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

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. Biotechnol. Bioeng. 2010;107: 964–973. © 2010 Wiley Periodicals, Inc.     

Quantitation of heparosan with heparin lyase III and spectrophotometry

Heparosan is Escherichia coli K5 capsule polysaccharide, which is the key precursor for preparing bioengineered heparin. A rapid and effective quantitative method for detecting heparosan is important in the large-scale production of heparosan. Heparin lyase III (Hep III) effectively catalyzes the heparosan depolymerization, forming unsaturated disaccharides that are measurable using a spectrophotometer at 232 nm. We report a new method for the quantitative detection of heparosan with heparin lyase III and spectrophotometry that is safer and more specific than the traditional carbazole assay. In an optimized detection system, heparosan at a minimum concentration of 0.60 g/L in fermentation broth can be detected.     

Escherichia coli K5 heparosan fermentation and improvement by genetic engineering

N-acetyl heparosan is the precursor for the biosynthesis of the important anticoagulant drug heparin. The E. coli K5 capsular heparosan polysaccharide provides a promising precursor for in vitro chemoenzymatic production of bioengineered heparin. This article explores the improvements of heparosan production for bioengineered heparin by fermentation process engineering and genetic engineering.     

Ozonolysis of the double bond of the unsaturated uronate residue in low-molecular-weight heparin and K5 heparosan

Ozone is known to add across and cleave carbon–carbon double bonds. Ozonolysis is widely used for the preparation of pharmaceuticals, for bleaching substances and for killing microorganisms in air and water sources. Some polysaccharides and oligosaccharides, such as those prepared using chemical or enzymatic β-elimination, contain a site of unsaturation. We examined ozonolysis of low-molecular-weight heparins (LMWHs), enoxaparin and logiparin, and heparosan oligo- and polysaccharides for the removal of the nonreducing terminal unsaturated uronate residue. 1D ¹H NMR showed that these ozone-treated polysaccharides retained the same structure as the starting polysaccharide, except that the C4–C5 double bond in the nonreducing end unsaturated uronate had been removed. The anticoagulant activity of the resulting product from enoxaparin and logiparin was comparable to that of the starting material. These results demonstrate that ozonolysis is an important tool for the removal of unsaturated uronate residues from LMWHs and heparosan without modification of the core polysaccharide structure or diminution of anticoagulant activity. This reaction also has potential applications in the chemoenzymatic synthesis of bioengineered heparin from Escherichia coli-derived K5 heparosan.     

Control of the heparosan <Emphasis Type="Italic">N</Emphasis>-deacetylation leads to an improved bioengineered heparin

The production of the anticoagulant drug heparin from non-animal sources has a number of advantages over the current commercial production of heparin. These advantages include better source material availability, improved quality control, and reduced concerns about animal virus or prion impurities. A bioengineered heparin would have to be chemically and biologically equivalent to be substituted for animal-sourced heparin as a pharmaceutical. In an effort to produce bioengineered heparin that more closely resembles pharmaceutical heparin, we have investigated a key step in the process involving the N-deacetylation of heparosan. The extent of N-deacetylation directly affects the N-acetyl/N-sulfo ratio in bioengineered heparin and also impacts its molecular weight. Previous studies have demonstrated that the presence and quantity of N-acetylglucosamine in the nascent glycosaminoglycan chain, serving as the substrate for the subsequent enzymatic modifications (C5 epimerization and O-sulfonation), can impact the action of these enzymes and, thus, the content and distribution of iduronic acid and O-sulfo groups. In this study, we control the N-deacetylation of heparosan to produce a bioengineered heparin with an N-acetyl/N-sulfo ratio and molecular weight that is similar to animal-sourced pharmaceutical heparin. The structural composition and anticoagulant activity of the resultant bioengineered heparin was extensively characterized and compared to pharmaceutical heparin obtained from porcine intestinal mucosa.     

Metabolic engineering of Escherichia coli BL21 for biosynthesis of heparosan, a bioengineered heparin precursor

As a precursor of bioengineered heparin, heparosan is currently produced from Escherichia coli K5, which is pathogenic bacteria potentially causing urinary tract infection. Thus, it would be advantageous to develop an alternative source of heparosan from a non-pathogeneic strain. In this work we reported the biosynthesis of heparosan via the metabolic engineering of non-pathogenic E. coli BL21 as a production host. Four genes, KfiA, KfiB, KfiC and KfiD, encoding enzymes for the biosynthesis of heparosan in E. coli K5, were cloned into inducible plasmids pETDuet-1 and pRSFDuet-1 and further transformed into E. coli BL21, yielding six recombinant strains as follows: sA, sC, sAC, sABC, sACD and sABCD. The single expression of KfiA (sA) or KfiC (sC) in E. coli BL21 did not produce heparosan, while the co-expression of KfiA and KfiC (sAC) could produce 63mg/L heparosan in shake flask. The strain sABC and sACD could produce 100 and 120mg/L heparosan, respectively, indicating that the expression of KfiB or KfiD was beneficial for heparosan production. The strain sABCD could produce 334mg/L heparosan in shake flask and 652mg/L heparosan in 3-L batch bioreactor. The heparosan yield was further increased to 1.88g/L in a dissolved oxygen-stat fed-batch culture in 3-L bioreactor. As revealed by the nuclear magnetic resonance analysis, the chemical structure of heparosan from recombinant E. coli BL21 and E. coli K5 was identical. The weight average molecular weight of heparosan from E. coli K5, sAC, sABC, sACD, and sABCD was 51.67, 39.63, 91.47, 64.51, and 118.30kDa, respectively. This work provides a viable process for the production of heparosan as a precursor of bioengineered heparin from a safer bacteria strain.     

Heparosan Chain Characterization: Sequential Depolymerization of E. Coli K5 Heparosan by a Bacterial Eliminase Heparin Lyase III and a Bacterial Hydrolase Heparanase Bp to Prepare Defined Oligomers

Heparosan is a non-sulfated polysaccharide and potential applications include, chemoenzymatic synthesis of heparin and heparan sulfates. Heparosan is produced using microbial cells (natural producers or engineered cells). The characterization of heparosan isolated from both natural producers and engineered-cells are critical steps towards the potential applications of heparosan. Heparosan is characterized using 1) analysis of intact chain size and polydispersity, and 2) disaccharide composition. The current paper describes a novel method for heparosan chain characterization, using heparin lyase III (Hep-3, an eliminase from Flavobacterium heparinum) and heparanase Bp (Hep-Bp, a hydrolase from Burkholderia pseudomallei). The partial digestion of E. coli K5 heparosan with purified His-tagged Hep-3 results in oligomers of defined sizes. The oligomers (degree of polymerization from 2 to 8, DP2-DP8) are completely digested with purified GST-tagged Hep-Bp and analyzed using gel permeation chromatography. Hep-Bp specifically cleaves the linkage between d -glucuronic acid (GlcA) and N-acetyl-d -glucosamine (GlcNAc) but not the linkage between 4-deoxy-α-L-threo-hex-4-enopyranosyluronic acid (deltaUA) and GlcNAc, and results in the presence of a minor resistant trisaccharide (GlcNAc-GlcA-GlcNAc). This method successfully demonstrated the substrate selectivity of Hep-BP on heparosan oligomers. This analytical tool could be applied towards heparosan chain mapping and analysis of unnatural sugar moieties in the heparosan chain.     

Effects of carbon sources and feeding strategies on heparosan production by Escherichia coli K5

This work aimed to develop an optimal carbon source feeding strategy to achieve maximal production of heparosan as a precursor of bioengineered heparin by Escherichia coli K5. Glycerol gave higher heparosan titer and productivity compared to glucose. The maximum heparosan production (187 mg/L) and heparosan productivity (5.19 mg/L/h) in glycerol-defined medium were 26.4% higher than the heparosan production (148 mg/L) and heparosan productivity (4.11 mg/L/h) in glucose-defined medium. DO-stat feeding approach as compared to pH-stat feeding, exponential feeding, exponential combined with pH-stat feeding, and constant rate feeding gave the highest heparosan titer at 8.63 g/L, which was nine times that of batch culture. The obtained optimal glycerol feeding strategy may be useful for the scaling-up of microbial heparosan production.     

Identification and molecular cloning of a heparosan synthase from Pasteurella multocida type D.

Pasteurella multocida Type D, a causative agent of atrophic rhinitis in swine and pasteurellosis in other domestic animals, produces an extracellular polysaccharide capsule that is a putative virulence factor. It was reported previously that the capsule was removed by treating microbes with heparin lyase III. We molecularly cloned a 617-residue enzyme, pmHS, which is a heparosan (nonsulfated, unepimerized heparin) synthase. Recombinant Escherichia coli-derived pmHS catalyzes the polymerization of the monosaccharides from UDP-GlcNAc and UDP-GlcUA. Other structurally related sugar nucleotides did not substitute. Synthase activity was stimulated about 7-25-fold by the addition of an exogenous polymer acceptor. Molecules composed of approximately 500-3,000 sugar residues were produced in vitro. The polysaccharide was sensitive to the action of heparin lyase III but resistant to hyaluronan lyase. The sequence of the pmHS enzyme is not very similar to the vertebrate heparin/heparan sulfate glycosyltransferases, EXT1 and 2, or to other Pasteurella glycosaminoglycan synthases that produce hyaluronan or chondroitin. The pmHS enzyme is the first microbial dual-action glycosyltransferase to be described that forms a polysaccharide composed of beta4GlcUA-alpha4GlcNAc disaccharide repeats. In contrast, heparosan biosynthesis in E. coli K5 requires at least two separate polypeptides, KfiA and KfiC, to catalyze the same polymerization reaction.     

Response surface optimization of the heparosan N-deacetylation in producing bioengineered heparin

The chemical step in the chemoenzymatic synthesis of bioengineered heparin has been examined and optimized statistically using a response surface methodology. A four factor, two level full factorial design experiment and a three factor Box-Behnken design were carried out. The goal was to establish a method to prepare N-sulfo, N-acetyl heparosan of the desired N-acetyl content, number average molecular weight, and in maximum yield by controlling the reactant concentrations, reaction time and reaction temperature. The response surface models obtained were used to predict the reaction conditions required to optimally prepare N-sulfo, N-acetyl heparosan from Escherichia coli generated heparosan starting material of different molecular weights.     

Photochemical Preparation of a Novel Low Molecular Weight Heparin

Commercial low molecular weight heparins (LMWHs) are prepared by several methods including peroxidative cleavage, nitrous acid cleavage, chemical ?-elimination, and enzymatic β-elimination. The disadvantages of these methods are that strong reaction conditions or harsh chemicals are used and these can result in decomposition or modification of saccharide units within the polysaccharide backbone. These side-reactions reduce product quality and yield. Here we show the partial photolysis of unfractionated heparin can be performed in distillated water using titanium dioxide (TiO(2)). TiO(2) is a catalyst that can be easily removed by centrifugation or filtration after the photochemical reaction takes place, resulting in highly pure products. The anticoagulant activity of photodegraded LMWH (pLMWH) is comparable to the most common commercially available LMWHs (i.e., Enoxaparin and Dalteparin). (1)H NMR spectra obtained show that pLMWH maintains the same core structure as unfractionated heparin. This photochemical reaction was investigated using liquid chromatography/mass spectrometry (LC/MS) and unlike other processes commonly used to prepare LMWHs, photochemically preparation affords polysaccharide chains of reduced length having both odd and even of saccharide residues.     

大肠杆菌K5产肝素前体heparosan的研究进展

肝素是一种被广泛临床应用的抗凝血药物多糖。Heparosan是某些细菌荚膜中的GAG成分,其二糖骨架结构与脊椎动物中的肝素类似,可以作为肝素和硫酸乙酰肝素的生物合成前体。本文综述了肝素及肝素前体heparosan的功能与应用,heparosan在大肠杆菌K5中合成转运相关酶的研究,以及发酵法生产heparosan的研究进展,并对其应用前景进行了展望。     

Metabolic engineering of Bacillus subtilis for biosynthesis of heparosan using heparosan synthase from Pasteurella multocida,PmHS1

Heparosan, the capsular polysaccharide discovered in many pathogenic bacteria, is a promising material for heparin preparation. In this study, the Pasteurella multocida heparosan synthase 1 (PmHS1) module was used to synthesize heparosan with controlled molecular weight, while tuaD/gtaB module or gcaD module was responsible for UDP-precursors production in Bacillus subtilis 168. After metabolic pathway optimization, the yield of heparosan was as high as 237.6 mg/L in strain containing PmHS1 module and tuaD/gtaB module, which indicated that these two modules were key factors in heparosan production. The molecular weight of heparosan varied from 39 to 53 kDa, which indicated that heparosan molecular weight could be adjusted by the amount of PmHS1 and the ratio of two UDP precursors. The results showed that it would be possible to produce safe heparosan with appropriate molecular weight which is useful in heparin production.

     

Production of N-sulfated polysaccharides using yeast-expressed N-deacetylase/N-sulfotransferase-1 (NDST-1)

Heparan sulfate/heparin N-deacetylase/N-sulfotransferase-1 (NDST-1) is a critical enzyme involved in heparan sulfate/heparin biosynthesis. This dual-function enzyme modifies the GlcNAc-GlcA disaccharide repeating sugar backbone to make N-sulfated heparosan. N-sulfation is an absolute requirement for the subsequent epimerization and O-sulfation steps in heparan sulfate/heparin biosynthesis. We have expressed rat liver (r) NDST-1 in Saccharomyces cerevisiae as a soluble protein. The yeast-expressed enzyme has both N-deacetylase and N-sulfotransferase activities. N-acetyl heparosan, isolated from Escherichia coli K5 polysaccharide, de-N-sulfated heparin (DNSH) and completely desulfated N-acetylated heparan sulfate (CDSNAcHS) are all good substrates for the rNDST-1. However, N-desulfated, N-acetylated heparin (NDSNAcH) is a poor substrate. The rNDST-1 was partially purified on heparin Sepharose CL-6B. Purified rNDST-1 requires Mn(2+) for its enzymatic activity, can utilize PAPS regenerated in vitro by the PAPS cycle (PAP plus para-nitrophenylsulfate in the presence of arylsulfotransferase IV), and with the addition of exogenous PAPS is capable of producing 60-65% N-sulfated heparosan from E. coli K5 polysaccharide or Pasteurella multocida polysaccharide.     

Photolytic depolymerization of alginate

A photochemical reaction has been developed for the partial de-polymerization of sodium alginate, a polysaccharide utilized in medicine, pharmacy, basic sciences and foods. An aqueous solution of sodium alginate was photochemically depolymerized to ∼40% of its average molecular weight using ultraviolet light in the presence of titanium dioxide catalyst at pH 7 over a period of 3 h. The products were separated giving four fractions all having an average molecular weight that was smaller than that of the starting material. Characterization of the guluronate (G) and mannuronate (M) contents, and determination of the M/G ratio of photochemically depolymerized alginate, were accomplished using ¹H NMR spectroscopy. The resulting M/G ratio was compared to that obtained for alginate fractions produced by acid hydrolysis. The M and G content, of each alginate fraction, was also assigned with regards to their occurrence in G-rich, M-rich or M/G heteropolymeric domains. This new depolymerization method might also be applicable in the preparation of alginate oligosaccharides for use in the food and pharmaceutical industries.     

Liquid chromatography/mass spectrometry sequencing approach for highly sulfated heparin-derived oligosaccharides

Liquid chromatography/mass spectrometry (LC/MS) is applied to the analysis of complex mixtures of oligosaccharides obtained through the controlled, heparinase-catalyzed depolymerization of heparin. Reversed-phase ion-pairing chromatography, utilizing a volatile mobile phase, results in the high resolution separation of highly sulfated, heparin-derived oligosaccharides. Simultaneous detection by UV absorbance and electrospray ionization-mass spectrometry (ESI-MS) provides important structural information on the oligosaccharide components of this mixture. Highly sensitive and easily interpretable spectra were obtained through post-column addition of tributylamine in acetonitrile. High resolution mass spectrometry afforded elemental composition of many known and previously unknown heparin-derived oligosaccharides. UV in combination with MS detection led to the identification of oligosaccharides arising from the original non-reducing end (NRE) of the heparin chain. The structural identification of these oligosaccharides provided sequence from a reading frame that begins at the non-reducing terminus of the heparin chain. Interestingly, 16 NRE oligosaccharides are observed, having both an even and an odd number of saccharide residues, most of which are not predicted based on biosynthesis or known pathways of heparin catabolism. Quantification of these NRE oligosaccharides afforded a number-averaged molecular weight consistent with that expected for the pharmaceutical heparin used in this analysis. Molecular ions could be assigned for oligosaccharides as large as a tetradecasaccharide, having a mass of 4625 Da and a net charge of -32. Furthermore, MS detection was demonstrated for oligosaccharides with up to 30 saccharide units having a mass of >10000 Da and a net charge of -60.     

Ozonolytic depolymerization of polysaccharides in aqueous solution.

The selective oxidation of beta-D-glycosidic linkages of polysaccharides by ozone has great utility as a general method for depolymerization of polysaccharides. Here we describe a 'one-step' method whereby polysaccharides dissolved in water or basic solutions are depolymerized by ozonolysis. The oxidation of glycosidic linkages of unprotected carbohydrates by ozone is complicated by several side reactions. We describe here optimized conditions for carrying out ozonolysis degradation. We also characterize the major pathways for unwanted degradation by various side reactions. In the preferred oxidation pathway, the aldosidic linkage is oxidized to an aldonic ester function that hydrolyzes under the basic conditions employed to give a free aldonate, with cleavage of the polysaccharide chain. Nonselective degradation pathways include oxidative degradation by radical species that oxidize glycosyl residues to formic, acetic, and oxalic acids. The nonselective degradation caused by acids is minimized by basic buffers. The products of polysaccharide depolymerization form a size distribution around a nominal molecular weight, and the average molecular weight of the products can be controlled by the rate or amount of ozone passed through the reaction mixture. The ozonolysis method described herein provides a convenient, inexpensive, and controllable means for generating small polysaccharides or large oligosaccharide fragments.     

N-acetyl-heparosan lyase of Escherichia coli K5: gene cloning and expression.

The structure of the capsular polysaccharide of Escherichia coli K5 is identical to that of N-acetyl-heparosan, a nonsulfated precursor of heparin, which makes this E. coli antigen an attractive starting point for the chemical synthesis of analogs of low-molecular-weight heparin. This polysaccharide is synthesized as a high-molecular-weight molecule that can be depolymerized by an enzyme displaying endo-beta-eliminase activity. The eliminase-encoding gene, designated elmA, has been cloned from E. coli K5 by expression in E. coli K-12. The K-12 genome is devoid of the elmA sequence. The elmA gene product is 820 amino acids long. Active recombinant eliminase is produced by K-12 cells in both cell-bound and secreted forms. Deletion analyses have shown that the C terminus and the N terminus are required for activity and secretion, respectively.     

Sulfonation of papain-treated chitosan and its mechanism for anticoagulant activity

The novel low-molecular-weight chitosan polysulfate (MW 5120-26,200 Da) was prepared using the depolymerization of chitosan with papain (EC. 3.4.22.2). The sulfonation of depolymerized products was performed using chlorosulfonic acid in N,N-dimethylformamide under semi-heterogeneous conditions. The structures of the products were characterized by FTIR, ¹³C NMR, and ¹H NMR (1D, 2D NMR) spectroscopy. The present study sheds light on the mechanism of anticoagulant activity of chitosan polysulfate. Anticoagulant activity was investigated by an activated partial thromboplastin assay, a thrombin time assay, a prothrombin time assay, and thrombelastography. Surface plasmon resonance also provided valuable data for understanding the relationship between the molecular binding of sulfated chitosan to two important blood clotting regulators, antithrombin III and heparin cofactor II. These results show that the principal mechanism by which this chitosan polysulfate exhibits anticoagulant activity is mediated through heparin cofactor II and is dependent on polysaccharide molecular weight.