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
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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-bage–nanA (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.
KeywordsN-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 (http://www.editage.com) for English language editing and Publication Support.
Compliance with Ethical Standards
Conflict of interest
The authors declare no conflict of interest.
- 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
- 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.CrossRefGoogle Scholar
- 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.CrossRefGoogle Scholar
- 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.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.CrossRefGoogle Scholar