Molecular analysis of the aureothin biosynthesis gene cluster from streptomyces thioluteus HKI-227 [Elektronische Ressource] : new insights into polyketide assemply / von Jing He

Molecular Analysis of the Aureothin Biosynthesis Gene
Cluster from Streptomyces thioluteus HKI-227;
New Insights into Polyketide Assembly

Dissertation

zur Erlangung des akademischen Grades
doctor rerum naturalium

vorgelegt dem Rat der Biologisch-Pharmazeutischen Fakultät der
Friedrich-Schiller-Universität Jena

von

Jing He
Geboren am 10.02.1977 in Wuhan, People’s Republic of China

Jena, im November 2004





























Gutachter

1. Prof. Dr. Susanne Grabley
2. Prof. Dr. Erika Kothe
3. Prof. Dr. Jörn Piel

Tag der Doktorprüfung: 12 January 2005

Tag der öffentlichen Verteidigung: 31 January 2005 Index I
Index
Abbreviation ..............................................................................................................................i
A. Introduction .......................................................................................................1
1. Streptomycetes as Producers of Bioactive Secondary Metabolites .................... 1
2. Microbial Polyketide Biosynthesis..........................................................................3
2.1 Genetic Contributions to Understanding Polyketide Biosynthesis.................................3
2.2 Molecular Diversity of Polyketides......................................................................................5
2.3 Classification of Polyketide Synthases ..............................................................................7
2.4 Modular Type I Polynthases.................................................................................8
2.4.1 The Erythromycin Polyketide Synthase .........................................................................10
2.4.2 Genetic Engineering of Modular Type I Polyketide Synthases ......................................11
2.5 Some Speculations on the Evolution of the Iterative and Non-Iterative PKS...............14
3. Aureothin ................................................................................................................. 15
4. Research Goals.......................................................................................................16
B. Materials and Methods.................................................................................... 18
1. Materials.. 18
1.1 Media..................................................................................................................................18
1.1.1 Media for the Cultivation of Escherichia coli Strains ......................................................18
1.1.2 MediaStreptomyces Strains .........................................................18
1.2 Buffers and Solutions.......................................................................................................19
1.2.1 Buffers for Plasmid DNA Preparation from E. coli..........................................................19
1.2.2 Buffers for Protoplast Transformation of Streptomyces .................................................20
1.2.3 Buffers for N-Oxidation Assay........................................................................................21
1.2.4 Buffers for Electrophoresis.............................................................................................21
1.2.5 Solutions for Preparation of Competent E. coli Cells by Chemical Method...................21
1.2.6 Buffers for Hybridization.................................................................................................22
1.3 Strains and Plasmids........................................................................................................23
1.4 Antibiotics and Enzymes..................................................................................................28
1.5 PCR Primers ......................................................................................................................30
1.6 Special Devices .................................................................................................................31 Index II
2. Methods.................................................................................................................... 33
2.1 Cultivation of E. coli Cells ................................................................................................33
2.2 Growth and Preservation of Streptomyces Strains.......................................................33
2.3 Amplification of DNA Fragments by PCR .......................................................................33
2.4 Purification of DNA Fragments from Solutions or Agarose Gel...................................34
2.5 Cloning of PCR Products with the pGEM-T Easy Vector System ................................34
2.6 Preparation High Quality Plasmid DNA from E. coli......................................................35
2.7 Introduction of DNA into E. coli.......................................................................................35
2.7.1 Preparation and Transformation of Competent Cells by the Chemical Method.............35
2.7.2 Transformation of E. coli Cells by Electroporation .........................................................35
2.8 Isolation of Genomic DNA from Streptomyces ..............................................................36
2.9 Plasmid DNA Isolation from Streptomyces ....................................................................36
2.10 Introduction of DNA into Streptomyces..........................................................................37
2.10.1 Protoplast Transformation of Streptomyces.................................................................37
2.10.2 Intergeneric Transfer of Plasmids from E. coli to Streptomyces by Conjugation.........37
2.11 Construction of Cosmid Library......................................................................................38
2.11.1 Insert DNA Preparation and End-Repair Reaction.......................................................38
2.11.2 Size Selection of Insert DNA ........................................................................................38
2.11.3 In-Gel Ligation ..............................................................................................................39
2.11.4 In Vitro Packaging39
2.12 Construction of a Random Shotgun Library for Sequencing.......................................40
2.12.1 Random Incision of Cosmid DNA by Sonication..........................................................40
2.12.2 Blunt End-Repair Reaction...........................................................................................40
2.12.3 Size Selection of Sheared DNA Fragments .................................................................40
2.12.4 Ligation with Sequencing Vector DNA .........................................................................41
2.12.5 Transformation into E. coli Cells ..................................................................................41
2.13 Southern Hybridization.....................................................................................................41
2.13.1 Capillary Transfer and Fixation of DNA........................................................................41
2.13.2 Labeling of the Probes .................................................................................................42
2.13.3 Hybridization.................................................................................................................42
2.13.4 Immunological Detection..............................................................................................43
2.14 Screening the Genomic Cosmid Library by PCR ..........................................................43
2.15 Gene Knock-out by the PCR Targeting System.............................................................44
2.16 Feeding Experiments........................................................................................................44
2.17 N-Oxidation Assay ............................................................................................................45 Index III
2.18 Fermentation and Detection of Metabolites ...................................................................45
C. Results and Discussion .................................................................................. 46
1. Cloning, Sequencing and Heterologous Expression of the Aureothin
Biosynthesis Gene Cluster .................................................................................... 46
1.1 Design of the Primers for Cloning...................................................................................46
1.2 Construction and Screening of a S. thioluteus HKI-227 Genomic Cosmid Library ...48
1.3 Heterologous Expression of the Aureothin Biosynthesis Gene Cluster.....................50
1.4 Sequence Analysis of the Genomic Region Involved in Aureothin Biosynthesis......51
1.4.1 The Aureothin PKS Genes.............................................................................................52
1.4.2 Genes Putative Involved in Starter Unit Synthesis and Post-PKS Processing..