Modulare Polyketidsynthasen (PKS) sind Multienzymsysteme des bakteriellen Sekundärstoffwechsels. An ihnen läuft eine schrittweise Biosynthese vielfältiger Kohlenstoff-Gerüste ab, die von einfachen Carbonsäure-Einheiten ausgeht. Polyketid-Verbindungen zeigen eine große Bandbreite pharmazeutisch interessanter Aktivitäten. In dieser Arbeit wurde eine Reihe von Evolutionsstudien durchgeführt. Zunächst wurden die phylogenetischen Beziehungen zwischen modularen PKS und anderen PKS-Systemen sowie Fettsäuresynthasen untersucht, wodurch ihre zentrale Stellung innerhalb eines langen Evolutionsprozesses gezeigt werden konnte. Eine detaillierte Analyse der Phylogenien von Domänen bakterieller modularer PKS ergab, dass das Ausmaß an Genduplikationen, Genverlusten und Ereignissen horizontalen Gentransfers zwischen den verschiedenen Bakteriengruppen beträchtlich variiert. Aus der Genomsequenz des Actinobakteriums Streptomyces avermitilis wurden die Phylogenien aller Domänentypen rekonstruiert. Der Vergleich dieser Einzelphylogenien ermöglichte es, eine Reihe von homologen Rekombinationsereignissen aufzufinden. Homologe Rekombination scheint der Hauptmechanismus zu sein, auf dem die Strukturvielfalt der Polyketide in Bakterien beruht. Mit Hilfe eines „genome mining“-Ansatzes konnte im Genom des Cyanobakteriums Nostoc punctiforme eine Reihe von Biosynthese-Clustern, die zu den PKS und nichtribosomalen Peptidsynthetasen gehören, identifiziert werden. Durch chromatographische und massenspektrometrische Analysen von Zellextrakten und Kulturüberständen konnten einige der Biosynthese-Cluster bestimmten Metaboliten zugeordnet werden. Eines der Cluster wurde hinsichtlich des produzierten Metaboliten und der Regulationsstruktur eingehender charakterisiert. Die Folgerungen aus den gewonnen Ergebnissen werden im allgemeinen Zusammenhang der Evolution metabolischer Diversität ausführlich diskutiert. / Modular polyketide synthases (PKS) are multienzym systems of bacterial secondary metabolism. They perform a stepwise biosynthesis of diverse carbon skeletons from simple carboxylic acid units. Polyketide compounds possess a wide range of pharmaceutically interesting activities. In this study, a series of evolutionary analyses was performed. Initially, the phylogenetic relationships between modular PKS and other PKS systems as well as fatty acid synthases were investigated revealing their central position within a long evolutionary process. In detail reconstruction of the phylogenies of bacterial modular PKS domains demonstrated that the extent of gene duplications, gene losses and horizontal gene transfer events varies considerably between different bacterial groups. Using the genome sequence of the actinobacterium Streptomyces avermitilis the phylogenies of all domain types were reconstructed. Comparison of these phylogenies allowed for detecting numerous events of homologous recombination, which appears to be the main mechanism underlying polyketide structural diversity in bacteria. A genome mining approach revealed a number of biosynthesis clusters of the PKS and nonribosomal peptide synthetase type in the genome of the cyanobacterium Nostoc punctiforme. Cell extracts and culture supernatants were analysed by means of liquid chromatography and mass spectrometry and some of the biosynthesis clusters could be assigned to specific metabolites. One of the clusters was characterised in greater detail regarding the produced metabolite and the cluster’s regulatory structure. The implications of the results are extensively discussed within the general context of the evolution of metabolic diversity.

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.

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 penicillique. / Aspergillus 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.

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.