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Analysis of the Melanin Biosynthesis In Regulation and evolution of the penicillin biosynthesis gene cluster of Aspergillus nidulans Dissertation zur Erlangung des akademischen Grades doctor rerum naturalium (Dr. rer. nat.) vorgelegt dem Rat der Biologisch-Pharmazeutischen Fakultät der Friedrich-Schiller-Universität Jena von Petra Spröte geboren am 07. April 1980 in Querfurt 2008 1. Gutachter: Prof. Dr. Axel A. Brakhage (FSU Jena) 2. Gutachter: Prof. Dr. Christian Hertweck (FSU Jena) 3. Gutachter: Prof. Dr. Ulrich Kück (Ruhr-Universität Bochum) Datum der öffentlichen Verteidigung: 08. Dezember 2008 'I will take the Ring,' he said, 'though I do not know the way.' J.R.R. Tolkien – The Lord of the Rings Summary i SUMMARY Penicillin is one of the most important antibiotics and consequently, its biosynthesis is probably the best understood pathway in fungal secondary metabolism. Several studies have indicated that the penicillin biosynthesis gene cluster of the filamentous fungus Aspergillus nidulans, comprising the genes acvA, ipnA and aatA, is regulated by a complex network. With AnCF (A. nidulans CCAAT-binding factor) and AnBH1 (A. nidulans basic region helix-loop- helix protein 1) main trans-acting factors were identified. However, there is little knowledge about the environmental signals and their transmission modulating the expression of the structural genes. Moreover, evolution of the gene cluster that contains genes of apparently both bacterial (acvA and ipnA) and eukaryotic (aatA) origin has not been elucidated. In this work, the light-dependent regulator velvet A (VeA) and a central protein kinase C (PkcA) were found to be part of the penicillin biosynthesis regulating network. Expression of an inducible alcAp-veA gene fusion indicated that under alcAp-inducing conditions both the penicillin titre and acvA gene expression were drastically reduced. The analysis of strains with different genetic backgrounds and with a different promoter system (niiAp) confirmed VeA as repressor of penicillin biosynthesis in A. nidulans, most likely via repression of acvA gene expression. A putative repressor binding site within the acvA promoter was shown not to be involved in VeA-mediated regulation. In conclusion, the obtained findings were assessed in the light of VeA being part of the recently identified velvet complex connecting light, development and secondary metabolism. AnBH1, a repressor of aatA gene expression, was shown by in silico analysis to contain a putative protein kinase C (PKC) phosphorylation site. Inhibition of PKC activity by application of calphostin C and knocking down of pkcA expression by a pkcA antisense construct led to cytoplasmic localisation of the usually in the nucleus located AnBH1-eGFP fusion protein, suggesting that PkcA acted upstream of this transcription factor. This finding linked regulation of penicillin biosynthesis to that of cell wall integrity since PkcA appeared to be involved in both signal transduction pathways. However, both nuclear localisation of AnBH1-eGFP and in vitro binding of AnBH1 to the aatA promoter was independent of the phosphorylated putative PKC site, indicating a rather indirect influence of PkcA. Analysis of the aatA encoded acyl-CoA : isopenicillin N acyltransferase (IAT) by different eGFP fusion proteins and A. nidulans mutant strains impaired in peroxisomal biogenesis or function revealed that the peroxisomal localisation of the IAT was dependent on both its atypical peroxisomal targeting signal 1 (PTS1) and the respective PTS1 receptor protein PexE. In contrast to Penicillium chrysogenum, in A. nidulans strains exhibiting a Summary ii mislocalised IAT, penicillin production was reduced but not eliminated. The same was true for strains without functional peroxisomes or with impaired PTS1 import, indicating a beneficial but not essential role of peroxisomes for penicillin production in this fungus. In contrast to acvA and ipnA that apparently are of bacterial origin and were most likely obtained by interkingdom horizontal gene transfer, there is no bacterial aatA homologue. Thus, the evolutionary origin of aatA showing all features of a eukaryotic gene remained obscure. This work showed that disruption of aatA still enabled penicillin production in A. nidulans. Genome mining led to the discovery of the aatB gene which had a similar structure and expression pattern as aatA. Phylogenetic analysis revealed putative aatB homologues widely distributed also within non-producing fungi, whereas aatA homologues were exclusively found as part of the penicillin biosynthesis gene cluster. Disruption of aatB in A. nidulans resulted in a reduced penicillin titre. In contrast to aatA, aatB did not encode a PTS1 sequence, resulting in cytoplasmic localisation of the encoded AatB protein. Overexpression of a mutated aatBPTS1 gene in an aatA-disruption strain, which led to peroxisomal localisation of AatB, increased the penicillin titre more than overexpression of the wild-type aatB. Electrophoretic mobility shift assays, surface plasmon resonance analyses and Northern blot analyses indicated that the promoters of both aatA and aatB were bound and regulated by the same transcription factors AnCF and AnBH1. Taken all these findings together, it was suggested that aatB was a paralogue of aatA. This thesis therefore extended the model of evolution of the penicillin biosynthesis gene cluster by recruitment of a biosynthesis gene and its cis-regulatory sites upon gene duplication. Kurzzusammenfassung iii KURZZUSAMMENFASSUNG Penicillin gehört zu den wichtigsten Antibiotika und dementsprechend ist die Biosynthese dieses pilzlichen Sekundärmetaboliten besonders gut untersucht. Verschiedene Studien haben gezeigt, dass das Penicillinbiosynthese-Gencluster des filamentösen Pilzes Aspergillus nidulans, bestehend aus den Genen acvA, ipnA und aatA, von einem komplexen regulatorischen Netzwerk kontrolliert wird. Dabei spielen globale Transkriptionsfaktoren wie AnCF (A. nidulans CCAAT-bindender Faktor) und AnBH1 (A. nidulans basische Region Helix-Loop-Helix-Protein 1) eine wichtige Rolle. Allerdings wurden die Umweltsignale, welche die Expression der Strukturgene mittels bislang ebenfalls unbekannter Signaltransduktionskaskaden beeinflussen, bisher kaum analysiert. Zudem existieren nur wenige Untersuchungen zur Evolution des Genclusters, welches sowohl Gene vermutlich bakteriellen (acvA und ipnA) als auch eukaryontischen (aatA) Ursprungs enthält. In dieser Arbeit wurden der lichtabhängige Regulator Velvet A (VeA) und eine zentrale Proteinkinase C (PkcA) als Teil des regulatorischen Netzwerks identifiziert. Die Expression einer regulierbaren alcAp-veA-Genfusion zeigte, dass unter alcAp-induzierenden Bedingungen sowohl der Penicillintiter als auch die acvA-Genexpression stark reduziert war. Die Analyse von Stämmen mit unterschiedlichem genetischen Hintergrund bzw. mit einem anderen regulierbaren Promotor (niiAp) bestätigte den reprimierenden Effekt von VeA auf die Penicillinbiosynthese von A. nidulans, welcher vermutlich durch Repression der acvA- Genexpression verursacht wurde. Die gewonnenen Erkenntnisse wurden auch vor dem Hintergrund diskutiert, dass VeA kürzlich als Teil des velvet-Komplexes identifiziert wurde, welcher lichtabhängig sowohl Entwicklung als auch Sekundärmetabolismus koordiniert. In silico-Analysen zeigten, dass AnBH1, ein Repressor der aatA-Genexpression, eine vermeintliche Phosphorylierungsstelle für eine Proteinkinase C (PKC) enthielt. Sowohl die Inhibition der PKC-Aktivität durch Zugabe von Calphostin C als auch die Expression eines pkcA-antisense-Konstrukts beeinflusste die nukleäre Lokalisation einer AnBH1-eGFP-Fusion. Dies ließ auf eine Position von PkcA upstream von AnBH1 in der Signalkette schließen. Somit ergab sich eine Verbindung der Regulation der Penicillinbiosynthese mit jener der Zellwandintegrität, da PkcA vermutlich Teil beider Signaltransduktionskaskaden ist. Sowohl die nukleäre Lokalisation von AnBH1-eGFP als auch die in vitro-Bindung von AnBH1 an den aatA-Promotor waren jedoch unabhängig von einer Phosphorylierung der putativen PKC- Erkennungsstelle, was auf einen indirekten Einfluss der PkcA schließen ließ. Die Analyse der aatA-kodierten Acyl-CoA : Isopenicillin N-Acyltransferase (IAT) mittels verschiedener eGFP-Proteinfusionen und A. nidulans-Stämmen mit eingeschränkter Kurzzusammenfassung iv Peroxisomenbildung bzw. -funktion zeigte, dass die peroxisomale Lokalisation der IAT sowohl von deren atypischer peroxisomaler Signalsequenz 1 (PTS1) als auch vom entsprechenden PTS1-Rezeptor PexE abhängig war. Anders als Penicillium chrysogenum produzierte ein A. nidulans-Stamm mit einer fehllokalisierten IAT immer noch Penicillin. Dies wurde ebenfalls für Stämme ohne funktionelle Peroxisomen bzw. mit eingeschränktem PTS1-Import beobachtet und ließ auf eine vorteilhafte, aber nicht essentielle Rolle der Peroxisomen für die Penicillinbiosynthese in A. nidulans schließen. Im Gegensatz zu acvA und ipnA, welche wahrscheinlich durch horizontalen Gentransfer aus Prokaryonten auf die Pilze übertragen wurden, gab es kein bakterielles aatA-Homolog. Der evolutionäre Ursprung dieses eindeutig eukaryontischen Gens war somit ungeklärt. Diese Arbeit zeigte, dass die Disruption des aatA-Gens in A. nidulans die Produktion geringer Mengen an Penicillin immer noch ermöglichte. Die anschließende Analyse des Genoms führte zur Entdeckung des strukturell ähnlichen aatB-Gens. Phylogenetische Untersuchungen ergaben, dass putative Homologe des aatB-Gens auch in nicht-produzierenden Pilzen vorkamen, wohingegen aatA-Homologe stets Teil
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