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Results for “OLIGOSACCHARIDES”

Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

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At least 109 records · Page 6
All sourcesNASA NTRSDOE OSTI
DOE OSTI2021

CRISPR/Cas9 disruption of glucan synthase in Nannochloropsis gaditana attenuates accumulation of β-1,3-glucose oligomers

Nannochloropsis species have garnered significant interest for biofuel production due to their ability to accumulate high levels of triacylglycerols (TAGs), especially following nitrogen-starvation. Furthermore, the first response to nutrient starvation is the synthesis of chrysolaminarin, a soluble β-1,3-glucan with β-1,6-branching. We employ CRISPR/Cas9 to knock out two key enzymes responsible for the synthesis of this oligosaccharide: a beta-glucan synthase (BGS) gene putatively responsible for the glucose β-1,3-linkages, and a transglycosylase (TGS) which putatively catalyzes β-1,6-branching. Analysis of the biomass from the generated mutants confirmed an ~5-fold decrease in the accumulation of soluble carbohydrate following nitrogen starvation, without an observed growth defect in a diel light-cycling regime compared to CRISPR-expressing controls.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

The pectin puzzle: Decoding the fine structure of rhamnogalacturonan-I (RG-I) in Arabidopsis thaliana uncovers new pectin features

Pectin is generally divided into four distinct structural categories, namely homogalacturonan, xylogalacturonan, rhamnogalacturonan I (RG-I) and rhamnogalacturonan II. While much of the structural diversity of homogalacturonan, xylogalacturonan and rhamnogalacturonan II has been elucidated, the structural features of RG-I are less well understood. In this work, we employed multiple complementary analytical techniques to present a detailed structural analysis of RG-I in the model species Arabidopsis thaliana . Starting with highly purified RG-I from different Arabidopsis tissues, we employed comparative linkage and nuclear magnetic resonance analysis along with mass spectrometry analysis of enzymatically digested RG-I oligosaccharides. Besides the presence of the canonical α-1,5-arabinan, β-1,4-galactan, β-1,6-galactan and arabinogalactan RG-I side chains of varying lengths, we show that a large portion of the β-1,6-galactan is terminated by either 4-O-methyl β-glucuronic acid (GlcA) residues or, to a smaller degree, β-GlcA that lacks the Me-ether group. Importantly, O-acetylation of RG-I GalA residues is a minor modification while 10 % of the backbone Rha residues are 3-O-acetylated, and most of the acetylated Rha is additionally branched with β-galactose substituents. Taken together, the combined results of these different analytical techniques present the most comprehensive structural overview of Arabidopsis thaliana RG-I to date.

25 ENERGY STORAGE↗
DOE OSTI2025

The unique architecture of umbrella toxins permits a two-tiered molecular bet-hedging strategy for interbacterial antagonism

Bacteria exist in competitive and rapidly changing environments in which the nature of future threats cannot be easily predicted. Streptomyces coelicolor produces three antibacterial umbrella particles that harbor distinct polymorphic toxin domains and an overlapping set of six diversified lectins. Here, we show that the exquisite specificity of umbrella particles derives from lectin-mediated species-specific binding to previously undescribed hypervariable surface glycoconjugates. A cryo-electron microscopy (cryo-EM) structure of one such lectin in complex with its oligosaccharide substrate defines the molecular basis for targeting through the coordinated recognition of multiple glycan features. Biochemical and genetic studies of several target species, in conjunction with lectin-swapping experiments, support a model whereby S. coelicolor umbrella toxin diversification at the levels of lectin composition and toxin polymorphism represents a unique, two-tiered bet-hedging strategy. Bioinformatic analyses support this as a means by which the unusual architecture of umbrella toxins offers Streptomyces a generalizable strategy to antagonize an unpredictable array of competitors.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Dynamic genetic adaptation of Bacteroides thetaiotaomicron during murine gut colonization

To understand how a bacterium ultimately succeeds or fails in adapting to a new host, it is essential to assess the temporal dynamics of its fitness over the course of colonization. Here, we introduce a human-derived commensal organism, Bacteroides thetaiotaomicron (Bt), into the guts of germ-free mice to determine whether and how the genetic requirements for colonization shift over time. Combining a high-throughput functional genetics assay and transcriptomics, we find that gene usage changes drastically during the first days of colonization, shifting from high expression of amino acid biosynthesis genes to broad upregulation of diverse polysaccharide utilization loci. Within the first week, metabolism becomes centered around utilization of a predominant dietary oligosaccharide, and these changes are largely sustained through 6 weeks of colonization. Spontaneous mutations in wild-type Bt also evolve around this locus. These findings highlight the importance of considering temporal colonization dynamics in developing more effective micro biome-based therapies.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Biotechnological advances in algae-based foods: applications in nutrition and microbiome health

Algae are a sustainable, nutrient-rich resource with growing potential in food biotechnology. Their ability to thrive in diverse environments makes them a promising alternative to conventional crops. Rich in proteins, essential fatty acids, and bioactive compounds, algae support the development of functional foods, including plant-based meat and seafood alternatives. Advances in synthetic biology and fermentation have enhanced algal nutrient profiles and enabled novel applications. Algae-derived polysaccharides, such as alginate, fucoidan, laminarin, and porphyran, exhibit prebiotic effects by modulating the gut microbiota and promoting SCFA production. Enzymatic hydrolysis efficiently produces bioactive oligosaccharides, while engineered microbial systems support scalable production. Algae also enable synbiotic food development by serving as both prebiotic substrates and probiotic carriers.

Yu, Sora↗
DOE OSTI2017

Raman and Infrared spectroscopies and X-ray diffraction data on bupivacaine and ropivacaine complexed with 2-hydroxypropyl-β-cyclodextrin

The data presented in this article are related to the research article entitled “Probing the dynamics of complexed local anesthetics via neutron scattering spectroscopy and DFT calculations. This work shows the molecular and structural behavior of the local anesthetics (LAs) bupivacaine (BVC, C 18 H 28 N 2 O) and ropivacaine (RVC, C 17 H 26 N 2 O) before and after complexation with the water-soluble oligosaccharide 2-hydroxypropyl–β–cyclodextrin (HP-β-CD).

71 CLASSICAL AND QUANTUM MECHANICS, GENERAL PHYSIC↗
DOE OSTI2020

Enhanced 2'-Fucosyllactose production by engineered Saccharomyces cerevisiae using xylose as a co-substrate

2'-Fucosyllactose (2'-FL), a human milk oligosaccharide with confirmed benefits for infant health, is a promising infant formula ingredient. Although Escherichia coli, Saccharomyces cerevisiae, Corynebacterium glutamicum, and Bacillus subtilis have been engineered to produce 2'-FL, their titers and productivities need be improved for economic production. Glucose along with lactose have been used as substrates for producing 2'-FL, but accu- mulation of by-products due to overflow metabolism of glucose hampered efficient production of 2' -FL regardless of a host strain. To circumvent this problem, we used xylose, which is the second most abundant sugar in plant cell wall hydrolysates and is metabolized through oxidative metabolism, for the production of 2'-FL by engi- neered yeast. Specifically, we modified an engineered S. cerevisiae strain capable of assimilating xylose to pro- duce 2' -FL from a mixture of xylose and lactose. First, a lactose transporter (Lac12) from Kluyveromyces lactis was introduced. Second, a heterologous 2'-FL biosynthetic pathway consisting of enzymes Gmd, WcaG, and WbgL from Escherichia coli was introduced. Third, we adjusted expression levels of the heterologous genes to maximize 2'-FL production. The resulting engineered yeast produced 25.5 g/L of 2'-FL with a volumetric productivity of 0.35 g/L∙h in a fed-batch fermentation with lactose and xylose feeding to mitigate the glucose repression. Interestingly, the major location of produced 2'-FL by the engineered yeast can be changed using different culture media. While 72% of the produced 2'-FL was secreted when a complex medium was used, 82% of the produced 2'-FL remained inside the cells when a minimal medium was used. In conclusion, as yeast extract is already used as food and animal feed ingredients, 2'-FL enriched yeast extract can be produced cost-effectively using the 2'-FL-accumu- lating yeast cells.

2′ -fucosyllactose↗
DOE OSTI2018

Direct Determination of Hydroxymethyl Conformations of Plant Cell Wall Cellulose Using 1 H Polarization Transfer Solid-State NMR

In contrast to the well-studied crystalline cellulose of microbial and animal origins, cellulose in plant cell walls is disordered due to its interactions with matrix polysaccharides. Plant cell wall (PCW) is an undisputed source of sustainable global energy; therefore, it is important to determine the molecular structure of PCW cellulose. The most reactive component of cellulose is the exocyclic hydroxymethyl group: when it adopts the tg conformation, it stabilizes intrachain and interchain hydrogen bonding, while gt and gg conformations destabilize the hydrogen-bonding network. So far, information about the hydroxymethyl conformation in cellulose has been exclusively obtained from 13 C chemical shifts of monosaccharides and oligosaccharides, which do not reflect the environment of cellulose in plant cell walls. Here, we use solid-state Nuclear Magnetic Resonance (ssNMR) spectroscopy to measure the hydroxymethyl torsion angle of cellulose in two model plants, by detecting distance-dependent polarization transfer between H4 and H6 protons in 2D 13 C– 13 C correlation spectra. We show that the interior crystalline portion of cellulose microfibrils in Brachypodium and Arabidopsis cell walls exhibits H4–H6 polarization transfer curves that are indicative of a tg conformation, whereas surface cellulose chains exhibit slower H4–H6 polarization transfer that is best fit to the gt conformation. Joint constraints by the H4–H6 polarization transfer curves and 13 C chemical shifts indicate that it is unlikely for interior cellulose to have a significant population of the gt and gg conformation mixed with the tg conformation, while surface cellulose may adopt a small percentage of the gg conformation. These results provide new constraints to the structure and matrix interactions of cellulose in plant cell walls, and represent the first direct determination of a torsion angle in an important noncrystalline carbohydrate polymer.

09 BIOMASS FUELS↗
DOE OSTI2018

Understanding the pH-Dependent Reaction Mechanism of a Glycoside Hydrolase Using High-Resolution X-ray and Neutron Crystallography

Glycoside hydrolases (GHs) commonly use the retaining or inverting mechanisms to hydrolyze carbohydrates, and the rates of catalysis are usually pH dependent. Deeper understanding of these pH-dependent reaction mechanisms is of great importance for protein engineering and drug design. We used high-resolution X-ray crystallography to analyze the sugar ring configurations of an oligosaccharide ligand during hydrolysis for the family 11 GH, and the results support the 1 S 3 → 4 H 3 → 4 C 1 conformational itinerary. These results indicate that sugar ring flexibility may help to distort and break the glycosidic bond. Constant pH molecular dynamics simulations and neutron crystallography demonstrate that the catalytic glutamate residue (E177) has alternate conformational changes to transfer a proton to cleave the glycosidic bond. Furthermore, a neutron crystallography analysis shows that the H-bond length between E177 and its nearby tyrosine residue (Y88) is shortened when the pH increases, preventing E177 from rotating downward and obtaining a proton from the solvent for catalysis. This result indicates that the H-bond length variation may play a key role in the pH-dependent reaction mechanism. Here, our results demonstrate that both sugar ring flexibility and protein dynamics are important in the pH-dependent reaction mechanism and may help to engineer GHs with different pH optima.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2020

Increasing the Carbohydrate Output of Bamboo Using a Combinatorial Pretreatment

Bamboo represents a family of highly lignified biomass with significant resistance to cellulolytic enzyme degradation, and typically, a severe thermochemical pretreatment is needed to overcome its recalcitrance for producing fermentable sugars. Combinatorial pretreatments with a low holding temperature and low alkali dosage were investigated in this study in an attempt to remove the impediments to the accessibility of bamboo cellulose to enzymes and enable the efficient conversion of hemicellulose. Upon autohydrolysis at 180 °C, more than half of the hemicellulose (~58%) was solubilized, contributing to further delignification in the subsequent dilute alkali extraction (DAE). The solubilized hemicellulose mainly existed in the form of soluble xylan, which, after a novel endo-β-1,4-xylanase hydrolysis, generated a large amount of xylo-oligosaccharides with the xylobiose and xylotriose proportion reaching 72.99%. After the fractionation with combinatorial pretreatments, cellulose and xylose enzymatic hydrolysis yields were 73.36 and 86.98%, respectively, representing increments of 22.42 and 43.15% compared with the sole autohydrolysis process. Fluorescence microscopy (FM) and confocal Raman microscopy (CRM) images revealed that these dramatic improvements were caused by the deconstruction of the bamboo cell walls, including separation of the adjacent cell walls, removal of hemicellulose and lignin, migration and redistribution of lignin, and the increased exposure of cellulose. In addition, lignin chemical structure variations during the combinatorial pretreatment were analyzed with gel permeation chromatography (GPC), 2D HSQC NMR, and 31 P NMR, and the correlation between the lignin structure and enzymatic hydrolysis efficiency was investigated. In summary, this study highlighted the application of combinatorial pretreatments to maximize the carbohydrate output of bamboo and unveiled their mechanism that enabled the high-efficiency utilization of bamboo.

09 BIOMASS FUELS↗
DOE OSTI2014

Effect of Divalent Cation Removal on the Structure of Gram-Negative Bacterial Outer Membrane Models

The Gram-negative bacterial outer membrane (GNB-OM) is asymmetric in its lipid composition with a phospholipid-rich inner leaflet and an outer leaflet predominantly composed of lipopolysaccharides (LPS). LPS are polyanionic molecules, with numerous phosphate groups present in the lipid A and core oligosaccharide regions. The repulsive forces due to accumulation of the negative charges are screened and bridged by the divalent cations (Mg 2+ and Ca 2+ ) that are known to be crucial for the integrity of the bacterial OM. Indeed, chelation of divalent cations is a well-established method to permeabilize Gram-negative bacteria such as Escherichia coli. Here, we use X-ray and neutron reflectivity (XRR and NR, respectively) techniques to examine the role of calcium ions in the stability of a model GNB-OM. Using XRR we show that Ca 2+ binds to the core region of the rough mutant LPS (RaLPS) films, producing more ordered structures in comparison to divalent cation free monolayers. Using recently developed solid-supported models of the GNB-OM, we study the effect of calcium removal on the asymmetry of DPPC:RaLPS bilayers. We show that without the charge screening effect of divalent cations, the LPS is forced to overcome the thermodynamically unfavorable energy barrier and flip across the hydrophobic bilayer to minimize the repulsive electrostatic forces, resulting in about 20% mixing of LPS and DPPC between the inner and outer bilayer leaflets. These results reveal for the first time the molecular details behind the well-known mechanism of outer membrane stabilization by divalent cations. This confirms the relevance of the asymmetric models for future studies of outer membrane stability and antibiotic penetration.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2015

Human gut Bacteroidetes can utilize yeast mannan through a selfish mechanism

Yeasts, which have been a component of the human diet for at least 7000 years, possess an elaborate cell wall α-mannan. The influence of yeast mannan on the ecology of the human microbiota is unknown. Here we show that yeast α-mannan is a viable food source for Bacteroides thetaiotaomicron (Bt), a dominant member of the microbiota. Detailed biochemical analysis and targeted gene disruption studies support a model whereby limited cleavage of α-mannan on the surface generates large oligosaccharides that are subsequently depolymerized to mannose by the action of periplasmic enzymes. Co-culturing studies showed that metabolism of yeast mannan by Bt presents a ‘selfish’ model for the catabolism of this recalcitrant polysaccharide. This report shows how a cohort of highly successful members of the microbiota has evolved to consume sterically-restricted yeast glycans, an adaptation that may reflect the incorporation of eukaryotic microorganisms into the human diet.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2017

Deciphering minimal antigenic epitopes associated with Burkholderia pseudomallei and Burkholderia mallei lipopolysaccharide O-antigens

Burkholderia pseudomallei (Bp) and Burkholderia mallei (Bm), the etiologic agents of melioidosis and glanders, respectively, cause severe disease in both humans and animals. Studies have highlighted the importance of Bp and Bm lipopolysaccharides (LPS) as vaccine candidates. Here we describe the synthesis of seven oligosaccharides as the minimal structures featuring all of the reported acetylation/methylation patterns associated with Bp and Bm LPS O-antigens (OAgs). Our approach is based on the conversion of an L-rhamnose into a 6-deoxy-L-talose residue at a late stage of the synthetic sequence. Using biochemical and biophysical methods, we demonstrate the binding of several Bp and Bm LPS-specific monoclonal antibodies with terminal OAg residues. Mice immunized with terminal disaccharide–CRM197 constructs produced high-titer antibody responses that crossreacted with Bm-like OAgs. Collectively, these studies serve as foundation for the development of novel therapeutics, diagnostics, and vaccine candidates to combat diseases caused by Bp and Bm.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2019

Visualizing conformation transitions of the Lipid II flippase MurJ

The biosynthesis of many polysaccharides, including bacterial peptidoglycan and eukaryotic N-linked glycans, requires transport of lipid-linked oligosaccharide (LLO) precursors across the membrane by specialized flippases. MurJ is the flippase for the lipid-linked peptidoglycan precursor Lipid II, a key player in bacterial cell wall synthesis, and a target of recently discovered antibacterials. However, the flipping mechanism of LLOs including Lipid II remains poorly understood due to a dearth of structural information. Here we report crystal structures of MurJ captured in inward-closed, inward-open, inward-occluded and outward-facing conformations. Together with mutagenesis studies, we elucidate the conformational transitions in MurJ that mediate lipid flipping, identify the key ion for function, and provide a framework for the development of inhibitors.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2019

The crystal structure of the chitinase ChiA74 of Bacillus thuringiensis has a multidomain assembly

There is no structural information about any chitinase synthesized by Bacillus thuringiensis , the most successful microbial insect larvicide used worldwide. In this study, we solved the 3D structure of the chitinase ChiA74 at 2.26 Å. The crystal structure shows that ChiA74 is composed of a modular arrangement formed by (i) a catalytic region (CD), (ii) a chitinase insertion domain (CID), (iii) a fibronectin type III domain (FnIII), and (iv) a chitin binding domain (CBD). The location of the CBD with respect to the CD has no structural similarity to other chitinases with known structures. The activity of a ChiA74 lacking its secretion signal peptide (ChiA74Δsp) and a truncated version lacking its CBD/FnIII domains (ChiA74Δsp-50) did not have statistical differences in activity against colloidal chitin. However, ChiA74Δsp exhibits 4.5 and 2.0 higher activity than versions lacking the CBD (ChiA74Δsp-60) and CBD/FnIII domains (ChiA74Δsp-50), respectively, when crystalline chitin was used as substrate. Our data suggest that the CBD might plays a significant role in crystalline chitin hydrolysis. We also demonstrated the importance of the catalytic E211 in the CD, as mutants ChiA74Δsp E211N and ChiA74Δsp D207N, E211N were inactive against colloidal and crystalline chitins, chitosan and 4-MU-GlcNAc 3 . ChiA74 has a processive activity producing oligosaccharides with degree of polymerization (DP) of 1 (GlcNAc) and 2 (GlcNAc 2 ).

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Neotropical termite microbiomes as sources of novel plant cell wall degrading enzymes

In this study, we used shotgun metagenomic sequencing to characterise the microbial metabolic potential for lignocellulose transformation in the gut of two colonies of Argentine higher termite species with different feeding habits, Cortaritermes fulviceps and Nasutitermes aquilinus. Our goal was to assess the microbial community compositions and metabolic capacity, and to identify genes involved in lignocellulose degradation. Individuals from both termite species contained the same five dominant bacterial phyla (Spirochaetes, Firmicutes, Proteobacteria, Fibrobacteres and Bacteroidetes) although with different relative abundances. However, detected functional capacity varied, with C. fulviceps (a grass-wood-feeder) gut microbiome samples containing more genes related to amino acid metabolism, whereas N. aquilinus (a wood-feeder) gut microbiome samples were enriched in genes involved in carbohydrate metabolism and cellulose degradation. The C. fulviceps gut microbiome was enriched specifically in genes coding for debranching- and oligosaccharide-degrading enzymes. These findings suggest an association between the primary food source and the predicted categories of the enzymes present in the gut microbiomes of each species. To further investigate the termite microbiomes as sources of biotechnologically relevant glycosyl hydrolases, a putative GH10 endo-β-1,4-xylanase, Xyl10E, was cloned and expressed in Escherichia coli. Functional analysis of the recombinant metagenome-derived enzyme showed high specificity towards beechwood xylan (288.1 IU/mg), with the optimum activity at 50 °C and a pH-activity range from 5 to 10. These characteristics suggest that Xy110E may be a promising candidate for further development in lignocellulose deconstruction applications.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Structural basis of superinfection exclusion by bacteriophage T4 Spackle

A bacterial cell infected with T4 phage rapidly establishes resistance against further infections by the same or closely related T-even-type bacteriophages – a phenomenon called superinfection exclusion. Here we show that one of the T4 early gene products and a periplasmic protein, Spackle, forms a stoichiometric complex with the lysozyme domain of T4 tail spike protein gp5 and potently inhibits its activity. Crystal structure of the Spackle-gp5 lysozyme complex shows that Spackle binds to a horseshoe-shaped basic patch surrounding the oligosaccharide-binding cleft and induces an allosteric conformational change of the active site. In contrast, Spackle does not appreciably inhibit the lysozyme activity of cytoplasmic T4 endolysin responsible for cell lysis to release progeny phage particles at the final step of the lytic cycle. Our work reveals a unique mode of inhibition for lysozymes, a widespread class of enzymes in biology, and provides a mechanistic understanding of the T4 bacteriophage superinfection exclusion.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2015

Extracellular vesicles from Paracoccidioides pathogenic species transport polysaccharide and expose ligands for DC-SIGN receptors

Extracellular vesicles (EVs) mediate non-conventional transport of molecules across the fungal cell wall. We aimed at describing the carbohydrate composition and surface carbohydrate epitopes of EVs isolated from the pathogenic fungi Paracoccidioides brasiliensis and P. lutzii using standard procedures. Total EV carbohydrates were ethanol-precipitated from preparations depleted of lipids and proteins, then analyzed by chemical degradation, gas chromatography-mass spectrometry, nuclear magnetic resonance and size-exclusion chromatography. EV glycosyl residues of Glc, Man, and Gal comprised most probably two major components: a high molecular mass 4,6-α-glucan and a galactofuranosylmannan, possibly an oligomer, bearing a 2-α-Manp main chain linked to β-Galf (1,3) and α-Manp (1,6) end units. The results also suggested the presence of small amounts of a (1→6)- Manp polymer, (1→3)-glucan and (1→6)-glucan. Glycan microarrays allowed identification of EV surface lectin(s), while plant lectin microarray profiling revealed terminal Man and GlcNAc residues exposed at the EVs surface. Mammalian lectin microarray profiling showed that DC-SIGN receptors recognized surface carbohydrate in Paracoccidioides EVs. Our results suggest that oligosaccharides, cytoplasmic storage, and cell wall polysaccharides can be exported in fungal EVs, which also expose surface PAMPs and lectins. As a result, the role of these newly identified components in the interaction with the host remains to be unraveled.

59 BASIC BIOLOGICAL SCIENCES↗