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Diversity of Pseudomonas aeruginosa Type IV Pilins and Identification of a Novel D-arabinofuranose Post-translational ModificationKus, Julianne 31 July 2008 (has links)
The opportunistic bacterial pathogen Pseudomonas aeruginosa uses type IV pili (T4P) for adherence to, and rapid colonization of, surfaces via twitching motility. T4P are formed from thousands of pilin (PilA) subunits. Two groups of P. aeruginosa pilins were described previously (I and II), distinguished by protein length and sequence. PilA_I was glycosylated with an O-antigen subunit through the action of PilO/TfpO, encoded downstream of pilA_I. To determine if additional pilin variants existed, analysis of the pilin locus of >300 P. aeruginosa strains from a variety of environments was conducted. Three additional pilin alleles were discovered, each of which was invariantly associated with a unique, previously unidentified, downstream gene(s): pilA_III+tfpY, pilAIV+tfpW+tfpX, pilA_V+tfpZ. This survey also revealed that strains with group I T4P were more commonly associated with respiratory infections than strains with other pilins, suggesting that glycosylated T4P may confer a colonization advantage in this environment. The newly identified group IV pilin, represented by strain Pa5196, migrated aberrantly through SDS-PA gels, suggesting it was also glycosylated, a hypothesis confirmed by periodic acid-Schiff staining and mass spectrometry (MS) analyses. Disruption of Pa5196 O-antigen biosynthesis did not prevent the production of glycosylated pilins, demonstrating that these pilins were modified in a novel manner, unlike group I pilins. Using MS, nuclear magnetic resonance spectroscopy and site-directed mutagenesis, the Pa5196 pilins were shown to be uniquely modified with homo-oligosaccharides of mycobacterial-like α-1,5-D-arabinofuranose at multiple locations. Residues Thr64 and Thr66, located on the αβ-loop region of the protein, appear to be the preferred, but not exclusive sites of modification, each being modified with up to four D-Araf sugars. This region of the pilin is partially surface-exposed in the pilus, therefore modification of these sites may influence the surface chemistry of the fibre. Residues Ser81, Ser82, Ser85 and Ser89, located in the β-strand region, were also modified, mainly with mono- and disaccharides. Bioinformatic analyses and mutagenesis of TfpW suggest that this novel protein is an arabinosyltransferase necessary for PilA_IV modification. This research has increased our understanding of the complexity of this virulence factor, and may aid in development of new therapeutics for P. aeruginosa and mycobacterial infections.
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Sinteza i detaljna biološka ispitivanja tiazolnih C-nukleozida / Synthesis and detailed biological testing of thiazole C-nucleozidesKojić Vesna 26 April 2013 (has links)
<p>U radu je ostvarena totalna sinteza novih acikličnih tiazolnih C-nukleozide sa dvostrukom vezom i 2′,3′-dideoksi funkcijom u šećernoj komponenti. Ostvarena višefazna sinteza pomenutih acikličnih analoga tiazofurina zasnovana je na D-arabinozi kao hiralnom prekursoru. Ispitana je in vitro citotoksična aktivnost novosintetizovanih nukleozida prema ćelijskim linijama K562, HL-60, HT-29, MCF-7, MDA-MB-231, HeLa, Raji, PC3, Jurkat, Hs 294T i MRC-5, kao i provera ćelijskih mehanizama koji su u osnovi uočenog citotoksičnog potencijala novosintetisanih analoga u odnosu na tiazofurin kao referentno jedinjenje.</p> / <p>A total synthesis of new acyclic thiazole C-nucleozides bearing a double bond or 2′,3′-dideoxy functionality in the sugar moiety was achieved in this work. The multi-step synthesis of the mentioned thiazofurin analogues is based on D-arabinose as a chiral precursor. In vitro cytotoxic activity of newly synthesized compounds was evaluated against the following cell lines: K562, HL-60, HT-29, MCF-7, MDA-MB-231, HeLa, Raji, PC3, Jurkat, Hs 294T and MRC-5. A study of cell mechanisms underlaying the significant cytotoxic potential of these molecules was caried out and the results were compared to thiazofurin that servad as a referent compound in all biological testings.</p>
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In Vitro Synthetic Biology Platform and Protein Engineering for BiorefineryKim, Jae Eung 17 July 2017 (has links)
In order to decrease our dependence on non-renewable petrochemical resources, it is urgently required to establish sustainable biomass-based biorefineries. Replacing fossil fuels with renewable biomass as a raw feedstock for the production of chemicals and biofuels is a main driving force of biorefinering. Almost all kinds of biomass can be converted to biochemicals, biomaterials and biofuels via continuing advances on conversion technologies. In vitro synthetic biology is an emergent biomanufacturing platform that circumvents cellular constraints so that it can implement some biotransformations better than whole-cell fermentation, which spends a fraction of energy and carbon sources for cellular duplication and side-product formation. In this work, the in vitro synthetic (enzymatic) biosystem is used to produce a future carbon-neutral transportation fuel, hydrogen, and two high-value chemicals, a sugar phosphate and a highly marketable sweetener, representing a new portfolio for new biorefineries.
Hydrogen gas is a promising future energy carrier as a transportation fuel, offering a high energy conversion efficiency via fuel cells, nearly zero pollutants produced to end users, and high mass-specific and volumetric energy densities compared to rechargeable batteries. Distributed production of cost-competitive green hydrogen from renewable biomass will be vital to the hydrogen economy. Substrate costs contribute to a major portion of the production cost for low-value bulk biocommodities, such as hydrogen. The reconstitution of 17 thermophilic enzymes enabled to construct an artificial enzymatic pathway converting all glucose units of starch, regardless of the branched and linear contents, to hydrogen gas at a theoretic yield (i.e., 12 H2 per glucose), three times of the theoretical yield from dark microbial fermentation. Using a biomimetic electron transport chain, a maximum volumetric productivity was increased by more than 200-fold to 90.2 mmol of H2/L/h at a high starch concentration from the original study in 2007.
In order to promote economics of biorefineries, the production of a sugar phosphate and a fourth-generation sweetener is under development. D-xylulose 5-phosphate (Xu5P), which cannot be prepared efficiently by regular fermentation due to the negatively charged and hydrophilic phosphate groups, was synthesized from D-xylose and polyphosphate via a minimized two-enzyme system using a promiscuous activity of xylulose kinase. Under the optimized condition, 32 mM Xu5P was produced from 50 mM xylose and polyphosphate, achieving a 64% conversion yield, after 36 h at 45 °C. L-arabinose, a FDA-approved zero-calorie sweetener, was produced from D-xylose via a novel enzymatic pathway consisting of xylose isomerase, L-arabinose isomerase and xylulose 4-epimerase (Xu4E). Promiscuous activity of Xu4E, a monosaccharide C4-epimerase, was discovered for the first time. Directed evolution of Xu4E enabled to increase the catalytic function of C4-epimerization on D-xylulose as a substrate by more than 29-fold from the wild-type enzyme. Together, these results demonstrate that the in vitro synthetic biosystem as a feasible biomanufacturing platform has great engineering, and can be used to convert renewable biomass resources to a spectrum of marketable products and renewable energy.
As future efforts are addressed to overcome remaining challenges, for example, decreasing enzyme production costs, prolonging enzyme lifetime, engineering biomimetic coenzymes to replace natural coenzymes, and so on. This in vitro synthetic biology platform would become a cornerstone technology for biorefinery industries and advanced biomanufacturing (Biomanufacturing 4.0). / Ph. D.
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Synthesis of ring A of (+)-Ambruticin S and bicyclic nucleosides for antisense drug technologyChen, Bin 08 1900 (has links)
La synthèse énantiosélective de la (+)-ambruticine S, un produit naturel antifongique a été effectuée au sein de notre groupe. Trois approches ont été développées pour la synthèse du fragment lactone (cycle A). Ces trois voies d’accès au cycle A ont pour intermédiaire commun le methyl α-D-glycopyranoside déjà porteur du diol requis et disponible commercialement à bon prix. Une désoxygénation de l’hydroxyle en C-4 et l’homologation d’un carbone de la chaine latérale en C-6 ont permis l’obtention du cycle lactonique A.
Le deuxième projet est une collaboration entre le groupe Hanessian et ISIS Pharmaceuticals afin de développer de nouveaux oligonucléosides antisens. Les nucléosides antisens [4.3.0]-bicycliques cis et trans ont été synthétisés avec succès à partir d’un monosaccharide naturel commun, L-arabinose, porteur des stéréocentres requis. Un réaction clé d’allylation de Sakurai a permis d’obtenir les diastéréoisomères cis et trans dans des conditions de contrôle de type Felkin-Ahn et de contrôle par chélation respectivement. Les composés bicycliques finaux cibles ont été obtenus par une réaction d’aldol intramoléculaire catalyzéé par la proline, par métathèse de fermeture de cycle et par l’application de la méthode de Vorbrüggen pour la synthèse de nucléosides. / An enantioselective synthesis of the antifungal natural product (+)-ambruticin S has been accomplished in our group. For the synthesis of a ring A lactone fragment, three approaches were developed. They all started from commercially available and inexpensive methyl α-D-glycopyranoside, which already possesses the required diol unit. A deletion of the hydroxyl group at C-4 and a one-carbon homologation of the C-6 side chain furnished the ring A lactone.
The second project is an ongoing collaboration between the Hanessian group and ISIS pharmaceuticals to develop new antisense oligonucleosides. The cis- and trans-[4.3.0]bicyclic antisense nucleosides were successfully synthesized from a common natural monosaccharide, L-arabinose, which bears the required stereocenters. A key Sakurai allylation led to the cis- and trans diastereomers under Felkin-Ahn and chelation-controlled conditions respectively. The final bicyclic targets were achieved by a practical proline-catalyzed intramolecular aldol reaction and ring-closing metathesis (RCM) strategy, and application of the Vorbrüggen method for nucleoside synthesis.
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Synthesis of ring A of (+)-Ambruticin S and bicyclic nucleosides for antisense drug technologyChen, Bin 08 1900 (has links)
La synthèse énantiosélective de la (+)-ambruticine S, un produit naturel antifongique a été effectuée au sein de notre groupe. Trois approches ont été développées pour la synthèse du fragment lactone (cycle A). Ces trois voies d’accès au cycle A ont pour intermédiaire commun le methyl α-D-glycopyranoside déjà porteur du diol requis et disponible commercialement à bon prix. Une désoxygénation de l’hydroxyle en C-4 et l’homologation d’un carbone de la chaine latérale en C-6 ont permis l’obtention du cycle lactonique A.
Le deuxième projet est une collaboration entre le groupe Hanessian et ISIS Pharmaceuticals afin de développer de nouveaux oligonucléosides antisens. Les nucléosides antisens [4.3.0]-bicycliques cis et trans ont été synthétisés avec succès à partir d’un monosaccharide naturel commun, L-arabinose, porteur des stéréocentres requis. Un réaction clé d’allylation de Sakurai a permis d’obtenir les diastéréoisomères cis et trans dans des conditions de contrôle de type Felkin-Ahn et de contrôle par chélation respectivement. Les composés bicycliques finaux cibles ont été obtenus par une réaction d’aldol intramoléculaire catalyzéé par la proline, par métathèse de fermeture de cycle et par l’application de la méthode de Vorbrüggen pour la synthèse de nucléosides. / An enantioselective synthesis of the antifungal natural product (+)-ambruticin S has been accomplished in our group. For the synthesis of a ring A lactone fragment, three approaches were developed. They all started from commercially available and inexpensive methyl α-D-glycopyranoside, which already possesses the required diol unit. A deletion of the hydroxyl group at C-4 and a one-carbon homologation of the C-6 side chain furnished the ring A lactone.
The second project is an ongoing collaboration between the Hanessian group and ISIS pharmaceuticals to develop new antisense oligonucleosides. The cis- and trans-[4.3.0]bicyclic antisense nucleosides were successfully synthesized from a common natural monosaccharide, L-arabinose, which bears the required stereocenters. A key Sakurai allylation led to the cis- and trans diastereomers under Felkin-Ahn and chelation-controlled conditions respectively. The final bicyclic targets were achieved by a practical proline-catalyzed intramolecular aldol reaction and ring-closing metathesis (RCM) strategy, and application of the Vorbrüggen method for nucleoside synthesis.
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