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.
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References
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 24:159–169
Datsenko KA, Wanner BL (2000) One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci USA 97:6640–6645
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 7:201–214
El Maarouf A, Petridis AK, Rutishauser U (2006) Use of polysialic acid in repair of the central nervous system. Proc Natl Acad Sci 103:16989–16994
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 280:3555–3563
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 526:122
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 97:4775–4784
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 35:1120–1134
Liu F, Lee HJ, Strynadka NCJ, Tanner ME (2009) Inhibition of Neisseria meningitidis sialic acid synthase by a tetrahedral intermediate analogue. Biochemistry 48:9194–9201
Rutishauser U (2008) Polysialic acid in the plasticity of the developing and adult vertebrate nervous system. Nat Rev Neurosci 9:26–35
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 38:809–816
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 383:83–89
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 35:2039–2044
Uehara T, Park JT (2004) The N-acetyl-d-glucosamine kinase of Escherichia coli and its role in murein recycling. J Bacteriol 186:7273–7279
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 7:697–701
Vimr ER, Kalivoda KA, Deszo EL, Steenbergen SM (2004) Diversity of microbial sialic acid metabolism. Microbiol Mol Biol Rev 68:132–153
Wang B (2012) Molecular mechanism underlying sialic acid as an essential nutrient for brain development and cognition. Adv Nutr 3:465S–472S
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 151:120–127
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 42:1073–1082
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.
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Zhu, D., Zhan, X., Wu, J. et al. Efficient whole-cell biocatalyst for Neu5Ac production by manipulating synthetic, degradation and transmembrane pathways. Biotechnol Lett 39, 55–63 (2017). https://doi.org/10.1007/s10529-016-2215-z
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DOI: https://doi.org/10.1007/s10529-016-2215-z