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Biotechnology Letters

, Volume 39, Issue 1, pp 55–63 | Cite as

Efficient whole-cell biocatalyst for Neu5Ac production by manipulating synthetic, degradation and transmembrane pathways

  • Deqiang Zhu
  • Xiaobei Zhan
  • Jianrong Wu
  • Minjie Gao
  • Zhongsheng Zhao
Original Research Paper

Abstract

Objective

To develop a strategy for producing N-acetyl-d-neuraminic acid (Neu5Ac), which is often synthesized from exogenous N-acetylglucosamine (GlcNAc) and pyruvate, but without using pyruvate.

Result

An efficient three-module whole-cell biocatalyst strategy for Neu5Ac production by utilizing intracellular phosphoenolpyruvate was established. In module I, the synthetic pathway was constructed by coexpressing GlcNAc 2-epimerase from Anabaena sp. CH1 and Neu5Ac synthase from Campylobacter jejuni in Escherichia coli. In module II, the Neu5Ac degradation pathway of E. coli was knocked out, resulting in 2.6 ± 0.06 g Neu5Ac l−1 after 72 h in Erlenmeyer flasks. In module III, the transmembrane mode of GlcNAc was modified by disruption of GlcNAc-specific phosphotransferase system and Neu5Ac now reached 3.7 ± 0.04 g l−1. In scale-up catalysis with a 1 l fermenter, the final Neu5Ac yield was 7.2 ± 0.08 g l−1.

Conclusion

A three-module whole-cell biocatalyst strategy by manipulating synthetic, degradation and transmembrane pathways in E. coli was an economical method for Neu5Ac production.

Keywords

Coexpressing vector GlcNAc-specific PTS Neu5Ac NeuNAc synthase Phosphoenolpyruvate Whole-cell biocatalyst 

Notes

Acknowledgments

This research was supported by the National High Technology Research and Development Program of China (2012AA021505), the National Natural Science Foundation of China No. 31171640, and the Program of Introducing Talents of Discipline to Universities (111-2-06), the Fundamental Research Funds for the Central Universities (JUSRP51504, JUSRP51632A).

Supporting information

Supplementary Table 1—Strains and plasmids used.

Supplementary Table 2—Primers used.

Supplementary material

10529_2016_2215_MOESM1_ESM.docx (19 kb)
Supplementary material 1 (DOCX 19 kb)

References

  1. Choi YH, Kim JH, Park JH, Lee N, Kim DH, Jang KS, Park IH, Kim BG (2014) Protein engineering of alpha 2,3/2,6-sialyltransferase to improve the yield and productivity of in vitro sialyllactose synthesis. Glycobiology 24:159–169CrossRefPubMedGoogle Scholar
  2. Datsenko KA, Wanner BL (2000) One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci USA 97:6640–6645CrossRefPubMedPubMedCentralGoogle Scholar
  3. Deng MD, Severson DK, Grund AD, Wassink SL, Burlingame RP, Berry A, Running JA, Kunesh CA, Song L, Jerrell TA, Rosson RA (2005) Metabolic engineering of Escherichia coli for industrial production of glucosamine and N-acetylglucosamine. Metab Eng 7:201–214CrossRefPubMedGoogle Scholar
  4. El Maarouf A, Petridis AK, Rutishauser U (2006) Use of polysialic acid in repair of the central nervous system. Proc Natl Acad Sci 103:16989–16994CrossRefPubMedPubMedCentralGoogle Scholar
  5. Gunawan J, Simard D, Gilbert M, Lovering AL, Wakarchuk WW, Tanner ME, Strynadka NCJ (2005) Structural and mechanistic analysis of sialic acid synthase NeuB from Neisseria meningitidis in complex with Mn2+, phosphoenolpyruvate, and N-acetylmannosaminitol. J Biol Chem 280:3555–3563CrossRefPubMedGoogle Scholar
  6. Lakdawala SS, Jayaraman A, Halpin RA, Lamirande EW, Shih AR, Stockwell TB, Lin X, Simenauer A, Hanson CT, Vogel L, Paskel M, Minai M, Moore I, Orandle M, Das SR, Wentworth DE, Sasisekharan R, Subbarao K (2015) The soft palate is an important site of adaptation for transmissible influenza viruses. Nature 526:122CrossRefPubMedPubMedCentralGoogle Scholar
  7. Lin BX, Zhang ZJ, Liu WF, Dong ZY, Tao Y (2013) Enhanced production of N-acetyl-d-neuraminic acid by multi-approach whole-cell biocatalyst. Appl Microbiol Biotechnol 97:4775–4784CrossRefPubMedGoogle Scholar
  8. Linton D, Karlyshev AV, Hitchen PG, Morris HR, Dell A, Gregson NA, Wren BW (2000) Multiple N-acetyl neuraminic acid synthetase (neuB) genes in Campylobacter jejuni: identification and characterization of the gene involved in sialylation of lipo-oligosaccharide. Mol Microbiol 35:1120–1134CrossRefPubMedGoogle Scholar
  9. Liu F, Lee HJ, Strynadka NCJ, Tanner ME (2009) Inhibition of Neisseria meningitidis sialic acid synthase by a tetrahedral intermediate analogue. Biochemistry 48:9194–9201CrossRefPubMedGoogle Scholar
  10. Rutishauser U (2008) Polysialic acid in the plasticity of the developing and adult vertebrate nervous system. Nat Rev Neurosci 9:26–35CrossRefPubMedGoogle Scholar
  11. Song W, Bahn SY, Cha HJ, Pack SP, Choi YS (2016) Recombinant production of a shell matrix protein in Escherichia coli and its application to the biomimetic synthesis of spherulitic calcite crystals. Biotechnol Lett 38:809–816CrossRefPubMedGoogle Scholar
  12. Sundaram AK, Pitts L, Muhammad K, Wu J, Betenbaugh M, Woodard RW, Vann WF (2004) Characterization of N-acetylneuraminic acid synthase isoenzyme 1 from Campylobacter jejuni. Biochem J 383:83–89CrossRefPubMedPubMedCentralGoogle Scholar
  13. Tabe-Bordbar S, Marashi S-A (2013) Finding elementary flux modes in metabolic networks based on flux balance analysis and flux coupling analysis: application to the analysis of Escherichia coli metabolism. Biotechnol Lett 35:2039–2044CrossRefPubMedGoogle Scholar
  14. Uehara T, Park JT (2004) The N-acetyl-d-glucosamine kinase of Escherichia coli and its role in murein recycling. J Bacteriol 186:7273–7279CrossRefPubMedPubMedCentralGoogle Scholar
  15. Vann WF, Tavarez JJ, Crowley J, Vimr E, Silver RP (1997) Purification and characterization of the Escherichia coli K1 neuB gene product N-acetylneuraminic acid synthetase. Glycobiology 7:697–701CrossRefPubMedGoogle Scholar
  16. Vimr ER, Kalivoda KA, Deszo EL, Steenbergen SM (2004) Diversity of microbial sialic acid metabolism. Microbiol Mol Biol Rev 68:132–153CrossRefPubMedPubMedCentralGoogle Scholar
  17. Wang B (2012) Molecular mechanism underlying sialic acid as an essential nutrient for brain development and cognition. Adv Nutr 3:465S–472SCrossRefPubMedPubMedCentralGoogle Scholar
  18. Yang LB, Zhan XB, Zheng ZY, Wu JR, Gao MJ, Lin CC (2014) A novel osmotic pressure control fed-batch fermentation strategy for improvement of erythritol production by Yarrowia lipolytica from glycerol. Bioresour Technol 151:120–127CrossRefPubMedGoogle Scholar
  19. Zhou Z, Wang C, Xu H, Chen Z, Cai H (2015) Increasing succinic acid production using the PTS-independent glucose transport system in a Corynebacterium glutamicum PTS-defective mutant. J Ind Microbiol Biotechnol 42:1073–1082CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2016

Authors and Affiliations

  • Deqiang Zhu
    • 1
  • Xiaobei Zhan
    • 1
  • Jianrong Wu
    • 1
  • Minjie Gao
    • 1
  • Zhongsheng Zhao
    • 1
  1. 1.The Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of BiotechnologyJiangnan UniversityWuxiChina

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