Wax ester fermentation and fatty acid biosynthesis in the facultatively anaerobic flagellate Euglena gracilis [Elektronische Ressource] / vorgelegt von Sara Tucci

Tucki

Sujets

• Biologie

Informations

• Publié par: heinrich-heine-universitat_dusseldorf
• Publié le: 01 janvier 2008
• Nombre de lectures: 15
• Langue: English
• Poids de l'ouvrage: 2 Mo

Extrait

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





Inaugural-Dissertation

zur

Erlangung des Doktorgrades der
Mathematisch-Naturwissenschaftlichen Fakultät
der Heinrich-Heine-Universität Düsseldorf





vorgelegt von
Sara Tucci
aus Neapel, Italien



Düsseldorf
2008
Aus dem Institut für Ökologische Pflanzenphysiologie
der Heinrich-Heine Universität Düsseldorf






















Gedruckt mit der Genehmigung der Mathematisch- Naturwissenschaftlichen Fakultät der
Heinrich-Heine Universität Düsseldorf



Referent: Prof. Dr. Peter Proksch
Korreferent: Prof. Dr. William Martin



Tag der mündlichen Prüfung: 9.12.2008
Teilergebnisse dieser Arbeit wurden mit Genehmigung des Betreuers in folgenden
Beiträgen vorab veröffentlicht:



Publikationen:

Tucci S, Proksch P and Martin W: Fatty acid biosynthesis in mitochondria
of Euglena gracilis. In: Advances in Plant Lipid Research: Proceedings of the 17th
International Symposium on Plant Lipids,East Lansing, Michigan, July 2006 (C.
Benning and J. Ohlrogge, eds.), Michigan State University Press. 133-136

Tucci S and Martin W. (2007) A novel prokaryotic trans-2-enoyl-CoA reductase from the
spirochete Treponema denticola. FEBS Letters. 581: 1561-1566



Tagungsbeiträge:

Tucci S, Proksch P and Martin W. Wax ester fermentation of the facultatively anaerobic
flagellate Euglena gracilis. SFB TR1, Munich, March 2006, Germany.

Tucci S, Proksch P and Martin W. Wax ester fermentation of the facultatively anaerobic
thflagellate Euglena gracilis. 17 International Symposium on Plants Lipids. East
Lansing, MI, July 2006, USA.

Tucci S, Proksch P and Martin W. Effect of Flufenacet on the wax ester fraction of the
thfalultatively anaerobic flagellate Euglena gracilis. 11 IUPAC International
Congress of Pesticide Chemistry, Kobe, August 2006, Japan.

Tucci S and Martin W. A novel prokaryotic trans-2-enoyl-CoA reductase from the
thspirochete Treponema denticola. 48 International Congress on the Bioscience of
Lipids. September 2007, Turku, Finnland

Tucci S, Proksch P and Martin W. Anaerobic metabolism and wax ester fermentation of
different Euglena gracilis species. Endosymbiosis: From Prokaryotic to Eukaryotic
Organelles, Munich, October 2008, Germany.
ABBREVIATIONS
Abbreviations
General abbreviations, chemicals and enzymes

A absorbance
Aa amino acid
ACCase acetyl-CoA carboxylase
ACP Acyl-Carrier-Protein
Amp ampicillin
APS ammoniumperoxidisulfate
ATP adenosine triphosphate
BLAST basic Local Alignment Search Tool
BSA bovine-serume albumine
CCR crotonoyl-CoA reductase
CHAPS 3-[(3-cholamidopropyl)- dimethylammonio]-1-propane-sulfonate
CHCl chlorophorm 3
CoA coenzym A
DE diethylether
DMSO dimethyl sulfoxide
DNase desoxyribonuclease
DTT dithiotreitol
EDTA ethylenediamintetraacetate
ECL enhanced chemoluminescence
ER Endoplasmic Reticulum
FAD Flavin Adenine Dinucleotide reduced
FADH ide oxidized
FAlcs Fatty Alcohols
FAMEs Fatty Acid Methyl Esters
FAS Fatty Acid Synthase
FPLC Fast-Protein-Liquid-Chromatography
Fig Figure
Ga billion of years
GC gas chromatography
HEPES 2-[4-(2-hydroxyethyl)1-1-piperazinyl)-ethansulfonic acid
HPLC High Performance Liquid Chromatography
IPTG isopropyl-D-thiogalactoside
KAS ketoacyl-ACP synthase
Km kanamycin ABBREVIATIONS
LB Luria-Bertani medium
Luminol 3-aminophtalhydrazide
Me CO acetone 2
MeOH methanol
MS mass spectroscopy
NAD nicotinamide adenine dinucleotide reduced
NADH nicotinamucleotide oxidized
NADP nicotinamide adenine dinucleotide phosphate reduced
NADPH nicotinamucleote oxidized
NCBI National Center for Biotechnology Information
NiNTA Nickel Nitriloacetic Acid
OD Optical Density
ORF Open Reading Frame
PAGE Polyacrylamide Gel Electrophoresis
PCR Polymerase Chain Reaction
PMSF Polymethyl Sulfonic Acid
PE petrol ether
PKS polyketide synthase
Pre- precursor
RNAse ribonuclease
RT Room Temperature
S substrate
SDS Sodium Dodecylsulfate
SiO Silica gel 2
Tab. table
TCA Trichloroacetic Acid
TE Tris-EDTA
TEMED N, N, N’,N’-Tetramethylethylendiamin
TER trans-2-enoyl-CoA reductase
TLC Thin Layer Chromatography
Tris 2-amino-2(hydroxymethyl) 1,3-propandione
Triton X-100 octylphenoxy poly-(8-10)-ethyleneglycol
Tween 20 polyoxyethylene sorbitan monolaureate
X-Gal 5-Bromo-4-Chlor-3-Indol-β-D-galactopyranoside


ABBREVIATIONS
Amino acids

A, Ala alanine
C, Cys cysteine
D, Asp aspartic acid
E, Glu glutamic acid
F, Phe phenylalanine
G, Gly glycine
H, His histidine
I, Ile isoleucine
K, Lys lysine
L, Leu leucine
M, Met methionine
N, Asn asparagine
P, Pro proline
Q, Gln glutamine
R, Arg arginine
S, Ser serine
T, Thr threonine
V, Val valine
W, Trp triptophane
Y, Tyr tyrosine

Nucleic acids

DNA deoxyribonucleic acid
RNA ribonucleic acid
dNTP desoxynucleoside-5’-triphosphate


Units

bp base pair
°C degree Celsius
g gram
g gravity
h hour ABBREVIATIONS
kb kilo base pair
kDa kilo Dalton
l litre
m meter
M molar
mg milligram
mm minute
μg microgram
μl microlitre
mM millimolar
μM micromolar
nm nanometer
rpm revolution per minute
s second
-1 -1U Unit: μmol mg min
v/v volume per volume
w/v weight per volume CONTENTS

1. Introduction ........................................................................................................... 1
1.1 Mitochondria: function and origin....................................................................... 1
1.1.1 Type I and type II eukaryotes........................................................................ 2
1.1.2 Anaerobic mitochondria................................................................................ 2
1.1.3 The mitochondrion of Euglena gracilis .......................................................... 4
1.2 The importance of fatty acids............................................................................. 6
1.3 Fatty acid biosynthesis in plants 7
1.4 Fatty acid biosynthesis in E. gracilis .................................................................11
1.5 Aims of this work ..............................................................................................12
2. Materials and Methods .......................................................................................14
2.1 Chemicals........................................................................................................14
2.2 Kits...................................................................................................................14
2.3 Chromatographic material ................................................................................14
2.4 Enzymes ..........................................................................................................15
2.5 Strains..............................................................................................................15
2.5.1 Euglena gracilis strains...............................................................................15
2.5.2 Treponema denticola...................................................................................16
2.5.3 Streptomyces avermitilis..............................................................................16
2.5.4 Escherichia coli strains ................................................................................16
2.6 Plasmids...........................................................................................................17
2.7 Oligonucleotides...............................................................................................17
2.8 Working with E. gracilis ....................................................................................19
2.9 E. gracilis bleaching..........................................................................................20
2.10 Working with nucleic acids................................................................................20
2.10.1 Isolation of total RNA from E. gracilis.......................................................20
2.10.2 cDNA synthesis .......................................................................................21
2.10.3 Plasmid DNA isolation ................................