyltransferase enzymes involved in expression of the lacto- N -neotetraose terminal lipopolysaccharide structure in Neisseria meningitidis [J ] . Journal of Biological Chemistry , 1996 , 271 ( 32 ): 19166 - 19173 .

ZHU Y Y , LUO G C , LI Z Y , et al . Efficient biosynthesis of lacto- N -neotetraose by a novel β-1,4-galactosyltransferase from Aggregatibacter actinomycetemcomitans NUM4039 [J ] . Enzyme and Microbial Technology , 2022 , 153 : 109912 .

LUO G C , ZHU Y Y , MENG J W , et al . A novel β-1,4-galactosyltransferase from Histophilus somni enables efficient biosynthesis of lacto- N -neotetraose via both enzymatic and cell factory approaches [J ] . Journal of Agricultural and Food Chemistry , 2021 , 69 ( 20 ): 5683 - 5690 .

MURATA T , INUKAI T , SUZUKI M , et al . Facile enzymatic conversion of lactose into lacto- N -tetraose and lacto- N -neotetraose [J ] . Glycoconjugate Journal , 1999 , 16 ( 3 ): 189 - 195 .

王建宇 , 向芷璇 , 刘丹 , 等 . 芽孢杆菌截短β-半乳糖苷酶的理性设计及在合成乳糖- N -新四糖中的应用 [J ] . 食品工业科技 , 2025 , 46 ( 17 ): 232 - 239 .

WANG J Y , XIANG Z X , LIU D , et al . Rational design of a truncated β-galactosidase from Bacillus sp. and its application in the synthesis of lacto- N -neotetraose [J ] . Science and Technology of Food Industry , 2025 , 46 ( 17 ): 232 - 239 .

WANG J Y , XIANG Z X , LIU D , et al . Protein engineering of a novel β-galactosidase from Thermus scotoductus for efficient synthesis of lacto- N -neotetraose from chitin powder [J ] . Journal of Agricultural and Food Chemistry , 2024 , 72 ( 16 ): 9289 - 9296 .

LI J , WANG J Y , YAN Q J , et al . Biochemical characterization of a novel C-terminally truncated β-galactosidase from Paenibacillus antarcticus with high transglycosylation activity [J ] . Journal of Dairy Science , 2024 , 107 ( 12 ): 10141 - 10152 .

LUO G C , HUANG Z L , ZHU Y Y , et al . Crystal structure and structure-guided tunnel engineering in a bacterial β-1,4-galactosyltransferase [J ] . International Journal of Biological Macromolecules , 2024 , 279 : 135374 .

LAU K , THON V , YU H , et al . Highly efficient chemoenzymatic synthesis of β1-4-linked galactosides with promiscuous bacterial β1-4-galactosyltransferases [J ] . Chemical Communications , 2010 , 46 ( 33 ): 6066 - 6068 .

ZHANG Z Q , KONG H C , BAN X F , et al . C-terminal domains of β-galactosidase from Paenibacillus macquariensis modulate product distribution by altering substrate binding conformation [J ] . International Journal of Biological Macromolecules , 2025 , 310 : 143412 .

CHOI J Y , HONG H , SEO H , et al . High galacto-oligosaccharide production and a structural model for transgalactosylation of β-galactosidase Ⅱ from Bacillus circulans [J ] . Journal of Agricultural and Food Chemistry , 2020 , 68 ( 47 ): 13806 - 13814 .

HE X Y , LI Y , TAO Y H , et al . Discovering and efficiently promoting the extracellular secretory expression of Thermobacillus sp. ZCTH02-B1 sucrose phosphorylase in Escherichia coli [J ] . International Journal of Biological Macromolecules , 2021 , 173 : 532 - 540 .

LIU X W , XIA C F , LI L , et al . Characterization and synthetic application of a novel beta1,3-galactosyltransferase from Escherichia coli O55: H7 [J ] . Bioorganic & Medicinal Chemistry , 2009 , 17 ( 14 ): 4910 - 4915 .

MCARTHUR J B , YU H , CHEN X . A bacterial β1-3-galactosyltransferase enables multigram-scale synthesis of human milk lacto- N -tetraose (LNT) and its fucosides [J ] . ACS Catalysis , 2019 , 9 ( 12 ): 10721 - 10726 .

SCHMÖLZER K , WEINGARTEN M , BALDENIUS K , et al . Lacto- N -tetraose synthesis by wild-type and glycosynthase variants of the β- N -hexosaminidase from Bifidobacterium bifidum [J ] . Organic & Biomolecular Chemistry , 2019 , 17 ( 23 ): 5661 - 5665 .

CASTEJÓN-VILATERSANA M , FAIJES M , PLANAS A . Transglycosylation activity of engineered Bifidobacterium lacto- N -biosidase mutants at donor subsites for lacto- N -tetraose synthesis [J ] . International Journal of Molecular Sciences , 2021 , 22 ( 6 ): 3230 .

VUILLEMIN M , HOLCK J , MATWIEJUK M , et al . Improvement of the transglycosylation efficiency of a lacto- N -biosidase from Bifidobacterium bifidum by protein engineering [J ] . Applied Sciences , 2021 , 11 ( 23 ): 11493 .

MIYAZAKI T , SATO T , FURUKAWA K , et al . Enzymatic synthesis of lacto- N -difucohexaose Ⅰ which binds to Helicobacter pylori [J ] . Methods in enzymology , 2010 , 480 : 511 - 524 .

LI T , LI J , YAN Q J , et al . Biochemical characterization of a novel β-galactosidase from Lacticaseibacillus zeae and its application in synthesis of lacto- N -tetraose [J ] . Journal of Dairy Science , 2023 , 106 ( 10 ): 6623 - 6634 .

WADA J , ANDO T , KIYOHARA M , et al . Bifidobacterium bifidum lacto- N -biosidase, a critical enzyme for the degradation of human milk oligosaccharides with a type 1 structure [J ] . Applied and Environmental Microbiology , 2008 , 74 ( 13 ): 3996 - 4004 .

FANG J L , TSAI T W , LIANG C Y , et al . Enzymatic synthesis of human milk fucosides α1, 2-fucosyl para-lacto- N -hexaose and its is omeric derivatives [J ] . Advanced Synthesis & Catalysis , 2018 , 360 ( 17 ): 3213 - 3219 .

VUILLEMIN M , LENGYEL M , MUSCHIOL J , et al . Enzymatic lacto- N -biose elongation of human milk oligosaccharides with the GH136 lacto- N -biosidase LnbX engineered for improved transglycosylation [J ] . Enzyme and Microbial Technology , 2025 , 189 : 110660 .

ZHU Y Y , WAN L , MENG J W , et al . Metabolic engineering of Escherichia coli for lacto- N -triose Ⅱ production with high productivity [J ] . Journal of Agricultural and Food Chemistry , 2021 , 69 ( 12 ): 3702 - 3711 .

PRIEM B , GILBERT M , WAKARCHUK W W , et al . A new fermentation process allows large-scale production of human milk oligosaccharides by metabolically engineered bacteria [J ] . Glycobiology , 2002 , 12 ( 4 ): 235 - 240 .

HU D D , WU H , ZHU Y Y , et al . Engineering Escherichia coli for highly efficient production of lacto- N -triose Ⅱ from N -acetylglucosamine, the monomer of chitin [J ] . Biotechnology for Biofuels , 2021 , 14 ( 1 ): 198 .

SUGITA T , KOKETSU K . Transporter engineering enables the efficient production of lacto- N -triose Ⅱ and lacto- N -tetraose in Escherichia coli [J ] . Journal of Agricultural and Food Chemistry , 2022 , 70 ( 16 ): 5106 - 5114 .

HU M M , ZHANG T . Metabolic engineering of the probiotic Escherichia coli Nissle 1917 for lacto- N -triose Ⅱ production [J ] . Food Bioscience , 2024 , 59 : 103959 .

LI J Z , HE T Y , ZHAO J J , et al . Combination of metabolic engineering and high-throughput screening to realize high-producing lacto- N -triose Ⅱ in Escherichia coli [J ] . Journal of Agricultural and Food Chemistry , 2025 , 73 ( 28 ): 17769 - 17775 .

WANG Z J , ZHANG Z M , LI Y , et al . Two-step production method for lacto- N -triose Ⅱ via cell-coupled biocatalytic strategy [J ] . Journal of Agricultural and Food Chemistry , 2025 , 73 ( 14 ): 8391 - 8400 .

ZHANG P , ZHU Y Y , LI Z Y , et al . Designing a highly efficient biosynthetic route for lacto- N -neotetraose production in Escherichia coli [J ] . Journal of Agricultural and Food Chemistry , 2022 , 70 ( 32 ): 9961 - 9968 .

LIAO Y X , WU J Y , LI Z K , et al . Metabolic engineering of Escherichia coli for high-level production of lacto- N -neotetraose and lacto- N -tetraose [J ] . Journal of Agricultural and Food Chemistry , 2023 , 71 ( 30 ): 11555 - 11566 .

HU M M , LI M L , LI C C , et al . High-level productivity of lacto- N -neotetraose in Escherichia coli by systematic metabolic engineering [J ] . Journal of Agricultural and Food Chemistry , 2023 , 71 ( 9 ): 4051 - 4058 .

TAO M T , YANG L H , ZHAO C H , et al . Implementation of a quorum-sensing system for highly efficient biosynthesis of lacto- N -neotetraose in engineered Escherichia coli MG1655 [J ] . Journal of Agricultural and Food Chemistry , 2024 , 72 ( 13 ): 7179 - 7186 .

LIAO C , XU X H , HUANG H Y , et al . Construction of a plasmid-free Escherichia coli strain for lacto- N -neotetraose biosynthesis [J ] . Systems Microbiology and Biomanufacturing , 2024 , 4 ( 3 ): 965 - 982 .

ZHANG M W , ZHANG K , LIU T L , et al . High-level production of lacto- N -neotetraose in Escherichia coli by stepwise optimization of the biosynthetic pathway [J ] . Journal of Agricultural and Food Chemistry , 2023 , 71 ( 43 ): 16212 - 16220 .

LIAO Y X , LAO C W , WU J Y , et al . High-yield synthesis of lacto- N -neotetraose from glycerol and glucose in engineered Escherichia coli [J ] . Journal of Agricultural and Food Chemistry , 2024 , 72 ( 10 ): 5325 - 5338 .

LI Z Y , ZHU Y Y , ZHANG P , et al . Pathway optimization and uridine 5′-triphosphate regeneration for enhancing lacto- N -tetraose biosynthesis in engineered Escherichia coli [J ] . Journal of Agricultural and Food Chemistry , 2022 , 70 ( 25 ): 7727 - 7735 .

QIAN Q Y , YANG L H , ZHAO C H , et al . Highly efficient production of lacto- N -tetraose in plasmid-free Escherichia coli through chromosomal integration of multicopy key glycosyltransferase genes [J ] . International Journal of Biological Macromolecules , 2025 , 284 : 137987 .

DONG X M , LI N , LIU Z M , et al . Modular pathway engineering of key precursor supply pathways for lacto- N -neotetraose production in Bacillus subtilis [J ] . Biotechnology for Biofuels , 2019 , 12 : 212 .

DONG X M , LI N , LIU Z M , et al . CRISPRi-guided multiplexed fine-tuning of metabolic flux for enhanced lacto- N -neotetraose production in Bacillus subtilis [J ] . Journal of Agricultural and Food Chemistry , 2020 , 68 ( 8 ): 2477 - 2484 .

YANG J , MUND N K , YANG L R , et al . Engineering glycolytic pathway for improved lacto- N -neotetraose production in Pichia pastoris [J ] . Enzyme and Microbial Technology , 2025 , 184 : 110576 .

刘丹 , 梁山泉 , 闫巧娟 , 等 . 基于模块优化强化大肠杆菌合成乳糖- N -新四糖的研究 [J ] . 食品科学技术学报 , 2024 , 42 ( 2 ): 75 - 83 .

LIU D , LIANG S Q , YAN Q J , et al . Study on enhancement of lacto- N -neotetraose synthesis in Escherichia coli based on module optimization [J ] . Journal of Food Science and Technology , 2024 , 42 ( 2 ): 75 - 83 .

LIU T L , ZHANG K , ZHANG M W , et al . De novo synthesis of lacto- N -neotetraose in Escherichia coli through metabolic engineering with glucose as the sole carbon source [J ] . Journal of Agricultural and Food Chemistry , 2025 , 73 ( 22 ): 13736 - 13745 .

TAO M T , YANG L H , ZHAO C H , et al . Rational modification of Neisseria meningitidis β1,3- N -acetylglucosaminyltransferase for lacto- N -neotetraose synthesis with reduced long-chain derivatives [J ] . Carbohydrate Polymers , 2024 , 345 : 122543 .

LIU Y F , LIU L , LI J H , et al . Synthetic biology toolbox and chassis development in Bacillus subtilis [J ] . Trends in Biotechnology , 2019 , 37 ( 5 ): 548 - 562 .

LIU H , ZENG Q Q , ZHU C L , et al . High-throughput screening and directed evolution of β-1, 3- N -acetylglucosaminyltransferase for enhanced LNnT production in engineered Saccharomyces cerevisiae [J ] . Journal of Agricultural and Food Chemistry , 2025 , 73 ( 13 ): 7966 - 7974 .

ZHU Y Y , LI Z Y , LUO G C , et al . Metabolic engineering of Escherichia coli for efficient biosynthesis of lacto- N -tetraose using a novel β-1, 3-galactosyltransferase from Pseudogulbenkiania ferrooxidans [J ] . Journal of Agricultural and Food Chemistry , 2021 , 69 ( 38 ): 11342 - 11349 .

HU M M , LI M L , MIAO M , et al . Engineering Escherichia coli for the high-titer biosynthesis of lacto- N -tetraose [J ] . Journal of Agricultural and Food Chemistry , 2022 , 70 ( 28 ): 8704 - 8712 .

YANG L H , ZHU Y Y , ZHAO C H , et al . Elimination of residual lacto- N -triose Ⅱ for lacto- N -tetraose biosynthesis in engineered Escherichia coli [J ] . Journal of Agricultural and Food Chemistry , 2023 , 71 ( 33 ): 12511 - 12518 .

WANG J , LAO C W , WU J Y , et al . Multimodular metabolic engineering strategy enables high-efficiency synthesis of lacto- N -fucopentaose Ⅰ in engineered Escherichia coli [J ] . Journal of Agricultural and Food Chemistry , 2025 , 73 ( 25 ): 15869 - 15879 .

ABRAHAMSON C H , PALMERO B J , KENNEDY N W , et al . Theoretical and practical aspects of multienzyme organization and encapsulation [J ] . Annual Review of Biophysics , 2023 , 52 : 553 - 572 .

BANANI S F , LEE H O , HYMAN A A , et al . Biomolecular condensates: organizers of cellular biochemistry [J ] . Nature Reviews Molecular Cell Biology , 2017 , 18 ( 5 ): 285 - 298 .

LYON A S , PEEPLES W B , ROSEN M K . A framework for understanding the functions of biomolecular condensates across scales [J ] . Nature Reviews Molecular Cell Biology , 2021 , 22 ( 3 ): 215 - 235 .

LAU Y H , GIESSEN T W , ALTENBURG W J , et al . Prokaryotic nanocompartments form synthetic organelles in a eukaryote [J ] . Nature Communications , 2018 , 9 : 1311 .

WAN L , ZHU Y Y , KE J T , et al . Compartmentalization of pathway sequential enzymes into syn thetic protein compartments for metabolic flux optimization in Escherichia coli [J ] . Metabolic Engineering , 2024 , 85 : 167 - 179 .

LI Y S , LI Y , LI P F , et al . Whole-cell biosynthesis of branched human milk hexasaccharide lacto- N -neohexaose [J ] . Journal of Agricultural and Food Chemistry , 2025 , 73 ( 28 ): 17814 - 17823 .

Views

下载量

CSCD

Alert me when the article has been cited

提交

Tools

Publicity Resources

Progress in oxygen-resistant and efficient organic triplet-triplet annihilation upconversion luminescence

Phenolic acid compounds： extraction from plants to biosynthesis

Advances in small-molecule biopesticides and their biosynthesis

Plant synthetic biology: new opportunities for large-scale culture of plant cells

Related Author

LIU Dan

QI Fang

PEI Chenxu

LI Jiayao

PENG Yi

LIN Wenyue

FENG Hongjuan

HUANG Ling

Related Institution

Tianjin Key Laboratory of Biosensing and Molecular Recognition， Research Center for Analytical Sciences， Frontiers Science Center for New Organic Matter， Haihe Laboratory of Sustainable Chemical Transformations， College of Chemistry， Nankai University

Department of Bioengineering， College of Chemistry and Chemical Engineering， the North University of China

Biotechnology Research Institute， Chinese Academy of Agricultural Sciences

Academy of National Food and Strategic Reserves Administration

College of Life Science and Technology， Beijing University of Chemical Technology

⁰