Synthesis of globopentaose using a novel β1,3-galactosyltransferase activity of the Haemophilus influenzae β1,3-N-acetylgalactosaminyltransferase LgtD

AbstractWe have previously described a bacterial system for the conversion of globotriaose (Gb3) into globotetraose (Gb4) by a metabolically engineered Escherichia coli strain expressing the Haemophilus influenzae lgtD gene encoding β1,3-N-acetylgalactosaminyltransferase [Antoine, T., Bosso, C., Heyraud, A. Samain, E. (2005) Large scale in vivo synthesis of globotriose and globotetraose by high cell density culture of metabolically engineered Escherichia coli. Biochimie 87, 197–203]. Here, we found that LgtD has an additional β1,3-galactosyltransferase activity which allows our bacterial system to be extended to the synthesis of the carbohydrate portion of globopentaosylceramide (Galβ-3GalNAcβ-3Galα-4Galβ-4Glc) which reacts with the monoclonal antibody defining the stage-specific embryonic antigen-3. In vitro assays confirmed that LgtD had both β1,3-GalT and β1,3-GalNAcT activities and showed that differences in the affinity for Gb3 and Gb4 explain the specific and exclusive formation of globopentaose

Spatio-temporal connectivity of the aquatic microbiome associated with cyanobacterial blooms along a Great Lake riverine-lacustrine continuum

Lake Erie is subject to recurring events of cyanobacterial harmful algal blooms (cHABs), but measures of nutrients and total phytoplankton biomass seem to be poor predictors of cHABs when taken individually. A more integrated approach at the watershed scale may improve our understanding of the conditions that lead to bloom formation, such as assessing the physico-chemical and biological factors that influence the lake microbial community, as well as identifying the linkages between Lake Erie and the surrounding watershed. Within the scope of the Government of Canada’s Genomics Research and Development Initiative (GRDI) Ecobiomics project, we used high-throughput sequencing of the 16S rRNA gene to characterize the spatio-temporal variability of the aquatic microbiome in the Thames River–Lake St. Clair-Detroit River–Lake Erie aquatic corridor. We found that the aquatic microbiome was structured along the flow path and influenced mainly by higher nutrient concentrations in the Thames River, and higher temperature and pH downstream in Lake St. Clair and Lake Erie. The same dominant bacterial phyla were detected along the water continuum, changing only in relative abundance. At finer taxonomical level, however, there was a clear shift in the cyanobacterial community, with Planktothrix dominating in the Thames River and Microcystis and Synechococcus in Lake St. Clair and Lake Erie. Mantel correlations highlighted the importance of geographic distance in shaping the microbial community structure. The fact that a high proportion of microbial sequences found in the Western Basin of Lake Erie were also identified in the Thames River, indicated a high degree of connectivity and dispersal within the system, where mass effect induced by passive transport play an important role in microbial community assembly. Nevertheless, some cyanobacterial amplicon sequence variants (ASVs) related to Microcystis, representing less than 0.1% of relative abundance in the upstream Thames River, became dominant in Lake St. Clair and Erie, suggesting selection of those ASVs based on the lake conditions. Their extremely low relative abundances in the Thames suggest additional sources are likely to contribute to the rapid development of summer and fall blooms in the Western Basin of Lake Erie. Collectively, these results, which can be applied to other watersheds, improve our understanding of the factors influencing aquatic microbial community assembly and provide new perspectives on how to better understand the occurrence of cHABs in Lake Erie and elsewhere

Structure of the Polysaccharide Secreted by Vibrio alginolyticus CNCM I-5035 (Epidermist 4.0TM)

Vibrio alginolyticus (CNCM I-5035) secretes an exopolysaccharide used as ingredient in cosmetic industry under the trademark Epidermist 4.0TM. It is appreciated for its ability to improve the physical and chemical barrier functions of the skin by notably increasing the keratinocyte differentiation and epidermal renewal. Composition analyses and in depth characterization of the polysaccharides as well as oligosaccharides obtained by mild acid hydrolyses revealed that it was composed of a repetition unit of three residues: d-galactose (d-Gal), d-N-acetylglucosamine (GlcNAc) and l-N-acetylguluronic acid, of which 30% (M/M) was acetylated in position 3. The complete structure of the polysaccharide was resolved giving the repetition unit: [→3)-α-d-Gal-(1→4)-α-l-GulNAcA/α-l-3OAc-GulNAcA-(1→4)-β-d-GlcNAc-(1→]

Structure of the Exopolysaccharide Secreted by a Marine Strain Vibrio alginolyticus

Vibrio alginolyticus (CNCM I-4151) secretes an exopolysaccharide whose carbohydrate backbone is decorated with amino acids, likely conferring its properties that are appreciated in cosmetics. Here, the secreted polysaccharide of another strain of V. alginolyticus (CNCM I-5034) was characterized by chromatography and one- and two-dimensional NMR spectroscopy experiments. The structure was resolved and shows that the carbohydrate backbone is made of four residues: D-galactose (Gal), D-galacturonic acid (GalA) D-N-acetylglucosamine (GlcNAc) and D-glucuronic acid (GlcA), forming a tetrasaccharide repetition unit [→4)-β-d-GlcA-(1→3)-α-d-Gal-(1→3)-α-d-GalA-(1→3)-β-GlcNAc(1→]. GlcA is derivatized with a lactate group giving ‘nosturonic acid’, and GalA is decorated with the amino acid alanine

Synthèse microbiologique des antigènes glucidiques des groupes sanguins

Les antigènes glucidiques des groupes sanguins ont été longtemps étudiés pour leur rôle crucial en matière de greffe et de transplantation. Ils ont été identifiés à la surface des globules rouges mais sont également présents dans les sécrétions et sur de nombreux tissus de l organisme et. L essor de la glycobiologie au cours des vingt dernières années a permis de définir leur implication dans d autres mécanismes biologiques importants notamment l infection, l oncogenèse ou l embryogenèse. De ces différentes implications découlent de nombreuses applications thérapeutiques. Les structures saccharidiques présentent un énorme potentiel pour des développements innovants dans le domaine de la santé. La fabrication de nouveaux médicaments à partir d oligosaccharides des groupes sanguins requiert leur disponibilité en grande quantité. Les méthodes de synthèse chimique et enzymatique ont été développées, mais elles restent difficiles et donnent un rendement final assez faible. Une approche alternative est la synthèse par voie microbiologique. Elle consiste en une production par culture à haute densité de souches recombinantes d Escherichia coli qui surexpriment des gènes codant pour des glycosyltransférases. Grâce à ce procédé, une cinquantaine d oligosaccharides des groupes sanguins ont été obtenus à l échelle du gramme lors de cette étude. Ces molécules appartiennent aux familles d antigènes sanguins principales possédant de réels potentiels biologiques, en l occurence les systèmes ABH (types 1, 2, 4, 5), Lewis et P.Saccharidic blood group antigens are well known for their crucial role in blood transfusion and organe transplantation. They were first discovered more than a century ago on red cells and were later found in various tissues and secretion fluids. The rise of glycobiology during the past twenty years demonstrates their involvment in other biological mechanisms such as the binding of bacteria, toxins, or viruses to mammalian cell surface glycans or the specific role in oncogenesis and embryogenesis. From these different implications derived several therapeutic applications, the blood group antigens are very promising targets for drug development. In this perspective, large amount of these molecules is required. Chemical and enzymatic syntheses are developed but not allow the obtaining of large scale preparative samples of pure well-characterized oligosaccharides for use in biological studies. An alternative approach called the living factory is proposed in this study, it is based on high cell density culture of Escherichia coli strain overexpressing the glycosyltransferase genes. Through this process, some fifty oligosaccharides with biological interest belong to three systems of blood group (ABH type 1, 2, 4, 5, Lewis and P) have been synthesized in gram scale.GRENOBLE1-BU Sciences (384212103) / SudocSudocFranceF

Bacterial synthesis of polysialic acid lactosides in recombinant Escherichia coli K-12

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NMR Analyses of the Enzymatic Degradation End-Products of Diabolican: The Secreted EPS of Vibrio diabolicus CNCM I-1629

Diabolican, or HE800, is an exopolysaccharide secreted by the non-pathogenic Gram-negative marine bacterium Vibrio diabolicus (CNCM I-1629). This polysaccharide was enzymatically degraded by the Bacteroides cellulosilyticus WH2 hyaluronan lyase. The end products were purified by size-exclusion chromatography and their structures were analyzed in depth by nuclear magnetic resonance (NMR). The oligosaccharide structures confirmed the possible site of cleavage of the enzyme showing plasticity in the substrate recognitions. The production of glycosaminoglycan-mimetic oligosaccharides of defined molecular weight and structure opens new perspectives in the valorization of the marine polysaccharide diabolican

Misincorporation of Galactose by Chondroitin Synthase of Escherichia coli K4: From Traces to Synthesis of Chondbiuronan, a Novel Chondroitin-Like Polysaccharide

International audienceChondroitin synthase KfoC is a bifunctional enzyme which polymerizes the capsular chondroitin backbone of Escherichia coli K4, composed of repeated β3N-acetylgalactosamine (GalNAc)-β4-glucuronic acid (GlcA) units. Sugar donors UDP-GalNAc and UDP-GlcA are the natural precursors of bacterial chondroitin synthesis. We have expressed KfoC in a recombinant strain of Escherichia coli deprived of 4-epimerase activity, thus incapable of supplying UDP-GalNAc in the bacterial cytoplasm. The strain was also co-expressing mammal galactose β-glucuronyltransferase, providing glucuronyl-lactose from exogenously added lactose, serving as a primer of polymerization. We show by the mean of NMR analyses that in those conditions, KfoC incorporates galactose, forming a chondroitin-like polymer composed of the repeated β3-galactose (Gal)-β4-glucuronic acid units. We also show that when UDP-GlcNAc 4-epimerase KfoA, encoded by the K4-operon, was co-expressed and produced UDP-GalNAc, a small proportion of galactose was still incorporated into the growing chain of chondroitin