title: High cell density cultivation of a recombinant E. coli strain expressing a key enzyme in bioengineered heparin production.
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).
authors: Department of Experimental Medicine, Second University of Naples, Via De Crecchio 7, 80138 Naples, Italy.
authors: Odile Francesca
authors: OF
authors: Restaino
authors: Ujjwal
authors: U
authors: Bhaskar
authors: Priscilla
authors: P
authors: Paul
authors: Lingyun
authors: L
authors: Li
authors: Mario
authors: M
authors: De Rosa
authors: Jonathan S
authors: JS
authors: Dordick
authors: Robert J
authors: RJ
authors: Linhardt
mesh_terms: D015239:Arylsulfotransferase / Q000235:genetics / Q000378:metabolism; D001698:Biomedical Engineering / Q000379:methods*; D004187:Disaccharides / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism*; D006493:Heparin / Q000096:biosynthesis*
chemical_list: D004187:Disaccharides; C560363:heparosan; D006493:Heparin; D015239:Arylsulfotransferase