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High Cell Density Cultivation of Recombinant Escherichia coli Strains Expressing 2-O-Sulfotransferase and C5-Epimerase for the Production of Bioengineered Heparin

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Abstract

Bioengineered heparin is being investigated as a potential substitute for the animal-sourced anticoagulant drug. One step in the current process to prepare bioengineered heparin involves the conversion of N-sulfo heparosan, rich in → 4)GlcNS(1 → 4) GlcA(1 → sequences (where S is sulfo, GlcN is α-d-glucosamine, and GlcA is β-d-glucuronic acid), to a critical intermediate, rich in → 4)GlcNS(1 → 4) IdoA2S(1 → sequences (where S is sulfo and IdoA is α-l-iduronic acid), using 2-O-sulfotransferase (2-OST) and C5 epimerase (C5-epi). Until now, these heparan sulfate biosynthetic enzymes have been expressed in Escherichia coli grown in shake flask culture as fusion proteins. The current study is focused on the high cell density fed-batch cultivation of recombinant E. coli strains expressing both enzymes. We report the high productivity expression of active 2-OST and C5-epi enzymes of 6.0 and 2.2 mg/g dry cell weight, respectively.

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Acknowledgments

The authors gratefully acknowledge funding from the NIH in the form of grant HL096972, from the National Natural Science Foundation of China under Grant No. 31171737 and the PRC grant 210208310507 supporting the visit of Jianhua Zhang, and from the Bioengineered Heparin Consortium.

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Authors and Affiliations

  1. Department of Food Science and Engineering, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai, 200240, People’s Republic of China

    Jianhua Zhang

  2. Department of Chemical and Biological Engineering, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY, 12180, USA

    Jianhua Zhang, Jonathan S. Dordick, Robert J. Linhardt & Fuming Zhang

  3. Department of Biology, Rensselaer Polytechnic Institute, Troy, NY, 12180, USA

    Matt Suflita, Jonathan S. Dordick & Robert J. Linhardt

  4. Department of Chemistry and Chemical, Biology, Biomedical Engineering, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY, 12180, USA

    Guoyun Li, Lingyun Li & Robert J. Linhardt

  5. College of Biological and Environmental Engineering, Zhejiang University of Technology, Hangzhou, 310032, People’s Republic of China

    Weihong Zhong

  6. Department of Biomedical Engineering, Rensselaer Polytechnic Institute, Troy, NY, 12180, USA

    Jonathan S. Dordick & Robert J. Linhardt

Authors
  1. Jianhua Zhang
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  2. Matt Suflita
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  3. Guoyun Li
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  5. Lingyun Li
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  6. Jonathan S. Dordick
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  7. Robert J. Linhardt
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  8. Fuming Zhang
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Correspondence to Jianhua Zhang or Fuming Zhang.

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Zhang, J., Suflita, M., Li, G. et al. High Cell Density Cultivation of Recombinant Escherichia coli Strains Expressing 2-O-Sulfotransferase and C5-Epimerase for the Production of Bioengineered Heparin. Appl Biochem Biotechnol 175, 2986–2995 (2015). https://doi.org/10.1007/s12010-014-1466-1

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  • DOI: https://doi.org/10.1007/s12010-014-1466-1

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