Molecular Biotechnology

, Volume 60, Issue 6, pp 427–434 | Cite as

Production of N-acetyl-d-neuraminic Acid by Recombinant Single Whole Cells Co-expressing N-acetyl-d-glucosamine-2-epimerase and N-acetyl-d-neuraminic Acid Aldolase

  • Chao-Hung Kao
  • Yih-Yuan Chen
  • Lian-Ren Wang
  • Yen-Chung Lee
Original Paper


N-acetyl-d-neuraminic acid (Neu5Ac) is a costly precursor for many drugs such as anti-influenza antivirals. In a previous study, a whole-cell process for Neu5Ac production was developed using a combination of two Escherichia coli cells expressing Anabaena sp. CH1 N-acetyl-d-glucosamine-2-epimerase (bage) and E. coli N-acetyl-d-neuraminic acid aldolase (nanA), respectively. In this study, we constructed a bAGE and NanA co-expression system to improve Neu5Ac production. Two recombinant E. coli strains, E. coli BL21 (DE3) pET-bagenanA (HA) and E. coli BL21 (DE3) pET-bage–2nanA (HAA), synchronously expressing bAGE and NanA were used as biocatalysts to generate Neu5Ac from N-acetyl-d-glucosamine (GlcNAc) and pyruvate. The HA biocatalysts produced 187.5 mM Neu5Ac within 8 h. The yield of GlcNAc was 15.6%, and the Neu5Ac production rate was 7.25 g/L/h. The most active HAA biocatalysts generated 412.6 mM Neu5Ac and a GlcNAc yield of 34.4%. HAA achieved a Neu5Ac production rate of 15.9 g/L/h, which surpassed those for all reported Neu5Ac production processes so far. The present study demonstrates that using recombinant E. coli cells synchronously expressing bAGE and NanA as biocatalysts could potentially be used in the industrial mass production of Neu5Ac.


N-acetyl-d-glucosamine-2-epimerase N-acetyl-d-neuraminic acid aldolase Co-expression Neu5Ac production Neu5Ac productivity Conversion yield 



Acknowledgment is made to the financial support of National Science Council of Taiwan (Grant No. NSC 101-2313-B-241-001-MY3) and Ministry of Science and Technology of Taiwan (Grant Nos. MOST 103-2313-B-415-005 and MOST 106-2313-B-241-001). We would like to thank Editage ( for English language editing and Publication Support.

Compliance with Ethical Standards

Conflict of interest

The authors declare no conflict of interest.


  1. 1.
    Varki, A., & Schauer, R. (2009). Sialic acids. In A. Varki, R. D. Cummings, J. D. Esko, H. H. Freeze, P. Stanley, C. R. Bertozzi, G. W. Hart & M. E. Etzler (Eds.), Essentials of glycobiology (2nd ed., pp. 199–218). Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.Google Scholar
  2. 2.
    Inoue, S., & Kitajima, K. (2006). KDN (deaminated neuraminic acid): Dreamful past and exciting future of the newest member of the sialic acid family. Glycoconjugate Journal, 23, 277–290.CrossRefPubMedGoogle Scholar
  3. 3.
    Schauer, R. (2009). Sialic acids as regulators of molecular and cellular interactions. Current Opinion in Structural Biology, 19, 507–514.CrossRefPubMedGoogle Scholar
  4. 4.
    Chen, X., & Varki, A. (2010). Advances in the biology and chemistry of sialic acids. ACS Chemical Biology, 5, 163–176.CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Varki, A. (2008). Sialic acids in human health and disease. Trends in Molecular Medicine, 14, 351–360.CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Wang, B., & Brand-Miller, J. (2003). The role and potential of sialic acid in human nutrition. European Journal of Clinical Nutrition, 57, 1351–1369.CrossRefPubMedGoogle Scholar
  7. 7.
    Kawai, N., Ikematsu, H., Iwaki, N., Maeda, T., Kawashima, T., Hirotsu, N., & Kashiwagi, S. (2009). Comparison of the effectiveness of zanamivir and oseltamivir against influenza A/H1N1, A/H3N2, and B. Clinical Infectious Diseases, 48, 996–997.CrossRefPubMedGoogle Scholar
  8. 8.
    von Itzstein, M. (2007). The war against influenza: Discovery and development of sialidase inhibitors. Nature Reviews Drug Discovery, 6, 967–974.CrossRefGoogle Scholar
  9. 9.
    Koketsu, M., Juneja, L. R., Kawanami, H., Kim, M., & Yamamoto, T. (1992). Preparation of N-acetylneuraminic acid from delipidated egg yolk. Glycoconjugate Journal, 9, 70–74.CrossRefPubMedGoogle Scholar
  10. 10.
    Juneja, L. R., Koketsu, M., Nishimoto, K., Kim, M., Yamamoto, T., & Itoh, T. (1991). Large-scale preparation of sialic acid from chalaza and egg-yolk membrane. Carbohydrate Research, 214, 179–186.CrossRefPubMedGoogle Scholar
  11. 11.
    Furuhata, K. (2004). Chemistry of N-acetylneuraminic acid (Neu5Ac). Trends in Glycoscience and Glycotechnology, 16, 143–169.CrossRefGoogle Scholar
  12. 12.
    Deninno, M. P. (1991). The synthesis and glycosidation of N-acetylneuraminic acid. Synthesis, 8, 583–593.CrossRefGoogle Scholar
  13. 13.
    Ferrero, M. A., Reglero, A., Fernandez-Lopez, M., Ordas, R., & Rodriguez-Aparicio, L. B. (1996). N-acetyl-D-neuraminic acid lyase generates the sialic acid for colominic acid biosynthesis in Escherichia coli K1. The Biochemical Journal, 317, 157–165.CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Maru, I., Ohnishi, J., Ohta, Y., & Tsukada, Y. (1998). Simple and large-scale production of N-acetylneuraminic acid from N-acetyl-D-glucosamine and pyruvate using N-acyl-D-glucosamine-2-epimerase and N-acetylneuraminate lyase. Carbohydrate Research, 306, 575–578.CrossRefPubMedGoogle Scholar
  15. 15.
    Kragl, U., Gygax, D., Ghisalba, O., & Wandrey, C. (1991). Enzymatic two-step synthesis of N-acetyl-neuraminic acid in the enzyme membrane reactor. Angewandte Chemie International Edition, 30, 827–828.CrossRefGoogle Scholar
  16. 16.
    Lee, J. O., Yi, J. K., Lee, S. G., Takahashi, S., & Kim, B. G. (2004). Production of N-acetylneuraminic acid from N-acetylglucosamine and pyruvate using recombinant human renin binding protein and sialic acid aldolase in one pot. Enzyme and Microbial Technology, 35, 121–125.CrossRefGoogle Scholar
  17. 17.
    Zimmermann, V., Hennemann, H. G., Daumann, T., & Kragl, U. (2007). Modelling the reaction course of N-acetylneuraminic acid synthesis from N-acetyl-D-glucosamine—New strategies for the optimisation of neuraminic acid synthesis. Applied Microbiology and Biotechnology, 76, 597–605.CrossRefPubMedGoogle Scholar
  18. 18.
    Wang, T. H., Chen, Y. Y., Pan, H. H., Wang, F. P., Cheng, C. H., & Lee, W. C. (2009). Production of N-acetyl-D-neuraminic acid using two sequential enzymes overexpressed as double-tagged fusion proteins. BMC Biotechnology, 9, 63.CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Hu, S. Y., Chen, J., Yang, Z. Y., Shao, L. J., Bai, H., Luo, J. L., Jiang, W. H., & Yang, Y. L. (2010). Coupled bioconversion for preparation of N-acetyl-d-neuraminic acid using immobilized N-acetyl-d-glucosamine-2-epimerase and N-acetyl-d-neuraminic acid lyase. Applied Microbiology and Biotechnology, 85, 1383–1391.CrossRefPubMedGoogle Scholar
  20. 20.
    Datta, A. (1970). Regulatory role of adenosine triphosphate on hog kidney N-acetyl-D-glucosamine-2-epimerase. Biochemistry, 9, 3363–3370.CrossRefPubMedGoogle Scholar
  21. 21.
    Tabata, K., Koizumi, S., Endo, T., & Ozaki, A. (2002). Production of N-acetyl-D-neuraminic acid by coupling bacteria expressing N-acetyl-D-glucosamine-2-epimerase and N-acetyl-D-neuraminic acid synthetase. Enzyme and Microbial Technology, 30, 327–333.CrossRefGoogle Scholar
  22. 22.
    Tao, F., Zhang, Y. A., Ma, C. Q., & Xu, P. (2010). Biotechnological production and applications of N-acetyl-d-neuraminic acid: Current state and perspectives. Applied Microbiology and Biotechnology, 87, 1281–1289.CrossRefPubMedGoogle Scholar
  23. 23.
    Zhang, Y., Tao, F., Du, M., Ma, C., Qiu, J., Gu, L., He, X., & Xu, P. (2010). An efficient method for N-acetyl-D-neuraminic acid production using coupled bacterial cells with a safe temperature-induced system. Applied Microbiology and Biotechnology, 86, 481–489.CrossRefPubMedGoogle Scholar
  24. 24.
    Tao, F., Zhang, Y., Ma, C., & Xu, P. (2011). One-pot bio-synthesis: N-acetyl-D-neuraminic acid production by a powerful engineered whole-cell catalyst. Scientific Reports, 1, 142.CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Lin, B. X., Zhang, Z. J., Liu, W. F., Dong, Z. Y., & Tao, Y. (2013). Enhanced production of N-acetyl-D-neuraminic acid by multi-approach whole-cell biocatalyst. Applied Microbiology and Biotechnology, 97, 4775–4784.CrossRefPubMedGoogle Scholar
  26. 26.
    Zhou, J. B., Chen, X. Z., Lu, L. P., Govender, A., Yang, H. Q., & Shen, W. (2016). Enhanced production of N-acetyl-D-neuraminic acid by whole-cell bio-catalysis of Escherichia coli. Journal of Molecular Catalysis B: Enzymatic, 125, 42–48.CrossRefGoogle Scholar
  27. 27.
    Zhu, D. Q., Zhan, X. B., Wu, J. R., Gao, M. J., & Zhao, Z. S. (2017). Efficient whole-cell biocatalyst for Neu5Ac production by manipulating synthetic, degradation and transmembrane pathways. Biotechnology Letters, 39, 55–63.CrossRefPubMedGoogle Scholar
  28. 28.
    Zhu, D. Q., Wu, J. R., Zhan, X. B., Zhu, L., Zheng, Z. Y., & Gao, M. J. (2017). Phosphoenolpyruvate-supply module in Escherichia coli improves N-acetyl-d-neuraminic acid biocatalysis. Biotechnology Letters, 39, 227–234.CrossRefPubMedGoogle Scholar
  29. 29.
    Ishikawa, M., & Koizumi, S. (2010). Microbial production of N-acetylneuraminic acid by genetically engineered Escherichia coli. Carbohydrate Research, 345, 2605–2609.CrossRefPubMedGoogle Scholar
  30. 30.
    Lee, Y. C., Chien, H. C. R., & Hsu, W. H. (2007). Production of N-acetyl-D-neuraminic acid by recombinant whole cells expressing Anabaena sp. CH1 N-acetyl-D-glucosamine-2-epimerase and Escherichia coli N-acetyl-D-neuraminic acid lyase. Journal of Biotechnology, 129, 453–460.CrossRefPubMedGoogle Scholar
  31. 31.
    Sambrook, J., & Russell, D. W. (2001). Molecular cloning: A laboratory manual (3rd ed.). Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.Google Scholar
  32. 32.
    Sun, W., Ji, W., Li, N., Tong, P., Cheng, J., He, Y., Chen, Y., Chen, X., Wu, J., Ouyang, P., Xie, J., & Ying, H. (2013). Construction and expression of a polycistronic plasmid encoding N-acetylglucosamine-2-epimerase and N-acetylneuraminic acid lyase simultaneously for production of N-acetylneuraminic acid. Bioresource Technology, 130, 23–29.CrossRefPubMedGoogle Scholar
  33. 33.
    Xu, J. W., Zhao, W., & Zhong, J. J. (2010). Biotechnological production and application of ganoderic acids. Applied Microbiology and Biotechnology, 87, 457–466.CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Chao-Hung Kao
    • 1
    • 2
  • Yih-Yuan Chen
    • 3
  • Lian-Ren Wang
    • 4
  • Yen-Chung Lee
    • 4
  1. 1.Department of BiotechnologyHungkuang UniversityTaichungTaiwan, Republic of China
  2. 2.Bachelor Degree Program in Animal HealthcareHungkuang UniversityTaichungTaiwan, Republic of China
  3. 3.Department of Biochemical Science and TechnologyNational Chiayi UniversityChiayiTaiwan, Republic of China
  4. 4.Department of Bioagricultural ScienceNational Chiayi UniversityChiayiTaiwan, Republic of China

Personalised recommendations