Dissertations / Theses on the topic 'Polyketide synthase genes'
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Walsh, Maura Stephanie. "Cloning fungal polyketide synthase genes." Thesis, University of Bristol, 1993. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.333957.
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Wiesmann, Kristen E. H. "Engineering of the erythromycin-producing polyketide synthase." Thesis, University of Cambridge, 1995. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.264505.
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Roberts, Gareth A. "The erythromycin-producing polyketide synthase from Saccharopolyspora erythraea." Thesis, University of Cambridge, 1993. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.308298.
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Arrowsmith, Teresa Jayne. "Characterisation of putative polyketide synthase genes from Streptomyces cinnamonensis." Thesis, University of Southampton, 1990. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.292909.
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Yu, Tin-Wein. "Physical and functional studies of polyketide synthase genes of Streptomyces." Thesis, University of East Anglia, 1995. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.260005.
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Chooi, Yit Heng, and not supplied. "Genetic potential of lichen-forming fungi in polyketide biosynthesis." RMIT University. Applied Sciences, 2008. http://adt.lib.rmit.edu.au/adt/public/adt-VIT20081027.161315.
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Lichens produce a diverse array of bioactive secondary metabolites, many of which are unique to the organisms. Their potential applications, however, are limited by their finite sources and the slow-growing nature of the organisms in both laboratory and environmental conditions. This thesis set out to investigate polyketide synthase genes in lichens, with the ultimate goal of providing a sustainable source of lichen natural products to support these applications. To expand the diversity of PKS genes that could be detected in lichens, new degenerate primers targeting ketoacylsynthase (KS) domains of specific clades of PKS genes have been developed and tested on various lichen samples. Using these primers, 19 KS domains from various lichens were obtained. Phylogenetic analysis of the KS domains was used to infer the function of the PKS genes based on the predicted PKS domain architecture and chemical analysis by TLC and/or HPLC. KS domains from PKS clades not previously known in lichens were identified; this included the clade III NR (non-reducing)-PKSs, PR (partially reducing)-PKSs and HR (highly reducing)-PKSs. The discovery of clade III NR-PKSs with C-methyltransferase (CMeT) domain and their wide occurrence in lichens was especially significant. Based on the KS domain phylogenetic analysis and compounds detected in the individual lichens, the clade III NR-PKSs were hypothesized to be involved in the biosynthesis of β-orsellinic acid and methylphloroacetopheno ne - the monoaromatic precursors for many lichen coupled phenolic compounds, such as β-orcinol depsides/depsidones and usnic acid. A strategy has been developed to isolate clade III NR-PKSs directly from environmental lichen DNA using clade III NR-type KS amplified from the degenerate primers (NR3KS-F/R) as homologous probes. Another pair of degenerate primers specific to the CMeT domain of NR-PKSs has also been developed to facilitate the cloning and probing of new clade III NR-PKS genes in lichens. A clade III NR-PKS gene (xsepks1) from X. semiviridis was cloned successfully. This is the first report of the isolation of a full-length PKS gene from environmental lichen DNA. The domain architecture of xsepks1 is KS-AT-ACP-CMeT, as expected for a clade III NR-PKS, suggesting that the newly developed clade-specific primers are useful for cloning new clade III NR-PKS genes and that KS domain phylogenetic analysis can predict the functional domains in PKSs. Attempts were made to characterize the function of xsepks1 by heterologous expression in Aspergillus species. Both A. nidulans (transformed with 5´partial xsepks1 including native promoter) and A. oryzae (transformed with full-length xsepks1 under the regulation of starch-inducible amyB promoter) were tested as potential hosts for the expression of lichen PKS genes. Transcriptional analysis showed that A. nidulans could potentially utilize the lichen PKS gene promoter and both fungal hosts could splice the introns of a lichen PKS gene. Several compounds unique to the A. oryzae transformants carrying xsepks1 were detected, but they could not be reproduced in subsequent fermentations even though the gene was transcribed into mRNA. None of the expected products (β-orsellinic acid, methylphloroacetophenone or similar methylated monoaromatic compounds) was detected in A. oryzae transformants, and the function of xsepks1 remains to be determined. The other clade III NR-PKS genes detected in X. semiviridis cou ld also be responsible for the biosynthesis of β-orsellinic acid or methylphloroacetophenone, as precursors of the major secondary metabolites detected in X. semiviridis (i.e. fumarprotocetraric acid, succinprotocetraric acid and usnic acid). Overall, the work in this thesis demonstrated the prospect of using a molecular approach to access the lichen biosynthetic potential without going through the cumbersome culturing stage.
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Morris, Nathan Z. "Molecular detection of type II polyketide synthase genes in Cuban soils." Thesis, University of Warwick, 2000. http://wrap.warwick.ac.uk/59431/.
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Molecular detection methods were developed to study the distribution of type II polyketide synthase (PKS) genes in Cuban soils. A PCR based detection method targeting the α and β ketosynthase genes was applied to a number of different total community DNA samples. These genes were detected in 43% of samples tested from a number of different locations. A botanical garden site located in Havana, Cuba, was found to show the greatest distribution of type II PKS genes across the sites tested. It was not possible to amplify type II PKS genes from a pristine island site off the coast of Cuba. Further investigation revealed that actinornycetes containing type II PKS were present in the soil community at a level above the detection limit of the PCR protocol. Further total community DNA cleanup steps failed to allow the detection of type II PKS genes within the DNA samples suggesting PCR inhibition was responsible for negative results. The molecular detection of type II PKS genes in total community DNA was compared to the detection of type II PKS genes in actinomycete isolates. A lack of correlation between these two approaches was observed with the molecular detection limit unable to amplify type II PKS genes in <50% of crop soils tested. Actinomycetes containing type II PKS genes could be isolated from all crop soils tested. No difference was seen in the detection of type II PKS genes between rhizosphere and bulk soil samples. Actinomycetes were isolated using a selective isolation procedure at a level of approximately 10(7) cfu g-1 soil compared to 10(8) cfu g-1 for total bacterial counts. Actinomycetes were isolated from Cuban crop soils and screened for the presence of type II PKS genes. Out of 100 isolates 26 were found to contain the genes of interest. Phylogenetic analysis of these isolates based on 16S rDNA and recA sequence data showed them to be closely grouped within the streptomycetes. Sequence data based on KSα genes from Cuban isolates showed them to be representative of both spore pigment and antibiotic polyketide genes. A representative clone library was constructed containing type II PKS genes amplified from total community DNA. Rhizosphere and bulk soil samples were compared from the same site. Sequences obtained from rhizosphere total community DNA appeared to be widely distributed when compared to published sequences and included examples of both spore pigment and antibiotic polyketide genes. A molecular method was developed to amplify near full length α and β KS genes from type II PKS gene clusters. Expression vectors were constructed to allow these genes to be expressed along with an ACP to give a functional minimal PKS for polyketide chain production. This method was used on total community DNA in an attempt to extract diverse genes from as yet uncultured organisms.
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Punya, Juntira. "Polyketide synthase genes from the wood-decaying fungus Xylaria sp. BCC1067." Thesis, University of Westminster, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.251721.
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Nicholson, Thomas Peter. "Design and development of oligonucleotide probes for novel fungal polyketide synthase genes." Thesis, University of Bristol, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.322607.
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Kim, Kwang Hyung. "Functional Analysis of Secondary Metabolite Biosynthesis-Related Genes in Alternaria brassicicola." Diss., Virginia Tech, 2009. http://hdl.handle.net/10919/39452.
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Alternaria brassicicola is a necrotrophic pathogen that causes black spot disease on virtually all cultivated Brassicas, A. brassicicola is renowned for its ability to prodigiously produce secondary metabolites. To test the hypothesis that secondary metabolites produced by A. brassicicola contribute to pathogenicity, we identified seven nonribosomal peptide synthetases (NPSs) and 10 polyketide synthases (PKSs) in the A. brassicicola genome. The phenotype resulting from knockout mutations of each PKS and NPS gene was investigated with an emphasis on discovery of fungal virulence factors. A highly efficient gene disruption method using a short linear double stranded DNA construct with minimal elements was developed, optimized, and used to functionally disrupt all NPS and PKS genes in A. brassicicola. Three NPS and two PKS genes, and one NPS-like gene appeared to be virulence factors based upon reduced lesion development of each mutant on inoculated green cabbage and Arabidopsis compared with the wild-type strain. Furthermore some of the KO mutants exhibited developmental phenotypic changes in pigmentation and conidiogenesis. To further characterize the roles of several genes of interest in A. brassicicola development and pathogenesis, the genes AbNPS2, AbPKS9, and NPS-like tmpL were selected for in-depth functional analysis. We provide substantial evidence that the AbNPS2-associated metabolite is involved in conidial cell wall construction, possibly as an anchor connecting two cell wall layers. We also characterized a biosynthetic gene cluster harboring the AbPKS9 gene and demonstrated that this cluster is responsible for the biosynthesis of depudecin, an inhibitor of histone deacetylases and a minor virulence factor. Finally, we demonstrated that a NPS-like protein named TmpL is involved in a filamentous fungi-specific mechanism for regulating levels of intracellular reactive oxygen species during conidiation and pathogenesis in both plant and animal pathogenic fungi. Collectively our results indicate that small molecule nonribosomal peptides and polyketides in A. brassicicola play diverse, but also fundamental, roles in fungal development and pathogenesis.Ph. D.
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Powell, N. G. "Cloning of the genes encoding the milbemycin polyketide synthase of Streptomyces hygroscopicus and S. griseochromogenes." Thesis, Swansea University, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.638560.
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Strains of both Streptomyces hygroscopicus and S. griseochromogenes produce the antiparasitic compound milbemycin. Milbemycin is a macrocyclic polyketide synthesised by a Polyketide Synthase (PKS) in a manner analogous to the synthesis of long chain fatty acids by Fatty Acid Synthases (FAS). DNA libraries were constructed of S. hygroscopicus and S. griseochromogenes genomic DNA and probed with oligonucleotide probes for key PKS activities. A number of overlapping cosmids were identified with inserts potentially encoding the milbemycin PKS. The cosmids derived from S. hygroscopicus seemed to form three distinct groups based on homology with each other, while all of the S. griseochromogenes cosmids appeared to be derived from a single cluster. The DNA sequence was determined for a single fragment from each group of cosmids and the derived amino acid sequences were use to search the SwissProt protein sequence database. This analysis revealed that all of the fragments appeared to encode modular PKSs. Recombinant bacteriophage and plasmids were engineered to disrupt the chromosomal homologues of all of the sequenced fragments. Potential recombinants were isolated and assayed for milbemycin production by Thin Layer Chromatography (TLC) and High Performance Liquid Chromatography (HPLC). Attempts to disrupt the S. griseochromogenes PKS genes were unsuccessful and the function of the cloned DNA remains unproven. In S. hygroscopicus disruption of two of the putative PKS clusters resulted in mutant phenotypes both exhibiting partial loss of milbemycin synthesis. Hence the milbemycin PKS gene cluster has been conclusively identified and the way paved for determination of its genetic organisation.
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Silva, Caroline Souza Pamplona da. ""Caracterização molecular de cianobactérias brasileiras e distribuição de genes de produtos naturais"." Universidade de São Paulo, 2006. http://www.teses.usp.br/teses/disponiveis/64/64133/tde-12072006-095306/.
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O espaço intergênico (IGS) juntamente com suas subunidades flanqueadoras (cpcB) e (cpcA) do operon do ficocianina foi usado para identificar linhagens de cianobactérias. Dentro do domínio Bacteria somente as cianobactérias possuem o operon da ficocianina e a região cpcBA-IGS é suficientemente variável para diferenciar linhagens desses microrganismos. No presente estudo 25 linhagens de cianobactérias isoladas de diversos locais brasileiros foram caracterizadas usando a seqüência cpcBA-IGS. DNA genômico foi extraído das ordens Chroococcales (oito linhagens), Oscillatoriales (duas linhagens), Nostocales (onze linhagens) e Stigonematales (quatro linhagens). Os oligonucleotídeos iniciadores PCβF/PCαR, específicos para a seqüência cpcBA-IGS, foram usados para amplificar fragmentos de DNA de aproximadamente 685 pb. Os produtos da PCR foram clonados, seqüenciados e as seqüências foram comparadas pela análise BLAST. Todas as seqüências de Microcystis e também as seqüências de Radiocystis fernadoi SPC736, Planktothrix mougeotii SPC788, Geitlerinema splendidum SPC923, Microchaete investiens CENA64 e Gloeotrichia UFV-B2 mostraram identidades com seqüências do GenBank. Entretanto, nenhuma identidade foi encontrada para as seqüências restantes. As relações filogenéticas das seqüências de cpcBA-IGS foram investigadas junto com outras seqüências de cianobactéria do Genbank usando a análise Neighbour Joining. A topologia da árvore foi congruente com outras árvores de cianobactérias, com exceção de todas as seqüências sem identidades no GenBank, as quais formaram um agrupamento separado. Os dados das seqüências de cpcBA-IGS analisadas confirmam que as cianobactéria heterocitadas formam um grupo monofilético. Estudos anteriores realizados com linhagens de cianobactéria mostraram que estes microrganismos são uma fonte rica de produtos naturais. No presente estudo conduzido com 59 linhagens de cianobactérias, sendo a maioria isolada de ambientes brasileiros, isto foi confirmado. Para alcançar esse objetivo, dois conjuntos de iniciadores degenerados foram usados para produzir seqüências amplificadas por PCR das sintetases de peptídeos não-ribossômicos (NRPSs), e de sintases policetídeos (PKSs) modulares, as quais são enzimas multifuncionais envolvidas na produção de produtos naturais. O sistema híbrido NRPS/PKS também foi amplificado por PCR usando uma combinação de iniciadores de NRPS e de PKS. Essa abordagem molecular mostrou a presença de genes de NRPS e de PKS em 93% e 81% linhagens de cianobactérias, respectivamente. Genes de NRPS/PKS foram encontrados em 87% das cianobactérias examinadas. Numa tentativa de atribuir funções a oito fragmentos de PKS identificados por PCR, estas seqüências foram clonadas, seqüenciadas e analisadas filogeneticamente. As seqüências de PKSs da Microcystis aeruginosa NPCD1 e Fischerella CENA62 mostraram correlação com a síntese de sideróforo e de microcistina, respectivamente. Todas as 59 linhagens foram analisadas para a produção do microcistinas e 20 linhagens apresentaram resultados positivos. Para a maioria das linhagens potencialmente produtoras de microcistinas os produtos de PCR esperados de NRPS, PKS e NRPS/PKS foram amplificados. A produção de sideróforos foi testada em 28 linhagens e somente cinco produziram resultados positivos. Em três linhagens produtoras de sideróforos todos os três sistemas moleculares analisados estavam presentes. Estes resultados serão altamente valiosos na exploração futura de cada peptídeo dessas cianobactérias e para a elucidação da bioatividade de tais produtos naturais.The intergenic spacer (IGS) together with its flanking subunits (cpcB) and (cpcA) of the phycocyanin operon has been used to identify cyanobacterial strains. Within the Bacteria domain only cyanobacteria present phycocyanin operon and the cpcBA-IGS region is variable enough to differentiate strains of these microorganisms. In the present study 25 cyanobacterial strains isolated from several Brazilian locations were characterized using the cpcBA-IGS sequence. Genomic DNA was extracted from the orders Chroococcales (eight strains), Oscillatoriales (two strains), Nostocales (eleven strains) and Stigonematales (four strains). The primers PCβF/PCαR targeting the cpcBA-IGS sequence were used to amplify DNA fragments of approximately 685 bp. The PCR products were cloned, sequenced and the sequences were compared by BLAST analysis. All Microcystis sequences and also sequences from Radiocystis fernadoi SPC736, Planktothrix mougeotii SPC788, Geitlerinema splendidum SPC923, Microchaete investiens CENA64 and Gloeotrichia UFV-B2 showed identities with sequences from GenBank. However, no identities were found for the remaining sequences. Phylogenetic relationships of the cpcBA-IGS sequences were investigated together with other cyanobacterial sequences from Genbank using the Neighbour Joining analysis. The tree topology was congruent with previous cyanobacterial trees, except for all sequences with no identities in the GenBank, which formed a separated cluster. The cpcBA-IGS sequences analysis data confirm that heterocyte-forming cyanobacteria are a monophyletic group. Previous studies carried out with cyanobacterial strains showed that these microorganisms are a rich source of natural products. This has been confirmed in the present study conducted with 59 cyanobacterial strains, with the majority of them isolated from Brazilian environment. To reach this goal, two sets of degenerate primers were used to generate PCR amplification sequences of nonribosomal peptide synthetases (NRPSs) and modular polyketide synthases (PKSs), which are multifunctional enzymes implicated in natural products production. Also, NRPS/PKS hybrid system was PCR amplified by using a combination of NRPS and PKS primers. This molecular approach revealed the presence of NRPS and PKS genes in 93% and 81% cyanobacterial strains, respectively. NRPS/PKS genes were found in 87% of cyanobacteria examined. In an attempt to attribute functions to eight PCR identified PKS fragments, these sequences were cloned, sequenced and phylogenetically analyzed. PKSs sequences of Microcystis aeruginosa NPCD1 and Fischerella CENA62 showed correlation with the synthesis of siderophore and microcystin, respectively. All 59 strains were analyzed for microcystin production and 20 strains presented positive results. For the majority of potentially producing-microcystin strains expected PCR products of NRPS, PKS and NRPS/PKS were amplified. The siderophores production was tested in 28 strains and only five gave positive results. In three producing-siderophore strains all three molecular systems analyzed were present. These results will be highly valuable for further exploring each of these cyanobacterial peptides and for elucidating the bioactivity of such natural products.
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Ward, Amber L. "Identification of Genes Required to Synthesize an Antibiotic-like Compound from the Soil Bacterium Rhodococcus sp. MTM3W5.2." Digital Commons @ East Tennessee State University, 2015. https://dc.etsu.edu/etd/2558.
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Rhodococcus is a soil bacterium, member of the Actinobacteria, and a close relative of the prolific small molecule producer Streptomyces. Recent interest in Rhodococcus as an under investigated source of possible bioactive secondary metabolites is sparked by the discovery of many polyketide synthase and non-ribosomal peptide synthetase genes of unknown function from sequenced Rhodococcus genomes. Rhodococcus species strain MTM3W5.2 was recently shown to produce a strong inhibitory compound with activity against most strains of Rhodococcus and closely related genera. A goal of this investigation is to discover the gene(s) required to synthesize this inhibitory molecule. The engineered Rhodococcus transposon, pTNR, was used to generate random insertional mutations in the genome of MTM3W5.2. The transposon insertion sites for 8 non-producing mutants were cloned and sequenced. Genes that encode polyketide synthases usually form parts of large biosynthetic gene clusters responsible for the production of small polyketide molecules.
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Rojas, Juan Diego Rojas. "Prospecção de genes biossintéticos de policetídeos a partir de fungos isolados de cana-de-açúcar." Universidade de São Paulo, 2010. http://www.teses.usp.br/teses/disponiveis/87/87131/tde-22122010-162031/.
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A partir de 280 isolados fúngicos de cana-de-açúcar, 18 cepas foram avaliadas quanto á presença de genes da policetídeo sintase por meio da técnica do PCR. Estes fungos foram identificados taxonomicamente por uma abordagem polifásica, classificando-os dentro de quatro ordens e nove gêneros. A avaliação da atividade biológica demonstrou a presença de metabolitos com propriedades antibióticas quando enfrentados a micro-organismos patogênicos. Segundo a análise de correspondência múltipla, esta atividade poderia estar associada com a local de isolamento dos fungos. Foram detectadas 36 seqüências similares a genes PKS a partir de 17 destes fungos. A análise filogenética do domínio KS, conduzida pelo método de neighbor-joining, indicou que 16 seqüências se acomodaram dentro do grupo monofilético dos PKS envolvidos na produção de policetídeos não reduzidos e as outras 10 seqüências se acomodaram dentro do grupo monofilético dos PKS envolvidos na produção de policetídeos reduzidos. A análise do domínio CMT também apontou que as seqüências podiam se acomodaram em grupos de PKS dependendo do grau de redução do policetídeo, todas as seqüências CMT se relacionaram com PKS envolvidos na produção de policetídeos reduzidos. As análises dos modelos estruturais também demonstraram que as seqüências estavam altamente relacionadas com estruturas protéicas da família das enzimas de condensação, destacando a presença de uma hélice característica que carrega o resíduo de cisteína, responsável pela atividade de condensação. Extratos orgânicos obtidos de cultivos dos fungos foram avaliados parar detectar a presença de compostos tipo lovastatina. Por meio de cromatografia CCDS, detectaram-se bandas de 10 extratos com o mesmo deslocamento que a lovastatina padrão, mas apenas 6 destas foram confirmados por CLAE. O isolado A. flavus CBMAI 1023, foi selecionado para a realização de experimentos de produção a maior escala onde foi possível isolar e caracterizar um novo policetídeo.From a group of 280 sugarcane-derived fungi 18 strains were assessed for the presence of polyketide synthase genes by PCR approaches. These fungi were identified taxonomically by a polyphasic approach classifying into four orders and nine genres. Biological activity tests showed the presence of antibiotic metabolites against pathogenic microorganisms and the relationship of this activity might be linked with the fungal isolate location by multiple correspondence analyses. 36 sequences similar to PKS genes fragments were detected from 17 of these fungi. A neighbor-joining phylogenetic analysis of the KS domain showed that 16 sequences fit on the monophyletic group of PKS evolved with production of non reduced polyketides, and the other 10 sequences fit on the monophyletic group of PKS evolved with the production of reduced polyketides. CMT domain analysis also pointed that the sequences fit with groups of PKS depending on polyketide reduction grade, all ten related to PKS evolved with the synthesis of reduced polyketides. Protein structural analysis also pointed out that these sequences are closely related with proteins from condensing enzyme family, highlighting the presence of a characteristic helix elbow that bears the cysteine residue responsible for the condensation activity. The fungi were also tested for their capacity of producing lovastatin compounds where chromatographic TLC detected bands from 10 extracts with the same dislocation compared to a lovastatin, but only 6 were confirmed by HPLC. The A. flavus CBMAI (1023) were selected for upscale production experiments, from where it was possible isolate and characterize a new polyketide compound.
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Rajgarhia, Vineet B. "Analysis of the polyketide synthase genes of the daunorbicin producer, Streptomyces sp. strain C5 : generation of PKS mutants, and analysis of the unusual anthracycline products made by these PKS mutants /." The Ohio State University, 1998. http://rave.ohiolink.edu/etdc/view?acc_num=osu1487950658546452.
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Bacha, Nafees. "Caractérisation des polycétones synthases intervenant dans la biosynthèse d’ochratoxine A, d’acide pénicillique, d’asperlactone et d’isoasperlactone chez aspergillus westerdijkiae." Thesis, Toulouse, INPT, 2009. http://www.theses.fr/2009INPT004A/document.
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Aspergillus westerdijkiaem qui est récemment démembré d'A. ochraceus est un producteur principal de plusieurs composés de type polycétone d'importance économique. Ces composés incluent l’ochratoxin A, mellein, l'acide penicillique, asperlactone et l’isoasperlactone et quelques intermédiaires comme l'acide 6- methylsalicylique et l’acide orsellinique. La biosynthèse de ces métabolites est catalysée par un groupe d'enzymes connues comme la polycétone synthases (PKSs). Ce travail a été visé pour cloner et a caractérisé fonctionnellement les différentes genes des PKS i.e. aoks1, aolc35-12 et aomsas, et de genes de polyketide synthases-non ribosomal peptide synthase (PKS-NRPS) i.e. aolc35-6, chez A. westerdijkiae. Ces gènes ont été inactivés par l'insertion du gène d’hygromycine B phosphotransferase d’Escherichia coli dans le génome d'A. westerdijkiae, pour obtenir les mutantes ao?ks1, ao?lc35-12, ao?msas et ao?lc35-6. Les mutants ao?ks1 et ao?lc35-12 ont été trouvés déficients dans la biosynthèse d’ochratoxin A, mais produisaient encore mellein. À notre connaissance, c’est la première fois que nous avons caractérisé les gènes impliquées dans la biosynthèse d’OTA, sachant que mellein, qui était proposé dans la littérature comme un intermédiaire, joue a cune role dans la biosynthesis de l'OTA. Ensuite le mutant ao?msas n'a pas seulement perdu la capacité de produire isoasperlactone et asperlactone, mais aussi il ne produit pas l’intermédiaire acide 6-methylsalicylique. Basé sur les expériences de la caractérisation génétique et de complémentation chimiques, nous avons proposé un shéma hypothétique de la biosynthèse d’asperlactone et isoasperlactone dans lequel l'acide 6-methylsalicylique, diepoxide et aspyrone jouent le rôle d’intermédiaires. La techniques de gène knock-out et de la reverse transcription PCR (RT-PCR) ont montré que seulle gène de type PKS-NRPS « aolc35-6 » identifié chez A. westerdijkiae codant pour un intermédiaire inconnu(s) qui pourrait inciter l'expression de gène aomsas et un gène impliqué dans la biosynthèse d'acide orsellinique et d'acide penicilliqueAspergillus westerdijkiaem which is recently dismembered from A. ochraceusm is the principal producer of several economically important polyketide metabolites. These metabolites include ochratoxin A, mellein, penicillic acid, asperlactone and isoasperlactone and some intermediates like orsellinic acid and 6-methylsalicylic acid. The biosynthesis of these metabolites is catalyzed by a group of enzymes known as polyketide synthases (PKSs). This work was aimed to clone and functionally characterized various PKS i.e. aoks1, aolc35-12 and aomsas, and polyketide synthasesnon ribosomal peptide synthase (PKS-NRPS) genes i.e. aolc35-6, in A. westerdijkiae. These genes were inactivated by the insertion of Escherichia coli hygromycin B phosphotransferase gene in the genome of A. westerdijkiae to obtain ao?ks1, ao?lc35-12, ao?msas and ao?lc35-6 mutants. ao?ks1, ao?lc35-12 mutants were found deficient in ochratoxin A biosynthesis but are still producing mellein. To our knowledge, we for the first time characterized a gene involved in OTA biosynthesis, with the information about mellein which was proposed in the literature to be an intermediate OTA. Further ao?msas mutant not only lost the capacity to produce isoasperlactone and asperlactone but also the intermediate nature product 6-methylsalicylic acid. Based on the genetic characterization and chemical complementation experiments, we have proposed a hypothetical pathway mentioning that 6-methylsalicylic acid, diepoxid and aspyrone are intermediates of isoasperlactone and asperlactone. Gene knockout technique and reverse transcription PCR (RT-PCR) shown that the only PKS-NRPS gene aolc35-6 so far identified in A. westerdijkiae encoding certain unknown intermediate(s) which induces the expression of aomsas gene and a gene involved in the biosynthesis of orsellinic acid and penicillic acid
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Rogers, David. "CIS-REGULATORY ANALYSIS OF THE PIGMENT CELL DIFFERENTIATION GENE POLYKETIDE SYNTHASE." Master's thesis, University of Central Florida, 2008. http://digital.library.ucf.edu/cdm/ref/collection/ETD/id/2701.
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The analysis of Gene Regulatory Networks (GRNs) is essential to understanding the complete process of embryo development. Elucidating every gene regulatory circuit from maternal regulatory inputs all the way to the activation of differentiation gene batteries is an important step in increasing our understanding of developmental biology. In this work I study the cis-regulatory architecture of a pigment cell differentiation gene, polyketide synthase (SpPks) in the sea urchin Strongylocentrotus purpuratus. SpPks encodes an enzyme that is responsible for the biosynthesis of the sea urchin pigment echinochrome in larval pigment cells. The analysis of the promoter of a differentiation gene will lead to identifying the direct upstream regulators and ultimately to elucidating the structure of the upstream gene regulatory network, which is mostly uncharacterized. From previous studies the transcription factors SpGcm and SpGatae are predicted to be positive regulators of SpPks. Here, I identify a minimal 1kb promoter region containing putative DNA-binding sites for both GCM and GATAE that is able to recapitulate the expression of SpPks. I further show by mutagenesis that a putative DNA-binding site for GCM located 1,179 base pairs upstream of the start of transcription is a direct target for the positive cis-regulation of SpPks. Quantitative analysis of the transcriptional regulatory function of the GCM-mutagenized construct suggests that GCM is not necessary for the start of SpPks transcription but is required for its maintenance. Several GATA E binding sites have been identified within the minimal promoter for SpPks by means of consensus sequence. My analysis suggests that GATA E may be a direct positive regulator and could potentially be required for the onset of transcription of SpPks, though further experimentation will be necessary to characterize the exact regulatory function of GATA E.M.S.
Department of Biology
Sciences
Biology MS
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Kirkpatrick, Clare Louise. "Genetic characterisation of a polyketide synthase gene cluster in Rhodococcus aetherivorans I24." Thesis, University of Cambridge, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.613937.
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Couch, Robin D. "Identification and disruption of the emodin anthrone polyketide synthase gene from Aspergillus terreus." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 2000. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape2/PQDD_0018/NQ49487.pdf.
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Stephens, Tricia. "CHARACTERIZATION OF PIGMENT CELL SPECIFIC GENES IN THE SEA URCHIN EMBRYO (STRONGYLOCENTROTUS PURPURATUS)." Master's thesis, University of Central Florida, 2007. http://digital.library.ucf.edu/cdm/ref/collection/ETD/id/2692.
Full textAbstract:
In sea urchin development, cell fate specification appears by the 60-cell stage embryo when several embryonic territories are recognized: the small micromeres, the large micromeres which will generate primary mesenchyme cells, the vegetal2 layer that will give rise to pigment cells, immunocytes, and muscle cells, the vegetal1 layer, as well as the oral and aboral ectoderm. A Delta-Notch signaling event is required for the differential specification of mesodermal cells that will give rise to secondary mesenchyme cells (SMCs). SMCs produce four cell types: pigment cells, blastocoelar cells, circumesophageal muscle cells, and coelomic pouch cells. Pigment cells are the first to be specified. During primary invagination at the gastrula stage, eight pigment cell progenitors delaminate from the archenteron into the blastocoel. By the pluteus stage, approximately 30 pigment cells are embedded in the ectoderm. Pigment cells produce echinochrome, a napthoquinone pigment. Previously, several genes in the sea urchin embryo were isolated that are expressed specifically in pigment cell precursors during the blastula stage. The goal of this research was to characterize a subset of these genes, which are highly similar to: the polyketide synthase gene (Pks), a sulfotransferase gene (Sult), three different members of the flavin-containing monooxygenase gene family (Fmo), and the transcription factor glial cells missing (Gcm). Polyketide synthases (PKSs) are a large family of multifunctional proteins mainly found in bacteria, fungi, and plants. They are responsible for the biosynthesis of a variety of polyketide compounds including antibiotics and mycotoxins. In the sea urchin, SpPks is required for echinochrome biosynthesis. Flavin-containing monooxygenases (FMOs) are NADPH-dependent flavoproteins mainly found in bacteria, plants, and higher metazoan. They are responsible for catalyzing the oxidation of several compounds including the detoxification of xenobiotics and activation of numerous metabolites. It is known that SpFmo1 is required for echinochrome biosynthesis. Sulfotransferases are found from bacteria through higher eukaryotes. These enzymes catalyze the sulfate conjugation of several substrates resulting in either compound detoxification or bioactivation.M.S.
Department of Biology
Sciences
Biology MS
21
Timsina, Brinda Adhikari. "Evolution and Expression of polyketide synthase gene in the lichen-forming fungal families Cladoniaceae and Ramalinaceae." NRC Research Press, 2012. http://hdl.handle.net/1993/23271.
Full textAbstract:
Fungal polyketides are synthesized by polyketide synthases (PKS) encoded by PKS genes. The function of many PKS genes is unknown and the number of PKS genes exceeds the number of polyketides in many genomes. The lichen-forming fungi, Cladonia and Ramalina have chemical variants separated by habitat suggesting that environmental conditions may influence polyketide production. The goal of this thesis was to examine evolutionary relationships as a framework to investigate PKS gene function in the lichen-forming fungal families Cladoniaceae and Ramalinaceae. A phylogenetic analysis of the genus Ramalina (Chapter 2) using nuclear and mitochondrial ribosomal DNA sequences showed monophyly for seven species and included three species, which were not examined in phylogenies prior to this study. One monophyletic species, R. dilacerata was chosen for further tests of the effect of growing conditions on PKS gene expression (Chapter 3). Growth media containing yeast extracts produced the largest colony diameters and the fewest number of polyketides. A significant negative relationship occurred between colony diameter and number of secondary metabolites. Expression of two types of PKS genes was correlated with pH-level and media conditions that produced larger numbers of secondary products in R. dilacerata. A PKS gene phylogeny was constructed for 12 paralogs detected in members of the C. chlorophaea complex (Chapter 4) and gene selection was inferred using dN/dS estimations. The gene phylogeny provided evidence for independent origins and purifying and positive selection of PKS paralogs. This research provided insight into the evolution of PKS genes in the C. chlorophaea complex and identified potential genes that produce non-reduced polyketides present in C. chlorophaea.
This thesis provided evidence for diversification of both morphological and chemical species and monophyly of previously unstudied Ramalina species. This research also supported theories of secondary metabolite synthesis based on growing conditions of R. dilacerata, and it revealed that PKS genes under selection in the Cladonia chlorophaea group provide the lichen with the adaptive capacity to survive under variable conditions. Knowledge of the ecological function of fungal polyketides can be valuable for conservation management and policy makers; and for understanding the potential pharmaceutical roles of these natural products.
22
Burson, Kim Katrina. "Gene shuffling of bacterial aromatic polyketide synthases ; Binding and fusion studies of vesicular stomatitis virus (VSV) /." May be available electronically:, 1998. http://proquest.umi.com/login?COPT=REJTPTU1MTUmSU5UPTAmVkVSPTI=&clientId=12498.
Full textAbstract:
Thesis (Ph. D.)--Stanford University, 1998.Submitted to the Department of Chemistry. Copyright by the author. No collective title. Part 1 Gene shuffling of bacterial aromatic polyketide synthases. Chapters 1-3. Part 2 Binding and fusion studies of vesicular stomatitis virus (VSV). Chapters 4-5.
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Metin, Banu. "Characterization And Functional Analysis Of A Novel Multicopper Oxidase And Associated Polyketide Biosynthesis Gene Cluster Of Aspergillus Fumigatus." Phd thesis, METU, 2007. http://etd.lib.metu.edu.tr/upload/12609029/index.pdf.
Full textAbstract:
In this study, novel polyketide biosynthesis gene cluster of Aspergillus
fumigatus was characterized and functionally analyzed. Analysis of the newly
sequenced A. fumigatus genome for laccases, which are involved in melanin
biosynthesis and detoxification in fungi, resulted in several putative laccase and
multicopper oxidase gene sequences, one of which, Afu4g14490 (tpnJ), was selected
for further characterization. The predicted amino acid sequence TpnJp showed 63%
identity with the dihydrogeodin oxidase of Aspergillus terreus, which is involved in
the biosynthesis of the antifungal geodin. When the genome region of tpnJ was
investigated, the presence of a polyketide biosynthesis gene cluster containing 13
genes, hypothesized to be responsible for the production of trypacidin and
monomethylsulochrin, was realized. By a comparative genomics approach, a putative
geodin biosynthesis gene cluster containing 13 genes, including dihydrogeodin
oxidase, in A. terreus and a putative trypacidin biosynthesis gene cluster containing
13 genes in N. fischeri were established. Targeted deletions of the polyketide
synthase (tpnC) and multicopper oxidase (tpnJ) genes confirmed the hypothesis that TpnCp, a three-domain minimal polyketide synthase, is involved in trypacidin and
monomethylsulochrin biosynthesis in A. fumigatus. TpnCp is the first fungal minimal
polyketide synthase whose functional role was experimentally identified. Moreover,
the fact that LC-MS analysis of DtpnJ strain showed the absence of trypacidin and
the presence of a higher amount of monomethylsulochrin in DtpnJ strain, confirmed
the hypothesis that TpnJp is involved in the oxidation of monomethylsulochrin into
trypacidin. This novel multicopper oxidase having high substrate specificity is given
the name monomethylsulochrin oxidase.
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Karppinen, K. (Katja). "Biosynthesis of hypericins and hyperforins in Hypericum perforatum L. (St. John’s wort) – precursors and genes involved." Doctoral thesis, University of Oulu, 2010. http://urn.fi/urn:isbn:9789514263101.
Full textAbstract:
Abstract
Hypericum perforatum L. (St. John’s wort) is a medicinal plant widely utilized for the treatment of depression. The antidepressant activity is mainly attributed to the phenolic compounds hypericins and hyperforins, which also have a wide range of other pharmacologically interesting properties. The biosynthetic routes leading to hypericins and hyperforins are poorly understood, although a polyketide pathway including type III polyketide synthases (PKSs) has been suggested to be involved. Furthermore, a gene called hyp-1 is assumed to attend to the final stages of the hypericin biosynthesis. In the present work, the biosynthesis of hypericins and hyperforins in H. perforatum was further studied by focusing on the elucidation of the precursors and genes involved.
The incorporation of isotopically labelled branched-chain amino acids into hyperforins was investigated as well as the possibilities to enhance the production of hyperforins in H. perforatum in vitro cultures by feeding them with amino acid precursors. Furthermore, two novel cDNAs encoding for type III PKSs were isolated from H. perforatum. The functions of these new genes, designated HpPKS1 and HpPKS2, as well as the role of hyp-1 were elucidated by comparing their expression with the levels of hypericins and hyperforins in H. perforatum tissues. The enzymatic activity of the recombinant HpPKS2 protein was also analyzed. To study Hyp-1 at a protein level, a protein extraction method was optimized for tissues of Hypericum species.
The results show the incorporation of valine and isoleucine into the acyl side chain of hyperforin and adhyperforin, respectively. Through the biotransformation of the amino acid precursors, it is possible to enhance the levels of adhyperforin, but not hyperforin, in H. perforatum shoot cultures, which demonstrates the tight regulation of the hyperforin biosynthesis. A correlation between HpPKS1 expression and hyperforins was detected in H. perforatum tissues. The localization of HpPKS2 mRNA in dark glands in which hypericins accumulate as well as the octaketide synthase activity of the recombinant HpPKS2 suggest that HpPKS2 is associated with possible co-operating tailoring enzymes in the biosynthesis of hypericins. The presence of both hyp-1 mRNA and Hyp-1 protein in distinct places compared with hypericins in H. perforatum tissues does not support the idea that Hyp-1 would be involved in the biosynthesis of hypericins in dark glands, although mobility of the Hyp-1 protein was shown to be possible.
The present thesis extends knowledge about the biosynthesis of hypericins and hyperforins in H. perforatum by providing new candidate genes for their biosynthesis and by identifying precursors for hyperforins. Moreover, new information was obtained about the role of hyp-1 in H. perforatum.
25
ZHUO, JING-WEI, and 卓靖爲. "Functional characterization of the polyketide synthase genes of Bacillus subtilis." Thesis, 2018. http://ndltd.ncl.edu.tw/handle/a296hf.
Full textAbstract:
碩士長榮大學
醫學研究所
106
Polyketides are synthesized by the polyketide synthases (PKSs) megacomplex. Many polyketides can inhibit the growth of fungi and bacteria. In addition to the PKS genes, sfp gene is also required for polyketide biosynthesis. Sfp converts apo-form PKSs into holo-from PKSs by posttranslational modification. This study was to investigate the function of the pksF gene cluster found in Bacillus subtilis F29-3. Mutagensis analysis shown that both pksF and sfp gene were required for a polyketide (Polyketide-F) synthesis. Observation by scanning electron microscopy revealed that the size of the pksF mutant was 60% of that of the wild strain. The sporulation of the wild-type strain and the pksF mutant strain was analyzed. The wild-type strain did not produce spores within 24 hours, and the pksF mutant strain had produced a large number of spores in 12 hours. B. subtilis F29-3 and mutant strains were co-cultured with Phaseolus vulgaris HV177 and Arabidopsis thaliana Col-0, and plants root length were longer when coculture B. subtilis F29-3. The results show that the pksF gene cluster is involved in Polyketide F synthesis and the genes of pksF affect the sporulation ability, root length in plants and the size of the bacteria.
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Jhang, Yu-Cheng, and 張育誠. "Cloning and heterologous expression of the polyketide synthase genes from Leptosphaeria sp. NTOU806." Thesis, 2016. http://ndltd.ncl.edu.tw/handle/30576638336676903153.
Full textAbstract:
碩士國立臺灣海洋大學
生命科學暨生物科技學系
104
Secondary metabolites produced from bacteria, fungi, plants,mollusks, algae, insects etc. have been found. The metabolites show pharmacological activities and have potential in the new drug development. Polyketides, a type of secondary metabolites, produced by polyketide synthase (PKS) have medicine application including immune inhibitor, antibiotics, anticholesterol, antitumor, etc. PKSs contain multiple functional domain to synthesize polyketides. Organic extract from fungus NTOU 806 have been demonstrated a biological activity in the inhibiting the production of nitric oxide and lipopolysaccharide-induced inflammation, but not containing cytotoxicity, in RAW264.7 macrophage cells. Whole genome sequencing of NTOU 806ws performed and six PKS genes were chosen: Hyb-zero, PKS2271, PKS2675, PKS3010, PKS3034A, and PKS3034B.We used long range PCR to obtain target genes and these DNA were cloned into pENTR/D-TOPO cloning vector. The PKS genes were cloned into yeast expression vectors using LR reaction technologyin the Gateway cloning system. These vector vectors contain GPD and GAL promoter and consist four different selection makers, The expression vectors were transformed into Saccharomyces cerevisiae, and the induction protein were detected by western blot. HPLC was performed to analyze new compounds in induction medium by NTOU 806 PKSs, observing the new products in the PKS2271, 3034A, 3034B and Hybrid-zero. In protein expression analysis, we found PKS3010, PKS2271, and PKS2675 (MT-ER-KR-ACP) by western blot using anti-GFP antibody.
27
Lin, Kui-Yo, and 林奎佑. "Molecular cloning of three genes encoding hypericin synthase, polyketide synthase and benzophenone synthase from Hypericum geminiflorum Hemsl. and Hypericum japonicum Thunb." Thesis, 2013. http://ndltd.ncl.edu.tw/handle/32874325814626502066.
Full textAbstract:
碩士國立屏東科技大學
生物科技系所
101
Hypericin and hyperforin are two biologically active substances in plants of Hypericum genus, and they have anti-depression, anti-bacterial, anti-viral and anti-oxidation effects. It is known that the biosynthesis of two compounds are related to the expression of HYP1, PKS1, and BPS genes which mediated the production of hypericin synthase, polyketide synthase and benzophenone synthase for the respected enzymatic reactions. Since there are fifteen species of Hypericum plants in local area of Taiwan, it is suggested to clone the HYP1, PKS1, BPS genes from H. geminiflorum Hemsl. and H. japonicum Thunb. for heterologously expression of recombinant proteins for the analysis of enzyme activity and the biosynthesis of compounds. Currently the HYP1, PKS1, BPS genes from H. geminiflorum Hemsl. and BPS gene from H. japonicum Thunb. are cloned and analyzed. It is suggested to clone HYP1 and PKS1 genes from H. japonicum Thunb. and to functional express the recombinant proteins for the biosynthesis of hypericin and hyperforin heterologously in the future. Keyword： Hypericin, Hyperforin, HYP1, PKS1, BPS
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Yu, Kai-Chieh, and 游凱傑. "Cloning and heterologous expression of the polyketide synthase genes from Leptosphaeria sp. NTOU 806." Thesis, 2015. http://ndltd.ncl.edu.tw/handle/shkuvu.
Full textAbstract:
碩士國立臺灣海洋大學
生命科學暨生物科技學系
103
Polyketides are belong to secondary metabolites obtained from bacteria, fungi, plants, mollusks, algae, insects, sponges etc. and contain diverse chemical structure and extensive bioactivity. They have been applied in the field of agriculture, industry and pharmaceuticals such as antibiotics erythromycin and tetracycline used in the human, veterinary antibiotic tylosine, anticancer drug doxorubicin, immunosuppressant rapamycin, or cholesterol-lowering drug lovastatin. Therefore, the discovery and producing of novel polyketides may have a high contribution in the development of new drugs. In order to establish a strategy for heterologous expression of polyketide synthases (PKS) that underline the biosynthesis of polyketide, we choice the PKS genes, pfaA, pfaB, pfaC, pfaD and pfaE, involving EPA biosynthesis in Shewanella oneidensis MR-1. These genes was analyzed by bioinformation to design cloning primers. For cloning EPA PKSs, we extracted genomic DNA and performed long-range PCR to obtain full-length EPA PKS genes. These genes were cloned into pENTER/D-TOPO cloning vector and then used gateway cloning system to construct yeast expression vectors in which harbor GAL1 promoter in their 5’ end and in-frame GFP in their 3’-end and contain ura3, leu, his or trp auxotrophic selection marker, respectively. These EPA PKS expression vectors were transformed into INVSc1 and BJ2168 Saccharomyces cerevisiae by lithium chloride method. After induction, the protein expression of EPA PKSs were analyzed by western blot using GFP antibody, and EPA production was detected by gas chromatography. A Leptosphaeria sp fungus, NTOU806, contains bioactive compounds that inhibited the production of nitric oxide and lipopolysaccharide-induced inflammation and showed low cytotoxicity in RAW264.7 cells. For identification of PKS genes and the biosynthesis of polyketides, we performed whole genome sequencing of NTOU806, finding that NTOU806 contains 10 genes of PKS and 3 genes of nonribosomal peptide synthetase. The PKS genes, not containing intron, of PKS2271, PKS2675, PKS3034A and PKS3034B were performed cloning from genomic DNA of NTOU806 and transformed into S. cerevisiae for expression. Moreover, PKS64 obtained from NTOU2362 were performed large scale culture to purify its polyketide. These yeast transformants were collected culture medium, extracted by ethyl acetate, evaporated to concentrate their products and dissolved by methanol. These collected material were analyzed and purified by high performance liquid chromatography, estimated molecular weight by mass spectrometry, and determined chemical structure by nuclear magnetic resonance. Accroding to analysis of HPLC which shows that gene PKS2271 and PKS2675 can produce new chemical compound. NTOU 2362(PKS 64 gene) can express the new protein by induction of S. cerevisiae BJ2168. We use HPLC analysis to ensure the production of the new compund. after the HPLC analysis,we use MS analysis to analyze the new compound. we found out that the new chemical compoud has the molecular weight 144 now we use the final analysis,NMR,and we found that the formula of the chemical compound is tryptophol.Our results show that PKS genes are successfully expressed in S. cerevisiae, and we establish a reliable and feasible strategy to purify and identify polyketides from heterologous expression.
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Yu, Po-Wei, and 余浡維. "Functional Characterization of Polyketide Synthase-encoding Genes and the Related Biosynthetic Pathway in Antrodia cinnamomea." Thesis, 2017. http://ndltd.ncl.edu.tw/handle/yaven2.
Full textAbstract:
博士國立臺灣大學
植物病理與微生物學研究所
105
Antrodia cinnamomea is a unique resupinate basidiomycete endemic to Taiwan. Besides the abundant triterpenoid metabolites, A. cinnamomea is known for producing antroquinonols, which were reported to have notable medicinal potential in oncology and immunology. However, neither the biosynthetic pathway of these compounds nor the corresponding genes are currently clear. To investigate the biosynthesis of antroquinonols in A. cinnamomea, we focused on the polyketide synthase (PKS) genes due to the similar structure of the cyclohexenone moiety of antroquinonols to the aromatic polyketide. Four putative PKS genes, including three reducing PKSs and one non-reducing PKS, pks63787, were characterized in A. cinnamomea based on the partially deciphered genome and the constructed fosmid library. For the first time, a gene disruption platform was established in A. cinnamomea via a protoplast-mediated transformation system. Our study showed that the pks63787 knock-out mutant of A. cinnamomea (∆pks63787) is deficient in the biosynthesis of several aromatic metabolites which are involved in the antioxidant activity and colony morphology. In the further study, we pointed out by phylogenetic analysis that pks63787 likely encodes an orsellinic acid synthase, whose function was double-confirmed with a complementary feeding test. The amendment of orsellinic acid not only restores the ability of ∆pks63787 in producing its deficient pigment, benzenoids and antroquinonols, but also enhances the productivity of several antroquinonols. These results provide direct evidence that the PKS63787 is involved in the biosynthesis of antroquinonols, and supported our hypothesis that the cyclohexenone moiety is a polyketide synthesized via the PKS63787-conducted polyketide pathway. Along with the identification of numerous PKS63787- and orsellinic acid-mediated components, six compounds, including two benzenoids 1, 2, two antroquinonols 3, 4 and two antrocinnanoates 5, 6, were reported for the first time. In conclusion, our study has contributed to the understanding of the PKS genes and the biosynthesis of antroquinonols in A. cinnamomea, and the adopted procedure may be conducive to genetics research focusing on natural products in A. cinnamomea and other basidiomycetes.
30
Yu, Fang-Yi, and 余芳儀. "Isolation of antibiotic compounds andcloning of polyketide synthase genes from the peach brown rot pathogen Monilinia fructicola." Thesis, 2011. http://ndltd.ncl.edu.tw/handle/09836132039871231808.
Full textAbstract:
碩士國立中興大學
植物病理學系所
99
Monilinia fructicola is a fungal pathogen which causes blossom blight and fruit rot of Prunnus species. In several early studies, M. fructicola was reported that could produce phenolic polyketides as antimicrobial compounds against bacterial and fungal pathogens of human. Among our M. fructicola collections, strain TW5-4 grew slowly, forms dark colony in agar medium and has strong antimicrobial activity against phytopathogenic fungi and bacteria. A spontaneous albino mutant of TW5-4 (TW5-4WM) with normal growth and less antimicrobial activity is also identified during subculturing in the lab. In this study, the differences of the two strains (TW5-4 and TW5-4MW) on fungal growth, sporulation, appressorium formation, melanin accumulation, pathogenicity and antimicrobial activity were investigated. Antimicrobial compounds were extracted by ethyl acetate and analyzed by thin layer chromatography (TLC) and LC-MS-MS. Furthermore, polyketide synthase genes, which are potentially involved in the biosynthesis of melanin and antimicrobial compounds of M. fructicola, were cloned by degenerate PCR and inverse PCR. Their expressions were detected by rt-PCR and real time-rt-PCR. Our data showed that compared with strain TW5-4MW and M1, a typical strain of M. fructicola, strain TW5-4 has less ability on growth, sporulation, appressorium formation and pathogenicity but has stronger ability on melanin accumulation and anti-fungal activity. The ethyl acetate extracts from mycelia of TW5-4 showed inhibitory activity against mycelial growth and spore germination of two target plant pathogens Penicillium digitatum and Botrytis cinerea. Antimicrobial compounds were localized with Rf=0.45 by bioassay after separated by TLC, which could react with FeCl3 and ninhydrin solution, indicating that the antimicrobial compound might be phenolic and/or contain amine(s). Two compounds which may contribute to antifungal activity were further determined by LC-MS-MS. Their molecular weights are 350 and 346. One full-length and 11 partial PKS genes of M. fructicola were cloned and analyzed. Some PKSs have high sequence similarity to fungal PKS involved in pigment formation and phytotoxin production. PKS gene LC1 and LC2, which are closely related to the PKS genes involved in melanin biosynthesis, expressed at higher levels in TW5-4 than TW5-4WM. No significant correlation was found between the expression levels of the other 10 genes and antimicrobial activity or melanin accumulation.
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Lin, Jyuan-Siou, and 林娟秀. "Cloning and heterologous expression of the polyketide synthase genes of endophytic Pseudallescheria boydii NTOU 2362 from mangrove plants." Thesis, 2014. http://ndltd.ncl.edu.tw/handle/9jpg7r.
Full textAbstract:
碩士國立臺灣海洋大學
生物科技研究所
102
Secondary metabolites, which come from microorganisms, have a lot of properties in pharmacology. In the process of synthesis, they are always produced polyketides by polyketide synthases. There have been using in medicine such as immunosuppressant, rapamycin; antibiotics, erythromycin and penicillin; cholesterol lowering agents, lovastatin; anticancer drugs, epothilone B. PKS has multiple functional domain property, and it can synthesize diversity natural compound. Thus, secondary metabolites have enormous potential. In our previous study, we discovered Pseudallescheria boydii NTOU2362 had antivirus activity and inhibited LPS to induce NO production in RAW 264.7. We also have finished the whole genome sequencing. However, we did not understand PKS genes of NTOU2362, which could produce compounds. In this study, we used bioinformatics tools to analyze 9 PKS genes from NTOU2362 such as PKS61, PKS64, PKS81, PKS128, PKS98, PKS306, PKS338, PKS484, and PKS506. PKS98, PKS306, and PKS484 were open reading frame (ORF), and the other six PKS genes had intron. Therefore, we had to use the software to analyze splicing site of the six genes, but we did not find correct splicing sites. We also used RT-PCR to check whether they had splicing sites or not. We used DNA sequencing method to check the products of RT-PCR, and discovered PKS64, PKS81, PKS128, PKS338, and PKS506 had splicing sites in NTOU2362. Thus, we used long range PCR to get eight completely ORFs, and used gateway system to get eight recombinant DNAs which have EGFP gene at N-site. We used lithium chloride method to transform recombinant DNAs into Saccharomyces cerevisiae expression system, and induced it. After we disrupted pellets, we used western blot method to detect GFP. The result was PKS64 had signal. We used HPLC to analyze supernatant, and discovered PKS64, PKS306, and PKS506 had activity of producing compound. Hence, we further analyzed the pellets that we had disrupted, and used western blot method to detect GFP. We discovered all of PKS98, PKS484, PKS 506, PKS64, and PKS338 had protein expression. We can know that cell walls of expression host, which come from fungi, are hard to disrupt. Thus, proteins are not released easily by disrupting pellets. In our study, we successfully got big fragment of PKS ORFs. We also detected each protein expression of all PKS genes, and each activity of producing compounds. In this result, we can understand that each PKS gene can produce what kind of compounds in NTOU2362. In the future, we will not only use the sequence of 5’GU~AG3’ to analyze the splicing site of fungi, but also use the sequence of 5’TA~AGG3’ to analyze it. In the compound analysis, we can use LC-MS to detect the compound production, and analyze the culture medium which is hard to analyze by HPLC method. Therefore, we can further discuss the synthesis pathway about compounds producing of PKS in NTOU2362.
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Grimm, Ann C. "Characterization of the doxorubicin polyketide synthase genes of streptomyces peucetius and the development of a system for consistent growth." 1995. http://catalog.hathitrust.org/api/volumes/oclc/32870288.html.
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Fang, Yi-Ting, and 方羿婷. "Antifungal Effect and Molecular Cloning of Polyketide Synthase Gene from Polygonum cuspidatum." Thesis, 2011. http://ndltd.ncl.edu.tw/handle/88547934646927560759.
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Wang, Li-Ping, and 王麗萍. "Heterologous Expression of Fungal-Derived Nonreducing Polyketide Synthase Gene in Aspergillus nidulans." Thesis, 2013. http://ndltd.ncl.edu.tw/handle/64338489583191341858.
Full textAbstract:
碩士嘉南藥理科技大學
生物科技系
101
Fungal secondary metabolism is very important in fungal growth and development. The production of secondary metabolites is relied on the changes of environment. In Aspergillus, gene cluster is involved in the regulation of secondary metabolism, and the major products of the gene cluster are belong to non-reducing polyketide synthases (NRPKSs). However, it is not very clear about the function and activity of the secondary metabolites catalyzed by NRPKSs. Cordyceps militaris is a useful herbal medicine grew on some insect larvae or pupae. Cordyceps militaris contains many kinds of functional components such as cordycepin, cordycepic acid, nucleosides, and Cordyceps polysaccharide. We assume that the secondary metabolic pathway is also existed in C. militaris. In order to clarify the function of NRPKSs in the regulation of secondary metabolism and the secondary metabolites in C. militaris, fusion PCR technique was used to the recombination of selective markers , alcA promoter, and target gene. Heterologous expression was performed in Aspergillus nidulans. In this study, the use of fusion PCR completed which NRPKS fusion gene, and seven of transformants were obtained. The variety of secondary metabolites was analyzed by high-performance liquid chromatography. The results indicated that some secondary metabolites were produced in transformants. This study showed a great potential for developing the application of fungal secondary metabolites and studying the function of NRPKS genes.
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Chi-NingLiu and 劉致寧. "Polyketide synthase gene sequence and expression analysis in Aurantiochytrium sp. strain L-BL10." Thesis, 2013. http://ndltd.ncl.edu.tw/handle/47386178203924645395.
Full textAbstract:
碩士國立成功大學
生物科技研究所碩博士班
101
Aurantiochytrium sp. strain L-BL10 is rich in C22 polyunsaturated fatty acids (PUFAs), DHA (C22:6n-3), and DPA (C22:5n-6); however, this species contains very few PUFAs with 18 and 20 carbons. Previous studies in our laboratory demonstrated that adding fatty acid synthase (FAS) inhibitor to a L-BL10 culture failed to decrease the percentages of DHA and DPA. These earlier results revealed that, in addition to the standard pathway, a secondary biosynthesis pathway is involved in the production of these two PUFAs in L-BL10. The purpose of our current research was to identify enzymes capable of catalyzing the synthesis of PUFAs along this specific biosynthetic pathway. First, several genes suspected of being involved in PUFA production were synthesized from the L-BL10 genome sequence and NCBI database. We then predicted the functional domains using SMART (Simple Modular Architecture Research Tool) software and investigated the enzyme classification according to functional domain arrangement and phylogenetic analysis. In addition, we examined gene expression over various incubation times using real-time PCR. These efforts revealed that 8 polyketide synthase (PKS)-like genes may be involved in the production of DHA and DPA in Schizochytrium sp. ATCC 20888. Previous researchers confirmed that the polyketide synthase of ATCC 20888 comprises three genes, pksA, pksB, and pksC. The results of this current study further demonstrated that L-BL10 has three complete polyketide synthase genes, which are similar to Schizochytrium sp. ATCC 20888. The lengths of these genes are 10068, 6117, and 4386 base pairs, respectively. We also identified the functional domains capable of synthesizing PUFAs, such as ketoacyl synthase, malonyl-CoA:ACP acyl transferase, acyl carrier protein, ketoacyl-ACP reductase, enoyl reductase, chain length factor, acyl transferase, and dehydratase functional domain. These results indicate that L-BL10 should have the ability to synthesize PUFAs. Moreover, the PKS of L-BL10 was shown to belong to Type I iterative PKS according to its arrangement of functional domains and the results of phylogenetic analysis related to KS domains. Conversely, we found that PKS gene expression reached the culminating point when L-BL10 was in the last stage of log phase (after culturing for 20 hours). At this point, the nitrogen source on the culture plate was depleted and L-BL10 began using the carbon source. Thus, we surmise that the PKS gene may be regulated by its nutrient source, such that the co-existence of a carbon source and nitrogen source on the culture plate prevents induction of the PKS gene. Nonetheless, these conditions could promote the accumulation of DHA and DPA when induced by only a carbon source on the culture plate.