Applied Microbiology and Biotechnology

, Volume 97, Issue 9, pp 3893–3900 | Cite as

High cell density cultivation of a recombinant E. coli strain expressing a key enzyme in bioengineered heparin production

  • Odile Francesca Restaino
  • Ujjwal Bhaskar
  • Priscilla Paul
  • Lingyun Li
  • Mario De Rosa
  • Jonathan S. Dordick
  • Robert J. Linhardt
Biotechnological products and process engineering

Abstract

A bioengineered heparin, as a replacement for animal-derived heparin, is under development that relies on the fermentative production of heparosan by Escherichia coli K5 and its subsequent chemoenzymatic modification using biosynthetic enzymes. A critical enzyme in this pathway is the mammalian 6-O-sulfotransferase (6-OST-1) which specifically sulfonates the glucosamine residue in a heparin precursor. This mammalian enzyme, previously cloned and expressed in E. coli, is required in kilogram amounts if an industrial process for bioengineered heparin is to be established. In this study, high cell density cultivation techniques were exploited to obtain recombinant 6-OST-1. Physiological studies were performed in shake flasks to establish optimized growth and production conditions. Induction strategies were tested in fed-batch experiments to improve yield and productivity. High cell density cultivation in 7-l culture, together with a coupled inducer strategy using isopropyl β-d-1-thiogalactopyranoside and galactose, afforded 482 mg l−1 of enzyme with a biomass yield of 16.2 mg gcdw−1 and a productivity of 10.5 mg l−1 h−1.

Keywords

Bioengineered heparin High cell density cultivations Heparosan Sulfotransferase 

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Copyright information

© Springer-Verlag Berlin Heidelberg 2013

Authors and Affiliations

  • Odile Francesca Restaino
    • 1
    • 2
  • Ujjwal Bhaskar
    • 2
    • 3
  • Priscilla Paul
    • 2
    • 3
  • Lingyun Li
    • 2
    • 4
  • Mario De Rosa
    • 1
  • Jonathan S. Dordick
    • 2
    • 3
    • 5
    • 6
    • 7
  • Robert J. Linhardt
    • 2
    • 3
    • 4
    • 5
    • 7
  1. 1.Department of Experimental MedicineSecond University of NaplesNaplesItaly
  2. 2.Center for Biotechnology and Interdisciplinary StudiesTroyUSA
  3. 3.Department of Chemical and Biological EngineeringTroyUSA
  4. 4.Department of Chemistry and Chemical BiologyRensselaer Polytechnic InstituteTroyUSA
  5. 5.Department of BiologyRensselaer Polytechnic InstituteTroyUSA
  6. 6.Department of Materials Science and EngineeringTroyUSA
  7. 7.Department of Biomedical EngineeringTroyUSA

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