Analysis of the genetic potential of the spongederived fungus Penicillium chrysogenum E01-10/3 for polyketide production [Elektronische Ressource] / Marija Avramović

Marija Avramović

Sujets

• Biologie

Informations

• Publié par: rheinische_friedrich-wilhelms-universitat_bonn
• Publié le: 01 janvier 2011
• Nombre de lectures: 23
• Langue: English
• Poids de l'ouvrage: 3 Mo

Extrait

Analysis of the genetic potential of the sponge-
derived fungus Penicillium chrysogenum E01-
10/3 for polyketide production






Dissertation
zur
Erlangung des Doktorgrades (Dr. rer. nat.)
der
Mathematisch-Naturwissenschaftlichen Fakultät
der Rheinischen Friedrich-Wilhelms-Universität Bonn


vorgelegt von
Marija Avramovi ć
aus
Belgrad



Bonn 2010

Angefertigt mit Genehmigung der Mathematisch-Naturwissenschaftlichen
Fakultät der Rheinischen Friedrich-Wilhelms-Universität Bonn


1. Gutachter: Prof. Dr. Jörn Piel
2. Gutachterin: Priv.-Doz. Dr. Gerhild van Echten-Deckert


Tag der Mündlichen Prüfung: 16.02.2011

Erscheinungsjahr: 2011




To the unconditional love of my parents





“In every job that must be done
There is an element of fun
You find the fun and snap!
The job’s a game
And every task you undertake
Becomes a piece of cake
A lark! A spree! It’s very clear to see that…
A Spoonful of sugar helps the medicine go down
The medicine go down
Just a spoonful of sugar helps the medicine go down
In a most delightful way…
The honey bees that fetch the nectar
From the flowers to the comb
Never tire of ever buzzing to and fro
Because they take a little nip
From every flower that they sip
And hence,
They find
Their task is not a grind …”

“A spoon full of sugar”, Disney (Mary Poppins)

Acknowledgements

I want to thank my supervisor, Prof. Dr. Jörn Piel for giving me the opportunity to work in his
lab and for introducing me into the puzzling world of polyketides. I am grateful for all
suggestions that I got from him during the practical work in the lab, as well as for the writing
of my thesis. Moreover, I want to thank him for his trust and unique experience in building up
a laboratory together with other “pioneers” of his group. This helped me to face easier other
challenges in the life.

Special thanks go to Priv. Doz. Dr. Gerhild van Echten-Deckert for very efficient and
dedicated review of my thesis as the second reviewer. Moreover, I appreciate a lot her kind
encouragement for the finalization of my dissertation.
I want to thank all members of the examination committee for their participation and taking
their time for reading of my thesis.

Furthermore, I would like to thank the collaborating groups of Prof. Dr. Bringmann
(University of Würzburg) and Prof. Dr. Imhoff (University of Kiel) for providing us with the
laboratory samples and valuable information. Special thank in this aspect goes to Dr. Tobias
Gulder and Dr. Rüdiger Stöhr.

I would like to thank all members of the lab for creating a pleasant and exciting environment
for scientific research.

Especially, I want to say thanks to my colleagues Dr. Cristian Gurgui, Zeynep Yunt, Dr.
Katrin Zimmerman, Dr. Jing He, Sinisa Hrvatin and Dr. Daniel Butzke for introducing me
into the diverse methods in molecular biology and biochemistry and for their helpful
discussions. This brought me always a step forward in my research.

Special thanks go to Nina Heycke for the help on preparation and screening of the genomic
library. Zeynep Yunt I thank for helping me to deal with the HPLC experiments. Dr. Katja
Fisch I thank for her assistance in designing degenerate primers for screening of the genomic
library.

Moreover, without fast and selfless assistance of Dr. Katrin Zimmerman and Dr. Kathrin
Reinhardt in administrative questions at the institute, I would not be able to proceed with my
work. I also thank them for encouraging me to speak the German language.

Thanks to all of my friends in Germany and Serbia who gave me support during all these
years. Especially to Zeynep Yunt and Cristian Gurgui who encouraged me in critical phases
of my research and life, and were always there when I had homesickness.

Members of my YES Medical Device Services GmbH team I want to thank for their
encouragement and support.

I want to express my deepest thanks to my parents for their absolute support and their belief
in me. I want to thank them for giving me strength in crucial phases of my life, also in critical
times when I was supposed to help and encourage them. I am grateful for their love.

The last but not least, I want to thank my husband Dr. Oliver Wingerter without whose
inexhaustible support and belief in me I would never be writing these acknowledgements. I
want to thank him not only for proof-reading of my dissertation and for giving his critical
opinion and suggestions, but also for all his motivation, love and care in the last few years,
including his patience.

SUMMARY ....................................................................................................................................................... - 1 -
INTRODUCTION ............................................................................................................................................. - 4 -
1 FUNGI AS PRODUCERS OF BIOLOGICALLY ACTIVE SECONDARY METABOLITES ....................................... - 4 -
1.1 Fungal mycotoxins ...................................................................................................................... - 5 -
1.2 Fungal secondary metabolites as approved pharmaceuticals ..................................................... - 8 -
1.3 Marine-derived fungal polyketide metabolites .......................................................................... - 13 -
1.4 The diversity of polyketides produced by fungus P. chrysogenum ............................................ - 20 -
2 POLYKETIDE BIOSYNTHESIS................................................................................................................. - 27 -
2.1 The molecular background to understand polyketide biosynthesis ........................................... - 28 -
2.2 Types of polyketide synthases .................................................................................................... - 29 -
2.3 Fungal polyketide synthases ...................................................................................................... - 31 -
2.4 Current challenges in the fungal polyketide research are a ....................................................... - 36 -
2.5 The perspectives in fungal poylketide research area ................................................................ - 44 -
3 BIOSYNTHESIS OF SORBICILLACTONE A .............................................................................................. - 45 -
4 RESEARCH GOALS ................................................................................................................................ - 48 -
RESULTS AND DISCUSSION ..................................................................................................................... - 50 -
5 DETECTION OF SORBICILLACTONE A FROM P. CHRYSOGENUM E01-10/3 LIQUID CULTURES ................. - 50 -
6 GENETIC POTENTIAL OF P. CHRYSOGENUM STRAIN E01-10/3 FOR POLYKETIDE PRODUCTION .............. - 51 -
6.1 Design of PCR primers and cloning of putative PKS gene fragments ...................................... - 51 -
6.2 Phylogenetic analysis of amino acid sequences from putative PKS gene fragments ................ - 85 -
7 SCREENING OF A GENOMIC LIBRARY FOR THE PUTATIVE SORBICILLACTONE GENE CLUSTER ............... - 92 -
7.1 Construction of P. chrysogenum genomic fosmid library ......................................................... - 92 -
7.2 Screening of the genomic library via hybridization .................................................................. - 92 -
7.3 Screening nomic library via PCR .............................................................................. - 101 -
7.4 Summary of P. chrysogenum E01-10/3 genomic library screening ........................................ - 117 -
8 DNA SEQUENCE ANALYSIS OF THE PUTATIVE SORBICILLACTONE GENE CLUSTER ............................. - 119 -
8.1 Shotgun sequencing of the putative sorbicillacone gene cluster ............................................. - 120 -
8.2 Identification of ORFs and prediction of enzyme functions ....................................................
8.3 Subcloning of fragment-2 from 49C8 fosmid clone ................................................................. - 125 -
8.4 KS domain phylogenetic analysis of cloned whole –length PKS genes ................................... - 136 -
8.5 Domain analysis of cloned whole-length PKS genes .............................................................. - 140 -
8.6 Proposed PKS biosynthetic route of sorbicillactones biosynthesis ......................................... - 149 -
9 ISOTOPE-LABELLED FEEDING EXPERIMENTS ...................................................................................... - 154 -
10 GENERAL DISCUSSION AND FURTHER PROSPECTIVES ......................................................................... - 157 -
MATERIALS AND METHODS ................................................................................................................. - 165 -
11 MATERIALS ......................