Metabolic engineering of Escherichia coli for production of lacto-N-neotetraose

Abstract

Lacto-N-neotetraose is an abundant human milk oligosaccharide with multifaceted physiological functions. In this study, Escherichia coli MG1655 has been used as the chassis strain for lacto-N-neotetraose production. First, the genes lacz, ugd, gcd, nagB, ushA, setA were deleted in MG1655, and the Neisseria meningitidis genes lgtA and lgtB encoding β-1,3-N-acetylglucosaminyltransferase and β-1,4-galactosyltransferase, respectively, were overexpressed, and the resulting strain WY011/pC-A/pR-B could produce 1.102 g/L lacto-N-neotetraose. Next, the rate-limiting gene overexpression, expression vector screening, and ribosome-binding site strength were optimized, and the resulting strain WY011/pR-BEA1R could produce 2.634 g/L lacto-N-neotetraose. Further optimization of fermentation parameters enabled the engineered strain WY011/pR-BEA1R to produce 3.533 g/L lacto-N-neotetraose, and a whole-cell catalysis process developed by using this strain with the mixed substrates of glycerol, lactose, and galactose boosted the titer to 10.845 g/L, which is the highest reported titer in small-scale systems so far. Ultimately, fed-batch fermentation of the strain WY011/pR-BEA1R in a 2-L bioreactor achieved an LNnT titer of 16.202 g/L.

Keywords

Human milk oligosaccharides / Lacto-N-neotetraose / LNnT / Escherichia coli / Metabolic engineering / Whole-cell catalysis

References

[1] Spicer SK, Gaddy JA, Townsend SD. Recent advances on human milk oligosaccharide antimicrobial activity. Curr Opin Chem Biol, 2022

[2] Zhang P, Zhu Y, Li Z, et al.. Recent advances on lacto-N-neotetraose, a commercially added human milk oligosaccharide in infant formula. J Agric Food Chem, 2022, 70(15): 4534-4547

[3] Puccio G, Alliet P, Cajozzo C, Janssens E, Corsello G, Sprenger N, Wernimont S, Egli D, Gosoniu L, Steenhout P. Effects of infant formula with human milk oligosaccharides on growth and morbidity. J Pediatr Gastroenterol Nutr, 2017, 64(4): 624-631

[4] Singh RP, Niharika J, Kondepudi KK, et al.. Recent understanding of human milk oligosaccharides in establishing infant gut microbiome and roles in immune system. Food Res Int, 2022

[5] Bode L. Human milk oligosaccharides: every baby needs a sugar mama. Glycobiology, 2012, 22(9): 1147-1162

[6] Vandenplas Y, Berger B, Carnielli V, et al.. Human milk oligosaccharides: 2′-fucosyllactose (2′-FL) and lacto-N-neotetraose (LNnT) in infant formula. Nutrients, 2018

[7] Bych K, Mikš MH, Johanson T, et al.. Production of HMOs using microbial hosts - from cell engineering to large scale production. Curr Opin Biotechnol, 2019, 56: 130-137

[8] Pérez-Escalante E, Alatorre-Santamaría S, Castañeda-Ovando A, et al.. Human milk oligosaccharides as bioactive compounds in infant formula: recent advances and trends in synthetic methods. Crit Rev Food Sci Nutr, 2020, 62(1): 181-214

[9] Thomas PG, Carter MR, Atochina O, Da'Dara AA, Piskorska D, McGuire E, Harn DA. Maturation of dendritic cell 2 phenotype by a helminth glycan uses a toll-like receptor 4-dependent mechanism. J Immunol, 2003, 171(11): 5837-5841

[10] Terrazas LI, Walsh KL, Piskorska D, McGuire E, Harn DAJr.. The schistosome oligosaccharide lacto-n-neotetraose expands Gr1⁺ cells that secrete anti-inflammatory cytokines and inhibit proliferation of naive CD4⁺ cells: a potential mechanism for immune polarization in helminth infections. J Immunol, 2001, 167(9): 5294-5303

[11] Moore RE, Xu LL, Townsend SD. Prospecting human milk oligosaccharides as a defense against viral infections. ACS Infect Dis, 2021, 7(2): 254-263

[12] Meng J, Zhu Y, Wang H, et al.. Biosynthesis of human milk oligosaccharides: enzyme cascade and metabolic engineering approaches. J Agric Food Chem, 2023, 71(5): 2234-2243

[13] Yang L, Zhu Y, Meng J, Zhang W, Mu W. Recent progress in fucosylated derivatives of lacto-N-tetraose and lacto-N-neotetraose. Crit Rev Food Sci Nutr, 2023, 64(28): 10384-10396

[14] Zhu Y, Luo G, Wan L, et al.. Physiological effects, biosynthesis, and derivatization of key human milk tetrasaccharides, lacto-N-tetraose, and lacto-N-neotetraose. Crit Rev Biotechnol, 2021, 42: 1-19

[15] Yamada A, Hatano K, Koyama T, Matsuoka K, Esumi Y, Terunuma D. Syntheses of a series of lacto-N-neotetraose clusters using a carbosilane dendrimer scaffold. Carbohydr Res, 2006, 341(4): 467-473

[16] Zhu Y, Luo G, Li Z, et al.. Efficient biosynthesis of lacto-N-neotetraose by a novel β-1,4-galactosyltransferase from Aggregatibacter actinomycetemcomitans NUM4039. Enzyme Microb Technol, 2022

[17] Dong X, Li N, Liu Z, et al.. Modular pathway engineering of key precursor supply pathways for lacto-N-neotetraose production in Bacillus subtilis. Biotechnol Biofuels, 2019

[18] Dong X, Li N, Liu Z, et al.. CRISPRi-guided multiplexed fine-tuning of metabolic flux for enhanced lacto-N-neotetraose production in Bacillus subtilis. J Agric Food Chem, 2020, 68(8): 2477-2484

[19] Liu Y, Yan Q, Ma J, et al.. Production of lacto-N-triose II and lacto-N-neotetraose from chitin by a novel β-N-acetylhexosaminidase expressed in Pichia pastoris. ACS Sustain Chem Eng, 2020, 8(41): 15466-15474

[20] Liu H, Zeng Q, Zhu C, et al.. High-throughput screening and directed evolution of β-1,3-N-acetylglucosaminyltransferase for enhanced LNnT production in engineered Saccharomyces cerevisiae. J Agric Food Chem, 2025, 73(13): 7966-7974

[21] Zhang M, Zhang K, Liu T, et al.. High-level production of lacto-N-neotetraose in Escherichia coli by stepwise optimization of the biosynthetic pathway. J Agric Food Chem, 2023, 71(43): 16212-16220

[22] Liao Y, Lao C, Wu J, et al.. High-yield synthesis of lacto-N-neotetraose from glycerol and glucose in engineered Escherichia coli. J Agric Food Chem, 2024, 72(10): 5325-5338

[23] Zhang W, Liu Z, Gong M, et al.. Metabolic engineering of Escherichia coli for the production of lacto-N-neotetraose (LNnT). Syst Microbiol Biomanuf, 2021, 1(3): 291-301

[24] Zhang P, Zhu Y, Li Z, et al.. Designing a highly efficient biosynthetic route for lacto-N-neotetraose production in Escherichia coli. J Agric Food Chem, 2022, 70(32): 9961-9968

[25] Tao M, Yang L, Zhao C, et al.. Implementation of a quorum-sensing system for highly efficient biosynthesis of lacto-N-neotetraose in engineered Escherichia coli MG1655. J Agric Food Chem, 2024, 72(13): 7179-7186

[26] Liao C, Xu X, Huang H, et al.. Construction of a plasmid-free Escherichia coli strain for lacto-N-neotetraose biosynthesis. Syst Microbiol Biomanuf, 2024, 4(3): 965-982

[27] Wang K, Wang X, Luo H, et al.. Synergetic fermentation of glucose and glycerol for high-yield N-acetylglucosamine production in Escherichia coli. Int J Mol Sci, 2022

[28] Cong L, Ran FA, Cox D, et al.. Multiplex genome engineering using CRISPR/Cas systems. Science, 2013, 339(6121): 819-823

[29] Jiang Y, Chen B, Duan C, et al.. Multigene editing in the Escherichia coli genome via the CRISPR-Cas9 system. Appl Environ Microbiol, 2015, 81(7): 2506-2514

[30] Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2^−ΔΔCt method. Methods, 2001, 25(4): 402-408

[31] Liu S, Li D, Qin Z, et al.. Enhancing glycosylation of flavonoids by engineering the uridine diphosphate glucose supply in Escherichia coli. J Agric Food Chem, 2023, 71(46): 17842-17851

[32] Zampieri M, Hörl M, Hotz F, et al.. Regulatory mechanisms underlying coordination of amino acid and glucose catabolism in Escherichia coli. Nat Commun, 2019, 10(13354

[33] Song J, Zhuang M, Fang Y, et al.. Self-regulated efficient production of L-threonine via an artificial quorum sensing system in engineered Escherichia coli. Microbiol Res, 2024

Funding

Jiangsu Basic Research Center for Synthetic Biology(Grant No. BK20233003)

RIGHTS & PERMISSIONS

Jiangnan University