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Enhanced production of  N-acetyl-d-neuraminic acid by multi-approach whole-cell biocatalyst

  • Biotechnological products and process engineering
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Abstract

N-Acetyl-d-neuraminic acid (Neu5Ac) has attracted considerable interest due to its promising potential applications in medicine. Significant efforts have been made in whole-cell biocatalyst for Neu5Ac production, but the processes often result in suboptimal performance due to poor expression of enzymes, imbalances of pathway components, disturbance of competing pathways, and barriers of mass transport. In this study, we engineered Escherichia coli strains capable of producing Neu5Ac by assembling a two-step heterologous pathway consisting of N-acetyl-d-glucosamine 2-epimerase (AGE) and Neu5Ac aldolase (NanA). Multiple approaches were used to improve the efficiency of the engineered pathway and process for enhanced Neu5Ac production. Firstly, we identified that NanA was the rate-controlling enzyme in this pathway. With increased expression of NanA, a ninefold increase in Neu5Ac production (65 mM) was observed. Secondly, knocking out nanTEK genes blocked Neu5Ac uptake and the competing pathway, which kept the reactions to the synthetic direction as the final product went outside of the cells and enhanced the Neu5Ac production by threefold, resulting in 173.8 mM of Neu5Ac. Thirdly, we improved the performance of the system by promoting substrate transport and optimizing concentrations of substrates. An overall whole-cell biocatalytic process was developed and a maximum titer of 240 mM Neu5Ac (74.2 g/L) was achieved, with productivity of 6.2 g Neu5Ac/L/h and conversion yield of 40 % from GlcNAc. The engineered strain could be reused for at least five cycles with a productivity of >6 g/L/h. It is a cost-effective process for Neu5Ac production with potential applications in large-scale industrial production.

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Acknowledgments

This work was supported by grants from Special Foundation of President of the Chinese Academy of Sciences for Yong Tao (China, Y129011EE1) and Postdoctoral Science Foundation of China for Baixue Lin (China, 2011M500420).

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Authors and Affiliations

  1. Department of Industrial Microbiology and Biotechnology, Institute of Microbiology, Chinese Academy of Sciences, NO.1 West Beichen Road, Chaoyang District, Beijing, 100101, People’s Republic of China

    Bai-Xue Lin, Zi-Juan Zhang, Wei-Feng Liu & Yong Tao

  2. State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100101, People’s Republic of China

    Zhi-Yang Dong

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  1. Bai-Xue Lin
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  2. Zi-Juan Zhang
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  3. Wei-Feng Liu
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  4. Zhi-Yang Dong
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  5. Yong Tao
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Correspondence to Yong Tao.

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Lin, BX., Zhang, ZJ., Liu, WF. et al. Enhanced production of  N-acetyl-d-neuraminic acid by multi-approach whole-cell biocatalyst. Appl Microbiol Biotechnol 97, 4775–4784 (2013). https://doi.org/10.1007/s00253-013-4754-8

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  • DOI: https://doi.org/10.1007/s00253-013-4754-8

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