Systematic pathway engineering of Escherichia coli for enhanced 3’-sialyllactose production

Abstract

3’-Sialyllactose (3’-SL), the simplest sialylated human milk oligosaccharide (HMO), is widely incorporated into infant formula due to its roles in shaping the gut microbiota, inhibiting pathogen adhesion, and supporting neurodevelopment. In this study, we developed an efficient microbial cell factory for 3’-SL production by enhancing carbon utilization and CTP regeneration. Following the identification of a highly active α-2,3-sialyltransferase, metabolic flux was redirected through pfkA deletion and fbaA overexpression. The precursor pool was strengthened by optimizing the expression of key enzymes involved in UDP-GlcNAc and CMP-Neu5Ac biosynthesis. To further improve pathway balance and stability, promoter engineering was applied to optimize the expression of four key genes (neuA, neuB, neuC and ST). The optimized multigene cassette was subsequently integrated into the chromosome using a one-step multicopy integration strategy, generating a plasmid-free and antibiotic marker-free production strain. The resulting strain achieved 3’-SL titers of 7.75 g L⁻¹ in shake flasks and 102.18 g L⁻¹ in a 5-L fed-batch bioreactor, representing, to the best of our knowledge, the highest reported level to date. This work demonstrates a robust strategy combining metabolic rewiring, adaptive gene expression tuning and multicopy genome integration, providing a versatile platform for the sustainable production of 3’-SL and other high-value HMOs.

Graphical abstract

Keywords

Metabolic engineering / Promoter optimization / Multicopy integration / α-2,3-sialyltransferase / Human milk oligosaccharides

References

[1] Bych K, Mikš MH, Johanson T, Hederos MJ, Vigsnæs LK, Becker P. Production of HMOs using microbial hosts - from cell engineering to large scale production. Curr Opin Biotechnol, 2019, 56: 130-137

[2] Chen Y, Zhu, Y., Wang H, Chen R, Liu Y, Zhang, W., Mu W. De novo biosynthesis of 2’-fucosyllactose in a metabolically engineered Escherichia coli using a novel ɑ1,2-fucosyltransferase from Azospirillum lipoferum. Bioresource Technol, 2023, 374: 128818

[3] Craft KM, Townsend SD. Mother knows best: deciphering the antibacterial properties of human milk oligosaccharides. Acc Chem Res, 2019, 52: 760-768

[4] Fang H, Gao J, Mund N, Tan Y, Shi P, Zhao C. Recent advances in metabolic engineering strategies for the production of human milk oligosaccharides in microbial hosts. ACS Synth Biol, 2025, 14(8): 2885-2905

[5] Fierfort N, Samain E. Genetic engineering of Escherichia coli for the economical production of sialylated oligosaccharides. J Biotechnol, 2008, 134: 261-265

[6] Han NS, Kim T, Park Y, Kim J, Seo J. Biotechnological production of human milk oligosaccharides. Biotechnol Adv, 2012, 30(6): 1268-1278

[7] Jiang Y, Sun T, Lin Y, Liu M, Wang X. Is it possible to obtain substitutes for human milk oligosaccharides from bovine milk, goat milk, or other mammal milks?. Compr Rev Food Sci F, 2024, 23: e70018

[8] Li Y, Sun M, Huang S, Yu H, Chokhawala HA, Thon V, Chen X. The Hd0053 gene of Haemophilus ducreyi encodes an α2,3-sialyltransferase. Biochem Biophys Res Commun, 2007, 361(2): 555-560

[9] Li Q, Sun B, Chen J, Zhang Y, Jiang Y, Yang S. A modified pCas/pTargetF system for CRISPR-Cas9-assisted genome editing in Escherichia coli. Acta Biochim Biophys Sin, 2021, 53(5): 620-627

[10] Li Z, Chen X, Ni Z, Yuan L, Sun L, Wang Y, Wu J, Yao J. Efficient Production of 3’-sialyllactose by single whole-cell in one-pot biosynthesis. Processes, 2021, 9(6): 932

[11] Li C, Li M, Gao W, Zhang T, Liu Z, Miao M. Biosynthesis of sialyllacto-N-tetraose c in engineered Escherichia coli. J Agric Food Chem, 2024, 72: 25836-25846

[12] Li C, Li M, Hu M, Miao M, Zhang T. Metabolic engineering of Escherichia coli for high-titer biosynthesis of 3’-Sialyllactose. J Agric Food Chem, 2024, 72(10): 5379-5390

[13] Lin Q, Liu Y, Wang H, Huang Z, Zhang W, Sheng M, Zhu Y, Wang R, Mu W. Improving 3′-sialyllactose biosynthesis in Escherichia coli by engineering Neisseria meningitidis 406Y α2,3-sialyltransferase. Process Biochem, 2025, 153: 11-17

[14] Lis-Kuberka J, Orczyk-Pawiłowicz M. Sialylated oligosaccharides and glycoconjugates of human milk. The impact on infant and newborn protection, development and well-being. Nutrients, 2019, 11(2): 306

[15] Liu C, Lv X, Li J, Liu L, Du G, Liu Y. Metabolic engineering of Escherichia coli for increased bioproduction of N-acetylneuraminic acid. J Agric Food Chem, 2022, 70: 15859-15868

[16] Lu M, Mosleh I, Abbaspourrad A. Engineered microbial routes for human milk oligosaccharides Synthesis. ACS Synth Biol, 2021, 10: 923-938

[17] Lv X, Chen X, Liu Y, Yuan L, Wu J, Yao J. Efficient production of 3’-Sialyllactose using Escherichia coli. J Agric Food Chem, 2024, 72(49): 27314-27325

[18] Lyons KE, Ryan CA, Dempsey EM, Ross RP, Stanton C. Breast milk, a source of beneficial microbes and associated benefits for infant health. Nutrients, 2020, 12(4): 1039

[19] Moore SJ, Lai HE, Kelwick RJR, Chee SM, Bell DJ, Polizzi KM, Freemont PS. EcoFlex: A multifunctional MoClo Kit for E. coli synthetic biology. ACS Synth Biol, 2016, 5(10): 1059-1069

[20] Schelch S, Eibinger M, Zuson J, Kuballa J, Nidetzky B. Modular bioengineering of whole-cell catalysis for sialo-oligosaccharide production: coordinated co-expression of CMP-sialic acid synthetase and sialyltransferase. Microb Cell Fact, 2023, 22: 241

[21] Stukenberg D, Hensel T, Hoff J, Daniel BJ, Inckemann R, Tedeschi JN, Nousch F, Fritz G. The marburg collection: a golden gate DNA assembly framework for synthetic biology applications in Vibrio natriegens. ACS Synth Biol, 2021, 10(8): 1904-1919

[22] Talafová K, Hrabárová E, Nahálka J. A semi-multifunctional sialyltransferase from Bibersteinia trehalosi and its comparison to the Pasteurella multocida ST1 mutants. J Biotechnol, 2015, 216: 116-124

[23] Thurl S, Munzert M, Boehm G, Matthews C, Stahl B. Systematic review of the concentrations of oligosaccharides in human milk. Nutr Rev, 2017, 75(11): 920-933

[24] Tian R, Liu Y, Liu LLiu L, Chen J. Microbial production of oligosaccharides and polysaccharides. Systems and Synthetic Biotechnology for Production of Nutraceuticals, 2019, Singapore, Springer

[25] Van Leeuwen SS, Te Poele EM, Chatziioannou AC, Benjamins E, Haandrikman A, Dijkhuizen L. Goat milk oligosaccharides: their diversity, quantity, and functional properties in comparison to human milk oligosaccharides. J Agric Food Chem, 2020, 68(47): 13469-13485

[26] Wu Y, Tang J, Wang S, Tian Z (2022) Expression element, genetic engineering bacteria and preparation method of N-acetylneuraminic acid. Chin Patent CN118308391B

[27] Xu P, Rizzoni EA, Sul S, Stephanopoulos G. Improving metabolic pathway efficiency by statistical model-based multivariate regulatory metabolic engineering. ACS Synth Biol, 2016, 6: 148-158

[28] Yang S, Zhang Y, Xu J, Zhang J, Zhang J, Yang J, Jiang Y, Yang S. Orthogonal CRISPR-associated transposases for parallel and multiplexed chromosomal integration. Nucleic Acids Res, 2021, 49(17): 10192-10202

[29] You R, Wang L, Hu M, Tao Y. Efficient production of 2’-fucosyllactose from fructose through metabolically engineered recombinant Escherichia coli. Microb Cell Fact, 2024, 23: 38

[30] Yu H, Chokhawala H, Karpel R, Yu H, Wu B, Zhang J, Zhang Y, Jia Q, Chen X. A multifunctional Pasteurella multocida Sialyltransferase: a powerful tool for the synthesis of sialoside libraries. J Am Chem Soc, 2005, 127(50): 17618-17619

[31] Yue S, Wang N, Chen J, Su C, Li H, Gong J, Xu Z, Shi J. Enhanced biosynthesis of 6’-sialyllactose in Escherichia coli via systematic metabolic engineering. J Agric Food Chem, 2025, 73(20): 12383-12391

[32] Zhang Y, Sun X, Wang Q, Xu J, Dong F, Yang S, Yang J, Zhang Z, Qian Y, Chen J, Zhang J, Liu Y, Tao R, Jiang Y, Yang J, Yang S. Multicopy chromosomal integration using CRISPR-associated transposases. ACS Synth Biol, 2020, 9(8): 1998-2008

[33] Zhang Y, Yang J, Yang S, Zhang J, Chen J, Tao R, Jiang Y, Yang J, Yang S. Programming cells by multicopy chromosomal integration using CRISPR-associated transposases. CRISPR J, 2021, 4(3): 350-359

[34] Zhang J, Zhu Y, Zhang W, Mu W. Efficient production of a functional human milk oligosaccharide 3’-Sialyllactose in genetically engineered Escherichia coli. ACS Synth Biol, 2022, 11(8): 2837-2845

[35] Zhang W, Zhu Y, Wang H, Huang Z, Liu Y, Xu W, Mu W. Highly efficient biosynthesis of 3’-sialyllactose in engineered Escherichia coli. Int J Biol Macromol, 2024, 269: 132081

[36] Zhang Z, Zhang H, Wang Z, You X, Mao Y, Li Z, Wu Z, Wu J, Zhao J, Chai W. A cellular coupling strategy leveraging pathway modularization and cofactor regeneration for the biosynthesis of 3’-Sialyllactose. J Agric Food Chem, 2025, 73(29): 18380-18389

[37] Zhao C, Li X, Guo L, Gao C, Song W, Wei W, Wu J, Liu L, Chen X. Reprogramming metabolic flux in Escherichia coli to enhance chondroitin production. Adv Sci, 2024, 11: 2307351

[38] Zhu Y, Cao H, Wang H, Mu W. Biosynthesis of human milk oligosaccharides via metabolic engineering approaches: current advances and challenges. Curr Opin Biotechnol, 2022, 78: 102841

Funding

National Key R&D Program of China(2025YFA0921000)

Science and Technology Commission of Shanghai Municipality(Science and Technology Commission of Shanghai Municipality)

RIGHTS & PERMISSIONS

The Author(s)