Production of K5 Polysaccharides of Different Molecular Weight by Escherichia Coli

Abstract

An investigation was made of the molecular weight of the Escherichia coli antigenic K5 polysaccharide. Two components with molecular weights of 16,000 and 1,500 were observed. The ratio of these two components depended on a lyase produced by the same E. coli. This lyase acts by a β-elimination mechanism to depolymerize the K5. At the end of the fermentation the thermally treated (to inactivate the lyase) and untreated twenty-four-hour-old cultures each contained two K5 components of different Mw, in different ratios. The two components were monitored over a fermentation time-course. "3C-NMR spectrometry was employed to verify the lyase mechanism of action.

E. coli K5 fermentation and the preparation of heparosan, a bioengineered heparin precursor

“…The K5 heparosan chain is believed to be anchored on the cell surface through lipid substitution at the reducing end of the polysaccharide to a phosphatidic acid molecule in the outer membrane of E. coli (Alexander and Schmidt, 1982; Jann and Jann, 1990). Portions of the heparosan polysaccharide can be shed from E. coli K5 through the action of K5 heparosan lyase, an enzyme originating from a bacterial phage that cleaves the heparosan chain through a β‐elimination mechanism (Hanfling et al, 1996; Manzoni et al, 1996, 2000). The gene encoding K5 lyase is integrated into the E. coli K5 DNA (Manzoni et al, 1996) and its expression may be inducible (Legoux et al, 1996; Manzoni et al, 1996, 2000).…”

Escherichia coliK5 heparosan fermentation and improvement by genetic engineering

“…Previous studies have shown that the activity of K5 lyase expressed by the Escherichia coli was affected by growth media and culture conditions. 10,15,32 However, the mechanism to control the lyase expression is not clear. It seems the lyase is present throughout the entire time course of the fermentation.…”

“…Previous studies have shown that the K5 lyase exist in both an intracellular form and an extracellular form. 15,32 This suggests that Escherichia coli has the machinery to export the lyase to the extracellular space. A new K5 expression system would be needed that would may be effective for increasing heparosan production.…”

“…31 The lyase gene is integrated into the Escherichia coli K5 DNA. 15 An improvement by genetic engineering and metabolic engineering may represent an excellent strategy to afford heparosan with ideal structural properties and in excellent yields for use as a precursor for bioengineered heparin production.…”

“…[11][12][13] While the heparosan produced by P. multicida is usually of high molecular weight ~200-300 KDa, 14 the Escherichia coli K5 heparosan is closer to the size of heparin, with an average molecular weight of 10 KDa to 20 KDa. 9,10,15 For this reason, we chose Escherichia coli K5 as our bacterial strain for further study. The K5 heparosan polysaccharide, comprises the Escherichia coli capsule and acts as molecular camouflage, rendering reduced immunogenicity 11,12 because of its identical structure to mammalian heparosan, the biosynthetic precursor of heparin and heparan sulfate.…”