Endogenous chondroitin extends the lifespan and healthspan in C. elegans.

Chondroitin, a class of glycosaminoglycan polysaccharides, is found as proteoglycans in the extracellular matrix, plays a crucial role in tissue morphogenesis during development and axonal regeneration. Ingestion of chondroitin prolongs the lifespan of C. elegans. However, the roles of endogenous chondroitin in regulating lifespan and healthspan mostly remain to be investigated. Here, we demonstrate that a gain-of-function mutation in MIG-22, the chondroitin polymerizing factor (ChPF), results in elevated chondroitin levels and a significant extension of both the lifespan and healthspan in C. elegans. Importantly, the remarkable longevity observed in mig-22(gf) mutants is dependent on SQV-5/chondroitin synthase (ChSy), highlighting the pivotal role of chondroitin in controlling both lifespan and healthspan. Additionally, the mig-22(gf) mutation effectively suppresses the reduced healthspan associated with the loss of MIG-17/ADAMTS metalloprotease, a crucial for factor in basement membrane (BM) remodeling. Our findings suggest that chondroitin functions in the control of healthspan downstream of MIG-17, while regulating lifespan through a pathway independent of MIG-17.

Reprogramming Metabolic Flux in Escherichia Coli to Enhance Chondroitin Production.

Reprogramming metabolic flux is a promising approach for constructing efficient microbial cell factories (MCFs) to produce chemicals. However, how to boost the transmission efficiency of metabolic flux is still challenging in complex metabolic pathways. In this study, metabolic flux is systematically reprogrammed by regulating flux size, flux direction, and flux rate to build an efficient MCF for chondroitin production. The ammoniation pool for UDP-GalNAc synthesis and the carbonization pool for UDP-GlcA synthesis are first enlarged to increase flux size for providing enough precursors for chondroitin biosynthesis. Then, the ammoniation pool and the carbonization pool are rematched using molecular valves to shift flux direction from cell growth to chondroitin biosynthesis. Next, the adaptability of polymerization pool with the ammoniation and carbonization pools is fine-tuned by dynamic and static valve-based adapters to accelerate flux rate for polymerizing UDP-GalNAc and UDP-GlcA to produce chondroitin. Finally, the engineered strain E. coli F51 is able to produce 9.2 g L-1 chondroitin in a 5-L bioreactor. This strategy shown here provides a systematical approach for regulating metabolic flux in complex metabolic pathways for efficient biosynthesis of chemicals.

Essential hyaluronan structure for binding with hyaluronan-binding protein (HABP) determined by glycotechnological approach.

Hyaluronan specifically binds to aggrecan globular domain 1, which is often referred to as just hyaluronan binding protein (HABP), however, the hyaluronan carbohydrate structure recognized by HABP had not been studied in detail. The aim of the present study was to investigate the important structure of hyaluronan for binding to HABP. We prepared hybrid oligosaccharides from hyaluronan and chondroitin, with or without modification of the reducing or non-reducing terminus, as tools to determine the preferred structure of hyaluronan for binding to the HABP by a competitive ELISA-like method. The non-reducing terminal structure was critical, especially, the glucuronic acid (GlcUA) and N-acetylglucosamine (GlcNAc) of the hyaluronan-unit are essential for complete HABP binding activity, and for any HABP binding activity, respectively. It is possible to replace GlcUAß-1-3GlcNAc of the internal disaccharide units with GlcUAß-1-3N-acetylgalactosamine (GalNAc), if the chain length is decasaccharide or larger.

Interactions between Sclerostin and Glycosaminoglycans.

Sclerostin (SOST) is a glycoprotein having many important functions in the regulation of bone formation as a key negative regulator of Wnt signaling in bone. Surface plasmon resonance (SPR), which allows for a direct quantitative analysis of the label-free molecular interactions in real-time, has been widely used for the biophysical characterization of glycosaminoglycan (GAG)-protein interactions. In the present study, we report kinetics, structural analysis and the effects of physiological conditions (e.g., salt concentrations, Ca2+ and Zn2+concentrations) on the interactions between GAGs and recombinant human (rh) and recombinant mouse (rm) SOST using SPR. SPR results revealed that both SOSTs bind heparin with high affinity (rhSOST-heparin, KD~36 nM and rmSOST-heparin, KD~77 nM) and the shortest oligosaccharide of heparin that effectively competes with full size heparin for SOST binding is octadecasaccharide (18mer). This heparin binding protein also interacts with other highly sulfated GAGs including, disulfated-dermatan sulfate and chondroitin sulfate E. In addition, liquid chromatography-mass spectrometry was used to characterize the structure of sulfated GAGs that bound to SOST.

Molecular weight determination of heparosan- and chondroitin-like capsular polysaccharides: figuring out differences between wild -type and engineered Escherichia coli strains.

Heparin and chondroitin sulfate are used as anti-thrombic and anti-osteoarthritis drugs, respectively, but their pharmacological actions depend on their structural characteristics such as their sulfation grade and their molecular weight. In the last years, new fermentation-based biotechnological approaches have tried to obtain heparin and chondroitin sulfate starting from the heparosan and chondroitin-like capsular polysaccharides produced by Escherichia coli K5 and K4. The study of the microbial capsular polysaccharide molecular weight is critical to obtain nature-like or structural tailor cut glycosaminoglycan homologues. However, so far, it has been scarcely investigated. In this paper, for the first time, a new protocol was set up to determine the molecular weights of the capsular polysaccharides of three wild-type and three engineered E. coli K5 and K4 strains. The protocol includes a small-scale downstream train to purify the intact polysaccharides, directly from the fermentation broth supernatants, by using ultrafiltration membranes and anion exchange chromatography, and it couples size exclusion chromatography analyses with triple detector array. In the purification high recovery (> 85.0%) and the removal of the main contaminant, the lipopolysaccharide, were obtained. The averaged molecular weights of the wild-type capsular polysaccharides ranged from 51.3 to 90.9 kDa, while the engineered strains produced polysaccharides with higher molecular weights, ranging from 68.4 to 130.6 kDa, but with similar polydispersity values between 1.1 and 1.5.

Semisynthesis of Chondroitin Sulfate Oligosaccharides Based on the Enzymatic Degradation of Chondroitin.

Chondroitin sulfate (CS) is a structurally complex polyanionic glycosaminoglycan that plays essential roles in physiological processes. Here we report a facile approach to a library of CS tetra- and hexasaccharides based on the enzymatic degradation of chondroitin over 10 or 11 steps, which is the shortest synthetic route toward size-defined CS oligosaccharides reported to date. Subsequent biotinylation enabled the investigation of their interactions with growth factors, filling in the gaps of the existing research, and providing probes for further exploration of the biological functions of CS.

Crataeva tapia bark lectin (CrataBL) is a chemoattractant for endothelial cells that targets heparan sulfate and promotes in vitro angiogenesis.

Formation of new blood vessels from preexisting ones, a process known as angiogenesis, is one of the limiting steps for success in treatment of ischemic disorders. Therefore, efforts to understanding and characterize new agents capable to stimulate neovascularization are a worldwide need. Crataeva tapia bark lectin (CrataBL) has been shown to have chemoattractant properties for endothelial cells through the stimulation of migration and invasiveness of human umbilical vein endothelial cells (HUVEC) because it is a positively charged protein with high affinity to glycosaminoglycan. In addition, CrataBL increased the production of chondroitin and heparan sulfate in endothelial cells. These findings orchestrated specific adhesion on collagen I and phosphorylation of tyrosine kinase receptors, represented by vascular endothelial growth factor receptor-2 (VEGFR-2) and fibroblast growth factor receptor (FGFR), whose downstream pathways trigger the angiogenic cascade increasing cell viability, cytoskeleton rearrangement, cell motility, and tube formation. Moreover, CrataBL inhibited the activity of matrix metalloproteases type 2 (MMP-2), a protein related to tissue remodeling. Likewise, CrataBL improved wound healing and increased the number of follicular structures in lesioned areas produced in the dorsum-cervical region of C57BL/6 mice. These outcomes altogether indicate that CrataBL is a pro-angiogenic and healing agent.

Knockdown of chondroitin-4-sulfotransferase-1, but not of dermatan-4-sulfotransferase-1, accelerates regeneration of zebrafish after spinal cord injury.

Glycosaminoglycans such as chondroitin sulfate (CS) and dermatan sulfate (DS) are long chains of repeating disaccharide units, covalently linked to core proteins to form proteoglycans. Proteoglycans can be cell membrane-bound or are part of the extracellular matrix. They are important in a wide range of biologic processes, including development, synaptic plasticity, and regeneration after injury, as well as modulation of growth factor signaling, cell migration, survival, and proliferation. Synthesis of CS and DS in the Golgi apparatus is mediated by sulfotransferases that modify sugar chains through transfer of sulfate groups to specific positions on the sugar moieties. To clarify the functions of CS and DS during nervous system regeneration, we studied the effect of chondroitin 4- O-sulfotransferase-1/carbohydrate sulfotransferase-11 (C4ST-1/Chst-11) and dermatan 4- O-sulfotransferase-1/Chst-14 (D4ST-1/Chst-14) down-regulation on spinal cord regeneration in larval and adult zebrafish. In our study, knockdown of C4ST1/Chst-11 accelerated regeneration after spinal cord injury in larval and adult zebrafish and knockdown of D4ST1/Chst-14 did not alter regenerative capacity. From these and previous observations, we drew the conclusion that different CS and DS expression patterns can be growth permitting, growth inhibiting, or neutral for regrowing or sprouting axons, depending on the tissue environment of a particular animal species.-Sahu, S., Li, R., Loers, G., Schachner, M. Knockdown of chondroitin-4-sulfotransferase-1, but not of dermatan-4-sulfotransferase-1, accelerates regeneration of zebrafish after spinal cord injury.

Genetic profiling of young and aged endothelial progenitor cells in hypoxia.

Age is a major risk factor for diseases caused by ischemic hypoxia, such as stroke and coronary artery disease. Endothelial progenitor cells (EPCs) are the major cells respond to ischemic hypoxia through angiogenesis and vascular remodeling. However, the effect of aging on EPCs and their responses to hypoxia are not well understood. CD34+ EPCs were isolated from healthy volunteers and aged by replicative senescence, which was to passage cells until their doubling time was twice as long as the original cells. Young and aged CD34+ EPCs were exposed to a hypoxic environment (1% oxygen for 48hrs) and their gene expression profiles were evaluated using gene expression array. Gene array results were confirmed using quantitative polymerase chain reaction, Western blotting, and BALB/c female athymic nude mice hindlimb ischemia model. We identified 115 differentially expressed genes in young CD34+ EPCs, 54 differentially expressed genes in aged CD34+ EPCs, and 25 common genes between normoxia and hypoxia groups. Among them, the expression of solute carrier family 2 (facilitated glucose transporter), member 1 (SLC2A1) increased the most by hypoxia in young cells. Gene set enrichment analysis indicated the pathways affected by aging and hypoxia most, including genes "response to oxygen levels" in young EPCs and genes involved "chondroitin sulfate metabolic process" in aged cells. Our study results indicate the key factors that contribute to the effects of aging on response to hypoxia in CD34+ EPCs. With the potential applications of EPCs in cardiovascular and other diseases, our study also provides insight on the impact of ex vivo expansion might have on EPCs.

Alternative substrate-bound conformation of bacterial solute-binding protein involved in the import of mammalian host glycosaminoglycans.

Glycosaminoglycans (GAGs), constituted by repeating uronate and amino sugar units, are major components of mammalian extracellular matrices. Some indigenous and pathogenic bacteria target GAGs for colonization to and/or infection of host mammalian cells. In Gram-negative pathogenic Streptobacillus moniliformis, the solute-binding protein (Smon0123)-dependent ATP-binding cassette (ABC) transporter incorporates unsaturated GAG disaccharides into the cytoplasm after depolymerization by polysaccharide lyase. Smon0123, composed of N and C domains, adopts either a substrate-free open or a substrate-bound closed form by approaching two domains at 47° in comparison with the open form. Here we show an alternative 39°-closed conformation of Smon0123 bound to unsaturated chondroitin disaccharide sulfated at the C-4 and C-6 positions of N-acetyl-d-galactosamine residue (CΔ4S6S). In CΔ4S6S-bound Smon0123, Arg204 and Lys210 around the two sulfate groups were located at different positions from those at other substrate-bound 47°-closed conformations. Therefore, the two sulfate groups in CΔ4S6S shifted substrate-binding residue arrangements, causing dynamic conformational change. Smon0123 showed less affinity with CΔ4S6S than with non-sulfated and monosulfated substrates. ATPase activity of the Smon0123-dependent ABC transporter in the presence of CΔ4S6S was lower than that in the presence of other unsaturated chondroitin disaccharides, suggesting that CΔ4S6S-bound Smon0123 was unpreferable for docking with the ABC transporter.

Expression of chondroitin-4-O-sulfotransferase in Escherichia coli and Pichia pastoris.

Chondroitin sulfates are linear sulfated polysaccharides called glycosaminoglycans. They are important nutraceutical and pharmaceutical products that are biosynthesized through the action of chondroitin sulfotransferases on either an unsulfated chondroitin or a dermatan polysaccharide precursor. While the enzymes involved in the biosynthesis of chondroitin sulfates are well known, the cloning end expression of these membrane-bound Golgi enzymes continue to pose challenges. The major chondroitin-4-sulfotransferase, Homo sapiens C4ST-1, had been previously cloned and expressed from mammalian CHO, COS-7, and HEK 293 cells, and its activity was shown to require glycosylation. In the current study, a C4ST-1 construct was designed and expressed in both Escherichia coli and Pichia pastoris in its non-glycosylated and glycosylated forms. Both constructs showed similar activity albeit different kinetic parameters when acting on a microbially prepared unsulfated chondroitin substrate. Moreover, the glycosylated form of C4ST-1 showed lower stability than the non-glycosylated form.

New insight into chondroitin and heparosan-like capsular polysaccharide synthesis by profiling of the nucleotide sugar precursors.

Escherichia coli K4 and K5 capsular polysaccharides (K4 and K5 CPSs) have been used as starting material for the biotechnological production of chondroitin sulfate (CS) and heparin (HP) respectively. The CPS covers the outer cell wall but in late exponential or stationary growth phase it is released in the surrounding medium. The released CPS concentration was used, so far, as the only marker to connect the strain production ability to the different cultivation conditions employed. Determining also the intracellular UDP-sugar precursor concentration variations, during the bacterial growth, and correlating it with the total CPS production (as sum of the inner and the released ones), could help to better understand the chain biosynthetic mechanism and its bottlenecks. In the present study, for the first time, a new capillary electrophoresis method was set up to simultaneously analyse the UDP-glucose (UDP-Glc), UDP-galactose (UDP-Gal), UDP-N-acetylgalactosamine (UDP-GalNAc), UDP-N-acetylglucosamine (UDP-GlcNAc) and UDP-glucuronic acid (UDP-GlcA) and the inner CPS portion, extracted at the same time from the bacterial biomasses; separation was performed at 18°C and 18 kV with a borate-based buffer and detection at 200 nm. The E. coli K4 and K5 UDP-sugar pools were profiled, for the first time, at different time points of shake flask growths on a glycerol-containing medium and on the same medium supplemented with the monosaccharide precursors of the CPSs: their concentrations varied from 0.25 to 11 µM·gcdw-1, according to strain, the type of precursor, the growth phase and the cultivation conditions and their availability dramatically influenced the total CPS produced.

Chondroitin 4-O-Sulfotransferase Is Indispensable for Sulfation of Chondroitin and Plays an Important Role in Maintaining Normal Life Span and Oxidative Stress Responses in Nematodes.

Chondroitin sulfate (CS)/chondroitin (Chn) chains are indispensable for embryonic cell division and cytokinesis in the early developmental stages in Caenorhabditis elegans and mice, whereas heparan sulfate (HS) is essential for axon guidance during nervous system development. These data indicate that the fundamental functions of CS and HS are conserved from worms to mammals and that the function of CS/Chn differs from that of HS. Although previous studies have shown that C. elegans produces HS and non-sulfated Chn, whether the organism produces CS remains unclear. Here, we demonstrate that C. elegans produces a small amount of 4-O-sulfated Chn and report the identification of C41C4.1, an orthologue of the human chondroitin 4-O-sulfotransferase gene. Loss of C41C4.1 in C. elegans resulted in a decline in 4-O-sulfation of CS and an increase in the number of sulfated units in HS. C41C4.1 deletion mutants exhibited reduced survival rates after synchronization with sodium hypochlorite. Collectively, these results show for the first time that CS glycans are present in C. elegans and that the Chn 4-O-sulfotransferase responsible for the sulfation plays an important role in protecting nematodes from oxidative stress.

Mutations in Biosynthetic Enzymes for the Protein Linker Region of Chondroitin/Dermatan/Heparan Sulfate Cause Skeletal and Skin Dysplasias.

Glycosaminoglycans, including chondroitin, dermatan, and heparan sulfate, have various roles in a wide range of biological events such as cell signaling, cell proliferation, tissue morphogenesis, and interactions with various growth factors. Their polysaccharides covalently attach to the serine residues on specific core proteins through the common linker region tetrasaccharide, -xylose-galactose-galactose-glucuronic acid, which is produced through the stepwise addition of respective monosaccharides by four distinct glycosyltransferases. Mutations in the human genes encoding the glycosyltransferases responsible for the biosynthesis of the linker region tetrasaccharide cause a number of genetic disorders, called glycosaminoglycan linkeropathies, including Desbuquois dysplasia type 2, spondyloepimetaphyseal dysplasia, Ehlers-Danlos syndrome, and Larsen syndrome. This review focused on recent studies on genetic diseases caused by defects in the biosynthesis of the common linker region tetrasaccharide.

A detailed quantitative outcome measure of glycosaminoglycans in human articular cartilage for cell therapy and tissue engineering strategies.

OBJECTIVE: Ideally, cartilage regenerative cell therapy should produce a tissue which closely matches the microstructure of native cartilage. Benchmark reference information is necessary to assess the quality of engineered cartilage. Our goal was to examine the variation in glycosaminoglycans (GAGs) in cartilage zones within human knee joints of different ages. DESIGN: Osteochondral biopsies were removed from the medial femoral condyles of deceased persons aged 20-50 years. Fluorophore-Assisted Carbohydrate Electrophoresis (FACE) was used to profile GAGs through the superficial, middle and deep zones of the articular cartilage. Differences were identified by statistical analysis. RESULTS: Cartilage from the younger biopsies had 4-fold more hyaluronan in the middle zone than cartilage from the older biopsies. The proportion of hyaluronan decreased with increasing age. Cartilage from the middle and deep zones of younger biopsies had significantly more chondroitin sulphate and keratan sulphate than the cartilage from older biopsies. This would suggest that chondrocytes synthesise more sulphated GAGs when deeper in the tissue and therefore in conditions of hypoxia. With increasing age, there was significantly more chondroitin-6 sulphate than chondroitin-4 sulphate. For the first time, unsulphated chondroitin was detected in the superficial zone. CONCLUSIONS: As an outcome measure, FACE offers the potential of a complete, detailed assessment of all GAGs and offers more information that the widely used 1,9-dimethylmethylene blue (DMMB) dye assay. FACE could be very useful in the evolving cartilage regeneration field.

Chondroitin-based nanoplexes as peptide delivery systems--Investigations into the self-assembly process, solid-state and extended release characteristics.

A new type of self-assembled polyelectrolyte complex nanocarrier composed of chondroitin (CHON) and protamine (PROT) was designed and the ability of the carriers to bind salmon calcitonin (sCT) was examined. The response of sCT-loaded CHON/PROT NPs to a change in the properties of the liquid medium, e.g. its pH, composition or ionic strength was studied and in vitro peptide release was assessed. The biocompatibility of the NPs was evaluated in Caco-2 cells. CHON/PROT NPs were successfully obtained with properties that were dependent on the concentration of the polyelectrolytes and their mixing ratio. X-ray diffraction determined the amorphous nature of the negatively charged NPs, while those with the positive surface potential were semi-crystalline. sCT was efficiently associated with the nanocarriers (98-100%) and a notably high drug loading (13-38%) was achieved. The particles had negative zeta potential values and were homogenously dispersed with sizes between 60 and 250 nm. CHON/PROT NPs released less than 10% of the total loaded peptide in the first hour of the in vitro release studies. The enthalpy of the decomposition exotherm correlated with the amount of sCT remaining in NPs after the release experiments. The composition of medium and its ionic strength was found to have a considerable influence on the release of sCT from CHON/PROT NPs. Complexation to CHON markedly reduced the toxic effects exerted by PROT and the NPs were compatible and well tolerated by Caco-2 cells.

A novel eliminase from a marine bacterium that degrades hyaluronan and chondroitin sulfate.

Lyases cleave glycosaminoglycans (GAGs) in an eliminative mechanism and are important tools for the structural analysis and oligosaccharide preparation of GAGs. Various GAG lyases have been identified from terrestrial but not marine organisms even though marine animals are rich in GAGs with unique structures and functions. Herein we isolated a novel GAG lyase for the first time from the marine bacterium Vibrio sp. FC509 and then recombinantly expressed and characterized it. It showed strong lyase activity toward hyaluronan (HA) and chondroitin sulfate (CS) and was designated as HA and CS lyase (HCLase). It exhibited the highest activities to both substrates at pH 8.0 and 0.5 m NaCl at 30 °C. Its activity toward HA was less sensitive to pH than its CS lyase activity. As with most other marine enzymes, HCLase is a halophilic enzyme and very stable at temperatures from 0 to 40 °C for up to 24 h, but its activity is independent of divalent metal ions. The specific activity of HCLase against HA and CS reached a markedly high level of hundreds of thousands units/mg of protein under optimum conditions. The HCLase-resistant tetrasaccharide Δ(4,5)HexUAα1-3GalNAc(6-O-sulfate)ß1-4GlcUA(2-O-sulfate)ß1-3GalNAc(6-O-sulfate) was isolated from CS-D, the structure of which indicated that HCLase could not cleave the galactosaminidic linkage bound to 2-O-sulfated d-glucuronic acid (GlcUA) in CS chains. Site-directed mutagenesis indicated that HCLase may work via a catalytic mechanism in which Tyr-His acts as the Brønsted base and acid. Thus, the identification of HCLase provides a useful tool for HA- and CS-related research and applications.

Identification of phosphatase that dephosphorylates xylose in the glycosaminoglycan-protein linkage region of proteoglycans.

Recently, we demonstrated that FAM20B is a kinase that phosphorylates the xylose (Xyl) residue in the glycosaminoglycan-protein linkage region of proteoglycans. The phosphorylation of Xyl residues by FAM20B enhances the formation of the linkage region. Rapid dephosphorylation is probably induced just after synthesis of the linker and just before polymerization initiates. Indeed, in vitro chondroitin or heparan sulfate polymerization does not occur when the Xyl residue of the tetrasaccharide linkage region is phosphorylated. However, the enzyme responsible for the dephosphorylation of Xyl remains unknown. Here, we identified a novel protein that dephosphorylates the Xyl residue and designated it 2-phosphoxylose phosphatase. The phosphatase efficiently removed the phosphate from the phosphorylated trisaccharide, Galß1-3Galß1-4Xyl(2-O-phosphate), but not from phosphorylated tetrasaccharide, GlcUAß1-3Galß1-3Galß1-4Xyl(2-O-phosphate). Additionally, RNA interference-mediated inhibition of 2-phosphoxylose phosphatase resulted in increased amounts of GlcNAcα1-4GlcUAß1-3Galß1-3Galß1-4Xyl(2-O-phosphate), Galß1-3Galß1-4Xyl(2-O-phosphate), and Galß1-4Xyl(2-O-phosphate) in the cells. Gel filtration analysis of the glycosaminoglycan chains synthesized in the knockdown cells revealed that these cells produced decreased amounts of glycosaminoglycan chains and that the chains had similar lengths to those in the mock-transfected cells. Transcripts encoding this phosphatase were ubiquitously, but differentially, expressed in human tissues. Moreover, the phosphatase localized to the Golgi and interacted with the glucuronyltransferase-I involved in the completion of the glycosaminoglycan-protein linkage region. Based on these findings, we conclude that transient phosphorylation of the Xyl residue in the glycosaminoglycan-protein linkage region controls the formation of glycosaminoglycan chains of proteoglycans.

IS2-mediated overexpression of kfoC in E. coli K4 increases chondroitin-like capsular polysaccharide production.

Transposons are developing molecular tools commonly used for several applications: one of these is the delivery of genes into microorganisms. These mobile genetic elements are characterised by two repeated insertion sequences that flank a sequence encoding one or more orfs for a specific transposase that moves these sequences to other DNA sites. In the present paper, the IS2 transposon of Escherichia coli K4 was modified in vitro by replacing the sequence coding for the transposase with that of the kfoC gene that codes for chondroitin polymerase. KfoC is responsible for the polymerisation of the bacterial capsular polysaccharide whose structure is analogous to that of chondroitin sulphate, a glycosaminoglycan with established and emerging biomedical applications. The recombinant construct was stably integrated into the genome of E. coli K4 by exploiting the transposase from endogenous copies of IS2 in the E. coli chromosome. A significant improvement of the polysaccharide production was observed, resulting in 80 % higher titres in 2.5-L fed-batch cultivations and up to 3.5 g/L in 22-L fed-batch cultures.

Methods for the Pasteurella glycosaminoglycan synthases: enzymes that polymerize hyaluronan, chondroitin, or heparosan chains.

Multiple glycosyltransferases (GTases) that produce glycosaminoglycans (GAGs) have been identified; -several distinct putative architectures and catalytic abilities have been noted from microbes and vertebrates. Here the preparation and use of a class of enzymes (Class II) from the gram-negative bacterium, Pasteurella multocida, with utility in chemoenzymatic synthesis of GAGs is described. The analyses of sugar products include thin layer chromatography (TLC), matrix-assisted laser desorption ionization mass spectroscopy (MALDI-ToF MS), and polyacrylamide gel electrophoresis (PAGE). The related Chapter 18 is focused on larger molecular weight GAGs analyses as well as the Class I membrane streptococcal and mammalian HA synthases.