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

Abstract

Heparosan is an acidic polysaccharide natural product, which serves as the critical precursor in heparin biosynthesis and in the chemoenzymatic synthesis of bioengineered heparin. Heparosan is also the capsular polysaccharide of Escherichia coli K5 strain. The current study was focused on the examination of the fermentation of E. coli K5 with the goal of producing heparosan in high yield and volumetric productivity. The structure and molecular weight properties of this bacterial heparosan were determined using polyacrylamide gel electrophoresis and Fourier transform-mass spectrometry. Fermentation of E. coli K5 in a defined medium using exponential fed batch glucose addition with oxygen enrichment afforded heparosan at 15 g/L having a number average molecular weight of 58,000 Da and a weight average molecular weight of 84,000 Da.

Operation of a Benchtop Bioreactor

“…New users of benchtop Bioreactors may have trouble determining initial setpoints for pH, pO 2 and temperature; however, published research can be referenced for this information 5,6,7,8,9 . With bacterial cultures in particular, it is recommended to start agitation at the same speed as the shaker flask and the temperature at the same setpoint.…”

Escherichia coliK5 heparosan fermentation and improvement by genetic engineering

“…[6][7][8][9] Therefore, the production of heparosan is the first step in making bioengineered heparin. 10 Heparosan is biosynthesized as a polysaccharide capsule in bacteria including Escherichia coli K5 and Pasteurella multicida. [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.…”

“…[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.…”

“…In our previous study, we have increased the Escherichia coli K5 heparosan fermentation yield and productivity, and extensively studied the structure and molecular weight of the heparosan produced. 10 High cell density culturing of Escherichia coli using an exponential feeding strategy, can afford up to 15 g/L heparosan at a productivity of 0.4 g/L*h. In traditional exponential feeding, active feedback is absent on the nutritional status of the culture, and there are risks of overfeeding glucose, resulting in the accumulation of toxic byproducts such as acetate, preventing the culture to growing to higher cell density. 24,25 In a 20 L fermentor with a 15 L working volume, a pH-stat feeding strategy was utilized with culture Minimally, cells must have the capacity to replenish the supply of the components of cell wall synthesis needed to support rapid cell growth.…”

“…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.…”