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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 (PAGE) 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.

SUBMITTER: Wang Z

PROVIDER: S-EPMC2974770 | biostudies-literature | 2010 Dec

REPOSITORIES: biostudies-literature

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

Wang Zhenyu Z Ly Mellisa M Zhang Fuming F Zhong Weihong W Suen Amy A Hickey Anne Marie AM Dordick Jonathan S JS Linhardt Robert J RJ

Biotechnology and bioengineering 20101201 6

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 d ...[more]

PMID: 20717972

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Controlled Photochemical Depolymerization of K5 Heparosan, a Bioengineered Heparin Precursor.

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Analysis of E. coli K5 capsular polysaccharide heparosan.

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Project description:A capsular polysaccharide from Escherichia coli K5 was previously found to have the same structure, [-(4)beta GlcA(1)----(4)alpha GlcNAc(1)-]n, as that of the non-sulphated precursor polysaccharide in heparin biosynthesis [Vann, Schmidt, Jann & Jann (1981) Eur. J. Biochem. 116, 359-364]. The K5 polysaccharide was N-deacetylated (by hydrazinolysis) and N-sulphated, and was then incubated with detergent-solubilized enzymes from a heparin-producing mouse mastocytoma, in the presence of adenosine 3'-phosphate 5'-phospho[35S] sulphate ([35S]PAPS). Structural analysis of the resulting 35S-labelled polysaccharide revealed the formation of all the major disaccharide units found in heparin. The identification of 2-O-[35S]sulphated IdoA (L-iduronic acid) as well as 6-O-[35S]sulphated GlcNSO3 units demonstrated that the modified K5 polysaccharide served as a substrate in the hexuronosyl C-5-epimerase and the major O-sulphotransferase reactions involved in the biosynthesis of heparin. The GlcA units of the native (N-acetylated) E. coli polysaccharide were attacked by the epimerase only when PAPS was present in the incubations, whereas those of the chemically N-sulphated polysaccharide were epimerized also in the absence of PAPS, in accord with the notion that N-sulphate groups are required for epimerization. With increasing concentrations of PAPS, the mono-O-sulphated disaccharide unit-IdoA(2-OSO3)-GlcNSO3- was progressively converted into the di-O-sulphated species -IdoA(2-OSO3)-GlcNSO3(6-OSO3)-. A small proportion of the 35S-labelled polysaccharide was found to bind with high affinity to the proteinase inhibitor antithrombin. This proportion increased with increasing concentration of PAPS up to a level corresponding to approximately 1-2% of the total incorporated 35S. The solubilized enzymes thus catalysed all the reactions required for the generation of functional antithrombin-binding sites.

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Project description:Heparin is a widely used drug for the control of blood coagulation. The majority of heparin that is produced commercially is derived from animal sources, is poly-disperse in nature and therefore ill-defined in structure. This makes regulation of heparin challenging with respect to identifying its absolute structural identity, purity, and efficacy. This raises the question as to whether there might be alternative methods of producing commercial grade heparin. The commentary highlights ways that we might manufacture heparin using bioengineering approaches to yield a successful therapeutic replacement for animal-derived heparin in the future.

| S-EPMC4140866 | biostudies-literature

Optimization of bioprocess conditions improves production of a CHO cell-derived, bioengineered heparin.

Project description:Heparin is the most widely used anticoagulant drug in the world today. Heparin is currently produced from animal tissues, primarily porcine intestines. A recent contamination crisis motivated development of a non-animal-derived source of this critical drug. We hypothesized that Chinese hamster ovary (CHO) cells could be metabolically engineered to produce a bioengineered heparin, equivalent to current pharmaceutical heparin. We previously engineered CHO-S cells to overexpress two exogenous enzymes from the heparin/heparan sulfate biosynthetic pathway, increasing the anticoagulant activity ∼100-fold and the heparin/heparan sulfate yield ∼10-fold. Here, we explored the effects of bioprocess parameters on the yield and anticoagulant activity of the bioengineered GAGs. Fed-batch shaker-flask studies using a proprietary, chemically-defined feed, resulted in ∼two-fold increase in integrated viable cell density and a 70% increase in specific productivity, resulting in nearly three-fold increase in product titer. Transferring the process to a stirred-tank bioreactor increased the productivity further, yielding a final product concentration of ∼90 μg/mL. Unfortunately, the product composition still differs from pharmaceutical heparin, suggesting that additional metabolic engineering will be required. However, these studies clearly demonstrate bioprocess optimization, in parallel with metabolic engineering refinements, will play a substantial role in developing a bioengineered heparin to replace the current animal-derived drug.

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