Investigations on

ABC and MFS transporters of spp.

A dissertation presented to the Faculty of Chemistry and Pharmacy of

Albert -Ludwigs University of Freiburg im Breisgau for the degree of Doctor rerum naturalium

Submitted by Irene Santillana Larraona

from Barcelona, Spain

- June 2015 -

Dean: Prof. Dr. Bernhard Breit

Chair of the doctoral committee: Prof. Dr. Stefan Weber

Referent: Prof. Dr. Andreas Bechthold

Co-referent: Jun. Prof. Dr. Stefan Günther

Third examiner: Prof. Dr. Oliver Einsle

Date of the examination: June 29, 2015

Date of promotion: July 2, 2015

I hereby declare that the presented work was completed independently by me and using only the sources cited in the list of references.

Freiburg, June 2015.

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______Irene Santillana Larraona

Parts of this work have been published:

Presentation

“Inactivation of transporters by Redirect Technology” PhD seminar. May 7, 2013.

Poster

Santillana, I.; Derochefort, J.; Zuo, C.; Bechthold, A.: “New insights into the rishirilide gene cluster: Minimal PKS, oxygenases, hydrolases and transporters”. DPhG, Annual meeting 2013. Drug discovery inspired by nature. Freiburg, October 09 – 11, 2013.

Santillana, I.; Yan, X.; Wunsch-Palasis, J.; Bechthold, A.: “Characterization of the rishirilide transporters and their influence on its production”. within soils. Symposium. Münster, October 25 - 28, 2012.

Gessner, A.; Kroeger, J; Santillana, I.; Bechthold, A.: “Function, specificity and use of transporters involved in of saccharide or involved in the efflux of antibiotics in ”. Membrane proteins and biological membranes. GRK 1478. Freiburg, January 10, 2012.

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This work was performed in the Institute of Pharmaceutical Biology and Biotechnology of the University of Freiburg in the research group of

Prof. Dr. Andreas Bechthold,

who I want to sincerely thank for giving me this opportunity. Thank you for opening me the doors of your working group and for supporting me during this time. Thank you for enabling me to develop my own research and to work independently on a topic that I enjoyed.

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I also want to thank:

. Jun. Prof. Dr. Stefan Günther for being my co-referent and for supporting me especially during the first months of this work. I want to express my gratitude for

the welcome into the pharmaceutical bioinformatics working group. | vii . Prof. Dr. Oliver Einsle for so kindly accepting to be my third examiner. . Dr. Gabriele Weitnauer for her help, organization and for the support with the MolBio Bachelor Praktikum. . The whole working group of pharmaceutical bioinformatics, and particularly Dr. Anika Erxleben, Dr. Xavier Lucas, Stephan Flemming and Kersten Döring for introducing me into the bioinformatics world and for the great start of my stay in Freiburg. Thank you for enabling my participation in the StreptomeDB project. . The former PhD students of the working group of Prof. Bechthold for all the initial support. I want to give a big thank-you to Dr. Julia Wunsch-Palasis for showing me everything necessary when I arrived to the lab and Dr. Theresa Siegl for her help, advices and also fun in our office. . The current working group for the good atmosphere and all cakes and Fanta we enjoyed together. I want to especially thank Denise Deubel for her help and for bringing her energy and good mood into the lab; Stefanie Hackl for all great conversations about travelling and interesting life stories; Astrid Erber for taking me into “uni-sports” and other cool activities; Yvonne Schmidt-Bohli for helping me with protein related troubles and for being always available for a good advice; and Suzan Samra for her huge humanity and for doing this working group a better place. . My “final phase” office colleagues Jasmin Kroeger and Tanja Heitzler for their daily motivation words and because despite the hard days, we also had fun! . Our TAs, Marcus Essing and Sandra Groß, for the computer and laboratory support. Elisabeth Welle for her big and immediate help, for teaching me about HPLC and for taking care of us and our lab. . My “super” bachelor and diploma students Veronika Brinschwitz and Jannis Brehm for their dedication, their help and their sense of humor. It was a pleasure to share my project with you both. . Dr. Tina Strobel and Stephan Flemming for all the time we enjoyed together, for the relax evenings on the sofa, for the trips and the parties, because you made possible that I never felt alone. Ihr seid so cool!

. Anja Greule for reading and correcting all this work. For being my “half of the orange” in the lab, having fun and great scientific discussions. For sharing your knowledge and for finding always time to help me and all the others. For being an amazing colleague and even a better friend. viii| . Familie Löppenberg, für euer Interesse, eure Hilfe beim Schreiben während den Wochenenden und für eure immer netten Worte. . A Pablo y Ana por disfrutar juntos de mis visitas a Barcelona y por venir a conocer Freiburg. A Mario, por ser la cosa más bonita, por sus enormes abrazos cuando llego de visita y porque, aunque él no lo sepa, siempre me saca una sonrisa. . A mis padres, por su amor y apoyo incondicional. Porque a pesar de la distancia, siempre os siento conmigo y sois mi punto de referencia. Por recordarme cada día que esta vida está para disfrutarla. . Marius for all the effort and time dedicated to enhance this work. For being always by my side and for taking me into your arms when everything felt “too much”. Thank you for being a huge motivation to improve myself every day.

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“Happiness only real when shared”

- Alexander Supertramp -

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Contents

I. SUMMARY / ZUSAMMENFASSUNG ...... 1

II. INTRODUCTION ...... 4

1. Natural products ...... 4

1.1. Polyketides ...... 4 Rishirilide B, a type II PKS from Streptomyces bottropensis Goe C4/4 ...... 7

1.2. Terpenoides ...... 8 Phenalinolactone, a terpenoide metabolite from Streptomyces sp. Tü6071 ...... 10

2. Ins and outs across the membranes ...... 11

2.1. Bacterial membranes ...... 11

2.2. ATP-binding cassette transporters ...... 13

2.3. Major facilitator superfamily transporters ...... 15

3. Aim of this work ...... 17

III. MATERIAL AND METHODS ...... 18

1. Material ...... 18

1.1. General manufacturer information ...... 18 Contents

1.2. Laboratory material, equipment and analytical instruments ...... 19

1.3. Chemicals and reagents ...... 21

1.4. Enzymes, antibodies and kits ...... 24 xii| 1.5. Solutions and buffers ...... 25

1.6. solutions ...... 31

1.7. Components of media ...... 32

1.8. Bacterial stem lines ...... 32

1.9. Vectors ...... 34

1.10. Software and database ...... 38

2. Methods ...... 40

2.1. Methods in microbiology ...... 40 2.1.1. Cultivation of bacterial strains ...... 40 2.1.2. Transfer of DNA ...... 42 2.1.3. Bacterial screening ...... 47 2.1.4. Minimal inhibitory concentration in E. coli ...... 48 2.1.5. Disc diffusion antibiotic sensitivity testing ...... 48

2.2. Methods in molecular biology ...... 49 2.2.1. Plasmid isolation from E. coli ...... 49 2.2.2. Genomic DNA isolation from Streptomyces spp...... 50 2.2.3. Precipitation and concentration of DNA ...... 50 2.2.4. Analysis and purification of DNA by agarose gel electrophoresis ...... 51 2.2.5. Restriction digestion of DNA ...... 51 2.2.6. Ligation ...... 52 2.2.7. Dephosphorylation of plasmid DNA ...... 52 2.2.8. DNA sequencing ...... 52 2.2.9. DNA amplification – Polymerase chain reaction ...... 53 2.2.10. Construction of plasmids ...... 62 2.2.11. Gene inactivation in Streptomyces spp...... 69

2.3. Methods in protein engineering ...... 75 2.3.1. Heterologous expression of recombinant proteins ...... 75 Contents

2.3.2. Isolation of proteins ...... 77 2.3.3. Purification of proteins ...... 78 2.3.4. Concentration of proteins...... 80 2.3.5. Analysis of protein ...... 81 | xiii 2.4. Isolation and analysis of secondary metabolites ...... 82 2.4.1. Extraction with ethyl acetate ...... 82 2.4.2. Solid phase extraction ...... 83 2.4.3. High Pressure Liquid / Electron Spray Ionization - Mass Spectrum analysis ...... 84 2.4.4. Preparative High Pressure Liquid Chromatography ...... 85

2.5. Methods in bioinformatics ...... 86 2.5.1. StreptomeDB ...... 86 2.5.2. Galaxy ...... 88

IV. RESULTS ...... 91

1. Investigations on the transporter genes of rishirilide gene cluster...... 91

1.1. Bioinformatics characterization of rishirilide gene cluster transporters ...... 91 1.1.1. Characterization of rslT1 ...... 94 1.1.2. Characterization of rslT2 ...... 95 1.1.3. Characterization of rslT3 ...... 96 1.1.4. Characterization of rslT4 ...... 98

1.2. Inactivation and expression of the ABC transporter system RslT1-T3 ...... 100 1.2.1. Construction of rslT1, rslT2 and rslT3 deletion cosmids by Red/ET ...... 100 1.2.2. Production analysis of S. albus::cos4ΔrslT123 and the complemented mutant ...... 105 1.2.3. Production analysis of rslT1 and rslT123 overexpression mutants ...... 109 1.2.4. Protein expression and purification of RslT1 ...... 111

1.3. Inactivation and expression of the MFS transporter RslT4 ...... 121 1.3.1. Construction of the deletion cosmid cos4ΔrslT4 by Red/ET ...... 121 1.3.2. Production analysis of S. albus::cos4ΔrslT4 and complementation ...... 122 1.3.3. Production analysis of rslT4 overexpression mutant ...... 125 1.3.4. Production analysis of rslR4 overexpression mutant ...... 126 1.3.5. MIC test in E. coli and disk diffusion assay in S. albus ...... 128 1.3.6. Purification of Rishirilide B ...... 130 Contents

2. Investigations on the phenalinolactone producer Streptomyces sp. Tü6071...... 132

2.1. Genome analysis and screening for transporters involved in phenalinolactone biosynthesis ...... 132 xiv| 2.2. Inactivation and expression of plaABC ...... 136 2.2.1. Gene inactivation of plaABC1-3 via double crossover ...... 136 2.2.2. Gene inactivation of plaABC1 via single crossover ...... 138

3. StreptomeDB contribution ...... 139

V. DISCUSSION ...... 141

1. Homologies of rishirilide gene cluster in other strains ...... 141

2. The abc importer system RslT123 ...... 144

2.1. From gene organization to quaternary structure ...... 144

2.2. RslT123, an importer? ...... 149 Purification of the substrate binding protein ...... 151

2.3. Influence of the ABC transporter system in rishirilide B production ...... 155 2.3.1. Lack of the amino acid importer ...... 155 2.3.2. Complementation and overexpression ...... 157

3. The major facilitator superfamily transporter RslT4...... 159

3.1. RslT4, a multidrug transporter ...... 161

3.2. Influence of the MFS transporter in rishirilide B production ...... 164

4. Transporter genes of Streptomyces sp. Tü6071 ...... 167

VI. BIBLIOGRAPHY ...... 169

VII. ABBREVIATIONS ...... 185

VIII. APPENDIX ...... 189 Contents

1. Plasmid maps ...... 189

1.1. pTOS-plaABC123 ...... 189

1.2. pUWL-H-plaABC123 ...... 190 | xv 1.3. pUWL-rslT123 ...... 190

1.4. pUWL-OriT-rslT1 ...... 191

1.5. pTOS-rslT1 ...... 191

1.6. pTOS-rslT123 ...... 192

1.7. pTOS-rslT4 ...... 192

1.8. pKCplaABC123 ...... 193

1.9. pKCplaABC1SCO ...... 193

1.10. pET28rslT1N ...... 194

1.11. pET28rslT1C ...... 194

1.12. pET28rslT1Ctrun ...... 195

1.13. pET28rslT123 ...... 195

1.14. pUWLrslT1C ...... 196

1.15. pUWLrslT1CT2T3 ...... 196

2. Galaxy workflows ...... 197

3. List of figures ...... 200

4. List of tables...... 205

5. Curriculum Vitae ...... 207

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I. Summary / Zusammenfassung

ABC (ATP binding cassette) and MFS (major facilitator superfamily) transporters are the two largest families of membrane proteins involved in the import and export of substances across biological membranes. Members of these families have been described for the up- take of a variety, necessary molecules which belong to cell or for the efflux of toxins and multiple structurally unrelated drugs. In this work ABC and MFS transporters of two Streptomyces spp. have been investigated for their implication in the biosynthesis of two secondary metabolites.

Rishirilide B is a type II PKS metabolite produced by Streptomyces bottropensis Goe C4/4 and its gene cluster includes four transporter genes, rslT1-4. An ABC transporter is encoded by rslT1-3 which were predicted to compose an amino acid importer system defined by the substrate binding protein (SBP) RslT1. The multidrug transporter encoded by rslT4 belongs to the MFS and its transcription is controlled by RslR4, a MarR regulator. Inactivation experiments of these transporter genes were performed by Redirect© Technology in the cosmid cos4 which contained rishirilide cluster, for its heterologous expression in Streptomyces albus J1074. Deletion and overexpression experiments of rslT1-3 indicated the capacity of the importer system to affect rishirilide B production. It was therefore suggested that the ABC transporter is responsible for the uptake of a precursor needed for the biosynthesis of rishirilide B. Isobutyryl was proposed as the starter unit for the PKS biosynthesis of rishirilide B and was described to derivate from the valine catabolism. Based on these assumptions, RslT123 was suggested to be involved in the import of this amino acid. Attempts to purify the SBP to prove its substrate specificity failed. Furthermore, deletion and overexpression experiments of the MFS transporter RslT4 and its regulator RslR4 led to the proposal of a regulatory mechanism for the biosynthesis and export of rishirilide B. However, rishirilide B production increased in the ΔrslT4 mutant indicating that another I. Summary / Zusammenfassung transport mechanism of the host took over the function. The multidrug efflux capacity of RslT4 was determined by MIC and disc diffusion assays.

In addition, analysis of the genome sequence of Streptomyces sp. Tü6071 was executed with

2 | the help of the bioinformatics platform Galaxy with a focus on transporter genes. S. sp. Tü6071 is responsible for the production of the terpenoids phenalinolactones A-D. Investigations on the genome sequence to identify transporter genes involved in the biosynthesis of these secondary metabolites led to the prediction of three ABC transporter genes, plaABC1-3. Inactivation experiments of plaABC1 by homologous recombination did not show a connection of the ABC transporter to phenalinolactones but implied a role in aerial mycelium formation.

Bei ABC- (ATP binding cassette) und MFS- (major facilitator superfamily) Transportern handelt es sich um zwei der größten Membranprotein-Familien. Transporter dieser Familien sind für die Aufnahme von zahlreichen, essentiellen Molekülen des Stoffwechsels der Zelle oder für den Efflux von Toxinen und anderen, strukturell unterschiedlichen Substanzen verantwortlich. In dieser Arbeit wurde die Bedeutung von ABC- und MFS- Transporter zweier Streptomyceten-Stämme in der Sekundärmetabolit-Biosynthese untersucht.

Rishirilid B ist ein PKS Typ II Metabolit, der von Streptomyces bottropensis Goe C4/4 gebildet wird. Das entsprechende Gencluster enthält die vier Transportergene rslT1-4. Die Gene rslT1-3 kodieren für einen ABC-Transporter. Das Auftreten des Substratbindeproteins (SBP) RslT1 lässt vermuten, dass es sich hierbei um ein Aminosäure-Importsystem handelt. Der von rslT4 kodierte Multidrug-Transporter zählt zur MFS-Familie wobei dessen Transkription vom MarR Regulator RslR4 kontrolliert wird. Das Rishirilid-Gencluster liegt auf dem Cosmid cos4 und wurde in Streptomyces albus J1074 heterolog exprimiert. Inaktivierungsexperimente der Transporter wurden mittels Redirected© Technologie durchgeführt. Die Deletion und Überexpression von rslT1-3 zeigen, dass dieses Importsystem die Rishirilid B-Produktion beeinflusst. Diese Ergebnisse führen zu der Annahme, dass es sich bei RslT123 um einen ABC-Transporter für die Aufnahme einer Vorstufe handelt, welche für die Biosynthese von Rishirilid B benötigt werden. Als Startereinheit für die PKS-Synthese von Rishirilid B wurde Isobutyryl postuliert, welches als Derivat des Valin-Katabolismus beschrieben wird. Anhand dieser Postulate liegt die I. Summary / Zusammenfassung Vermutung nahe, dass RslT123 am Import von Aminosäuren beteiligt ist. Diverse Versuche der Aufreinigung des SBP zur Bestimmung dessen Substratspezifität schlugen fehl. Durch die Deletion und Überexpression von RslT4 sowie MarR Regulator RslR4 konnte ein Regulationsmechanismus der Rishirilid B–Biosynthese postuliert werden. Da die | 3 Deletionmutante ∆rslT4 jedoch Rishirilid überproduziert, übernehmen vermutlich andere Transporter von S. albus dessen Aufgabe. Die Fähigkeit des MFS Transporter RslT4, Antibiotika aus der Bakterienzelle auszuschleusen, wurde mittels MIC- und Agardiffusionstests bestimmt.

Zusätzlich wurde die Genomsequenz von Streptomyces sp. Tü6071 mittels der bioinformatischer Plattform Galaxy ausgewertet. Ziel war die Identifizierung von Transportergenen, wobei der Fokus auf solchen lag, die an der Biosynthese von Sekundärmetaboliten beteiligt sind. S. sp. Tü6071 produziert die Terpenoide Phenalinolacton A-D. Die Untersuchung der Genomsequenz lässt die Beteiligung der drei ABC Transportergene plaABC1-3 an derer Synthese vermuten. Die Inaktivierung von plaABC1 durch homologe Rekombination zeigte keinen Zusammenhang des ABC- Transporters mit der Phenalinolacton-Biosynthese. Allerdings konnte ein Einfluss von plaABC1 auf die Bildung des Luftmyzels von S. sp. Tü6071 gezeigt werden.

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II. Introduction

1. Natural products Natural products have been the main source of compounds for the development of drugs (Harvey et al. 2015). Plants and microbes provide most of the leads from natural products with promising activities useful in a broad range of therapeutic categories (Harvey 2008). Among the microbial area, the main sources have been fungi and terrestrial actinomycetes. There are more than 22000 known secondary metabolites of microbial origin, 70 % of which are produced by actinomycetes, 20 % from fungi, 7 % from Bacillus spp. and 1 – 2 % by other . Streptomycetes, a genus of the actinomycetes, are source of more than a half of over 10000 known antibiotics (Subramani & Aalbersberg 2012). The genetic capability of these soil microorganisms is enormous. The biosynthetic steps for each secondary metabolite are ruled by specific enzymes which are normally organized in clusters. They generally utilized intermediates of the primary metabolism as precursors to achieve the specific moieties of the secondary metabolites (Martin 1992). A big amount of bacterial secondary metabolites are synthesized by polyketide synthases, ribosomal and nonribosomal syntethases, terpene synthases or by a combination of these enzymes.

1.1. Polyketides Polyketide synthases (PKSs) are responsible for the synthesis of a broad range of natural products with pharmacological properties (O’Hagan 1992). The biosynthesis of these compounds includes sequential Claisen condensations of extender units derived from malonyl-coenzyme A (CoA) with an activated carboxylic acid starter unit. Depending on the starter and extender units, the length of the carbon chain, folding, reductions and termination determined by the PKS a variety of structures are reached. Post-PKS modifications by tailoring enzymes lead to glycosylation, acylation, alkylation and/or oxidation of the polyketide carbon chain. Bacterial PKSs are classified in three groups, II. Introduction type I – III, on the basis of molecular genetics, PKS structure and biochemistry (Hopwood 1997; Moore & Hopke 2001).

Type I PKSs are divided into two subgroups, the modular type I PKSs of bacteria and the iterative type I PKSs of fungi. Modular type I PKSs are exemplified with the biosynthesis of | 5 erythromycin, an antibiotic produced by Saccharopolyspora erythraea (Woodward et al. 1981) in Figure 1-1.

Figure 1-1 | Representation of the modular polyketide synthase of erythromycin, a type I PKS metabolite. Extracted from (Weber et al. 2015). The multifunctional enzyms DEBS 1 – 3 and its coding genes eryAI-III are drawn with thick arrows. The catalytic domains are represented in colored circles under the annotated modules. The domains include the ketosynthase (KS), acyl transferase (AT), acyl carrier protein (ACP), ketoreductase (KR), dehydratase (DH) and enoyl reductase (ER). The last module incorporates as well the terminal thiostherase (TE). The structures of the intermediate 6-deoxyerythronolide B and the final erythromycin A are also shown.

Modular type I PKSs consist of one or more multifunctional polypeptides organized in catalytic modules. Each module contains a set of non-iteratively active sites responsible for the catalysis of one cycle of chain extension. It includes three essential domains, a ketosynthase (KS), an acyltransferase (AT), and an acyl carrier protein (ACP). In addition, variable domains are associated for the modification of the keto group, for example ketoreductase (KR), dehydratase (DH) and enoyl reductase (ER) (Staunton & Weissman II. Introduction

2001). Type I PKS are also known to enclose a loading module with an ATL and ACPL activity.

The ATL (the loading AT) of this didomain accepts an acetate or propionate starter unit, commonly utilized by type I PKS, in an activated form, attached to coenzym A (CoA).

Following, the propionyl-CoA or acetyl-CoA are transferred to the ACPL (the loading ACP) 6 | which transferred the starter unit to the KS of the first module. The last intermediate is transferred to a thioesterase (TE) in the final module for release through hydrolysis or cyclization (Moore & Hertweck 2002). The iterative type I PKSs differ from the modular type I PKSs in the capacity of some of the constituent enzymes to catalyze successive steps of the cycle for the formation of the carbon chain.

Type II PKSs generally catalyze the formation of phenolic aromatic compounds. Figure 1-2 exemplifies this catalytic pathway with the biosynthesis of actinorhodin, a blue pigment produced by Streptomyces coelicolor A3(2).

Figure 1-2 | Representation of the type II PKS biosynthesis of actinorhodin. Extracted from Kim and Yi (2012). The KS domain is represented by a KS subunit (in yellow) and the CLF (in green) and catalizes the initiation of the chain and condensation through Claisen condensations. The activated malonyl-CoA is supplied by an ACP transacylase, MCAT (purple) to the ACP (blue) which delivers it to the KS heterodimer. The KR domain reduces specific carbonyl groups for aromatization and cyclation of the carbon chain. Varios tailoring enzymes modified the aromatic structure to yield the final structure.

The biosynthetic reactions lead to a polyketone chain by repeated decarboxylative condensations (Claisen condensations). These elongations of the carbon chain are catalyzed

by three iterative independent proteins, KSα, KSβ and ACP (minimal PKS). The KS domain II. Introduction of type II PKSs consists of a heterodimer formed by a KSα subunit (active site) and a KSβ or chain length factor (CLF). CLF subunit is an important determinant of the acyl chain limiting the number of extender units that it incorporates (Fischbach & Walsh 2006). The

KSα-CLF dimer catalyzes the chain initiation and it grows by two or three carbons in each | 7 cycle while remains anchored to the same ACP domain (Staunton & Weissman 2001). The polyketone chain is modified by tailoring enzymes through carbonyl reduction of carbonyl groups followed by cyclization and aromatization processes to result in an aromatic compound. The precursor is released from the ACP and can be further modified by glycosyltransferases and other various tailoring enzymes to generate the final metabolite (Kim & Yi 2012). In general, aromatic polyketides are synthesized from an acetyl-CoA starter unit although well-known compounds like actinorhodin utilized malonyl-CoA (Figure 1-2). The malonyl-CoA is decarboxylated to yield and acetyl-S-KS intermediate which is further elongated by the minimal PKS (Moore & Hertweck 2002).

Type III PKSs, also known as chalcone synthase-like PKS, lead normally to monocyclic or bicyclic aromatic polyketides. They consist of homodimeric enzymes where a single active site is used iteratively for the loading of the start unit, each Claisen condensation step, and the final off-loading/cyclization of the polyketide chain. They do not utilize substrates covalently linked to an ACP, like type I - II PKSs, but condense malonyl-CoA derivatives with acyl-CoA esters (Chemler et al. 2012).

Rishirilide B, a type II PKS secondary metabolite from Streptomyces bottropensis Goe C4/4 Rishirilide A and its precursor rishirilide B (Figure 1-3) were firstly isolated in 1984 from the fermentation broth of Streptomyces rishiriensis ORF-1056 by Iwaki et al. (1984). In 2002, Arnold isolated rishirilide A and mensacarcin (Figure 1-3) from different production cultures of Streptomyces bottropensis Goe C4/4. This strain was identified from a soil sample nearby the canteen of the Universtiy of Göttingen, Göttingen (Arnold 2002). II. Introduction

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Figure 1-3 | Structures of mensacarcin and rishirilides A and B, type II PKS secondary metabolites produced by S. bottropensis Goe C4/4 (Arnold 2002; Linnenbrink 2009)

Linnenbrink (2009) constructed a genomic cosmid library of S. bottropensis Goe C4/4 in order to identify and heterologously express the secondary metabolites gene clusters. Rishirilide gene cluster was identified in the cosmid named cos4 and its heterologous expression in Streptomyces albus J1074 led to a robust production of rishirilide B (Yan et al. 2012).

Rishirilide B is a type II PKS metabolite with an anthracene structure consisting of a tricyclic, partially aromatic backbone and a C-4 isopentyl side chain. The compound showed

antithrombotic activity by a selective inhibition of α2-macroglobulin (Iwaki et al. 1984). This mode of action is interesting for the treatment and prevention of thrombosis by fibrinolytic accentuation (Aoki 1979). In addition, rishirilide B was described for its glutathione S-transferase inhibitory activity (Komagata et al. 1992). This mechanism has a potential therapeutic utility in combination with anticancer drugs due to the enhancement of the chemotherapy resistance.

Rishirilide gene cluster contains 28 putative ORFs spaning 35 kb. It includes 20 structural genes which showed high homology to known aromatic PKS genes and 10 putative oxidoreductases for the post-PKS modifications. In addition, four regulatory genes and four transporter genes were annotated (Yan et al. 2012) which have been the spotlight of the thesis.

1.2. Terpenoids Among the secondary metabolites, terpenes represent one of the largest and most diverse class of secondary metabolites (Breitmaier 2006). Terpenes show a variety of chemical structures including linear functionalized hydrocarbons or chiral, carbocyclic skeletons with a diverse chemical modification. Those terpenes with functionally modified forms are II. Introduction commonly named terpenoids or isoprenoids (Harrewijn et al. 2002). More than 55000 terpenes have been isolated and include a variety of activities such hormones, pigments, and communication and defense mechanisms like toxins (Ajikumar et al. 2008). These compounds are known to be widespread in plants and fungi but an increasing number of | 9 terpene synthases are being identified in bacteria thanks to bioinformatics analysis (Yamada et al. 2015). The huge majority of Streptomyces spp. produce geosmin, a C12 degraded sesquiterpene alcohol. However, the number of terpenoid natural products in Streptomyces spp. is limited. Terpenoids are synthesized by consecutive condensations of a five-carbon- unit isopentenyl pyrophosphate (IPP) and its isomer, dimethylallyl diphosphate (DMAPP) (Figure 1-4). For many years, it was believed that these compounds were only achieved by the mevalonate pathway (Figure 1-4). However, an alternative mevalonate-independent pathway (Figure 1-4) was proved by (Kuzuyama & Seto 2003). Streptomyces spp. are described to utilize the nonmevalonate pathway, also named methylerythritol phosphate (MEP) or deoxyxylulose-5-phosphate (DOXP) pathway, on their primary metabolism. In addition, some of them are able to synthesize secondary metabolites thanks to the mevalonate pathway.

Following the formation of the IPP, three prenyltransferases generate the direct precursors of the terpene structure, geranyl diphosphate (GPP, C10), farnesyl diphosphate (FPP, C15) and geranylgeranyl diphosphate (GGPP, C20). Subsequently, the terpene synthases (TPS) catalyze the formation of hemiterpenes (C5), monoterpenes (C10), sesquiterpenes (C15) or diterpenes (C20) from the substrates DMAPP, GPP, FPP or GGPP, respectively (Tholl 2006) (Figure 1-3). The terpene structure is then modified by secondary enzymatic reactions like hydroxylation, reduction or glycosylation. II. Introduction

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Figure 1-4 | Representation of the mevalonate pathway and the nonmevalonate pathway for the synthesis of IPP and the following reactions for the terpene biosynthesis. Both pathways lead to the formation of isopentenyl pyrophosphate (IPP) and its isomer dimethylallyl pyrophosphate (DMAPP). Mevalonate pathway uses as initial substrates acetyl-CoA and acetoacetyl-CoA while MEP requires pyruvate and glycealdehyde polyprenyl diphosphate (PPP). Abbreviations: HMG-CoA = hydroxymethylglutaryl-CoA; MVA = mevalonate; MVAP = 5-phosphomevalonate; MVAPP = mevalonate-5-diphosphate; DOXP = deoxyxylulose-5- phosphate; MEP = methylerythritol phosphate; CDP ME = 4-diphosphocytidyl methylerythritol; CDP MEP = 4- diphosphocytidyl methylerythritol phosphate; MEcPP = methylerythritol cyclodiphosphate; HMBPP = hydroxymethylbutenyl 4-diphosphate.

Phenalinolactone, a terpenoid metabolite from Streptomyces sp. Tü6071 Streptomyces sp. Tü6071 was first isolated from a soil sample in Cape Coast, Ghana on a glycerol – arginine agar (El-Nakeeb & Lechevalier 1963). The strain was characterized and described to produce streptocidins A-D by Gebhardt, Pukall, and Fiedler (2001). Later, Gebhardt (2002) identified four new terpene glycoside antibiotics, phenalinolactones A-D, produced by this strain and demonstrated their activity against Gram-positive microorganisms. The structures of phenalinolactones A-D were elucidated by Meyer (2003) (Figure 1-5). II. Introduction

| 11

Figure 1-5 | Structures of phenalinolactones A-D (1-4) (Meyer 2003)

The gene cluster responsible of their production was annotated by Dürr et al. (2006) and since then further research to elucidate the biosynthetic pathway of the compounds has been done (Binz et al. 2008, Daum et al. 2010, Gebhardt et al. 2011). Phenalinolactone A-D consist of a tricyclic anti/anti/syn-configured perhydrophenanthrene backbone. It has been proposed that it is formed by condensation of four isoprene units, where the fourth unit comprises the β carbon of a singular γ-butyrolactone moiety. Phenalinolactone gene cluster contains two genes which showed high similarity to the HMBPP (hydroxymethylbutenyl 4- diphosphate) and DOXP (deoxyxylulose-5-phosphate) synthases from the nonmevalonate pathway (Figure 1-4). These findings seemed to be the first example of this kind of genes located in a secondary metabolite gene cluster (Dürr et al. 2006).

2. Ins and outs across the membranes

2.1. Bacterial membranes All living organisms need membranes to stay alive. Membranes control the movement of substances into and out the cells by being selectively permeable. They provide the cells with a boundary to the extracellular space (plasma membranes) as well as with inner compartments (organelles). Moreover, membranes play an active role in cell life providing transduction systems to sense the environment and to communicate with other cells. They are also involved in the capture and release of energy, e. g. photosynthesis and oxidative phosphorylation (Brown 1996).

Bacteria are distinguished in two classes based on the capacity of their cell walls to retain a crystal-violet dye, a method that was stablished by Hans Christian Gram over 100 years ago. Gram-positive cell walls are composed of a thick peptidoglycan layer (20 – 80 nm) outside II. Introduction the cytoplasmic membrane and include teichoic and lipoteichoic acids (Figure 2-1 A). In contrast, Gram-negative cell walls consist of a thin peptidoglycan layer (1 – 7 nm) in-between the outer and inner membranes creating a periplasmic space. The outer membrane and the peptidoglycan layer are connected though lipoproteins providing a solid structure for the 12 | maintenance of the cell shape (Figure 2-1 B) (Cabeen & Jacobs-Wagner 2005).

A)

B)

Figure 2-1 | Representation of Gram-positive and Gram-negative cell wall. Extracted from Cabeen and Jacobs-Wagner (2005). A) The cell wall of Gram-positive bacteria contains a thick peptidoglycan layer over a cytoplasmic membrane. The peptidoglycan layer incorporates teichoic acids and lipoteichoic acids which extend to the cytoplasmic membrane. B) The cell wall of Gram-negative bacteria consists of a thin peptidoglycan layer in a periplasmic space surrounded by an outer and a cytoplasmic membranes. The peptidoglycan layer is linked to the outer membrane through lipoproteins. The outer membrane includes lipopolysaccharides which extend to the extracellular space and porins which create a channel through the membrane to the periplasmic space.

For the control of substrates across the cytoplasmic membrane, nature has developed a variety of systems which can be divided in channels and transporter proteins. Channels catalyze facilitated diffusion (by an energy-independent process) through a transmembrane pore. This diffusion-limited transport of the substrate occurs without evidence for a carrier- mediated mechanism. It implies a weak interaction between the membrane protein and the substrate. In contrast, transporter proteins undergo conformational changes to achieve the translocation of the specific substrates (Lodish et al. 2000). II. Introduction Bacterial transport proteins include “primary” transporters, “secondary” transporters and “group translocation” systems. Primary transporters comprise P-type ATPases and ATP- binding cassette (ABC) transporters which drive ions and solutes, respectively, across the membrane. For their operation, these transporters require energy which is generated from | 13 the hydrolysis of ATP. Secondary transporter conduct the substrate translocation thanks to the free energy stored in the ion or solute gradients generated by primary transporters. This group include one of the largest family of transporters, the major facilitator superfamily (MFS). Group translocation transport systems function by coupling the translocation of a substrate to its chemical modification. This translocation results in a modified substrate released to the other side of the membrane (Law et al. 2008). ABC and MFS transporters represent the main focus of this work and are therefore described more in detail.

2.2. ATP-binding cassette transporters ATP-binding cassette (ABC) transporters couple the energy of ATP hydrolysis to the translocation of specific substrates across the cytoplasmic membrane. They contain a highly conserved ATP binding cassette which is the most characteristic feature of this family (Higgins et al. 1986). ABC transporters are classified into importers and exporters depending on their architecture and mechanism. Exporters move substrates from the cytosolic side of the membrane to the extracellular or periplasmic space as opposed to importers. All ABC transporters have a core consisting of four functional units including two ATP or nucleotide binding domains (NBD) and two transmembrane domains (TMD). In addition, importers require an accessory domain for the recognition and delivery of the transported substrate to the TMD. In Gram-negative bacteria these substrate binding proteins (SBP) are soluble in the periplasmic space. In Gram-positive bacteria, due to the lack of an outer membrane to retain the SBP, it is necessary a linkage of the protein to the cytoplasmic membrane. This phenomenon can take place via lipid anchor or a transmembrane peptide. Alternatively, the SBP can be fused to the TMD. In bacteria, all variety of combinations of genes encoding the TMDs, NBDs and the SBP (in case of importers) have been described (Figure 2-2). II. Introduction A)

14 | B)

C)

Figure 2-2 | Representation of the different architecture of the ABC transporters. Modified from Biemans-Oldehinkel, Doeven, and Poolman (2006). The SBP is indicated with a moon shape, the TMD with squares and the NBD with ovals. A) Organization of importers in Gram-negative bacteria. For each transporter system, different upper-case letters indicate that the proteins are encoded by different genes. B) Organization of importers in Gram-positive bacteria. For each transporter system, different upper-case letters indicate that the proteins are encoded by different genes. C) Organization of exporters in Gram-positive and Gram-negative bacteria. For each transporter system, different upper-case letters indicate that the proteins are encoded by different genes.

Importers are divided in type I and type II based on the substrate uptake and the transmembrane segments of the TMDs. Type I are responsible for the transport of ions, sugars, amino acids and other small substrates. The transport depends on the specific binding to the SBP which deliver substrates to the TMDs. Each TMD involves the formation of 6 transmembrane helixes leading to a total of 12 in the ABC system. Type II importers consist of 10 helixes in each TMD generating a total of 20 transmembrane segments. They mediate the transport of metal chelates, larger substrates compared to those of type I ABC importers. No defined binding pockets have been described in the TMDs of type II importers suggesting that all specificity of the translocation depends on the SBP (Locher 2009).

Although a conserved architecture of two TMDs and two NBDs has been identified among all ABC transporters, an agreed mechanism has been long discussed (van der Heide & Poolman 2002; van der Does & Tampé 2004; Biemans-Oldehinkel et al. 2006; Locher 2009). A raising consensus model encloses four steps which is exemplified in Figure 2-3. II. Introduction

| 15

Figure 2-3 | Representation of the transport mechanism of a Gram-negative ABC importer. Modified from Locher (2009). The SBP binds to the substrate and interacts with the closed conformation of the TMDs and signals the NBDs to bind ATP. A conformational change of the TMDs exposes a binding site to where the substrate is transferred. The ATP hydrolysis leads to the dissociation of the NBD dimer and the substrate is translocated to the cytosol.

First the SBP recognizes the substrate and the complex substrate-SBP interacts with the closed conformation of the TMDs. This sends a signal to the NBDs to cooperatively bind ATP leading to a change on the TMDs which exposes a binding site to the outside. The SBP transfers the substrate to the binding site and hydrolysis of ATP takes place. This yields to the dissociation of the NBD dimer what causes the inward-facing conformation of the TMDs. The substrate reaches the cytosolic space and the SBP is then released from the TMDs (Biemans-Oldehinkel et al. 2006).

ABC transporters can participate in a wide variety of cellular processes. They are involved in nutrient uptake, secretion of signal peptide independent proteins, transport across intracellular membranes, regulation, and drug resistance (Higgins 1992).

2.3. Major facilitator superfamily transporters Transporters of the major facilitator superfamily (MFS) represent one of the largest group of secondary transporters. The number of families for the classification of MFS transporter based on their function and phylogeny, have been raising over the time. They were first divided into 17 families (Pao et al. 1998) and expanded to 29 (Saier et al. 1999) and 74 (Reddy et al. 2012). Nowadays (June 2015), the MFS includes 82 families which transport a set of related compounds (Saier et al. 2014). They are responsible of the transport of specific compounds like sugars, nucleotides, drugs, , neurotransmitters and siderophores. Furthermore, they cause the efflux of multiple structurally unrelated drugs (Saier et al. 1999; Reddy et al. 2012).

The transport mechanism display three different kinetics, uniport, symport and antiport (Figure 2-4). Uniport implies the transport of one single solute through the membrane along II. Introduction its gradient. Symport and antiport are co-transporter that move two solutes at the same time, one against its gradient and the other along its gradient. In symport the molecules move in the same direction while in antiport they do it in the opposite.

16 |

Figure 2-4 | Representation of the uniport, symport and antiporter mechanisms of transporters. The triangles show the gradient of the substrates transported.

The drug efflux is only associated with antiport which couples the transport of the drug and the electrochemical gradient of protons or sodium ions across a membrane in opposite directions (Law et al. 2008). Well described multidrug transporters belong to three different drug:H+ antiporter families named DHA1-3. Examples of these families include the EmrD and MdfA (DHA1) multidrug transporters identified in E. coli (Adler & Bibi 2004; Yin et al. 2006). QacA (DHA2) was described as a multidrug efflux protein of which mediates the resistance to a wide range of antimicrobial compounds (Brown & Skurray 2001). Moreover, MefA (DHA3) identified in Streptococcus pyrogenes is responsible for the macrolide efflux (Clancy et al. 1996).

MFS transporter share a conserved common structure consisting of two symmetric groups of 6 helixes (N and D domain) encoded by a single polypeptide. Some exceptions have been described with 6, 14 or 24 TMD (Pao et al. 1998). For the translocation of the substrates, three conformational states are needed in the MFS transporter: outward-open for substrate upload, substrate-bound occluded, and inward-open for substrate release. This mechanism implies the exposure of the binding site to both sites of the membrane during one transport cycle (Jardetzky 1966). Therefore, the architecture of an MFS antiporter was proposed to require (i) a gating mechanism which is able to open and close. This conformational change is promote by the interaction with the substrate and H+ or Na+ ions. (ii) A substrate binding site which can alter its affinity state. And (iii) a H+ or Na+ transfer site which markedly enhances the substrate binding affinity (Yamaguchi et al. 1993). This mechanism have been sustained by the crystallization of several MFS transporters in different conformational states (Shi 2013). The substrate binding site is located between the N and D domain which create a central cavity around TMD 1, 4, 7 and 10. These transmembrane regions provide the majority of residues essential for substrate coordination and co-transport coupling (Law et II. Introduction al. 2008). Recently, Masureel et al. (2014) have proposed a detailed transport cycle of LmrT, a multidrug transporter from . Thanks to double electron-electron resonance measurements they investigated how protons and ligands shift this equilibrium to enable transport. Figure 2-5 shows their proposed mechanism where the translocation of | 17 protons involves protonation and deprotonation of specific residues (Glu and Asp) of the central cavity.

Figure 2-5 | Proposed mechanism for the translocation of substrate/H+ of LmrP transporter. Extracted from (Masureel et al. 2014). The figure shows only 6 TMD of LmrP and the extracellular side is represented on the top. In the resting state the transporter is outward-closed but allows the entry of the substrate to the central cavity. The binding to the active site results in the outward-open form permitting the proton entrance. The protons protonate the carboxylic residues of the Glu which were coordinated with the substrate leading to its release. The protons migrate to the Asp what yields to an inward-open form allowing the deprotonation of the residues and the reset of the resting state.

3. Aim of this work The aim of this project was to identify the role that transporter proteins play in the biosynthesis of natural products.

The biosynthetic gene cluster of rishirilide B, a type II PKS metabolite produced by Streptomyces bottropensis Goe C4/4, contains four transporter genes. They encode an ABC and a MFS transporter, which were investigated for elucidation of their structure, function and influence in rishirilide B production.

Furthermore, the genome sequence of Streptomyces sp. Tü6071 was analyzed for identification of transporter genes. An ABC transporter was investigate for its relation with the produced secondary metabolites phenalinolactones A-D.

18 |

III. Material and methods

1. Material Detailed information about the companies that provided this work with laboratory equipment, analytical instruments, chemicals, reagents and other substances is summarized in Table 1.1-1. Following reference to the companies on this thesis is done with abbreviated names.

1.1. General manufacturer information

Table 1.1-1 | Detailed information of manufacturers and abbreviation

Abbreviation Detailed information Agilent Agilent Technologies, Santa Clara, USA Amersham Biosciences Europe GmbH (GE Healthcare), Amersham Freiburg, Germany Analytik Jena Analytik Jena Aktiengesellschaft, Jena, Germany Andreas Hettich Andreas Hettich GmbH & Co.KG, Tuttlingen, Germany AppliChem AppliChem, Darmstadt, Germany Applied Bio Applied Biosystems, Carlsbad, USA Beckmann Beckmann Coulter, Krefeld, Germany Bioline Bioline GmbH, Luckenwalde, Germany Bio-Rad 1 Bio-Rad Laboratories GmbH, Munich, Germany Bio-Rad 2 Bio-Rad Laboratories, Inc., California, USA Biotium Biotium Inc., Hayward, USA BMG BMG LABTECH GmbH, Ortenberg, Germany Braun Braun, Melsungen, Germany Büchi Büchi Labortechnik AG, Flawil, Switzerland Carl Zeiss Carl Zeiss, Jena, Germany Cell Signaling Cell Signalling Technology, Inc., Danvers, USA Difco Difco, Voigt Global Distribution Inc., Lawrence, USA Eppendorf Eppendorf, Wesseling-Berzdorf, Germany III. Material and methods

Abbreviation Detailed information GE Healthcare GE Healthcare UK Ltd, Buckinghamshire, UK Gilson Gilson, Inc., Middleton, USA Glycosynth Glycosynth Limited, Warrington, UK Greiner Greiner Bio-One BioScience, Frickenhausen, Germany | 19 Heidolph Heidolph Instruments GmbH & Co. KG, Schwabach, Germany Helmut Helmut Saur Laborbedarf, Reutlingen, Germany Infors Infors AG, Bottmingen, Switzerland KPL KPL, Inc., Maryland, USA Merck Merck, Darmstadt, Germany NEB New England Biolabs Inc., Ipswich, USA Peqlab Peqlab, VWR International GmbH, Erlangen, Germany Pharmacia Pharmacia, Freiburg, Germany Promega Promega, Madison, USA Qiagen Qiagen, Hilden, Germany Roth Carl Roth GmbH & Co. KG, Karlsruhe, Germany Sartorius analytic, Altmann Analytik GmbH & Co. KG, Sartorius Munich, Germany Schoenenberger W. Schoenenberger GmbH & Co. KG, Magstadt, Germany Schott Schott Instruments, SI Analytics GmbH, Mainz, Germany Sigma Sigma-Aldrich Co., Seelze, Germany Stratagene Stratagene, Heidelberg, Germany Südzucker Südzucker, Mannheim, Germany Systec Systec GmbH, Labor-Systemtechnik, Wettenberg, Germany Thermo 1 Thermo Fisher Scientific Inc., Wilmington, USA Thermo 2 Thermo Spectronic, Wuppertal, Germany Thermo 3 Thermo Electron Corporation, Vantaa, Finland TPP TPP Techno Plastic Products AG, Trasadingen, Switzerland Waters 1 Waters, Milford, USA Waters 2 Waters, Eschborn, Germany X-Gluc Direct X-Gluc Direct, www.x-gluc.com, UK

1.2. Laboratory material, equipment and analytical instruments

Table 1.2-1 | Laboratory material and manufacturer

Laboratory material Manufacturer Blotting papers Rotilabo®, thickness 0.35 mm Roth Centrifuge tubes 15 mL and 50 mL Roth and TPP Electroporation cuvette, 0.1 cm gap, sterile Sigma Filter paper, Solvent Filter, Supor PES, 47 mm caliber, 0.2 μm Waters 1 pore size III. Material and methods

Laboratory material Manufacturer Immun-Blot® PVDF membrane for protein Blotting Bio-Rad 2 Insuline syringe Omnican® F, 2 mL Braun Micro centrifuge tubes 1.5 mL and 2 mL (eppi) Roth and Eppendorf Multichannel digital pipette, Finnpipette® Thermo 3 20 | Petri dishes Rotilabo®, diameter 10 cm Roth Pipette 2 µL, 20 µL, 200 µL, 1000 µL, Pipetman classicTM Gilson Protein concentration tube, Corning® Spin-X® UF concentrators Sigma PS-Microplate, V-bottom, clear, 96 well Greiner Syringe filter Rotilabo®, 0.45 µm, PVDF Roth Syringe Injekt®, 10 mL Braun Syringe sterile filter Rotilabo®, sterile, 0.2 µm, PVDF Roth

Table 1.2-2 | Laboratory equipment and manufacturer

Equipment Manufacturer Autoclave, Tuttnauer Systec 5075 ELV Systec Centrifuge 5417R and 5415R Eppendorf Centrifuge Avanti J-6000, Rotor JA-10 Beckmann Centrifuge Rotina 35 R Andreas Hettich Electrophoresis Power Supply EPS 601 Amersham Electroporation E. coli PulserTM Bio-Rad 1 French® Pressure Cell Press Thermo 2 Microscope Carl Zeiss PCR Cycler, 2720 Thermal Cycler Applied Bio PCR Mastercycler, epgradient Eppendorf Protein electrophoresis chamber and power supply Bio-Rad 1 Rota vapor ROTAVAPOR-R and LABOROTA 4000 Büchi and Heidolph Shaker Multitron HT, Typ AJ 118 Infors Thermomixer Eppendorf UV Transilluminator Image Master VDS (312 nm) Peqlab Illuminator, Pharmacia; UV Transilluminator with CCD camera Camera, Stratagene Vacuum concentrator BA-VC-300 U Helmut Vortex Reax Top Heidolph Weighing scales Sartorius

III. Material and methods

Table 1.2-3 | Analytical instruments and manufacturer

Instrument Manufacturer ÄktaTMFPLC (Fast Protein Liquid Chromatography) Column: HisTrap FF 5 mL Column material: Ni Sepharose (Agarose) 6 Fast Flow | 21 Column particle size: 90 μm diameter Column volume: 5 mL Amersham UV detector: 280 nm Fraction collector: FRAC-901 Pump: P-920 Monitor: UPC-900 Preparative HPLC Automatic injection system: 717 plus Autosampler Column oven: Waters Jetstream 2 (23 °C) Waters 2 Pre-column: 50 mm x 9.4 mm; Particle size: 5 μm

Column: 150 mm x 9.4 mm; Particle size: 5 μm Two pumps with control module: Waters 515 HPLC Pump Detector: Waters 2996 Photodiode Array Detector (λ = 254 nm) LC/MS Autosampler: G1313A TM Pre-column: XBridge C18 (20 mm × 4.6 mm; Particle size: 3.5 μm) TM Column: Xbridge C18 (100 mm × 4.6 mm; Particle size: 3.5 μm) Agilent Degasser: G1322A Quarternary pump: G1311A Diode array detector (DAD) G1315B (λ = 254 nm and 400 nm) Quadrupole mass detector (MSD) G1946D (2 – 3000 m/z) NanoDrop 2000 Spectrophotometer Thermo 1 pH meter, pH electrode BlueLine 14 pH Schott SPE column Oasis® HLB 20 35 cc (6g) Waters Ultrospec 2100 pro UV/Visible Spectrophotometer Amersham

1.3. Chemicals and reagents

Table 1.3-1 | General chemicals, reagents and manufacturer

Chemical/Reagents Manufacturer 1 kb DNA Ladder Promega and NEB 1,4-Dithiothreit (DTT) Roth 5-Bromo-4-chloro-3-indolyl phosphate disodium salt (BCiP) Roth 5-Bromo-4-chloro-3-indolyl-β-D-galacto-pyranoside (X-Gal) Roth 5-Bromo-4-chloro-3-indolyl-β-D-glucurone acid (X-Gluc) X-Gluc Direct Acetic acid Roth Acrylamide Rotiphorese® Gel 30 Roth III. Material and methods

Chemical/Reagents Manufacturer Adenine ribonucleotide triphosphates (rATP) NEB Agar-Agar Roth Agarose Roth Ammonium peroxydisulfate (APS) Roth 22 | Bovine Serum Albumin (BSA) Promega and NEB Bromophenol blue sodium salt Roth CASO broth – TSB medium Roth Chloric acid Roth CutSmart buffer NEB Deoxynucleotide (dNTP) solution mix NEB

Disodium hydrogen phosphate (Na2HPO4) Merck D-Mannitol Roth Ethidium bromide Roth Ethylenediaminetetraacetic acid disodium salt (EDTA) Roth GelRed Nucleic Acid Gel Stain 10000X in water Biotium Roth Glycerol Roth Imidazol Roth Isopropyl-β-D-thiogalactopyranoside (IPTG) Roth L-Arabinose Roth LB-Medium, Lennox Roth Malt extract Roth Methylene blue Merck N, N, N´,N´-Tetramethylethan-1,2-diamin (TEMED) Roth Natriumdihydrogenphosphate monohydrate Roth n-Dodecyl-β-D-maltopyranoside (DDM) Roth Ni-NTA agarose Qiagen P7702S protein marker, broad range (2–212 kDa) Biolabs p-Nitrophenyl-β-D-glucuronid Glycosynth p-Nitrotetrazolium blue chloride (NBT) Roth Potassium acetate (KAc) Roth Powdered milk Roth Protease Inhibitor Cocktail Promega Protein-Marker IV Prestained Peqlab Roti®-Mark Standard Roth Sodium chloride (NaCl ) Roth Sodium dodecyl sulfate (SDS) Roth Sodium hydroxide (NaOH) Roth Soluble starch Difco Soy flour, Hensel Voll-Soja Schoenenberger Sucrose Südzucker III. Material and methods

Chemical/Reagents Manufacturer Tris(hydroxymethyl)-aminomethan (TRIS) Roth TRIS-HCl Roth

Triton-X 100 Roth Trypton/Pepton from Casein Roth | 23 Tween® 1000 Roth Tween® 20 Roth Xylene cyanol Merck Yeast extract Roth

Table 1.3-2 | Organic solvents and manufacturer

Organic solvent Manufacturer Acetone Roth Acetonitrile (ACN) Roth Chloroform Roth Dichloromethane (DCM) Roth Dimethyl sulfoxide (DMSO) Roth Dimethylformamide (DMF) Roth Ethanol (EtOH) Roth Ethyl acetate Roth Isopropanol Roth Methanol HPLC quality (MeOH) Roth

Table 1.3-3 | Antibiotics and manufacturer

Antibiotic Manufacturer Ampicillin sodium salt Sigma Apramycin sulfate salt AppliChem Bacitracin Sigma Carbenicillin disodium salt Roth Sigma Gentamycin sulfate salt Roth Hygromycin B Roth Kanamycin sulfate salt Roth Nalidixic acid sodium salt Sigma Norfloxacin Sigma Novobiocin sodium salt Sigma Phosphomycin disodium salt Sigma Spectinomycin dihydrochloride pentahydrate AppliChem sulfate salt Sigma hydrochloride Roth III. Material and methods

Antibiotic Manufacturer Thiostrepton Sigma Sigma

24 | 1.4. Enzymes, antibodies and kits

Table 1.4-1 | Enzymes and manufacturer

Enzyme Manufacturer Antarctic Phosphatase NEB DNase NEB Lysozyme Roth Pfu polymerase Purified by P. Schneider Phusion polymerase Purified by S. Maier Proteinase K Promega Restriction enzymes Promega, NEB RNase A Qiagen T4-DNA-Ligase 1 Promega T4-DNA-Ligase 2 NEB Taq polymerase Purified by P. Schneider TM Velocity DNA Polymerase Bioline

Table 1.4-2 | Antibodies and manufacturer

Antibody Manufacturer

His6-Tag monoclonal antibody from mouse IgG1 Roche Anti-Mouse IgG (H+L) Antibody, Human Serum Adsorbed and KPL Peroxidase Labeled

Table 1.4-3 | Kits and manufacturer

Kit name Manufacturer innuPREP Plasmid Mini Kit Analytik Jena Pure YieldTM Plasmid Midiprep System Kit Promega Pure YieldTM Plasmid Miniprep System Kit Promega Rapid DNA Dephos & Ligation Kit Roche AG Wizard®SV Gel and PCR Clean-up System Kit Promega

III. Material and methods 1.5. Solutions and buffers Solutions and buffers used in this work are organized according to the experiments performed in the following tables (Table 1.5-1 to Table 1.5-12). Composition and relevant remarks for their preparation and/or conservation are included. Unless indicated, distillated | 25 water was used as solvent. Sterile solutions were achieved by sterile filtration with syringe sterile filter Rotilabo® (Table 1.2-1) or by autoclave (20 min, 121 °C, and 1 bar).

Table 1.5-1 | Solutions and buffers used for agarose electrophoresis and DNA visualization

Buffer/solution Composition Notes Agarose 0.7 % (m/V) Boiled for solving agarose and store at Agarose in 1x TAE buffer 60 °C Ethidium Protect from light. Submerge the gel Ethidium bromide 1 μg/mL bromide bath 20 – 30 min depending on the thickness GelRedTM 1x staining Protect from light. Submerge the gel GelRed bath solution 20 – 30 min depending on the thickness DNA fragments of 10 kb, 8 kb, 6 kb, 5 Ladder 1 1 kb NEB ladder 5 µL/lane kb, 4 kb, 3 kB, 2 kb, 1.5 kb, 1 kb and 0.5 kb Xylene cyanol 0.25 % (m/V) Visible cloud on the 0.7 % agarose gel Loading Sucrose 40 % (m/V) migrating like a 5 kb DNA fragment. buffer 1 in TE buffer pH 7.6 Storage at 8 °C Bromophenol blue 0.25 % Visible cloud on the 0.7 % agarose gel Loading (m/V) migrating like 1 kb DNA fragment. buffer 2 Sucrose 40 % (m/V) Storage at 8 °C in TE buffer pH 7.6 Agarose gels stained for 5 min in the Methylene Methylene blue 0.2 % (m/V) bath. Distained in water until bands blue bath were visible TRIS 40 mM TAE buffer EDTA 1 mM Adjusted to pH 8 with acetic acid Acetic acid (96 % V/V) TRIS 10 mM TE buffer Adjusted to pH 7.6 with HCl 1 M EDTA 1 mM

Table 1.5-2 | Solutions and buffers used for polymerase chain reaction (PCR)

Solution/buffer Composition Notes DMSO DMSO 50 % (V/V) 100μL of dATP, dCTP, dGTP and Final concentration of each dNTPs mixture dTTP (100 mM) dNTP 10 mM. in 1 mL water final volume Storage at -20 °C III. Material and methods

Solution/buffer Composition Notes TRIS-HCl (pH 8.8) 200 mM KCl 100 mM (NH4)2SO4 100 mM Pfu buffer MgSO4 20 mM 26 | Triton® X-100 1 % (V/V) BSA 1 mg/mL TRIS (pH 9.0) 100 mM KCl 500 mM Taq buffer MgCl2 15 mM Triton® X-100 1 % (V/V)

Table 1.5-3 | Solutions and buffers used for genomic DNA isolation of Streptomyces

Solution/Buffer Composition Notes Chloroform Chloroform Ethanol solution Ethanol 70 % (V/V) Storage at -20 °C Isopropanol Isopropanol Storage at -20 °C Lysozyme solution Lysozyme 50 mg/mL Storage at -20 °C

NaCl2 solution NaCl2 5 M Autoclaved Proteinase K solution Proteinase K 20 mg/mL Storage at -20 °C RNase solution RNase 1 μg/mL Storage at -20 °C SDS solution SDS 10 % (m/V) Autoclaved TRIS-HCl 20 mM SET buffer EDTA 25 mM Adjusted to pH 8 NaCl 75 mM Sucrose solution Sucrose 25 % (m/V) Autoclaved

Table 1.5-4 | Solutions and buffers used for plasmid DNA isolation of E. coli (Alkaline lysis)

Solution/Buffer Composition Notes Ethanol solution Ethanol 70 % (V/V) Storage at -20 °C Isopropanol Isopropanol Storage at -20 °C TRIS 30 mM Adjusted to pH 8. After P1 Alkaline Lysis EDTA 3 mM autoclave add RNAse. Storage RNAse A 100 µg/mL at 8 °C Preparation of NaOH and SDS NaOH 200 mM P2 Alkaline Lysis solutions independently. Mix SDS 1 % (m/V) 1:1 afterwards. No autoclaved Adjusted to pH 5.2 with acetic P3 Alkaline Lysis KAc 3 M acid and stored at 8 °C

III. Material and methods

Table 1.5-5 | Solutions and buffers for SDS-PAGE analysis

Solution/Buffer Composition Notes TRIS-HCl 60 mM (pH 8.6) Glycerol 10 % (m/V) 2 x SDS-PAGE Mixed 1:1 with the sample SDS 5 % (m/V) loading buffer to be analyzed | 27 Bromphenol blue 0.02 % (m/V) DTT 100 mM Coomassie Brilliant Blue R-250 2.5 g/L Gels stained for 30 – 60 brilliant blue Ethanol 450 mL/L minutes tracking dye Acetic acid 100 mL/L Distaining Ethanol 450 mL/L Gels distained for 60 – 90 solution Acetic acid 100 mL/L minutes Myosin (beef) 212 kDa; ß-galactosidase (rec. E. coli) 118 kDa; serumalbumin (beef glycosylated) 66 kDa; ovalbumin Roti®-Mark Standard. 8 µL Protein ladder 1 (chicken) 43 kDa; carbonic anhydrase per lane were used 29 kDa; Trypsin-inhibitor (soya) 20 kDa; Lysozym (chicken) 14 kDa Ten recombinant proteins of 170 kDa, peqGOLD protein marker 130 kDa, 100 kDa, 70 kDa (orange Protein ladder 2 IV Prestained. 7 µL per colored), 55 kDa, 40 kDa, 35 kDa, 25 lane were used kDa, 15 kDa and 10 kDa (green colored) Myosin (rabbit muscle) 212 kDa; MBP-β-galactosidase (E. coli) 158 kDa; β-galactosidase (E. coli) 116 kDa; phosphorylase b (rabbit muscle) 97 kDa; serum albumin (bovine) 66 kDa; glutamic dehydrogenase (bovine liver) 55 kDa; MBP2 (E. coli) P7702S protein marker, Protein ladder 3 42 kDa; thioredoxin reductase (E. coli) broad range (2 – 212 kDa). 34 kDa; triosephosphate isomerase 10 µL per lane were used (E. coli) 26 kDa; trypsin inhibitor2 (soybean) 20 kDa; lysozyme (chicken egg white) 14 kDa; aprotinin3 (bovine lung) 6 kDa; insulin A4 (bovine pancreas) 3.4 kDa; B chain4 (bovine pancreas) 2 kDa SDS 25 mM SDS-PAGE Adjusted to pH 8.3 with TRIS-HCl 0.1 % (m/V) running buffer HCl 1 M Glycine 19.2 mM Amounts indicated for Water 2.68 mL preparation of 2 gels. APS TRIS-HCl solution (1.5 M; pH 8.8) 2 mL and TEMED start the Separation gel SDS 10 % (m/V) 80 µL polymerization reaction. (12 % (V/V) Rotiphorese-acrylamide gel 30 % (m/V) Isopropanol was used to acrylamide) 3.2 mL obtain a smooth surface APS 10 % (m/V) 40 µL on the top of the gel and TEMED 4 µL remove bubbles. 30 min III. Material and methods

Solution/Buffer Composition Notes until complete polymerization were necessary Amounts indicated for Water 3.66 mL 28 | preparation of 2 gels. TRIS-HCl solution (1.5 M; pH 8.8) Polymerization reaction 1.5 mL Stacking gel (4 % was started when adding SDS 10 % (m/V) 60 µL (V/V) APS and TEMED. When Rotiphorese-acrylamide gel 30 % (m/V) acrylamide) the stacking gel was 798 µL added, the comb was APS 10 % (m/V) 30 µL places on the top to create TEMED 6 µL the wells

Table 1.5-6 | Buffers used for affinity chromatography

Buffer Composition Notes Adjusted to pH 8 with HCl 1 M. TRIS-HCl 50 mM Free of particles by filtration Buffer A1 NaCl 300 mM through 0.45 µm pore. Buffer Imidazole 10 mM used for ÄktaTMFPLC system Adjusted to pH 8 with HCl 1 M. TRIS-HCl 125 mM Free of particles by filtration Buffer A2 NaCl 150 mM through 0.45 µm pore. Buffer (Imidazole 10 mM) used for manual Ni-NTA TRIS-HCl 50 mM Adjusted to pH 8. Free of Buffer B1 NaCl 300 mM particles. Buffer used for Imidazole 500 mM ÄktaTMFPLC system Adjusted to pH 8 with HCl 1 M. TRIS-HCl 125 mM Free of particles by filtration Buffer B2 NaCl 150 mM through 0.45 µm pore. Buffer Imidazole 250 mM used for manual Ni-NTA NaCl 137 mM KCl 2.7 mM PBS buffer Adjusted to pH 8 with HCl 1 M Na2HPO4 10 mM

KH2PO4 1.8 mM

III. Material and methods

Table 1.5-7 | Solutions and buffers used for Western Blot

Solution Composition Notes TRIS-HCl 20 mM NaCl 0.5 M Freshly prepared before Blocking buffer Tween 20 0.05 % (V/V) use | 29 Triton X-100 0.1 % (V/V) Powdered milk 5 % (m/V) TRIS-HCl 100 mM (pH 9.5) NaCl 100 M

Staining solution MgCl2 5 mM Protected from light BCiP 0.033 % (m/V) NBT 0.0165 % (m/V) TRIS-HCl 20 mM NaCl 0.5 M TBS buffer Tween 20 0.05 % (V/V) Triton X-100 0.1 % (V/V) TRIS-HCl 25 mM (pH 7.5) Glycine 192 mM Methanol was added Transfer buffer SDS 0.1 % (m/V) shortly before use Methanol 20 % (V/V)

Table 1.5-8 | Solutions use for blue/white screening of E. coli

Solution Composition Notes 20 µL per agar plate. Sterile filtered with syringe IPTG solution ITPG 1 M sterile filter Rotilabo®. Storage at -20 °C X-Gal 100 mg/mL 20 µL per agar plate. Auto sterile. Storage at -20 °C. X-Gal solution in DMF Protected from light

Table 1.5-9 | Solutions used for blue/white screening of Streptomyces

Solution Composition Notes X-Gluc 100 mM X-Gluc solution Auto sterile. Storage at -20 °C. Protect from light in DMF

Table 1.5-10 | Solutions for permanent cultures

Solution Composition Notes Glycerin Glycerin 30 % (m/V) Autoclaved. Conservation of E. coli at -20 °C solution Autoclaved. Conservation of Streptomyces at Sucrose solution Sucrose 25 % (m/V) -20 °C III. Material and methods

Table 1.5-11 | Solutions for preparation of CaCl2 E. coli competent cells

Solution Composition Notes

CaCl2 solution CaCl2 1 M Autoclaved. Storage at 8 °C

CaCl2 1 M CaCl2 Glycerin solution Autoclaved. Storage at 8 °C 30 | Glycerin 15 % (m/V) MgCl2 solution MgCl2 1 M Autoclaved. Storage at 8 °C

Table 1.5-12 | Solutions used for preparation of electrocompetent cells

Solution Composition Notes Sucrose 0.65 % (m/V) Electroporation PEG 1000 3 %(m/V) Autoclaved buffer Glycerol 1 % (m/V) Glycerin solution Glycerin 10 % (m/V) Autoclaved Sterile filtered with syringe sterile Lysozyme solution Lysozyme 50 mg/mL filter Rotilabo®. Storage at -20 °C Sucrose solution Sucrose 10 % (m/V) Autoclaved

Table 1.5-13 | Solvents for preparative HPLC and LC/MS

Solution Composition Notes Acetic acid 0.5 % (V/V) in Sterile filtered with syringe sterile Solvent A acetonitrile filter Rotilabo® Acetic acid 0.5 % (V/V) in Sterile filtered with syringe sterile Solvent B distillated water filter Rotilabo®

Table 1.5-14 | Solutions needed for protein expression

Solution Composition Notes Sterile filtered with syringe sterile IPTG solution IPTG 1 M filter Rotilabo® L-Arabinose Sterile filtered with syringe sterile L-Arabinose 1 M solution filter Rotilabo® Glucose solution Glucose 2 % (m/V) Autoclaved

Table 1.5-15 | Detergents used for membrane protein solubilization

Detergent Composition Notes N-Lauroylsarcosine N-Lauroylsarcosine sodium salt 10 % - DDM n-Dodecyl-β-D-maltopyranoside 10 % -

III. Material and methods 1.6. Antibiotic solutions

Table 1.6-1 | Antibiotics and their concentration used in this work

Stock Final concentration Antibiotic Abbreviation Solvent concentration in media | 31 Distillated Acid nalidixic Nali 30 mg/mL See 2.1.4 water Distillated Ampicillin Amp 50 mg/mL 50 µL/mL water Distillated Ampicillin Amp 50 mg/mL 50 µL/mL water Apramycin Distillated Apra 100 mg/mL 50 µL/mL sulfate water Distillated Bacitracin Baci 100 mg/mL See 2.1.4 water Distillated Carbenicillin Carb 50 mg/mL 50 µL/mL water Chloramphenicol Cam 30 mg/mL Ethanol 30 µL/mL Distillated Gentamycin Genta 100 mg/mL See 2.1.4 water Distillated Hygromycin B Hyg 100 mg/mL 100 µL/mL water Kanamycin Distillated Kana 50 mg/mL 50 µL/mL sulfate water Distillated Norfloxacin Norflo 100 mg/mL See 2.1.4 water Distillated Novobiocin Novo 100 mg/mL See 2.1.4 water Phosphomycin Distillated Phospho 200 mg/mL 200 mg/mL disodium salt water Spectinomycin Distillated 150 µL/mL, for dihydrochloride x Spec 150 mg/mL water MIC test see 2.1.4 5 H2O Tetracycline 20 µL/mL, for MIC Tetra 5 mg/mL Ethanol hydrochloride test see 2.1.4 Thiostrepton Thio 50 mg/mL DMSO 50 µL/mL Distillated Vancomycin Van 100 mg/mL See 2.1.4 water

III. Material and methods 1.7. Components of media All media were prepared as shown in Table 1.7-1 using distillated water and adjusting pH with HCl 1 M. All media was autoclaved for sterilization (20 min, 121 °C, and 1 bar). When solid media was needed 21 g/L of agar-agar was added after setting the pH. 32 | Table 1.7-1 | Media used in this work

Medium Composition Notes Adjusted to pH 7.4 Yeast extract 4 g/L Medium used for secondary HA medium Malt extract 10 g/L metabolite production of Glucose 4 g/L Streptomyces spp. Lysogenic Broth Adjusted to pH 7.2 LB medium LB 20 g/L Medium used for cultivation of E. coli Adjusted to pH 7.2

MgCl2 or CaCl2 10 mg/L can be Soy flour 20 g/L added after sterilization to favor MS medium D-mannitol 20 g/L sporulation of Streptomyces spp. Medium used for intergeneric conjugation Tryptic Soy Broth Adjusted to pH 7.2 TSB medium CASO Boullion 30 g/L Cultivation medium for Streptomyces spp.

1.8. Bacterial stem lines

Table 1.8-1 | E. coli strains used in this work

Relevant phenotype and/or Reference of Strain Notes characteristics source

− − − F ompT hsdSB(rB mB ) gal Protein (Studier & E. coli BL21 (DE3) dcm (DE3) expression Moffatt 1986) E. coli BL21 (DE3) – − − + r F ompT hsdS(rB mB ) dcm Tet Protein (Moncrieffe codon plus gal endA Hte [argU proL Camr] expression et al. 2012) RP/pETcoco-2-L1SL2 – − − E. coli BL21 (DE3) F ompT hsdSB (rB mB ) gal dcm Protein InvitrogenTM pLysS (DE3) pLysS (Camr) expression – − − E. coli BL21 (DE3) F ompT hsdSB (rB mB ) gal dcm Protein InvitrogenTM StarTM rne131 (DE3) expression III. Material and methods

Relevant phenotype and/or Reference of Strain Notes characteristics source – − − F ompT hsdSB (rB mB ) gal dcm Protein (Wagner et E. coli C43 (DE3) (DE3) expression al. 2008) F– φ80/lacZΔM15 Δ(lacZYA- argF)U169 recA1 endA1 hsdR17 | 33 E. coli DH5α Cloning InvitrogenTM (rk−, mk+) phoA supE44 λ− thi-1 gyrA96 relA1 E. coli DH5α containing a deletion of acrA encoding a (Simm et al. E. coli DH5α ∆acrAB membrane fusion protein and MIC test 2012) acrB encoding a multidrug efflux system protein E. coli DH5α carrying pBADαβγ E. coli Cloning (Gust et al. plasmid for high recombination DH5α/pBADαβγ/cos4 Red/ET 2002) efficiency and the cosmid cos4 F–, dam-13::Tn9 dcm-6 hsdM hsdR zij-202::Tn10 recF143 galK2 (MacNeil et E. coli ET12567 GalT22 ara-14 lacY1 xyl-5 leuB6 Cloning al. 1992) thi-1 tonA31 rpsL136 HisG4 tsx-78 mtl-1 glnV44 E. coli E. coli ET12567 carrying pUB307 (Flett et al. Conjugation ET12567/pUB307 helper plasmid 1997) E. coli E. coli ET12567 carrying pUZ8002 (Bierman et Conjugation ET12567/pUZ2008 helper plasmid al. 1992) – − − F ompT hsdSB (rB mB ) gal dcm Protein E. coli RosettaTM 2 Novagen (DE3) pRARE2 (Camr) expression F– proA+B+ lacIq ΔlacZM15/ fhuA2 Δ(lac-proAB) glnV gal R(zgb- E. coli Turbo Cloning NEB 210::Tn10)TetS endA1 thi-1 Δ(hsdS- mcrB)5 F´::Tn10 proA+B+ lacIqΔ(lacZ)M15/ recA1 endA1 E. coli XL1Blue Cloning Stratagene gyrA96(Nalr) thi hsdR17 (rK–mK+) glnV44 relA1 lac

Table 1.8-2 | Actinomycetes strains used in this work

Relevant phenotype and/or Strain Reference of source characteristics Streptomyces albus J1074 Heterologous expression host (Yan et al. 2012) Mensacarcin and rishirilide A Streptomyces bottropensis (Arnold 2002) and B producer Streptomyces lividans TK24 Protein expression host (Kieser et al. 2000) Streptomyces sp. Tü6071 Phenalinolactone producer (Dürr et al. 2006) III. Material and methods 1.9. Vectors

Table 1.9-1 | Cosmids and plasmids needed for this work

Plasmid/ Antibiotic Description Reference cosmid Resistant 34 | Cosmid containing rishirilide gene cluster (rsl) (Linnenbrink cos4 Apra which was cloned into pOJ436 2009) Replicative vector pAL1 derivate with sce(a) Hyg (Siegl et al. pALSceI under tipA promoter Thio 2010) Vector containing code-optimized protein for the λ-Red recombination. red-α, red-β, red-γ (Zhang et al. pBADαβγ Tetra from the λ-phage; temperature sensitive 2003) replicon from the plasmid pSC101 Cloning vector, template for spectinomycin pcdfDuet resistant cassette amplification in Red/ET Spec Novagen protocol Suicide vector. gusA marker gene and I-SceI pKCXY02 sites. Used for construction of the inactivation Apra (Yan 2012) plasmids by homologous recombination Protein expression vector carrying an N-

terminal His6-Tag/thrombin/T7-Tag pET28a(+) Kana Novagen configuration plus an optional C-terminal

His6-Tag sequence Cloning vector, template for spectinomycin pLERE- A. cassette amplification needed for gene Spec Kobylyanskyy Spec-OriT inactivation via double cross-over (unpublished) (Bierman et pOJ436 pUC replicon, Aprar, (cos)3λ, intΦC31, IncPα oriT Apra al. 1992) (Bierman et pSET152 Integrative vector (intΦC31) Apra al. 1992) pTOS(z)- pTOS vector where Aprar cassette has been T. Heitzler Spect spec replaced for Specr cassette. Integrase vwb (unpublished) Non-transmissible mediator of intergeneric (Bennett et al. pUB307 conjugation through oriT. RP4TnAc, IncPα Kana 1977) replicon Cloning vector, with bla, lacZ'α, pMB1- pUC19 Carb NEB Replicon Modification of pUWLoriT with hph instead of Hyg pUWL-H (Petzke 2010) tsr Carb pUWL-H- pUWL-H containing the regulatory gene rslR4 Hyg (Yan 2012) rslR4 from rishirilide gene cluster Carb III. Material and methods

Plasmid/ Antibiotic Description Reference cosmid Resistant (Doumith et Conjugative vector derivate from pUWL201. pUWL- Thio al. 2000; Replicative in Actinomycetes with ermE oriT Carb Luzhetskyy et promoter al. 2005) | 35 pUWL- Plasmid derivate from pUWL-H containing Hyg (Petzke 2010) tnp5-H tnp5 gene Carb Non-transmissible mediator of intergeneric (Kieser et al. pUZ8002 conjugation through oriT, RK2-derived (IncP- Kana 2000; Blaesing 1α group), tra1 and tra2 region. et al. 2005) Cosmid from S. sp.Tü6071 cosmid library (Dürr et al. Sbe01h10 carrying the sequence 1674391 – 1708609. It Apra 2006) contained the transporter genes plaABC1-3

Table 1.9-2 | Relevant cloning plasmids created in this work

Cloning Plasmid Vector Description details 2.2.10.2.b pUC19rslT1 pUC19 Blunt ends rslT1 PCR fragment and c Blunt ends PCR of rslT1, rslT2 and rslT3 2.2.10.2.a pUC19rslT123 pUC19 genes and d Blunt ends rslT1 PCR fragment for C- pUC19rslT1C pUC19 2.2.10.4.b terminal His-tag expression Blunt ends PCR product of rslT1 60 nt pUC19rslT1Ctrun pUC19 truncated on the start codon side for C- 2.2.10.4.b terminal His-tag expression Blunt ends rslT1 PCR fragment for N- pUC19rslT1N pUC19 2.2.10.4.a terminal His-tag expression pUC19rslT4 pUC19 Blunt ends rslT4 PCR fragment 2.2.10.2.e

Table 1.9-3 | Cosmids created in this work

Cloning Cosmid Vector Description details Cosmid cos4 where rslT1, rslT2 and rslT3 cos4∆rslT123 cos4 2.2.11.3.b were deleted Cosmid cos4 carrying a Specr cassette cos4∆rslT123S cos4 instead of rslT1, rslT2 and rslT3 exchanged 2.2.11.3.b by homologous recombination III. Material and methods

Cloning Cosmid Vector Description details Cosmid cos4 carrying a Specr cassette cos4∆rslT1S cos4 instead of rslT1 exchanged by homologous 2.2.11.3.a recombination 36 | Cosmid cos4 carrying a Specr cassette cos4∆rslT2S cos4 instead of rslT2 exchanged by homologous 2.2.11.3.a recombination Cosmid cos4 carrying a Specr cassette cos4∆rslT3S cos4 instead of rslT3 exchanged by homologous 2.2.11.3.a recombination cos4∆rslT4 cos4 Cosmid cos4 where rslT4 was deleted 2.2.11.3.c Cosmid cos4 carrying a Specr cassette cos4∆rslT4S cos4 instead of rslT4 exchanged by homologous 2.2.11.3.c recombination

Table 1.9-4 | Complementation and overexpression plasmids created in this work

Cloning Plasmid Vector Description details pTOS(z)- Complementation/overexpression pTOS-plaABC 2.2.10.1.a spec integrative plasmid containing plaABC1-3 pTOS(z)- Complementation/overexpression pTOS-rslT1 2.2.10.2.c spec integrative plasmid containing rslT1 Complementation/overexpression pTOS(z)- pTOS-rslT123 integrative plasmid containing rslT1, rslT2 2.2.10.2.d spec and rslT3 pTOS(z)- Complementation/overexpression pTOS-rslT4 2.2.10.2.e spec integrative plasmid containing rslT4 pUWL-H- pUWL- Complementation/overexpression 2.2.10.1.b plaABC tnp5-H replicative plasmid containing plaABC1-3 Complementation/overexpression pUWL- pUWL-rslT123 replicative plasmid containing rslT1, rslT2 2.2.10.2.a tnp5-H and rslT3 pUWL-OriT- Complementation/overexpression pUWL-OriT 2.2.10.2.b rslT1 replicative plasmid containing rslT1

III. Material and methods

Table 1.9-5 | Inactivation plasmids created in this work

Cloning Plasmid/Cosmid Vector Description details Intermediate of the inactivation plasmid for deletion of plaABC1-3 from S. sp. Tü6071 via pKC-3T1 pKCXY02 2.2.10.3.a | 37 double crossover with homologous regions upstream the target genes Intermediate of the inactivation plasmid for deletion of plaABC1-3 from S. sp. Tü6071 via pKC-3T1-3T2 pKC-3T1 2.2.10.3.a double crossover with homologous regions upstream and downstream the target genes Inactivation plasmid for deletion of plaABC1- 3 from S. sp. Tü6071 via double crossover pKC-3T1- pKCplaABC123 with homologous regions upstream and 2.2.10.3.a 3T2 downstream the target genes and the spectinomycin cassette marker gene Inactivation plasmid for deletion of plaABC1 from S. sp. Tü6071 via single crossover with pKCplaABC1SCO pKCXY02 2.2.10.3.b ca. 1000 bp homologous region of the target gene

Table 1.9-6 | Protein expression plasmids for E. coli created in this work

Cloning Plasmid Vector Description details rslT1, rslT2 and rslT3 cloned into protein pET- pET28rslT123 expression vector without His6-tag but still 2.2.10.4.c 28a(+) under influence of the inducible T7 system pET- rslT1 cloned into protein expression vector pET28rslT1C 2.2.10.4.b 28a(+) with C-terminal His6-tag rslT1 60 bp truncated on the start codon site pET- pET28rslT1Ctrun cloned into protein expression vector for C- 2.2.10.4.b 28a(+) terminal His6-tag pET- rslT1 cloned into protein expression vector pET28rslT1N 2.2.10.4.a 28a(+) for N-terminal His6-tag

Table 1.9-7 | Plasmids for protein analysis in S. lividans created in this work

Cloning Plasmid/Cosmid Vector Description details

rslT1 His6-tag on the C-terminal pUWL- pUWLrslT1C amplified from pET28rslT1C and cloned 2.2.10.4.d tnp5-H into pUWL-H III. Material and methods

Cloning Plasmid/Cosmid Vector Description details

rslT1 His6-tag on the C-terminal pUWL- amplified from pET28rslT1C cloned pUWLrslT1CT2T3 2.2.10.4.e tnp5-H together into pUWL-H with rslT2 and 38 | rslT3 obtained from pUC19rslT123

1.10. Software and database

Table 1.10-1 | Software and databases used in this work

Resource Provider Reference Unicorn 4.11 ÄKTATMFPLC software

Amersham Biosciences AB, Uppsala, Sweden SignalP 4.0 Server for prediction of presence and location of signal peptide cleavage sites in amino acid sequences (Petersen et al. Center for Biological Sequence Analysis at the 2011) Technical University of Denmark http://www.cbs.dtu.dk/services/SignalP/ Phobius 1.0 A combined transmembrane topology and signal peptide predictor (Käll et al. 2004) Stockholm Bioinformatics Centre http://phobius.sbc.su.se/ LipoP Prediction of lipoproteins and signal peptides in Gram negative bacteria (Juncker et al. Center for Biological Sequence Analysis at the 2003) Technical University of Denmark http://www.cbs.dtu.dk/services/LipoP/ AntiSMASH Antibiotics and Secondary Metabolite Analysis Shell University of Groningen (Netherlands), University of Tübingen (Germany), University of Manchester (Blin et al. 2013) (England), and University of California, San Francisco (USA) http://antismash.secondarymetabolites.org BLAST NCBI Basic Local Alignment Search Tool National Center for Biotechnology Information, (Acland et al. National Library of Medicine, National Institutes of 2014) Health, Bethesda, USA http://www.ncbi.nlm.nih.gov/BLAST.cgi ChemStation Agilent Technologies, Waldbronn, Germany Rev. A.09.03 III. Material and methods

Resource Provider Reference Clone Manager Scientific and Educational Software, Cary, USA Suite 7 ExPASy Expert Protein Analysis System (Artimo et al. Swiss Institute of Bioinformatics Resources Portal 2012) http://expasy.org | 39 Galaxy Pharmaceutical Bioinformatics, Institute for Pharmaceutical Sciences, University of Freiburg, (Ramírez et al. Germany 2014)

MarvinSketch Advanced chemical editor for drawing chemical structures, queries and reactions. Free access. ChemAxon Limited, Budapest, Hungary http://www.chemaxon.com/products/marvin/marv insketch/ PubChem NCBI Compound database (Bolton et al. https://pubchem.ncbi.nlm.nih.gov/ 2008) Pubmed National Center for Biotechnology Information, National Library of Medicine, National Institutes of (Acland et al. Health, Bethesda, USA 2014) http://www.ncbi.nlm.nih.gov/pubmed RCSB Protein Center for Integrative Proteomics Research, New Data Bank Yersey and University of California, San Diego, USA (Berman 2000) www.rcsb.org StreptomeDB Pharmaceutical Bioinformatics, Institute for Pharmaceutical Sciences, University of Freiburg, (Lucas et al. Germany 2013) http://www.pharmaceutical- bioinformatics.de/streptomedb TransportDB Genomic comparisons of Membrane Transport Systems Macquarie University, Sydney, Australia (Ren et al. 2007) www.membranetransport.org Transporter University of California, San Diego, USA (Saier et al. classification http://www.tcdb.org 2014) database Uniprot Universal Protein Resource European Bioinformatics Institute (EMBL-EBI), SIB (The UniProt Swiss Institute of Bioinformatics and Protein Consortium Information Resource (PIR) 2014) http://www.uniprot.org/blast Waters Empower® 2002 Waters Corporation Milford, USA Analytical HPLC software

III. Material and methods 2. Methods

2.1. Methods in microbiology

40 | 2.1.1. Cultivation of bacterial strains 2.1.1.1. Cultivation of E. coli strains and preparation of permanent cultures

E. coli strains (Table 1.8-1) were cultivated in LB liquid medium (Table 1.7-1). For pre- cultures, cells were incubated 8 – 15 h in 20 mL antibiotic LB using 100 mL Erlenmeyer shake flasks with agitator. For main cultures, cells were cultivated in 100 mL antibiotic LB using 250 mL with agitator. Liquid cultures of E. coli were incubated at 37 °C and 180 rpm between 8-15 h. Antibiotic concentrations used in the cultures are listed in Table 1.6-1.

Specific conditions used for heterologous protein expression in E. coli cultivated in LB medium are summarized in section 2.3.1.

The Red/ET protocol included the use of E. coli DH5α containing the thermo-sensitive plasmid pBADαβγ. Specific growth conditions were used for its efficiency which can be found in section 2.2.11.3.

For cultivation in solid medium, 20 mL of melted LB agar containing the appropriate antibiotics added when the medium was cooled down (approx. 60 °C) were poured on petri dishes. Cells were carefully spread on the top using a sterile Drigalski spatula and incubated at 37 °C between 12 – 15 h.

For preparation of permanent cultures, the desired strain was cultivated in 100 mL antibiotic LB medium at 37 °C and 180 rpm overnight. The culture was centrifuged (586 g, 20 min, RT) and the pellet resuspended in 10 mL glycerol solution (Table 1.5-10). Permanent cultures were stored at -20 °C for 6 months or -80 °C for longer periods.

2.1.1.2. Cultivation of Streptomyces strains and preparation of permanent cultures

Streptomyces strains (Table 1.8-2) were cultivated at 28 °C. For liquid pre-cultures, cells were cultivated in 30 mL of TSB medium (Table 1.7-1) in 100 mL Erlenmeyer shake flasks containing a metal coil at 180 rpm until a thick suspension of fine particles was visible. Antibiotics were added to the media when needed (Table 1.6-1). For cultivation on solid medium, 30 mL of melted TSB agar containing the appropriate antibiotics added when the III. Material and methods medium was cooled down (approx. 60 °C) were poured on petri dishes. Cells were carefully spread on the top using a sterile inoculation loop or picked into the agar with a sterile toothpick to favor sporulation. Plates were incubated for 2 – 5 days until sporulation was visible covering the plate with a white surface. | 41 For passaging (2.2.11.2), the cells were picked from the conjugation plates and transferred into a 30 mL TSB cultures containing spectinomycin 50 µg/mL (Table 1.6-1) which were incubated at 180 rpm for 2 – 3 days. 1 mL of this pre-culture was inoculated into a fresh 30 mL Spec TSB culture under the same conditions. This process was repeated 10 – 35 times. To verify the success of the passage by achieving the double crossover mutants, single clones were needed. Dilutions 1:10 were performed starting with 100 µL of the Streptomyces culture and mixed with 900 µL sterile water. 5 – 10 of these dilution steps were prepared and from them 0.5 mL were spread with an inoculation loop over antibiotic TSB agar plates in order to get the adequate dilution which led to the growth of single colonies.

HA medium (Table 1.7-1) was chosen for production of secondary metabolite from Streptomyces mutants (2.4). 1 mL of pre-culture was transferred into 100 mL of HA medium in 500 mL Erlenmeyer shake flasks with metal coil and cultivated for 4 – 7 days under agitation at 180 rpm.

Petri dished containing 30 mL of MS agar (Table 1.7-1) were used for intergeneric conjugation (2.1.2.3) in order to favor the growth of Streptomyces over E. coli. MS agar medium was also used to obtain big amounts of sporulated Streptomyces by spreading 1 mL of a pre-culture on the top of the medium with an inoculation loop and incubation for 2 – 4 days. Addition of antibiotics on MS plates was done over the cells spread on the agar, not directly on the warm medium as described before. The appropriate amount of antibiotics per plate was added to 1 mL of water or liquid TSB. The liquid was carefully spread over the cells with a pipette to ensure that the whole surface was covered.

For permanent cultures, the desired strain was cultivated in 30 mL of antibiotic TSB medium until obtain a thick suspension of fine particles. The culture was then centrifuged (586 g, 30 min) and the pellet was washed with 30 mL sucrose solution (Table 1.5-10). After a second centrifugation step the pellet was resuspended in 10 mL sucrose solution. Permanent cultures were stored at -20 °C for 6 months or -80 °C for longer periods. III. Material and methods 2.1.2. Transfer of DNA Transformation and conjugation mediate the transport of single-stranded DNA (ssDNA) through the bacterial membranes. Transformation involves the direct uptake of environmental DNA whereas conjugation means the transfer of DNA between a donor cell 42 | and a recipient cell (Chen et al. 2005). These processes take place naturally in some bacterial strains and was firstly observed in Streptococcus pneumoniae by Griffith in 1928 (Griffith 1928). This ability is used under laboratory conditions to bring artificial competence. The following methods are based on the ones described by Sambrook, Fritsch and Maniatis (1989).

2.1.2.1. Transformation of E. coli strains

Transformation of CaCl2 competent cells by heat shock

. Preparation of CaCl2 competent cells

A pre-culture of LB medium (Table 1.7-1) containing the appropriate antibiotics (Table 1.6-1) and the desired stem line (Table 1.8-1) was prepared and the cells were incubated at 37 °C, 180 rpm overnight. A 100 µL aliquot was transferred into 100 mL fresh LB medium and

cultivated until an OD600 nm of 0.4 - 0.6 was reached (approx. 5 h). Culture was cooled down on ice and centrifuged using two 50 mL centrifuge tubes (586 g, 20 min, and 4 °C). The

pellets were resuspended together in 50 mL ice cold 0.1 M MgCl2 solution (Table 1.5-11). It was incubated on ice for 20 min and centrifugation was repeated. The pellet was washed

with 50 mL ice cold 0.1 M CaCl2 solution (Table 1.5-11) and incubated on ice for 20 min. Cells were centrifuged once more and another washing step was performed. Final pellet was

resuspended in 3 mL ice cold 0.1 M CaCl2 glycerin solution (Table 1.5-11). The 1.5 mL micro centrifuge tubes were previously labelled and precooled at -80 °C. The resuspended cells were pipetted in 150 µL aliquots into the frozen micro centrifuge tubes and stored immediately at -80 °C.

. Heat shock

Frozen CaCl2 competent cells were thawed on ice for 4 minutes, at which point 2 – 10 μL of plasmid were added to the cell suspension and carefully mixed. Afterwards the mixture was incubated on ice for 15 min. Heat shock was administered for 90 seconds in a 42 °C water bath and cells were placed back onto ice for some seconds. 700 μL of LB were added under sterile conditions. Cells were regenerated in a 37 °C water bath for 1 h and then centrifuged III. Material and methods for 3 min at 2655 g. The supernatant was poured and the remaining liquid (approx. 200 µL) was used to resuspend the pellet for plating on the applicable antibiotic medium.

Transformation by electroporation

. Preparation of competent cells for electroporation | 43 A pre-culture of LB medium (Table 1.7-1) containing the appropriate antibiotics (Table 1.6-1) and the desired stem line (Table 1.8-1) was prepared and the cells were incubated at 37 °C, 180 rpm overnight. A 100 µL aliquot was transferred into 100 mL fresh LB medium and cultivated until an OD600 nm of 0.4 - 0.6 was reached (approx. 5 h). Culture was cooled down on ice and centrifuged using two 50 mL centrifuge tubes (586 g, 20 min, and 4 °C). The pellets were resuspended together in 50 mL ice cold 0.1 M MgCl2 solution (Table 1.5-11). It was incubated on ice for 20 min and same steps were performed three times. Final pellet was resuspended in 2 mL ice cold 10 % glycerin solution. The 1.5 mL micro centrifuge tubes were previously labelled and precooled at -80 °C. The resuspended cells were pipetted in 70 µL aliquots into the frozen micro centrifuge tubes and stored immediately at -80 °C.

. Electroshock

Electrocompetent cells were retrieved from -80 °C storage and thawed for 4 min. 2 - 10 μL of plasmid or cosmid were added carefully into the cell suspension, mixed and taken onto the notch of the electroporation cuvette which was then placed on ice for 15 min. The cuvette was wiped down on the metal contacts to remove moisture and inserted into the holder of the “E. coli pulser” electroporator. A shock pulse of 1.8 kV was administered. 1 mL LB medium was used to resuspend the cells from inside the cuvette and the mixture was taken out into a new 1.5 mL micro centrifuge tube. The cells were allowed to recover at 37 °C (water bath or 180 rpm incubation shaker) for 1 - 3 h. Cells were centrifuged at 2655 g for 3 min, the supernatant was poured and the pellet resuspended in the remaining medium (approx. 200 μL). Cells were plated on the appropriated antibiotics LB agar and incubated at 37 °C.

Transformation of E. coli DH5α/pBADαβγ/cos4 for Red/ET An aliquot of E. coli DH5α/pBADαβγ/cos4 (Table 1.8-1) from -80 °C permanent stock was grown overnight in 20 mL LB Apra/Tetra at 28 °C. 1 mL of the overnight culture was transferred into a new 100 mL LB Apra/Tetra shaker flask and 1 mL of 1 M L-arabinose (Table 1.5-14) was added. The plasmid pBADαβγ (Table 1.9-1) contained the genes necessary for the λ-Red recombination and also a heat-sensitive replicon at 28 °C. By addition of fresh arabinose solution, the expression of the λ-Red operon was induced. Cells were grown at III. Material and methods

28 °C until an OD600 0.4 - 0.6 was reached (approx. 6 h). Culture was cooled down on ice and centrifuged using two 50 mL centrifuge tubes (centrifuge Rotina R 35, 586 g, 20 min, and 4 °C). The pellets were resuspended together in 50 mL ice cold 10 % glycerin solution (Table 1.5-12). It was incubated on ice for 20 min and same steps were performed three times. Final 44 | pellet was resuspended in 1 mL ice cold 10 % glycerin solution. Two 1.5 mL micro centrifuge tubes were previously labelled and precooled at -80 °C. The resuspended cells were pipetted in a 70 µL aliquot for immediate use and the rest was freeze for permanent storage at -80 °C. 15 µL of the PCR product were added carefully into the 70 µL cell aliquot, mixed and taken onto the notch of the electroporation cuvette which was then placed on ice for 15 min. The cuvette was wiped down on the metal contacts to remove moisture and inserted into the holder of the “E. coli pulser” electroporator. A shock pulse of 1.8 kV was administered. 1 mL LB medium was used to resuspend the cells from inside the cuvette and the mixture was taken out into a new 1.5 mL micro centrifuge tube. The cells were allowed to recover at 28 °C (water bath) for 3 h and the desired recombination should take place. Cells were centrifuged at 2655 g for 3 min, the supernatant was poured and the pellet resuspended in the remaining medium (approx. 200 μL). Cells were plated on the appropriated antibiotics LB agar and incubated at 37 °C.

2.1.2.2. Transformation of Streptomyces spp.

. Preparation of Streptomyces spp. competent cells

A 20 mL pre-culture was prepared with the desired stem line and was cultivated at 28 °C, 180 rpm in antibiotic TSB medium until a thick fine particles growth was obtained. 1 mL of the pre-culture was transferred into a fresh 100 mL antibiotic TSB media. It was grown at 28 °C, 180 rpm for 24 – 30 h. The mycelium was centrifuged (586 g, 30 min, 4 °C) and the pellet was washed twice with 10 mL of ice cold 10% sucrose (Table 1.5-12). Afterwards the pellet was resuspended in 50 mL of ice cold 15 % glycerol (Table 1.5-12) and centrifuged. The pellet was resuspended in 10 mL ice cold 15% glycerol and 100 µg/mL lysozyme (Table 1.5-12). The mixture was incubated at 28 °C, 180 rpm for 30 min and centrifuged. To remove the lysozyme, the cells were washed twice with 10 mL of ice cold 15% glycerol. The final pellet was resuspended in 2 mL of electroporation buffer for Streptomyces (Table 1.5-12) and 50 µL aliquots were pipetted in precooled 1.5 mL micro centrifuge tubes. Permanent stock was stored immediately at -80 °C.

III. Material and methods . Electroshock

The competent cells prepared for electroporation were mixed with 10 µL of DNA and taken onto the notch of the electroporation cuvette which was then placed on ice for 15 min. The cuvette was wiped down on the metal contacts to remove moisture then inserted into the | 45 holder of the “E. coli pulser” electroporator. A shock pulse of 2 kV was administered. 1 mL TSB medium was used to resuspend the cells from inside the cuvette and the mixture was taken out into a new 1.5 mL micro centrifuge tube. The cells were allowed to recover at 28 °C (water bath) for 3 h. Cells were plated on the appropriated antibiotics TSB agar and incubated at 28 °C.

2.1.2.3. Intergeneric conjugation

Intergeneric conjugation was used to transfer the desired vector (Table 1.9-3, Table 1.9-4, Table 1.9-5, Table 1.9-7) carrying an origin of transfer (oriT) from a donor strain (E. coli ET12567/pUZ8002 or pUZ307, Table 1.8-1) into a recipient strain (Streptomyces spp., Table 1.8-2). The process is based on the capacity of this E. coli strain to build a sex pilus thanks to a RP4 derivative plasmid (pUZ8002 or pUB307, Table 1.9-1) and to mobilize the non- methylated ssDNA into Streptomyces spp.

. Preparation of donor cells

Competent E. coli ET12567/pUZ8002 or pUB307 cells were transformed with the desired plasmid or cosmid (Table 1.9-3, Table 1.9-4, Table 1.9-5, Table 1.9-7). Selection was carried out with Kana (50 μg/mL) to ensure the presence of the helper plasmid pUZ8002 or pUB307 plus the antibiotic the vector to be transferred gave resistance to. Resulting clones expressing double resistance were plated onto fresh antibiotic medium and spread across the entire surface of the plate with a Drigalsky spatula and grown for 8 – 12 h at 37 °C. With the help of an inoculating loop all cell material was taken from the plate and mixed together with the pertinent Streptomyces strain.

Alternatively, the resulting clones were picked into 100 mL antibiotic LB and grown until reaching OD600 nm of 0.6 – 0.8. The culture was then centrifuged (586 g, 15 min, RT) and the pellet was washed twice with 20 mL of sterile water. The final pellet was resuspended in 600 µL of sterile water (enough for 3 conjugation plates) and mixed together with the pertinent Streptomyces strain.

III. Material and methods . Preparation of recipient cells

Streptomyces spp. (Table 1.8-2) and their mutants were used for conjugation from mycelium liquid cultures or from spore suspension. In the first case, 30 mL of antibiotic TSB media were inoculated with appropriate strain and incubated at 28 °C for 30 – 35 h until a fine 46 | mycelia was visible. Small aliquots depending on the strain (Table 2.1.2-1), one per conjugation plate, were taken from the pre-culture into 2 mL micro centrifuge tubes and washed twice with fresh TSB medium. The final pellet was ready then to be mixed with the donor cells.

Spores from one MS agar spore plate, one per conjugation plate, were scraped from the surface with 3 mL TSB medium and filtered through a sterile cotton piece. The spore suspension was collected with a 1 mL pipette (end cut tip) into a 2 mL micro centrifuge tube and washed twice with fresh TSB medium. The final spore pellet was resuspended in 300 µL of TSB medium.

. Conjugation

Donor and recipient cells were shortly mixed in the 2 mL preparation micro centrifuge tubes, plated together on MS agar (Table 1.7-1) and distributed evenly with an inoculating loop. Positive and negative controls were made by the same procedure without the addition of the donor strain. Each conjugation plate was incubated for 8 – 15 h at 28 °C (Table 2.1.2-1). After incubation all plates except the positive control were overlayed with appropriate antibiotic of the transferred construct (Table 1.9-3, Table 1.9-4, Table 1.9-5, and Table 1.9-7) and as well as phosphomycin. Plates were estimated to contain 30 mL of agar. Antibiotics (Table 1.6-1) were solved in 1.5 mL sterile water or TSB medium and distributed evenly over the plate with a Drigalsky spatula or the 1 mL pipette. Plates were allowed to dry for 30 min before being placed back into the 28 °C incubator and were incubated for 2 – 5 days at 28 °C until exconjugants colonies were visible.

Table 2.1.2-1 | Conjugation volumes and incubation times

Incubation time before Strain Volume per conjugation antibiotic overlay Streptomyces albus J1074 300 μL – 500 μL 6 – 8 h Streptomyces species Tü6071 1 mL 12 – 15 h Streptomyces lividans TK24 300 μL – 500 μL 12 – 15 h Streptomyces bottropensis 300 μL – 500 μL 8 – 15 h

III. Material and methods 2.1.3. Bacterial screening 2.1.3.1. Blue-white screening in E. coli

Blue-white screening (Cronan et al. 1988) is an efficient and rapid tool for identification of recombinant bacteria. It is based on the ability of the enzyme β-galatosidase to hydrolyze | 47 X-Gal to form 5-bromo-4-chloro-indoxyl, which spontaneously dimerizes to produce an insoluble blue pigment called 5,5’-dibromo-4,4’-dichloro-indigo. E. coli DH5α, E. coli XL1Blue and NEB Turbo E. coli (Table 1.8-1) express functional β-galatosidase when its α-subunit is complemented with a vector carrying the lacZα gene. A multiple cloning site (MCS) is located within the lacZα so that a successful insertion of foreign DNA into the plasmid will avoid the α-complementation. IPTG is a lactose analogue used as an inducer of the expression of lacZ gene.

For performing blue/white screening, antibiotic LB agar plates (Table 1.7-1) were evenly overlayed with 20 µL X-Gal solution and 20 µL IPTG solution (Table 1.5-8). After transformation, as described in 2.1.2.1, cells were plated onto the prepared LB agar without direct light and incubated overnight at 37 °C. To intensify the blue color of the non- recombinant colonies, the plates were incubated for 4 – 6 h at 8 °C. The white colonies were picked for further plasmid isolation (2.2.1).

2.1.3.2. GusA assay

The reporter gene gusA encodes for the enzyme β-D-glucuronidase (GUS) that hydrolyzes the substrate X-Gluc (5-Bromo-4-chloro-3-indolyl-β-D-glucurone acid) and gives a visual color reaction turning the colonies into a blue indigo color. Most Streptomyces spp. do not carry an endogenous GUS activity therefore screening is easily possible without need of special mutations and cofactors (Myronovskyi et al. 2011).

For a blue/white screening, the recombinant plasmids of pKCXY02 (Table 1.9-5) were conjugated as described in 2.1.2.3. For the analysis of GUS activity, the exconjugants were picked onto a new antibiotic TSB agar plate (Table 1.6-1 and Table 1.7-1) and incubated at 28 °C until spores were visible (2 – 5 days). 60 µL of X-Gluc solution (Table 1.5-9) were solved in 1.5 mL of sterile water and the mixture was used to overlay the exconjugants plate. The cells were incubated at 28 °C for somehoursuntil the color reaction was visible. Notice that X-Gluc is solved in DMF therefore after this screening cells were not viable anymore. III. Material and methods 2.1.4. Minimal inhibitory concentration in E. coli For testing the minimal inhibitory concentration (MIC) of different antibiotics a mutated strain of E. coli was used. E. coli DH5α ΔacrAB carries the deletion of acrA, encoding a membrane fusion protein and deletion of acrB, encoding a multidrug efflux system protein 48 | that causes an inactive RND-transporter complex. These mutations increase the sensibility of the cells towards toxic compounds and therefore useful for exogenous transporter characterization (Simm et al. 2012).

The recombinant pTOS(z)-spec plasmids carrying different transporter genes were transformed into E. coli DH5α ΔacrAB as described in 2.1.2.1. Pre-cultures of E. coli DH5α ΔacrAB mutants were prepared (2.1.1.1) and 50 µL were transferred into 20 mL antibiotic LB

medium (Table 1.6-1) that was grown until an OD600 nm of 0.7 – 0.8 was reached.

For preparation of the 96 well microplates, 50 µL of LB medium (50 µg/mL Spec) were pipetted into each well of the microplate beside the first well of each row. The first column was filled with 99 µL of LB medium (50 µg/mL Spec) and 1 µL of the antibiotic stock solution (Table 1.6-1) to be tested. Following, 50 µL of the first well were pipetted into the second and carefully mixed. This dilution step (1:2) was repeated for the rest of the columns and the remaining 50 µL of the last wells were discarded. Next, E. coli DH5α ΔacrAB mutants were

added to the microplate. A dilution of the grown culture was preformed to reach an OD600 nm of 0.04 in LB medium (50 µg/mL Spec). 50 µL of the cell suspension were added into each

well of the microplate to achieve a final OD600 nm of 0.02. The microplates were incubated at 37 °C and 180 rpm for 15 h.

2.1.5. Disc diffusion antibiotic sensitivity testing Another method to simply visualize the sensibility of bacteria to different antibiotics consists on the application of filter paper discs impregnated with the substances to be tested over a homogenous lay of the desired strain (Herrmann et al. 1960). In this work, the bacteria investigated were mutants of Streptomyces albus J1074 (Table 1.8-2) carrying cos4, cos4ΔrslT4 or pOJ436 (Table 1.9-1 and Table 1.9-3). Pre-cultures were prepared in 30 mL of TSB medium cultivated for 3 days at 28 °C and 180 rpm. Samples of 1 mL were centrifuged (2655 g, 3 min, RT) in order to obtain comparable amount of cells between the mutants. The harvested cells were washed with 1 mL of fresh TSB medium and after centrifugation (2655 g, 3 min, RT) another 1 mL was used to resuspend and equally spread the cells with the help of a Drigalski spatula onto the MS agar plates. The sterile filter paper discs were placed on the III. Material and methods surface keeping around 3 cm distance between them and the borders of the plate. A volume of 10 µL of the antibiotic solutions (Table 1.6-1) was carefully pipetted over the disc. In the case the antibiotic was not solved in water but in an organic solvent, the solution was pipetted on the disc before placing it on the agar and let the solvent evaporate for some | 49 minutes. The MS plates were incubated for 3 days at 28 °C until defined inhibition zones were visible.

2.2. Methods in molecular biology

2.2.1. Plasmid isolation from E. coli Depending on the desired purity and concentration of the required plasmid DNA, isolation was performed following different protocols. The purified plasmid DNA was solved in water and stored at -20 °C.

2.2.1.1. Alkaline lysis

The following method is based on Birnboim and Doly (1979). All necessary solutions and buffers for the procedure are summarized in Table 1.5-4.

Single clones were picked into 2 mL micro centrifuge tubes containing 1.5 mL antibiotic LB medium (Table 1.7-1 and Table 1.6-1) and were cultivated overnight at 37 °C, 180 rpm. The grown bacteria were centrifuged (20817 g, 5 min, RT) and the pellet was resuspended in 200 µL of buffer P1. Next, 200 µL of buffer P2 were added, the micro centrifuge tubes were carefully inverted 4 – 6 times and incubated at room temperature for 5 min. Following, 200 µL of buffer P3 were added, mixed carefully by inverting and the tubes were incubated 5 min in ice. The samples were centrifuged at 4 °C, 20817 g, for 15 min. The supernatant was transferred into a new 1.5 mL micro centrifuge tube and 400 µL of ice cold isopropanol (stored at -20 °C) were added. The mixture was vortexed and centrifuged 25 – 40 min, 20817 g, 4 °C. The supernatant was discarded, 200 µL ice cold 70 % ethanol (stored at -20 °C) were added and micro centrifuge tubes were centrifuged again (10 min, 20817 g, 4 °C). Ethanol was poured from the micro centrifuged tube and the remaining liquid was carefully taken out by inverting the tube over a paper. The pellet was dried completely for 10 – 20 min at 60 °C. Finally, the DNA was rehydrated with 30 µL of water.

III. Material and methods 2.2.1.2. Purification kits

When high concentrated and pure extrachromosomal DNA was required, isolation was performed with the help of different kits (Table 1.4-3). innuPREP Plasmid Mini Kit was used

50 | for cosmid isolation from 20 – 50 mL cultures. Pure YieldTM Plasmid Midiprep System Kit was used for plasmid and cosmid isolation of 100 mL main cultures. Pure YieldTM Plasmid Miniprep System Kit was utilized for plasmid purification from 20 mL cultures. The procedure was done following the instructions of the manufactures.

2.2.2. Genomic DNA isolation from Streptomyces spp. Genomic DNA from Streptomyces spp. and mutants were isolated to be used as template for control PCRs. A fast protocol was modified from Kieser et al. (2000). All solutions and buffers needed for this isolation are described on Table 1.5-3.

Mycelia from a 30 mL pre-culture (2.1.1.2) were pelleted in a 50 mL centrifuge tube (586 g, 20 min), washed once with 10 mL 25 % sucrose solution, pelleted again and resuspended in 2 mL 25 % sucrose solution. 1 mL of the resulting solution was transferred to a 2 mL micro centrifuge tube and centrifuged for 3 min at 2655 g and RT. The pellet was resuspended in 500 µL SET-Buffer and 10 µL of lysozyme (50 mg/mL), 0.5 µL RNase (10 µg/mL) were added. The mixture was incubated for 30 min at 37 °C in the shaker at 180 rpm, following which 14 µL of proteinase K (50 mg/mL) and 50 µL SDS (10 %) were added. Samples were incubated for 1 hour at 55 °C. Next, 200 µL of NaCl solution were added and mixed before the addition of 500 μL chloroform. Each sample was vortexed for 2 min and then centrifuged at 20817 g for 20 min at 4 °C. The upper phase was collected with a cut 1 mL tip and transferred to a new 1.5 mL micro centrifuge tube. 500 μL of ice cold isopropanol (-20 °C) were added and samples were vortexed for 1 min. DNA was allowed to precipitate for 10 min at RT and finally centrifuged (20817 g, 10 min, 4 °C). The supernatant was carefully poured and 1 mL 70 % ethanol was added for a second centrifugation step (20817 g, 10 min, 4 °C). The pellet was finally dry until the ethanol was evaporated and resuspended in 30 – 50 µL of water. DNA concentration and purity was measured with NanoDrop (Table 1.2-3).

2.2.3. Precipitation and concentration of DNA Concentration of DNA was performed by precipitation with isopropanol (Sambrook et al. 1989). The DNA solution was vortexed with 400 µL of ice cold isopropanol (Table 1.5-3) and centrifuged 25 min, 20817 g and 4 °C. The supernatant was carefully poured and the pellet was washed with 200 µL of ice cold 70 % ethanol (Table 1.5-3). The pellet was evaporated III. Material and methods 15 – 20 min at RT and was dried at 60 °C for some minutes. Finally, the DNA was rehydrated with water (20 – 50 µL) to achieve the desired concentration.

2.2.4. Analysis and purification of DNA by agarose gel electrophoresis Gels for agarose gel electroporation were made with 0.7 % agarose (Table 1.5-1) dissolved in | 51 1x TAE buffer (Table 1.5-1). DNA separation was carried out at 100 V in a electrophoresis chamber with 1x TAE buffer at RT. Identification of fragment size was guided by a 1 kb DNA ladder (Table 1.5-1), 5 – 10 µL per lane.

In case of analytical gels, ethidium bromide bath or 3x GelRed bath were used for staining (Table 1.5-1). Gels were incubated for 20 – 30 min. DNA fragments were visualized with a UV-transilluminator and photographed with a CCD camera or UV Transilluminator Image Master VDS (312 nm) (Table 1.2-3).

For preparative gels with the intended purpose of collecting a specific fragment, gels were stained for 10 – 20 min with methylene blue (Table 1.5-1) and distained in water. The desired fragment was excised with a scalpel and DNA was isolated from the gel using the Wizard® SV Gel and PCR Clean-up System (Table 1.4-3) according to manufacturer’s instructions. The isolated DNA was eluted with 50 μL sterile water and stored at -20 °C.

2.2.5. Restriction digestion of DNA Digestion of DNA by restriction endonucleases (Table 1.4-1) was carried out according to manufacturer´s instructions at enzyme specific incubation temperature in the appropriate buffer supplied for 30 min to 2 h.

Table 2.2.5-1 | Volumes for analytical and preparative digestions

Component Analytical digestions Preparative digestions DNA 4 µL 20 µL Restriction enzyme A 0.5 µL 2 µL Restriction enzyme B 0.5 µL 2 µL BSA (if needed) 1 µL 5 µL NEB buffer 1 µL 5 µL Water ad 10 µL ad 50 µL

Digestions with restriction enzymes allowed the qualitative analysis of DNA based on the size of the obtained fragments and also made possible the cloning of DNA fragments into vectors. Digestions mixtures (Table 2.2.5-1) were cleaned up with Wizard® SV Gel and PCR III. Material and methods Clean-Up System (Table 1.4-3) following manufacturer’s instructions. Alternatively, by precipitating DNA with isopropanol (2.2.3) or by agarose gel electrophoresis (2.2.4).

2.2.6. Ligation 52 | T4-DNA-ligase from Promega (1) and NEB (2) (Table 1.4-1) were used for ligation of DNA fragments. Reaction mixture with 15 µL final volume was prepared with 2 µL of buffer, 1 µL of T4-DNA ligase 1 or 0.5 µL of T4-DNA ligase 2, 10 µL of vector solution and 1 µL of insert solution. Ligations proceeded at RT for 2 – 4 h with T4-DNA ligase 1 and for 15 min at RT with T4-DNA ligase 2.

Ligation using T4-DNA ligase 2 could be performed directly after restriction digestion when using did CutSmart buffer (Table 1.3-1) and restriction enzymes could be heat-inactivated. After digestion, the enzyme was heat-inactivated as described by the manufacturer and afterwards 1.5 µL of ATPr (Table 1.3-1) was added to the mixture, as well as T4-DNA ligase 2 and the other DNA fragment to be ligated. This procedure was special useful for ligation of digested vectors where to clone an insert (2.2.10) and for the religation step of the digested cosmid cos4 in Red/ET protocol (2.2.11.3).

2.2.7. Dephosphorylation of plasmid DNA Dephosphorylation of a linearized vector, digested with one restriction enzyme, was removed in order to prevent self-ligation and enhance the integration of the desired fragment. For this purpose, Antarctic Phosphatase (Table 1.4-1) and its buffer were added to the digested vector as described in Table 2.2.5-1 and were incubated at 37 °C for one hour. Ligation could be performed directly after dephosphorylation although better results were achieved by purification of DNA with Wizard® SV Gel and PCR Clean-up System Kit (Table 1.4-3).

2.2.8. DNA sequencing Plasmid sequencing was delegated to 4base lab GmbH (Reutlingen) and GATC Biotech AG (Cologne). Samples were required to contain 20 µL solution with 30 – 100 ng/mL concentration of DNA in a 1.5 mL micro centrifuge tube. Concentration of DNA was calculated with NanoDrop (Table 1.2-3). The primers used for sequencing were self-designed or were chosen from “free universal primer list” (http://www.gatc-biotech.com/en/support/ support/free-universal-primers.htmL). The most common sequencing primers are III. Material and methods summarized in Table 2.2.8-1. Sequencing data were analyzed with Clone Manager Suit 7 (Table 1.10-1) by alignment of the sequences.

Table 2.2.8-1 | Universal primers used for plasmid sequencing

Plasmid Universal Sequence Notes | 53 sequenced Primer pKCXY02 M13-FP TGTAAAACGACGGCCAGT lacZα gene sequence constructs binding site pKCXY02 M13-RP CAGGAAACAGCTATGACC lacZα gene sequence constructs binding site pET28a(+) pET-RP CTAGTTATTGCTCAGCGG T7 terminator region constructs binding site pUC19 pUC-F GCCAGTGAATTCGAGCTCGG lacZα gene sequence constructs binding site pUC19 pUC-R TGCCTGCAGGTCGACTCTAG lacZα gene sequence constructs binding site pUWL-H T3 ATTAACCCTCACTAAAGGGA Downstream MCS constructs region binding site pET28a(+) and T7 TAATACGACTCACTATAGGG T7 promoter region of pTOS(Z)-spec lacZα gene binding site constructs

2.2.9. DNA amplification – Polymerase chain reaction Polymerase chain reaction (PCR) technic was used for amplification of desired regions of DNA (Mullis et al. 1986). Primers were designed with the help of Clone Manager Suit 7 (Table 1.10-1) and were ordered from Eurofins MWG Operon (Ebersberg, Germany). All solution, buffers and polymerases needed are detailed in Table 1.5-2. A variety of PCR programs were set up as summarized in Table 2.2.9-1 to Table 2.2.9-4. Elongation time was calculated depending of the length of the target fragment and the synthesis speed of the applied polymerase (Table 2.2.9-13). Annealing temperatures were calculated 5 °C underneath the highest melting temperatures of the primers (Table 2.2.9-13) although different program combinations were done in order to increase the amount of amplified target.

III. Material and methods

Table 2.2.9-1 | PCR program 1. Standard PCR

PCR step Temperature Time Number of cycles

Initiation 94 °C 5 min 1 Denaturation 94 °C 45 sec 54 | Annealing X1 45 sec 25 Elongation 72 °C X2

Termination 72 °C 10 min 1 Note. X1 stands for the calculated annealing temperature and X2 the calculated elongation time

Table 2.2.9-2 | PCR program 2

PCR step Temperature Time Number of cycles

Initiation 94 °C 5 min 1 Denaturation 94 °C 30 sec

Annealing 65 °C 30 sec 10 Elongation 72 °C X2 Denaturation 94 °C 30 sec

Annealing 60 °C 30 sec 10 Elongation 72 °C X2 Denaturation 94 °C 45 sec

Annealing 55 °C 45 sec 10 Elongation 72 °C X2

Termination 72 °C 10 min 1 Note. X2 stands for the calculated elongation time

Step down PCR (also known as Touch down PCR) (Zhang & Gurr 2000) increases the outcome of product by creating a temperature gradient with every cycle increasing the specificity of the reaction at higher temperatures on the first cycles and increasing the efficiency towards the end by lowering the annealing temperature.

Table 2.2.9-3 | PCR program 3. Step down.

PCR step Temperature Time Number of cycles

Initiation 94 °C 5 min 1 Denaturation 94 °C 30 sec

Annealing Step down 30 sec 10 (X1 + 5 °C to X1 - 5 °C) III. Material and methods

PCR step Temperature Time Number of cycles Elongation 72 °C X2 Denaturation 94 °C 30 sec

Annealing X1 – 5 °C 30 sec 15 | 55 Elongation 72 °C X2

Termination 72 °C 10 min 1 Note. X1 stands for the calculated annealing temperature and X2 the calculated elongation time. The annealing temperature started at X1 + 5 °C and decreased 1 °C for the first 10 cycles. For the following 20 cycles, the annealing temperature was set X1 – 5 °C.

Gradient PCR was especially useful to amplify fragments where the melting temperatures of the primer were too disparate from each other. Due to the fact that each PCR tube was brought to different annealing temperatures, only some of them showed successful amplification. For further amplifications, the effective parameters of successful PCR reactions were chosen to perform standard PCRs.

Table 2.2.9-4 | PCR program 4a. Gradient PCR.

PCR step Temperature Time Number of cycles

Initiation 94 °C 5 min 1 Denaturation 94 °C 30 sec Annealing Gradient from X1 + 5 30 sec 25 °C to X1 - 5 °C Elongation 72 °C X2

Termination 72 °C 10 min 1 Note. X1 was the calculated annealing temperature and X2 the calculated elongation time. The device created a gradient of annealing temperature on the PCR plate (from left to right) in every cycle.

Table 2.2.9-5 | PCR program 4b. Gradient PCR.

PCR step Temperature Time Number of cycles

Initiation 94 °C 5 min 1 Denaturation 94 °C 30 sec Annealing Gradient from X1 + 5 30 sec 10 °C to X1 - 5 °C Elongation 72 °C X2 Denaturation 94 °C 30 sec 20 Annealing X1 – 5 °C 30 sec III. Material and methods

PCR step Temperature Time Number of cycles Elongation 72 °C X2

Termination 72 °C 10 min 1 Note. X1 was the calculated annealing temperature and X2 the calculated elongation time. The device created a 56 | gradient of annealing temperature on the PCR plate (from left to right) in every cycle for 10 cycles and afterwards a single annealing temperature was set for the next 20 cycles.

For analytical PCR Taq polymerase (Table 1.4-1) was used. This enzyme has 5’  3’ endonuclease activity, creates sticky ends by adding an adenine (A) to the 3’ end and is able to synthesize 1000 bp per min. To achieve amplification of DNA fragments with blunt ends, for direct cloning into pUC19 cloning vector, Pfu polymerase, Phusion polymerase or VelocityTM polymerase were utilized (Table 1.4-1). These enzymes have 5’  3’ endonuclease activity and 3’  5’exonuclease activity what makes them more suitable for cloning proposes avoiding undesired mutations. Analytical and standard PCR reactions were designed as described in Table 2.2.9-6 and Table 2.2.9-7.

Table 2.2.9-6 | Design of analytical PCR reactions

Component Volume Concentration / Unit equivalent DNA template 0.5 µL approx. 2 – 4 μg/μL Taq-Buffer 10x 2 µL x1 DMSO 50 % 2 µL 5 % dNTPs 0.5 µL per nucleotide 20 nmol Forward primer 10 µM 1 µL 20 pmol Reverse primer 10 µM 1 µL 20 pmol

Distillated H2O ad 20 µL Taq-polymerase 0.5 µL ca. 5 Units

Table 2.2.9-7 | Design of standard PCR reactions

Component Volume Concentration / Unit equivalent DNA template 1 µL approx. 2 – 4 μg/μL Pfu buffer 10x / 5 µL 1x Taq buffer 10x 10 µL 2x DMSO 50 % 5 µL 5 % dNTPs 10 µM 2 µL per nucleotide 20 nmol Forward primer 10 µM 2 µL 20 pmol Reverse primer 10 µM 2 µL 20 pmol

Distillated H2O ad 50 µL Pfu polymerase / 1 µL ca. 5 Units Phusion polymerase

III. Material and methods 2.2.9.1. Primers designed in this work

Table 2.2.9-8 | Primer design for Red/ET gene inactivation

Restriction Primer Sequence site | 57 Primer for inactivation of rslT1 Msc18RedET-F TGTCCCTTTTTCTGTCTATTTGTCCCTGTTGCCGGGTC NdeI ACATATGCTTGAACGAATTGTTAGAC Msc18RedET-R GTCCCAGAACAGAGCCCCGTACCGTCGAGGAGACCCC NdeI ATGCATATGGTCCGCAGCGCCCGCCGCAG Primer for inactivation of rslT1, rslT2 and rslT3 rslT1- TGTCCCTTTTTCTGTCTATTTGTCCCTGTTGCCGGGTC NheI 3RedETFw AGCTAGCGGAGCGTAGCGACCGAGTG rslT1- GCACTACGCAGCGAACGACTGAGGGACGGACCGCCGA NheI 3RedETRv TGGCTAGCGGCTATTTAACGACCCTGC Primer for inactivation of rslT2 rslT2RedETFw CCTCCCGTCGAGCGCGGGCGGCGGCGCGGCCTCCGCG NheI TCGCTAGCGGAGCGTAGCGACCGAGTG rslT2RedETRv CTTCGTCGAACGGCGCTACGCGACGGGGGACCGAGGA NheI TGGCTAGCGGCTATTTAACGACCCTGC Primer for inactivation of rslT3 rslT3RedETFw GCCCGCCGAACGACTTGTGGACACCGACTGCCTCGAT NheI CAGCTAGCGGAGCGTAGCGACCGAGTG rslT1- GCACTACGCAGCGAACGACTGAGGGACGGACCGCCGA NheI 3RedETRv TGGCTAGCGGCTATTTAACGACCCTGC Primer for inactivation of rslT4 rslT4RedETRv CAGACCGCTCCAGCGGCGGCGGAGAGCGCCGCCGAGG NheI TGGCTAGCGGAGCGTAGCGACCGAGTG rslT4RedETFw GCGATGAGGGGCGGGCCGGTGCCGGGGGCGCGGCGG NheI TCAGCTAGCGGCTATTTAACGACCCTGC

Table 2.2.9-9 | Primer design for amplification of homologous regions (HR) for gene inactivation and marker (Spectinomycin cassette)

Primer Sequence Restriction site Primer for inactivation of plaABC1-3 amplification of homologous region upstream plaABC1 Eco-3T1-F GGCGATGAATTCGGAGTCCCAGCTCCACTG EcoRI Xba-3T1-R CCTGCGTCTAGACCGTGATCCGTTTGTCC XbaI Primer for inactivation of plaABC1-3 amplification of homologous region downstream plaABC3 Xba-3T2-F CAAGACGGTCTAGATGGCCGGTTCGGTCGTCCTC XbaI Pst-3T2-R GAACGCCTGCAGTTGACCTTCCGGCACCTTG PstI III. Material and methods

Primer Sequence Restriction site Primer for inactivation of plaABC1 via single crossover plaABC1SCOFw CAAGAATTCGACGGCTCTG EcoRI plaABC1SCORv TAATGGATCCCTCGGAGTTC BamHI

58 |

Table 2.2.9-10 | Primer design for gene amplification

Primer Sequence Restriction site Primer for amplification of rslT1 rslT1-Fw-Hind ATAAGGATCCCTTCCTGGCCCGTGGCTGAC BamHI HindIII, rslT1-Rv-Bam TTACAAGCTTAGAGACGGGATCCGGATGAC BamHI Primer for amplification of rslT1, rslT2 and rslT3 rslT123BamHI-R GATGACGGATCCGCGCCGGTGGCGCATG BamHI rslT123HindIII-F CCATAAGCTTGTTCGCCGAGCACCTTCTGCC HindIII Primer for amplification of rslT4 T4inpTOSfor CCGAATTCTACGCATGACACGCGGAAC EcoRI T4inpTOSrev CCATCGATAGGAGGGAGCGCTGGATTC XbaI Primer for amplification of plaABC1-3 3T Fw Hind GCGGAAGCTTGATGCTGAGATGCGTTGACGATC HindIII 3T Rv BamHI AATTGGATCCCATGATCCACGGCTGTCGCCGTAC BamHI

For expression of His6-Tag proteins, special considerations were required for the primer design in order to clone the gene with the appropriate restriction sites and correct frame in the MCS of pET28a(+) (Table 1.9-1, Figure 2-1).

Figure 2-1 | Detailed view on the MCS of pET28a(+) vector (Novagen). Possible restriction sites are marked in black. Black arrows show the direction of transcription

In case of a C-terminal tag, the forward primer included the NcoI restriction site (ccATGg) which already contained a start codon in its sequence. Thus, the original start codon of the III. Material and methods target protein was skipped as well as one more base in order to keep the correct frame. For the forward primer, the sequence was chosen in a way that the stop codon of the natural gene was omitted to allow the transcription to go on including the histidine triplets as part of the sequence. In case of an N-terminal tag, NheI (notice a mismatch in the primer name) | 59 and XhoI sites were included in the forward and reverse primer, respectively. Unlike for the C-terminal tag, the stop codon of the natural gene sequence was indispensable.

Table 2.2.9-11 | Primer design for His-Tag protein expression

Primer Sequence Restriction site Primer for amplification of rslT1 N-terminal His-Tag rslT1_His_FwNde TTAAGCTAGCATGTCCCGAACTCCG NheI rslT1_His_RvXhoI TAATCTCGAGTCAGGGCTGCGCCG XhoI Primer for amplification of rslT1 C-terminal His-Tag rslT1_His_FwNco2 TAATCCATGGCCCGAACTCCGGC NcoI rslT1_HisC_RvXhoI TAATCTCGAGGGGCTGCGCCG XhoI Primer for amplification of rslT1 C-terminal His-Tag and truncated N-terminal rslT1_HisC_FwNco3 TAATCCATGGCTCGGGCGCTCAC NcoI rslT1_HisC_RvXhoI TAATCTCGAGGGGCTGCGCCG XhoI Primer for amplification of rslT1 C-terminal His-Tag in pUWL-H rslT1CHispUWL_Fw CGCGAAGCTTGCGGATAACAATTC HindIII rslT1CHispUWL_Rv CATAGGATCCGCTTTGTTAGCAGC BamHI

Table 2.2.9-12 | Primer design for control PCRs

Primer Sequence Comments Msc18Control-F CGTTCGGTCGGTCACCTCGG Control of rslT1 deletion. Msc18Control-R CTTCGACGAGCCGACCAGCG Expected size 300 bp Control T123-F CGCGTTCCTCGCCGAGTCGG Control of rslT123 deletion. Control T123-R CCAACCGGGCGCTCGCGTC Expected size 300 bp Control_rslT2Fw GATGGCGGATTCCTCGTGGAC Control of rslT2 deletion. Control_rslT2Rv GTCGGACGCGACCAGACCGAG Expected size 300 bp Control_rslT3Fw CTCGTGGCCGACGAGTTCGC Control of rslT3 deletion. Control T123-R CCAACCGGGCGCTCGCGTC Expected size 300 bp CtrT4Red-Prm-f CCCTCCACTATAACCAACTC Control of rslT4 deletion. CtrT4Red-Prm-r GCCTTCCTCAGTGCGTAGAG Expected size 800 bp 3T Fw Hind GCGGAAGCTTGATGCTGAGATGC Control of plaABC1-3 GTTGACGATC inactivation via homologous 3T Rv BamHI AATTGGATCCCATGATCCACGGCT recombination. Expected size GTCGCCGTAC 3.5 kb and 1.7 kb in single crossover, 1.7 kb in double crossover III. Material and methods

2.2.9.2. Detailed PCR parameters

Components of the PCR mixtures and programs used for amplification of the target DNA 60 | fragments are described in Table 2.2.9-1 to Table 2.2.9-7. Table 2.2.9-13 includes all necessary information for the amplification of every single target.

Table 2.2.9-13 | Detailed information of main PCR amplifications

Annealin Elongation PCR Amplification Primer pair* DNA Template g temp. time/Poly program** product Amplification of Specr cassette for inactivation of rishirilide gene cluster transporters Msc18RedET- 1 min / aadA-T1 pcdfDuet 55 °C 3 F/R (Taq) (1.1 kb) rslT2RedET 25 sec aadA-T2 pcdfDuet 66 °C 1 Fw/Rv (Velocity) (1.1 kb) rslT3RedET 25 sec aadA-T3 pcdfDuet 66 °C 1 Fw/Rv (Velocity) (1.1 kb) rslT1-3RedET 25 sec aadA-T123 pcdfDuet 65 °C 4b Fw/Rv (Velocity) (1.1 kb) rslT4RedET 25 sec aadA-T4 pcdfDuet 66 °C 1 Fw/Rv (Velocity) (1.1 kb) Amplification of rishirilide gene cluster transporters rslT1-Fw- 25 sec rslT1 Hind/rslT1-Rv- cos4 68 °C 1 (Velocity) (1.2 kb) Bam rslT123BamHI- 55 sec rslT123 R/rslT123 cos4 65 °C 4b (Velocity) (2.9 kb) HindIII-F T4inpTOSfor/ 3 min 20 rslT4 cos4 62 °C 2 T4inpTOSrev sec (Pfu) (1.7 kb) Amplification of S. sp. Tü6071 transporter genes 3T Fw Hind/ 3T 7 min plaABC1-3 Sbe01h10 60 °C 3 Rv BamHI (Pfu) (3.5 kb) Amplification of HR for inactivation of S. sp. Tü6071 transporter genes Eco-3T1-F/ 5 min 30 3T1 Sbe01h10 62 °C 4b Xba-3T1-R sec (Pfu) (3.1 kb) Xba-3T2-F/ 4 min 30 3T2 Sbe01h10 65 °C 4b Pst-3T2-R sec (Pfu) (2.4 kb) plaABC1SCO 2 min plaABC1SCO Fw/plaABC1SC Sbe01h10 50 °C 1 (Pfu) (1.3 kb) ORv

III. Material and methods

Annealin Elongation PCR Amplification Primer pair* DNA Template g temp. time/Poly program** product Amplification for heterologous protein expression rslT1_His_FwNd Table 2 min rslT1N e/rslT1_ cos4 2 2.2.9-2 (Pfu) (1 kb) | 61 His_RvXhoI rslT1_His_FwNc Table 2 min rslT1C o2/rslT1_HisC_ cos4 2 2.2.9-2 (Pfu) (1 kb) RvXhoI rslT1_HisC_FwN Table 2 min rslT1Ctrun co3/rslT1_HisC_ cos4 2 2.2.9-2 (Pfu) (1 kb) RvXhoI rslT1CHispUWL rslT1C for pET28rslT1C 20 sec _Fw/rslT1CHisp 55 °C 1 pUWL (Table 1.9-6) (Phusion) UWL_Rv (1 kb) Control PCRs of gene inactivation Control T123F/ 3 min 30 Cos4ΔrslT123 62 °C 1 600 bp ControlT123-R sec (Taq) CtrT4Red-Prm- Table 40 sec f/CtrT4 Red- Cos4ΔrslT4 2 800 bp 2.2.9-2 (Phusion) Prm-r 3T Fw Hind/ 3T S. sp. Tü6071:: 7 min Rv BamHI pKCplaABC 60 °C 3 3.5 kb / 1.7 kb (Pfu) (2.2.11.2) Note. The table contains the primer pair name, DNA template (Table 1.9-1, unless indicated), annealing temperature, elongation time calculated for depending on the polymerase used (Table 1.4-1), PCR program and name of the product amplified with its expected length. *Complete primer pair information is detailed in Table 2.2.9-8 to Table 2.2.9-10. **PCR program information is referred in Table 2.2.9-1 to Table 2.2.9-5.

2.2.9.3. Colony PCR

Colony PCR (Hofmann & Brian 1991) is a method for rapidly screen of colonies which carry the successful plasmid without previous DNA isolation. Single E. coli colonies from a transformation plate (2.1.2.1) were picked with a toothpick onto a new plate for saving a possible right clone. The toothpicks were immediately dipped into a 1.5 mL micro centrifuge tube containing 50 µL of sterile water to solve the cell material. The tubes containing the toothpick were incubated with the lid open on a heating block at 95 °C for 5 min. The toothpick was discarded and the samples shortly incubated on ice. After centrifugation (20817 g, 2 min, RT), 1 µL of the supernatant was used as PCR template.

III. Material and methods 2.2.10. Construction of plasmids 2.2.10.1. Cloning of Streptomyces sp. Tü6071 transporter genes

a. Construction of pTOS-plaABC

62 | The primer pair 3T Fw Hind/3T Rv BamHI (Table 2.2.9-10) was used for PCR amplification of plaABC1-3as described in Table 2.2.9-13. The resultant DNA fragment was purified by performing a preparative agarose gel electrophoresis (2.2.4) and ligated with a previous SmaI digested (2.2.5) pUC19 vector (Table 1.9-1) as described in 2.2.6. After transferring (2.1.2.1) the ligation product into competent E. coli (Table 1.8-1), blue-white screening was carried out (2.1.3.1). White clones were picked for plasmid DNA isolation by performing alkaline lysis (2.2.1.1) and the purified product was qualified with restriction digestions (2.2.5) and visualized through analytical agarose gel electrophoresis (2.2.4). The correct construct was isolated again from a 20 mL culture using Pure YieldTM Plasmid Miniprep System Kit (2.2.1.2). The purified plasmid was digested with XbaI and EcoRI (2.2.5) and the target genes (3.5 kb fragment) were cleaned up by preparative agarose electrophoresis (2.2.4). The plasmid pTOS(Z)-spec (Table 1.9-1) was digested and purified following the same procedure. Insert and vector were ligated (2.2.6), transferred into E. coli (Table 1.8-1) as described in 2.1.2.1 and the resultant colonies were prepared for DNA purification by alkaline lysis (2.2.1.1). The isolated plasmids were verified by control digestion (2.2.5) and agarose gel electrophoresis (2.2.4). The final construct was purified once more from a main culture (2.1.1.1) using Pure YieldTM Plasmid Midiprep System Kit (2.2.1.2).

b. Construction of pUWL-H-plaABC The primer pair 3T Fw Hind/3T Rv BamHI (Table 2.2.9-10) was used for PCR amplification of plaABC1-3as described in Table 2.2.9-13. The resultant DNA fragment was purified by performing a preparative agarose gel electrophoresis (2.2.4) and ligated with a previous SmaI digested (2.2.5) pUC19 vector (Table 1.9-1) as described in 2.2.6. After transferring (2.1.2.1) the ligation product into competent E. coli (Table 1.8-1), blue-white screening was carried out (2.1.3.1). White clones were picked for plasmid DNA isolation by performing alkaline lysis (2.2.1.1) and the purified product was qualified with restriction digestions (2.2.5) and visualized through analytical agarose gel electrophoresis (2.2.4). The correct construct was isolated again from a 20 mL culture using Pure YieldTM Plasmid Miniprep System Kit (2.2.1.2). The purified plasmid was digested with BamHI and HindIII (2.2.5) and the target genes (3.5 kb fragment) were cleaned up by preparative agarose electrophoresis (2.2.4). The plasmid pUWL-H-tnp5 (Table 1.9-1) was digested and purified following the same III. Material and methods procedure. Insert and vector were ligated (2.2.6), transferred into E. coli (Table 1.8-1) as described in 2.1.2.1 and the resultant colonies were prepared for DNA purification by alkaline lysis (2.2.1.1). The isolated plasmids were verified by control digestion (2.2.5) and agarose gel electrophoresis (2.2.4). The final construct was purified once more from a main culture | 63 (2.1.1.1) using Pure YieldTM Plasmid Midiprep System Kit (2.2.1.2).

2.2.10.2. Cloning of rishirilide gene cluster transporter genes

a. Construction of pUWL-rslT123 The primer pair CompT123BamHI/CompT123HindIII (Table 2.2.9-10) was used for PCR amplification of rslT1, rslT2 and rslT3 (also referred as rslT123) as described in Table 2.2.9-13 using double amount of dNTPs (4 µL) as standardized (Table 2.2.9-7). The resultant DNA fragment was purified by performing a preparative agarose gel electrophoresis (2.2.4) and ligated with a previous SmaI digested (2.2.5) pUC19 vector (Table 1.9-1) as described in 2.2.6. After transferring (2.1.2.1) the ligation product into competent E. coli (Table 1.8-1), blue- white screening was carried out (2.1.3.1). White clones were picked for plasmid DNA isolation by performing alkaline lysis (2.2.1.1) and the purified product was qualified with restriction digestions (2.2.5) and visualized through analytical agarose gel electrophoresis (2.2.4). The correct construct (pUC19rslT123) was isolated again from a 20 mL culture using Pure YieldTM Plasmid Miniprep System Kit (2.2.1.2). The following cloning steps to achieve the construction of pUWL-rslT123 (Table 1.9-4) were carried out by V. Brinschwitz and were described in the Bachelor thesis (Brinschwitz 2013).

b. Construction of pUWL-OriT-rslT1 The primer pair rslT1-Fw-Hind/rslT1-Rv-Bam (Table 2.2.9-10) was used for PCR amplification of rslT1 as described in Table 2.2.9-13. The resultant DNA fragment was purified by performing a preparative agarose gel electrophoresis (2.2.4) and ligated with a previous SmaI digested (2.2.5) pUC19 vector (Table 1.9-1) as described in 2.2.6. After transferring (2.1.2.1) the ligation product into competent E. coli (Table 1.8-1), blue-white screening was carried out (2.1.3.1). White clones were picked for plasmid DNA isolation by performing alkaline lysis (2.2.1.1) and the purified product was qualified with restriction digestions (2.2.5) and visualized through analytical agarose gel electrophoresis (2.2.4). The correct construct was isolated again from a 20 mL culture using Pure YieldTM Plasmid Miniprep System Kit (2.2.1.2). The purified plasmid was digested with BamHI (2.2.5) and the target genes (1.2 kb fragment) were cleaned up by preparative agarose electrophoresis (2.2.4). The plasmid pUWL-OriT (Table 1.9-1) was digested and purified following the same III. Material and methods procedure. Insert and vector were ligated (2.2.6), transferred into E. coli (Table 1.8-1) as described in 2.1.2.1 and the resultant colonies were prepared for DNA purification by alkaline lysis (2.2.1.1). The isolated plasmids were verified by control digestion (2.2.5) and agarose gel electrophoresis (2.2.4). The final construct was purified once more from a main culture 64 | (2.1.1.1) using Pure YieldTM Plasmid Midiprep System Kit (2.2.1.2).

c. Construction of pTOS-rslT1 The primer pair rslT1-Fw-Hind/rslT1-Rv-Bam (Table 2.2.9-10) was used for PCR amplification of rslT1 as described in Table 2.2.9-13. The resultant DNA fragment was purified by performing a preparative agarose gel electrophoresis (2.2.4) and ligated with a previous SmaI digested (2.2.5) pUC19 vector (Table 1.9-1) as described in 2.2.6. After transferring (2.1.2.1) the ligation product into competent E. coli (Table 1.8-1), blue-white screening was carried out (2.1.3.1). White clones were picked for plasmid DNA isolation by performing alkaline lysis (2.2.1.1) and the purified product was qualified with restriction digestions (2.2.5) and visualized through analytical agarose gel electrophoresis (2.2.4). The correct construct was isolated again from a 20 mL culture using Pure YieldTM Plasmid Miniprep System Kit (2.2.1.2). The following cloning steps to achieve the construction of pTOS-rslT1 (Table 1.9-4) were carried out by V. Brinschwitz and were described in the Bachelor thesis (Brinschwitz 2013).

d. Construction of pTOS-rslT123 The primer pair CompT123BamHI/CompT123HindIII (Table 2.2.9-10) were used for PCR amplification of rslT1, rslT2 and rslT3 (also referred as rslT123) as described in Table 2.2.9-13. The resultant DNA fragment was purified by performing a preparative agarose gel electrophoresis (2.2.4) and ligated with a previous SmaI digested (2.2.5) pUC19 vector (Table 1.9-1) as described in 2.2.6. After transferring (2.1.2.1) the ligation product into competent E. coli (Table 1.8-1), blue-white screening was carried out (2.1.3.1). White clones were picked for plasmid DNA isolation by performing alkaline lysis (2.2.1.1) and the purified product was qualified with restriction digestions (2.2.5) and visualized through analytical agarose gel electrophoresis (2.2.4). The correct construct (pUC19rslT123) was isolated again from a 20 mL culture using Pure YieldTM Plasmid Miniprep System Kit (2.2.1.2). The following cloning steps to achieve the construction of pTOS-rslT123 (Table 1.9-4) were carried out by V. Brinschwitz and were described in the Bachelor thesis (Brinschwitz 2013). III. Material and methods e. Construction of pTOS-rslT4 The primer pair T4inpTOSfor/T4inpTOSrev (Table 2.2.9-10) was used for PCR amplification of rslT4 as described in Table 2.2.9-13. The resultant DNA fragment was purified by performing a preparative agarose gel electrophoresis (2.2.4) and ligated with a previous SmaI | 65 digested (2.2.5) pUC19 vector (Table 1.9-1) as described in 2.2.6. After transferring (2.1.2.1) the ligation product into competent E. coli (Table 1.8-1), blue-white screening was carried out (2.1.3.1). White clones were picked for plasmid DNA isolation by performing alkaline lysis (2.2.1.1) and the purified product was qualified with restriction digestions (2.2.5) and visualized through analytical agarose gel electrophoresis (2.2.4). The correct construct was isolated again from a 20 mL culture using Pure YieldTM Plasmid Miniprep System Kit (2.2.1.2). The following cloning steps to achieve the construction of pTOS-rslT4 (Table 1.9-4) were carried out by J. Brehm and were described in the Diploma thesis (Brehm 2014).

2.2.10.3. Cloning of inactivation plasmids

a. Construction of pKCplaABC123 The primer pairs Eco-3T1-F/Xba-3T1-R and Xba-3T2-F/Pst-3T2-R (Table 2.2.9-9) were used for PCR amplification of homologous regions upstream (3T1) and downstream (3T2) of plaABC1-3 as described in Table 2.2.9-13. The resultant DNA fragments (3T1 and 3T2) were purified by performing a preparative agarose gel electrophoresis (2.2.4) and directly digested with EcoRI/XbaI and XbaI/PstI (2.2.5), respectively. The digestion products were cleaned up with Wizard® SV Gel and PCR Clean-Up System (Table 1.4-3) following manufacturer’s instructions. The vector pKCXY02 (Table 1.9-1) was first digested with EcoRI and XbaI and purified following the same procedure. Upstream homologous region (3T1) and the linearized pKCXY02 were ligated (2.2.6) and transferred into E. coli (Table 1.8-1) as described in 2.1.2.1. Blue-white screening was carried out (2.1.3.1), white clones were picked for plasmid DNA isolation by performing alkaline lysis (2.2.1.1) and the purified product was qualified with restriction digestions (2.2.5) and visualized through analytical agarose gel electrophoresis (2.2.4). The correct construct pKC-3T1 (Table 1.9-5) was isolated again from a 20 mL culture using Pure YieldTM Plasmid Miniprep System Kit (2.2.1.2). pKC-3T1 was then digested with XbaI and PstI, ligated with 3T2, transferred and isolated following the same procedure as described above. The resultant plasmid pKC3T1-3T2 (Table 1.9-5) was digested with XbaI and ligated with the digested aadA cassette following the same cloning steps. aadA was obtained from preparative digestion of pLERE-Spec-OriT (Table 1.9-1) with SpeI and NheI. The achievement of the final construct pKCplaABC123 was verified by control III. Material and methods digestion (2.2.5) and visualized with the help of agarose gel electrophoresis (2.2.4). The process is exemplified in Figure 2-2.

66 |

Figure 2-2 | Scheme of the construction of pKCplaABC123 with the restriction sites used, homologous regions upstream (3T1) and downstream (3T2) and the marker (spectinomycin cassette, aadA)

b. Construction of pKCplaABC1SCO The primer pair plaABC1SCOFw/plaABC1SCORv (Table 2.2.9-10) was used for PCR amplification of an inner homologous region of orf01987 (also named in this work plaABC1) as described in Table 2.2.9-13. The resultant DNA fragment was purified by performing a preparative agarose gel electrophoresis (2.2.4) and ligated with a previous SmaI digested (2.2.5) pUC19 vector (Table 1.9-1) as described in 2.2.6. After transferring (2.1.2.1) the ligation product into competent E. coli (Table 1.8-1), blue-white screening was carried out (2.1.3.1). White clones were picked for plasmid DNA isolation by performing alkaline lysis (2.2.1.1) and the purified product was qualified with restriction digestions (2.2.5) and visualized through analytical agarose gel electrophoresis (2.2.4). The correct construct was isolated again from a 20 mL culture using Pure YieldTM Plasmid Miniprep System Kit (2.2.1.2). The purified plasmid was digested with BamHI and EcoRI (2.2.5) and the target DNA fragment (1.3 kb fragment) was cleaned up by preparative agarose electrophoresis (2.2.4). The plasmid pKCXY02 (Table 1.9-1) was digested and purified following the same procedure. Insert and vector were ligated (2.2.6), transferred into E. coli (Table 1.8-1) as described in 2.1.2.1 and the resultant colonies were prepared for DNA purification by alkaline lysis (2.2.1.1). The isolated plasmids were verified by control digestion (2.2.5) and agarose electrophoresis (2.2.4). The final construct was purified once more from a main culture (2.1.1.1) using Pure YieldTM Plasmid Midiprep System Kit (2.2.1.2). III. Material and methods 2.2.10.4. Cloning of plasmids for protein expression analysis

a. Construction of pET28rslT1N

For expression of RslT1 His6-Tag on the N-terminal end, rslT1 was amplified using the primer pair rslT1_His_FwNde/rslT1_His_RvXhoI (Table 2.2.9-11) and performance of PCR as | 67 described in Table 2.2.9-13. The resultant DNA fragment was purified by performing a preparative agarose gel electrophoresis (2.2.4) and ligated with a previous SmaI digested (2.2.5) pUC19 vector (Table 1.9-1) as described in 2.2.6. After transferring (2.1.2.1) the ligation product into competent E. coli (Table 1.8-1), blue-white screening was carried out (2.1.3.1). White clones were picked for plasmid DNA isolation by performing alkaline lysis (2.2.1.1) and the purified product was qualified with restriction digestions (2.2.5) and visualized through analytical agarose gel electrophoresis (2.2.4). The correct construct was isolated again from a 20 mL culture using Pure YieldTM Plasmid Miniprep System Kit (2.2.1.2). The purified plasmid was digested with NheI and XhoI (2.2.5) and the target DNA fragment (1 kb fragment) was cleaned up by preparative agarose electrophoresis (2.2.4). The plasmid pET28a(+) (Table 1.9-1) was digested and purified following the same procedure. Insert and vector were ligated (2.2.6), transferred into E. coli (Table 1.8-1) as described in 2.1.2.1 and the resultant colonies were prepared for DNA purification by alkaline lysis (2.2.1.1). The isolated plasmids were verified by control digestion (2.2.5) and agarose electrophoresis (2.2.4). The final construct was purified once more from a main culture (2.1.1.1) using Pure YieldTM Plasmid Midiprep System Kit (2.2.1.2).

b. Construction of pET28rslT1C and pET28rslT1Ctrun

For expression of RslT1 His6-Tag on the C-terminal end, rslT1 was amplified using the primer pair rslT1_His_FwNco2/rslT1_HisC_RvXhoI and rslT1_HisC_FwNco3/rslT1_HisC_RvXhoI (Table 2.2.9-11) and performance of PCR as described in Table 2.2.9-13. The resultant DNA fragment were purified by performing a preparative agarose gel electrophoresis (2.2.4) and ligated with a previous SmaI digested (2.2.5) pUC19 vector (Table 1.9-1) as described in 2.2.6. After transferring (2.1.2.1) the ligation product into competent E. coli (Table 1.8-1), blue- white screening was carried out (2.1.3.1). White clones were picked for plasmid DNA isolation by performing alkaline lysis (2.2.1.1) and the purified product was qualified with restriction digestions (2.2.5) and visualized through analytical agarose gel electrophoresis (2.2.4). The correct constructs were isolated again from 20 mL cultures using Pure YieldTM Plasmid Miniprep System Kit (2.2.1.2). The purified plasmids were digested with NcoI and XhoI (2.2.5) and the target DNA fragments (1 kb fragment) were cleaned up by preparative III. Material and methods agarose electrophoresis (2.2.4). The plasmid pET28a(+) (Table 1.9-1) was digested and purified following the same procedure. Insert and vector were ligated (2.2.6), transferred into E. coli (Table 1.8-1) as described in 2.1.2.1 and the resultant colonies were prepared for DNA purification by alkaline lysis (2.2.1.1). The isolated plasmids were verified by control 68 | digestion (2.2.5) and agarose electrophoresis (2.2.4). The final constructs were purified once more from main cultures (2.1.1.1) using Pure YieldTM Plasmid Midiprep System Kit (2.2.1.2).

c. Construction of pET28rslT123 For construction of pET28rslT123 the plasmid pUC19rslT123 (Table 1.9-2) was requiered. pUC19rslT123 was preparative digested with EcoRI and XbaI (2.2.5) and the target DNA fragment (2.9 kb fragment) was cleaned up by preparative agarose electrophoresis (2.2.4). The vector pET28a(+)(Table 1.9-1) was digested and purified following the same procedure. Insert and vector were ligated (2.2.6), transferred into E. coli (Table 1.8-1) as described in 2.1.2.1 and the resultant colonies were prepared for DNA purification by alkaline lysis (2.2.1.1). The isolated plasmids were verified by control digestion (2.2.5) and agarose electrophoresis (2.2.4). The final constructs were purified once more from main cultures (2.1.1.1) using Pure YieldTM Plasmid Midiprep System Kit (2.2.1.2).

d. Construction of pUWLrslT1C

For expression of RslT1 His6-Tag on the C-terminal end in Streptomyces lividans TK24 (Table 1.8-2), rslT1 was amplified using the primer pair rslT1CHispUWL_Fw/rslT1CHispUWL_Rv (Table 2.2.9-11), and the plasmid pET28rslT1C (Table 1.9-7) as template following the PCR conditions described in Table 2.2.9-13. The resultant DNA fragment was purified by performing a preparative agarose gel electrophoresis (2.2.4) and directly cut with BamHI and HindIII. The digestion was cleaned up with Wizard® SV Gel and PCR Clean-Up System (Table 1.4-3) following manufacturer’s instructions. The vector pUWL-H-tnp5 (Table 1.9-1) was digested with the same restriction enzymes and was purified by preparative agarose gel electrophoresis (2.2.4). Insert and vector were ligated (2.2.6), transferred into E. coli (Table 1.8-1) as described in 2.1.2.1 and the resultant colonies were prepared for DNA purification by alkaline lysis (2.2.1.1). The isolated plasmids were verified by control digestion (2.2.5) and agarose gel electrophoresis (2.2.4). The final construct was purified once more from a main culture (2.1.1.1) using Pure YieldTM Plasmid Midiprep System Kit (2.2.1.2).

e. Construction of pUWLrslT1CT2T3 For construction of the plasmid pUWLrslT1CT2T3 the previously cloned plasmids pUWLrslT1C (Table 1.9-7) and pUC19rslT123 (Table 1.9-2) were required. pUWLrslT1C was III. Material and methods digested with ClaI (2.2.5) and a preparative agarose gel electrophoresis (2.2.4) was performed in order to purify the big fragment (8.5 kb) containing the backbone of pUWL-H and the end part of rslT1 with the C-terminal His6-Tag. pUC19rslT123 was digested as well with ClaI following the same procedures. The small DNA fragment (2.2 kb) which | 69 contained rslT2, rslT3 and the beginning of rslT1 was purified from the preparative agarose gel. Both purified fragments were ligated (2.2.6), transferred into E. coli (Table 1.8-1) as described in 2.1.2.1 and the resultant colonies were prepared for DNA purification by alkaline lysis (2.2.1.1). The isolated plasmids were verified by control digestion (2.2.5) and agarose gel electrophoresis (2.2.4). The ligation could lead to two constructs differing in the orientation of the integrated inserts. That one achieving the reconstruction of rslT1 was useful for further experiments (Figure 2-3). The final construct was purified once more from a main culture (2.1.1.1) using Pure YieldTM Plasmid Midiprep System Kit (2.2.1.2).

Figure 2-3 | Representation of the cloning plasmids required for the construction on pUWLrslT1CT2T3

2.2.11. Gene inactivation in Streptomyces spp. In order to identify the function of the transporter genes, deletion mutants of Streptomyces sp. Tü6071 were created by homologous recombination of the inactivation plasmids pKCplaABC1SCO via single crossover (2.2.11.1) and pKCplaABC123 via double crossover (2.2.11.2). Deletion mutants of the cosmid cos4 heterologously expressed in Streptomyces albus J1074 were achieved by Redirect Technology (2.2.11.3).

2.2.11.1. Gene inactivation via single crossover

Homologous recombination is a natural DNA repair process that has been widely used for the last forty years as an engineering tool (Smithies 2001). This method consists on the gene disruption by integration of a suicide vector thanks to the recombination of an internal fragment of the target gene. When homologous recombination takes places, the vector is integrated leading to two non-functional copies of the gene, one truncated at the 3’ end and the other at the 5’ end (Kieser et al. 2000). The event is represented in Figure 2-4. It is III. Material and methods necessary to ensure that both truncated copies of the gene will not give an active protein, therefore it is recommended a minimum gene size of 1 kb.

70 |

Figure 2-4 | Integration of the inactivation plasmid in a single crossover

For inactivation of plaABC1 in Streptomyces sp. Tü6071 the plasmid pKCplaABC1SCO (Table 1.9-5) was constructed as described in a. It was then transferred to S. sp. Tü6071 by intergeneric conjugation (2.1.2.3). Those mutants that integrated the suicide inactivation plasmid and therefore disrupted the target gene would be able to grow under antibiotic selection (Table 1.6-1).

2.2.11.2. Gene inactivation via double crossover

Homologous recombination is a natural DNA repair process that has been widely used as an engineering tool (Smithies 2001). Inactivation via double crossover consists in the replacement of a target gene by a marker in two crossover steps. It is used for multiple gene deletion or when deletion via single crossover of a single gene is not possible due to its small size for the success of the recombination. The inactivation vector should contain a second antibiotic marker gene beside the one to be exchanged for the target gene. In this way, the transformants carrying single or double crossover could be distinguished by antibiotic selection (Kieser et al. 2000). The inactivation plasmid pKCplaABC123 (Table 1.9-5) was cloned as described in 2.2.10.3 for inactivation of plaABC1-3. By intergeneric conjugation (2.1.2.3) the plasmid was transferred into Streptomyces sp. Tü6071. The recombination III. Material and methods process is represented in Figure 2-5. Single crossover exconjugants were selected by antibiotic resistance (Aprar and Specr) and were also screened with GUS assay (2.1.3.2). Genomic DNA of single crossover mutants was isolated (2.2.2) and control PCR was performed with primer pair 3T Fw Hind/3T Rv BamHI (Table 2.2.9-12) as described in | 71 section 2.2.9.2.

In order to enhance the second crossover, the cultures were passage through fresh cultures selecting on the resistant gene inserted into the target gene as explained in 2.1.1.2. I-SceI technology, described in 0, was also applied in order to increase the chances of success of the recombination.

Figure 2-5 | Integration of the inactivation plasmid in a double crossover. Event A and B show the two possibilities for the second crossover to happen. Event “A” would lead to the desired inactivation and event “B” would lead back to the wild type.

III. Material and methods I-SceI endonuclease to enhance double cross-over Siegl et al. (2010) developed a system consisting in the expression of I-SceI endonuclease to create controlled genomic DNA double-strand breaks. I-SceI was cloned into the plasmid pALSceI (Table 1.9-1) which allowed its expression under the control of the thiostrepton- 72 | inducible promoter tipA (Table 1.6-1). pALSceI was transferred into the single crossover mutant S. sp. Tü6071::pKCplaABC123 (2.2.11.2) by intergeneric conjugation (2.1.2.3). I-SceI expression was induced with thiostrepton permitting that the synthesized endonuclease recognizes I-SceI sites located in the integrated pKCplaABC123 vector. By cutting the double-strand genomic DNA, homologous recombination mechanisms should be activated in order to repair the broken DNA causing a double crossover (Siegl et al. 2010)

2.2.11.3. Via Redirect Technology

Redirect Technology (Red/ET) was used for inactivation of rslT1-4 transporter genes in the cosmid cos4 for its heterologous expression in Streptomyces albus J1074. The method was first developed by Gust, Kieser, and Chater (2002). Modifications were done to adapt the protocol to this work. The aim of this technology is to exchange in an efficient recombination E. coli strain (Table 1.8-1) a desired DNA region (one or more genes) by a marker, which could be easily removed afterwards.

Firstly, the marker gene (spectinomycin cassette) was amplified using special designed “Red/ET” primers (Table 2.2.9-8). The forward and reverse Red/ET primers consisted of three sections (Figure 2-6). At the 5’ end, the primers should contain 39 bp homologous to the upstream and downstream regions, including start and stop codon, of the target gene to be deleted. At the 3’ end, the primers contained 20 bp homologous to the upstream and downstream regions of the Specr cassette. In between these two HR a restriction sites was added (NheI) so that the Specr could be removed if needed. III. Material and methods

| 73

Figure 2-6 | Representation of the Red/ET primer design. The homologous regions upstream and downstream the target genes are drawn in green. The NheI restriction site is drawn in yellow and the necessary homologous regions for the amplification of the spectinomycin marker gene are represented in blue.

PCR was performed under conditions detailed in Table 2.2.9-13. The resultant amplified fragment was purified by preparative agarose gel electrophoresis (2.2.4) and digested with DpnI (2.2.5) to ensure the absence of DNA template. This reaction mixture was cleaned up with Wizard® SV Gel and PCR Clean-Up System (Table 1.4-3) following manufacturer’s instructions. Fresh electrocompetent E. coli DH5α/pBADαβγ/cos4 cells (Table 1.8-1) were prepared and transformed with the PCR product as described in 2.1.2.1 “Transformation of E. coli DH5α/pBADαβγ/cos4 for Red/ET”. Cells were selected with two antibiotics (Apra/Spec) and the appearing clones were cultivated for isolation of the cosmids by alkaline lysis (2.2.1.1). Control digestions were performed with BamHI (2.2.5) and visualized by agarose gel electrophoresis (2.2.4). The correct constructs were transferred (2.1.2.1) into E. coli XL1Blue (Table 1.8-1) to guarantee the loss of the recombination plasmid pBADαβγ. The transformants were selected once more on Apra and Spec and the cosmids (cos4ΔgeneS, Figure 2-7) were isolated from 20 mL pre-cultures with Pure YieldTM Plasmid Midiprep System Kit (2.2.1.2). Purified DNA was checked one more time with BamHI. A preparative digestion with NheI (2.2.5) was performed to cut out the Specr cassette and the sample was either heated for inactivation of the enzyme as described from the manufacturer or purified with Wizard® SV Gel and PCR Clean-Up System (Table 1.4-3). Religation was carried out with ligase 1 or ligase 2 (Table 1.4-1) as described in 2.2.6. After transformation of E. coli XL1Blue with the religated cosmid, the cells were plated on a selection medium containing Apra. To rapidly screen for those clones that lost the Specr cassette, appearing colonies were picked in parallel onto two plates containing Apra and Apra/Spec. Those growing on Apra but not on Apra/Spec were picked into liquid cultures for cosmid isolation (2.2.1.2). The purified DNA was controlled by digesting with BamHI (2.2.5) and by PCR amplification (Table 2.2.9-13). The final construct (cos4Δgene, Figure 2-7) was then transferred into E. coli ET124567/pUZ8002 or pUB307 (Table 1.8-1) for further conjugation into Streptomyces albus III. Material and methods (Table 1.8-2) as described in 2.1.2.3. Exconjugants integrated the cosmid cos4 carrying the desired gene deletion for further analysis of rishirilide production.

74 |

Figure 2-7 | Overview of the Red/ET protocol. First line represents the design of the primer pair for amplification of the marker gene with its homologous regions (blue), the restriction site (yellow) and the homologous regions upstream and downstream of the target gene to be deleted. Second line represents the resultant PCR product. Third line describes the target gene in cos4 and the possible recombination between homologous regions of the PCR fragment. Fourth line illustrates the resultant sequence after recombination where Specr cassette has been exchanged for the the target gene (cos4ΔgeneS). Fifth line represents the final construct were Specr cassette has been removed after digestion and religation steps.

a. Inactivation of rslT1, rslT2 and rslT3 independently Inactivation of rslT1, rslT2 and rslT3 was carried out independently by using the primer pairs Msc18RedET-F/Msc18RedET-R, rslT2RedETFw/rslT2RedETRv and rslT3RedETFw/rslT1- 3RedETRv (Table 2.2.9-8) for amplification of the Specr cassette following the PCR conditions detailed in Table 2.2.9-13. The protocol was performed as described in 2.2.11.3.

III. Material and methods b. Inactivation of rslT1, rslT2 and rslT3 at once The genes rslT1, rslT2 and rslT3 were inactivated at once by using the primer pair rslT1- 3RedETFw/rslT1-3RedETRv (Table 2.2.9-8) for amplification of the Specr cassette following the PCR conditions described in Table 2.2.9-13. The protocol was performed as detailed in | 75 2.2.11.3.

c. Inactivation of rslT4 Inactivation of rslT4 was carried out by using the primer pair rslT4RedETRv/rslT4RedETFw (Table 2.2.9-8) for amplification of the Specr cassette following the PCR conditions described in Table 2.2.9-13 by J. Brehm (Brehm 2014). Further steps of the protocol were performed in this work as detailed in 2.2.11.3.

2.3. Methods in protein engineering

2.3.1. Heterologous expression of recombinant proteins 2.3.1.1. Expression system in E. coli

Protein expression test The constructed plasmids pET28rslT1N, pET28rslT1C and pET28rslT1Ctrun (Table 1.9-6) were transferred into different E. coli strains (Table 1.8-1). E. coli BL21 (DE3) codon plus RP/pETcoco-2-L1SL2 carry a helper plasmid encoding for three chaperons from S. coelicolor (GroES, GroEL1 and GroEL2) which should increase the amount of soluble protein produced and its right folding (Moncrieffe et al. 2012). E. coli BL21 (DE3) pLysS carry the pLysS plasmid which encodes T7 lysozyme, an T7 RNA polymerase inhibitor that lowers the background expression level of the target gene but does not interfere with its expression after IPTG induction. E. coli BL21 (DE3) StarTM carry a mutation in the RNaseE gene (rne131) that reduces the level of endogenous RNases and therefore increases the stability of mRNA transcripts and protein yield. E. coli C43 (DE3) carry an unknown mutation that increases the membrane surface, what made them more likely to highly express membrane proteins (Miroux & Walker 1996). The chosen cells were transformed with the protein expression plasmids (Table 1.9-6) by heat shock (2.1.2.1) and plated on their appropriate selection antibiotic LB agar plates (Table 1.6-1). Appearing clones were picked and cultivated overnight in 20 mL antibiotic LB medium (2.1.1.1). 100 µL of the pre-cultures were transferred into fresh 100 mL antibiotic LB and incubated in the shaker at 37 °C until an OD600 nm 0.4 – 0.6 was reached. The cultures were cooled down on ice for 10 min and protein expression was induced by adding 0.1 – 1 mM IPTG (Table 1.5-14). After induction the cells III. Material and methods were grown for 3 – 15 hours at 37 °C, 28 °C or 20 °C for testing the level of protein expression at different time points and growing conditions. In any case, 2 samples of 1 mL were taken in parallel at different time points, one to obtain the cell pellet by centrifugation (20817 g,

2 min) and the other to measure the OD600 nm in order to quantify the amount of cells. With 76 | an aim to compare the amount of protein produced at different time points, the pellets were

resuspended by adding 20 µL of water per 0.1 units of OD600 nm (OD600 nm = 1 equates to 5 x 108 cells/mL). Standardized cell suspensions were analyzed by SDS-PAGE or Western Blot as described in 2.3.5 and 2.3.5.2. Those culture conditions showing the most intense

protein band were tested for His6-Tag purification using manual Ni-NTA system as detailed in 2.3.3.1.

Special cultivation conditions were applied to E. coli BL21 (DE3) codon plus RP/pETcoco-2- L1SL2 cultures for optimization of soluble protein expression as described in Betancor et al. (2008). Glucose 0.2 % (m/V) was added to the 100 mL cultures next to the antibiotics to maintain a low copy number of pETcoco-2-L1SL2 plasmid (Moncrieffe et al. 2012). Cultivation of the bacterial culture after IPTG induction was recommended at 20 °C, 180 rpm for 20 h.

Preparative expression for protein purification Expression of RslT1 using pET28rslT1N and pET28rslT1C was performed in larger scale with those cells lines and cultivation conditions that showed stronger protein production on the expression tests. For this purpose, 1 – 3 L of LB medium were prepared and were inoculated

with 1 mL of the adequate overnight pre-culture. When the OD600 nm of the cell suspension reached 0.4 – 0.6, cells were cooled down on ice and protein expression was induced by adding the optimal amount of IPTG previously observed. Cells were cultivated under the tested conditions and the cell suspension was centrifuged at 17700 g, 4 °C for 20 min. Further protein purification steps by FLPC were proceeded as explained in 2.3.3.2.

2.3.1.2. Expression system in Streptomyces lividans

Protein expression test The constructed plasmids pUWLrslT1C and pUWLrslT1CT2T3 (Table 1.9-7) were transferred into E. coli ET12567/pUB307 (Table 1.8-1) by heat shock (2.1.2.1). Cells were prepared as described in 2.1.2.3 for conjugation into Streptomyces lividans (Table 1.8-2). Exconjugants were pre-cultured (2.1.1.2) and 1 mL was transferred into 100 mL antibiotic TSB medium (Table 1.7-1). Due to the lack of an inducible system, S. lividans/pUWLrslT1C or III. Material and methods /pUWLrslT1CT2T3 were grown for 2 – 3 days as well as the wild type that was used as a reference. The harvested cells of a 30 mL culture were then resuspended in 10 mL buffer A1 (without imidazole, Table 1.5-6) and cells were disrupted by French® Pressure Cell Press as described in 2.3.2.1. Protein analysis was performed by SDS-PAGE as detailed in 2.3.5.1. | 77 Preparative expression for protein purification S. lividans/pUWLrslT1C was cultivated for 2 – 3 days in 500 mL flasks with 100 mL TSB. A total of 1.5 L culture was cultivated and afterwards centrifuged (17700 g, 4 °C, 30 min) for cell harvest. The pellet was resuspended in 120 mL buffer A1 (without imidazole, Table 1.5-6) and cells were disrupted as described in 2.3.2.1. Further steps for protein purification by using ÄKTATM FLPC system are described in 2.3.3.2.

2.3.2. Isolation of proteins Disruption of bacterial cells was mandatory for purification of the target proteins in order to obtained cytosolic soluble proteins and broken cell membranes. During these procedures all samples were kept on ice, all buffers were used ice cold and centrifuges were previously set up to 4 °C.

2.3.2.1. Cell disruption

Lysozyme. When working with 100 mL cultures of E. coli expression cells for protein purification with manual Ni-NTA (2.3.3.1), lysozyme (Table 1.3-1) was added to the resuspended pellet. A small spatula of the powder was added, gently mixed and the mixture was incubated for 30 min on ice while shaking. For disruption of Streptomyces cells, lysozyme was as well added before proceeding with French® Pressure Cell Press.

French® Pressure Cell Press. French® Pressure Cell Press is a piston type pump, which disrupts the cells by pumping them through a narrow aperture under a high pressure about 1100 psi, so that the cells underlie high shear forces and burst open. To treat the samples as cold as possible, the piston of the Cell Press was stored at 8 °C overnight. For cleaning the system before cell disruption, the tubes were clean with 50 mL buffer A (Table 1.5-6). E. coli cells were disrupted twice to ensure the release of intracellular material. S. lividans cells was disrupted three times and, in addition, lysozyme was previously added to the sample. To separate the disrupted cells and soluble proteins from the unbroken cells and other insoluble components, the suspension was centrifuged at 586 g and 4 °C for 30 min. The supernatant was stored on ice for further experiments. III. Material and methods 2.3.2.2. Membrane protein isolation

Use of detergents. In order to solubilize membrane proteins from the cell membrane two detergents were tested (Table 1.5-15). A detergent solution of n-Dodecyl-β-D-

78 | maltopyranoside (DDM) was added to the samples to a final concentration of 1 %. DDM consists of a hydrophilic maltose and a hydrophobic alkyl chain. A second detergent solution tested was N-lauroylsarcosine, an anionic surfactant, which was added to the samples to a final concentration of 1 %. Detergents were added to the supernatant after centrifugation of disrupted cells or after membrane protein isolation. The mixtures were incubated for one hour on ice and constant shaking.

Isolation of membrane proteins by ultracentrifugation. For isolation of membrane proteins, 1 L culture of E. coli BL21 (DE3) StarTM/pET28rslT1N was cultivated as described in 2.3.1.1 and protein expression was induced with 0.5 mM IPTG. After 12 h incubation, the culture was centrifuged (17700 g, 4°C, 20 min) and the pellet was resuspended in 50 mL of PBS (Table 1.5-6). Cells were washed two times more with 20 mL of PBS by centrifugation at 17700 g, 4 °C for 20 min. The final pellet was resuspended with 20 mL buffer A2 (Table 1.5-6). The cells were disrupted by French® Pressure Cell Press as explained in 2.3.2.1 and the supernatant was diluted to 40 mL by adding buffer A2 before ultracentrifugation at 40000 rpm. The sample was centrifuged at 4 °C, 40000 rpm for 70 min. The supernatant was stored and the small pellet containing the cell membranes was resuspended in 7 mL buffer buffer A2 using a potter and Protease Inhibitor Cocktail (Table 1.3-1) was added. The membrane suspension was tested with the two different detergent solutions described above. The mixtures were incubated in a shaker on ice for 90 min. Following, purification with manual Ni-NTA was performed (2.3.3.1) and the fractions were analyzed by SDS-PAGE (2.3.5.1) and Western Blot (2.3.5.2).

2.3.3. Purification of proteins Within the aim of protein purification, affinity chromatography was performed. It is based on the occurrence of very specific biological interactions between a column matrix and the protein to be isolated. The protein is capable to bind to a specific ligand that is attached to the insoluble matrix of the column. Nowadays, a big variety of commercial ligands are available and the selection depends on the protein to be purified. In this work a Ni-NTA agarose (Table 1.3-1) and a Ni-HisTrap affinity column were used (Table 1.2-3). A metal (Ni2+) is chelated by the nitriloacetic acid (NTA) which is immobilized to the column material consisting of agarose beads. Basic protein residues like the imidazole functional group of III. Material and methods histidine are able to bind to the free coordination units of the Ni-atoms of the NTA matrix. Constructed plasmids for protein purification encoded recombinant proteins with an N- or

C-terminal polyhistidine tail (His6-Tag) which binds with high affinity to the Ni-NTA column because of its six imidazole groups. Elution of the bounded protein was enhanced | 79 by the addition of a competitor compound. Increasing concentrations of imidazole in an elution buffer running through the column released the target protein due to the higher affinity to the Ni-atoms.

2.3.3.1. Manual Ni-NTA

Cultures of 100 mL of E. coli or 30 mL of S. lividans were tested for protein purification by using a Ni-NTA agarose column. The cell pellet from the protein expression cultures (2.3.1) were resuspended in 8 mL buffer A2 (10 mM imidazole, Table 1.5-6). The sample was divided into four 2 mL micro centrifuge tubes and centrifuged at 20817 g, 4 °C for 10 min. The supernatant was discarded and the pellets were resuspended in the same amount of buffer A2 and combined in a 15 mL centrifuge tube. Cells were disrupted by adding lysozyme as described in 2.3.2.1. The sample was divided again into four 2 mL micro centrifuge tubes and centrifuged at 20817 g, 4 °C for 15 min.

In parallel, 1 mL of 50 % Ni-NTA agarose suspension in 30 % ethanol was washed twice using 2 mL of buffer A2 by centrifugation. The protein sample and the clean Ni-NTA soil were incubated in a small glass surrounded by ice and agitation using a magnetic fly for one hour. The column was set up with a small filter on the bottom and the mixture was poured in. The sample was allowed to flow through the column, and the follow-through was collected. Two or three washing steps were performed by carefully adding fractions of 4 mL of buffer A2 (10 mM imidazole) to the wall of the column. Afterwards, four elution steps with 500 µL of buffer B2 each were performed. All collected fraction were checked by SDS-PAGE (2.3.5.1) with or without previous concentration by protein precipitation (2.3.4).

2.3.3.2. ÄKTATM FPLC purification system

Fast protein liquid chromatography was performed for purification of proteins out of 1 – 3 L cultures by using the ÄKTATM system (Table 1.2-3). After disruption of the cells by French® Pressure Cell Press and centrifugation (2.3.2.1), the samples were incubated with DDM detergent to release the membrane proteins (2.3.2.2). Before charging the super loop with the cell debris, it was centrifuged once more at 586 g, 4 °C for 30 min. The column was equilibrated with 5 column volumes of ddH2O and 5 column volumes of buffer A1. Then, III. Material and methods the protein solution was loaded onto the column with a flow rate of 0.5 mL/min. To collect the proteins, fractions of 2 – 10 mL were automatic or manually taken using 15 mL centrifuge tubes on the fraction collector. The elution methods and volumes used for washing and elution steps for the heterologously expressed proteins in E. coli or S. lividans are shown in 80 | Figure 2-8. After purification, the column was washed with 60 mL water and 60 mL 20 % EtOH at a flow rate of 0.2 mL/min for its storage.

A)

C)

B)

Figure 2-8 | ÄKTATMFLPC set up methods for protein purification. Methods described in A) and B) were used for purification of N- and C- terminal RslT1 heterologously expressed in E. coli. Method described in C) was used for C-terminal RslT1 heterologously expressed in S. lividans.

2.3.4. Concentration of proteins To visualize the proteins separated via SDS-PAGE after Ni-NTA purification, concentration of the collected fractions was performed if necessary. Proteins were precipitated by adding trichloroacetic acid to a final concentration of 1 % (V/V) and incubated on ice for 30 min. The samples were centrifuged at 20817 g, 4 °C for 10 min and the supernatant was carefully discarded. 15 µL of SDS loading dye were added (a change of the color from blue to orange could be observed) together with 2 µL of TRIS base (1 M) to neutralize the acid pH of the III. Material and methods solution (blue color was reestablished). Samples were incubated for 10 min at 37 °C and 300 rpm for protein solubilization.

2.3.5. Analysis of protein 2.3.5.1. Sodium Dodecyl Sulfate – Polyacrylamide Gel Electrophoresis | 81 (SDS-PAGE)

To analyze a mixture of proteins after expression test (2.3.1) or after a purification procedure (2.3.3), a sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was used. SDS-PAGE separates a protein mixture in an electric field through a polyacrylamide matrix according to the molecular weight of the unstructured linear protein. SDS is an anionic detergent that surrounds the proteins and builds complexes hiding the original protein charge so that the proteins separate only according to their size. Due to net created with the polyacrylamide matrix the migration speed of the proteins is slower as bigger they are (LaemmLi 1970). All solutions and buffers used for SDS-PAGE are described in Table 1.5-5. A volume of 10 µL of protein sample was mixed with 10 µL of SDS loading dye and the solution was heated up at 100 °C for 10 min at 300 rpm. The SDS gels were prepared (Table 1.5-5) and set up in the vertical electrophoresis system and the chamber was filled with running buffer. The samples were pipetted into the wells as well as the ladder (Table 1.5-5) which served as reference to guess the size of the studied proteins. The electrophoresis was run at 130 V for 80 – 100 min, until the bromphenol blue was clearly running out from the bottom part of the gels. The gel was carefully taken out of the glass plates and incubated on agitation with 50 mL of Coomassie blue tracking dye (Table 1.5-5). After 30 – 60 min the gels were distained with 100 mL distaining solution (Table 1.5-5) and conserved for analysis.

2.3.5.2. Western Blot

By using a western blot, it was possible to identify specific proteins from a complex mixture due to separation by size and visualization by marking the target protein with a primary and secondary antibody. A modified protocol from Mahmood and Yang (2012) was used. All solutions and buffers used for Western Blot procedure are described in Table 1.5-7.

The protein mixture was first separated based on molecular weight by SDS-PAGE (2.3.5.1) and transferred from the polyacrylamide gel to a polyvinylidene fluoride (PVDF) membrane. For the electrotransfer it was necessary to prepare a sandwich as shown in Figure 2-9. The filter sheets and the PDVF membrane were adjusted to the polyacrylamide gel dimension. The sponges and the filter papers were moistened with transfer buffer and the PDVF III. Material and methods membrane with methanol. The sandwich was introduced in the transfer apparatus which was placed on ice. Transfer buffer was added until covering the sandwich completely and the electrodes were placed on the top ensuring that the PDVF membrane was located between the gel and the positive electrode (Figure 2-9). 82 |

Figure 2-9 | Western blot sandwich and electrode position for the correct transfer of the proteins

Due to the negative charged SDS-protein complex and the electric field applied for 50 – 90 min at 60 V, the proteins migrated to the PVDF membrane which was then blocked with a solution of milk proteins (blocking buffer). After incubation for 1 hour, the primary antibody (Table 1.6-1) was added and membrane was incubated overnight at 4 °C under agitation. Afterwards, the membrane was washed three times with TBS buffer for 5 min keeping agitation at RT. The secondary antibody (Table 1.6-1) was added and it was incubated with the PDVF membrane for one hour. The membrane was washed 3 times with TBS buffer and 10 mL of the staining solution were added. The colored bands appeared on the membrane after 10 – 15 min and the reaction was stopped by washing with water. The membranes were scanned for data analysis.

2.4. Isolation and analysis of secondary metabolites

2.4.1. Extraction with ethyl acetate Streptomyces strains and mutants were cultivated in HA medium (Table 1.7-1) for secondary metabolite production. Cells were cultured as described in 2.1.1.2 and centrifuged (20 min, III. Material and methods 586 g) before extraction in order to independently analyze the content of the media and the cells.

To extract rishirilide from the medium, the supernatant was adjusted to pH 3.2 with HCl

1 M to ensure the protonated form of rishirilide carboxyl group so that it occurs on the | 83 organic phase due to a decrease of the polarity. The sample was transferred to a separation funnel, mixed with ethyl acetate 1:1 (V/V) and shacked for 20 – 30 min. After that, the funnel was settled until the two phases were clearly separated. The aqueous phase was discarded and the organic phase was collected through a cellulose filter into a 100 mL or 250 mL evaporating flask. Solvents were evaporated in the rotatory evaporator and the crude extract was prepared for LC/MS analysis (2.4.2) or preparative HPLC (2.4.4).

To analyze the content of the cells, the harvested pellet was resuspended in 20 mL of acetone and shacked for 10 min. The sample was centrifuged (586 g, 20 min, RT) and the supernatant was taken into a clean flasks and solved in 80 mL of ddH2O. The sample was adjusted to pH 3.2 and extracted as described above, by adding ethyl acetated and evaporating the organic phase. When dried, the cell crude extract was prepared for LC/MS analysis (2.4.2).

2.4.2. Solid phase extraction Solid phase extraction (SPE) is based on the capacity to separate compounds solved in a liquid (mobile phase) by interaction within a matrix (stationary phase) depending on the polarity. The desired analytes and some impurities are retained on the stationary phase and by using the appropriate eluent the separated compounds can be collected in fractions. SPE was used for purification of rishirilide B after ethyl acetate extraction (2.4.1). It consisted of and a hydrophilic-lipophilic-balanced reversed-phase cartridge (Oasis® HLB20 35 cc (6g), Table 1.2-3) was chosen for the purification. For equilibration, 100 mL of 100 % methanol (V/V) were run through the column followed by 100 mL 70 % methanol (V/V) and finally 100 mL of 50 % methanol (V/V). Following the equilibration, the cartridge was sank in 50 % methanol overnight. The eluent fractions (100 mL) were prepared with a methanol gradient from 50 % to 100 % in 10 % steps. The crude extract after ethyl acetate extraction (2.4.1) was solved in 10 mL of the 100 mL 50 % methanol (V/V) solution prepared and centrifuged for 10 min at 586 g. The supernatant was loaded in fractions of 1 mL slowly on the equilibrated cartridge and the pellet was saved. The matrix was eluted with the remaining 90 mL 50 % methanol (V/V). The centrifugation pellet was mixed with 10 mL of the next eluent fraction (60 % methanol), vortexed and centrifuged (586 g, 10 min, RT). The supernatant was carefully loaded on the cartridge and the second elution with the rest of 60 % methanol was III. Material and methods flow though. These steps were repeated with the increasing concentrations of the methanol elution fractions. The flow through of each elution fraction was collected in different evaporation flasks. The resulting samples were evaporated with the rotatory evaporator and analyzed by LC/MS as described in 2.4.3. The fraction containing a clean separation of 84 | rishirilide B was further purify by preparative HPLC (2.4.4).

2.4.3. High Pressure Liquid Chromatography / Electron Spray Ionization - Mass Spectrum analysis For analysis of the extracts a High Pressure Liquid Chromatography / Electron Spray Ionization - Mass Spectrum (HPLC/ESI-MS or LC/MS) was used (Table 1.2-3). The dried extracts to be analyzed were solved in 1 mL methanol and filtered (Syringe filter Rotilabo®, 0.45 µm, Table 1.2-1). The method used was named MENS04R or MENS04RX (without MS analysis). The detailed parameters are summarized in Table 2.4.3-1 to Table 2.4.3-3.

Table 2.4.3-1 | HPLC/ESI-MS parameters set for rishirilide analysis. Composition of the solvents, flow rate, column temperature, detection UV length and MS detector are given in the table.

Parameter Settings Solvent A Acetonitrile with 0.5 % acetic acid (V/V)

Solvent B H2O with 0.5 % acetic acid (V/V) Flow rate 0.5 mL/min Column temperature 28 °C 254 nm (Ref. 400 nm) Detection 400 nm (Ref. 600 nm) MSD Scan 100 – 500 Da; MSD(+), MSD(-) Modus

Table 2.4.3-2 | Parameters of ESI source

Parameter Settings Dry gas flow 12 L/min Dry gas temperature 350 °C Nebulizer pressure 50 psi Spray capillary voltage (Vcap positive) 3.000 V Spray capillary voltage (Vcap negative) 3.000 V

III. Material and methods

Table 2.4.3-3 | HPLC gradient for analysis of rishirilide with MENS04R and MENS04RX methods

Time Solvent A (%) Solvent B (%)

(min) (Acetonitrile with 0.5 % acetic acid (V/V)) (H2O with 0.5 % acetic acid (V/V)) 0 20 80 6 20 80 | 85 7 30 70 25 95 5 28 95 5 30 20 80 35 20 80

2.4.4. Preparative High Pressure Liquid Chromatography Preparative HPLC was used as final purification step of rishirilide B after SPE (2.4.2) with the fraction eluted at a gradient of 80 % methanol (V/V). The sample was diluted in methanol and filtered (Syringe filter Rotilabo®, 0.45 µm, Table 1.2-1) before injection on the HPLC described in Table 1.2-3. The parameters of the set up method are detailed in Table 2.4.4-1 and Table 2.4.4-2. Rishirilide B was eluted from the column at minute 5 and collected in a pear shape evaporating flask. The sample was evaporated and stored at – 20 °C. For quantification of the compound, the dry extract was solved in 1 mL methanol and transferred into a 2 mL micro centrifuge tube previously weighted. The sample was evaporated with the help of a vacuum concentrator and the amount of rishirilide B obtained was weighted.

Table 2.4.4-1 | Parameters of the preparative HPLC for rishirilide B analysis. Solvents, flow rate, column temperature and detection UV length are given in the table.

Parameter Settings Solvent A Acetonitrile with 0.5 % acetic acid (V/V)

Solvent B H2O with 0.5 % acetic acid (V/V) Flow rate 0.5 mL/min Column temperature 20 °C Detection 254 nm (Ref. 400 nm)

Table 2.4.4-2 | HPLC gradient for the analysis of rishirilide B

Time Solvent A (%) Solvent B (%)

(min) (Acetonitrile with 0.5 % acetic acid (V/V)) (H2O with 0.5 % acetic acid (V/V)) 0 50 50 4 50 50 11 5 95 III. Material and methods

Time Solvent A (%) Solvent B (%)

(min) (Acetonitrile with 0.5 % acetic acid (V/V)) (H2O with 0.5 % acetic acid (V/V)) 12 5 95 12.1 50 50 15 50 50 86 |

2.5. Methods in bioinformatics Part of this work was dedicated to the development of StreptomeDB in collaboration with Jun. Prof. Stefan Günther, Pharmaceutical Bioinformatics, Institute for Pharmaceutical Sciences (University of Freiburg). In addition, a stay of two months in this working group was utilized to learn the use of Galaxy, a platform developed for the analysis of large DNA sequence.

2.5.1. StreptomeDB Lucas et al. (2013) developed StreptomeDB (Table 1.10-1), the largest database of natural products isolated from Streptomyces spp. In 2013 it contained more than 2400 unique and diverse compounds from more than 1900 different Streptomyces strains. Names, chemical structures, source organism, references, biological role, activities and synthesis routes can be found in this database. Information was extracted from thousands of articles within an automatic text mining followed by a manual curation. In order to keep this database updated, a new search on more recent publications has been done. Participation on this process was part of this work. A selection of 150 articles previously screened for potential Streptomyces related investigations were examined. The curation software (Figure 2-10) was used for collection of the desired information as well as an excel file. III. Material and methods

| 87

Figure 2-10 | Screenshot of the curation software. On the left side the list of articles and Pubmed ID are collected; in the middle, the complete article title and the abstract appeared when choosing one article; on the right, the table for collection of the relevant information found was filled; down, the linkage of the information previously collected had to be introduced.

Figure 2-11 | Information collected in excel sheet for further inclusion into the database. Pubmed ID, compound name found on the article, Pubchem ID of the compound, structure found in the article, “weitere CIDs” (other CIDs) in case of more Pubchem ID for the same compound name and link for fast access to the article.

The complete articles were read in order to find information about a named compound and its Streptomyces strain producer. More detailed information about its activity, target organism, synthetic pathway and other entities were included when available. In the case of finding a compound-Streptomyces relationship the articles was tagged as “complete”. After collecting all compound names in the excel sheet, identification of compounds not included III. Material and methods in Pubchem database was performed. The compound was searched by name in PubChem data base and when a hit was found, the Pubchem ID was included in the excel table. “Weitere CIDs” was annotated when more than one CID appeared for further manual search of the correct structure. 88 | Those compounds not described in Pubchem database by name but the structure was available in the article were further investigated. To ensure that the same compound was previously described but named in a different way, the structures were manually draw with the help of MarvinSketch. The drawn structures were converted into “SMILES”. The generated SMILES were introduced in Pubchem database searching by “structure search/identical structure”. When no hit was obtained, the structure was kept for being included into StreptomeDB. When a match was found, the Pubchem ID of the hit was introduced into the excel sheet. (Figure 2-12)

Figure 2-12 | Overview of the process of the analyzed publications for the introduction of the information in StreptomeDB. The personal participation in this process is indicated in yellow. A selection of 150 publications were handled for the whole process and, in addition, 200 compounds belonging to other sources were overtaken for the last steps.

2.5.2. Galaxy The web-based platform Galaxy was firstly developed in the working group of Jun. Prof. Stefan Günther, Pharmaceutical Bioinformatics (University of Freiburg). Nowadays, it is III. Material and methods part of the “Galaxy project” which includes new bioinformatics tools, Galaxy/deepTools (Ramírez et al. 2014).

Galaxy is a platform consisting of a set of tools for the analysis of DNA sequences, generation of heatmaps or summary plots. For this work, it was especially useful the possibility of | 89 analysis of a complete genome sequence for gene prediction and identification of secondary metabolite gene clusters. Galaxy integrated several BLAST tools for search of the predicted genes in the desired database (non-redundant (NR) database, Uniprot Swissprot, Protein Data Bank (PDB), Transporter classification database or Refseq_protein). The server also allowed the direct comparison of complete genome sequences of two desired microorganisms. In addition, it was possible to develop workflows in order to perform an established genome analysis with different inputs within “one click.

Galaxy was utilized for the deep analysis of genome sequenced of Streptomyces sp. strain Tü6071 published by Erxleben et al. (2011). The investigation was focus on the identification of a transporter involved in the biosynthesis of phenalinolactone. Different workflows were created for (i) identification of secondary metabolite gene clusters, (ii) identification of transporter genes and their location on the genome, (iii) prediction of proteins containing transmembrane domains from a complete genome, and (iv) comparison of predicted genes of three different Streptomyces sp. and the identities between them. Figure 2-13 shows an example of the designed workflow with a genome sequence as input. It provided information about secondary metabolite gene clusters, putative proteins, putative proteins with transmembrane domains and putative transporters with transmembrane domains. Other workflows created can be found in Appendix 2. III. Material and methods

90 |

Figure 2-13 | Galaxy workflow for identification of secondary metabolite gene clusters and putative transporter genes

Moreover, another workflow allowed the analysis of the genomes of Streptomyces coelicolor, Streptomyces avermitilis and S. sp.Tü6071 to search for transporter genes in their genomes and further comparison between the strains. The following information was obtained for each strain: name of the ORF predicted, DNA sequence, description of the predicted protein, ID number from TransporttDB classification depending on the nature of the identified transporter (Ren et al. 2007), number of identical transporters in the genome and location of the gene within the as well predicted gene clusters. The descriptions of all the putative annotated transporter genes were compared between the strains in order to identify those singular for each strain and therefore maybe related to their secondary metabolites.

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IV. Results

1. Investigations on the transporter genes of rishirilide gene cluster The putative annotated transporter genes rslT1-T4 in rishirilide gene cluster (Yan et al. 2012) were investigated in order to described their role on the biosynthetic pathway. An initial bioinformatics characterization of the genes and their proteins was performed by using different BLAST databases and prediction software (III. Table 1.10-1). Gene inactivation, complementation and overexpression experiments of the target genes were carried out in the heterologous expression host Streptomyces albus J1074. The fermentation broth of the created mutants as well as the cell pellet were extracted and analyzed for the production of secondary metabolites. For further characterization of the substrate specificity of one of the transporter systems, RslT1 was heterologously expressed in E. coli and Streptomyces lividans and protein purification was performed.

1.1. Bioinformatics characterization of rishirilide gene cluster transporters DNA sequence analysis of the annotated rslT1, rslT2, rslT3 and rslT4 transporter genes of the rishirilide gene cluster was performed by using different bioinformatics tools. Figure 1-1 shows the organization of the cluster where rslT1, rslT2 and rslT3 are located in a row and in the same orientation between the minimal PKS genes (rslK1-K4 and rslA) and rslO1-O2 oxygenases. The genes rslT2 (750 bp) and rslT3 (960 bp) are overlap by four base pairs indicating that they are probably transcribed under the influence of the same promoter. The gene rslT1 (966 bp) is situated 110 bp downstream rslT2. Figure 1-1 shows as well rslT4 in the rishirilide cluster, a transporter gene located between the regulator rslR4 and the oxygenases rslO7-O10 and it consists of 1593 bp. RslR4 was identified to belong to the MarR regulatory family and has been proposed as a negative regulator of rslT4 by J. Wunsch-Palasis (Wunsch-Palasis 2013). Overexpression experiments of rslR4 in S. albus::cos4 are described in 1.3.4. IV. Results

92 |

Figure 1-1 | Organization of the rishirilide gene cluster (rsl). The transporter genes are labeled in the red boxes.

Blastx search from NCBI with non-redundant (NR) database and Uniprot database of the gene sequences led to the identification of RslT1, RslT2 and RslT3 as an ABC transporter system and RslT4 as a MFS multidrug transporter. The three highest hits of the individual search of the four genes were all found in the same strains, Streptomyces bottropensis ATCC 25435 (Hongyu Zhang et al. 2013), Streptomyces scabiei strain NCPPB 4086 (Harrison et al. 2014) and Micromonospora lupini strain Lupac 08 (Alonso-Vega et al. 2012) as shown in Table 1.1-1.

Table 1.1-1 | Results of blastx search of rslT1, rslT2, rslT3 and rslT4 in NCBI NR database and Uniprot database.

Query Description First hit Second hit Third hit gene Query coverage 100 % 100 % 83 % Identities 100 % 90 % 60 % Positives 100 % 95 % 77 % Gene name SBD_4864 IQ62_28420 MILUP08_44626 rslT1 Extracellular ABC transporter Extracellular ABC ABC transporter Protein name substrate- transporter substrate- substrate- binding protein binding protein binding protein S. bottropensis S. scabiei M. lupini strain Lupac Organism ATCC 25435 NCPPB 4086 08 Query coverage 99 % 99 % 99 % Identity 100 % 94 % 62 % Positives 100 % 96 % - Gene name SBD_4863 IQ62_28415 MILUP08_44627 Arginine ABC Putative amino acid rslT2 Uncharacterized transporter ABC-type transport Protein name protein ATP-binding system, ATPase protein component S. bottropensis S. scabiei M. lupini strain Lupac Organism ATCC 25435 NCPPB 4086 08 IV. Results

Query Description First hit Second hit Third hit gene Query coverage 99 % 99 % 74 % Identity 100 % 90 % 68 % Positives 100 % 93 % 78 % | 93 Gene name SBD_4862 IQ62_28410 MILUP08_44628 rslT3 Putative ABC Amino acid ABC Putative aminoacid Protein name transporter ATP- transporter ABC transporter binding protein permease permease S. bottropensis S. scabiei M. lupini strain Lupac Organism ATCC 25435 NCPPB 4086 08 Query coverage 99 % 99 % 90 % Identity 100 % 90 % 66 % Positives 100 % 94 % 79 % Gene name SBD_4846 IQ62_28340 MILUP08_44615 EmrB/QacA Multidrug resistance rslT4 subfamily Multimultidrug transporter, Protein name Multidrug transporter EmrB/QacA resistance subfamily transporter S. bottropensis S. scabiei M. lupini strain Lupac Organism ATCC 25435 NCPPB 4086 08 Note. Query coverage describes which percentage of the query sequence is being compared. Identities describe which percentage of the amino acids covered are identical. Positives describe which percentage of the amino acids covered are identical or similar in charge. Gene name, protein name and organism of the hit are also included in the table.

This match led to the suspicious that very similar cluster of rishirilide could be found in the genome of these three species. AntiSMASH analysis of these strains showed nearly identical gene cluster organization to rsl (Figure 1-2). IV. Results

94 |

Figure 1-2 | AntiSMASH outcome from rishirilide cluster search. Similar rsl clusters of Streptomyces bottropensis ATCC 25435, Streptomyces scabiei ATCC 25435 and Micromonospora lupini strain Lupac 08. The genes rslT1, rslT2 and rslT3 are highlighted with a red shadow while rslT4 is showed in grey.

1.1.1. Characterization of rslT1 AntiSMASH provides, beside the identification of similar secondary metabolites gene cluster, a smCOG annotation (secondary metabolism Clusters of Orthologous Groups) of the individual genes that form part of it. The predicted gene corresponding with the annotated rslT1 was in this case identified as an ABC transporter glutamine-binding protein GlnH.

Similar information was obtained when RslT1 protein sequence was introduced in PDB Protein Data Bank (Berman 2000). Crystal structures of the resultant matches belong to ABC transporter proteins complexed with , ornithine, glutamine or valine indicating their involvement in amino acid uptake. However, alignment of the sequences of these crystallized proteins with RslT1 did not show higher results than 31 % identities1 and 48 % positives2.

Assuming that RslT1 is the substrate binding protein (SBP) of an importer ABC transporter system, the protein sequence should present some conserve regions in order to be retained within the cell envelope (Sankaran & Wu 1995). In Gram-positive bacteria, membrane proteins like SBP are described to be anchored to the membrane through a lipid linkage. Lipoproteins have been described to contain a signal sequence in the N-terminal end of the pre-lipoprotein followed by a cysteine (Hayashi & Wu 1990). This crucial cysteine residue is

1 Identity = the extent to which two (nucleotide or amino acid) sequences have the same residues at the same positions in an alignment, often expressed as a percentage (Madden 2011). 2 Positive = the extent to which two (nucleotide or amino acid) sequences have residues with similar physico-chemical properties at the same positions in an alignment, often expressed as a percentage (Madden 2011). IV. Results located within a conserved sequence named “lipobox” (Sankaran and Wu 1995; Qi et al. 1995). The lipobox sequence is recognized by signal peptidase II (SPaseII) or lipoprotein signal peptidase (Lsp) (Tjalsma et al. 1999). To determine if RslT1 shares conserve motifs with lipoprotein structures, three software described below were utilized. For the signal | 95 peptide prediction, Phobius 1.01 (Cramer et al. 2011) and SignalP 4.1 (Petersen et al. 2011) were considered. Phobius predicted the first 37 amino acids to be a signal peptide and the rest of the sequence as non-cytosolic (Figure 1-3 A). SignalP 4.1 did not predict a defined signal peptide (Figure 1-3 B).

A) B)

Figure 1-3 | Outcome of the analysis of RslT1 in Phobius and SignalP software in search of a signal peptide. A) Phobius prediction. The red line represent the signal peptide, the blue the non-cytoplasmic domain. B) SignalP prediction. The green line represents the signal peptide score which should appear over the purple line.

For identification of a “lipobox” in RslT1 sequence, the prediction software LipoP (Juncker et al. 2003) was utilized. LipoP is considered to perform a consensus approach compare to other online tools as reviewed by Rahman et al. (2008). LipoP evidenced a cleavage site in amino acids 35 and 36 which correspond with the conserved cysteine previously described within the lipobox sequence by Sutcliffe and Harrington (2002). The sequence LLAAC was identified in RslT1 as the lipobox.

1.1.2. Characterization of rslT2 Blastx search identified rslT2 as a gene encoding an ATP-binding protein. ATP binding proteins are described for the ATP-hydrolyzing domains, also referred as nucleotide-binding domain (NBD). These domains have three highly conserve motifs Walker A, Walker B and ABC signature. Other characteristic motifs, Q-, D- and H-loop, contain just one highly conserved residue that interacts with the γ-phosphate of ATP (Figure 1-4) (Davidson et al. 2008). IV. Results

Figure 1-4 | Conserved regions of the NBD of an ABC transporter system responsible of the hydrolyzation of ATP.

96 | Walker motif A (GxxGxGK(T/S), “x” can be varied) and Walker motif B (hhhhD, h stands for hydrophobic amino acid) form the P-loop (phosphate binding-loop) which surrounds the nucleotides and utilizes its highly conserved residues of lysine and to bind to the phosphate oxygen atoms (Ramakrishnan et al. 2002). It is immediately precede by the signature motif (LSGGQQ/R/KQR) also referred as Walker C or Linker peptide that is unique to the ABC transport family (Schneider & Hunke 1998). Q-loop and H-loop (also called switch region) consist of glutamine (Q) and histidine (H) amino acids, respectively. In the context of an active form of the NBD as a dimer (NBD1 and NBD2), the D-loop of NBD1 interacts with Walker motif A of NBD2 and vice versa (Davidson & Chen 2004).

Manual analysis of RslT2 sequence identified all these conserve regions described for an ATP-hydrolyzing domain (Figure 1-5).

Figure 1-5 | RslT2 amino acid sequence with labelled conserved motifs. Walker A in red (GPSGSGKS); Q- loop, in blue (Q); ABC signature, in green (LSGGEQ); Walker B, in purple (AMLFD); H-loop, in orange (H).

RslT2 amino acid sequence was search for similar proteins in PDB Protein Data Bank (Berman 2000). The resulting hits showed crystal structures of proteins complexed with AMP, ADP or ATP. All of them were described as NBD. Alignment of the amino acid sequence of these crystallized proteins with RslT2 showed significant similarities with results around 52 % identities and 67 % positives.

1.1.3. Characterization of rslT3 The third component of the ABC transporter system, RslT3, was identified as a permease. This protein is made of two hydrophobic membrane-spanning or integral membrane (IM) domains or proteins, which create the channel through which the substrate passes during translocation (Biemans-Oldehinkel et al. 2006). This two IM domains typically consist of six IV. Results transmembrane helixes and interact with the two hydrophilic domains carrying the ATP- hydrolyzing domains on the cytosolic side of the membrane (Davidson et al. 2008).

RslT3 was analyzed with two software for prediction of membrane proteins, SOSUI server

(Hirokawa et al. 1998) and TMHMM 2.0 server (Krogh et al. 2001). In both cases, RslT3 was | 97 predicted to form 6 helix through the membrane with C- and N-terminus ends on the cytosolic side as shown in Figure 1-6. The SOSUI prediction differentiates between primary and secondary helixes. These are described to be involved in structure stability through the membrane and formation of active sites, respectively (Hirokawa et al. 1998). First, second and sixth helixes were predicted as primary helixes, the other three as secondary helixes. Between the fourth and fifth helix, a conserved EAA motif located at a distance of ~100 residues of the C-terminal end was identified which is characteristic of cytoplasmic membrane proteins of bacterial binding protein-dependent transporters (Dassa & Hofnung 1985; Mourez et al. 1997). A) C)

B)

Figure 1-6 | Outcome of RslT3 analysis with SOSUI software. A) RslT3 amino acid sequence with the six 23 AA transmembrane regions labelled in green and the EAA motif in red. B) Output of SOSUI server. The graphic shows the representation of the 6 helixes, in black hydrophobic amino acids, in blue polar amino acids, in bold blue positive charged amino acids and in red negative charged amino acids. C) Output of SOSUI server. The graphic shows the representation of the 6 transmembrane domains with primary and secondary helixes. The upper part indicates the cytosolic side where N- and C-terminus ends were predicted to be present.

No significant matches were found by comparing RslT3 sequence on the Protein Data Bank (Berman 2000) due to the lack of crystal structure of membrane proteins. IV. Results 1.1.4. Characterization of rslT4 The sequence of rslT4 has been different annotated by A. Linnenbrink (Linnenbrink 2009) and X. Yan (Yan et al. 2012). The DNA sequence proposed by X. Yan was 1446 bp with a GTG as start codon (encoding for valine). However, A. Linnenbrink proposed a 1539 bp gene, 93 98 | bp longer on the start codon side, with an ATG (encoding for methionine) as a start codon. These different annotations were not crucial for the prediction of rslT4 as a gene encoding a Major Facilitator Superfamily (MFS) transporter. MFS transporter are known for a uniform topology of 12 transmembrane α-helixes connected by hydrophilic loops, with the N- and C- terminal located in the cytoplasm (Pao et al. 1998). MFS proteins typically consist of 400 – 600 amino acids even though they share low sequence identity or similarity. The conserved sequence DRxxRR (where “x” is any amino acid) has been proposed as a signature sequences that could be found in both N- and C-halves of the protein (Maiden et al. 1987).

The translated 481 and 512 amino acid sequences resultant from the two proposed annotation (Figure 1-7 C) are characterized in this section. Inactivation and expression of rslT4 in the experimental part of this work, the gene sequence proposed by X. Yan (Yan et al. 2012) was utilized.

The RslT4 was analyzed with two software for prediction of membrane protein, SOSUI server (Hirokawa et al. 1998) and TMHMM 2.0 server (Krogh et al. 2001). In both cases, RslT4 was predicted to form 14 helixes through the membrane (TM) with the C- and N-terminal expressed towards the cytosolic side. Figure 1-7 shows the SOSUI prediction which differentiates as well between primary and secondary helixes which are described to be involved in structure stability through the membrane and formation of active sites, respectively (Hirokawa et al. 1998). TM2-3, TM5-6, TM8-9 and TM14 were defined as primary helixes, the other transmembrane regions as secondary helix. The annotation of rslT4 by X. Yan was predicted to create the same topology in the membrane with the only different of a shorter N-terminal cytosolic arm. The starting amino acid valine is pointed with a red arrow in Figure 1-7 A. The organization of the amino acids in the 14 transmembrane helixes and its location on the primary sequence are shown in Figure 1-7 B and C. IV. Results A)

| 99

B)

C)

Figure 1-7 | Outcome of RslT4 analysis with SOSUI software. A) Graphic representation of 14 transmembrane domains with primary and secondary helixes. The upper part represents the cytosolic side where N- and C- terminal ends were predicted. B) Graphic representation of the 14 helixes the hydrophobic amino acids in black, polar amino acids in blue, positive charged amino acids in bold blue and negative charged amino acids in red. C) RslT4 amino acid sequence with the 14 transmembrane regions labelled in green. The first 31 amino acids on the N-terminal end not present in the shorter annotation of rslT4 are given in brackets.

Only four similar crystallized protein were found when searching for the sequence of RslT4 on the Protein Data Bank (Berman 2000). However, none of them showed high E-value3 nor significant alignment with the query sequence.

The protein sequence was investigated in order to find a signal peptide sequence by using the prediction tools Phobius 1.01 (Cramer et al. 2011) and SignalP 4.1 (Petersen et al. 2011). No

3 E-value = the Expectation value or Expect value represents the number of different alignments with scores equivalent to or better than S that is expected to occur in a database search by chance. The lower the E value, the more significant the score and the alignment (Madden 2011). IV. Results signal peptide was identified on the RslT4 512 amino acid sequence although Phobius 1.01 identified a 16 amino acid signal peptide for the shorter annotation of RslT4 (Figure 1-8).

A) 100 |

B)

Figure 1-8 | Outcome of the analysis of RslT4 in Phobius and SignalP software for identification of a signal peptide. A) Results of the analysis of the long annotated protein RslT4. On the left, the graphical output analysis from Phobius prediction software. The red line represent the probability (0 – 1) of a signal peptide and the blue the non-cytoplasmic domain. On the right, the graphical output from SignalP of RslT4 analysis. The green line represents the signal peptide score which should be over the purple line. B) Results of the analysis of the short annotated protein RslT4. On the left, the graphical output from Phobius prediction software. The red line represent the probability of a signal peptide and the blue line the non-cytoplasmic domain. On the right, the graphical output from SignalP of RslT4 analysis. The green line represents the signal peptide score which should be over the purple line.

1.2. Inactivation and expression of the ABC transporter system RslT1-T3

1.2.1. Attempts to construct rslT1, rslT2 and rslT3 deletion cosmids by Red/ET With the aim of identifying the role of RslT1, RslT2 and RslT3 in rishirilide metabolism, the genes rslT1, rslT2 and rslT3 were independently and simultaneously inactivated by following Redirect© Technology procedure (III.2.2.11.3). The target genes were exchanged by homologous recombination for a marker gene (spectinomycin resistance cassette, aadA, Specr) which was amplified by PCR with the help of special primers (III. Table 2.2.9-8). In order to avoid polar effects on the expression of the clustered genes the marker was excised IV. Results by restriction digest. The modified cosmids were heterologously expressed in Streptomyces albus J1074 for production evaluation of the secondary metabolites after ethyl acetate extraction and LC/MS analysis.

1.2.1.1. Inactivation of rslT1 | 101

The fragment containing the spectinomycin cassette and the homologous regions upstream and downstream of rslT1 for its inactivation was successfully amplified by PCR as described in III. Table 2.2.9-13 (Figure 1-9 A). The resultant 1.1 kb DNA was transferred into E. coli DH5α/pBADαβγ containing cos4 for its recombination to achieve the deletion construct cos4ΔrslT1S. The purified cosmid of a clone Aprar and Specr was controlled by digestion with BamHI. The agarose gel showed the 13 expected fragment size on a 0.7 % agarose gel (Figure 1-9 B and C). Although cos4ΔrslT1S and cos4 showed the same size of the digested fragments due to the similar size of rslT1 and the exchanged marker gene, it was useful to confirm the correct cosmid isolation. Following steps would have included the digestion of the Specr cassette to release it from the cosmid. However, the chosen restriction sites (NdeI) included in the primers for the marker amplification were not appropriate due to the presence of a third NdeI site somewhere else in the cosmid. Alternatively, the correct inactivation of rslT1 was achieved in combination with the deletion of rslT2 and rslT3 leading to the construct cos4ΔrslT123 as explained in section 1.2.1.4.

A) B) C)

Figure 1-9 | Agarose gels from rslT1 inactivation experiments. A) Lane 1 – 4 show the spectinomycin cassette amplified by PCR with its expected size of 1.1 kb. Lane L contains 1 kb ladder B) Lanes 1 – 2 show the resulting digestions of cos4 and cos4ΔrslT1S with BamHI, respectively. The size of the expected fragments can be seen in C. Lane L contains the 1 kb ladder. C) The table contains the size of the expected fragments after digestion of cos4 and cos4ΔrslT1S with BamHI in base pairs. The distinctive fragment of the digestion between the control and the inactivation mutant is highlighted with a grey shadow.

IV. Results 1.2.1.2. Inactivation of rslT2

The fragment containing the spectinomycin cassette and the homologous regions upstream and downstream of rslT2 for its inactivation was successfully amplified by PCR as described

102 | in III. Table 2.2.9-13. The resultant 1.1 kb DNA was transferred into E. coli DH5α/pBADαβγ containing cos4 for its recombination to achieve the deletion construct cos4ΔrslT2S. The purified cosmid of a clone Aprar and Specr was controlled by digestion with BamHI. The agarose gel showed the 13 expected fragment size on a 0.7 % agarose gel (Figure 1-10 A). Although cos4ΔrslT2S and cos4 showed the same size of the digested fragments due to the similar size of rslT2 and the exchanged marker gene, it was useful to confirm the correct cosmid isolation. Following steps were performed in order to release the marker gene by digesting with NheI from the cosmid. The correct digestion with NheI was visualized by agarose gel electrophoresis (Figure 1-10 B) and the religated cosmid was transferred into E. coli XL1Blue. The isolated DNA from selected clones Aprar but not Specr was controlled by digestion with BamHI. None of the examined cosmids was detected to contain the correct fragment size as shown in Figure 1-10 C. Alternatively, inactivation of rslT2 was achieved in combination with the deletion of rslT1 and rslT3 leading to the construct cos4ΔrslT123 as explained in section 1.2.1.4.

A) B) C) D)

Figure 1-10 | Agarose gels from rslT2 inactivation experiments. A) Lane 1 – 2 show the digestions of cos4 and cos4ΔrslT2S with BamHI, respectively. The size of the expected fragments can be seen in D in base pairs. Lane L contains the 1 kb ladder. B) Lane 1 shows cos4ΔrslT2 digested with NheI which releases the spectinomycin cassette (960 bp) from the cosmid. Lane L contains the 1 kb ladder C) Lanes 1 – 3 and 5 – 6 show the digestions with BamHI of the isolated cosmids cos4ΔrslT2 after religation. Lane 4 shows the control digestion with BamHI of cos4. The size of the expected fragments can be seen in D. Lane L contains the 1 kb ladder. D) The table contains the size of the expected fragments after digestion of cos4, cos4ΔrslT2S and cos4ΔrslT2 with BamHI in base pairs. The distinctive fragment of the digestion between the control and the inactivation mutant is highlighted with a grey shadow.

IV. Results 1.2.1.3. Inactivation of rslT3

The fragment containing the spectinomycin cassette and the homologous regions upstream and downstream of rslT3 for its inactivation was successfully amplified by PCR as described in III. Table 2.2.9-13 (Figure 1-11). The resultant 1.1 kb DNA was transferred into E. coli | 103 DH5α/pBADαβγ containing cos4 for its recombination to achieve the deletion construct cos4ΔrslT3S. The purified cosmid from a clone Aprar and Specr was controlled by digestion with BamHI. The agarose gel showed the 13 expected fragments visible on a 0.7 % agarose gel. Although no difference between cos4ΔrslT3S and cos4 showed the same size of the digested fragments due to the similar size of rslT3 and the exchanged marker gene, it was useful to confirm the correct cosmid isolation. Following steps were performed in order to release the marker gene by digesting with NheI from the cosmid. The correct digestion with NheI was visualized by agarose gel electrophoresis (Figure 1-11) and the religated cosmid was transferred into E. coli XL1Blue. The isolated DNA from selected clones Aprar but not Specr was controlled by digestion with BamHI. None of the examined cosmids was detected to contain the correct fragment size as shown in Figure 1-11. Alternatively, inactivation of rslT3 was achieved in combination with the deletion of rslT1 and rslT2 leading to the construct cos4ΔrslT123 as explained in section 1.2.1.4.

A) B) C)

Figure 1-11 | Agarose gels from rslT3 inactivation experiments. A) Lane 1 the digestion of cos4ΔrslT2S with BamHI, Lane L contains the 1 kb ladder. The size of the expected fragments can be seen in C in base pairs. B) Lanes 1 – 5 show the digestions of the isolated cosmids after religation digested with BamHI. Line 6 shows the control digestion with BamHI of cos4. The size of the expected fragments can be seen in C in base pairs. Lane L contains the 1 kb ladder. C) The table contains the size of the expected fragments after digestion of cos4, cos4ΔrslT3S and cos4ΔrslT3 with BamHI in base pairs. The distinctive fragment of the digestion between the control and the inactivation mutant is highlighted with a grey shadow.

IV. Results 1.2.1.4. Inactivation of rslT123

Due to the difficulties of the individual inactivation of rslT1, rslT2 and rslT3, inactivation of these three genes at once was performed. With this aim, a marker gene containing the Specr

104 | cassette and the homologous regions upstream rslT3 and downstream rslT1 was amplified by PCR as described in III. Table 2.2.9-13. The resultant 1.1 kb DNA was transferred into E. coli DH5α/pBADαβγ containing cos4 for its recombination to achieve the deletion construct cos4ΔrslT123S. The purified cosmid from a clone Aprar and Specr was controlled by digestion with BamHI. The agarose gel showed the 13 expected fragments visible on a 0.7 % agarose gel and an evident difference with cos4 was observed (Figure 1-12 A). In order to release the marker gene from the cosmid a digestion with NheI was performed. This was correctly visualized by agarose gel electrophoresis and the religated cosmid was transferred into E. coli XL1Blue. The isolated DNA was digested with BamHI and the correct resultant fragments were verified on the agarose gel (Figure 1-12 B). In addition, control PCR using the primer pair Control T123-F/Control T123-R was performed as described in III. Table 2.2.9-13 and the expected fragment of 680 bp was obtained compared to the control cos4 (3467 bp) (Figure 1-12 C). The correct construct of cos4ΔrslT123 was confirmed.

A) B) C) D)

Figure 1-12 | Agarose gels from rslT1, rslT2 and rslT3 inactivation experiments. A) Lanes 1 – 2 show the digestions of cos4ΔrslT123S and cos4 with BamHI, respectively. The size of the expected fragments can be seen in D, the different fragments are pointed with the arrows on the agarose gel. Lane L contains the 1 kb ladder. B) Lane 1 shows cos4ΔrslT123 and cos4 digested with BamHI, respectively. The size of the expected fragments can be seen in D, the different fragments are pointed with the arrows on the agarose gel. Lane L contains 1 kb ladder. C) Lanes 1 – 2 show the amplified product of the control PCRs of cos4ΔrslT123 and cos4, respectively. The correct size of the expected fragments are pointed with the arrows. Lane L contains 1 kb ladder. D) The table contains the size of the expected fragments in base pairs after digestion of cos4, cos4ΔrslT123S and cos4ΔrslT123 with IV. Results

BamHI. The distinctive fragment of the digestion between the control and the inactivation mutant is highlighted with a grey shadow.

1.2.2. Production analysis of S. albus::cos4ΔrslT123 and the complemented mutant | 105 To determine the importance of the lack of the ABC transporter system in cos4 on rishirilide metabolism, the recombinant cosmid cos4ΔrslT123 was transferred into Streptomyces albus J1074 by intergeneric conjugation as described in III.2.1.2.3. The mutant was cultivated in HA production medium (III. Table 1.7-1) for 4 days. The harvested cells and the supernatant were extracted with ethyl acetate (III.2.4.1) for analysis by LC/MS (III.2.4.3). As control, S. albus::cos4 and S. albus::pOJ463 were cultivated and analyzed in the same way.

Figure 1-13 shows the HPLC chromatograms achieved after the analysis of the described extractions. They show the comparison of the production cultures of the ABC transporter system deletion mutant to the controls. IV. Results A)

106 |

B)

Figure 1-13 | HPLC chromatograms (λ = 254 nm) achieved from the analysis of the production cultures of S. albus::pOJ436 (negative control), S. albus::cos4 (positive control) and S. albus::cos4ΔrslT123. A) Chromatograms obtained from the HPLC analysis of the medium. Rishirilide B peak is highlighted with a blue shadow. B) Chromatograms obtained from the HPLC analysis of the extract from inside the cells. Rishirilide B peak is highlighted with a blue shadow.

The HPLC chromatograms of the production cultures of S. albus::cos4ΔrslT123 and S. albus::cos4 showed comparable profile in the analysis of the medium and the harvested cells. However, rishirilide B peak (20.7 min) reached a smaller maximum absorbance in the inactivation mutant in comparison to the positive control. Because of this, quantification of the secondary metabolite was necessary.

To quantify rishirilide B production of the deletion mutant in comparison with the positive control, three different S. albus::cos4ΔrslT123 exconjugants and three cultures of S. albus::cos4 were analyzed. The area under the curve (AUC) of rishirilide B peak in the HPLC chromatograms obtained was divided by the weight of the dried cell pellet in mg. In this IV. Results case, calculation of the dried pellet was done from extrapolation of 1 mL sample of the production culture. Figure 1-14 shows the comparison of the calculated relation of the amount of rishirilide found in the supernatant of S. albus::cos4ΔrslT123 and S. albus::cos4.

B) | 107

Figure 1-14 | Average of the calculated AUC of rishirilide B peak divided by the weight of the dry cell pellet (mg) from S. albus::cos4ΔrslT123 and S. albus::cos4 cultures. The maximum and minimum calculations are drawn with a thin line over the bars. A) Calculation of rishirilide B detected in the media. B) Calculation of rishirilide B detected inside the cells.

The ABC transporter system deletion mutants showed a decreasing tendency of the amounts of rishirilide B detected in the media as well as inside the cells with values of 0.94 and 0.67- fold, respectively, in comparison with the control S. albus::cos4.

Complementation of the deletion mutant S. albus::cos4ΔrslT123 was performed transferring the constructed plasmid pUWL-rslT123 (III. Table 1.9-4) by intergeneric conjugation as described in III.2.1.2.3. The exconjugants were cultivated in HA production medium (III. Table 1.7-1) and after 4 days the cell pellet and supernatant were extracted with ethyl acetate (III.2.4.1) for analysis by LC/MS (III.2.4.3). As control, cultures of S. albus::cos4 and S. albus::cos4ΔrslT123 were cultivated and processed under the same conditions.

Figure 1-15 shows the HPLC chromatograms obtained from the analysis of the media from S. albus::cos4ΔrslT123/pUWL-rslT123 fermentation cultures compared to the positive control S. albus::cos4 and the deletion mutant S. albus::cos4ΔrslT123. IV. Results A)

108 |

B)

Figure 1-15 | HPLC chromatograms (λ = 254 nm) achieved from the analysis of the production cultures of S. albus::cos4ΔrslT123, S. albus::cos4 (positive control) and S. albus::cos4ΔrslT123/pUWL-rslT123. A) Chromatograms obtained from the HPLC analysis of the medium. Rishirilide B peak is highlighted with a blue shadow. B) Chromatograms obtained from the HPLC analysis of the extract from inside the cells. Rishirilide B peak is highlighted with a blue shadow.

The HPLC chromatograms obtained from the analysis of the media as well as inside the cells showed comparable profile between the mutants. In contrast with the inactivation mutant S. albus::cos4ΔrslT123, rishirilide B peak (20.7 min) of the complemented exconjugants seemed to reach the same maximum absorbance as in the positive control. Because of this, quantification of the secondary metabolite was necessary.

To quantify the amount of rishirilide B produced, three cultures of each mutant were analyzed. The area under the curve (AUC) of rishirilide peak was divided by the weight of IV. Results the dried cell pellets in mg. In this case, calculation of the dried pellet was done by from the remaining debris of the 100 mL production culture after acetone extraction. Figure 1-16 shows the calculated amounts of S. albus::cos4ΔrslT123/pUWL-rslT123 and the control S. albus::cos4. | 109 A)

Figure 1-16 | Average of the calculated AUC of rishirilide B peak divided by the weight of the dry cell pellet (mg) from S. albus::cos4ΔrslT123/pUWL-rslT123 and S. albus::cos4 cultures. The maximum and minimum calculations are drawn with a thin line over the bars. A) Calculation of rishirilide B detected in the crude extract. B) Calculation of rishirilide B detected inside the cells.

The complementation mutants showed a significant higher amount of rishirilide B secreted to the medium and detected inside the cells, about 1.2 and 1.9-fold respectively, in comparison with control S. albus::cos4.

1.2.3. Production analysis of rslT1 and rslT123 overexpression mutants Streptomyces albus J1074 containing the cosmid cos4 was manipulated in order to overexpress the ABC transporter system RslT1-T3 as well as its substrate binding protein RslT1. The constructed plasmids pUWL-rslT123, pTOS-rslT123, pUWL-OriT-rslT1 and pTOS- rslT1 (III. Table 1.9-4) were transferred by intergeneric conjugation into S. albus::cos4. The overexpression mutants were cultivated in HA production medium (III. Table 1.7-1) and after 4 days the cell pellet and supernatant were extracted with ethyl acetate (III.2.4.1) for analysis by LC/MS (III.2.4.3). As control, S. albus::cos4 and S. albus::pOJ436 were cultivated and processed in the same way.

The HPLC chromatograms achieved after analysis of the production cultures showed a comparable profile for the extract from the media and inside the cells as shown in the previous section (Figure 1-13 and Figure 1-15).

In order to quantify and compare the production rishirilide B, the amount of cells in the cultures was measured by drying the complete harvested pellet after extraction. The IV. Results integrated area under the curve (AUC) of rishirilide peak was divided by the weight of the dried cell pellets. The resulting calculation is shown in Figure 1-17

A)

110 |

B)

Figure 1-17 | Average of the calculated AUC of rishirilide B peak divided by the weight of the dry cell pellet (mg) from S. albus::cos4, S. albus::cos4::pTOS-rslT1, S. albus::cos4::pTOS-rslT123 and S. albus::cos4/pUWL-rslT123 cultures. The maximum and minimum calculations are drawn with a thin line over the bars. A) Calculation of rishirilide B detected in the media of the production cultures of S. albus::cos4, S. albus::cos4::pTOS-rslT1 and S. albus::cos4::pTOS-rslT123. This extraction was performed by V. Brinschwitz (Brinschwitz 2013). B) Calculation of rishirilide B detected in the media of the production cultures of S. albus::cos4 and S. albus::cos4/pUWL-rslT123 (left) and inside the cells (right).

A significant higher amount of rishirilide B was observed when overexpressing the ABC transporter system as well as the substrate binding subunit RslT1. The amount of rishirilide B detected in the media of the cultures of S. albus::cos4::pTOS-rslT1 and S. albus::cos4::pTOS-rslT123 was 1.21 and 1.71-fold higher than in S. albus::cos4, respectively. The amount of rishirilide B detected in the media and inside the cells of S. albus::cos4/pUWL-rslT123 cultures was 1.25- and 1.68-fold higher, respectively, compare to S. albus::cos4.

In average, the production cultures of the mutants containing pTOS-rslT123 and pUWL- rslT123 showed similar amounts of rishirilide B compared to the controls although pTOS showed more pronounced deviation. pUWL-rslT123 led to a significant increase of rishirilide B production even though the stability of the plasmid in S. albus was compromised. Stocks IV. Results that were stored for long periods were not able to reproduce the same results therefore new conjugations were performed before the production assays. This problem was not observed when working with the integrative plasmid pTOS(z). On the other hand, conjugation was not always efficient due to the fast development of resistance against spectinomycin in S. | 111 albus::cos4.

1.2.4. Protein expression and purification of RslT1 With the aim of characterizing the substrate specificity of the ABC transporter system RslT1-3, the substrate binding subunit RslT1 (34 kDa) should be overexpressed and purified. The gene rslT1 was cloned into the vectors pET28a(+) and pUWL-H for its heterologous expression in E. coli and Streptomyces lividans, respectively. The gene was mutated in order to introduce an N- or C-terminal tag consisting of 6 histidines (His6-Tag) for its further affinity chromatography purification. The gene was also truncated at the 3’ end as an attempt to produce a cytosolic protein due to the lack of the possible signal peptide (1.1). RslT1 was also expressed together with the other two components of the ABC transporter system, RslT2 and RslT3, to ensure the right formation of the protein complex. Cloning steps for the construction of the protein expression plasmids pETrslT1N, pETrslT1C, pETrslT1Ctrun, pETrslT123, pUWLrslT1C and pUWLrslT1CT2T3 are detailed in III.2.2.10.4. Section III.2.3.1 describes the procedure followed for the protein expression in E. coli and S. lividans.

1.2.4.1. Heterologous expression of RslT1 in E. coli

Protein expression of RslT1 with N and C-terminal His6-Tag First attempt for the expression of RslT1 in E. coli included the construction of the plasmids pET28rslT1N and pET28rslT1C in order to work with the inducible T7 system and a His6-Tag on the N or C-terminal of the target gene. rslT1 was successfully amplified by PCR and the resultant fragments were processed as explained in III.2.2.10.4 to achieve the insertion into the protein expression plasmid pET28a(+). Figure 1-18 shows the agarose gel with the PCR amplification of rslT1 for an N- or C-terminal His6-Tag (about 1 kb) and the control digestions of the final constructs with the expected size of the fragments. Digestion of pETrslT1N with ClaI led to two fragments of 4209 bp and 2059 bp while pETrslT1C led to a two fragments of 4141 bp and 2059 bp. IV. Results

A) B)

112 |

Figure 1-18 | Agarose gels with the resultant amplification of rslT1 by PCR and the control digestions of pET28rslT1N and pET28T1C. A) PCR amplification of rslT1 for its cloning into the protein expression vector pET28a(+). Lanes 1 – 3 show rslT1 amplification for an N-terminal His6-Tag. Lanes 4 – 5 show rslT1 amplification for a C-terminal His6-Tag. Lane L contains 1 kb ladder. B) Agarose gel with the control digestions of pET28slT1N and pETrslT1C. Lanes 1 and 3 show the uncut plasmids. Lanes 2 and 4 show the resultant digestions of pET28slT1N and pETrslT1C with ClaI, respectively, with the expected fragment sizes of 4209 + 2059 bp and 4141 + 2059 bp.

The T7 system compatible strains E. coli BL21 (DE3), E. coli BL21 (DE3) codon plus RP/pETcoco-2-L1SL2, E. coli BL21 (DE3) pLysS, E. coli BL21 (DE3) StarTM, E. coli RossetaTM 2 and E. coli C43 (DE3) (III. Table 1.8-1) were transformed with pETrslT1N and pETrslT1C for an expression test. Different combinations of cultivation temperature and amounts of IPTG were tested in order to find out the optimal conditions for protein expression as explained in III.2.3.1.1. RslT1, a protein consisting of 321 amino acid, has an estimated molecular weight of 35 kDa.

Figure 1-19 shows the SDS gels of the expression test with E. coli BL21 (DE3) StarTM and E. coli BL21 (DE3) codon plus RP/pETcoco-2-L1SL2 containing pET28rslT1N which revealed an increasing amount of protein with a molecular weight of ca. 42 kDa.

A) B)

Figure 1-19 | SDS gels of the expression tests with pET28rslT1N. A) Expression test of pET28rslT1N in E. coli BL21 (DE3) StarTM induced with 1 mM IPTG and cultivated at 28 °C after induction. Lane L contains the protein ladder 2. Lane 1 – 5 show the protein separation of the cells from samples taken before induction and 1 – 4 h after induction, respectively. An increasing band of ca. 42 kDa compared to the ladder is marked with an arrow (RslT1 = 35 kDa). B) Expression test of pET28rslT1N in E. coli BL21 (DE3) codon plus RP/pETcoco-2-L1SL2 induced with 1 mM IPTG and cultivated at 37 °C after induction. Lane 1 – 5 show the protein separation of the cells from samples taken before induction and 1 – 4 h after induction, respectively. An increasing band of ca. 42 kDa IV. Results compared to the ladder is marked with an arrow (RslT1 = 35 kDa). Lane L contains the protein ladder 2. Lanes 6 and 7 show the protein separation of the control E. coli BL21 (DE3) codon plus RP/pETcoco-2-L1SL2/pET28a(+) from samples taken 4 h after induction and before induction, respectively.

Due to the difference of the size of the visualized protein (ca. 42 kDa) to the expected size of RslT1 (35 kDa), a western blot was performed as described in III.2.3.5.2 to ensure that the | 113 overexpressed protein corresponds to the tagged RslT1. Expression test was carried out in E. coli RossetaTM 2 and E. coli BL21 (DE3) codon plus RP/pETcoco-2-L1SL2 with cultivation temperatures of 28 °C and 37 °C after inducing the expression with 1 mM IPTG. Results are shown in Figure 1-20 where a clear increasing band around 40 kDa compared to the ladder was again detected in both cell lines. This band was especially intense for the cultures of E. coli BL21 (DE3) codon plus RP/pETcoco-2-L1SL2/pET28rslT1N cultivated at 37 °C.

A)

B)

Figure 1-20 | Western blot of the cell cultures expressing RslT1 with an N-terminal His6-Tag. A) Western blot gel containing the separated proteins from samples of the expression test of E. coli BL21 (DE3) codon plus RP/pETcoco-2-L1SL2/pET28rslT1 cultivated at 28 °C and 37 °C after 1 mM IPTG induction. Samples were taken before induction (t = 0) and 2, 3 and 4 h after induction (t = 2 – 4 h). B) Western blot of the separated proteins from samples of the expression test E. coli RossetaTM 2/pET28rslT1 cultivated at 28 °C and 37 °C after 1 mM IPTG induction. Samples were taken before induction (t = 0) and 2, 3 and 4 h after induction (t = 2 – 4 h).

After verification of the expression of RslT1 His6-Tag and assuming that the protein was bounded to the membrane, the protocol for isolation of membrane proteins by IV. Results ultracentrifugation was performed to obtain a pure protein fraction (III.2.3.2.2). Therefore, 1 L of cultured E. coli BL21 (DE3) StarTM/pET28rslT1N prepared for protein expression which was induced with 0.5 mM IPTG and further cultivation of the cells at 28 °C for 12 h. The harvested cells were lysed by using French® Pressure cell press and the supernatant was 114 | further processed after centrifugation. Samples were prepared for ultracentrifugation as detailed in III.2.3.2.2 and the resulting membrane pellet was resuspended in buffer A2. DDM detergent was added to the resuspended membranes. The supernatant after ultracentrifugation and the resuspended membranes were analyzed by SDS-PAGE (Figure 1-21 A). Both fractions showed a protein band over 40 kDa although it was notably more intense in the supernatant which contained the soluble proteins. With the purpose to reveal which fraction contained the bigger amount of RslT1 able to bind the Ni2+ for its purification, they were tested in small scale with manual Ni-NTA agarose column (III.2.3.3.1). The collected samples were examined on a SDS-PAGE and RslT1 was visualized in both cases on the elution fractions.

B)

A)

C)

Figure 1-21 | SDS gels of protein samples after ultracentrifugation and manual Ni-NTA purification of the cultured E. coli BL21 (DE3) StarTM/pET28rslT1N. A) Lane 1 contains the separated proteins of the resuspended membrane pellet obtained after ultacentrifugation. Lane 2 contains the supernatant fraction of the ultracentrifugation sample. Lane L contains the protein ladder 2. B) SDS gel of the collected samples from the manual Ni-NTA purification of the resuspended membrane pellet after ultracentrifugation. Lane 1 contains the flow through, lanes 2 – 4 contain the three washing fractions and lane 6 – 8 contain the elution fractions. Lane L contains the protein ladder 2. C) SDS gel of the collected samples from the manual Ni-NTA purification of the IV. Results supernatant after ultracentrifugation. Lane 1 contains the flow through, lanes 2 – 4 contain the three washing fractions and lane 6 – 8 contain the elution fractions. Lane L contains the protein ladder 2.

Due to the detection of RslT1 in the membrane pellet fraction as well as in the supernatant after ultracentrifugation another attempt to purify RslT1 by manual Ni-NTA was carried out.

The step of membrane isolation by ultracentrifugation was omitted. Protein expression | 115 culture was prepared as described above and after centrifugation of the cell debris the supernatant was treated with DDM detergent as detailed in (III.2.3.2.2). Ni-NTA purification was performed (III.2.3.3.1) and the collected fractions were analyzed by SDS-PAGE leading to comparable gels to the ones shown in Figure 1-21 B and C. A protein of ca. 40 kDa was visible on the elution fractions. Thus, a big scale purification with ÄKTATMFPLC system was executed. With this purpose, 3 L culture of E. coli BL21 (DE3) codon plus RP/pETcoco-2- L1SL2/pET28rslT1N was cultivated overnight at 20 °C after induction with 0.5 mM IPTG. After cell lysis, as described above, the soluble fraction was incubated with DDM detergent and after centrifugation, the supernatant was loaded into the pre-column of the ÄKTATMFPLC system. The system was run with a manual program which included three elution steps. Proteins were eluted with the help of a gradient of buffer B2 at 5 % (at 65 mL), 15 % (at 95 mL) and 50 % (at 125 mL). Figure 1-22 shows the UV chromatogram (λ = 280 nm) of the purification process and the SDS-PAGE analysis of the collected samples.

A)

B)

Figure 1-22 | ÄKTATMFPLC chromatogram (λ = 280 nm) and SDS gels from the analysis of the collected samples for RslT1 purification. A) Chromatogram of the ÄKTATMFPLC system. In red the collection fractions of the system are marked. The fractions taken for SDS-PAGE analysis are numbered in green and correspond with the lanes of the SDS gels in B. Small increments on the absorbance were observed when increasing the percentage of buffer B2 B) The SDS gels show the protein separation of the samples collected from the ÄKTATMFPLC system that are numbered in green in A (Lanes 1 – 14). Lane L contains the protein ladder 1 (left) and ladder 2 (right). A notable elution of proteins was observed when the changing conditions to 50 % of buffer IV. Results

B2. A thick band corresponding to a protein of about 70 kDa compared to the ladder was visible. None of the elution fractions showed the expected band of ca. 42 kDa, but the flow through fraction.

The increase of buffer B2 percentage led to the detection of small signals on the chromatogram. The collected fractions were analyzed by SDS-PAGE revealing a constant

116 | elution of proteins along the process. A considerable band with the size of RslT1 was observed in the flow through but not in the late fractions where the amounts of imidazole increased. The procedure was repeated with another cell line, E. coli BL21 (DE3) StarTM, moreover several elution conditions were tested which led to comparable chromatograms to those shown in Figure 1-22 A.

2+ RslT1, carrying an N-terminal His6-Tag, seemed not to bind efficiently to the Ni in the ÄKTATMFPLC system for its purification. Alternatively, expression tests were performed with

the construct pET28rslT1C which contained rslT1 for its expression with a C-terminal His6- Tag (Figure 1-18). Diverse E. coli strains transformed with the plasmid were examined. Figure 1-23 A shows the SDS gels containing the samples from the expression test of E. coli BL21 (DE3) StarTM/pET28rslT1C. An increasing band with a size of ca. 42 kDa was detected which

suggested an overexpression of RslT1 with the C-terminal His6-Tag. A manual Ni-NTA purification was performed with the cells cultivated for 4 h after induction. The harvested cells were resuspended in buffer A1 and incubated with lysozyme (III.2.3.2.1). Subsequently DDM detergent was added to the mixture to release the proteins bounded to the membrane (III.2.3.2.2). After centrifugation, purification was carried out with Ni-NTA column (III.2.3.3.1) and the collected fractions were analyzed by SDS-PAGE which can be seen in Figure 1-23 B. Elution fractions revealed a noteworthy band with the same size as visualized

in the expression test which suggested an effective purification of the C-terminal His6-Tag RslT1

A) B)

Figure 1-23 | SDS gel of RslT1 C-terminal expression test and manual Ni-NTA purification. A) The SDS gel shows the analysis of protein samples from the expression test of E. coli BL21 (DE3) StarTM/pET28rslT1C cultivated at 28 °C for 4 h after induction with 1 mM IPTG. Lane 1 contains the sample taken before induction. IV. Results

Lanes 2 – 4 contain the samples taken 2 – 4 h after induction, respectively. Lane L contains the protein ladder 1. An increasing band of ca. 42 kDa is pointed with an arrow. B) SDS gel of the analysis of the collected samples from manual Ni-NTA purification of the culture E. coli BL21 (DE3) StarTM/pET28rslT1C cultivated for 4 h after 1 mM IPTG induction. Lane 1 contains the samples from the flow through, lanes 2 – 3 contain the two washing fractions and lane 4 – 7 contain the elution fractions. Lane L contains the protein ladder 2. Two bands were observed on the elution fractions, one corresponding to the expected band of ca. 42 kDa which is pointed with an arrow. | 117 Protein expression was carried out in large scale with E. coli BL21 (DE3) StarTM/pET28rslT1C reproducing the same valuable conditions of the expression test as described above. Preparation of the samples for purification with the ÄKTATMFPLC system was performed as explained in III.2.3.3.2. The system was run with different proportions of buffer B2 with the aim of finding the optimal conditions to achieve a clean elution of the target protein. All attempts showed comparable chromatograms as shown in Figure 1-22. The chromatograms obtained from ÄKTATMFPLC system evidenced tiny increments of the absorbance when increasing the amount of imidazole. However, analysis of the collected fractions by SDS- PAGE evidenced that the target protein was lost in the flow through.

Protein expression of an N-terminus truncated RslT1 with C-terminal His6-Tag Gao et al. (2012) described the purification of a substrate binding protein (OppA) which is part of an ABC transporter system involved in nutrient uptake, sporulation and other biological processes in Thermoanaerobacter tengcongensis. Purification of OppA was only successful when expressed in E. coli as a soluble protein by truncating 30 amino acids on the N-terminus, a fact that avoid the need of using detergents. Based on this finding, rslT1 was amplified by PCR omitting the first 60 bp (III. Table 2.2.9-13) as an attempt to delete the putative signal peptide. Under this conditions, it was expected an accumulation of the protein on the cytosolic compartment. The correct amplified gene fragment of ca. 1 kb (Figure 1-24 A) was cloned through several steps to the final construct pET28rslT1Ctrun (III.2.2.10.4). Figure 1-24 B shows the control digestion which corroborated the correct cloning of the plasmid. IV. Results A) B)

118 |

Figure 1-24 | Agarose gels with the resultant truncated rslT1 amplified by PCR and the control digestions of pET28rslT1Ctrun. A) PCR amplification of the truncated rslT1 for its cloning into the protein expression vector pET28a(+). Lanes 1 – 2 show the expected size of ca. 1 kb of the PCR product which is pointed with an arrow. The empty lane 3 contains the negative control where no template was added to the PCR mixture and therefore no amplification should be observed. Lane L contains the 1 kb ladder. B) The agarose gel shows the correct control digestions of pET28slT1Ctrun. Lane 1 contains the digestion with XbaI which led to a 6142 bp fragment. Lane 2 contains the digestion with ClaI which led to a 4082 bp and 2056 bp fragments. Lane 3 contains the digestion with XhoI and NcoI which led to 5231 bp and 911 bp fragments. Lane L contains the 1 kb ladder.

E. coli BL21 (DE3) StarTM and E. coli BL21 (DE3) codon plus RP/pETcoco-2-L1SL2 were transformed with pET28rslT1Ctrun. Expression tests were performed to investigate the influence of different amounts of IPTG and diverse cultivation temperatures on the expression of the protein. Figure 1-25 shows the SDS gels from the analysis of the samples

taken during the expression test of the truncated RslT1 C-terminal His6-Tag.

A) B)

Figure 1-25 | SDS gels of the expression test of the truncated RslT1 C-terminal His6-Tag. A) The gel contains the separated proteins from samples of the expression test of pET28rslT1Ctrun in E. coli BL21 (DE3) StarTM at time 0, 2 and 4 h after 0.5 mM IPTG induction and cultivation at 28 °C (Lanes 1 – 3) and cultivation at 37 °C (Lanes 4 – 6). Lane L contains the protein ladder 1. No increasing band was detected B) The gel contains the separated proteins from samples of the expression test of pET28rslT1Ctrun in E. coli BL21 (DE3) codon plus RP/pETcoco-2-L1SL2 at time 0, 3 and 20 h and cultivation at 20 °C after 0.1 mM (Lanes 1 – 3), 0.5 mM (Lanes 4 – 6) and 1 mM (Lanes 7 – 9) IPTG induction. Lane L contains the protein ladder 1. No increasing band was detected.

Even though different cultivation conditions did not lead to a remarkable increase of an induced protein visible on the SDS gels, the harvested cells of the induced cultures were prepared for a manual Ni-NTA purification (III.2.3.3.1). The collected fractions were IV. Results analyzed by SDS-PAGE which indicated a lack of an overexpressed protein able to specifically bind the Ni2+.

Protein expression of RslT1-T3

Due to the weak expression of RslT1 in E. coli, rslT1 was cloned together with rslT2 and rslT3 | 119 into the protein expression vector pET28a(+) with the intention to obtain a steady production of the ABC transporter system. As described in III.2.2.10.4, pET28rslT123 was cloned carrying the three transporter genes without linked histidine residues but still under the influence of the inducible T7 system. Expression test were executed as previously described (III.2.3.1.1) in E. coli BL21 (DE3) and E. coli C43 (DE3). Protein expression was induced with 0.1 – 1 mM IPTG and the cultures were further cultivated at 28 °C or 37 °C. The collected samples were analyzed by SDS-PAGE and RslT1 was detected in comparable amounts as when expressed apart.

1.2.4.2. Heterologous expression in Streptomyces lividans

E. coli is the most commonly used host for production of recombinant proteins (Nakashima et al. 2005). However, expression and isolation of heterologous proteins is sometimes by problems of insolubility, cytotoxicity, post-translational modifications, or inefficient translation. Alternative host-vector systems have been developed over the last decades in other strains such as Bacillus spp. (Kashima & Udaka 2004), Lactococcus lactis (Kashima & Udaka 2004) or Streptomyces spp. (Enguita et al. 1996). As an approach to express RslT1 in a more similar host to the original strain, Streptomyces lividans was preferred for the heterologous expression of RslT1. For this purpose, the plasmids pUWLrslT1C and pUWLrslT1CT2T3 were cloned as detailed in III.2.2.10.4.

For the construction of pUWLrslT1C, the target gene was amplified from the already successfully cloned pETrslT1C in order to obtain the restriction sites BamHI and HindIII. These sites were necessary for the cloning into pUWL-H and the expression of RslT1 with a

His6-Tag in S. lividans. The control digestions of the final construct which generated DNA fragments with the expected size are shown in Figure 1-26 A.

For the construction of pUWLrslT1CT2T3, the previously cloned pUWLT1C was digested with ClaI to released part of rslT1. The big digested fragment containing the backbone of the vector and the C-terminal end of rslT1 with the histidine cue attached was further utilized. To obtain the missing part of the sequence of rslT1 and the complete rslT2 and rslT3, the previous constructed plasmid pUC19rslT123 (III. Table 1.9-2) was digested with ClaI. The IV. Results fragment containing the desired sequence of rslT1, rslT2 and rslT3 was ligated with the previously digested pUWLT1C. Taking into account the correct orientation of the ligated fragments, the final plasmid was achieved. The control digestions visualized on the agarose gels showed the expected size of the fragments (Figure 1-26 B). 120 | A) B)

Figure 1-26 | Agarose gels with the control digestions of the constructed plasmids pUWLrslT1C and pUWLrslT1CT2T3. A) The agarose gel shows the control digestion of the isolated plasmid pUWLrslT1C with XhoI (Lanes 1 – 2) which resulted in 3 fragments of 7057, 1348 and 430 bp. Lane L contains the 1 kb ladder. B) The agarose gel shows the correct control digestions of the plasmid pUWLrslT1CT2T3. Lane 1 contains the digestion with KpnI which led to two fragments of 9275 pb and 1328 bp. Lane 2 contains the digestion with NcoI which led to three fragments of 6475 bp, 3122 bp and 1251 bp. Lane L contains the 1 kb ladder.

The verified plasmids, as well as the empty vector for control, were transferred into S. lividans by intergeneric conjugation as described in III.2.1.2.3. The achieved exconjugants were cultivated in TSB medium for 2 – 3 days (III.2.1.1.2) and the harvested cells were lysed by French® Pressure cell press (III.2.3.2.1). The supernatants of the centrifuged cell debris were analyzed by SDS-PAGE in order to visualize an overproduction of RslT1 compared to the control. Due to the huge amount of proteins produced by S. lividans it was not possible to distinguished single bands. Therefore affinity chromatography was performed with the

aim to identify RslT1 thanks to the C-terminal His6-Tag. The collected fractions from the manual Ni-NTA purification were analyzed by SDS-PAGE leading to the detection of a protein band with the expected size of ca. 42 kDa (Figure 1-27). IV. Results

| 121

Figure 1-27 | SDS gel of the collected fractions from manual Ni-NTA purification from S. lividans/pUWLrslT1C cultures. The gel contains samples from the flow though (Lane 1), two washing steps (Lanes 2 – 3) and four elution fractions (Lanes 4 – 7). Lane L contains the protein ladder 3. A tinny band on the second elution fraction was observed with a size of ca. 42 kDa compared to the ladder.

S. lividans containing pUWLrslT1C, where the target gene is cloned in the plasmid directly in front of the promoter, led to a more distinct detection of RslT1 on the SDS gels after manual Ni-NTA purification compared to S. lividans/pUWLrslT1CT2T3. The whole gel showed weak protein detection even though a band of ca. 42 kDa was revealed on the second elution fraction. This detection indicated the binding of the C-terminal His6-Tag RslT1 to the Ni2+.

Preparation of 1.5 L of S. lividans/pUWLrslT1C cultures was carried out in order to purify the target protein by ÄKTATMFPLC system. The bacteria were cultivated for 3 days and the harvested cells were resuspended in 150 mL of buffer A2 for disruption with French® Pressure cell press and by addition of lysozyme (III.2.3.2.1). After centrifugation, 80 mL of the supernatant were loaded into the ÄKTATMFPLC system. The pressure of the column raised up very easily indicating an overload of the column which impeded the purification. Therefore, a smaller volume (20 mL) was injected into the system leading to no detection of the desired protein on the elution fractions.

1.3. Inactivation and expression of the MFS transporter RslT4

1.3.1. Construction of the deletion cosmid cos4ΔrslT4 by Red/ET The gene rslT4 was inactivated by Redirect© Technology procedure as described in (III.2.2.11.3). The target gene was exchanged by homologous recombination for a maker which contained a spectinomycin resistance (Specr) cassette (aadA) and the upstream and IV. Results downstream homologous regions of rslT4 as detailed in III. Table 2.2.9-13. PCR was performed by J. Brehm (Brehm 2014) and the resultant 1.1 kb DNA was transferred into E. coli DH5α/pBADαβγ containing cos4 for its recombination to achieve the inactivated construct cos4ΔrslT4S. The purified cosmid from a clone Aprar and Specr was verified by 122 | digestion with BamHI. The agarose gel showed the 13 expected fragment visible on a 0.7 % agarose gel and a self-evident difference with cos4 (Figure 1-28 A). In order to release the marker gene from the cosmid a digestion with NheI was performed. This was correctly visualized by agarose gel electrophoresis and the religated cosmid was transferred into E. coli. The isolated DNA was digested with BamHI and the resultant fragments were verified by agarose gel electrophoresis (Figure 1-28 B). In addition, control PCR using the primer pair CtrT4Red-Prm-f/CtrT4Red-Prm-r was performed as described in III. Table 2.2.9-13 and the expected band of 886 bp, in contrast with the 5332 bp of the control cos4, confirmed the right construct of cos4ΔrslT4.

A) B) C)

Figure 1-28 | Agarose gels from rslT4 inactivation experiments. A) Lanes 1 – 2 show the digestions of cos4ΔrslT4S and cos4 with BamHI, respectively. The size of the expected fragments can be seen in D, the different fragments are pointed with the arrows. Lane L contains the 1 kb ladder. B) Lanes 1 – 2 show cos4ΔrslT4 and cos4 digested with BamHI, respectively. The size of the expected fragments can be seen in D, the different fragments are pointed with the arrows. Lane L contains 1 kb ladder. C) The table contains the size of the expected fragments after digestion of cos4, cos4ΔrslT4S and cos4ΔrslT4 with BamHI in base pairs. The difference between the control and the inactivation mutant is highlighted with a grey shadow.

1.3.2. Production analysis of S. albus::cos4ΔrslT4 and complementation The recombinant cosmid cos4ΔrslT4 was transferred into Streptomyces albus J1074 by intergeneric conjugation as described in III.2.1.2.3. The mutant was cultivated in production medium (III. Table 1.7-1) and after 4 days the cell pellet and the supernatant were extracted with ethyl acetate (III.2.4.1) for analysis by LC/MS (III2.4.3). As control, S. albus::cos4 and IV. Results S. albus::pOJ463 were cultivated and analized in the same way. Figure 1-29 shows the HPLC chromatograms achieved after analysis of the extractions of the media and inside the cells of the investigated mutants.

A) | 123

B)

Figure 1-29 | HPLC chromatograms (λ = 254 nm) achieved from the analysis of the production cultures of S. albus::cos4 (positive control) and S. albus::cos4ΔrslT4. A) Chromatograms obtained from the HPLC analysis of the medium. Rishirilide B peak is highlighted with a blue shadow. B) Chromatograms obtained from the HPLC analysis of the extract from inside the cells of S. albus::cos4 and S. albus::cos4ΔrslT4. Rishirilide B peak is highlighted with a blue shadow.

The HPLC chromatogram of the extracted media of S. albus::cos4ΔrslT4 showed a comparable rishirilide B peak to the positive control S. albus::cos4. To statistically quantify rishirilide production three different S. albus::cos4ΔrslT4 exconjugants were compared to three cultures of S. albus::cos4. The area under the curve (AUC) of rishirilide B peak was divided by the weight of the dried cell pellet. In this case, calculation of the dried pellet was done by evaporating the whole pellet from the 100 mL production culture after acetone extraction. Figure 1-30 shows the average of the calculated relation of the amount of rishirilide B detected in the supernatant and inside the cells. IV. Results

124 |

Figure 1-30 | Average of the calculated AUC of rishirilide B divided by the weight of the dry cell pellet (mg) from S. albus::cos4 and S. albus::cos4ΔrslT4. The graphic on the left shows the calculation of rishirilide B detected in the media of the cultures. The graphic on the right shows the calculation of rishirilide B detected inside the cells of the cultures. The maximum and minimum of the calculated values obtained are drawn with a thin line over the bars.

A significant higher amount of rishirilide B was observed in the MFS transporter deletion mutants compared to the controls. S. albus::cos4ΔrslT4 showed 1.41- and 3.25-fold increase of rishirilide B detected in the media and inside the cells, respectively, compare to the control.

Complementation of the deletion mutant S. albus::cos4ΔrslT4 was performed by transferring the constructed plasmid pTOS-rslT4 (III. Table 1.9-4) by intergeneric conjugation as described in III.2.1.2.3. The exconjugants were cultivated in HA production medium (III. Table 1.7-1) and after 4 days the cell pellet and supernatant were extracted with ethyl acetate (III.2.4.1) for analysis by LC/MS (III.2.4.3). As control, S. albus::cos4 and S. albus::cos4ΔrslT4 were cultivated and processed under the same conditions. IV. Results

| 125

Figure 1-31 | HPLC chromatograms (λ = 254 nm) achieved from the analysis of the production cultures of S. albus::cos4ΔrslT4::pTOS-rslT4, S. albus::cos4 and S. albus::cos4ΔrslT4. Chromatograms obtained from the HPLC analysis of the media after extraction with ethyl acetate. Rishirilide B peak is highlighted with a blue shadow.

The HPLC chromatogram of the extracted media of S. albus::cos4ΔrslT4::pTOS-rslT4 showed a comparable rishirilide B peak to S. albus::cos4ΔrslT4 and higher than in S. albus::cos4. The HPLC chromatograms obtained from the analysis of the cell pellet showed the same tendency. To statistically quantify rishirilide production three different S. albus::cos4ΔrslT4 exconjugants were compared to S. albus::cos4. The area under the curve (AUC) of rishirilide B peak was divided by the weight of the dried cell pellet. In this case, calculation of the dried pellet was done by evaporating the whole pellet from the 100 mL production culture after acetone extraction. Due to some difficulties experienced in the process, only two measurements could be performed for S. albus::cos4ΔrslT4::pTOS-rslT4. The values indicated lower amounts of rishirilide B produced by the complemented mutant. It is important to emphasize that the cell pellet determined was about 3-fold higher than the control. For a solid comparison these quantifications should be repeated in the future.

1.3.3. Production analysis of rslT4 overexpression mutant Streptomyces albus J1074 containing the cosmid cos4 was manipulated in order to overexpress the MFS transporter RslT4. The constructed plasmid pTOS-rslT4 (III. Table 1.9-4) was transferred by intergeneric conjugation into S. albus::cos4 (III.2.1.2.3). Three exconjugants of the overexpression mutant were cultivated in HA production medium (III. Table 1.7-1). After 4 days, the cell pellet and supernatant were extracted with ethyl acetate IV. Results (III.2.4.1) for analysis by LC/MS (III.2.4.3). As control, S. albus::cos4 and S. albus::cos4ΔrslT4 were cultivated and processed in the same way.

The HPLC chromatograms obtained from the analysis of the cultures of the mutants

126 | S. albus::cos4::pTOS-rslT4 provided comparable profiles to those achieved for the complementation mutant S. albus::cos4ΔrslT4::pTOS-rslT4 (Figure 1-31). Similar tendency was observed on the HPLC chromatograms achieved from the analysis of the cell pellet. To quantify and compare the amounts of rishirilide B produced by the overexpression mutants, three different exconjugants were analyzed. The area under the curve (AUC) of rishirilide B peak was divided by the weight of the dried cell pellet. In this case, calculation of the dried pellet was done by evaporating the whole pellet from the 100 mL production culture after acetone extraction. As control, S. albus::cos4 was cultivated and processed under the same conditions. Due to contamination of one of the cultures, only two quantifications could be performed for S. albus::cos4::pTOS-rslT4. The values indicated similar amounts of rishirilide B produced by the complemented mutant compare to the control. It is important to mention that the weight of the cell pellet determined was about 3.5-fold higher than the control. For a solid comparison of the overexpression mutants, these quantifications should be repeated in the future.

1.3.4. Production analysis of rslR4 overexpression mutant The regulator gene rslR4 is located right next to rslT4 in rishirilide gene cluster. Previous investigations on the regulatory system of rishirilide biosynthetic pathway have been done (X. Yan 2012, Wunsch-Palasis 2013). J. Wunsch-Palasis identified rslR4 as a negative regulator of rslT4 performing different assays based on the gusA reporter gene (Myronovskyi et al. 2011). With this assumption, overexpression experiments of the negative regulator gene were performed in order to observe the influence on rishirilide production.

The previously constructed plasmid pUWL-H-rslR4 by X. Yan (Yan 2012) (III. Table 1.9-1) was transferred into S. albus::cos4 by intergeneric conjugation (III.2.1.2.3) and the obtained exconjugants were cultivated in HA production medium for 4 days (III.2.1.1.2). The harvested cells and the supernatant were extracted with ethyl acetate (III.2.4.1) for analysis of the extract by LC/MS (III.2.4.3). As control, S. albus::cos4 was cultivated and processed in the same way. Figure 1-32 shows the HPLC chromatogram obtained from the analysis of the medium where very similar profile to the control was observed. Similar results were obtained for the HPLC chromatograms achieved from the analysis of the extract of the cell pellet. IV. Results

| 127

Figure 1-32 | HPLC chromatograms (λ = 254 nm) achieved from the analysis of the extract of the production cultures of S. albus::cos4/pUWL-H-rslR4 and S. albus::cos4. The chromatograms obtained from the HPLC analysis of the extracted media showed rishirilide B peak (highlighted with a blue shadow).

In order to quantify and compare the amount of rishirilide B produced between the mutants, three production cultures of S. albus::cos4/pUWL-H-rslR4 and S. albus::cos4 were prepared for extraction of the medium and the harvested cells. The amount of cells was quantified measuring the weight of the dry pellet after its extraction. The integrated area under the curve (AUC) of rishirilide B peak calculated from the HPLC chromatograms was divided by the weight of the dry pellet. The resulting calculation is shown in Figure 1-33 were a higher production of rishirilide B in the rslR4 overexpression mutant is evidenced in comparison to S. albus::cos4.

Figure 1-33 | Average of the calculation AUC of rishirilide B peak divided by the weight of the dry cell pellet (mg) from S. albus::cos4 and S. albus::cos4/pUWL-H-rslR4 cultures. The maximum and minimum calculations are drawn with a thin line over the bars. The graphic on the left shows the calculation of rishirilide IV. Results

B detected in the media. The graphic on the right shows the calculated relation of rishirilide B detected inside the cells.

The calculated amounts of rishirilide B detected in the media and inside the cells of the rslR4 overexpression mutants were 1.48- and 1.83-fold higher, respectively, in comparison to

128 | the control.

1.3.5. MIC test in E. coli and disk diffusion assay in S. albus 1.3.5.1. MIC test in E. coli expressing RslT123 and RslT4

To characterize the capability of the transporter genes of rishirilide gene cluster to provide resistance against different antibiotics they were express in E. coli to determine a modification on the MIC. The utilized strain E. coli DH5α ΔacrAB carries the deletion of acrA and acrB genes (Simm et al. 2012). These genes encode two membrane proteins part of a major MFS transporter (Sulavik et al. 2001). The deletion makes the strain more sensitive to toxic compounds what allows the characterization of heterologous multidrug transporters. Thus, E. coli DH5α ΔacrAB was utilized for testing the sensibility to 8 different antibiotics when expressing the ABC transporter system RslT123 and the MFS transporter RslT4. The cells were transformed with the previous constructed plasmids pTOS-rslT123 (III. Table 1.9-4) and pTOS-rslT4 (III. Table 1.9-1) and with the empty vector pTOS(z)-spec (III. Table 1.9-1) as a control. Preparation of the pre-cultures and the 96 well microplates was performed as described in (III.2.1.4). The microplates contained the 8 antibiotics in successive dilutions 1:10 and the same amount of cells of the E. coli DH5α ΔacrAB mutants in each well. After overnight incubation of the microplates, they were analyzed to identify the higher concentration where the cells, carrying the different transporter genes, were able to grow. Figure 1-34 shows a representation of the growth of E. coli DH5α ΔacrAB/pTOS- rslT123, E. coli DH5α ΔacrAB/pTOS-rslT4 and E. coli DH5α ΔacrAB/pTOS(z)-spec in the wells of the microplates. IV. Results

| 129

Figure 1-34 | MIC test in E. coli DH5α ΔacrAB/pTOS-rslT123, E. coli DH5α ΔacrAB/pTOS-rslT4 and E. coli DH5α ΔacrAB/pTOS(z)-spec of 8 different antibiotics in dilutions 1:2. The antibiotics tested in the rows A – H were A: bacitracin, B: gentamycin, C: nalidixic acid, D: norfloxacin, E: novobiocin, F: streptomycin, G: tetracycline, H: vancomycin. The concentrations of the antibiotics in each well is shown inside the circles expressed in (mg/mL). Wells where the three mutants grew equally are painted in dark orange. Wells with further growth of E. coli DH5α ΔacrAB/pTOS-rslT4 and E. coli DH5α ΔacrAB/pTOS(z)-spec are painted with light orange and those wells where only E. coli DH5α ΔacrAB/pTOS-rslT4 were still capable to grow are painted in yellow.

The MIC test indicated the capability of E. coli DH5α ΔacrAB which expressed MFS transporter RslT4, to grow in higher antibiotic concentrations. These cells showed MICs for nalidixic acid, streptomycin, tetracycline and vancomycin at least 2-fold higher in comparison to the control as well as to those expressing the ABC transporter system RslT123. In the case of spectinomycin, E. coli DH5α ΔacrAB/pTOS-rslT4 revealed a MIC of 500 mg/mL in contrast to a MIC of the control of 62.5 mg/mL. All mutants grew on bacitracin selection (1000 mg/mL) and none on gentamycin (0.49 mg/mL) and norfloxacin (0.49 mg/mL) selection. No difference was observed in case of novobiocin for which all mutants showed a MIC of 0.97 mg/mL.

1.3.5.2. Disk diffusion assay in S. albus mutants

To elucidate the role of RslT4 in drug tolerance, the knockout mutant S. albus::cos4ΔrslT4 was compared to S. albus::cos4 in a susceptibility assay. Pre-cultures of these mutants were cultivated for 4 days and similar amount of harvested cells were suspended in 1 mL TSB medium. The cells were equally spread on a MS plates. The antibiotic disks were prepared as described in (III.2.1.5) and placed carefully over the cells. The plates were cultivated for 2 – 3 days until the surface was covered with a homogeneous growth of sporulated Streptomyces. Figure 1-35 shows the calculated diameter of inhibition zone in cm of the eight tested antibiotics against the mutants. IV. Results

130 |

Figure 1-35 | Representation of the inhibition area of the eight antibiotics tested in a disc diffusion assay with S. albus::cos4 and S. albus::cos4ΔrslT4. The inhibition area (in cm) of the tested antibiotics for S. albus::cos4 is shown in blue, for S. albus::cos4ΔrslT4 is shown in orange.

Despite the constraints within the assay, it could be observed a tendency to bigger inhibition zones of five of the antibiotics in the ΔrslT4 mutants in comparison to S. albus::cos4. The difference was especially obvious for nalidixic acid, novobiocin and norfloxacin. Streptomycin, tetracycline and vancomycin did not show a significant alteration.

1.3.6. Purification of Rishirilide B

Rishirilide B was praised for its antithrombotic activity through a selective α2-macroglobulin inhibition leading to the activation of plasmin, an enzyme involved in insoluble fibrin degradation (Iwaki et al. 1984). Thus, rishirilide B mechanism of action might be useful in the treatment and prevention of thrombosis by fibrinolytic accentuation (Aoki 1979). Furthermore, Komagata et al. (1992) discovered on the search for novel inhibitors of glutathione S-transferase from microbial origin a potential therapeutic utility of rishirilide B in combination with anticancer drugs enhancing their resistance. Due to this promising therapeutical activities but a lack of an in depth characterization, rishirilide B was purified with the aim to obtaining a detailed activity profile. The assays should be performed in collaboration with Prof. Dr. Rolf Müller, Department of Pharmaceutical Biotechnology, Saarland University, Saarbrücken,Germany. IV. Results A total of 4 L of HA production medium of S. albus::cos4::pTOS-rslT123 was cultivated for 4 days before extraction of the supernatant with ethyl acetate (III.2.4.1). After evaporation of the organic solvent, the crude extract was prepared for further purification with solid phase extraction (SPE) as detailed in III.24.1. The fractions collected from the SPE column with | 131 50%, 60 %, 70 %, 80 % and 90 % methanol were analyzed by LC/MS in order to identify the fraction containing rishirilide B. Figure 1-36 shows the analyzed fractions of the SPE purification which indicates the presence of rishirilide B in the 80 % methanol fraction.

Figure 1-36 | HPLC chromatograms of the SPE fractions on the process of rishirilide B purification. The fraction eluted with 80 % methanol showed a clear peak of rishirilide B. This fraction was further use for purification.

Although 80 % methanol fraction appeared to be pure enough for activity test, the crude extract was further processed for preparative HPLC purification as described in III.2.4.4 with the purpose to get rid of impurities. The final extract was verified once more by LC/MS analysis which confirmed the presence of the single rishirilide B peak with the expected UV spectrum and mass. IV. Results 2. Investigations on the phenalinolactone producer Streptomyces sp. Tü6071 Streptomyces sp. Tü6071 was first isolated from a soil sample in Cape Coast, Ghana (El- Nakeeb & Lechevalier 1963) and it is known to produce phenalinolactoenes A –D, terpene glycosides with antibiotic activity (Gebhardt 2002). Phenalinolactone gene cluster was 132 | annotated by Dürr et al. (2006) and the biosynthetic pathway has been done (Binz et al. 2008, Daum et al. 2010, Klaus Gebhardt et al. 2011). In this work, investigations with to identify the transporter genes involved in phenalinolactone biosynthesis were carried out. The recently published genome sequence of S. sp. Tü6071 (Erxleben et al. 2011) and the new developed Galaxy platform (Ramírez et al. 2014) were the starting point to screen for possible transporter target genes.

2.1. Genome analysis and screening for transporters involved in phenalinolactone biosynthesis The genome sequence of Streptomyces sp. Tü6071 published by Erxleben et al. (2011) was used as input for its investigation with Galaxy server (III. Table 1.10-1). This platform made possible the one-step analysis of putative protein-coding sequences of the complete genome thanks to the created workflows (III.2.5.2, Appendix 2).

A Venn diagram was created to compare the description of the predicted transporter genes of Streptomyces coelicolor, Streptomyces avermitilis and S. sp.Tü6071. It was possible to identify those transporter present in all strains or those which are singular for one or two of the compared Streptomyces. Figure 2-1 shows the Venn diagram with a total of 305 transporter genes predicted in S. coelicolor, 307 genes in S. avermitilis and 265 genes in S. sp.Tü6071. All three strains share 159 transporter genes with identical description. On the other hand S. coelicolor contains 77 singular transporter genes, while S. avermitilis has 69 and S. sp.Tü6071 50. IV. Results

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Figure 2-1 | Venn diagram of the transporter description of the identified transporter genes from Streptomyces coelicolor, Streptomyces avermitilis and S. sp.Tü6071.

Analysis of the genome sequence of S. sp. Tü6071 provided information about putative ORFs, transporter genes and secondary metabolite gene clusters which was useful to create a chromosome map (Figure 2-2). All these features are characterized in relation to their distribution in the genome sequence. IV. Results

A B

C 134 | D

Figure 2-2 | Representation of S. sp. Tü6071 genome with ORFs, transporter genes and secondary metabolite gene clusters. The different levels of the circles show the following described features. Circle A indicates the position in kb of the sequenced S. sp. Tü6071 genome. Circle B corresponds to the organization of the predicted ORFs. Circle B indicates the transporter genes, in green the predicted ABC transporter and in red all other families of transporter. Circle C contains the predicted secondary metabolite gene clusters. The cluster identified for the production of phenalinolactone is pointed in red.

Using Galaxy server, 17 putative clusters were identified in the genome of S. sp. Tü6071 thanks to the integrated tool linked to antiSMASH database (Blin et al. 2013). The location of the predicted clusters in the genome is shown in Figure 2-2. Cluster number 9 was the only cluster identified to contain the genes necessary to produce a terpenoid structure. Therefore the cluster was predicted to code for the terpenoglycoside antibiotics phenalinolactones A-D (Meyer 2003; Dürr et al. 2006).

The phenalinolactone gene cluster as well as the 25 kb region upstream and downstream were compared to the whole genome of Streptomyces coelicolor and Streptomyces IV. Results avermitilis using Galaxy tools. All genes were identified with similarities between 25 % and 95 % and notable groups of genes were identically organized. Additional information about this comparison can be find in Appendix 2.

The predicted cluster was compared to published data (Dürr et al. 2006) and small | 135 differences were observed on the predicted genes (Figure 2-3).

Figure 2-3 | Organization of the phenalinolactone gene cluster. The genes annotated by Dürr et al. (2006) are drawn in orange. ORF1 – 3 were annotated as transporter genes. In red arrows, the additional predicted genes obtained with Galaxy analysis are drawn.

Three genes of the phenalinolactone cluster (ORF1 – 3) were predicted to encode transporter proteins which were putatively identified to be involved in potassium (ORF1 and 2) and amino acid permease (ORF3) (Dürr et al. 2006). In order to find out other transporter genes which could be involved in the export of the phenalinolactones, a detailed analysis 25 kb upstream and downstream the cluster was performed. Right upstream the cluster (2 kb away from the last annotated gene) a group of three genes described as ABC transporters were identified (orf01987, orf01988 and orf01990). They showed maximum identities of 30 % to S. coelicolor and S. avermitilis annotated proteins. Another group of four genes (orf01959, orf01960, orf01962 and orf01963) were identified 20 kb away with identities around 40 % to S. coelicolor and 80 % to S. avermitilis. About 15 kb downstream the cluster, an integral membrane protein (orf02069) was predicted with similarities of 75 % to proteins from S. coelicolor and S. avermitilis.

Orf01987, orf01988 and orf01990 were chosen to be investigated by inactivation experiments in the wild type (2.2). This decision was based on the closer location of the transporter genes to phenalinolactone cluster and their higher singularity. These genes were named plaABC1, plaABC2 and plaABC3, respectively, with the assumption to assembly an ABC transporter system (PlaABC123).

BLAST search of the target genes showed low identities to proteins available in the databases (NCBI, Uniprot and PDB). Unfortunately, the function of the similar proteins was not investigated so far. The gene plaABC1 could only be identified as putative substrate binding protein and plaABC2-3 as two putative integral membrane binding protein dependent transport proteins. IV. Results 2.2. Inactivation and expression of plaABC

2.2.1. Gene inactivation of plaABC1-3 via double crossover To evidence the implication of the three transporter genes (plaABC1-3) in phenalinolactone 136 | production, gene deletion experiments were carried out. For this purpose, the inactivation plasmid pKCplaABC123 was constructed. A spectinomycin cassette surrounded by homologous regions upstream and downstream of the target genes were cloned into pKCXY02 (III. Table 1.9-1) as described in III.2.2.10.3.

The homologous regions were amplified from the cosmid Sbe01h10, which is part of the S. sp. Tü6071 genomic DNA cosmid library, as verified by agarose gel electrophoresis (Figure 2-4 A). The resulting PCR products were named 3T1 (homologous region upstream the target genes) and 3T2 (homologous region downstream the target genes) and showed the expected sizes of 3.1 kb and 2.4 kb, respectively. The homologous regions and the Specr cassette were sequentially introduced into the suicide vector pKCXY02. First 3T1 was cloned to obtain pKC3T1, subsequently 3T2 to gain pKC3T1T2 and finally the Specr cassette to achieve the product pKCplaABC123. The construct was controlled by restriction digest showing the expected size of the fragments on the agarose gel (Figure 2-4 B). Digestion of pKCplaABC123 with DraIII resulted in two fragments of 6900 bp and 4714 bp visible on the gel and a band of 620 which was too weakly stained to be seen on the picture. Digestion with PstI led to two fragments of 8430 bp and 3804 bp, SpeI to a fragment of 12234 bp and HindIII to 11699 bp and 535 bp fragments.

A) B)

Figure 2-4 | Agarose gels of PCR amplification of 3T1 and 3T2 and control digestions of the final construct pKCplaABC123. A) The agarose gel on the left shows the correct amplification of 3T1 with the expected fragment size of 3.1 kb (lanes 1 – 2). The gel on the right shows the correct amplification of 3T2 with the expected fragment size of 2.4 kb. Lane L contains the 1 kb ladder. B) The agarose gel on the left contains the control digestion of pKCplaABC123 with DraIII in lane 1 (6900 bp and 4714 bp fragments. The missing 620 bp fragment was not visible IV. Results on the picture) and with PstI (8430 bp and 3804 bp fragments) in lane 2. The gel on the right contains the uncut plasmid (lane 1) and the control digestions with SpeI (12234 bp) in lane 2 and with HindIII (11699 bp and 535 bp fragments) in lane 2. Lanes L contain the 1 kb ladder.

The plasmid was transferred into S. sp. Tü6071 by intergeneric conjugation (III.2.1.2.3) and those cells which integrated the inactivation plasmid in its genome by homologous | 137 recombination were able to grow on Apra and Spec selective antibiotic medium. After more than 20 conjugation attempts changing the preparation procedure of the donor and recipient cells and cultivation time before antibiotic selection, 5 exconjugants were obtained. Conditions leading to single crossover mutants were achieved when working with sporulated recipient cells, 8 h cultivated donor cells on solid medium and 15 h of cultivation after conjugation (III.2.2.11.2). The cells where overlaid on the conjugation plate with Apra and phosphomycin but not Spec, which was omitted in that selection point. Exconjugants were further cultivated on medium containing the three antibiotics and its genomic DNA was isolated (III.2.2.2) for control PCR. The PCR amplification confirmed the success of the single crossover due to the presence of the two expected bands (3525 bp and 1730 bp). The fragments correspond to the amplification of the target genes (3525 bp) and the integrated Specr cassette (1730 bp) in the mutants and only the 3.5 kb band on the control (genomic DNA from the wild type).

Figure 2-5 | Agarose gel of the control PCR of isolated genomic DNA of single crossover exconjugants. The control PCR of the 5 exconjugants after single crossover (lanes 1 – 5) showed the correct amplification of the two expected fragments of 3.5 kb and 1.7 kb which correspond to the target genes and the Specr cassette, respectively. Lane 6 contains the expected band (3.5 kb) corresponding to the target genes which was amplified under the same PCR conditions using isolated genomic DNA from the wild type as template. Lane L contains the 1 kb ladder.

Two of the exconjugants were further proceeded for passaging in order to favor the double crossover event. The cultures were cultivated under Spec selection and were inoculated into IV. Results fresh medium every 1 or 2 days. Between the 10th passage and until the 35th, an aliquot of the cultures was obtained and diluted with the aim to analyze the genomic DNA of single colonies (III.2.2.11.2). Therefore, the single colonies were firstly selected on solid media containing Apra/Spec and only Spec. In sum, over 1500 colonies were screened on the 138 | antibiotic plates and those few which did not grow on Apra/Spec plates were further process for genomic DNA isolation and control PCR. In the case the double crossover happens, the control PCR should lead to the amplification of one band (1730 bp) indicating the loss of the target genes (3525 bp fragment) thanks to a second recombination step. The agarose gels of the control PCR hardly evidence the amplification of the bigger fragment due to an intense 1730 bp band. Therefore, GusA assay (III.2.1.3.2) was utilized to identify the double crossover recombinant mutants (Myronovskyi et al. 2011). First of all, it was controlled that the wild type is not able to metabolize X-Gluc (III. Table 1.5-9). When adding this compound, no color reaction occurred. In contrast, single crossover mutants which should integrate gusA as part of the inactivation plasmid provided a blue colorful reaction when adding X-Gluc over the colonies. After the second recombination step, the gusA gene should be lost, as well as the Aprar cassette, generating colonies which should not further metabolize X-Gluc. Unfortunately, the occurrence of the loss of the target genes was luckless.

As an attempt to favor the success of the double crossover, the I-SceI tool developed by Siegl et al. (2010) was applied. The utilized plasmid pKCplaABC123 contained the necessary I-SceI sequence for the recognition of the expressed I-SceI endonuclease when transferring the pAL-SceI plasmid by intergeneric conjugation into the single crossover mutants (III.2.2.11.2). I-SceI endonuclease should generate a DNA-double strand brake activating repairing mechanism enhancing the probabilities of a second recombination of the homologous regions. Unluckily, no colonies were obtained on the conjugation plates.

2.2.2. Gene inactivation of plaABC1 via single crossover To overcome the difficulties of inactivation of the three transporter genes plaABC1-3 via double crossover, the putative substrate binding protein plaABC1 was chosen for its individual deletion via single crossover (III.2.2.11.1). For this purpose, the inactivation plasmid pKCplaABC1SCO was constructed by cloning an inner homologous region (1.3 kb) of the target gene into the suicide vector pKCXY02 (III. Table 1.9-1). The correct amplification of the homologous region was verified by sequencing. The control digestions of the final construct indicated the proper ligation into the inactivation plasmid. The plasmid was transferred into S. sp. Tü6071 by intergeneric conjugation as detailed in IV. Results III.2.1.2.3. The negative control remained without any growth and the positive control sporulated and generated a characteristic change of the color of the agar. Conjugation plates suggested the growth of Streptomyces mutants because of the production of a brown pigment under antibiotic selection, as for the positive control. However it was not possible | 139 to pick single colonies from the surface of the solid medium (Figure 2-6). No sporulation was observed on the conjugation plates as it easily occurs with the wild type. The material scratched from the surface to be cultivated in liquid did not procure a growing culture.

Figure 2-6 | Conjugation plates of S. sp. Tü6071::pKCplaABC1SCO. The MS plates of the positive control contains S. sp. Tü6071, the negative control contains S. sp. Tü6071 which was overlaid with Apra and Phospho. Two conjugation plates contain the donor and recipient cells which were overlaid with the same antibiotics as the negative control.

3. StreptomeDB contribution The participation in the update of the StreptomeDB (Lucas et al. 2013) involved the analysis of 150 publications and, in addition, the identification of 200 compounds. The minimum information included in the Curation software required the identification of a Streptomyces strain producer of a named compound. Activity of the compound, target microorganism, biosynthetic pathway and other relevant data of the compound were introduced when available. In addition, information about the identified compound was collected in an excel sheet for further inclusion into the database. Among the 150 publications, 300 compounds were identified which were further detailed investigated. From the 300 compounds, 141 were identified by name search in PubChem database. Out of the other 159, the structure of 121 compounds was available in the publications and were manually drawn with MarvinSketch IV. Results for further analysis. The structures were converted into SMILE format and searched in PubChem database. The structures of 41 molecules were found in the database while 70 were not included so far. The remaining 10 compounds could not be drawn due to lack of detailed information about the structure in the publication. Figure 3-1 shows the distribution of the 140 | identified compounds from the analyzed publications.

Figure 3-1 | Graphic representation of the distribution of the 300 identified compounds out of 150 publications. ID = compound ID found by name search in PubChem database. No ID = compound ID was not found by name search in PubChem database. Structure = the structure was available in the publication. No structure = the structure was not available in the publication. New structure = the compound was not found in the PubChem database when searching by name nor by SMILE annotation. Found ID = the compound ID was found when searching by SMILE annotation in PubChem database. Excluded = the compound could not be drawn due to a lack of information in the publication.

From the additional 200 compounds investigated for a PubChem ID by name or SMILE annotation, 23 compounds were directly identified by name search in PubChem database. 59 structures were drawn with MarvinSketch for conversion into SMILE. 43 out of 59 were not identified in PubChem database and therefore the drawn structure was included in StreptomeDB. The remaining 118 named compounds were wrongly identified as a secondary metabolite on the previous analysis of the publication or the structure was not available.

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V. Discussion

1. Homologies of rishirilide gene cluster in other strains The bioinformatics analysis of the four transporter genes rslT1-4 from the rishirilide gene cluster of Streptomyces bottropensis Goe C4/4 (Yan et al. 2012), led to an interesting finding. A high homology was identified, not only of these four genes but nearly the whole rishirilide (rsl) gene cluster in three other strains, Streptomyces bottropensis ATCC 25435, Streptomyces scabiei NCPPB 4086, and Micromonospora lupini strain Lupac 08 (Figure 1-1).

Figure 1-1 | Gene cluster prediction from antiSMASH search of cos4, S. bottropensis ATCC 25435, S. scabiei NCPPB 4086 and M. lupini strain Lupac 08

Curiously, none of these strains has been so far described as rishirilide producers but other secondary metabolites (Figure 1-2). This suggests the presence of a silent rsl cluster in their genomes. Previous investigations performed on Streptomyces bottropensis Goe C4/4 would support the idea of the existence of cryptic clusters since the strain was firstly identified as producer of mensacarcin, a type II PKS metabolite (Arnold 2002). Arnold isolated a novel secondary metabolite, rishirilide A, only by cultivation of the strain in a specific medium. This change on the growing conditions woke up the silent rishirilide cluster, a well-known V. Discussion strategy to discover new metabolites (Masuma et al. 1986; Bode et al. 2002; Weber et al. 2015).

The genome sequence of Streptomyces bottropensis ATCC 25435 was published by Hongyu

142 | Zhang et al. (2013), a strain known for producing bottromycins A2, B2 and C2 (Figure 1-2) (Nakamura et al. 1967; Kaneda 1992; Kaneda 2002). The current version of antiSMASH (Blin et al. 2013) analysis led to the identification of 35 putative gene clusters including two type II PKS. One of these two clusters showed identical genes to those coding for rishirilide B what led to the suspicious that it could be the same strain. However, analysis of the second type II PKS cluster of S. bottropensis ATCC 25435 did not show similarities to mensacarcin cluster of S. bottropensis Goe C4/4 indicating that they are different species.

Streptomyces scabiei NCPPB 4086 is known to produce thaxtomine A (Figure 1-2), a compound used as pesticide (Hongbo Zhang et al. 2013). The draft genome sequence of the strain was recently published (Harrison et al. 2014) although no annotation about secondary metabolites gene clusters is available so far.

The anthraquinone derivates lupinacidins A-C (Figure 1-2) were isolated from the culture broth of Micromonospora lupini strain Lupac 08 (Igarashi et al. 2007). It is a Gram-positive bacteria from the order , and its genome sequence was firstly published in 2012 (Alonso-Vega et al. 2012). Further genome characterization has been done by Trujillo et al. (2014) who annotated fifteen cluster to be responsible of secondary metabolite production. Two of these gene clusters (number 7 and 10) were described as type II PKS, one of them reported as a putative cluster for the production of granaticin (Figure 1-2). Surprisingly, no specific annotation about lupinacidin cluster was done in this publication. However, due to the similarity of these compounds to rishirilide B and its intermediates (Figure 1-2) it could be assume that the responsible cluster is the one obtained by antiSMASH search (Figure 1-1). This cluster would correspond with the “cluster number 10” annotated by Trujillo et al. (2014). Lupinacidin A was also isolated from the fermentation broth of the marine-derived Streptomyces spinoverrucosus beside the novel compounds galvaquinones A-C, 5,8-dihydroxy-6-isopentyl-2,2,4-trimethylanthra[9,1-de] [1,3]oxazin- 7(2H)-one and islandicin (Figure 1-2) (Hu et al. 2012). Curiously, the structures of galvaquinones A and B match exactly the same structure of rishi2a (Yan 2012) and JW-2 (Wunsch-Palasis 2013), respectively. These compounds were isolated from the mutated rishirilide cluster heterologously expressed in S. albus. Unfortunately, no genome mining or V. Discussion biosynthesis investigations have been done so far in S. spinoverrucosus what could provide more information about the synthetic pathway of rishirilide A and B.

The finding of high similar transporter genes conserved within the different clusters identified in these strains which code, or would code if not silent, for secondary metabolites | 143 with such similar structures suggest a close relation of RslT1-4 to the biosynthesis of rishirilide. A deeper comparison of single genes of these lupinacidin and galvaquinone gene clusters could contribute to solve the biosynthesis of rishirilides A and B in the future.

Figure 1-2 | Structures of bottromycins A2, B2 and C2 (Kaneda 1992; Kaneda 2002), thaxtomine A (Hongbo Zhang et al. 2013), granaticin (Deng et al. 2011), lupinacidins A-C (Igarashi et al. 2007; Igarashi V. Discussion

et al. 2011), galvaquinones A-C, islandicin and 5,8-dihydroxy-6-isopentyl-2,2,4-trimethylanthra[9,1- de][1,3]oxazin-7(2H)-one (Hu et al. 2012), rishi2a (Yan 2012), JW-2 (Wunsch-Palasis 2013) and rishirilide B (Iwaki et al. 1984).

2. The ABC importer system RslT123

144 | 2.1. From gene organization to quaternary structure Many actinomycetes which are known to produce antibiotics contain in their biosynthetic cluster at least one ABC transporter. These transporters confer resistance to the drug when it is expressed in antibiotic-sensitive heterologous host (Méndez & Salas 1998; Méndez & Salas 2001). Martín, Casqueiro, and Liras (2005) discussed different secretion system for secondary metabolites and compared the four transporter families which belong to multidrug transporter class: (i) ABC transporters; (ii) major facilitator superfamily (MFS); (iii) small multidrug resistance (SMR); and (iv) resistance nodulation determinants (RND). They also described ABC transporters as multicomponent proteins able to transport a broad range of molecule sizes. In contrast, MFS transporters were reported to be encoded by a single polypeptide and to be able to transport only small molecules (Martín et al. 2005). Table 2.1-1 shows ABC transporters located in a secondary metabolite gene cluster and have been identified to be involved in its export.

Table 2.1-1 | Examples of ABC transporters located in secondary metabolite gene clusters. Information modified from Martín, Casqueiro, and Liras (2005).

ABC transporter Microorganism Secondary metabolite carA Streptomycs thermotolerans Carbomycin dirB Streptomyces peucetius Daunorubicin nocH Nocardia uniformis Nocardicin A mtrA, mtrB Streptomyces argillaceus Mithramycin oleB, oleC Streptomyces antibioticus Oleandomycin orf7, orf8, orf10 Lysobacter lactamgenus Cephabacin pimA, pimB Streptomyces natalensis Pimaricin srmB Streptomyces ambophaciens Spiramycin tlrC Streptomyces fradiae Tylosin

These findings firstly suggested a similar role for the ABC transporter genes in rishirilide cluster. Nevertheless, the exemplified transporter genes encode exclusively ATP binding proteins (nucleotide binding domain, NBD) and hydrophobic membrane components (transmembrane domain, TMD) (Martín et al. 2005). In contrast, rslT1-3 were predicted to encode additionally to a NBD and a TMD, a substrate binding protein (SBP). Those V. Discussion combinations of ABC transporter genes leading to NBDs, TMDs, and a SBP are mainly found in ABC importers (Alkhatib et al. 2012). LolD2CE, an ABC transporter of E. coli, is the only exception described in literature of an exporter containing a SBP (Narita 2011). The Lol system and its analogues are involved in the export of lipoproteins in Gram-negative from | 145 the cytoplasmic membrane to the outer membrane (Tanaka et al. 2007; Liechti & Goldberg 2012). Generally, SBP are expose to the extracellular space and should capture the substrate to be imported (Dassa & Bouige 2001; Berntsson et al. 2010; Alkhatib et al. 2012). Therefore, the prediction of RslT1 as a SBP was determinant to propose that RslT123 is involved in nutrient up-take as discussed in section 2.2. Bioinformatics approach to identify the imported substrate by RslT1-3 led to the hypothesis that the system might be involved in amino acid up-take. The particularities of the subunits of the ABC transporter system RslT123 are discussed below. The proposed organization of the protein complex in the membrane is shown in Figure 2-3.

In the absence of a retentive outer membrane in Gram-positive bacteria, SBPs have evolved distinct mechanisms for being retained within the cell envelopes. They include a transmembrane peptide, a lipid anchored or a fusion to the transmembrane domain of the ABC transporter system (Gilson et al. 1988; Sutcliffe & Russell 1995; Biemans-Oldehinkel et al. 2006). The amino acid analysis of RslT1 provided the evidenced of a conserved lipobox sequence (typically L-3–(A/S/T)-2– (G/A)-1–C+ (Rahman et al. 2008)), a domain which contains an indispensable cysteine residue to which a lipid moiety is attached in lipoproteins (von Heijne 1989; Sutcliffe & Harrington 2002). This lipobox is preceded by a signal peptide at the N-terminal end which would be cleaved by a type II signal peptidase (SPase II) (Tjalsma et al. 1999). Although the presence of a signal peptide in RslT1 was only identified by analysis of the protein sequence with Phobius software (Käll et al. 2004), the finding of a lipobox sequence supports the existence of a signal peptide. This conserved domains indicate that RslT1 is an extracellular protein anchored to the membrane as a lipoprotein. Therefore it is firstly transcribed as a pre-lipoprotein which translocates to the extracellular space via Sec (secretory) or Tat (twin arginine protein transport) pathway thanks to its signal peptide (Freudl 2013) (Figure 2-1). V. Discussion

146 |

Figure 2-1 | Scheme extracted from Natale, Brüser, and Driessen (2008) which represents the Sec- and Tat translocases of E. coli. The routes “a” and “b” show the co-translational and post-translational targeting in Sec-translocase, respectively. Route “c” shows the translocation of the pre-folded protein through Tat translocase.

In the extracellular space the pre-lipoprotein is acylated by a diacylglyceryl transferase to the conserved lipobox cysteine via a thioether linkage (Hutchings et al. 2009). Exceptionally, the existence of N-acylated lipoproteins in Bacillus subtilis and Staphylococcus aureus has been described although the responsible enzyme was not identified (Hayashi et al. 1985; Navarre et al. 1996). The SPase II cleaves the signal peptide by recognition of the diacylated lipobox leaving the lipid modified cysteine at the N-terminus of the mature lipoprotein (Hutchings et al. 2006). Thompson et al. (2010) reported the first complete analysis of the lipoproteome and lipoprotein biogenesis in Streptomyces and proposed the Tat as the common pathway for lipoprotein export in these strains, as previously described by Widdick et al. (2006). However, the described distinctive (S/T)-R-R-x-F-L-K “twin-arginine” amino acid motif (Berks 1996) was not identified in RslT1 sequence what suggests that the protein is probably translocated by Sec translocase.

Lipoproteins of Gram-positive bacteria have been proposed to develop comparable functions to periplasmic proteins in Gram-negative bacteria (Hutchings et al. 2009), an assumption especially based on the study of substrate binding proteins of ABC transporters (Nielsen & Lampen 1982; Sutcliffe & Russell 1995). The first example was demonstrated in Streptococcus pneumoniae with the ami operon, which shows strong homology to the oligopeptide (opp) up-take system of Salmonella typhimurium (Hiles et al. 1987; Alloing et V. Discussion al. 1990). RslT1 was compared by BLAST search to SBP of Gram-negative bacteria leading to identities around 30 – 40 %. For example, it showed 36 % identity to a periplasmic component of an amino acid ABC transporter of Rhizobium sp. CF142, a Gram-negative soil bacteria. These results support the amino acid recognition function proposed for RslT1 in | 147 this work. Nevertheless, characterization of a SBP by sequence analysis is hazardous as demonstrated by Quiocho and Ledvina (1996), who compared a dozen of crystallographic structures of SBPs. The investigated proteins shared low sequence homology even so their comparison led to the discovery of a high tertiary structure similarity. This finding opened a discussion if this fact would imply the interaction with alike ligands. The family of SBP were described as a ‘gold mine’ in sense of structure and molecular recognition (Quiocho & Ledvina 1996).

(i)

(ii)

(iii)

Figure 2-2 | Scheme of the lipidation process of RslT1 leading to its anchoring into the membrane. The membrane is represented with two fatty acids (shadowed in yellow) covalently bounded to a glycerol molecule V. Discussion

(shadowed in purple) which is as well phorylated (shadowed in blue). (i) RslT1, translocated to the extracellular space probably by Sec translocase, is shown with its lipobox (orange square) and the signal peptide (in green) on the N-terminal. (ii) After acylation of the cystein residue contained in the lipobox sequence by a thioester linkage, the protein is attached to the membrane. (iii) In a second reaction, the signal peptide is released by the signal peptidase type II leading to the active form of RslT1 as the substrate binding protein of the ABC transporter system.

148 | RslT2 was characterized as the nucleotide (ATP) binding domain (NBD) of the ABC transporter system due to the evidence of all distinctive conserved motifs of this sort of proteins such as Walker A – B and the ABC signature (IV.1.1.2). ABC systems are defined by the ATPase domain, which is highly conserved, and have been demonstrated to bind and hydrolyze ATP providing energy for a large number of biological processes (Dassa & Bouige 2001). Chrystal structures of isolated NBDs like the maltose transporter (Chen et al. 2003), Rad50 (a DNA repair protein) (Hopfner et al. 2000) or the archaeal MJ0796 transporter (Smith et al. 2002) showed that the P-loop and the signature sequence interact with two ATP molecules which are sandwiched in a NBD dimer. Comparison of the crystal structures of the ATP-bound sandwich dimers to the observation of nucleotide-free NBDs suggested that the dimerization of the NBDs implies a conformational change in the TMDs (Chen et al. 2003). Similar mechanism can be expected for RslT2, which should as well interact with the TMDs of RslT3 in the process of translocation of the substrate. The NBDs are expressed as separate polypeptides or fused as multifunctional polypeptides to another NBD subunit (e. g. the E. coli ribose transporter RbsA (Buckel et al. 1986)). Alternatively, the NBD can be fused to the TMD (e. g. the peptide transporter MHC (Trowsdale et al. 1990; Powis et al. 1992)). RslT2 and RslT3 do not seem to yield a gene fusion but to be organized in an operon. Qiu et al. (2011) showed similar gene organization for avtA and avtB which encode an ABC efflux pump of avermectins, potent antiparasite metabolites produced by Streptomyces avermitilis. The avtA stop codon overlaps the avtB start codon in ATGA, exactly as it occurs between rslT3 and rslT2. In addition, Qiu et al. (2011) proved by RT-PCR the generation of a combined avtAB mRNA.

RslT3 was predicted to have 6 membrane spanning segments with the N- and C-terminal on the cytoplasmic face and three extracellular and two intracellular loops. This structure was previously described for other permeases such as LacY or BtuC involved in up-take of maltose and , respectively (Yamato 1992; Korkhov et al. 2012). LacY and BtuC, as well as RslT3, seemed to function as a homodimer providing a total of 12 transmembrane helixes which expand through the membrane. It has been discussed that inner membrane domains from binding protein-dependent transport systems share a conserved sequence located approximately 90 residues away from the C-terminal (EAAxxxGxxxxxxxxxIxLP) V. Discussion (Dassa & Hofnung 1985). This EAA region is located between the fourth and fifth transmembrane helixes and has been described to be essential for the constitution of a functional transporter (Mourez et al. 1997) and to interact with the ATP-binding domains (Pearce et al. 1992). This sequence was likewise identified in RslT3 what consolidates the | 149 characterization of RslT3 as a permease and its interaction mechanism with RslT2.

Figure 2-3 | Representation of the ABC transporter system formed by RslT1-3. The twelve (6 x 2) transmembrane helixes built by the homodimer RslT3 are drawn with dark and light green cylinders as well the cytosolic N- and C-terminal. The cytosolic loop between the fourth and fifth helix of each subunit is in contact with the NBD RslT2 drawn in red. The SBP (acylated RslT1) is represented in orange, with a lipid cue attached that serves as an anchor into the membrane.

2.2. RslT123, an amino acid importer? Bioinformatics predictions of RslT123 and available literature about the structure and mechanism of ABC transporters do strongly support the assignment of RslT123 as an importer system. In addition, the results of the expression of RslT123 in E. coli DH5α ΔacrAB in MIC test showed that this system is not involved in drug efflux (IV.1.3.5.1). So then… what does RslT123 import?

Transporters are known for their big capacity to recognize and translocate specific compounds or a wide range of substances. In many cases, it has been shown their expendable presence due to the capacity of other transporter to assume their functions, a fact that is discussed in section 2.3. The deletion of rslT1-3 was not crucial for the normal production of rishirilide B. In addition, it also seemed not to influence the correct activity of any gene of rishirilide cluster as discussed in section 2.3. However, overexpression V. Discussion experiments showed an increase of the amounts of the rishirilide B produced indicating at least a relation of RslT123 with the metabolic pathway (2.3). In this work, the ABC transporter system RslT123 is proposed to be an amino acid importer. RslT1 showed similarities around 40 % to amino acid transporters described in the databases (NCBI, 150 | Uniprot, and Transporter Classification Database). Transporter Classification Database identified similar proteins to transport substrates like arginine, ornithine, glutamine, lysine, histidine, D-alanine and D-valine. Amino acids are essential for the living of the cell, therefore… what is the sense of an amino acid transporter in a secondary metabolite gene cluster? Rishirilide B is a type II PKS metabolite what means its metabolic pathway includes the formation of a long carbon chain by Claisen condensations of extender units derived from malonyl coenzymeA (malonyl-CoA) (Hertweck et al. 2007). However, X. Yan (2012) proposed that the very first starter unit for rishirilide B biosynthesis is not malonyl-CoA but isobutyryl-CoA. Isobutyryl-CoA would be necessary for the formation of the long carbon side chain present in the final structure. This assumption was based on the investigations performed by Marti et al. (2000) who investigated the biosynthetic pathway of R1128A-D (Figure 2-5). These anthraquinones were isolated from the fermentation broth of Streptomyces sp. No. 1128 and were previously reported as potent antagonists of the non- steroidal estrogen receptor (Hori, Abe, et al. 1993; Hori, Takase, et al. 1993). By feeding experiments with different amino acids, the unusual 4-methylvaleryl side chain of R1128C, like in rishirilide B, was identified to derive from valine (Marti et al. 2000). Valine catabolism has been previously investigated in microbes and higher animals (Massey et al. 1976; Sherman et al. 1986; Denoya et al. 1995) leading to the agreement that it includes oxidative deamination and decarboxylation steps to yield isobutyryl-CoA (Figure 2-4).

Figure 2-4 | Hypothesis of valine catabolism in bacteria, adapted from Y.-X. Zhang, Tang, and Hutchinson (1996)

V. Discussion Marti et al. (2000) proved also the decarboxylation of valine by feeding experiments with [1-13C]valine. No detection of peak in the 13C NMR spectrum of HU235 (Figure 2-5), another compound isolated with identical carbon chain backbone to that of R1128C (Figure 2-5).

| 151

Figure 2-5 | Structures of R1128A-D and HU235 (Marti et al. 2000)

In the future, similar experiments could be performed in order to demonstrate that isobutyryl-CoA is the starter unit for the biosynthesis of rishirilide B with an origin in valine catabolism. Considering this assumption, the presence of an amino acid transporter in rishirilide cluster could be a mechanism to ensure the up-take of valine to enhance rishirilide biosynthesis. Information extracted from TransportDB (Ren et al. 2007) showed three ABC transporter systems involved in the up-take of branched-amino acids in the genome of Streptomyces avermitilis MA-4680 and Streptomyces coelicolor A3(2). Unfortunately, this data is not available in TransportDB for the rishirilide producer Streptomyces bottropensis Goe C4/4 or the heterologous expression host Streptomyces albus J1074. In any case, similar numbers of transporter genes could be expected due to the similar genome size of the strains. In the view of these few branched-amino acid transporter systems in a complete whole genome, the existence of RslT1-3 in rishirilide gene cluster is extraordinary.

Purification of the substrate binding protein Several attempts to purify RslT1 were performed (IV.1.2.4) to prove its substrate specificity under laboratory conditions. Heterologous expression of the SBP in Streptomyces lividans TK24 and E. coli strains led to a close approach. The cloning of rslT1 into pET28a(+) allowed the introduction of a N- and C-terminal His6-Tag for affinity chromatography purification. In silico characterization of RslT1, could explain the defeat of the expression with an N- terminal tag. For the synthesis of the mature form of the lipoprotein RslT1, all amino acids V. Discussion on the N-terminal side of the crucial cysteine of the lipobox will be cleaved by the signal peptidase as prior discussed (2.1). Therefore, it is proposed in this work that detection of the SBP with the N-terminal histidine cue could be due to an ineffective mechanism of E. coli to express correctly a Gram-positive bacterium protein (Figure 2-6). The most critical step 152 | would be the efficient recognition of the signal peptide which would guide the protein through the Sec translocase to the periplasmic space. If the pre-lipoprotein of RslT1 would reach the cytoplasmic membrane, acylation of the cysteine residue by the prolipoprotein diacylglycerol transferase should take place. Right after, the cleavage of the signal peptide should also not pose a problem. These two events are likely to happen due to the high analogy of the responsible enzymes (Lsp and Lnt) in Gram-positive and Gram-negative bacteria (Hutchings et al. 2009).

A) B)

Figure 2-6 | Scheme extracted from Hutchings et al. (2009) representing the lipoprotein biogenesis in Gram-positive and negative bacteria. A) In Gram-negative bacteria (i) the unfolded or folded pre-lipoprotein are directed to the cytoplasmic membrane through the Sec or Tat translocases, respectively, depending on their signal peptide (painted in blue). (ii) The pre-lipoprotein is covalently lipidated at the cysteine of the lipobox by the prolipoprotein diacylglycerol transferase (Lgt). (iii) The signal peptide is released by a signal peptidase type II (Lsp) and a second lipid group is attached to the amino group of the cysteine residue by Lnt (lipoprotein N- acyl transferase) (iv). The lipoprotein stays on the cytoplasmic membrane (v) or is directed to the outer membrane thanks to the Lol (lipoprotein localisation) pathway (vi). B) In Gram-positive bacteria the pre- lipoproteins are guided unfolded or folded to the extra cytosolic space by the Sec or Tat translocases, respectively (i). After acylation of the lipobox cysteine with a membrane phospholipid through a thioester linkage by an Lgt analogue (ii), the signal peptide is cleaved by a signal peptidase II (Lsp analogue).

The results of this work suggest that the diacylation of the lipoprotein might be responsible for the detection of RslT1 in analytical SDS gels with a higher molecular weight than expected. Covalent linkage of the diacylglycerol to the cysteine residue should be stable enough to be maintained through the purification steps. The signal peptide cleaved and the lipid attached showed nearly identical molecular weight. However, it is possible that the carbon chain hindered the normal run of the protein on a SDS gels. V. Discussion

Expression tests of the recombinant RslT1 with a C-terminal His6-Tag in E. coli showed correct amounts of the overexpressed protein. However, purification by ÄKTATMFPLC system under the tested conditions did not yield to a clear elution of the desired protein. Purification of RslT1 was probably hindered by the low protein expression level in the | 153 heterologous host and the use of detergents during the purification process which disturbed the binding of the protein to the Ni2+.

Related to a low protein expression level, the toxicity of the overexpression of lipoproteins in E. coli has been discussed in literature. K. Nakamura and Inouye (1980) reported the instability of a lipoprotein gene from Serratia marcescens cloned in E. coli. They indicated the loss of part of the gene sequence during the cloning process. Furthermore, overexpression of the major E. coli lipoprotein (Lpp) led to lyses of the bacteria (Nakamura et al. 1982). Expression of malX and amiA which encode SBP lipoproteins responsible for the transport of maltose and oligopeptides, respectively, resulted in deleterious effects in E. coli (Martin et al. 1989). Sequence similarity of the prolipoproteins of Lpp, MalX and AmiA and therefore a similar activation of the export machinery is proposed to explain their toxic effects (Martin et al. 1989). Comparable investigations were carried out by Gómez, Ramón, and Sanz (1994) on the LplA lipoprotein of Bacillus cereus. Alike deleterious effects were observed in E. coli when LplA was overexpressed leading to the suggestion that this is due to its similarity to Lpp, as previously described for MalX and AmiA. Gómez, Ramón, and Sanz (1994) mentioned that the increment of anchored protein is altering the properties of the membrane. On this basis, overexpression of RslT1 as a lipoprotein could be limited to small amounts which would avoid damaging effects to the E. coli host.

To avoid the translocation machinery and the resulting attachment of RslT1 to the membrane, a truncated version of the protein was explored. Thus, rslT1 was amplified skipping the first 20 amino acids what was thought to be enough to disrupt the signal peptide. Gao et al. (2012) reported protein aggregation problems when purifying the full- length OppA. This protein was described as an ABC transport substrate-binding protein from Thermoanaerobacter tengcongensis. An N-terminal 30 amino acids truncated OppA was successfully expressed as a soluble protein in E. coli and purified without the use detergents (Gao et al. 2012). Unfortunately, no RslT1 truncated protein was detected in the expression test of this work probably due to the instability of the truncated protein in the cytosolic space. Singh and Röhm (2008) purified the SBP of the ABC transporter system AatJMQP required for the up-take of glutamate and aspartate in Pseudomonas putida V. Discussion KT2440. Overexpression and purification of the SBP AatJ was performed with a modified gene sequence where the first 72 bp were truncated, lacking the natural signal peptide (24 amino acids). The fragment was then cloned into a vector which contained the ompA signal peptide and a small sequence responsible of the transport into the periplasmic space (Singh 154 | & Röhm 2008). OmpA is a major protein of the outer membrane of E. coli which plays an essential role on its stability (Wang 2002). After induced expression of the truncated AatJ, it was released from the periplasmic space by osmotic shock and further purified (Singh & Röhm 2008). This procedure is an interesting alternative for future attempts to highly express RslT1 in E. coli as well as the assays described for quantification of the binding to the substrate.

Cloning of RslT1 with a C-terminal His6-Tag to achieve the constructs pUWLrslT1C and pUWLrslT1CT2T3 allowed the expression of the protein in the heterologous host S. lividans. However, some difficulties were experienced using this system. Due to the lack of an inducible expression system, identification of the desired protein band was difficult as a precise negative control was absence. S. lividans/pUWL-H cultivated under the same conditions was used as reference. Unfortunately, the broad protein content of Streptomyces entangled the analysis on the SDS-PAGE. In addition, lyses of Streptomyces cells involved the use of French® Pressure cell press what implied the cultivation of more voluminous cultures and a longer procedure compared to E. coli. Expression of the target protein was detected after performing affinity chromatography by manual Ni-NTA purification. A protein band with the expected size of ca. 42 kDa was visualized on the elution fractions by SDS-PAGE. However, purification using the ÄKTATMFPLC system did not showed a detectable elution of the protein on the chromatogram. Further attempts to purify RslT1 should include an ultracentrifugation step for isolation of the cell membranes as proceed with E. coli. Application of an inducible system for protein expression in Streptomyces could be considered. Enguita et al. (1996) reported an inducible thiostrepton expression system

for the expression of His6-Tag proteins from Nocardia lactamdurans in S. lividans. Horbal, Fedorenko, and Luzhetskyy (2014) recently published new inducible expression systems for Streptomyces spp. which could be help for the purification of RslT1.

Finally, purification of RslT1 was probably hindered by the use of detergents which was necessary to release the lipoprotein from the surrounding membrane. Nickel matrices are normally able to tolerate a limited amount of nonionic detergents although it should be determined experimentally for every protein and purification system (Bornhorst & Falke V. Discussion 2000). The data file of the Ni Sepharose matrix used from Amersham described several detergent which have been tested (2% Triton™ X-100 (nonionic), 2% Tween™ 20 (nonionic), 2% NP-40 (nonionic), 2% cholate (anionic), 1% CHAPS (zwitterionic)). An alternative for purification of RslT1 would be the search for other detergents more appropriate for the | 155 sample and the ÄKTATMFPLC system.

2.3. Influence of the ABC transporter system in rishirilide B production

2.3.1. Lack of the amino acid importer To investigate the role of the ABC transporter system in the biosynthesis of rishirilide B, rslT1-3 were deleted from the cosmid cos4 by Redirect© Technology (Gust et al. 2002) and the recombinant construct was heterologously expressed in Streptomyces albus J1074. In a first attempt, the transporter genes were attempted to be independently inactivated as explained in IV.1.2.1. rslT1, rslT2 and rslT3 were successfully exchanged for the Specr marker gene by recombination of the upstream and downstream homologous regions of the target genes flanking the antibiotic cassette. However, difficulties were experienced when erasing the Specr cassette by digestion with NheI and the cosmid had to be religated. Control digestion of the isolated DNA after religation and transformation into E. coli led to the identification of undesired conformation of the cosmid. Gust et al. (2004) summarized some common problems of the PCR-targeting in Streptomyces. It included degradation of the isolated recombinant cosmid DNA or the occasional presence of pseudo-resistant colonies on selective plates. Results of this work suggest that after digestion of the Specr cassette of the recombinant cos4 with NheI, undesired recombination events occurred. It is possible that thy led to a more stable although incomplete conformation of the cosmid. Fortunately, the complete deletion of the three ABC transporter genes was achieved with the construction of cos4ΔrslT123. Although the lack of the single inactivation, expression of cos4ΔrslT123 in S. albus was sufficient to identify the role of the ABC transporter system as discussed below.

Quantification of the amounts of rishirilide produced in the fermentation extracts of S. albus::cos4ΔrslT123 demonstrated a slight decrease compared to the control S. albus::cos4. The calculated amounts showed a decreased of 0.97- and 0.67-fold in the media and inside the cells, respectively, in ΔrslT123 mutants. These results suggested that rslT1-3 was not required for rishirilide B production when its cluster gene cluster was heterologously expressed in S. albus under the tested conditions. Assuming that RslT123 is an amino acid V. Discussion transporter, it is likely that other transporter systems present in the genome of S. albus could overtake the same function. Similar conclusions were proposed by D’Orazio et al. (2015) who performed investigations on ZnuABC transporter, an ABC transporter system involved in the up-take of zinc in Pseudomonas aeruginosa. Quantification of the intracellular zinc 156 | content was not affected by deletion of its defined ZnuABC transporter system what suggested the existence of redundant mechanisms for the acquisition of zinc. Comparable observations were described by Shang et al. (2013) investigating AroP, an histidine transporter of Corynebacterium glutamicum RES167. Feeding experiments with 14C-labeled aromatic amino acid showed residual up-take activities in a ΔaroP mutant suggesting the existence of additional up-take systems besides AroP. Alloing, de Philip, and Claverys (1994) performed inactivation experiments of the oligopeptide transport lipoprotein AmiA in Streptococcus pneumoniae. Gene deletion of amiA did not abolish the transport of oligopeptides demonstrating the dispensability of the binding protein. Other additional lipoprotein highly homologous, AliA and AliB, were identified and proposed to overtake the transport of AmiA substrate. A triple gene deletion of amiA, aliA and aliB, generated a mutant deficient for oligopeptide transport (Alloing et al. 1994).

The amounts of rishirilide B detected in ΔrslT123 mutants also indicates that the ABC transporter system is not complexed with other enzymes of the biosynthetic pathway. The lack of the ABC transporters allowed the normal synthesis of the metabolite suggesting that the expression and function of other enzymes were not affected. Pearson et al. (2004) worked on the characterization of mcyH, a gene encoding an ABC transport system proposed to be involved in the export of the hepatotoxin microcystin. Disruption of mcyH led to a complete loss of the hepatotoxin in the cyanobacterium Microcystis aeruginosa PCC 7806. Pearson et al. (2004) suggested that the ABC exporter might serve as an anchor to the membrane stabilizing the microcystin biosynthesis multienzyme complex. Thus, the lack of mcyH would cause the detachment of the protein complex and degradation of its components impeding the synthesis of microcystin (Pearson et al. 2004). Siegers et al. (1996) investigated the biosynthesis of the lantibiotic nisin which led to the identification of a synthetase complex consisting of NisB, NisC and NisT. NisT was described as an ABC transporter that translocates the mature nisin peptide over the cytoplasmic membrane in association with the other two biosynthetic enzymes (NisB-C). Disruption of NisT abolished nisin production indicating the ABC transporter is essential for the lantibiotic biosynthesis (Siegers et al. 1996). McyH (Pearson et al. 2004) and NisT (Siegers et al. 1996) differ from RslT123 in their gene organization. One single gene, mcyH and nisT, V. Discussion leads to a homodimer with ATPase activity on the N-terminal side of its protein sequence. In contrast to RslT123, these discussed ABC transporters are proposed to be exporters and essential for the secondary metabolite production due to their participation in the biosynthetic complex. | 157

2.3.2. Complementation and overexpression The rslT1-3 deletion mutant was complemented with the replicative plasmid pUWL-rslT123 which contained the transporter genes cloned under the influence of the strong promoter ermE*. The calculated amounts of rishirilide B detected in fermentation extracts of S. albus::cos4ΔrslT123/pUWL-rslT123 demonstrated a higher production compare to the control S. albus::cos4 in the media as well as inside the cells (1.2- and 1.9-fold, respectively). Nearly the same increment was detected when pUWL-rslT123 was expressed in S. albus containing the complete cos4 (1.25- and 1.68-fold in the media and inside the cells, respectively). This result supports the triviality of rslT1-3 within the rsl gene cluster as shown with its inactivation, yet at the same time they are able to enhance rishirilide production. Alike influence was detected when analyzing the overexpression mutants of S. albus::cos4 containing pTOS-rslT123, which carried the genes under the influence of ermEp1* promoter. Comparison of the capacity of pTOS(z)-spec and pUWL-H to overexpress rslT1-3 was analyzed in relation to their influence on rishirilide B production. Despite the weaker strength of the promoter sequence contained in pTOS-rslT123 (Siegl et al. 2013) and the integration of a single copy of the plasmid in the genome of S. albus did not impede a stronger impact on rishirilide B production. Increments of 1.71- and 1.25-fold of rishirilide B amounts were calculated for pTOS-rslT123 and pUWL-rslT123 mutants, respectively. Similar experiences were observed by Kalan et al. (2013) during the investigation of the bldA gene in S. calvus. This strain carries a mutation in bldA that makes the bacteria deficient in the formation of aerial mycelium and spores. Complementation with a functional copy of bldA restored sporulation and it was more prominent when using an integrative plasmid comparable to pTOS(z)-spec than when using pUWL-H (Kalan et al. 2013). This could be due to a higher stability of the integrative plasmids in these Streptomyces spp. compared to those which replicate autonomously. However, pTOS(z)-spec required the use of spectinomycin for its selection and, unfortunately, fast development of resistance was experienced when working with S. albus::cos4. Other integrative plasmids were considered but their use was limited to the antibiotic or the integrase due to the presence of an Aprar cassette and the integrase of phage VWB in the backbone of cos4. Despite these difficulties, the S. albus::cos4 RslT1 and RslT123 overexpression mutants were achieved. V. Discussion Overexpression of the ABC transporter system, which has been discussed in section 2.2 to be responsible for the up-take of valine, enhances rishirilide B production. A higher synthesis of the ABC importer could favor the translocation of its substrate into the cell. Intracellular increased amounts of valine which is proposed to provide the starter unit for 158 | the formation of the polyketide chain of rishirilide B could lead to its overproduction. Production of secondary metabolites has been described to be affected by the composition of the media (Martín et al. 2005). Even usual amino acids have been described as potential inducers of secondary metabolites, for example tryptophan for the biosynthesis of ergo alkaloids (Robbers & Floss 1968). Also, supplementation of the media with L-proline, L-tyrosine and L-alanine led to a 10 – 20 % increase of lincomycin production in Streptomyces lincolnensis 55-20 (Ye et al. 2009). Based on the same principle, rishirilide B production could be influenced by the overexpression of the amino acid importer system what would imply an increase of the intracellular valine. If RslT123 does not influence the expression of other genes as demonstrated by its inactivation, an increment of rishirilide B precursors is the most likely argument to explain the enhancement of its production. Similar findings were described by Li et al. (2010) when overexpressing malEFG-a, which encode an ABC transporter system involved in maltose up-take in Streptomyces avermitilis. They observed that overexpression of the transporter yield to an increase of avermectin production and the fermentation periods were reduced. It was proposed that the overexpression of malEFG-a enhances the utilization rate of starch providing more precursors for the biosynthesis of the secondary metabolite (Li et al. 2010).

The calculated amounts of rishirilide B detected in the fermentation extracts of S. albus::cos4::pTOS-rslT1 was compared to the amounts detected in S. albus::cos4:: pTOS- rslT123 and the control S. albus::cos4. With these experiments showed the influence of the overexpression of the SBP compared to the overexpression of the whole ABC transporter system. S. albus::cos4::pTOS-rslT1 and S. albus::cos4::pTOS-rslT123 led to an increase of rishirilide amounts in the media of 1.21- and 1.71-fold compared to the control. This calculation suggested that the SBP of the importer system is sufficient to enhance the production of the secondary metabolite. However, the SBP per se could not translocate the substrate through the membrane what indicates that RslT1 had to interact with other transporter proteins. It is possible that RslT1 is able to cooperate with other membrane proteins beside RslT2 and RslT3 to perform its function. Literature shows that nucleotide- binding domains of ABC transporters are capable to interact with other ABC-type permeases beside their original associated transporter (Quentin et al. 1999; Singh & Röhm 2008). V. Discussion However, this could be due to the highly conserved regions in the ATPases protein sequence which interacts with the TMDs as previously discussed in this work for RslT2 and RslT3. Another possibility is that the increase of the expression of RslT1 implies a more efficient recognition of its substrate leading to a more efficient up-take. Overexpression of OppA, the | 159 SBP of the oligopeptide ABC transport system of Lactococcus lactis, was investigated by Picon et al. (2000). An increment of OppA resulted in a highly increased peptide binding capacity and a slight affectation of the up-take rate. Van der Heide and Poolman (2002) discussed the possibility of one, two or four SBP participating in ABC transporter system. In this review it is suggested that multiple identical SBP could increase the translocation capacity or influence the kinetics of the transport. Apparently, nearly all SBP-dependent ABC transporter of Gram-negative bacteria present an excess of SBP per translocator (Ames et al. 1996). Van der Heide and Poolman (2002) proposed that additional SBPs could capture the substrate while the first SBP is still docked to the membrane translocator. This cooperative work of more than one SBP would lead to an obvious increase of the transport capacity of the system.

3. The Major Facilitator Superfamily transporter RslT4 Aside the ABC transporter RslT1-3 discussed above, rishirilide gene cluster was annotated to contain a fourth transporter, rslT4 (Yan et al. 2012). BLAST analysis of this gene led to the identification of rslT4 to encode a multidrug resistant transporter from the major facilitator superfamily (MFS). This superfamily of transporters have been described to be capable to translocate a variety of substrate by uniport, antiport or symport mechanisms. The substrate specificity, number of transmembrane domains and mechanism served as criteria for the classification in smaller families (Pao et al. 1998; Saier et al. 1999; Reddy et al. 2012; Yan 2013). The majority of MFS transporter have been described to form 12 helixes what differs from RslT4, which was predicted to form 14 (Krogh et al. 2001). Law, Maloney, and Wang (2008) proposed that these two extra helixes come from an insertion of the central cytoplasmic loop into the membrane. These transmembrane regions were not altered in both of the different annotations proposed for rslT4 by X. Yan et al. (2012) and Linnenbrink (2009). The experimental part of this work was performed with the shorter sequence annotated from X. Yan (93 bp shorten on the start codon site). Cloning of this sequence into different vectors led to an active form of the protein as showed with the overexpression experiments and MIC assays indicating that the first 31 amino acids do not play an essential role. V. Discussion BLAST search of RslT4 proposed the transporter to be part of the EmrB/QacA family. This family was described as a drug:H+ antiport with 14 transmembrane domains (DHA2) based on the similarity to the firstly studied transporters EmrB and QacA. EmrB is a MFS transporter part of the EmrAB–TolC system in E. coli, a three protein complex creating a 160 | continuous channel for the export of hydrophobic toxins from the cytoplasm to the exterior of the cell (Tanabe et al. 2009). QacA is a multidrug transporter from Staphylococcus aureus which has been shown to provide resistance against a broad range of antibiotics. Alignment of RslT4 with EmrB and QacA in Uniprot (The UniProt Consortium 2014) led to identities of 23 % and 20 %, respectively. MFS transporters predicted to have 14 transmembrane domains were described to share some conserved sequences named motifs A, B, C , D1, E and H located among the first 7 TMD (Paulsen & Skurray 1993; Paulsen et al. 1996). Figure 3-1 shows the alignment and the conserved regions of RslT4, EmrB and QacA. Although the identity between the proteins appears to be low it can be observed that they share these conserved motifs.

Figure 3-1 | Alignment of the first 298 amino acids of RslT4 with EmrB and QacA which described the EmrB/QacA family of the MFS transporters. The characteristic motifs are shadowed in different colors corresponding to the colored boxes which the motif name and conserved sequences. In the color boxes, the x represents any amino acid, the upper-case letters indicate a high occurrence of the residue (> 70%) and the small V. Discussion letters indicate an occurrence of 40 – 70 % (Paulsen & Skurray 1993; Paulsen et al. 1996). Sequence numbers on the right refer to the position of the last residue on each line.

The presence of MFS transporters in secondary metabolite gene cluster has been identified in several strains as exemplified in Table 3-1 (Martín et al. 2005). Many of these transporter have been described to play a regulatory role on the production of the exported antibiotic. | 161 This implication is discussed in section 3.2.

Table 3-1 | Examples of MFS transporters located in secondary metabolite gene clusters. Information modified from Martín, Casqueiro, and Liras (2005).

MFS transporter genes Strain Antibiotic exported actII-orf2, actII-orf3, actVA Streptomyces coelicolor Actinorhodin cmcT Streptomyces clavuligerus Cephamycin C entC Streptomyces maritimus Enterocin frnF Streptomyces roseofulvum Frenolicin lmrA Streptomyces lincolnensis Lincomycin mmr Streptomyces coelicolor Methylenomycin otrB Streptomyces rimosus Tetracyclin ptr Streptomyces pristinaespiralis Pristinamycin pur8 Streptomyces alboniger Puromycin tcmA Streptomyces glaucescens Tetracenomycin thnJ Streptomyces cattleya Thienamycin

3.1. RslT4, a multidrug transporter The capacity of RslT4 to provide resistance against different antibiotics by leading to an efflux pump was proved in a MIC assay. The utilized E. coli DH5α strain which carries the deletion of a major multi efflux system (AcrAB) was transformed to express RslT4. Eight different antibiotic were tested (nalidixic acid, bacitracin, tetracycline, gentamicin, norfloxacin, streptomycin, vancomycin and novobiocin) at different concentrations by performing 12 dilution steps 1:2 (IV.1.3.5.1). Expression of RslT4 lead to higher MIC values compare to the control for four of them: spectinomycin, vancomycin, tetracyclin and nalidixic acid. Gentamicine and norfloxacin inhibited the growth of E. coli mutants at all of the concentration tested. Bacitracin did not inhibit the growth at any of the concentrations tested. Novobiocin showed no difference in its MIC compared to the control. In order to find a relation of those compounds which MIC was affected by the expression of RslT4, the structures and their mechanism of action was studied. Curiously, the eight tested antibiotics did not show similarity in structure as can be seen in Figure 3-2. V. Discussion

162 |

Figure 3-2 | Chemical structure of the antibiotics tested in MIC assay against E. coli DH5α ΔacrAB expressing the MFS transporter RslT4

The mechanism of action of the four antibiotics which showed an increase of the MIC is discussed below (Figure 3-3). Vancomycin, a glycopeptide which targets the cell wall is rarely active against Gram-negative bacteria (Toscano & Storm 1982; Loll & Axelsen 2003). However, the compound showed inhibitory activity at a concentration of 250 µg/mL for the control and doubled it with the expression of RslT4. This is probably due to the higher sensibility of the mutated E. coli DH5α ΔacrAB to toxic compounds. The quinolone nalidixic acid, which inhibits the DNA gyrase showed a 2-fold higher MIC with the overexpression of the MFS transporter compared to the control. The aminoglycoside streptomycin and tetracycline target the 30S subunit of the ribosome and showed 8- and 2-fold higher MIC, respectively, in the RslT4 mutants compared to the control. These values revealed the capability of RslT4 to direct the compounds outside the cell as described for multidrug efflux pumps as part of antibiotic resistance mechanism (Lewis 2013). V. Discussion

| 163

Figure 3-3 | Representation of the different mechanism of antibiotic families. The scheme was extracted from the review published by Wilson (2014).

The same eight antibiotics were tested in a disc diffusion assay comparing their activity against S. albus::cos4 and S. albus::cos4ΔrslT4. The deletion mutant showed bigger inhibition zones for bacitracin, gentamicin, nalidixic acid, norfloxacin and novobiocin, compared to the control. Streptomycin, tetracycline and vancomycin caused no remarkable difference between the mutants. These results indicate that the lack of rslT4 makes the bacteria more sensible to some of the tested antibiotics. This supports the identification of RslT4 as a multidrug transporter.

Similar experiments were performed by Simm et al. (2012) investigating the MFS multidrug resistance transport protein BC4707 from Bacillus cereus. The transporter gene was described to be implicated in fluoroquinolone tolerance. The Δbc4707 mutant showed a higher sensibility to norfloxacin compared to the wild type. Expression of bc4707 in E. coli DH5α ΔacrAB resulted in higher tolerance to ciprofloxacin and norfloxacin. In comparison to these studies, RslT4 shows an extraordinary capability to efflux different compounds. V. Discussion 3.2. Influence of the MFS transporter in rishirilide B production To investigate the role that RslT4 plays in the export of rishirilide B and its ability to regulate the metabolite biosynthetic pathway, rslT4 was deleted from the cosmid cos4 and overexpressed in the heterologous host Streptomyces albus J1074. 164 | The transporter gene rslT4 was deleted from the cosmid cos4 by Redirect© Technology (Gust et al. 2002). The recombinant cosmid was transferred into the heterologous host for the analysis of rishirilide B production. Surprisingly, the calculated amounts of rishirilide B from the fermentation extracts of S. albus::cos4ΔrslT4 demonstrated higher production compare to the control S. albus::cos4. An increase of 1.41- and 3.25-fold of rishirilide amounts were calculated in the media and inside the cells, respectively. This increment on the production could be related to the involvement of the MFS transporter in a regulatory system of the biosynthesis of rishirilide B which is discuss below.

Several MFS antibiotic transporters have been described to be transcribed under the influence of a regulator. For example, the regulatory gene qacR was suggested to repress the transcription of qacA which encodes a MFS transporter in Staphylococcus aureus (Grkovic et al. 1998). The gene qacA is constitutively expressed in ΔqacR mutants while expression of qacR prevents qacA transcription. Furthermore, the export complex actII-ORF1-3, in actinorhodin gene cluster of Streptomyces coelicolor A3(2) were described to consist of an antibiotic exporter/repressor system (Caballero et al. 1991). The regulator actII-ORF1 and the operon actII-ORF2-3 share overlapping, divergent promoter regions which actII-ORF1 represses. Guilfoile and Hutchinson (1992) identified the genes tcmR and tcmA in Streptomyces glaucescens, encoding a regulator and a MFS transporter, respectively. The described that the TcmR protein represses the transcription of tcmR and tcmA, which are divergently oriented, by binding to an intergenic operator region. These genes are involved in in the regulation of tetracenomycin C (TCM C) resistance. Guilfoile and Hutchinson (1992) also demonstrated that increasing amounts of TMC C reduced the quantity of TcmR interacting with the promoter region. They suggested that TCM C interacts with TcmR reducing its capacity to bind the DNA allowing the expression of TcmA. These regulatory mechanisms involving MFS transporters seem to be closely coupled to the biosynthesis of the secondary metabolites.

Wunsch-Palasis (2013) described the repressor effect of RslR4 on the transcription of rslT4. RslR4 was identified to be a member of the MarR family, which is known to regulate multiple antibiotic resistance (Yan et al. 2012). In addition, rslR4 and rslT4 share the same V. Discussion organization divergently oriented as described for the examples above (qacR/qacA and tcmR/tcmA). On the other hand, X. Yan (2012) identified RslR4 as an activator of the transcription of rslR1, a SARP regulator (Streptomyces antibiotic regulatory proteins) of rishirilide gene cluster (Yan et al. 2012). X. Yan discussed the regulatory network of the four | 165 regulatory genes present in rishirilide cluster, RslR1-4. It was proposed a hierarchy in a regulatory cascade where RslR4 would be located on a higher lever for the control of RslR1 (Yan 2012). SARP regulators have been described to positively influence the transcription of biosynthetic genes and activate the production of secondary metabolites (Martín & Liras 2010). The positive regulatory effect of RslR1 on the biosynthesis of rishirilide B was proved by Wunsch-Palasis (2013). Results of this work therefore indicate that the inactivation of rslT4 led to a shift of the regulated system. Rishirilide B production was increased in S. albus::cos4ΔrslT4 due to the lack of a negative regulation of RslR4 over rslT4 but still a positive regulation over rslR1. The absence of RslT4 would imply the capability of another transporter from the host, S. albus, to overtake the efflux. As previously discussed for the ABC transporter system RslT123, this is a very common event in transporters. Particularly in S. albus, this occurs when the strain is used for heterologous expression of a secondary metabolite gene cluster which does not contain its own transporters. For example, heterologous expression of didesmethylmensacarcin (Yan et al. 2012) or iso-migrastatin (Feng et al. 2009) gene clusters led to steady productions of the metabolite in S. albus. An MFS transporter annotated as multidrug resistance protein in the genome sequence of S. albus J1074, SSHG_05318, showed the highest similarity to RslT4 with 38.2 % identity. This transporter could be responsible of the export of rishirilide B in the ΔrslT4 mutants.

Overexpression of rslR4 in S. albus::cos4 led to the detection of higher production of rishirilide B in the media as well as inside the cells. The calculated amounts showed an increase of 1.48- and 1.83-fold, respectively, compare to the control. These results support the role of RslR4 as an activator of the transcription of the positive regulator RslT1 leading to a higher amounts of rishirilide B produced. Concerning the repressor activity of RslR4 over rslT4, it is proposed that this negative regulation is taking place until some intermediates or rishirilide B is produced. This hypothesis is supported by the findings of the transport/regulator mechanism identified for tcmR/tcmA and actII-ORF1/actII-ORF2-3 described above. Guilfoile and Hutchinson (1992) proved that the DNA-binding of the repressor tcmR to the promoter region of tcmA (encoding the MFS transporter) is inhibited by tetracenomycin C, the substrate to be transported. Tahlan et al. (2007) showed that intermediates of actinorhodin biosynthetic pathway relieve the repression of actII-ORF1 V. Discussion over actII-ORF2-3. In this way, RslR4 probably activates the biosynthetic pathway of rishirilide B by influencing rslR1 and contains the expression of rslT4 until the production of the metabolite is achieve (Figure 3-4). This assumption should be proved to understand the regulatory mechanism. Similar analysis of the transcription of rslR4 and rslT4 could be 166 | performed as described by Guilfoile and Hutchinson (1992) and Tahlan et al. (2007).

Figure 3-4 | Representation of the proposed regulatory mechanism of RslR4 over rslR1 and rslT4. (i) The MarR regulator RslR4 activates the expression of rslR1 (Yan 2012), what should begin the biosynthesis of rishirilide B, and represses rslT4 (Wunsch-Palasis 2013). (ii) RslR4 binds to the promotor region in between the divergent genes rslR4 and rslT4. (iii) Rishirilide B or maybe some intermediates inhibits the DNA-binding of RslR4 to the promoter region of rslT4. (iv) The release of RslR4 from the promoter region activates the transcription of rslT4. (v) The expressed MFS transporter RslT4 located in the cell membrane allows the export of rishirilide B by antiport of H+.

The analysis of the fermentation broth of the complemented and overexpressed mutants of rslT4 led to reduced or equivalent amounts of rishirilide B detected. It is necessary to reproduce these calculations because only two repetitions of S. albus::cos4::pTOS-rslT4 and S. albus::cos4Δ::pTOS-rslT4 could be analyzed. It is important to consider that the weight of the dried harvested cells was 3 to 3.5-fold higher in the mutants compared to the control. Considering all production cultures generated in this work for all S. albus::cos4 mutants, the V. Discussion values for the cell pellet were in a range of 40 – 65 mg. The reason of this increase is still unclear. It is possible that RlsT4 influences other processes of the cell metabolism when it is not expressed under the influence of RslR4.

4. Transporter genes of Streptomyces sp. Tü6071 | 167 Investigations on the genome sequence of Streptomyces sp. Tü6071 using Galaxy as a main tool allowed the identification of all putative transporter genes. A total of 265 transporter genes were described and 50 of them appeared to be singular for S. sp. Tü6071 in comparison to Streptomyces coelicolor and Streptomyces avermitilis. Analysis of these other two strains for transporter genes provided disparate data compared to TransportDB (Ren et al. 2007). In this database S. coelicolor is annotated to contain 442 transporter genes and S. avermitilis 430. On the other hand, Bentley et al. (2002) identified 614 proteins with predicted transport function after genome analysis of S. coelicolor. In view of these different numbers it difficult conclude the certainty of the data. However, prediction tools are fast developing with the impact of increasing amounts of sequenced genomes available. Taking this into account, it is possible that Galaxy provided the most accurate result.

Sequence analysis of the phenalinolactone gene cluster and its upstream and downstream regions provided some transporter genes that were considered for their involvement in the biosynthesis of the secondary metabolites. Due to the proximity of plaABC1-3 to phenalinolactone cluster and the low identities detected to other annotated proteins these three genes were considered for experimental characterization. In order to identify their relevance on the secondary metabolite production, the genes were planned to be inactivated. The first attempt consisted in the delete all three genes via double crossover. This method required the construction of an inactivation plasmid, pKCplaABC123, which should be integrated in the genome by a first recombination step. After achieving the first single crossover, a second recombination step should occur for the deletion of the target genes. This process is promote by the repetitively cultivation of the single crossover bacteria in a selection medium with spectinomycin (passage). After more than 35 passages and more than 1200 single clones analyzed, the double crossover was not achieve. Similar experiments were previously performed in this strain by Myronovskyi et al. (2011) which successfully inactivate the regulatory of phenalinolactone gene cluster. They utilized comparable inactivation plasmids with homologous regions of about 700 bp necessary for the recombination. pKCplaABC123 contained homologous regions of 2500 – 3400 bp, what should enhance this event. The construction of the plasmid was positively controlled by V. Discussion restriction digestion and sequencing of the first 1000 bp upstream and downstream the integrated fragments. Although sequencing of these regions was correct, it is possible that some undesired conformation of the plasmid occurred. However, control PCR of the single crossover mutants showed the expected amplified bands as shown in IV.2.2.1. In addition, 168 | the double crossover was promote by using the I-SceI endonuclease tool (Siegl et al. 2010). Expression of I-SceI after conjugation of the pALSceI plasmid generates a DNA double- strand break which should activate the repair mechanism leading to the desired recombination. Nevertheless, expression of the endonuclease did not provide any growing colony. It could be due to the incapacity of the cells to survive after the brake of the DNA or that conjugation of the plasmid was not successful. Further attempts could be performed with another inactivation vector different from pKCXY02 (III. Table 1.9-1).

Alternatively, inactivation of plaABC1, the substrate binding protein, was planned via single crossover. The inactivation plasmid was successfully cloned and conjugation of S. sp. Tü6071 with pKCplaABC1SCO led to a strange appearance of the MS plates. No single colonies were obtained on the surface but a changed on color of the MS agar was remarkable (IV.2.2.2). A color change of the cultivation medium is characteristic of Streptomyces spp. due to their ability to produce pigments. These results suggest that plaABC1, and therefore plaABC2-3 as an ABC transporter system, could be involved in the formation of aerial mycelium. Akanuma et al. (2011) investigated the role of bldK-g in the formation of aerial mycelium. BldK-g was described as an ABC transporter predicted to transport oligopeptides from its amino acid sequence. By inactivation of bldKB-g, the gene encoding the substrate binding protein, they proved that the operon encodes an oligopeptide ABC transporter involved in the formation of aerial mycelium. Further experiments should be performed with plaABC1-3to clarify their involvement in this cell mechanism.

| 169

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VII. Abbreviations

Physical values A Absorbance g Earth's gravitational acceleration m Mass

OD600 nm Optical density at 600 nm t Time V Volume

Units % Per cent °C Celsius degree bp Base pairs Da Dalton g Gram h Hour kb Kilo base pairs L Liter M Molar m Meter min Minute Mol Mole, unit for amount of substance Pa Pascal psi Pound per square inch rpm Revolutions per minut s Second u Unit. For enzyme activity, the amount that catalyzed the conversion of one µmol of substrate per minute V Volt

VII. Abbreviations Prefix µ micro (10-6) c centi (10-2) G Giga (109) k Kilo (103) 186 | m Milli (10-3) n nano (10-9) p pico (10-12)

Nucleotide A Adenine C Cytosine G Guanine T Thiamine

Amino acids Alanine Ala A Arginine Arg R Asparagine Asn N Aspartic acid Asp D Cystein Cys C Glutamic acid Glu E Glutamine Gln Q Glycine Gly G Histidine His H Isoleucine Ile I Leucine Leu L Lysine Lys K Methionine Met M Phenylalanine Phe F Proline Pro P Ser S Threonine Thr T Tryptophan Trp W Tyrosine Tyr T Valine Val V

Bacterial strains B. subtillis Bacillus subtilis E. coli Escherichia coli S. albus Streptomyces albus J1074 VII. Abbreviations S. avermitilis Streptomyces avermitilis S. bottropensis Streptomyces bottropensis Goe C4/4 S. coelicolor Streptomyces coelicolor A3(2) S. sp. Tü6071 Streptomyces sp. Tü6071

| 187 Others aac(3)IV Apramycin resistance gene aadA Spectinomycin resistance gene ABC ATP-binding cassette ADP Adenosine diphosphate aphII Kanamycin resistance cassette APS Ammonium persulfate ATP Adenosine triphosphate bla β-Lactam resistance gene BLAST Basic local alignment search tool BSA Bovine serum albumin CoA Coenzyme A DMF Dimethylformamide DMSO Dimethyl sulfoxide DNA Deoxyribonucleic acid dNTP Deoxynucleotide triphosphate dTTP Deoxythiamine triphosphate EDTA Ethylenediaminetetraacetic acid ESI Electrospray ionization et al. et alii, and others FPLC Fast protein liquit chromatography

His6-Tag 6 histidine residue tagged IPTG Isopropyl β-D-1-thiogalactopyranoside MarR Multiple antibiotic resistance regulator MFS Major facilitator superfamily MS Mass spectrometry MSD Quadrupole mass detector NAD+ Nicotinamide adenine dinucleotide, oxidized form NADH Nicotinamide adenine dinucleotide, reduced form NBD Nucleotide binding domain NCBI National Center for Biotechnology Information Ni2+-NTA Nickel-Nitrilotriacetic acid ORF Open reading frame oriT Origin of transfer PAGE Polyacrylamide agarose gel electrophoresis PCR Polymerase chain reaction PKS Polyketide synthase RNA Ribonucleic acid VII. Abbreviations rRNA ribosomal RNA rsl Rishirilide RT Room temperature SARP Streptomyces antibiotic regulatory protein SBP Substrate binding protein 188 | SDS Sodium dodecyl sulfate sp. species SPE Solid phase extraction ssp. Subspecies tcr Tetracycline resistance gene TEMED N,N,N',N'-Tetramethylethylendiamin TMD Transmembrane domain TRIS tris(hydroxymethyl) aminomethane tsr Thiostreptone resistance gene UV Ultraviolet X-Gal 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside X-Gluc 5-Bromo-4-chloro-1H-indol-3-yl β-D-glucopyranosiduronic acid Δ Indicates a gene deletion

| 189

VIII. Appendix

1. Plasmid maps

1.1. pTOS-plaABC123

Figure 1-1 | Plasmid map of pTOS-plaABC123, for expression of the 3 transporter genes plaABC1-3 from Streptomyces sp. Tü6071. Cloning steps are detailed in III.2.2.10.1.

VIII. Appendix 1.2. pUWL-H-plaABC123

190 |

Figure 1-2 | Plasmid map of pUWL-plaABC123, for expression of the 3 transporter genes plaABC1-3 from Streptomyces sp. Tü6071. Cloning steps are detailed in III.2.2.10.1.

1.3. pUWL-rslT123

Figure 1-3 | Plasmid map of pUWL-rslT123, for expression of the 3 transporter genes rslT1-3 of rishirilide gene cluster from Streptomyces bottropensis Goe C4/4. Cloning steps are detailed in III.2.2.10.2

VIII. Appendix 1.4. pUWL-OriT-rslT1

| 191

Figure 1-4 | Plasmid map of pUWL-rslT1, for expression of the SBP RslT1 of rishirilide gene cluster from Streptomyces bottropensis Goe C4/4. Cloning steps are detailed in III.2.2.10.2

1.5. pTOS-rslT1

Figure 1-5 | Plasmid map of pTOS-rslT1, for expression of the SBP RslT1 of rishirilide gene cluster from Streptomyces bottropensis Goe C4/4. Cloning steps are detailed in III.2.2.10.2

VIII. Appendix 1.6. pTOS-rslT123

192 |

Figure 1-6 | Plasmid map of pTOS-rslT123, for expression of the 3 transporter genes rslT1-3 of rishirilide gene cluster from Streptomyces bottropensis Goe C4/4. Cloning steps are detailed in III.2.2.10.2

1.7. pTOS-rslT4

Figure 1-7 | Plasmid map of pTOS-rslT4, for expression of the MFS transporter gene rslT4 of rishirilide gene cluster from Streptomyces bottropensis Goe C4/4. Cloning steps are detailed in III.2.2.10.2 VIII. Appendix 1.8. pKCplaABC123

| 193

Figure 1-8 | Plasmid map of pKCplaABC123, for inactivation of the 3 transporter genes plaABC1-3 of Streptomyces sp. Tü6071 by homologous recombination via double crossover. Cloning steps are detailed in III.2.2.10.3.

1.9. pKCplaABC1SCO

Figure 1-9 | Plasmid map of pKCplaABC1SCO, for inactivation of the transporter gene plaABC1 of Streptomyces sp. Tü6071 by homologous recombination via single crossover. Cloning steps are detailed in III.2.2.10.3. VIII. Appendix 1.10. pET28rslT1N

194 |

Figure 1-10 | Plasmid map of pET28rslT1N, for the inducible expression of rslT1 with an N-terminal His6- Tag in E. coli.

1.11. pET28rslT1C

Figure 1-11 | Plasmid map of pET28rslT1C, for the inducible expression of rslT1 with a C-terminal His6- Tag in E. coli.

VIII. Appendix 1.12. pET28rslT1Ctrun

| 195

Figure 1-12 | Plasmid map of pET28rslT1Ctrun, for the inducible expression of rslT1 in E. coli with a C-terminal His6-Tag and 60 bp truncated at the N-terminal.

1.13. pET28rslT123

Figure 1-13 | Plasmid map of pET28rslT123, for the inducible expression of rslT1-3 in E. coli.

VIII. Appendix 1.14. pUWLrslT1C

196 |

Figure 1-14 | Plasmid map of pUWL-H-rslT1C, for the expression of rslT1 with a C-terminal His6-Tag in Streptomyces lividans TK24.

1.15. pUWLrslT1CT2T3

Figure 1-15 | Plasmid map of pUWL-H-rslT1CT2T3, for the expression of rslT1 with a C-terminal His6-Tag and rslT2-3 in Streptomyces lividans TK24.

VIII. Appendix 2. Galaxy workflows

| 197

Figure 2-1 | Workflow to achieve the comparison of the description of the predicted transporters from S. sp. Tü6071, S. coelicolor and S. avermitilis. As input: list of predicted transporters genes after BLAST search VIII. Appendix

(filtered >38 % identity) of the three strains. As outcome: a file with the number of transporter genes which were obtained with the same exact description and the description itself. The workflow continues in Figure 2-2.

198 |

Figure 2-2 | Continuation of the workflow in Figure 2-1 to achieve the comparison of the description of the predicted transporters from S. sp. Tü6071, S. coelicolor and S. avermitilis. As input, the file with the number of transporter genes which were obtained with the same exact description and the description itself. As outcome, a file with the overlapping and non-overlapping transporters between the three strains S. sp.Tü6071, S. coelicolor and S. avermetilis.

VIII. Appendix

A)

| 199

B)

Figure 2-3 | Workflow to achieve a desired organization of the information to continue with the workflow in Figure 2-4. As input, the list of the predicted transporter of S. sp. Tü6071 after BLAST search (filtered >38 % identity). As outcome, a file with tabular information of ORF name, ID number from TCDB (Transporter Classification Database), the description of the transporter, percentage of their identity to the best hit. VIII. Appendix

200 |

Figure 2-4 | Workflow for the identification of transporter genes inside secondary metabolite gene clusters. As input, (i) a file with non-overlapping transporters of S. sp. Tü6071; (ii) a file with the the description and ID number of all the transporters; and (iii) a file with the predicted proteins belonging to secondary metabolite gene clusters. As outcome, a file with the ORF name, ID number of the transporters, their description, the percentage of identity to the best BLAST search hit, the secondary metabolite gene cluster where they are located and the protein sequence.

3. List of figures Section II Figure 1-1 | Representation of the modular polyketide synthase of erythromycin, a type I PKS metabolite. Extracted from (Weber et al. 2015) ...... 5 Figure 1-2 | Representation of the type II PKS biosynthesis of actinorhodin. Extracted from Kim and Yi (2012) ...... 6 Figure 1-3 | Structures of mensacarcin and rishirilides A and B, type II PKS secondary metabolites produced by S. bottropensis Goe C4/4 (Arnold 2002; Linnenbrink 2009) ...... 8 Figure 1-4 | Representation of the mevalonate pathway and the nonmevalonate pathway for the synthesis of IPP and the following reactions for the terpene biosynthesis ...... 10 Figure 1-5 | Structures of phenalinolactones A-D (1-4) (Meyer 2003) ...... 11 Figure 2-1 | Representation of Gram-positive and Gram-negative cell wall. Extracted from Cabeen and Jacobs-Wagner (2005) ...... 12 Figure 2-2 | Representation of the different architecture of the ABC transporters. Modified from Biemans-Oldehinkel, Doeven, and Poolman (2006) ...... 14 Figure 2-3 | Representation of the transport mechanism of a Gram-negative ABC importer. Modified from Locher (2009) ...... 15 VIII. Appendix Figure 2-4 | Representation of the uniport, symport and antiporter mechanisms of transporters ...... 16 Figure 2-5 | Proposed mechanism for the translocation of substrate/H+ of LmrP...... 17 Section III | 201 Figure 2-1 | Detailed view on the MCS of pET28a(+) vector (Novagen) ...... 58 Figure 2-2 | Scheme of the construction of pKCplaABC ...... 66 Figure 2-3 | Representation of the cloning plasmids required for the construction on pUWLrslT1CT2T3 ...... 69 Figure 2-4 | Integration of the inactivation plasmid in a single crossover ...... 70 Figure 2-5 | Integration of the inactivation plasmid in a double crossover ...... 71 Figure 2-6 | Representation of the Red/ET primer design ...... 73 Figure 2-7 | Overview of the Red/ET protocol ...... 74 Figure 2-8 | ÄKTATMFLPC set up methods for protein purification ...... 80 Figure 2-9 | Western blot sandwich and electrode position for the correct transfer of the proteins ...... 82 Figure 2-10 | Screenshot of the curation software ...... 87 Figure 2-11 | Information collected in excel sheet for further inclusion into the database .. 87 Figure 2-12 | Overview of the process of the analyzed publications for the introduction of the information in StreptomeDB ...... 88 Figure 2-13 | Galaxy workflow for identification of secondary metabolite gene clusters and putative transporter genes ...... 90 Section IV Figure 1-1 | Organization of the rishirilide gene cluster (rsl) ...... 92 Figure 1-2 | AntiSMASH outcome from rishirilide cluster search ...... 94 Figure 1-3 | Outcome of the analysis of RslT1 in Phobius and SignalP software in search of a signal peptide ...... 95 Figure 1-4 | Conserved regions of the NBD of an ABC transporter system responsible of the hydrolyzation of ATP...... 96 Figure 1-5 | RslT2 amino acid sequence with labelled conserved motifs ...... 96 Figure 1-6 | Outcome of RslT3 analysis with SOSUI software ...... 97 Figure 1-7 | Outcome of RslT4 analysis with SOSUI software ...... 99 Figure 1-8 | Outcome of the analysis of RslT4 in Phobius and SignalP software for identification of a signal peptide ...... 100 Figure 1-9 | Agarose gels from rslT1 inactivation experiments ...... 101 VIII. Appendix Figure 1-10 | Agarose gels from rslT2 inactivation experiments ...... 102 Figure 1-11 | Agarose gels from rslT3 inactivation experiments ...... 103 Figure 1-12 | Agarose gels from rslT1, rslT2 and rslT3 inactivation experiments ...... 104 Figure 1-13 | HPLC chromatograms (λ = 254 nm) achieved from the analysis of the production 202 | cultures of S. albus::pOJ436 (negative control), S. albus::cos4 (positive control) and S. albus::cos4ΔrslT123 ...... 106 Figure 1-14 | Average of the calculated AUC of rishirilide B peak divided by the weight of the dry cell pellet (mg) from S. albus::cos4ΔrslT123 and S. albus::cos4 cultures ...... 107 Figure 1-15 | HPLC chromatograms (λ = 254 nm) achieved from the analysis of the production cultures of S. albus::cos4ΔrslT123, S. albus::cos4 (positive control) and S. albus::cos4ΔrslT123/pUWL-rslT123 ...... 108 Figure 1-16 | Average of the calculated AUC of rishirilide B peak divided by the weight of the dry cell pellet (mg) from S. albus::cos4ΔrslT123/pUWL-rslT123 and S. albus::cos4 cultures ...... 109 Figure 1-17 | Average of the calculated AUC of rishirilide B peak divided by the weight of the dry cell pellet (mg) from S. albus::cos4, S. albus::cos4::pTOS-rslT1, S. albus::cos4::pTOS- rslT123 and S. albus::cos4/pUWL-rslT123 cultures ...... 110 Figure 1-18 | Agarose gels with the resultant amplification of rslT1 by PCR and the control digestions of pET28rslT1N and pET28T1C ...... 112 Figure 1-19 | SDS gels of the expression tests with pET28rslT1N ...... 112

Figure 1-20 | Western blot of the cell cultures expressing RslT1 with N-terminal His6-Tag 113 Figure 1-21 | SDS gels of protein samples after ultracentrifugation and manual Ni-NTA purification of the cultured E. coli BL21 (DE3) StarTM/pET28rslT1N ...... 114 Figure 1-22 | ÄKTATMFPLC chromatogram (λ = 280 nm) and SDS gels from the analysis of the collected samples for RslT1 purification ...... 115 Figure 1-23 | SDS gel of RslT1 C-terminal expression test and Ni-NTA purification ...... 116 Figure 1-24 | Agarose gels with the resultant truncated rslT1 amplified by PCR and the control digestions of pET28rslT1Ctrun ...... 118

Figure 1-25 | SDS gels of the expression test of the truncated RslT1 C-terminal His6-Tag. . 118 Figure 1-26 | Agarose gels with the control digestions of the constructed plasmids pUWLrslT1C and pUWLrslT1CT2T3 ...... 120 Figure 1-27 | SDS gel of the collected fractions from manual Ni-NTA purification from S. lividans/pUWLrslT1C cultures ...... 121 Figure 1-28 | Agarose gels from rslT4 inactivation experiments ...... 122 VIII. Appendix Figure 1-29 | HPLC chromatograms (λ = 254 nm) achieved from the analysis of the production cultures of S. albus::cos4 (positive control) and S. albus::cos4ΔrslT4 ...... 123 Figure 1-30 | Average of the calculated AUC of rishirilide B divided by the weight of the dry cell pellet (mg) from S. albus::cos4 and S. albus::cos4ΔrslT4 ...... 124 | 203 Figure 1-31 | HPLC chromatograms (λ = 254 nm) achieved from the analysis of the production cultures of S. albus::cos4ΔrslT4::pTOS-rslT4, S. albus::cos4 and S. albus::cos4ΔrslT4 ...... 125 Figure 1-32 | HPLC chromatograms (λ = 254 nm) achieved from the analysis of the extract of the production cultures of S. albus::cos4/pUWL-H-rslR4 and S. albus::cos4 ...... 127 Figure 1-33 | Average of the calculation AUC of rishirilide B peak divided by the weight of the dry cell pellet (mg) from S. albus::cos4 and S. albus::cos4/pUWL-H-rslR4 cultures.... 127 Figure 1-34 | MIC test in E. coli DH5α ΔacrAB/pTOS-rslT123, E. coli DH5α ΔacrAB/pTOS- rslT4 and E. coli DH5α ΔacrAB/pTOS(z)-spec of 8 different antibiotics in dilutions 1:2 ... 129 Figure 1-35 | Representation of the inhibition area of the eight antibiotics tested in a disc diffusion assay with S. albus::cos4 and S. albus::cos4ΔrslT4 ...... 130 Figure 1-36 | HPLC chromatograms of the SPE fractions on the process of rishirilide B purification ...... 131 Figure 2-1 | Venn diagram of the transporter description of the identified transporter genes from Streptomyces coelicolor, Streptomyces avermitilis and S. sp.Tü6071...... 133 Figure 2-2 | Representation of S. sp. Tü6071 genome with ORFs, transporter genes and secondary metabolite gene clusters ...... 134 Figure 2-3 | Organization of the phenalinolactone gene cluster ...... 135 Figure 2-4 | Agarose gels of PCR amplification of 3T1 and 3T2 and control digestions of the final construct pKCplaABC ...... 136 Figure 2-5 | Agarose gel of the control PCR of isolated genomic DNA of single crossover exconjugants ...... 137 Figure 2-6 | Conjugation plates of S. sp. Tü6071::pKCplaABC1SCO ...... 139 Figure 3-1 | Graphic representation of the distribution of the 300 identified compounds out of 150 publications ...... 140 Section V Figure 1-1 | Gene cluster prediction from antiSMASH search of cos4, S. bottropensis ATCC 25435, S. scabiei NCPPB 4086 and M. lupini strain Lupac 08 ...... 141 Figure 1-2 | Structures of bottromycins A2, B2 and C2, thaxtomine A, granaticin, lupinacidins A-C, galvaquinones A-C, islandicin and 5,8-dihydroxy-6-isopentyl-2,2,4- trimethylanthra[9,1-de][1,3]oxazin-7(2H)-one, rishi2a, JW-2 and rishirilide B ...... 143 VIII. Appendix Figure 2-1 | Scheme extracted from Natale, Brüser, and Driessen (2008) which represents the Sec- and Tat translocases of E. coli ...... 146 Figure 2-2 | Scheme of the lipidation process of RslT1 leading to its anchoring into the membrane ...... 147 204 | Figure 2-3 | Representation of the ABC transporter system formed by RslT1-3 ...... 149 Figure 2-4 | Hypothesis of valine catabolism in bacteria, adapted from Y.-X. Zhang, Tang, and Hutchinson (1996) ...... 150 Figure 2-5 | Structures of R1128A-D and HU235 (Marti et al. 2000) ...... 151 Figure 2-6 | Scheme extracted from Hutchings et al. (2009) representing the lipoprotein biogenesis in Gram-positive and negative bacteria ...... 152 Figure 3-1 | Alignment of the first 298 amino acids of RslT4 with EmrB and QacA which described the EmrB/QacA family of the MFS transporters ...... 160 Figure 3-2 | Chemical structure of the antibiotics tested in MIC assay against E. coli DH5α ΔacrAB expressing the MFS transporter RslT4 ...... 162 Figure 3-3 | Representation of the different mechanism of antibiotic families. The scheme was extracted from the review published by Wilson (2014)...... 163 Figure 3-4 | Representation of the proposed regulatory mechanism of RslR4 over rslR1 and rslT4 ……………………………………………………………………………………………………………………………………..166

Section VII Figure 1-1 | Plasmid map of pTOS-plaABC123 ...... 189 Figure 1-2 | Plasmid map of pUWL-plaABC123 ...... 190 Figure 1-3 | Plasmid map of pUWL-rslT123 ...... 190 Figure 1-4 | Plasmid map of pUWL-OriT-rslT1 ...... 191 Figure 1-5 | Plasmid map of pTOS-rslT1 ...... 191 Figure 1-6 | Plasmid map of pTOS-rslT123 ...... 192 Figure 1-7 | Plasmid map of pTOS-rslT4 ...... 192 Figure 1-8 | Plasmid map of pKCplaABC ...... 193 Figure 1-9 | Plasmid map of pKCplaABC1SCO...... 193 Figure 1-10 | Plasmid map of pET28rslT1N ...... 194 Figure 1-11 | Plasmid map of pET28rslT1C ...... 194 Figure 1-12 | Plasmid map of pET28rslT1Ctrun ...... 195 Figure 1-13 | Plasmid map of pET28rslT123 ...... 195 Figure 1-14 | Plasmid map of pUWL-H-rslT1C...... 196 Figure 1-15 | Plasmid map of pUWL-H-rslT1CT2T3 ...... 196 VIII. Appendix Figure 2-1 | Workflow to achieve the comparison of the description of the predicted transporters from S. sp. Tü6071, S. coelicolor and S. avermitilis ...... 197 Figure 2-2 | Continuation of the workflow in Figure 2-1 ...... 198 Figure 2-3 | Workflow to achieve a desired organization of the information to continue with | 205 the workflow in Figure 2-4 ...... 199 Figure 2-4 | Workflow for the identification of transporter genes inside secondary metabolite gene clusters...... 200

4. List of tables Section III Table 1.1-1 | Detailed information of manufacturers and abbreviation ...... 18 Table 1.2-1 | Laboratory material and manufacturer ...... 19 Table 1.2-2 | Laboratory equipment and manufacturer ...... 20 Table 1.2-3 | Analytical instruments and manufacturer ...... 21 Table 1.3-1 | General chemicals, reagents and manufacturer ...... 21 Table 1.3-2 | Organic solvents and manufacturer ...... 23 Table 1.3-3 | Antibiotics and manufacturer ...... 23 Table 1.4-1 | Enzymes and manufacturer ...... 24 Table 1.4-2 | Antibodies and manufacturer ...... 24 Table 1.4-3 | Kits and manufacturer ...... 24 Table 1.5-1 | Solutions and buffers used for agarose electrophoresis ...... 25 Table 1.5-2 | Solutions and buffers used for polymerase chain reaction (PCR) ...... 25 Table 1.5-3 | Solutions and buffers used for genomic DNA isolation of Streptomyces ...... 26 Table 1.5-4 | Solutions and buffers used for plasmid DNA isolation of E. coli ...... 26 Table 1.5-5 | Solutions and buffers for SDS-PAGE analysis ...... 27 Table 1.5-6 | Buffers used for affinity chromatography ...... 28 Table 1.5-7 | Solutions and buffers used for Western Blot ...... 29 Table 1.5-8 | Solutions use for blue/white screening of E. coli ...... 29 Table 1.5-9 | Solutions used for blue/white screening of Streptomyces ...... 29 Table 1.5-10 | Solutions for permanent cultures ...... 29

Table 1.5-11 | Solutions for preparation of CaCl2 E. coli competent cells ...... 30 Table 1.5-12 | Solutions used for preparation of electrocompetent cells ...... 30 Table 1.5-13 | Solvents for preparative HPLC and LC/MS ...... 30 Table 1.5-14 | Solutions needed for protein expression ...... 30 VIII. Appendix Table 1.5-15 | Detergents used for membrane protein solubilization ...... 30 Table 1.6-1 | Antibiotics and their concentration used in this work ...... 31 Table 1.7-1 | Media used in this work ...... 32 Table 1.8-1 | E. coli strains used in this work ...... 32 206 | Table 1.8-2 | Actinomycetes strains used in this work ...... 33 Table 1.9-1 | Cosmids and plasmids needed for this work ...... 34 Table 1.9-2 | Relevant cloning plasmids created in this work ...... 35 Table 1.9-3 | Cosmids created in this work ...... 35 Table 1.9-4 | Complementation and overexpression plasmids created in this work ...... 36 Table 1.9-5 | Inactivation plasmids created in this work ...... 37 Table 1.9-6 | Protein expression plasmids for E. coli created in this work ...... 37 Table 1.9-7 | Plasmids for protein analysis in S. lividans created in this work ...... 37 Table 1.10-1 | Software and databases used in this work ...... 38 Table 2.1.2-1 | Conjugation volumes and incubation times ...... 46 Table 2.2.5-1 | Volumes for analytical and preparative digestions ...... 51 Table 2.2.8-1 | Universal primers used for plasmid sequencing ...... 53 Table 2.2.9-1 | PCR program 1. Standard PCR ...... 54 Table 2.2.9-2 | PCR program 2 ...... 54 Table 2.2.9-3 | PCR program 3. Step down...... 54 Table 2.2.9-4 | PCR program 4a. Gradient PCR...... 55 Table 2.2.9-5 | PCR program 4b. Gradient PCR...... 55 Table 2.2.9-6 | Design of analytical PCR reactions ...... 56 Table 2.2.9-7 | Design of standard PCR reactions ...... 56 Table 2.2.9-8 | Primer design for Red/ET gene inactivation ...... 57 Table 2.2.9-9 | Primer design for amplification of homologous regions (HR) for gene inactivation and marker (Spectinomycin cassette) ...... 57 Table 2.2.9-10 | Primer design for gene amplification ...... 58 Table 2.2.9-11 | Primer design for His-Tag protein expression ...... 59 Table 2.2.9-12 | Primer design for control PCRs ...... 59 Table 2.2.9-13 | Detailed information of main PCR amplifications ...... 60 Table 2.4.3-1 | HPLC/ESI-MS parameters set for rishirilide analysis ...... 84 Table 2.4.3-2 | Parameters of ESI source ...... 84 Table 2.4.3-3 | HPLC gradient for analysis of rishirilide with MENS04R and MENS04RX methods ...... 85 VIII. Appendix Table 2.4.4-1 | Parameters of the preparative HPLC for rishirilide B analysis ...... 85 Table 2.4.4-2 | HPLC gradient for the analysis of rishirilide ...... 85 Section IV Table 1.1-1 | Results of blastx search of rslT1, rslT2, rslT3 and rslT4 in NCBI NR database and | 207 Uniprot database...... 92 Table 2.1-1 | ABC transporters located in secondary metabolite gene clusters ...... 144 Table 3-1 | MFS transporters located in secondary metabolite gene clusters ...... 161

5. Curriculum Vitae