Strain development for heterologous expression of secondary metabolite clusters in actinobacteria

133 Pages·2016·5.84 MB·English

Strain development
for heterologous expression of secondary
metabolite clusters in actinobacteria

Dissertation
zur Erlangung des Grades
des Doktors der Naturwissenschaften
der Naturwissenschaftlich-Technischen Fakultät III
Chemie, Pharmazie, Bio- und Werkstoffwissenschaften
der Universität des Saarlandes

von
Niko Manderscheid
Saarbrücken
2015
Tag des Kolloquiums: 05.02.2016
Dekan: Prof. Dr.-Ing. Dirk Bähre
Berichterstatter: Prof. Dr. Andriy Luzhetskyy
Prof. Dr. Rolf W. Hartmann
Vorsitz: Prof. Dr. R. Müller
Akad. Mitarbeiter: Dr. M. Kohlstedt
Danksagung

Ich möchte mich ganz herzlich bei allen bedanken, die zur Entstehung dieser Arbeit beigetragen
haben.

Mein besonderer Dank gilt

Herrn Prof. Dr. Andriy Luzhetskyy für die Möglichkeit der Promotion in seiner Arbeitsgruppe, die
Überlassung des interessanten Themas und für seine ständige Hilfs- und Diskussionsbereitschaft.

Herrn Professor Dr. Rolf W. Hartmann für seine Tätigkeit als Zweitgutachter.

Herrn Dr. Thomas Paululat für die Strukturaufklärung der Substanzen.

Herrn Dr. Maksym Myronovskyi für seine Hilfsbereitschaft und ein immer offenes Ohr für alle Fragen
und Probleme sowie seine konstruktive Kritik.

Herrn Dr. Yuriy Rebets für seinen Ideenreichtum.

Frau Dr. Elke Brötz für die Analysen mittels HPLC-MS.

Frau Birgit Rosenkränzer für ihre Hilfe bei kleinen sowie großen Problemen.

Der Gruppe Metabolisches Engineering von Aktinomyzeten für eine tolle Arbeitsatmosphäre.

Herrn Prof. Dr. Hans-Peter Fiedler dafür, dass er mich auf diesen Weg gebracht hat.

Meiner lieben Freundin Steffi, die immer für mich da war.

Meinen Eltern Bodo und Marianne Manderscheid sowie meiner Schwester Kim, die mich immer

unterstützt haben.
Publications
Complete genome sequence of Streptomyces fulvissimus
Journal of biotechnology (2013)
M. Myronovskyi, B. Tokovenko, N. Manderscheid, L. Petzke, A. Luzhetskyy

Insights into the Pamamycin Biosynthesis
Angewandte Chemie (2015)
Dr. Yuriy Rebets, Dr. Elke Brötz, Niko Manderscheid, Dr. Bogdan Tokovenko, Dr. Maksym
Myronovskyi, Prof. Dr. Peter Metz, Dr. Lutz Petzke, and Dr. Andriy Luzhetskyy

An influence of the copy number of biosynthetic gene clusters on the production level of
antibiotics in a heterologous host.
Niko Manderscheid, Bohdan Bilyk, Thomas Paululat, Lutz Petzke and Andriy Luzhetskyy
Submitted to Journal of Biotechnology (2015)

Patents
Gene Cluster for Biosynthesis of Pamamycin
Pub. No.: WO/2015/092575 International Application No.: PCT/IB2014/066284
Petzke, Lutz; Herold, Andrea; Fleck, Christian; Treier-Marxen, Katrin; Ödman, Peter; Dickhaut,
Joachim; Weingarten, Melanie; Luzhetskyy, Andriy; Rebets, Yuriy; Brötz, Elke; Manderscheid, Niko;
Myronovskyi, Maksym

Improved microorganisms for the biosynthesis of pamamycins
European Patent: 15183922.2

Conference participation
1st European Conference on Natural Products (2013)
Identification and activation of cryptic gene clusters in Streptomyces fulvissimus
Niko Manderscheid, Andriy Luzhetskyy

VAAM workshop (2014)
Engineering precursor supply for polyketide synthesis in Streptomyces albus
Niko Manderscheid, Yuriy Rebets, Maksym Myronovskyi, Elke Brötz, Joachim Schmid, Klaus
Mauch, Andriy Luzhetskyy

2nd European Conference on Natural Products (2015)
Engineering precursor supply for Pamamycin biosynthesis in Streptomyces albus
Niko Manderscheid, Yuriy Rebets, Maksym Myronovskyi, Elke Brötz, Joachim Schmid, Klaus
Mauch, Andriy Luzhetskyy
List of contents

List of contents
Contents
Abbreviations: .......................................................................................................................................... i
A. Summary ............................................................................................................................................ 1
B. Zusammenfassung .............................................................................................................................. 1
I. Introduction .......................................................................................................................................... 2
1. Natural products .............................................................................................................................. 2
2. Antibiotics ....................................................................................................................................... 2
3. Antibiotic resistance ........................................................................................................................ 3
4. Screening for new compounds ........................................................................................................ 3
5. Streptomyces ................................................................................................................................... 5
6. How to access the hidden potential ................................................................................................. 6
7. Heterologous expression ................................................................................................................. 7
8. Streptomyces albus J1074 ............................................................................................................... 8
9. Strain development ......................................................................................................................... 8
10. Pamamycin .................................................................................................................................... 9
11. PKS ............................................................................................................................................. 10
12. Aim of this work ......................................................................................................................... 11
II. Complete genome sequence of Streptomyces fulvissimus ........................................................ 12
III. Insights into the pamamycin biosynthesis ....................................................................................... 14
1. Introduction ................................................................................................................................... 14
2. Materials and methods .................................................................................................................. 16
2.1. Strains, plasmids and culture conditions ............................................................................... 16
2.2. Preparation and manipulation of DNA .................................................................................. 16
2.3. Sequencing of genomes of S. alboniger DSMZ and S. sp. HKI118 and their analysis ........ 16
2.4. Deletion of the pamD gene in the S. alboniger genome ........................................................ 17
2.5. Pamamycin production analysis ............................................................................................ 17
2.6. Gene library preparation and cloning of the pam-gene cluster .............................................. 18
List of contents

2.7. Construction of the mutant strains ......................................................................................... 18
2.8. Complementation of pam gene mutations ............................................................................. 18
2.9. Feeding of mutant strains with hydroxyl acids S and L ........................................................ 19
2.10. Deletion of 3-oxoadipate:succinyl-CoA transferase gene in S. albus J1074 ...................... 19
2.11. Cloning, expression, purification and characterization of PamA ........................................ 19
3. Results and discussion .................................................................................................................. 25
4. Conclusion .................................................................................................................................... 30
IV. Strain development of Streptomyces albus J1074 for pamamycin production ............................... 31
1. Introduction ................................................................................................................................... 31
2. Materials and Methods .................................................................................................................. 35
2.1. Materials ................................................................................................................................ 35
2.1.1. Equipment ...................................................................................................................... 35
2.1.2. Consumables .................................................................................................................. 36
2.1.3. Chemicals ....................................................................................................................... 36
2.1.4. Cultivation Media ........................................................................................................... 37
2.1.5. Solutions and buffers ...................................................................................................... 38
2.1.6. Enzymes and kits ............................................................................................................ 39
2.1.7. Antibiotic solutions ........................................................................................................ 39
2.1.8. Synthetic oligonucleotides ............................................................................................. 39
2.1.9. Plasmids and Cosmids .................................................................................................... 40
2.1.10. Bacterial strains ............................................................................................................ 41
2.2. Methods ................................................................................................................................. 42
2.2.1. Methods in microbiology ............................................................................................... 42
2.2.1.1. Strain maintenance .................................................................................................. 42
2.2.1.2. Cultivation conditions ............................................................................................. 42
2.2.1.2.1. Preculture ........................................................................................................... 42
2.2.1.2.2. Main culture ...................................................................................................... 42
2.2.1.2.3. Optimization of NL SGG for pamamycin production ....................................... 42
2.2.1.2.4. Pamamycin production dependency on O level ............................................... 43
2
2.2.1.2.5. Pamamycin production curve ............................................................................ 43
List of contents

2.2.1.2.6. Influence of ammonium ions and L-Valine on VDH activity ........................... 43
2.2.1.2.7. Influence of succinate on pamamycin production ............................................. 43
2.2.2. Secondary metabolite analytics ...................................................................................... 43
2.2.2.1. Extraction of pamamycin ........................................................................................ 43
2.2.2.2. Dry weight calculation ............................................................................................ 44
2.2.2.3. HPLC Data Analysis ............................................................................................... 44
2.2.3. Methods in molecular biology ........................................................................................ 44
2.2.3.1. Plasmid isolation ..................................................................................................... 44
2.2.3.2. BAC isolation by MAX BAC ................................................................................. 45
2.2.3.3. Measurement of DNA concentration ...................................................................... 45
2.2.3.4. Chemical transformation ......................................................................................... 45
2.2.3.4.1. Preparation of chemically competent E. coli cells ............................................ 45
2.2.3.4.2. Transformation of chemically competent cells by heat shock........................... 46
2.2.3.5. Intergenic Conjugation of E. coli and Streptomyces ............................................... 46
2.2.3.5.1. Strain Preparation .............................................................................................. 46
2.2.3.5.2. Conjugation ....................................................................................................... 47
2.2.3.6. Agarose gel electrophoresis .................................................................................... 47
2.2.3.7. DNA purification from agarose gels ....................................................................... 47
2.2.3.8. Polymerase chain reaction (PCR) ........................................................................... 47
2.2.3.9. Red / ET Recombineering ....................................................................................... 48
2.2.3.9.1. Fragment preparation for cosmid targeting ....................................................... 48
2.2.3.9.2. Δ-red mediated recombination in E. coli GB05red ........................................... 48
2.2.3.9.3. Transfer of the recombined cosmid into S. albus J1074 and selection of
recombinants ..................................................................................................................... 49
3. Results ........................................................................................................................................... 51
3.1. Pamamycin production in Streptomyces albus J1074 ............................................................ 51
3.1.1. Heterologous expression of pamamycin cosmids R1 – R4 ............................................ 51
3.1.2. Optimization of NL SGG for pamamycin production .................................................... 53
3.1.3. Pamamycin production dependency on O level ............................................................ 53
2
3.1.4. Pamamycin production curve ......................................................................................... 54
3.2. Streptomyces albus J1074 strain development for pamamycin production ........................... 55
3.2.1. Identified genes in S. albus J1074 .................................................................................. 55
List of contents

3.2.2. InSilico simulations ........................................................................................................ 56
3.2.3. Knockout of the identified genes .................................................................................... 57
3.2.4. Heterologous expression of the pamamycin producing cosmid R2 in selected knockout
mutants ..................................................................................................................................... 57
3.2.4.1. Measurements of pamamycin production in the CCR mutants............................... 57
3.2.4.2. Measurement of pamamycin production in the triple knockout mutants ................ 59
3.2.4.3. Measurement of pamamycin production in the penta knockout mutant ................. 61
3.2.4.4. Ammonium feeding to S. albus J1074 / R2 and Del1ΔccrD1-1 ΔPCC2 ΔMCM
ΔPCC1 / R2 .......................................................................................................................... 61
3.2.4.5. Measurement of pamamycin production in the VDH knockout mutants ................ 63
3.2.4.6. Influence of succinate on pamamycin production .................................................. 64
4. Discussion ..................................................................................................................................... 65
4.1. Pamamycin production in Streptomyces albus J1074 ............................................................ 65
4.1.1. Heterologous expression of pamamycin cosmids R1 – R4 ............................................ 65
4.1.2. Optimization of NL SGG for pamamycin production .................................................... 65
4.1.3. Pamamycin production dependency on the O level ...................................................... 66
2
4.1.4. Pamamycin production curve ......................................................................................... 66
4.2. Streptomyces albus J1074 strain development for pamamycin production ........................... 67
4.2.1. Identified genes in S. albus J1074 .................................................................................. 67
4.2.2. InSilico simulations ........................................................................................................ 68
4.2.3. Knockout of the identified genes .................................................................................... 68
4.2.4. Heterologous expression of the pamamycin producing cosmid R2 in selected knockout
mutants ..................................................................................................................................... 68
4.2.4.1. Measurements of pamamycin production in the CCR mutants............................... 68
4.2.4.2. Measurement of pamamycin production in the triple knockout mutants ................ 70
4.2.4.3. Measurement of pamamycin production in the penta knockout mutant ................. 70
4.2.4.4. Ammonium feeding to S. albus J1074 / R2 and Del1ΔccrD1-1 ΔPCC2 ΔMCM
ΔPCC1 / R2 .......................................................................................................................... 71
4.2.4.5. Measurement of pamamycin production in the VDH knockout mutants ................ 71
4.2.4.6. Influence of succinate on pamamycin production .................................................. 71
List of contents

5. Summary of the strain development of Streptomyces albus J1074 for pamamycin production ... 72
V. An influence of the copy number of biosynthetic gene clusters on the production level of antibiotics
in a heterologous host ............................................................................................................................ 73
1. Introduction ................................................................................................................................... 73
2. Materials and methods .................................................................................................................. 75
2.1. Construction of plasmids for transposon mutagenesis .......................................................... 75
2.2. Introduction of additional attB-sites into S. albus genome .................................................... 75
2.3. Heterologous expression of the secondary metabolite clusters ............................................. 76
2.3.1. Pamamycin production ................................................................................................... 77
2.3.2. Didesmethylmensacarcin and griseorhodin production ................................................. 77
2.3.3. Aranciamycin production ............................................................................................... 78
2.4. Determination of production .................................................................................................. 78
2.4.1. Determination of pamamycin production ....................................................................... 78
2.4.2. Determination of demethoxyaranciamycinone production ............................................ 79
2.5. Production and isolation of 2-(1-hydroxyethyl)-1,8-dihydroxy-3-methylanthraquinone ...... 79
2.6. Structure elucidation of 2-(1-hydroxyethyl)-1,8-dihydroxy-3-methylanthraquinone ........... 79
3. Results and Discussion.................................................................................................................. 80
3.1. Introduction of additional attB-sites into the genome of Streptomyces albus J1074 ............. 80
3.2. Heterologous expression of pamamycin, aranciamycin, mensacarcin and griseorhodin....... 81
3.3. Production and isolation of 2-(1-hydroxyethyl)-1,8-dihydroxy-3-methylanthraquinone ...... 84
3.4. Structure elucidation of 2-(1-hydroxyethyl)-1,8-dihydroxy-3-methylanthraquinone ........... 85
4. Summary ....................................................................................................................................... 86
VI. Final conclusion .............................................................................................................................. 87
Literature ............................................................................................................................................... 89
Appendix ............................................................................................................................................. 103
1. Complete genome sequence of Streptomyces fulvissimus .......................................................... 103
2. Insights into the pamamycin Biosynthesis .................................................................................. 107
3. Strain development of Streptomyces albus J1074 for pamamycin production ........................... 118
4. An influence of the copy number of biosynthetic gene clusters on the production level of
antibiotics in a heterologous host .................................................................................................... 121
Abbreviations

Abbreviations:
% percent
°C degree Celsius
µ- micro-
ACN Acetonitrile
ad up to
bidest. double distilled
BLAST Basic Local Alignment Search Tool
Bp base pairs
ca. circa
CCR Crotonyl-CoA carboxylase / reductase
DAD Diode-Array-Detection
DCM Dichloromethane
dest. distilled
DNA Desoxyribonucleic acid
dNTP 2‘-Desoxyribonukleosid-5’triphosphate
EDTA Ethylenediaminetetraacetic acid
ESI-MS Elektrospray ionization mass spectroscopy
et al. et alii
FT-ICR-MS Fourier transformation ion cyclotron resonance mass spectrometry
g gram (weight unit)
h hour
HPLC High performance liquid chromatography
l Liter (volume unit)
m Meter (length unit)
m- milli-
M Molar
mAU milli absorbance units
MCM Methylmalonyl-CoA mutase
MeOH Methanol
Min Minutes
MM Minimal media
MS Mass spectrometry
MW Molecular weight
n- nano-

i