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

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255 Pages
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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.

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Published 01 January 2011
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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 ....................................................................................................................................... - 165 -
11.1 Media ...................................................................................................................................... - 165 -
11.2 Buffers and solutions ...............................................................................................................
11.3 Strains, vectors and plasmids .................................................................................................. - 169 -
11.4 Antibiotics and enzymes .......................................................................................................... - 171 -
11.5 PCR primers ............................................................................................................................ - 172 -
11.6 Equipment and expendable materials ..................................................................................... - 175 -
11.7 Chemicals and solvents ........................................................................................................... - 177 -
11.8 Commercial kits ...................................................................................................................... - 178 -
11.9 Special computer programs and internet resources ................................................................
12 METHODS............ - 180 -
12.1 Centrifugation ......................................................................................................................... - 180 -
12.2 Sterilization .............................................................................................................................
12.3 Cultivation of fungus P. chrysogenum E01-10/3 ..................................................................... - 180 -
12.4 Cultivation of E. coli cells .......................................................................................................
12.5 Isolation of chromosomal DNA from P. chrysogenum ............................................................ - 181 -
12.6 Determination of DNA concentration and purity .................................................................... - 181 -
12.7 Restriction enzyme digestion of DNA ...................................................................................... - 182 -
12.8 Amplification of DNA using polymerase chain reaction (PCR) .............................................. - 182 -
12.9 Agarose-gel-electrophoresis ................................................................................................... - 186 -
12.10 Purification of DNA fragments from solutions or agarose gel ................................................ - 186 -
- I - 12.11 Preparation of high quality plasmid DNA from E. coli ........................................................... - 187 -
12.12 Cloning of PCR products with the pGEM-T Easy Vector System ........................................... - 187 -
12.13 Preparation of the TA cloning vector ...................................................................................... - 188 -
12.14 Introduction of DNA into E. coli ............................................................................................. - 189 -
12.15 Preparation of genomic library of P. chrysogenum ................................................................ - 190 -
12.16 Screening the genomic P. chrysogenum fosmid library via PCR ............................................ - 194 -
12.17 Screening of the genomic fosmid library via hybridization ..................................................... - 195 -
12.18 Automated DNA sequencing .................................................................................................... - 199 -
12.19 In silico sequence analysis ...................................................................................................... - 200 -
12.20 Detection of sorbicillactone A from P. chrysogenum liquid cultures ...................................... - 201 -
REFERENCES .............................................................................................................................................. - 203 -
APPENDIX .................................................................................................................................................... - 218 -
13 UV SPECTRRUM OF SORBICILLACTONE A .......................................................................................... - 218 -
14 DEGENERATE PRIMER DESIGN ........................................................................................................... - 219 -
15 NUCLEOTIDE SEQUENCES OF AMPLIFIED PKS GENE FRAGMENTS ...................................................... - 226 -
16 NUCLEOTIDE SEQUENCE OF SHOT-GUN SEQUENCED 42H12 FOSMID CLONE ...................................... - 228 -
17 FGENESH IDENTIFICATION OF ORFS WITHIN CONTING-7 DNA SEQUENCES ....................................... - 239 -
18 LISTS OF FIGURES, TABLES AND STRUCTURES .................................................................................... - 242 -
18.1 List of figures........................................................................................................................... - 242 -
18.2 tables ............................................................................................................................
18.3 List of structures ...................................................................................................................... - 243 -
19 CURRICULUM VITAE ......................................................................................................................... - 246 -

- II - Abbreviations

% Percent F Fragmen
°C Degree FAD Flavin adenine dinucleotide
µ Micro FAS Fatty acid synthases(s)
6-MSA 6-Methylsalicylic acid FASTA DNA and protein sequence
A Adenosin alignment software package
A Ampere FDA Food and Drug Administration
A. Aspergillus FGSC Fungal Genomic Stock Center
aa Amino acid(s) FMN Flavin mononucleotid
ACV δ(L-a-aminoadipyl)-L-cysteinyl- FUMS Fumonisin synthase
D-valane FUSS Fusarisynthase
ad. Adapted h Hour
AMP Adenylation HCl Hydrochloric acid
approx. Approximately HMG-CoA 3-Hydroxy-3-methylglutaryl-
ARO Aromatase(s) coenzyme A
ARSs Autonomously replicating HMM Hidden Markov model
sequences HMW High molecular weight
ATP Adenosine triphosphate HR Highly reducing
BAC Bacterial Artificial Chromosome HR Highly-reduced
BBE Berberinbridge enzyme I Inosine
BLAST Basic Local Alignment Search i.e. That is
Tool IARC International Agency for
BMBF Bundesministerium für Bildung Research on Cancer
und Forschung (Federal Ministry IDH Isoepoxydon dehydrogenase
of Education and Research – IPTG Isopropylthio- β-D-galactoside
Germany) k Kilo
bp Base pair kDa Kilo Daltons
BSA Bovin Serum Albumin KS ketosynthase
C Cytosin l Liter
C/CON Condensation (domain of LDKS Lovastatindiketide synthase
PKS/NRPS) LDL Low-density lipoprotein
CCC Covalently closed circular DNA LED Light emiting diode
CDKS Compactin diketide synthase LMP Low meting point
cDNA Complementary DNA LNKS Lovastatin nonaketide synthase
CHS Chalcone synthase m Mili
CitS Citrinin synthase m Meter
CLC-TE Claisen-cyclase–thioesterase(s) M Molar
CNKS Compactin nonaketide synthase MAT Malonyl-CoA:ACP acyl
CNS Central nervous system transferase(s)
CoA Coenzyme A max. Maximum
conc. ConcentrationMb Megabases
CYC Cyclase(s) ME Minimumevolution
deg. Degenerate MEGA Molecular Evolutionary Genetics
DNA Desoxyribonucleic acid Analysis
DPPH 1,1-Diphenyl-2-picrylhydrazyl MEM Malt extract media
ds Double strandMeTs Methyl transferases
e.g. Example given MeTs Methyltransferases
EA Ergot alkaloidsmin Minute
EDTA Ethylendiamine tetraacetic acid M. Monascus
EH EPS15 homology MOPS 3-(N-Morpholino) propane
EMBL-EBI European Molecular Biology sulphonic acid
Laboratory- European MOS 3-Methylorcinaldehyde synthase
Bioinformatics Institute MP Microtiter plate
EMBnet European Molecular Biology MSAS 6-Methylsalicylic acid synthase
network MT/ME Methyl transferase(s)
EQS Equisetin synthase MWCO Molecular weight cut off
es End sequencen Nano
EST Expressesequencetag NA Norsolorinic acid
etc. Et cetera NADP Nicotinamide-adenine
- III - dinucleotide phosphate integration
NCBI National Center for RNA Ribonucleic acid
Biotechnology Information ROS Reactive oxygen species
NDA New Drug Application rpm Revolutions per minute
NEB New EnglandBiolabs RT Room temperature
NJ Neighbour-joining S. Saccharomyces
NLS Nuclear localization signal SAM S-adenosyl methionine
NR Non-reducing SDS Sodium dodecylsulfate
NRP Non-ribosomal peptide sec Second
NRPS Non-ribosomapeptide SQTKS Squalestatin tetraketide synthase
synthetase(s) SSH-PCR Subtractive hybridization PCR-
NSAS Norsolorinic acid synthase based approach
ORF Open reading frame(s) T Thymin
OSAS Orsollinic acid synthase TE Tris-EDTA solution
P. Penicillium TENS Tenellinsynthase
PBS Phosphate-buffered salane THNS Tetrahydroxynaphthalene
PEG Polyethylene glycol synthase
PKS Polyketide synthase(s) Tm Melting temperature
PLP Pyridoxal phosphate TNF- α Tumor necrosis factor-alfa
Pol Polymerase Tris Tris-(hydroxymethyl)-
PP Phosphopantetheine aminomethane
PPTase 4-PhosphopantetheinylTTS1 T-toxin synthase 1
transferase TTS2oxisyntha2
PR Partially reducing USA United States of America
PR Partially-reduced UV Ultra violet
PSI-BLAST Position-Specific Iterative Basic V Volt
Local Alignment Search Tool WA Naphthopyrone
PSSM Position specific score matrix WAS Naphthopyronsynthase
PT Product template WG Working group
PTGS Post-transcriptional gene WH2 Wiskott-Aldrich homology 2
silencing YWA1 Naphthopyrone
R Reductase ZS-A Zearalenone synthase A
R PKS Reducing polyketide synthase ZS-B B
R/RED Reducing/ reductase
rDNA Ribosomaldesoxyribonucleic
acid
REMI Restriction-enzyme-mediated
- IV - Summary
SUMMARY
The main goal of the presented dissertation was to evaluate the genetic potential of P.
chrysogenum E01-10/3 strain for the production of polyketides.
This marine-derived P. chrysogenum strain was isolated from the Mediterranean sponge
Ircinia fasciculata in the course of a research program focused on the discovery and
characterization of novel natural products. This led to the isolation and characterisation of two
novel and structurally highly similar polyketides: sorbicillactone A and sorbicillactone B.
Consistent with their structural similarity, it was proposed that the biosynthesis of both
compounds might be encoded by the same gene cluster. Consequently, the identification of
the sorbicillactone A gene cluster was in the focus of this dissertation as this compound was
previously shown to posses promising antileukaemic, antiviral and neuroprotective properties.
The iterative type I PKSs, which have only one multidomain protein with all the enzyme
activities covalently bound together, are responsible for the biosynthesis of fungal
polyketides. The single multifunctional protein is used to iteratively catalyze multiple rounds
of chain elongation and appropriate β-keto processing of a polyketide.
Since all of the fungal PKSs belong to the iterative type I PKS enzymes, degenerate primers
and hybridization probes fitting to this type of fungal PKS systems were used in PCR and
hybridization experiments. Of special help for the present study was the possibility to
differentiate between subtypes of fungal iterative type I PKSs on the amino acid level:
Nonreducing (NR), partially reducing (PR), and highly reducing (HR), in respect to level of
reduction of their polyketide products. Accordingly, PCR and hybridization experiments were
set up in order to take advantage of this fact.
During the course of this study, use of PCR enabled the amplification of partial PKS
sequences from nine putatively distinct fungal type I PKS gene loci from P. chrysogenum
E01-10/3. Six partial KS domain DNA sequences were used to reconstruct evolutionary
relationships in respect to other iterative type I PKSs. The results of the phylogenetic analysis
for KS domains illustrated that P. chrysogenum E01-10/3 strain has the genetic potential to
produce all three main categories of fungal polyketides – HR, PR (i.e. 6-MSA type) and NR.
The results of the phylogenetic analysis of PCR-amplified partial KS domains were valuable
to judge on good candidates for the screening of genomic library for putative sorbicillactone
gene cluster: Three partial PKS sequences shown to be putative members of the NR clade III
- 1 -