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, and particularly 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.

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

“…Concentrations of glucose and acetic acid in the medium during the fermentation were measured by HPLC as previously described [13]. …”

High cell density cultivation of a recombinant Escherichia coli strain expressing a 6-O -sulfotransferase for the production of bioengineered heparin

“…The 6‐OST activity assay was performed as previously described with some modification (Restaino et al …”

“…We recently reported the fed‐batch production of 6‐ O ‐sulfotransferase isoform‐1 (Restaino et al . ). In the current manuscript, we now report the fed‐batch production of 6‐ O ‐sulfotransferase isoform‐3 (6‐OST‐3).…”

Heparin and related polysaccharides: synthesis using recombinant enzymes and metabolic engineering

“…While protein engineering offers opportunities to improve the stability and activity of these recombinant enzyme catalysts, the lack of crystal structures for many of these enzymes posses a barrier to progress. Further efforts to scale-up the production of these enzymes in fed-batch fermenters are underway, and have been demonstrated for 4 out of 5 of the HS sulfotransferases including 2OST-1, C5 epimerase, 6OST-1 and 6OST-3 (Restaino et al 2013a; Zhang et al 2015a; Zhang et al 2015b). This opens the way for the industrial scale production of GAGs.…”