Publikationsserver der Universitätsbibliothek Marburg

Titel:Molekularbiologische und biochemische Untersuchungenvon Prenyltransferasen und NRPS-Enzymen aus Ascomyceten
Autor:Wunsch, Carsten
Weitere Beteiligte: Li, Shu-Ming (Prof. Dr.)
Veröffentlicht:2014
URI:https://archiv.ub.uni-marburg.de/diss/z2015/0036
DOI: https://doi.org/10.17192/z2015.0036
URN: urn:nbn:de:hebis:04-z2015-00365
DDC: Medizin
Titel (trans.):Molecular biological and biochemical investigations of prenyltransferases and NRPS-enzymes of Ascomycetes
Publikationsdatum:2015-07-28
Lizenz:https://rightsstatements.org/vocab/InC-NC/1.0/

Dokument

Schlagwörter:
Prenyltransferasen, Aspergillus, NRPS, cyclic dipeptides, NRPS, Aspergillus, zyklische Dipeptide, Nichtribosomale Peptidsynthetasen, Ascomyceten

Zusammenfassung:
Die Produktion sekundärer Stoffwechselprodukte durch Mikroorganismen stellt ein beachtliches Reservoir an biologisch-aktiven Naturstoffen da, die auch pharmazeutische Bedeutung haben können. Darunter fallen Antibiotika, Zytostatika oder auch Immunsuppressiva. Diese sind im Gegensatz zu Metaboliten des Primärstoffwechsels zunächst für Wachstum und Entwicklung entbehrlich, jedoch auf den zweiten Blick essentiell für die Bewältigung zahlreicher Wechselwirkungen des Individuums mit seiner Umwelt. Auch die filamentösen Pilze tragen ihren Teil zur Diversität der Naturstoffe bei. Die Grundstrukturen solcher Substanzen beruhen häufig auf der Mitwirkung von Nichtribosomalen Peptidsynthetasen (NRPS), NRPS-ähnlichen Enzymen, Polyketidsynthasen (PKS) und Hybridenzymen aus NRPS/PKS. Durch den Einfluss weiterer Modifikationsenzyme (tailoring enzymes), wie Prenyltransferasen, wird die biologische Vielfalt und Aktivität deutlich gesteigert. Zur gezielten chemoenzymatischen Synthese neuer Naturstoffe sind daher fundierte Kenntnisse über die Reaktionsmechanismen, sowie der biochemischen Eigenschaften dieser Enzymklassen von zentraler Bedeutung. Mit dieser Arbeit sollten fünf putative Prenyltransferasen aus A. terreus charakterisiert werden. Für zwei Vertreter CdpC7PT und EAU34068 konnte dieses Vorhaben bisher erfolgreich absolviert werden. CdpC7PT ist hierbei zum einen für die C7-Prenylierung von tryptophanhaltigen zyklischen Dipeptiden verantwortlich und stellt zum anderen die erste beschriebene Prenyltransferase der DMATS-Superfamilie dar, welche eine O-Prenylierung in Anwesenheit von tyrosinhaltigen zyklischen Dipeptide vollführt. Bei der Suche nach geeigneten Substraten für EAU34068 konnten trotz der Sequenzidentitäten von bis zu 42 % auf Aminosäureebene zu zyklischen Dipeptid-akzeptierenden Prenyltransferasen, diese Subtratgruppe leider nicht bestätigt werden. Erst eine ausgedehnte Suche zeigte die Flavanone Eriodictyol und Naringenin, die Hydroxynaphthaline 1-Naphtol und 1,7-Dihydroxynaphthalin, sowie das Xanthon 1,3,6-Trihydroxyxanthon als Substrate für eine Geranylierung auf. Dabei wurden regioselektive Prenylierungen bei allen diesen Substanzen beobachtet. Zwei weitere putative Prenyltransferasegene ATEG_02823 und ATEG_01730 befinden sich derzeit im Klonierungsstadium und eine weitere bedarf weiterer Expressionsoptimierungen ATEG_06111 zum Erhalt des gewünschten rekombinanten Proteins (EAU33872). Ein weiterer Schwerpunkt dieser Arbeit war die Charakterisierung der NRPS EAA61517 und EAW25548, sowie des NRPS-ähnlichen Enzyms EAU36364. Die zugehörigen Gene AN9226, NFIA_043670 und ATEG_03090 stammen entsprechend der Reihenfolge aus A. nidulans, N. fischeri und A. terreus und sollten in ein geeignetes Konstrukt zur Expression in A. nidulans TN02A7 überführt werden. Für AN9226, sowie NFIA_043670 konnten positive Transformanten erhalten werden. Durch den Vergleich mit dem untransformierten Stamm A. nidulans TN02A7 wurde anschließend die Sekundärmetabolitproduktion analysiert. Insbesondere für AN9226 wurden zahlreiche Strategien zur Optimierung der Expression und damit verbundener Produktzunahme verfolgt, wie beispielsweise die Mitwirkung des konstitutiven gpdA Promotors. Zum jetzigen Zeitpunkt konnte jedoch kein Produkt in direkten Zusammenhang mit dem jeweiligen inserierten Gen gebracht werden, aber in jedem Fall wurde die unerwartete Akkumulation des Polyketides Sterigmatocystin beobachtet. Durch die Kombination der NRPS FtmPS aus N. fischeri, dessen Ortholog aus A. fumigatus cyclo-L-Trp-L-Pro synthetisiert, mit verschiedenen Prenyltransferasen der DMATS-Superfamilie in A. nidulans sollten zum einen die in vitro erhaltenen Ergebnisse bestätigt werden und zum anderen auf diesem Wege ein System zur gezielten in vivo Synthese gewünschter prenylierter Derivate etabliert werden. Das NRPS-Gen NFIA_093690 wurde zu diesem Zweck unter die Kontrolle des konstitutiven gpdA Promotors, sowie des trpC Terminators gestellt und in den zur Transformation geeigneten Vektor pJW24 integriert. Nach erfolgreicher Transformation von A. nidulans TN02A7 konnte durch Analyse der Sekundärmetabolitproduktion das erstrebte cyclo-L-Trp-L-Pro bestätigt werden. Anschließend erfolgte die Koexpression der beiden reversen C2-Prenyltransferasen CdpC2PT und BrePT aus N. fischeri und A. versicolor, sowie der reversen C3-Prenyltransferasen CdpC3PT und CdpNPT aus N. fischeri und A. fumigatus. Die kodierenden Gene wurden ebenfalls mit den bereits erwähnten regulatorischen Elementen und dem Selektionsmarker pyroA in einem Vektor zusammengefügt und in den ftmPS-Transformanten A. nidulans CaW03 (ftmPS) eingebracht. Durch weiterführende Arbeiten von Frau Dr. Kathrin Mundt konnten vergleichend zu A. nidulans CaW03 (ftmPS) zusätzliche Produkte im Sekundärmetabolitspektrum identifiziert werden. Die Strukturaufklärung erbrachte im Fall von CdpC2PT und BrePT die Bestätigung einer reversen C2-Prenylierung von cyclo-L-Trp-L-Pro, für CdpNPT ein syn-cis konfiguriertes reverses C3-prenyliertes Derivat und bei CdpC3PT sowohl reverse C3- und C2-prenylierte Derivate, als auch eine reguläre Prenylierung an Position N1.

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