Biosynthesis of aminocoumarin antibiotics in Streptomyces [Elektronische Ressource] : generation of structural analogues by genetic engineering and insights into the regulation of antibiotic production = Biosynthese von Aminocoumarinantibiotika in Streptomyces / vorgelegt von Alessandra da Silva Eustáquio

Salix

Sujets

• Gesundheit

Informations

• Publié par: eberhard_karls_universitat_tubingen
• Publié le: 01 janvier 2004
• Nombre de lectures: 34
• Poids de l'ouvrage: 1 Mo

Extrait

Biosynthesis of aminocoumarin antibiotics in
Streptomyces: Generation of structural analogues
by genetic engineering and insights into the
regulation of antibiotic production

Biosynthese von Aminocoumarinantibiotika in
Streptomyces: Herstellung von Analoga durch
genetische Manipulation und Einblick in die
Regulation der Antibiotikaproduktion



DISSERTATION


der Fakultät für Chemie und Pharmazie
der Eberhard-Karls-Universität Tübingen
zur Erlangung des Grades eines Doktors
der Naturwissenschaften




2004



vorgelegt von
Alessandra da Silva Eustáquio
































Tag der mündlichen Prüfung: 7.12.2004

Dekan: Prof. Dr. S. Laufer
1. Berichterstatter: Prof. Dr. L. Heide
2. Berichterstatter: Prof. Dr. P. Ruth































Für Oliver CONTENTS I
CONTENTS

PUBLICATIONS AND PRESENTATIONS………………………………………………V
ABBREVIATIONS………………………………………………………………………….VI
SUMMARY……………………………………..…………………………………………….1
ZUSAMMENFASSUNG…………………………………..………………………………...4
I. INTRODUCTION...............................................................................................................7
I.1. THE SEARCH FOR NEW ANTIBIOTICS: AN OVERVIEW OF GENE TIC APPROACHES ........7
I.2. STREPTOMYCES – THE LARGEST ANTIBIOTIC-PRODUCING GENUS.............................9
I.3. AMINOCOUMARIN ANTIBIOTICS ..................................................................................11
I.3.1. Chemical structure11
I.3.2. Mechanism of action and clinical application................13
I.3.3. Structure-activity relationships.........................................................................16
I.3.4. Biosynthesis and identification of the biosynthetic gene clusters ..............17
I.4. REGULATION OF ANTIBIOTIC PRODUCTION24
I.5. AIMS OF THIS STUDY..................................................................................................26
II. MATERIALS AND METHODS..................................................................................29
II.1. CHEMICALS................................................29
II.2. MATERIALS FOR CHROMATOGRAPHY........30
II.3. ENZYMES AND KITS....................................................................................................31
II.4. MEDIA, BUFFERS AND SOLUTIONS.............31
II.4.1. Media for bacterial cultivation..........31
II.4.1.1. Cultivation of E. coli....................................................................................32
II.4.1.2. Streptomyces.......32
II.4.1.3. Clorobiocin production medium................................34
II.4.1.4. Novobiocin production medium ................................34
II.4.1.5. Protoplast transformation of Streptomyces.............................................35
II.4.2. Antibiotic solutions.............................................................36
II.4.3. Buffers and solutions.........................................................37
II.4.3.1. Buffers and Solutions for DNA isolation..................37
II.4.3.2. Buffers for DNA gel electrophoresis38
II.4.3.3. Buffers and solutions for Southern blot analysis....................................39
II.4.3.4. Solutions for blue/white selection of E. coli.............39
II.4.3.5. Buffers for preparation of protoplasts and transformation of
Streptomyces...............................................................................................40
II.4.3.6. Buffers for protein purification by nickel affinity chromatography........41
II.4.3.7. Buffers and solutions for protein gel electrophoresis (SDS-PAGE) and
for Coomassie staining...............................................................................41
II.4.3.8. Buffers and solutions for gel mobility-shift assay...42
II.5. PLASMIDS, BACTERIAL STRAINS, PRIMERS AND PROBES..........43
II.5.1. Vectors, cosmids and plasmids.......................................................................43
II.5.2. PCR primers used for construction of plasmids............49
II.5.3. Bacterial strains..................................50 CONTENTS II
II.5.4. Probes used in Southern blot analysis...........................................................52
II.6. CULTURE CONDITIONS ...............................................................52
II.6.1. Cultivation of E. coli52
II.6.2. Cultivation of Streptomyces .............................................................................53
II.6.2.1. General cultivation......................53
II.6.2.2. Production of secondary metabolites.......................53
II.6.2.3. Preparation of mycelia for storage and of spore suspensions of
Streptomyces...............................................................................................54
II.6.2.4. Sensitivity of S. rishiriensis, S. roseochromogenes and S. spheroides
to different antibiotics..................................................................................55
II.7. METHODS OF MOLECULAR BIOLOGY..........56
II.7.1. Purification, concentration and quantification of DNA..................................56
II.7.2. Agarose gel electophoresis of DNA................................................................56
II.7.3. DNA manipulation with enzymes.....56
II.7.4. DNA isolation......................................57
II.7.4.1. Isolation of plasmids from E. coli..............................................................57
II.7.4.2. Streptomyces................57
II.7.4.3. Isolation of genomic DNA from ........................................58
II.7.5. DNA denaturation for ssDNA transformation in Streptomyces ..................59
II.7.5.1. Alkaline treatment .......................................................................................59
II.7.6. PCR amplification..............................59
II.7.6.1. General conditions......................59
II.7.6.2. Conditions for amplification of the apramycin resistance cassette from
pIJ773 and from pUG019 ..........................................................................60
II.7.7. Southern blot analysis.......................61
II.7.7.1. Probe preparation ................................................................62
II.7.7.2. Southern blot preparation..........62
II.7.7.3. Prehybridization and hybridization...........................62
II.7.7.4. Detection ......................................................................................................62
II.7.7.5. Removal of probe........................63
II.7.8. Introduction of DNA in E. coli...........63
II.7.8.1. CaCl -mediated transformation.................................................................63 2
II.7.8.2. Electroporation............................................................64
II.7.9. Introduction of DNA in Streptomyces.............................65
II.7.9.1. PEG-mediated protoplast transformation................................................65
II.7.9.2. Conjugation from E. coli.............................................67
II.7.10. DNA sequencing and computer-assisted sequence analysis.................68
II.8. METHODS OF BIOCHEMISTRY AND BIOLOGY..............................................................69
II.8.1. Denaturing Polyacrylamide Gel Electrophoresis (SDS-PAGE)..................69
II.8.2. Overexpression and purification of recombinant protein from E. coli........69
II.8.2.1. Cultivation.....................................................................................................70
II.8.2.2. Preparation of cell-free extract..70
II.8.2.3. Purification by nickel affinity chromatography........70
II.8.3. Gel mobility-shift assays ...................................................................................71
II.8.3.1. Preparation of 3´-end DIG-labeled DNA fragments...............................71 CONTENTS III
II.8.3.2. Gel mobility-shift assay..............................................................................72
II.8.4. Bioassay with Bacillus subtilis.........73
II.9. CONSTRUCTION OF DELETION MUTANTS OF S. ROSEOCHROMOGENES AND S.
SPHEROIDES ..............................................73
II.9.1. Inactivation of clo-hal in S. roseochromogenes............................................73
II.9.2. Inactivation of cloZ in S. roseochromogenes................74
II.9.3. Inactivation of novE in S. spheroides .............................................................74
II.10. HETEROLOGOUS EXPRESSION OF THE NOVOBIOCIN AND CLOROBIOCIN
BIOSYNTHETIC GENE CLUSTERS................................................................................75
II.10