Genetic Transformation of Trichoderma spp.

The production of biofuels from plant biomass is dependent on the availability of enzymes that can hydrolyze the plant cell wall polysaccharides to their monosaccharides. These enzyme mixtures are formed by microorganisms but their native compositions and properties are often not ideal for application. Genetic engineering of these microorganisms is therefore necessary, in which introduction of DNA is an essential precondition. The filamentous fungus Trichoderma reesei-the main producer of plant-cell-wall-degrading enzymes for biofuels and other industries-has been subjected to intensive genetic engineering toward this goal and has become one of the iconic examples of the successful genetic improvement of fungi. However, the genetic manipulation of other enzyme-producing Trichoderma species is frequently less efficient and, therefore, rarely managed. In this chapter, we therefore describe the two potent methods of Trichoderma transformation mediated by either (a) polyethylene glycol (PEG) or (b) Agrobacterium. The methods are optimized for T. reesei but can also be applied for such transformation-resilient species as T. harzianum and T. guizhouense, which are putative upcoming alternatives for T. reesei in this field. The protocols are simple, do not require extensive training or special equipment, and can be further adjusted for T. reesei mutants with particular properties.

Phylogenomic Analyses of Nucleotide-Sugar Biosynthetic and Interconverting Enzymes Illuminate Cell Wall Composition in Fungi.

The fungi are an enormously successful eukaryotic lineage that has colonized every aerobic habitat on Earth. This spectacular expansion is reflected in the dynamism and diversity of the fungal cell wall, a matrix of polysaccharides and glycoproteins pivotal to fungal life history strategies and a major target in the development of antifungal compounds. Cell wall polysaccharides are typically synthesized by Leloir glycosyltransferases, enzymes that are notoriously difficult to characterize, but their nucleotide-sugar substrates are well known and provide the opportunity to inspect the monosaccharides available for incorporation into cell wall polysaccharides and glycoproteins. In this work, we have used phylogenomic analyses of the enzymatic pathways that synthesize and interconvert nucleotide-sugars to predict potential cell wall monosaccharide composition across 491 fungal taxa. The results show a complex evolutionary history of these cell wall enzyme pathways and, by association, of the fungal cell wall. In particular, we see a significant reduction in monosaccharide diversity during fungal evolution, most notably in the colonization of terrestrial habitats. However, monosaccharide distribution is also shown to be varied across later-diverging fungal lineages.IMPORTANCE This study provides new insights into the complex evolutionary history of the fungal cell wall. We analyzed fungal enzymes that convert sugars acquired from the environment into the diverse sugars that make up the fundamental building blocks of the cell wall. Species-specific profiles of these nucleotide-sugar interconverting (NSI) enzymes for 491 fungi demonstrated multiple losses and gains of NSI proteins, revealing the rich diversity of cell wall architecture across the kingdom. Pragmatically, because cell walls are essential to fungi, our observations of variation in sugar diversity have important implications for the development of antifungal compounds that target the sugar profiles of specific pathogens.

The heteropolysaccharide of Mangifera indica fruit: Isolation, chemical profile, complexation with ß-lactoglobulin and antioxidant activity.

A 91 kDa heteropolysaccharide (F2) was isolated from Mangifera indica fruit via extraction with H2O, purification by C2H5OH, starch removal and ion exchange chromatography. This polymer was made up mostly of Ara, Gal, Glc, Rha, Xyl, and GalA in a 37: 29: 9:3:2:19 molar proportion. It inherited a small backbone containing GalpA and Rhap units substituted with very large side chains containing differently linked Ara and Gal units plus esterified gallic acid (GA) residue. Several enzymes generated oligosaccharides including (i) Ara2-10Ac6-22, (ii) Gal1-8Ac5-26 and (iii) GA1Gal1Ac7 were characterized. This polysaccharide, which showed dose dependent antioxidant activity, exhibited synergism with gallic acid, and formed a complex (K = 1.2 × 106 M-1) with ß-lactoglobulin. Accordingly, H2O treatment produces a polysaccharide with desired biochemical properties; this could be effective in designing innovative functional food with flexible makeup.

Using a PCR instrument to hydrolyze polysaccharides for monosaccharide composition analyses.

Monosaccharide composition analysis after acid hydrolysis is the first step towards structural characterization of the polysaccharides. To modernize the hydrolytic procedure, we used a polymerase chain reaction (PCR) instrument to accomplish the task, which allows to generate monosaccharide products from up to 96 samples simultaneously within 30 min. Fucoidan, chitosan and propylene glycol alginate sodium sulfate (PSS) were chosen as representatives of complex, basic and acidic polysaccharides to optimize the hydrolytic conditions, respectively, through the orthogonal L9 (34) experiments. The hydrolysis loss ratio for monosaccharide standards were also measured. Using this assay, the hydrolysis plus 1-phenyl-3-methyl-5-pyrazolone (PMP) labeling of the monosaccharide products could be accomplished in 90 min with the RSD values less than 5 % based on HPLC analysis. We further confirmed the reliability of the assay by HPLC coupled MS analysis. In conclusion, PCR instrument-based hydrolysis assay is suitable for monosaccharide composition analysis of complex, acidic and basic polysaccharides.

Effects of lignin, cellulose, hemicellulose, sucrose and monosaccharide carbohydrates on soybean physical stem strength and yield in intercropping.

Soybean (Glycine max L.) has been extensively cultivated in maize-soybean relay intercropping systems in southwest China. However, during the early co-growth period, soybean seedlings suffer from severe shading by maize resulting in lodging and significant yield reduction. The purpose of the present research was to investigate the reasons behind severe lodging and yield loss. Therefore, four different soybean genotypes (B3, B15, B23, and B24) having different agronomic characteristics were cultivated in intercropping and monocropping planting patterns. The results showed that under different planting patterns, the stem resistance varied among genotypes (P < 0.01). The lodging resistance index of B3, B15, B23, and B24 genotypes was 70.9%, 60.5%, 65.2%, and 57.4%, respectively, under intercropping, among which the B24 genotype was less affected by the shade environment as there was little decrease in the lodging resistance index of this genotype under intercropping. The lignin content of B23 and B24 was significantly higher than that of B3 and B15 under both planting patterns. Under intercropping, the hemicellulose content of B23 and B24 stems was significantly higher than that of B3 and B15. Compared to the monocropping, the content of mannose in the structural carbohydrate of soybean stems was decreased in all genotypes except B23, but the difference was not significant. The content of xylose in the structural carbohydrate of soybean stems was significantly higher than that in B3 and B15. Mannose content showed no significant difference among genotypes. The arabinose content of B24 was significantly higher than that of B3, B15, and B23. The effective pod number, seed number per plant, seed weight per plant and yield of soybean plants were significantly decreased under intercropping. Conclusively, manipulation of structural and nonstructural carbohydrate rich soybean genotypes in intercropping systems could alleviate the yield loss due to lodging.

Microbial origin of bioflocculation components within a promising natural bioflocculant resource of Ruditapes philippinarum conglutination mud from an aquaculture farm in Zhoushan, China.

Ruditapes philippinarum conglutination mud (RPM) is a byproduct from the aquiculture of an important commercially bivalve mollusk R. philippinarum and has been recently reported as a promising natural bioflocculant resource. However the origin of bioflocculation components within RPM is still a pending doubt and impedes its effective exploitation. This study investigated the probability that RPM bioflocculation components originate from its associated microbes. RPM samples from an aquaculture farm in Zhoushan of China were applied to characterize its microbial community structure, screen associated bioflocculant-producing strains, and explore the homology between extracellular polysaccharides (EPS) from bioflocculant-producing isolates and RPM flocculation components. Results showed that RPM exhibited high bacterial biodiversity, with Proteobacteria, Bacteroidetes and Actinobacteria as the most abundant phyla; hgcI_clade, CL500_29_marine_group, Fusibacter, MWH_UniP1_aquatic_group and Arcobacter as the dominant genera. Fourteen highly efficient bioflocculant-producing strains were screened and phylogenetically identified as Pseudoalteromonas sp. (5), Psychrobacter sp. (3), Halomonas sp. (2), Albirhodobacter sp. (1), Celeribacter sp. (1), Kocuria sp. (1) and Bacillus sp. (1), all of which except Bacillus sp. were reported for the first time for their excellent flocculation capability. Furthermore, EPS from the bioflocculant-producing strains exhibited highly similar monosaccharide composition to the reported flocculation-effective RPM polysaccharides. On the other hand, the existence of fungi in RPM was rare and showed no flocculation functionality. Findings from Zhoushan RPM strongly supported that RPM flocculation components were of bacterial origin and make RPM reproduction possible by fermentation approach.

Glycosylation Profile of the Transferrin Receptor in Gestational Iron Deficiency and Early-Onset Severe Preeclampsia.

OBJECTIVE: To examine the expression of hypoxia-inducible factor-1α (HIF-1α), TfR1, and TfR1-attached terminal monosaccharides in placentas of women with IDAP and severe preeclampsia. METHODS: TfR1 and HIF-1α were detected by western blot. Immunoadsorption of TfR1 was performed to characterize the terminal monosaccharides by specific lectin binding. RESULTS: There was no difference in the expression of TfR1 and HIF-1α between groups. Lectin blot analysis pointed out an overexpression of galactose ß1-4 N-acetylglucosamine (Gal-GlcNAc) and mannose in severe preeclampsia. CONCLUSION: The increase in Gal-GlcNAc may be due to the increased presence of antennary structures and the mannose glycans of TfR1 may indicate the presence of misfolded or incomplete proteins. These findings may be associated with the low expression of placental TfR1 in women with preeclampsia.

Exogenous RNA interference exposes contrasting roles for sugar exudation in host-finding by plant pathogens.

Plant parasitic nematodes (PPN) locate host plants by following concentration gradients of root exudate chemicals in the soil. We present a simple method for RNA interference (RNAi)-induced knockdown of genes in tomato seedling roots, facilitating the study of root exudate composition, and PPN responses. Knockdown of sugar transporter genes, STP1 and STP2, in tomato seedlings triggered corresponding reductions of glucose and fructose, but not xylose, in collected root exudate. This corresponded directly with reduced infectivity and stylet thrusting of the promiscuous PPN Meloidogyne incognita, however we observed no impact on the infectivity or stylet thrusting of the selective Solanaceae PPN Globodera pallida. This approach can underpin future efforts to understand the early stages of plant-pathogen interactions in tomato and potentially other crop plants.

Probing sialoglycans on fetal bovine fetuin with azido-sugars using glycosyltransferases.

Sialic acids are negatively charged sugar residues commonly found on the terminal positions of most glycoproteins. They play important roles in the stability and solubility of these proteins. Due to their unique positioning, they also frequently act as receptors for various ligands, and therefore are involved in numerous cell-cell and cell-pathogen interactions. Here, using in vitro incorporation of clickable monosaccharides with various glycosyltransferases, we probed the sialoglycans on fetal bovine fetuin. The incorporated monosaccharides were detected with chemiluminescence via click chemistry in a format of western blotting. The results indicate that the non-reducing end Gal residues on both N- and O-glycans are fully sialylated, but the peptide-linked GalNAc residues in O-glycans are not. The presence of sialyl core-1 glycan was repeatedly confirmed by probing with α-2,3-sialyltransferases, N-acetylgalactosaminide α-2,6-sialyltransferases and a ß-1,6-N-acetylglucosaminyltransferase that is specific for core-1 glycan. The results also suggest the presence of a minute amount of sialyl Tn antigen on the protein.

Global microarray analysis of carbohydrate use in alkaliphilic hemicellulolytic bacterium Bacillus sp. N16-5.

The alkaliphilic hemicellulolytic bacterium Bacillus sp. N16-5 has a broad substrate spectrum and exhibits the capacity to utilize complex carbohydrates such as galactomannan, xylan, and pectin. In the monosaccharide mixture, sequential utilization by Bacillus sp. N16-5 was observed. Glucose appeared to be its preferential monosaccharide, followed by fructose, mannose, arabinose, xylose, and galactose. Global transcription profiles of the strain were determined separately for growth on six monosaccharides (glucose, fructose, mannose, galactose, arabinose, and xylose) and four polysaccharides (galactomannan, xylan, pectin, and sodium carboxymethylcellulose) using one-color microarrays. Numerous genes potentially related to polysaccharide degradation, sugar transport, and monosaccharide metabolism were found to respond to a specific substrate. Putative gene clusters for different carbohydrates were identified according to transcriptional patterns and genome annotation. Identification and analysis of these gene clusters contributed to pathway reconstruction for carbohydrate utilization in Bacillus sp. N16-5. Several genes encoding putative sugar transporters were highly expressed during growth on specific sugars, suggesting their functional roles. Two phosphoenolpyruvate-dependent phosphotransferase systems were identified as candidate transporters for mannose and fructose, and a major facilitator superfamily transporter was identified as a candidate transporter for arabinose and xylose. Five carbohydrate uptake transporter 1 family ATP-binding cassette transporters were predicted to participate in the uptake of hemicellulose and pectin degradation products. Collectively, microarray data improved the pathway reconstruction involved in carbohydrate utilization of Bacillus sp. N16-5 and revealed that the organism precisely regulates gene transcription in response to fluctuations in energy resources.

Sugar-binding and crystallographic studies of an arabinose-binding protein mutant (Met108Leu) that exhibits enhanced affinity and altered specificity.

In addition to hydrogen bonds, van der Waals forces contribute to the affinity of protein-carbohydrate interactions. Nonpolar van der Waals contacts in the complexes of the L-arabinose-binding protein (ABP) with monosaccharides have been studied by means of site-directed mutagenesis, equilibrium and rapid kinetic binding techniques, and X-ray crystallography. ABP, a periplasmic transport receptor of Escherichia coli, binds L-arabinose, D-galactose, and D-fucose with preferential affinity in the order of Ara greater than Gal much greater than Fuc. Well-refined, high-resolution structures of ABP complexed with the three sugars revealed that the structural differences in the ABP-sugar complexes are localized around C5 of the sugars, where the equatorial H of Ara has been substituted for CH3 (Fuc) or CH2OH (Gal). The side chain of Met108 undergoes a sterically dictated, ligand-specific, conformational change to optimize nonpolar interactions between its methyl group and the sugar. We found that the Met108Leu ABP binds Gal tighter than wild-type ABP binds Ara and exhibits a preference for ligand in the order of Gal much greater than Fuc greater than Ara. The differences in affinity can be attributed to differences in the dissociation rates of the ABP-sugar complexes. We have refined at better than 1.7-A resolution the crystal structures of the Met108Leu ABP complexed with each of the sugars and offer a molecular explanation for the altered binding properties.

The functional role of the carbohydrate in human melanoma associated antigens. I. Antigen-antibody complex formation.

Messenger ribonucleic acids (mRNA) for tumor associated antigens (TAA) were isolated from human melanotic melanoma (HMMC-ShA, HMMC-WJP) and amelanotic melanoma (HMMC-Sr, HMMC-KM) cells. The melanoma cells were cultured in standard Eagle's MEM and in MEM supplemented with tunicamycin (Tu). The mRNAs were translated in vitro using wheat germ system. Using a standard immunodiffusion system, TAA from melanoma cells harvested from standard MEM differed from the TAA obtained from cells harvested from MEM supplemented with Tu, whereas, TAA pretreated with endo-beta-N-acetylglucosaminidase resembled TAA of cells harvested from MEM supplemented with Tu. The translation products synthesized by mRNA of melanotic melanoma cells resembled TAA extracted directly with 3 M KCl from the cells, but differed from TAA isolated directly from the cells or synthesized by mRNA from amelanotic melanoma cells. Therefore, the melanotic melanoma differed from the amelanotic melanoma cells in their genetic expression. The TAA-immunological reactivities are modulated by a post-translation step regulating genetic expression.