An acidic exopolysaccharide α-D-galacturono-ß-D-glucan produced by the cyanobacterium Scytonema sp.

Some cyanobacteria produce a wide range of secondary metabolites, some of which are of industrial interest. Exopolysaccharides, particularly interesting among them, represent relatively complex primary structures with interesting bioactivity, biodegradability and specific applications. Cultivation of the freshwater cyanobacterium Scytonema sp. provided a proteoglycan-type exopolysaccharide with a relatively low yield and a wide spectrum of molecular weights (Mw) ranging from 2.2 to 1313 × 103 g/mol. Chemical analyses detected the presence of carbohydrates (46 wt%), proteins (10 wt%) and uronic acids (8 wt%). Monosaccharide analysis revealed up to seven neutral sugars with a dominance of glucose (23.6 wt%), galactose (7.4 wt%) and fucose (5.0 wt%) residues, while the others had a much lower content (0.9-3.4 wt%). The presence of galacturonic acid (8.0 wt%) indicated the appearance of ionic type of exopolysaccharide. A preliminary structural study indicated that the α-D-galacturono-ß-D-glucan forms a dominant part of Scytonema sp. exopolymer. Its backbone is composed of two 1,6-linked and one 1,2-linked ß-D-Glcp residues, which is branched at O6 by side chains composed of α-D-GalAp(1 â†’ 2)-ß-D-Glcp(1→ dimer or monomeric ß-D-Glcp(1→ residue.

Chemical Synthesis of Δ-4,5 Unsaturated Heparan Sulfate Oligosaccharides for Biomarker Discovery.

A methodology is described that can provide heparan sulfate oligosaccharides having a Δ4,5-double bond, which are needed as analytical standards and biomarkers for mucopolysaccharidoses. It is based on chemical oligosaccharide synthesis followed by modification of the C-4 hydroxyl of the terminal uronic acid moiety as methanesulfonate. This leaving group is stable under conditions used to remove temporary protecting groups, O-sulfation, and hydrogenolysis. Treatment with NaOH results in elimination of the methanesulfonate and formation of a Δ4,5-double bond.

An arabinose-rich heteropolysaccharide isolated from Belamcanda chinensis (L.) DC treats liver cancer by targeting FAK and activating CD40.

An undisclosed polysaccharide, BCP80-2, was isolated from Belamcanda chinensis (L.) DC. Structural investigation revealed that BCP80-2 consists of ten monosaccharide residues including t-α-Araf-(1→, →3,5)-α-Araf-(1→, →5)-α-Araf-(1→, →4)-ß-Xylp-(1→, →3)-α-Rhap-(1→, →4)-ß-Manp-(1→, t-ß-Glcp-(1→, →6)-α-Glcp-(1→, t-ß-Galp-(1→, and→3)-α-Galp-(1→. In vivo activity assays showed that BCP80-2 significantly suppressed neoplasmic growth, metastasis, and angiogenesis in zebrafish. Mechanistic studies have shown that BCP80-2 inhibited cell migration of HepG2 cells by suppressing the FAK signaling pathway. Moreover, BCP80-2 also activated immunomodulation and upregulated the secretion of co-stimulatory molecules CD40, CD86, CD80, and MHC-II. In conclusion, BCP80-2 inhibited tumor progression by targeting the FAK signaling pathway and activating CD40-induced adaptive immunity.

Structural characterization of an exopolysaccharide produced by two strains of Lacticaseibacilluscasei.

Exopolysaccharides (EPSs) were isolated and purified from Lacticaseibacillus casei strains type V and RW-3703M grown under various fermentation conditions (carbon source, incubation temperature, and duration). Identical 1H NMR spectra were obtained in all cases. The molar mass determined by size-exclusion chromatography coupled with multi-angle light scattering was different for the two strains and in different culture media. The primary structure was elucidated using chemical and spectroscopic techniques. Monosaccharide and absolute configuration analyses gave the following composition: d-Glc, 1; d-Gal, 2; l-Rha, 2; d-GlcNAc, 1. Methylation analysis indicated the presence of 4-linked Glc, terminal and 6-linked Gal, terminal and 3-linked Rha, and 3,4,6-linked GlcNAc. On the basis of one- and two-dimensional 1H and 13C NMR data, the structure of the EPS was consistent with the following hexasaccharide repeating unit: {4)[Rhap(α1-3)][Galp(α1-6)]GlcpNAc(ß1-6)Galp(α1-3)Rhap(ß1-4)Glcp(ß1-}n. Complete 1H and 13C NMR assignments are reported.

Total Synthesis of a Conjugation-Ready Tetrasaccharide Repeating Unit of Vibrio cholerae O:3 O-antigen Polysaccharide.

Herein, we report the first total synthesis of the tetrasaccharide repeating unit of Vibrio cholerae O:3 O-antigen polysaccharide. The highly complex tetrasaccharide contains rare amino sugars such as d-bacillosamine and l-fucosamine, highly labile sugar ascarylose, and higher carbon sugar d-d-heptose. Stereoselective glycosylation of the notoriously reactive ascarylose with d-d-heptose, poor nucleophilicity of the axial C4-OH of l-fucosamine, and amide coupling are the key challenges encountered in the total synthesis, which was completed via a longest linear sequence of 23 steps in 4.2% overall yield.

Antigenic and Structural Properties of the Lipopolysaccharide of the Uropathogenic Proteus mirabilis Dm55 Strain Classified to a New O85 Proteus Serogroup.

The aim of the study was the serological and structural characterization of the lipopolysaccharide (LPS) O antigen from P. mirabilis Dm55 coming from the urine of a patient from Lodz. The Dm55 LPS was recognized in ELISA only by the O54 antiserum, suggesting a serological distinction of the Dm55 O antigen from all the 84 Proteus LPS serotypes described. The obtained polyclonal rabbit serum against P. mirabilis Dm55 reacted in ELISA and Western blotting with a few LPSs (including O54), but the reactions were weaker than those observed in the homologous system. The LPS of P. mirabilis Dm55 was subjected to mild acid hydrolysis, and the obtained high-molecular-mass O polysaccharide was chemically studied using sugar and methylation analyses, mass spectrometry, and 1H and 13C NMR spectroscopy, including 1H,1H NOESY, and 1H,13C HMBC experiments. The Dm55 O unit is a branched three-saccharide, and its linear fragment contains α-GalpNAc and ß-Galp, whereas α-GlcpNAc occupies a terminal position. The Dm55 OPS shares a disaccharide epitope with the Proteus O54 antigen. Due to the structural differences of the studied O antigen from the other described Proteus O polysaccharides, we propose to classify the P. mirabilis Dm55 strain to a new Proteus O85 serogroup.

Structural Characterization and Anti-Osteoporosis Effects of a Novel Sialoglycopeptide from Tuna Eggs.

Several sialoglycopeptides were isolated from several fish eggs and exerted anti-osteoporosis effects. However, few papers have explored sialoglycopeptide from tuna eggs (T-ES). Here, a novel T-ES was prepared through extraction with KCl solution and subsequent enzymolysis. Pure T-ES was obtained through DEAE-Sepharose ion exchange chromatography and sephacryl S-300 gel filtration chromatography. The T-ES was composed of 14.07% protein, 73.54% hexose, and 8.28% Neu5Ac, with a molecular weight of 9481 Da. The backbone carbohydrate in the T-ES was →4)-ß-D-GlcN-(1→3)-α-D-GalN-(1→3)-ß-D-Glc-(1→2)-α-D-Gal-(1→2)-α-D-Gal-(1→3)-α-D-Man-(1→, with two branches of ß-D-GlcN-(1→ and α-D-GalN-(1→ linking at o-4 in →2,4)-α-D-Gal-(1→. Neu5Ac in the T-ES was linked to the branch of α-D-GlcN-(1→. A peptide chain, Ala-Asp-Asn-Lys-Ser*-Met-Ile that was connected to the carbohydrate chain through O-glycosylation at the -OH of serine. Furthermore, in vitro data revealed that T-ES could remarkably enhance bone density, bone biomechanical properties, and bone microstructure in SAMP mice. The T-ES elevated serum osteogenesis-related markers and reduced bone resorption-related markers in serum and urine. The present study's results demonstrated that T-ES, a novel sialoglycopeptide, showed significant anti-osteoporosis effects, which will accelerate the utilization of T-ES as an alternative marine drug or functional food for anti-osteoporosis.

Mechanisms of Formation of Antibodies against Blood Group Antigens That Do Not Exist in the Body.

The system of the four different human blood groups is based on the oligosaccharide antigens A or B, which are located on the surface of blood cells and other cells including endothelial cells, attached to the membrane proteins or lipids. After transfusion, the presence of these antigens on the apical surface of endothelial cells could induce an immunological reaction against the host. The final oligosaccharide sequence of AgA consists of Gal-GlcNAc-Gal (GalNAc)-Fuc. AgB contains Gal-GlcNAc-Gal (Gal)-Fuc. These antigens are synthesised in the Golgi complex (GC) using unique Golgi glycosylation enzymes (GGEs). People with AgA also synthesise antibodies against AgB (group A [II]). People with AgB synthesise antibodies against AgA (group B [III]). People expressing AgA together with AgB (group AB [IV]) do not have these antibodies, while people who do not express these antigens (group O [0; I]) synthesise antibodies against both antigens. Consequently, the antibodies are synthesised against antigens that apparently do not exist in the body. Here, we compared the prediction power of the main hypotheses explaining the formation of these antibodies, namely, the concept of natural antibodies, the gut bacteria-derived antibody hypothesis, and the antibodies formed as a result of glycosylation mistakes or de-sialylation of polysaccharide chains. We assume that when the GC is overloaded with lipids, other less specialised GGEs could make mistakes and synthesise the antigens of these blood groups. Alternatively, under these conditions, the chylomicrons formed in the enterocytes may, under this overload, linger in the post-Golgi compartment, which is temporarily connected to the endosomes. These compartments contain neuraminidases that can cleave off sialic acid, unmasking these blood antigens located below the acid and inducing the production of antibodies.

Structure elucidation and gene cluster of the O-antigen of Shewanella xiamenensis strain DCB-2-1 containing an amide of d-glucuronic acid with d-alanine and its bonding with U, Cr and V.

The aim of this work was to examine the structure and gene cluster of O-OPS of S. xiamenensis strain DCB-2-1 and survey its conceivability for chelating uranyl, chromate and vanadate ions from solution. O-polysaccharide (OPS, O-antigen) was isolated from the lipopolysaccharide of Shewanella xiamenensis DCB-2-1 and studied by 1D and 2D nuclear magnetic resonance (NMR) spectroscopy and sugar analysis. The following structure of the brunched pentasaccharide was established: where d-ß-GlcpA(d-Ala) is d-glucuronic acid acylated with NH group of d-Ala. The OPS structure established is unique among known bacterial polysaccharide structures. Interestingly, that dN-(d-glucuronoyl)-d-alanine derivative is not found in bacterial polysaccharides early. The O-antigen gene cluster of Shewanella xiamenensis strain DCB-2-1 has been sequenced. The gene functions were tentatively assigned by comparison with sequences in the available databases and found to be in agreement with the OPS structure. Based on the analysis of the IR spectra of the isolated polysaccharide DCB-2-1 and the products of its interaction with UO2(NO3)2 ∗ 6H2O, NH4VO3 and K2Cr2O7, a method of binding them can be proposed. Laboratory experiments show that the use of polysaccharide can be effective in removing uranyl, chromate and vanadate from solution.

Structural properties of the biologically active Dictyosphaerium chlorelloides exopolysaccharide α-d-manno-α-l-rhamno-α-d-(2-O-methyl)-galactan.

Structure of biopolymers produced by microalgae plays an important role for their potential biological activity prediction and applications. Previously isolated and well characterized dominant fractions (Dch5-8) from ion-exchange chromatography separation of the biologically active microalga Dictyosphaerium chlorelloides exopolysaccharide (Dch) were pooled and partially acid hydrolyzed. The dominant sugar components in the combined Dch5-8 fraction were Gal and its 2-O-methyl derivative, Rha and Man, all accounting for about 94 mol% of total amount of sugars. Separation of obtained hydrolysate on Bio-Gel P-2 afforded ten fractions. Their main components were identified by NMR. Based on oligosaccharide structures, the repeating unit of the polysaccharide backbone was identified as →2)-α-L-Rhap-(1→4)-2-O-methyl-[3-O-ß-D-Galp]-α-D-Galp-(1→ branched by Man. Furthermore, the higher molecular weight fraction contained glucuronorhamnan. NMR data indicate 1,4-linked Rha units in the backbone in α and ß configuration, branched at O2 by 2,4-di-O-methyl-ß-d-glucuronic acid.

Structural Characterization and Anticomplement Activity of an Acidic Heteropolysaccharide from Lysimachia christinae Hance.

A novel acidic heteropolysaccharide (LCP-90-1) was isolated and purified from a traditional "heat-clearing" Chinese medicine, Lysimachia christinae Hance. LCP-90-1 (Mw, 20.65 kDa) was composed of Man, Rha, GlcA, Glc, Gal, and Ara, with relative molar ratios of 1.00: 3.00: 11.62: 1.31: 1.64: 5.24. The backbone consisted of 1,4-α-D-GlcpA, 1,4-α-D-Glcp, 1,4-ß-L-Rhap, and 1,3,5-α-L-Araf, with three branches of ß-D-Galp-(1 → 4)-ß-L-Rhap-(1→, α-L-Araf-(1→ and α-D-Manp-(1→ attached to the C-5 position of 1,3,5-α-L-Araf. LCP-90-1 exhibited potent anticomplement activity (CH50: 135.01 ± 0.68 µg/mL) in vitro, which was significantly enhanced with increased glucuronic acid (GlcA) content in its degradation production (LCP-90-1-A, CH50: 28.26 ± 0.39 µg/mL). However, both LCP-90-1 and LCP90-1-A were inactivated after reduction or complete acid hydrolysis. These observations indicated the important role of GlcA in LCP-90-1 and associated derivatives with respect to anticomplement activity. Similarly, compared with LCP-90-1, the antioxidant activity of LCP-90-1-A was also enhanced. Thus, polysaccharides with a high content of GlcA might be important and effective substances of L. christinae.

Characterization of the major surface glycoconjugates of Trypanosoma theileri.

Trypanosoma theileri maintains a long-term extracellular infection with a low parasitaemia in bovids. The surface of this parasite is predicted to be decorated with several surface molecules including membrane surface proteases (MSPs), trans-sialidases and T. theileri putative surface proteins (TTPSPs). However, there are no experimental data to verify this hypothesis. Here, we have purified and partially characterized the surface glycoconjugates of T. theileri using biochemical and mass spectrometry-based approaches. The glycoconjugates fall into two classes: glycoproteins and glycolipids. Proteomic analysis of the glycoprotein fraction demonstrated the presence of MSPs and abundant mucin-like TTPSPs, with most predicted to be GPI-anchored. Mass spectrometric characterization of the glycolipid fraction showed that these are mannose- and galactose-containing glycoinositolphospholipids (GIPLs) that are larger and more diverse than those of its phylogenetic relative T. cruzi, containing up to 10 hexose residues and carrying either alkylacyl-phosphatidylinositol or inositol-phospho-ceramide (IPC) lipid components.

Structural analysis of a water insoluble polysaccharide from pearl millet and evaluating its prebiotic activity.

Epidemiological studies have shown an inverse correlation between dietary intake of prebiotics and the risk of chronic diseases. Pearl millet is a potential economic source to develop a new class of prebiotics in the form of its polysaccharide. In the present study, the chemical structure of a water insoluble homopolysaccharide (PMG), and its prebiotic properties were investigated. The structure of PMG was elucidated on the basis of total hydrolysis, methylation analysis, and 1D/2D NMR (1H, 13C, DEPT-135, HSQC, DQF-COSY, NOESY and ROESY) experiments. The results indicated that PMG was a glucan with an average molecular weight ~ 361 kDa having a backbone of (1 â†’ 3) α-d-glucopyranosyl residues. Hydrolysis of PMG by salivary and pancreatic α amylase was 1.75 % ± 0.34 and 1.99 % ± 0.18 respectively. A positive prebiotic score of PMG with both L. acidophilus and L. brevis (0.446 ± 0.031 & 0.427 ± 0.016) hints towards its prebiotic potential. These observations suggest that PMG might be used as a potential prebiotic component in the food and pharmaceutical applications.

Structural elucidation of hemicelluloses from oil-tea camellia fruit shell.

Oil-tea camellia fruit shell (CFS) is a very abundant waste lignocellulosic resource. The current treatments of CFS, i.e. composting and burning, pose a severe threat on environment. Up to 50 % of the dry mass of CFS is composed of hemicelluloses. However, chemical structures of the hemicelluloses in CFS have not been extensively studied, which limits their high-value utilization. In this study, different types of hemicelluloses were isolated from CFS through alkali fractionation with the assistance of Ba(OH)2 and H3BO3. Xylan, galacto-glucomannan and xyloglucan were found to be the major hemicelluloses in CFS. Through methylation, HSQC and HMBC analyses, we have found that the xylan in CFS is composed of →4)-ß-D-Xylp-(1→ and →3,4)-ß-D-Xylp-(1→ linked by (1→4)-ß glycosidic bond as the main chain; the side chains are α-L-Fucp-(1→, →5)-α-L-Araf-(1→, ß-D-Xylp-(1→, α-L-Rhap-(1→ and 4-O-Me-α-D-GlcpA-(1→, connected to the main chain through (1→3) glycosidic bond. The main chain of galacto-glucomannan in CFS consists of →6)-ß-D-Glcp-(1→, →4)-ß-D-Glcp-(1→, →4,6)-ß-D-Glcp-(1→ and →4)-ß-D-Manp-(1→; the side chains are ß-D-Glcp-(1→, →2)-ß-D-Galp-(1→, ß-D-Manp-(1→ and →6)-ß-D-Galp-(1→ connected to the main chain through (1→6) glycosidic bonds. Moreover, galactose residues are connected by α-L-Fucp-(1→. The main chain of xyloglucan is composed of →4)-ß-D-Glcp-(1→, →4,6)-ß-D-Glcp-(1→ and →6)-ß-D-Glcp-(1→; the side groups, i.e. ß-D-Xylp-(1→ and →4)-ß-D-Xylp-(1→, are connected to the main chain by (1→6) glycosidic bond; →2)-ß-D-Galp-(1→ and α-L-Fucp-(1→ can also connect to →4)-ß-D-Xylp-(1→ forming di- or trisaccharide side chains.

Chemical synthesis of oligosaccharide derivatives with partial structure of ß1-3/1-6 glucan, using monomeric units for the formation of ß1-3 and ß1-6 glucosidic linkages.

ß1-3/1-6 Glucans, known for their diverse structures, comprise a ß1-3-linked main chain and ß1-6-linked short branches. Laminarin, a ß1-3/1-6 glucan extracted from brown seaweed, for instance, includes ß1-6 linkages even in the main chain. The diverse structures provide various beneficial functions for the glucan. To investigate the relationship between structure and functionality, and to enable the characterization of ß1-3/1-6 glucan-metabolizing enzymes, oligosaccharides containing the exact structures of ß1-3/1-6 glucans are required. We synthesized the monomeric units for the synthesis of ß1-3/1-6 mixed-linked glucooligosaccharides. 2-(Trimethylsilyl)ethyl 2-O-benzoyl-4,6-O-benzylidene-ß-d-glucopyranoside served as an acceptor in the formation of ß1-3 linkages. Phenyl 2-O-benzoyl-4,6-O-benzylidene-3-O-(tert-butyldiphenylsilyl)-1-thio-ß-d-glucopyranoside and phenyl 2,3-di-O-benzoyl-4,6-di-O-levulinyl-1-thio-ß-d-glucopyranoside acted as donors, synthesizing acceptors suitable for the formation of ß1-3- and ß1-6-linkages, respectively. These were used to synthesize a derivative of Glcß1-6Glcß1-3Glcß1-3Glc, demonstrating that the proposed route can be applied to synthesize the main chain of ß-glucan, with the inclusion of both ß1-3 and ß1-6 linkages.

Total Synthesis of the Repeating Units of Proteus penneri 26 and Proteus vulgaris TG155 via a Common Disaccharide.

Herein, we report the first total synthesis of the trisaccharide and tetrasaccharide repeating units of P. penneri 26 and P. vulgaris TG155, respectively, having a common disaccharide unit, 3-α-l-QuipNAc-(1 → 3)-α-d-GlcpNAc-(1 →. Striking features of the targets are the presence of rare sugar units, l-quinovosamine and l-rhamnosamine, all joined through α-glycosidic linkages. Major challenges in the formation of 1,2-cis glycosidic linkages in the case of d-glucosamine, l-quinovosamine, and d-galactosamine have been addressed.

Total Synthesis of 6-Deoxy-l-talose Containing a Pentasaccharide Repeating Unit of Acinetobacter baumannii K11 Capsular Polysaccharides.

Herein, we report a concise synthetic approach for the first total synthesis of a pentasaccharide repeating unit of Acinetobacter baumannii K11 capsular polysaccharides containing a rare sugar 6-deoxy-l-talose. The pentasaccharide was synthesized in a convergent manner using a [3 + 2] block glycosylation strategy. During this synthetic strive, we used a 2,2,2-trichloroethoxycarbonyl (Troc)-protected monosaccharide unit to achieve a high yield during the glycosylation to synthesize a trisaccharide, and chemoselective deprotection of the Troc group from the trisaccharide was carried out under a mild, pH-neutral condition, keeping the O-glycosidic bond, azido, and acid/base sensitive group intact. A thiotolylglycoside disaccharide donor containing 6-deoxy-l-talose was synthesized for the first time by the armed-disarmed glycosylation method between two thiotolylglycosides.

TargeTron Inactivation of Chlamydia trachomatis gseA Results in a Lipopolysaccharide 3-Deoxy-d-Manno-Oct-2-Ulosonic Acid-Deficient Strain That Is Cytotoxic for Cells.

All members of the family Chlamydiaceae have lipopolysaccharides (LPS) that possess a shared carbohydrate trisaccharide antigen, 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo) that is functionally uncharacterized. A single gene, genus-specific epitope (gseA), is responsible for attaching the tri-Kdo to lipid IVA. To investigate the function of Kdo in chlamydial host cell interactions, we made a gseA-null strain (L2ΔgseA) by using TargeTron mutagenesis. Immunofluorescence microscopy and immunoblotting with a Kdo-specific monoclonal antibody demonstrated that L2ΔgseA lacked Kdo. L2ΔgseA reacted by immunoblotting with a monoclonal antibody specific for a conserved LPS glucosamine-PO4 epitope, indicating that core lipid A was retained by the mutant. The mutant strain produced a similar number of inclusions as the parental strain but yielded lower numbers of infectious elementary bodies. Transmission electron microscopy of L2ΔgseA-infected cells showed atypical developmental forms and a reduction in the number of elementary bodies. Immunoblotting of dithiothreitol-treated L2ΔgseA-infected cells lysates revealed a marked reduction in outer membrane OmcB disulfide cross-linking, suggesting that the elementary body outer membrane structure was affected by the lack of Kdo. Notably, lactic acid dehydrogenase release by infected cells demonstrated that L2ΔgseA was significantly more cytotoxic to host cells than the wild type. The cytotoxic phenotype may result from an altered outer membrane biogenesis structure and/or function or, conversely, from a direct pathobiological effect of Kdo on an unknown host cell target. These findings implicate a previously unrecognized role for Kdo in host cell interactions that facilitates postinfection host cell survival.

Structure and gene cluster annotation of the O-antigen of Aeromonas sobria strain K928 isolated from common carp and classified into the new Aeromonas PGO1 serogroup.

Aeromonas sobria strain K928 was isolated from a common carp during a Motile Aeromonas Infection/Motile Aeromonas Septicaemia disease outbreak on a Polish fish farm and classified into the new provisional PGO1 serogroup. The lipopolysaccharide of A. sobria K928 was subjected to mild acid hydrolysis, and the O-specific polysaccharide, which was isolated by gel-permeation chromatography, was studied using sugar and methylation analyses and 1H and 13C NMR spectroscopy. The following structure of the branched O-specific polysaccharide repeating unit of A. sobria K928 was established. →2)[α-D-Fucp3NRHb-(1→3)]-α-L-Rhap-(1→3)-ß-L-Rhap-(1→4)-α-L-Rhap-(1→3)-ß-D-FucpNAc-(1→ The O-antigen gene cluster was identified and characterized in the genome of the A. sobria K928 strain after comparison with sequences in the available databases. The composition of the O-antigen genetic region was found to be consistent with the O-polysaccharide structure, and its organization was proposed.

CRISPR-Cas9 driven structural elucidation of the heteroexopolysaccharides from Paenibacillus polymyxa DSM 365.

Paenibacillus polymyxa is a Gram-positive soil bacterium known for producing a wide range of exopolysaccharides. However, due to the biopolymer's complexity, structural elucidation has so far been inconclusive. Combinatorial knock-outs of glycosyltransferases were generated in order to separate distinct polysaccharides produced by P. polymyxa. Using a complementary analytical approach consisting of carbohydrate fingerprints, sequence analysis, methylation analysis as well as NMR spectroscopy, the structure of the repeating units of two additional heteroexopolysaccharides termed paenan I and paenan III were elucidated. Results for paenan I identified a trisaccharide backbone consisting of 1➔4-ß-d-Glc, 1➔4-ß-d-Man and a 1,3,4-branching ß-d-Gal residue with a sidechain comprising of a terminal ß-d-Gal3,4-Pyr and 1➔3-ß-d-Glc. For paenan III, results indicated a backbone consisting of 1➔3-ß-d-Glc, 1,3,4-linked α-d-Man and 1,3,4-linked α-d-GlcA. NMR analysis indicated monomeric ß-d-Glc and α-d-Man sidechains for the branching Man and GlcA residues respectively.