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Avaliação do efeito de moduladores epigenéticos na biossíntese de produtos naturais em fungos; Evaluation of the effect of epigenetic modifiers in the biosynthesis of fungal natural products.

Almeida, Marília Oliveira de
Fonte: Biblioteca Digitais de Teses e Dissertações da USP Publicador: Biblioteca Digitais de Teses e Dissertações da USP
Tipo: Tese de Doutorado Formato: application/pdf
Publicado em 02/07/2014 PT
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A manipulação seletiva de alvos epigenéticos usando pequenas moléculas inibidoras das enzimas histona-desacetilases (HDACs) e DNA metiltransferases (DNMTs) é uma estratégia para estimular a expressão das vias biossintéticas e a produção de novos metabólitos secundários em fungos. Neste trabalho, inibidores de histonadesacetilases (butirato de sódio, ácido hidroxâmico suberoilanilida e ácido valproico) e inibidores de DNA metiltransferases (5-azacitidina, hidralazina, procaína e procainamida) foram suplementados em culturas líquidas e sólidas dos fungos endofíticos Fusarium oxysporum SS46, Hyphodermella corrugata FLe8.2 e Chaetomium globosum VR10, das linhagens comerciais Fusarium oxysporum ATCC MYA 4623 e Chaetomium globosum ATCC 56726 e do fitopatógeno Botrytis cinerea B0510. O fungo endofítico H. corrugata FLe8.2, em meio PDB, produziu o composto 2-(2-metoxifenil)-4H-piran-4-ona, e sua cultura em meio Czapek suplementada com hidralazina levou ao isolamento de 3-metil-1,2,4-triazolo[3,4-a]-ftalazina. O tratamento de F. oxysporum SS46 e F. oxysporum ATCC MYA 4623 com hidralazina em meio Czapek levou ao isolamento de um novo composto, 2H-[1,2,4]triazino[3,4- a]-ftalazina. Nestas culturas houve biotransformação da hidralazina pelos fungos...

A defective mutant of Salmonella enterica Serovar Gallinarum in cobalamin biosynthesis is avirulent in chickens

Paiva, Jacqueline Boldrin de; Penha Filho, Rafael Antonio Casarin; Arguello, Yuli Melisa Sierra; Berchieri Junior, Ângelo; Lemos, Manuel Victor Franco; Barrow, Paul A.
Fonte: Sociedade Brasileira de Microbiologia Publicador: Sociedade Brasileira de Microbiologia
Tipo: Artigo de Revista Científica Formato: 495-504
ENG
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Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP); Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq); Salmonella enterica serovar Gallinarum (SG) é o agente do tifo aviário, doença severa que provoca mortalidade em até 80% do plantel de aves. SG encontra-se entre os poucos sorotipos de Salmonella que são agentes etiológicos de enfermidade específica, à semelhança de Salmonella Typhi em seres humanos podendo, portanto, servir de modelo experimental para outras salmoneloses hospedeiro especíifcas. Além dos mecanismos de virulência, a bactéria utiliza mecanismos de sobrevivência para permanecer no hospedeiro. A ativação desses mecanismos pode ou não estar associada à ativação dos mecanismos de virulência. Entre os mecanismos fisiológicos, está a produção de vitamina B12 que Salmonella spp. realiza em ambientes anaeróbicos, como quando encontra-se intracelularmente no organismo hospedeiro. Neste estudo, analisou-se a infecção de aves por cepas de SG, que tiveram genes alterados que participam da biossíntese de vitamina B12. Foram produzidos mutantes de SG contendo os genes cbiA e cobS alterados e um terceiro, contendo ambos os genes alterados. A sobrevivência e a ação patogênica de SG não foi modificada pela alteração simples de um dos genes...

Genome-wide comparison of genes involved in the biosynthesis, metabolism, and signaling of juvenile hormone between silkworm and other insects

Cheng,Daojun; Meng,Meng; Peng,Jian; Qian,Wenliang; Kang,Lixia; Xia,Qingyou
Fonte: Sociedade Brasileira de Genética Publicador: Sociedade Brasileira de Genética
Tipo: Artigo de Revista Científica Formato: text/html
Publicado em 01/06/2014 EN
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Juvenile hormone (JH) contributes to the regulation of larval molting and metamorphosis in insects. Herein, we comprehensively identified 55 genes involved in JH biosynthesis, metabolism and signaling in the silkworm (Bombyx mori) as well as 35 in Drosophila melanogaster, 35 in Anopheles gambiae, 36 in Apis mellifera, 47 in Tribolium castaneum, and 44 in Danaus plexippus. Comparative analysis showed that each gene involved in the early steps of the mevalonate (MVA) pathway, in the neuropeptide regulation of JH biosynthesis, or in JH signaling is a single copy in B. mori and other surveyed insects, indicating that these JH-related pathways or steps are likely conserved in all surveyed insects. However, each gene participating in the isoprenoid branch of JH biosynthesis and JH metabolism, together with the FPPS genes for catalyzing the final step of the MVA pathway of JH biosynthesis, exhibited an obvious duplication in Lepidoptera, including B. mori and D. plexippus. Microarray and real-time RT-PCR analysis revealed that different copies of several JH-related genes presented expression changes that correlated with the dynamics of JH titer during larval growth and metamorphosis. Taken together, the findings suggest that duplication-derived copy variation of JH-related genes might be evolutionarily associated with the variation of JH types between Lepidoptera and other insect orders. In conclusion...

A defective mutant of Salmonella enterica Serovar Gallinarum in cobalamin biosynthesis is avirulent in chickens

Paiva,Jacqueline Boldrin de; Penha Filho,Rafael Antonio Casarin; Arguello,Yuli Melisa Sierra; Berchieri Junior,Ângelo; Lemos,Manuel Victor Franco; Barrow,Paul A.
Fonte: Sociedade Brasileira de Microbiologia Publicador: Sociedade Brasileira de Microbiologia
Tipo: Artigo de Revista Científica Formato: text/html
Publicado em 01/09/2009 EN
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Salmonella enterica serovar Gallinarum (SG) is a fowl typhoid agent in chickens and is a severe disease with worldwide economic impact as its mortality may reach up to 80%. It is one of a small group of serovars that typically produces typhoid-like infections in a narrow range of host species and which therefore represents a good model for human typhoid. The survival mechanisms are not considered to be virulent mechanisms but are essential for the life of the bacterium. Mutants of Salmonella Gallinarum containing defective genes, related to cobalamin biosynthesis and which Salmonella spp. has to be produced to survive when it is in an anaerobic environment, were produced in this study. Salmonella Gallinarum is an intracellular parasite. Therefore, this study could provide information about whether vitamin B12 biosynthesis might be essential to its survival in the host. The results showed that the singular deletion in cbiA or cobS genes did not interfere in the life of Salmonella Gallinarum in the host, perhaps because single deletion is not enough to impede vitamin B12 biosynthesis. It was noticed that diluted SG mutants with single deletion produced higher mortality than the wild strain of SG. When double mutation was carried out...

Nonactin Biosynthesis: the Product of nonS Catalyzes the Formation of the Furan Ring of Nonactic Acid

Woo, Anton J.; Strohl, William R.; Priestley, Nigel D.
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /07/1999 EN
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Nonactin is the parent compound of a group of ionophore antibiotics, known as the macrotetrolides, produced by Streptomyces griseus subsp. griseus ETH A7796. Nonactin is a significant compound because of its inhibitory effects on the P170 glycoprotein-mediated efflux of chemotherapeutic agents in multiple-drug-resistant cancer cells. Nonactin is also significant in that it is a highly atypical polyketide. Very little is presently known about the genes of the nonactin biosynthesis cluster. In this paper we describe our efforts to establish a connection between the product of a gene from the nonactin biosynthesis cluster and a known biochemical transformation in nonactin biosynthesis. Nonactate synthase is the enzyme which catalyzes the formation of nonactic acid from an acyclic precursor in nonactin biosynthesis. We have synthesized the substrate for this enzyme and have detected the in vitro cyclization activity of the substrate in cell-free preparations of S. griseus subsp. griseus ETH A7796. Previous studies by R. Plater and J. A. Robinson (Gene 112:117–122, 1992) had suggested, based on sequence homology, that the product of a partial open reading frame found close to the tetranactin resistance gene of S. griseus could be the nonactate synthase. We have therefore cloned...

Enzymes Catalyzing the Early Steps of Clavulanic Acid Biosynthesis Are Encoded by Two Sets of Paralogous Genes in Streptomyces clavuligerus

Jensen, Susan E.; Elder, Kenneth J.; Aidoo, Kwamena A.; Paradkar, Ashish S.
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /03/2000 EN
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Genes encoding the proteins required for clavulanic acid biosynthesis and for cephamycin biosynthesis are grouped into a “supercluster” in Streptomyces clavuligerus. Nine open reading frames (ORFs) associated with clavulanic acid biosynthesis were located in a 15-kb segment of the supercluster, including six ORFs encoding known biosynthetic enzymes or regulatory proteins, two ORFs that have been reported previously but whose involvement in clavulanic acid biosynthesis is unclear, and one ORF not previously reported. Evidence for the involvement of these ORFs in clavulanic acid production was obtained by generating mutants and showing that all were defective for clavulanic acid production when grown on starch asparagine medium. However, when five of the nine mutants, including mutants defective in known clavulanic acid biosynthetic enzymes, were grown in a soy-based medium, clavulanic acid-producing ability was restored. This ability to produce clavulanic acid when seemingly essential biosynthetic enzymes have been mutated suggests that paralogous genes encoding functionally equivalent proteins exist for each of the five genes but that these paralogues are expressed only in the soy-based medium. The five genes that have paralogues encode proteins involved in the early steps of the pathway common to the biosynthesis of both clavulanic acid and the other clavam metabolites produced by this organism. No evidence was seen for paralogues of the four remaining genes involved in late...

Identification and Expression of Genes Involved in Biosynthesis of l-Oleandrose and Its Intermediate l-Olivose in the Oleandomycin Producer Streptomyces antibioticus

Aguirrezabalaga, Ignacio; Olano, Carlos; Allende, Nerea; Rodriguez, Leticia; Braña, Alfredo F.; Méndez, Carmen; Salas, José A.
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /05/2000 EN
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A 9.8-kb DNA region from the oleandomycin gene cluster in Streptomyces antibioticus was cloned. Sequence analysis revealed the presence of 8 open reading frames encoding different enzyme activities involved in the biosynthesis of one of the two 2,6-deoxysugars attached to the oleandomycin aglycone: l-oleandrose (the oleW, oleV, oleL, and oleU genes) and d-desosamine (the oleNI and oleT genes), or of both (the oleS and oleE genes). A Streptomyces albus strain harboring the oleG2 glycosyltransferase gene integrated into the chromosome was constructed. This strain was transformed with two different plasmid constructs (pOLV and pOLE) containing a set of genes proposed to be required for the biosynthesis of dTDP-l-olivose and dTDP-l-oleandrose, respectively. Incubation of these recombinant strains with the erythromycin aglycon (erythronolide B) gave rise to two new glycosylated compounds, identified as l-3-O-olivosyl- and l-3-O-oleandrosyl-erythronolide B, indicating that pOLV and pOLE encode all enzyme activities required for the biosynthesis of these two 2,6-dideoxysugars. A pathway is proposed for the biosynthesis of these two deoxysugars in S. antibioticus.

Engineering Anthracycline Biosynthesis toward Angucyclines

Metsä-Ketelä, Mikko; Palmu, Kaisa; Kunnari, Tero; Ylihonko, Kristiina; Mäntsälä, Pekka
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /04/2003 EN
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The biosynthesis pathways of two anthracyclines, nogalamycin and aclacinomycin, were directed toward angucyclines by using an angucycline-specific cyclase, pgaF, isolated from a silent antibiotic biosynthesis gene cluster. Addition of pgaF to a gene cassette that harbored the early biosynthesis genes of nogalamycin resulted in the production of two known angucyclinone metabolites, rabelomycin and its precursor, UWM6. Substrate flexibility of pgaF was demonstrated by replacement of the nogalamycin minimal polyketide synthase genes in the gene cassette with the equivalent aclacinomycin genes together with aknE2 and aknF, which specify the unusual propionate starter unit in aclacinomycin biosynthesis. This modification led to the production of a novel angucyclinone, MM2002, in which the expected ethyl side chain was incorporated into the fourth ring.

Cloning, Sequencing, and Functional Analysis of an Iterative Type I Polyketide Synthase Gene Cluster for Biosynthesis of the Antitumor Chlorinated Polyenone Neocarzilin in “Streptomyces carzinostaticus”

Otsuka, Miyuki; Ichinose, Koji; Fujii, Isao; Ebizuka, Yutaka
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /09/2004 EN
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Neocarzilins (NCZs) are antitumor chlorinated polyenones produced by “Streptomyces carzinostaticus” var. F-41. The gene cluster responsible for the biosynthesis of NCZs was cloned and characterized. DNA sequence analysis of a 33-kb region revealed a cluster of 14 open reading frames (ORFs), three of which (ORF4, ORF5, and ORF6) encode type I polyketide synthase (PKS), which consists of four modules. Unusual features of the modular organization is the lack of an obvious acyltransferase domain on modules 2 and 4 and the presence of longer interdomain regions more than 200 amino acids in length on each module. Involvement of the PKS genes in NCZ biosynthesis was demonstrated by heterologous expression of the cluster in Streptomyces coelicolor CH999, which produced the apparent NCZ biosynthetic intermediates dechloroneocarzillin A and dechloroneocarzilin B. Disruption of ORF5 resulted in a failure of NCZ production, providing further evidence that the cluster is essential for NCZ biosynthesis. Mechanistic consideration of NCZ formation indicates the iterative use of at least one module of the PKS, which subsequently releases its product by decarboxylation to generate an NCZ skeleton, possibly catalyzed by a type II thioesterase encoded by ORF7. This is a novel type I PKS system of bacterial origin for the biosynthesis of a reduced polyketide chain. Additionally...

Structure and Biosynthesis of Heat-Stable Antifungal Factor (HSAF), a Broad-Spectrum Antimycotic with a Novel Mode of Action▿

Yu, Fengan; Zaleta-Rivera, Kathia; Zhu, Xiangcheng; Huffman, Justin; Millet, Jeffrey C.; Harris, Steven D.; Yuen, Gary; Li, Xing-Cong; Du, Liangcheng
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
EN
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A screen for antifungal compounds from Lysobacter enzymogenes strain C3, a bacterial biological control agent of fungal diseases, has previously led to the isolation of heat-stable antifungal factor (HSAF). HSAF exhibits inhibitory activities against a wide range of fungal species and shows a novel mode of antifungal action by disrupting the biosynthesis of a distinct group of sphingolipids. We have now determined the chemical structure of HSAF, which is identical to that of dihydromaltophilin, an antifungal metabolite with a unique macrocyclic lactam system containing a tetramic acid moiety and a 5,5,6-tricyclic skeleton. We have also identified the genetic locus responsible for the biosynthesis of HSAF in strain C3. DNA sequencing of this locus revealed genes for a hybrid polyketide synthase-nonribosomal peptide synthetase (PKS-NRPS), a sterol desaturase, a ferredoxin reductase, and an arginase. The disruption of the PKS-NRPS gene generated C3 mutants that lost the ability to produce HSAF and to inhibit fungal growth, demonstrating a hybrid PKS-NRPS that catalyzed the biosynthesis of the unique macrolactam system that is found in many biologically active natural products isolated from marine organisms. In addition, we have generated mutants with disrupted sterol desaturase...

Characterization of the Biosynthesis Gene Cluster for the Pyrrole Polyether Antibiotic Calcimycin (A23187) in Streptomyces chartreusis NRRL 3882▿

Wu, Qiulin; Liang, Jingdan; Lin, Shuangjun; Zhou, Xiufen; Bai, Linquan; Deng, Zixin; Wang, Zhijun
Fonte: American Society for Microbiology (ASM) Publicador: American Society for Microbiology (ASM)
Tipo: Artigo de Revista Científica
EN
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The pyrrole polyether antibiotic calcimycin (A23187) is a rare ionophore that is specific for divalent cations. It is widely used as a biochemical and pharmacological tool because of its multiple, unique biological effects. Here we report on the cloning, sequencing, and mutational analysis of the 64-kb biosynthetic gene cluster from Streptomyces chartreusis NRRL 3882. Gene replacements confirmed the identity of the gene cluster, and in silico analysis of the DNA sequence revealed 27 potential genes, including 3 genes for the biosynthesis of the α-ketopyrrole moiety, 5 genes that encode modular type I polyketide synthases for the biosynthesis of the spiroketal ring, 4 genes for the biosynthesis of 3-hydroxyanthranilic acid, an N-methyltransferase tailoring gene, a resistance gene, a type II thioesterase gene, 3 regulatory genes, 4 genes with other functions, and 5 genes of unknown function. We propose a pathway for the biosynthesis of calcimycin and assign the genes to the biosynthesis steps. Our findings set the stage for producing much desired calcimycin derivatives using genetic modification instead of chemical synthesis.

The Extracellular Biosynthesis of Cadmium-Free Quantum Dots by the Fungus Fusarium oxysporum

Ballou, Colton
Fonte: Quens University Publicador: Quens University
Tipo: Tese de Doutorado
EN
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This thesis considers the extracellular biosynthesis of quantum dots by the fungus Fusarium oxysporum. It is particularly concerned with the biosynthesis of novel zinc and indium semiconducting quantum dots through biosynthetic pathways. Previous research has confirmed the successful biosynthesis of CdS, CdSe, CdTe, and ZnS quantum dots. This research provides evidence that F. oxysporum may be capable of biosynthesizing novel ZnSe, ZnTe, InS, InSe, and InTe quantum dots from elements of groups II-VI and III-V of the periodic table. There was also strong evidence that F. oxysporum secretes NADPH into its environment that may be required to drive the biosynthesis of quantum dots. Blocking the nitrate reductase enzyme through the addition of sodium nitrite to the reaction mixture had a significant impact on CdTe and ZnTe biosynthesis. The addition of glutathione reductase (EC 1.8.1.7) and glutathione disulfide (GSSG) to the reaction mixture on day 4 decreased both trapped (520 nm) and excitonic (460 nm) emissions. In the absence of NADPH, quantum dot biosynthetic yield is significantly reduced (paired t-test, p < 0.05). This thesis presents for the first time an economical and environmentally sustainable means of biosynthesizing a wide variety of cadmium-free quantum dots using F. oxysporum.; An economical and environmentally sustainable means of biosynthesizing a wide variety of cadmium-free quantum dots using F. oxysporum.

Plant cell wall biosynthesis: genetic, biochemical and functional genomics approaches to the identification of key genes

Farrokhi, N.; Burton, R.; Brownfield, L.; Hrmova, M.; Wilson, S.; Bacic, A.; Fincher, G.
Fonte: Blackwell Publishing Ltd. Publicador: Blackwell Publishing Ltd.
Tipo: Artigo de Revista Científica
Publicado em //2006 EN
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Cell walls are dynamic structures that represent key determinants of overall plant form, plant growth and development, and the responses of plants to environmental and pathogen-induced stresses. Walls play centrally important roles in the quality and processing of plant-based foods for both human and animal consumption, and in the production of fibres during pulp and paper manufacture. In the future, wall material that constitutes the major proportion of cereal straws and other crop residues will find increasing application as a source of renewable fuel and composite manufacture. Although the chemical structures of most wall constituents have been defined in detail, the enzymes involved in their synthesis and remodelling remain largely undefined, particularly those involved in polysaccharide biosynthesis. There have been real recent advances in our understanding of cellulose biosynthesis in plants, but, with few exceptions, the identities and modes of action of polysaccharide synthases and other glycosyltransferases that mediate the biosynthesis of the major non-cellulosic wall polysaccharides are not known. Nevertheless, emerging functional genomics and molecular genetics technologies are now allowing us to re-examine the central questions related to wall biosynthesis. The availability of the rice...

Molekularbiologische und biochemische Untersuchungen zur Biosynthese von Novobiocin in Streptomyces spheroides NCIMB 11891; Genetic and biochemical investigations on the biosynthesis of novobiocin from Streptomyces spheroides NCIMB 11891

Steffensky, Marion
Fonte: Universidade de Tubinga Publicador: Universidade de Tubinga
Tipo: Dissertação
DE_DE
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Das Aminocumarin-Antibiotikum Novobiocin gehört zur Gruppe der DNA-Gyrase-Inhibitoren und wird durch Streptomyces spheroides produziert. In den Vereinigten Staaten ist Novobiocin (Albamycin®, Pharmacia & Upjohn) als Antibiotikum zur Behandlung von Infektionen mit multiresistenten, Gram-positiven Bakterien zugelassen. Die Erforschung der Novobiocin-Biosynthese auf molekularbiologischer Ebene und die Charakterisierung der beteiligten Enzyme könnte ein nützliches Mittel zur Entwicklung neuer anti-infektiver Substanzen darstellen. Im ersten Teil dieser Arbeit wurde eine Dimethylallyldiposphat:4-Hydroxyphenylpyruvat Dimethylallyltransferase, die sehr wahrscheinlich an der Biosynthese von Novobiocin beteiligt ist, in S. spheroides identifiziert, partiell gereinigt und charakterisiert. Das Enzym war löslich und spezifisch für die Substrate 4-Hydroxyphenylpyruvat und Dimethylallyldiphosphat. Das Novobiocin-Biosynthese-Cluster aus S. spheroides NCIMB 11891 wurde durch Screening mit homologen dNDP-Glucose-4,6-Dehydratase Sonden kloniert. In dieser Arbeit konnte nach Sequenz-Analyse von 53,1 kb 42 offene Leserahmen (ORFs) identifiziert werden. 14 ORFs konnte eine mögliche Rolle innerhalb der Novobiocin-Biosynthese zugeordnet werden. Die Klonierung und Expression eines Schlüsselenzyms der Novobiocin-Biosynthese...

Biosynthesis of aminocoumarin antibiotics in Streptomyces : Generation of structural analogues by genetic engineering and insights into the regulation of antibiotic production; Biosynthesis of aminocoumarin antibiotics in Streptomyces : Generation of structural analogues by genetic engineering and insights into the regulation of antibiotic production; Biosynthese von Aminocoumarinantibiotika in Streptomyces : Herstellung von Analoga durch genetische Manipulation und Einblick in die Regulation der Antibiotikaproduktion

Silva, Alessandra Eustaquio da
Fonte: Universidade de Tubinga Publicador: Universidade de Tubinga
Tipo: Dissertação
EN
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The aminocoumarin antibiotics novobiocin and clorobiocin are potent inhibitors of bacterial DNA gyrase produced by different Streptomyces strains. Since they are closely related in structure, and their biosynthetic gene clusters have been cloned and sequenced, they represent interesting starting compounds for the development of new antibacterial agents by genetic engineering. However, the functional analysis of biosynthetic genes and the availability of genetic tools for manipulation of producer strains are pre-requisites for such approaches. Clorobiocin is more potent than novobiocin. It differs from the latter, clinically approved drug in the substitution pattern at C-8´ of the aminocoumarin moiety, carrying a chlorine atom instead of a methyl group, and in the presence of a 5-methylpyrrole-2-carboxyl moiety instead of a carbamoyl group at 3´´-OH of the deoxysugar unit. One aim of this thesis was the production of hybrid antibiotics, combining structural features of these two compounds. By gene inactivation, clo-hal was identified as the gene of the halogenase responsible for the introduction of the chlorine atom of clorobiocin. Expression of the methyltransferase gene novO in the clo-hal- mutant of the clorobiocin producer S. roseochromogenes led to the very efficient formation of a clorobiocin analog with -CH3 instead of -Cl at C-8´ (= novclobiocin 102). However...

Molekularbiologische und biochemische Untersuchungen zur Biosynthese der Aminocoumarin-Antibiotika Clorobiocin und Coumermycin A1; Molecular biological and biochemical investigations of the biosynthesis of the aminocoumarin antibiotics clorobiocin and coumermycin A1

Freitag, Anja
Fonte: Universidade de Tubinga Publicador: Universidade de Tubinga
Tipo: Dissertação
DE_DE
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Die Aminocoumarin-Antibiotika Novobiocin, Clorobiocin und Coumermycin A1 werden von verschiedenen Streptomycetenarten gebildet und sind Hemmstoffe der bakteriellen DNA-Gyrase. Ihre therapeutische Anwendung blieb jedoch durch ihre schlechte Wasserlöslichkeit, die geringe Aktivität gegenüber gram-negativen Bakterien und ihre relativ hohe Toxizität in Eukaryonten eingeschränkt. Für die Gewinnung neuer strukturell modifizierter Aminocoumarin-Antibiotika durch genetische Manipulation benötigt man genaue Informationen über die Biosynthese dieser Antibiotika. Die Biosynthesegencluster dieser drei Antibiotika sind bereits kloniert, sequenziert und die Funktion vieler Gene aufgeklärt. Dennoch bedarf es immer noch weiteren gezielten Untersuchungen, um die Biosynthese vollständig zu verstehen. Clorobiocin enthält einen Pyrrol-2-carboxylteil, der über eine Esterbindung mit dem Desoxyzucker verbunden ist. Dieser Pyrrolteil ist wichtig für die Bindung des Antibiotikums an sein biologisches Target. Die Gene, die an der Biosynthese dieser 5-Methylpyrrol-2-carbonsäure-Einheit beteiligt sind sowie die für die Übertragung der Acylkomponente auf den Desoxyzucker und dessen Modifizierung verantwortlich sind, konnten bereits durch Geninaktivierung...

Molekularbiologische und biochemische Untersuchungen der Fumigaclavinbiosynthese in Aspergillus fumigatus AF 293 / B 5233 und Penicillium commune NRRL 2033; Molecular and biochemical investigation of fumigaclavine biosynthesis in Aspergillus fumigatus AF 293 / B 5233 and Penicillium commune NRRL 2033

Unsöld, Inge
Fonte: Universidade de Tubinga Publicador: Universidade de Tubinga
Tipo: Dissertação
DE_DE
Relevância na Pesquisa
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Fumigaclavin A, B und C sind Ergotalkaloide des Clavin-Typs und werden von Aspergillus- und Penicillium-Arten produziert. Neueste Untersuchungen zeigen, dass Fumigaclavin C experimentell induzierte Leberschäden und Kolitis bei Mäusen bessern kann und gefäßrelaxierend wirkt. Fumigaclavine haben dieselbe Grundstruktur wie Lysergsäure und Dihydrolysergsäure, die Säurekomponenten der pharmazeutisch wichtigen Ergotalkaloide aus Claviceps purpurea bzw. derer semisynthetischen Derivate. Aufgrund der ähnlichen Strukturen und der gleichen biogenetischen Herkunft sind die Untersuchungen zur Biosynthese von Fumigaclavinen auch für das Verständnis der Biosynthese von Ergotalkaloiden aus C. purpurea von Bedeutung. Ein putatives Biosynthesegencluster von Fumigaclavin C wurde in der Genomsequenz von Aspergillus fumigatus AF 293 identifiziert. Das putative Gencluster ist 22 kb groß und enthält 11 ORFs. Sequenzanalyse und Vergleich mit Datenbankeinträgen der Gene aus dem Cluster ermöglichte die Aufstellung eines hypothetischen Biosyntheseweges für Fumigaclavin C. Der experimentelle Beweis für die Identität des Clusters von Fumigaclavin C aus A. fumigatus wurde durch heterologe Überexpression der beiden Prenyltransferasegene fgaPT1 (=Afu2g17990) und fgaPT2 (=Afu2g18040) in E. coli bzw. in Saccharomyces cerevisiae und anschließende biochemische Charakterisierung der Genprodukte erbracht. FgaPT2 katalysiert die Prenylierung von L-Tryptophan zu Dimethylallyltryptophan und somit den ersten Schritt in der Ergotalkaloid-Biosynthese. FgaPT1 katalysiert den letzten Schritt der Fumigaclavin C-Biosynthese...

MbtH and Adenylate-Forming Enzymes in the Biosynthesis of Aminocoumarin Antibiotics and Vancomycin; MbtH und Adenylat-Bildende Enzyme in der Biosynthese von Aminocoumarin-Antibiotika und Vancomycin

Boll, Björn
Fonte: Universidade de Tubinga Publicador: Universidade de Tubinga
Tipo: Dissertação
EN
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Streptomycetes possess biosynthetic gene clusters that allow them to produce a multiplicity of secondary metabolites. These include pharmaceutically relevant compounds like prenylated phenazines as well as aminocoumarin- and glycopeptide antibiotics. For specific alterations in the structure of these substances an exact knowledge of the steps of their biosynthesis is of central importance. In this work, enzymes of the biosynthesis of different compounds were biochemically characterized. This allows structural modifications and creation of new derivatives in the future plus an enhancement of the production yield. The first part of this work describes the identification and characterization of the prenyltransferase PpzP from Streptomyces anulatus which transfers a prenyl moiety to a phenazine. PpzP is encoded in a cluster with genes for the biosynthesis of phenazine-1-carboxylic acid (PCA) and the prenyl donor dimethylallyldiphosphate (DMAPP). Cloning and expression of the gene and purification of PpzP resulted in a 37 kDa soluble protein. Activity assays and mass spectrometric analyses confirmed the formation of a C-C bond between the C-1 atom of the isoprenoid substrate and the C-9 atom of the aromatic compound. In contrast to many other prenyltransferases the reaction of PpzP is independent of magnesium or other divalent cations. The Km values for the substrates were determined as 116 µM for DMAPP and 35 µM for PCA with a turnover number kcat of 0...

Molecular biological and biochemical investigations of the biosynthesis of the aminocoumarin antibiotics; Molecularbiologische und biochemische Untersuchungen zur Biosynthese von Aminocoumarin-Antibiotika

Xu, Hui
Fonte: Universität Tübingen Publicador: Universität Tübingen
Tipo: Dissertation; info:eu-repo/semantics/doctoralThesis
EN
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The structurally related aminocoumarin antibiotics novobiocin, clorobiocin and coumermycin A1 are potent inhibitors of bacterial gyrase and represent interesting starting compounds for the development of new antibacterial agents. X-ray crystallographic analysis has shown that the acyl moieties at the 3''-hydroxy group of the deoxysugars of these antibiotics, namely the carbamoyl group in novobiocin and 5-methylpyrrole-2-carboxylic acid moiety in clorobiocin and coumermycin A1, are particularly important for their binding to the biological target, i.e. the B subunit of gyrase. In this thesis, genes involved in the biosynthesis of the methylpyrrole moiety and those responsible for acyl transfer to the deoxysugar were identified by gene inactivation, heterologous expression as well as biochemical experiments. Several aminocoumarin derivatives were obtained from different defective mutants of the coumermycin and clorobiocin producers. Based on these findings, a series novel carbamoylated aminocoumarin antibiotics were generated by in vivo overexpression of carbamoyltransferase NovN from the novobiocin gene cluster and in vitro chemoenzymatic biosynthesis using NovN. Coumermycin A1 contains a central and two terminal pyrrole moieties. The coumermycin gene cluster from Streptomyces rishiriensis DSM 40489 contains three genes (couN3...

Ascorbic acid biosynthesis: a precursor study on plants

Barata-Soares,Anderson D.; Gomez,Maria Luiza P. A.; Mesquita,Carlos Henrique de; Lajolo,Franco M.
Fonte: Brazilian Journal of Plant Physiology Publicador: Brazilian Journal of Plant Physiology
Tipo: Artigo de Revista Científica Formato: text/html
Publicado em 01/12/2004 EN
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Since the first isolation of ascorbic acid (AsA) in 1928, few papers have been published regarding the biosynthesis of AsA in plants, especially in fruits. It took as long as 1998, before Wheeler, Jones and Smirnoff, based on a study with Arabidopsis leaves, proposed what can be considered the main pathway of biosynthesis of AsA, in which L-galactose (L-GAL) is a key precursor. This paper reports the effectiveness of some precursors (cold or radiolabeled) in the biosynthesis of AsA in different plants: green sweet pepper, white-pulp guava, red-pulp guava, papaya and strawberry at two ripening stages (mature green and ripe for papaya and mature green and half red for strawberry) and broccoli. The 'Smirnoff-Wheeler' pathway was functioning and active in all sources studied, as demonstrated by the increase in AsA contents and incorporation of labeled precursors into AsA. In papaya, the AsA content in the ripe fruit was higher than in the mature green, indicating the synthesis of AsA during ripening. On the other hand, the AsA content in the mature green strawberry was similar to that of the half red fruits. Our data demonstrate that L-GAL and L-Galactono-1,4-lactone (L-GL) are effective precursors for the biosynthesis of AsA in fruits and also provided additional evidence for the participation of D-mannose (D-MAN) and D-glucose-1P in the biosynthesis of AsA in plants.