Characterization and Heterologous Expression of UDP-Glucose 4-Epimerase From a Hericium erinaceus Mutant with High Polysaccharide Production

Hericium erinaceus is an important medicinal fungus in traditional Chinese medicine because of its polysaccharides and other natural products. Compared terpenoids and polyketides, the analysis of synthetic pathway of polysaccharides is more difficult because of the many genes involved in central metabolism. In previous studies, A6180, encoding a putative UDP-glucose 4-epimerase (UGE) in an H. erinaceus mutant with high production of active polysaccharides, was significantly upregulated. Since there is no reliable genetic manipulation technology for H. erinaceus, we employed Escherichia coli and Saccharomyces cerevisiae to study the function and activity of A6180. The recombinant overexpression vector pET22b-A6180 was constructed for heterologous expression in E. coli. The enzymatic properties of the recombinant protein were investigated. It showed that the recombinant A6180 could strongly convert UDP-α-D-glucose into UDP-α-D-galactose under optimal conditions (pH 6.0, 30°C). In addition, when A6180 was introduced into S. cerevisiae BY4742, xylose was detected in the polysaccharide composition of the yeast transformant. This suggested that the protein coded by A6180 might be a multifunctional enzyme. The generated polysaccharides with a new composition of sugars showed enhanced macrophage activity in vitro. These results indicate that A6180 plays an important role in the structure and activity of polysaccharides. It is a promising strategy for producing polysaccharides with higher activity by introducing A6180 into polysaccharide-producing mushrooms.

Comparison of Cell Wall Polysaccharide Composition and Structure Between Strains of Sporothrix schenckii and Sporothrix brasiliensis

Sporothrix schenckii, Sporothrix brasiliensis, and Sporothrix globosa are the main causative agents of sporotrichosis, a human subcutaneous mycosis. Differences in virulence patterns are associated with each species but remain largely uncharacterized. The S. schenckii and S. brasiliensis cell wall composition and virulence are influenced by the culturing media, with little or no influence on S. globosa. By keeping constant the culturing media, we compared the cell wall composition of three S. schenckii and two S. brasiliensis strains, previously described as presenting different virulence levels on a murine model of infection. The cell wall composition of the five Sporothrix spp. strains correlated with the biochemical composition of the cell wall previously reported for the species. However, the rhamnose-to-β-glucan ratio exhibits differences among strains, with an increase in cell wall rhamnose-to-β-glucan ratio as their virulence increased. This relationship can be expressed mathematically, which could be an important tool for the determination of virulence in Sporothrix spp. Also, structural differences in rhamnomannan were found, with longer side chains present in strains with lower virulence reported for both species here studied, adding insight to the importance of this polysaccharide in the pathogenic process of these fungi.

Structural Variation and Polysaccharide Profiling of Intervessel Pit Membranes

Functional roles of intervessel pit membrane (PM) depend on its structure and polysaccharide composition, which are mostly unknown or lack of accurate information. This study uses grapevine as a model plant and an immunogold-scanning electron microscopy technique to simultaneously analyze structures and polysaccharide compositions of intervessel PMs in relation to their functions. Intervessel PMs with different structural integrity were found in functional xylem with about 90 % of them being intact with a smooth or relatively smooth surface and the rest 10 % with progressively degraded structures. The results also elucidated details of the removal process of wall materials from surface toward its depth during the natural intervessel PM degradation. Four groups of pectic and hemicellulosic polysaccharides were present in intervessel PMs but displayed differential spatial distributions and quantities: weakly methyl-esterified homogalacturonans abundant in the surficial layers, heavily methyl-esterified homogalacturonans and xylans mostly in deep layers, and fucosylated xyloglucans relatively uniform in presence at different depths of an intervessel PM. This information is crucial to reveal the polysaccharide profiling of primary cell wall and to understand intervessel PM's roles in the safety and regulation of water transport as well as the plant susceptibility to vascular diseases.

Carbon Source-Dependent Changes of the Structure of Streptococcus pneumoniae Capsular Polysaccharide with Serotype 6F

The structure of the exopolysaccharide capsule of Streptococcus pneumoniae is defined by the genetic arrangement of the capsule operon allowing the unequivocal identification of the pneumococcal serotype. Here, we investigated the environment-dependent composition of the polysaccharide structure of S. pneumoniae serotype 6F. When grown in a chemically defined medium (CDM) with glucose versus galactose, the exopolysaccharide capsule of the serotype 6F strains reveals a ratio of 1/0.6 or 1/0.3 for galactose/glucose in the capsule by 1H-NMR analyses, respectively. Increased production of the capsule precursor UDP-glucose has been identified by 31P-NMR in CDM with glucose. Flow cytometric experiments using monoclonal antibodies showed decreased labelling of Hyp6AG4 (specific for serotype 6A) antibodies when 6F is grown in glucose as compared to galactose, which mirrors the 1H-NMR results. Whole-genome sequencing analyses of serotype 6F isolates suggested that the isolates evolved during two different events from serotype 6A during the time when the 13-valent pneumococcal conjugate vaccine (PCV-13) was introduced. In conclusion, this study shows differences in the capsular structure of serotype 6F strains using glucose as compared to galactose as the carbon source. Therefore, 6F strains may show slightly different polysaccharide composition while colonizing the human nasopharynx (galactose rich) as compared to invasive locations such as the blood (glucose rich).

Microbial responses to herbivory-induced vegetation changes in a high-Arctic peatland

Herbivory by barnacle geese (Branta leucopsis) alters the vegetation cover and reduces ecosystem productivity in high-Arctic peatlands, limiting the carbon sink strength of these ecosystems. Here we investigate how herbivory-induced vegetation changes affect the activities of peat soil microbiota using metagenomics, metatranscriptomics and targeted metabolomics in a comparison of fenced exclosures and nearby grazed sites. Our results show that a different vegetation with a high proportion of vascular plants developed due to reduced herbivory, resulting in a larger and more diverse input of polysaccharides to the soil at exclosed study sites. This coincided with higher sugar and amino acid concentrations in the soil at this site as well as the establishment of a more abundant and active microbiota, including saprotrophic fungi with broad substrate ranges, like Helotiales (Ascomycota) and Agaricales (Basidiomycota). A detailed description of fungal transcriptional profiles revealed higher gene expression for cellulose, hemicellulose, pectin, lignin and chitin degradation at herbivory-exclosed sites. Furthermore, we observed an increase in the number of genes and transcripts for predatory eukaryotes such as Entomobryomorpha (Arthropoda). We conclude that in the absence of herbivory, the development of a vascular vegetation alters the soil polysaccharide composition and supports larger and more active populations of fungi and predatory eukaryotes.

Changes in the flavan-3-ol and polysaccharide content during the fermentation of Vitis vinifera Cabernet-Sauvignon and cold-hardy Vitis varieties Frontenac and Frontenac blanc

Grape variety has a significant impact on wine flavan-3-ol and polysaccharide profile. The main objective of this work was to study differences in flavan-3-ol and polysaccharide diffusion from grape to wine during the fermentative alcoholic maceration of three Vitis sp. varieties: the cold-hardy hybrid varieties Frontenac and Frontenac blanc, and the V. vinifera variety Cabernet-Sauvignon. Polysaccharides from must and wine were precipitated by ethanol and quantified using the phenol-sulfuric method of Dubois. Flavan-3-ol concentration and profile were analysed by HPLC-FLD. Results showed that wines from Frontenac and Frontenac blanc had less oligomeric and polymeric flavan-3-ols than those from V. vinifera Cabernet-Sauvignon. Wines made from Frontenac also had a higher concentration in total polysaccharides. Preliminary results from GPC/SEC analyses suggested that Frontenac wine had a higher content in mannoproteins and rhamnogalacturonan-2 polysaccharides compared to the other studied varieties. Overall, wines of Frontenac showed the highest content in total polysaccharides, and the lowest content in condensed tannins. As polysaccharides are known to negatively impact wine perceived astringency, these results suggest that significant attention should be given to the polysaccharide composition of cold-hardy cultivars in the context of cold climate wine production. Such knowledge may help winemakers from cold climate areas to improve the winemaking processes and final wine composition when working with cold-hardy Vitis sp. varieties.Knowledge on interspecific hybrid polysaccharide and flavan-3-ol kinetic during the alcoholic fermentative maceration may help the winemakers from cold climate areas to improve winemaking processes and final wine composition.

Antioxidative study of polysaccharides extracted from red (Kappaphycus alvarezii), green (Kappaphycus striatus) and brown (Padina gymnospora) marine macroalgae/seaweed

Sterile and fresh tissues of three marine macroalgae red, green and brown (Kappaphycus alvarezii, Kappaphycus striatus and Padina gymnospora) collected from Malaysia east costal seas were compared for the antioxidants and polysaccharide composition of sugars as well as the active components. Results obtained showed that polysaccharides isolated from Kappaphycus alvarezii, Kappaphycus striatus and Padina gymnospora) can be used as a source of natural antioxidant compounds as they possess antioxidant potential in which the Padina gymnospora showed 15.56 ± 0.12 mg/mL to be the best antioxidants among all the polysaccharides studied. The hot water extraction method is effective in isolating polysaccharides from studied seaweeds. The GC–MS analysis revealed that there is presence of chemical compounds such as furfural was 25.53% in Kappaphycus alvarezii and 21.04% in Kappaphycus striatus also Padina gymnospora incorporates n- Hexadecanoic acid about 26.31% in seaweed polysaccharides that contribute to their antioxidant activities. Further studies can be done on determining the seaweed species that are available abundantly with the best source of natural antioxidant compounds.

Cell Wall Polysaccharide Composition of Grafted ‘Liberty’ Watermelon With Reduced Incidence of Hollow Heart Defect

Grafting watermelon scions to interspecific squash hybrids has been found to increase fruit firmness. Triploid (seedless) watermelon are prone to hollow heart (HH), an internal fruit disorder characterized by a crack in the placental tissue expanding to a cavity. Although watermelon with lower tissue firmness tend to have a higher HH incidence, associated differences in cell wall polysaccharide composition are unknown. Grafting “Liberty” watermelon to “Carnivor” (interspecific hybrid rootstock, C. moschata × C. maxima) reduced HH 39% and increased tissue firmness by 3 N. Fruit with and without severe HH from both grafted and non-grafted plants were analyzed to determine differences in cell wall polysaccharides associated with grafting and HH. Alcohol insoluble residues (AIR) were sequentially extracted from placental tissue to yield water soluble (WSF), carbonate soluble (CSF), alkali soluble (ASF), or unextractable (UNX) pectic fractions. The CSF was lower in fruit with HH (24.5%) compared to those without HH (27.1%). AIRs were also reduced, hydrolyzed, and acetylated for GC-MS analysis of monosaccharide composition, and a portion of each AIR was methylated prior to hydrolysis and acetylation to produce partially methylated alditol acetates for polysaccharide linkage assembly. No differences in degree of methylation or galacturonic and glucuronic acid concentrations were found. Glucose and galactose were in highest abundance at 75.9 and 82.4 μg⋅mg–1 AIR, respectively, followed by xylose and arabinose (29.3 and 22.0 μg⋅mg–1). Mannose was higher in fruit with HH (p < 0.05) and xylose was highest in fruit from grafted plants (p < 0.05). Mannose is primarily found in heteromannan and rhamnogalacturonan I side chains, while xylose is found in xylogalacturonan or heteroxylan. In watermelon, 34 carbohydrate linkages were identified with galactose, glucose, and arabinose linkages in highest abundance. This represents the most comprehensive polysaccharide linkage analysis to date for watermelon, including the identification of several new linkages. However, total pectin and cell wall composition data could not explain the increased tissue firmness observed in fruit from grafted plants. Nonetheless, grafting onto the interspecific hybrid rootstock decreased the incidence of HH and can be a useful method for growers using HH susceptible cultivars.