Dokument

Zusammenfassung:
Die ungesättigte Dicarbonsäure Itaconsäure wird durch mikrobielle Fermentation erzeugt und dient als Ausgangsstoff für die Produktion von Kosmetika, Klebstoffen oder sogar Biokraftstoff. Der phytopathogene Basidiomycet Ustilago maydis produziert unter bestimmten Bedingungen eine Vielzahl an Sekundärmetaboliten, zu denen auch die Itaconsäure gehört. Der Biosyntheseweg der Itaconsäure war jedoch in diesem Pilz bisher noch nicht bekannt. In dieser Arbeit wurden die für die Itaconsäure-Biosynthese in U. maydis verantwortlichen Gene identifiziert und charakterisiert. Alle beteiligten Gene sind in einem Gencluster organisiert, der durch den Transkriptionsfaktor Ria1 spezifisch reguliert wird. Anhand von Deletionsanalysen der entsprechenden Gene konnte gezeigt werden, dass in U. maydis zwei Enzyme essentiell für die Synthese von Itaconat sind. Im Verlauf dieser Arbeit wurde die enzymatische Aktivität dieser Enzyme bestimmt. Dabei wurde gezeigt, dass das PrpF-ähnliche Protein Aconitat-Δ-Isomerase (Adi1) die Umwandlung von cis- zu trans-Aconitat und damit den ersten Schritt der Itaconsäure-Biosynthese in U. maydis katalysiert. Der zweite Schritt wird durch das zweite essentielle Enzym trans-Aconitat-Decarboxylase (Tad1) katalysiert, das die Decarboxylierung von trans-Aconitat zu Itaconat vermittelt. Basierend auf diesen Daten konnte ein Modell für den Itaconsäure-Biosyntheseweg in U. maydis aufgestellt werden. Es wird angenommen, dass cis-Aconitat durch den mitochondriellen Transporter Ctp1 vom Mitochondrium ins Cytosol exportiert wird. Dort dient es als Substrat für Adi1, das die Isomerisierung zu trans-Aconitat katalysiert. Trans-Aconitat wird anschließend durch Tad1 zu Itaconat decarboxyliert, das vermutlich durch den Plasmamembran-Transporter Itp1 aus der Zelle transportiert wird. Der Itaconsäure-Gencluster ist während der pathogenen Entwicklung von U. maydis stark exprimiert. In dieser Arbeit konnte jedoch gezeigt werden, dass die Produktion von Itaconsäure während der biotrophen Phase nicht essentiell ist. Damit bleibt die biologische Rolle der Itaconsäure-Produktion für U. maydis weiterhin unklar. Im Verlauf dieser Arbeit wurde noch ein zweiter Gencluster in U. maydis identifiziert, der Ähnlichkeiten zum Itaconat-Gencluster aufweist. Dieser Gencluster enthält ein Gen für eine Aconitat Δ-Isomerase (adi2 ), die in der Lage ist, die Funktion von Adi1 vollständig zu ersetzen. Durch Wachstumstests konnte gezeigt werden, dass der Adi2-Gencluster für die Verstoffwechselung von cis- und trans-Aconitat essentiell ist, das sich auch in der Wirtspflanze Zea mays findet. Die Fähigkeit, trans-Aconitat als Kohlenstoffquelle zu verwenden, ist für U. maydis während der pathogenen Entwicklung möglicherweise von Vorteil, es konnte jedoch gezeigt werden, dass diese Fähigkeit nicht essentiell für die pathogene Entwicklung von U. maydis ist.

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