Dokument: Wax ester fermentation and fatty acid biosynthesis in the facultatively anaerobic flagellate Euglena gracilis

Titel:	Wax ester fermentation and fatty acid biosynthesis in the facultatively anaerobic flagellate Euglena gracilis
URL für Lesezeichen:	https://docserv.uni-duesseldorf.de/servlets/DocumentServlet?id=10166
URN (NBN):	urn:nbn:de:hbz:061-20090120-103307-8
Kollektion:	Dissertationen
Sprache:	Englisch
Dokumententyp:	Wissenschaftliche Abschlussarbeiten » Dissertation
Medientyp:	Text
Autor:	Tucci, Sara [Autor]
Dateien:	[Dateien anzeigen] Adobe PDF [Details] 2,09 MB in einer Datei [ZIP-Datei erzeugen] Dateien vom 19.01.2009 / geändert 19.01.2009
Dewey Dezimal-Klassifikation:	500 Naturwissenschaften und Mathematik » 570 Biowissenschaften; Biologie
Beschreibung:	The mitochondrion of the flagellate Euglena gracilis is a facultatively anaerobic organelle that is able to produce ATP in the presence and in the absence of oxygen. The shift of Euglena cells from aerobic to anaerobic conditions induces a mitochondrial wax ester fermentation coupled to a net synthesis of ATP. The mitochondrial fatty acid synthesis system uses acetyl-CoA and propionyl-CoA as primer and as C2- and C3-donor, respectively. A portion of fatty acids is reduced to alcohols, esterified with another fatty acid and deposited in the cytosol as waxes. E. gracilis strain Z cultures were grown under different conditions. The wax esters were isolated, transesterified and analysed by gaschromatography (GC) as fatty acid methyl esters and fatty alcohols. In this work it has been demonstrated that oxygen play a key role in the accumulation of wax esters which are mostly synthesized under anaerobic conditions. The synthesis of odd-number chain fatty acids and alcohols during anoxia and their lack by addition of the herbicide flufenacet, which inhibits the elongation but not the synthesis of fatty acids, confirm the involvement of the methylmalonyl-CoA pathway. Bleached mutants were generated by treatment with ofloxacin and streptomycin and their wax ester fraction was isolated and analyzed. In contrast to the data reported in the literature to date, the bleached mutants did not survive anaerobiosis. Growth curves of eleven different E. gracilis strains demonstrated that in absence of oxygen not all Euglena strain were able to survive anoxia and to perform wax ester fermentation. Their wax esters were isolated and analyzed. Northern blot analysis with RNA samples of the different Euglena strains showed that pyruvate:NAPD+ oxidoreductase homologues were present in all analyzed strains, while the trans-2-enoyl-CoA reductase was apparently not expressed in all cultures. The amino acid sequence of a putative mitochondrial thiolase/condensing enzyme of E. gracilis strain Z was identified and the protein was heterologously expressed in Saccharomyces cerevisiae and Escherichia coli. However, the recombinant protein was not active and it could not be characterized. The trans-2-enoyl-CoA reductase of E. gracilis strain Z which catalyzes the reduction of crotonoyl-CoA and hexenoyl-CoA during the mitochondrial fatty acid synthesis shares sequence similarity only with prokaryotic homologues. Two of these sequences, TDE0597 from Treponema denticola and SAV2368 from Streptomyces avermitilis, were chosen for further characterization. SAV2368 was heterologously expressed in E. coli and S. cerevisiae. However, the recombinant protein was not active and it could not be characterized. The TDE0597 was successfully expressed in E. coli. The protein was purified by affinity chromatography and biochemically characterized.
Lizenz:	Urheberrechtsschutz
Fachbereich / Einrichtung:	Mathematisch- Naturwissenschaftliche Fakultät » WE Biologie » Ökologische Pflanzenphysiologie
Dokument erstellt am:	20.01.2009
Dateien geändert am:	19.01.2009
Promotionsantrag am:	31.10.2008
Datum der Promotion:	09.12.2008