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Publié par | Thesee |
Nombre de lectures | 53 |
Langue | English |
Poids de l'ouvrage | 4 Mo |
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THÈSE
En vue de l'obtention du
DOCTORAT DE L’UNIVERSITÉ DE TOULOUSE DOCTORAT DE L’UNIVERSITÉ DE TOULOUSE
Délivré par Institut National Polytechnique De Toulouse
Discipline ou spécialité : Microbiologie & Biocatalyse Industrielles
Présentée et soutenue par BACHA NAFEES
Le 15/09/2009
Titre : CARACTERISATION DES POLYCETONES SYNTHASES INTERVENANT DANS
LA BIOSYNTHESE D’OCHRATOXINE A, D’ACIDE PENICILLIQUE, D’ASPERLACTONE ET
D’ISOASPERLACTONE CHEZ ASPERGILLUS WESTERDIJKIAE
JURY
MATHIEU Florence Professeur, Université de Toulouse Président
FORGET Florence Directeur de recherche,INRA, Bordeaux Rapporteur
RATOMAHENINA Robert Ingénieur de recherche,INRA, Montpellier Rapporteur
BARREAU Christian Chargé de Recherche CNRS, Bordeaux Examinateur
PUEL Olivier Ingénieur de recherche,INRA, Toulouse Exam
LEBRIHI Ahmed Professeur, Université de Toulouse Directeur de these
Ecole doctorale : École doctorale: Sciences Ecologiques, Vétérinaires, Agronomiques et
Bioingénieries
Unité de recherche : Laboratoire de Génie Chimique
Directeur de Thèse : M. LEBRIHI Ahmed (Pr. ENSAT-INPT)
Co-Directeur de Th Mme MATHIEU Florence (Pr. ENSAT-INPT)
Rapporteurs : Florence Richard-Forget et Robert Ratomahenina
Acknowledgements
First of all I am thankful to the alm ighty ALLAH, whose blessing and guidance let me
windup m y thesis with good health. Second, m y sincere appreciation goes to m y
supervisor Professor Ahmed LEBRIHI and co-supervisor Professor Florence MATHIEU,
whose scientific approach, careful reading and constructive comments were valuable.
Their timely and efficient contributions helped me shape my research work into its final
form and I express m y sincerest ap preciation for their assistance in an y way that I m ay
have asked.
I also wish to thank the “Higher Educa tion Comm ission (HEC)” of Pakistan, its
leadership and the staf f who in their limited resources supported me financially for my
studies in F rance. I m ust also acknowledge the way HEC rem ained in contac t with me
and addressed what so ever problem I have faced during my stay. The placement of new
doctorate degree holders was not an easy task. I appreciate the efforts of HEC to provide
me an opportunity to serve m y country. I mst also mu ention the services provided by
SFERE (Société française d'exportation des ressources éducatives) to facilitate m y living
in France.
I am also indebted to my colleag ues at “Laboratoire de Genie Chem ique (LGC)”,
particularly Patricia NOUVET, Thierry LIBOZ, Morie Cormen MONJE, Alaoui Lamrani
HAFSA, Rafik ERRAKHI and Samia AFOULOUSE who al ways welcomed, helped and
appreciated me. I am also thankful to Ali ATOUI, Anne-Claire Chorin Gwenaelle JARD,
Muhammad SHAHID a nd Muhammad ARSHAD for their guidance and support. I a m
grateful to all my Pakistani friends speci ally “Muhammad Ali Nizam ani” for reminding
me that I am not away from my fam ily. Sp ecial thanks, tribute and appreciation to all
those their names do not appear here who have contributed to the successful com pletion
of this study.
Finally, I’m forever indebted to m y family, who prioritized my studies over all f amily
related duties. My humble thanks to my mother, late father, wife Asma, brother Hafiz-ul-
Asad, Cousin Syed Tamiz-ud-din, sisters Afsheena, Neelam, Nighat and Farkhanda and
last but not the least my daughter (Aiza) eyes bab. Table of Contents
Introduction Générale ....................................................................................1
1- Literature Review ................................................................................4
1.1 Fungi and secondary metabolites ................................................................ 4
1.2 Aspergillus ochraceus / Aspergillus westerdijkiae NRRL 3174 ................ 11
1.2.1 Ochratoxin A................................................................................................. 13
1.2.2 Penicillic acid................................................................................................ 16
1.2.3 Methylsalicylic acid, asperlactone and isoasperlactone................................ 18
1.2.4 Mellein and Hydroxymellein........................................................................ 21
1.3 Fungal polyketides synthases..................................................................... 23
1.3.1 PKS producing reduced polyketides............................................................. 27
1.3.2 PKS producing partially reduced polyketides............................................... 27
1.3.3 PKS producing non-reduced polyketides...................................................... 28
1.4 Architectur base classification of PKSs .................................................... 29
1.5 Nonribosomal Peptide Synthetases ........................................................... 31
1.6 Hybrid between PKS and NRPS ............................................................... 36
1.7 Biosynthetic pathways of different fungal polyketide metabolites......... 37
1.7.1 Biosynthetic pathway of OTA ...................................................................... 37
1.7.2 Biosynthetic pathway of aflatoxin B1........................................................... 39
1.7.3 Biosynthetic pathway of Lovastatin.............................................................. 42
1.7.4 Biosynthetic pathway of Patulin ................................................................... 45
1.8 Organization of polyketide gene clusters.................................................. 47
2- Materials and Methods ......................................................................53
2.1 Materials ...................................................................................................... 53
2.1.1 Liste of kits utilized ...................................................................................... 53
2.1.2 List of products utilised ................................................................................ 54
i
Table of contents
2.1.3 Apparatus used.............................................................................................. 56
2.1.4 Culture medium............................................................................................ 57
a. Czapek Yeast Extract Agar (CYA)............................................................... 57
b. Czapek Dox Agar (CZ)................................................................................. 58
c. Synthetic Medium (SM)............................................................................... 58
d. Malt Extract Agar (MEA)............................................................................. 59
e. Yeast Extract Saccharose (YES)................................................................... 59
f. LB Broth....................................................................................................... 59
2.1.5 Oligonucleotide primers used....................................................................... 60
2.2 Methodology ................................................................................................ 61
2.2.1 Inoculums preparation and conservation ...................................................... 61
2.2.2 Extraction of ochratoxin A and other fungal secondary metabolites from
solid culture medium......................................................................................................... 62
2.2.3 Extraction of ochratoxin A and other fungal secondary metabolites from
liquid culture medium....................................................................................................... 62
2.2.4 Fluorescence and UV based HPLC Analyse ................................................ 62
2.2.5 Fungal nucleic acid extraction ...................................................................... 63
a. Preparation of fungal material ...................................................................... 63
b. Genomic DNA extraction............................................................................. 63
i. High molecular weight DNA extraction....................................................... 64
ii. CTAB method............................................................................................... 64
iii. Quick method of DNA extraction............................................................. 65
c. Total RNA extraction.................................................................................... 65
d. Quantification of DNA and RNA ...............................