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Studies Towards Secondary Metabolite Formation in Myxobacteria: Elucidation of Biosynthetic Pathways and Yield Optimization

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Studies Towards Secondary Metabolite Formation in Myxobacteria: Elucidation of Biosynthetic Pathways and Yield Optimization Studies towards secondary metabolite formation in Myxobacteria: Elucidation of biosynthetic pathways and yield optimization Dissertation zur Erlangung des Grades des Doktors der Naturwissenschaften der Naturwissenschaftlich-Technischen Fakultät III Chemie, Pharmazie, Bio- und Werkstoffwissenschaften der Universität des Saarlandes von Stefan Müller Saarbrücken 2015 Tag des Kolloquiums: 31.07.2015 Dekan: Prof. Dr. Dirk Bähre Berichterstatter: Prof. Dr. Rolf Müller Prof. Dr. Rolf W. Hartmann Vorsitz: Prof. Dr. Uli Kazmaier Akad. Mitarbeiter: Dr. Judith Becker Diese Arbeit entstand unter der Anleitung von Prof. Dr. Rolf Müller in der Fachrichtung 8.2 Pharmazeutische Biotechnologie der Naturwissenschaftlich-Technischen Fakultät III der Universität des Saarlandes in der Zeit von Mai 2011 bis April 2015. Veröffentlichungen der Dissertation Veröffentlichungen der Dissertation Teile dieser Arbeit wurden vorab mit Genehmigung der Naturwissenschaftlich-Technischen Fakultät III, vertreten durch den Mentor der Arbeit, in folgenden Beiträgen veröffentlicht: Müller S, Rachid S, Hoffmann T, Surup F, Volz C, Zaburannyi N, & Müller R (2014) Biosynthesis of crocacin involves an unusual hydrolytic release domain showing similarity to condensation domains. Chemistry & Biology, 21 (7), 855-865. Hoffmann T, Müller S, Nadmid S, Garcia R, & Müller R (2013) Microsclerodermins from terrestrial myxobacteria: An intriguing biosynthesis likely connected to a sponge symbiont. Journal of the American Chemical Society, 135 (45), 16904-16911. Darüber hinaus konnte zu einer weiteren Publikation, welche nicht Teil dieser Arbeit ist, beigetragen werden: Jahns C, Hoffmann T, Müller S, Gerth K, Washausen P, Höfle G, Reichenbach H, Kalesse M, & Müller R (2012) Pellasoren: Structure elucidation, biosynthesis, and total synthesis of a cytotoxic secondary metabolite from Sorangium cellulosum. Angewandte Chemie International Edition, 51 (21), 5239-5243. Table of contents Table of contents Zusammenfassung 1 Abstract 2 Introduction 3-28 Chapter I: 29-66 Biosynthesis of crocacin involves an unusual hydrolytic release domain showing similarity to condensation domains Chapter II: 67-124 Microsclerodermins from terrestrial myxobacteria: An intriguing biosynthesis likely connected to a sponge symbiont Chapter III: 125-204 Studies towards enhanced production of ambruticin and derivatives thereof via genetic engineering Final Discussion 205-226 Author’s effort 227 Danksagung 228 Zusammenfassung Zusammenfassung Von Myxobakterien produzierte Naturstoffe weisen eine enorme strukturelle Vielfalt mit bemerkenswerten biologischen Aktivitäten auf. In diesem Kontext befasst sich die vorliegende Dissertation sowohl mit der Identifizierung und Charakterisierung von Naturstoff- Biosynthesewegen, als auch mit der Optimierung eines Produktionsstammes. Die Arbeiten zum Sekundärmetaboliten Crocacin zeigen, dass die eingehende Erforschung bisher unbekannter Biosynthesewege großes Potential zur Entdeckung beispielloser Aspekte von naturstoffproduzierenden Multienzymkomplexen birgt. Die erstmalige Beschreibung einer Kondensationsdomäne mit hydrolytischer Aktivität ebnet somit den Weg für Untersuchungen zum Mechanismus der Katalyse. Ein weiterer Teil dieser Arbeit befasste sich mit der vergleichenden Charakterisierung der Microsclerodermin Gencluster aus Sorangium cellulosum So ce38 und Jahnella sp. MSr9139. Hierbei konnten die strukturellen Unterschiede der einzelnen Derivate auf Unterschiede in den jeweiligen Genclustern zurückgeführt werden. Des Weiteren wurden terrestrische Produzenten des bisher nur aus marinen Schwämmen bekannten Naturstoffs Microsclerodermin D identifiziert. Im Rahmen der Optimierung des Ambruticinproduzenten S. cellulosum strain #8405 wurden mehrere Faktoren, die im Zusammenhang mit der Regulation der Biosynthese stehen, charakterisiert und modifiziert. Zudem konnten für diese Bakteriengattung neuartige genetische Methoden entwickelt und erfolgreich angewandt werden. 1 Abstract Abstract Natural products produced by myxobacteria exhibit an enormous structural diversity with noticeable biological activities. In this context, the present dissertation deals with both, the identification and characterization of natural product biosynthetic pathways and the optimization of a production strain. The work on the secondary metabolite crocacin illustrates that the detailed exploration of so far unknown biosynthetic pathways bears huge potential to reveal unprecedented aspects of multienzyme complexes which assemble natural products. The primal depiction of a condensation domain with hydrolytic activity consequently paves the way for investigations concerning the mechanism of catalysis. Another part of this work covers the comparative characterization of the microsclerodermin gene clusters from Sorangium cellulosum So ce38 und Jahnella sp. MSr9139. Hereby, the structural differences among the respective derivatives could be linked to differences in corresponding gene clusters. Furthermore, terrestrial producers of the natural product microsclerodermin D, previously only known from marine sponges, were identified. In the course of the optimization of the ambruticin producer S. cellulosum strain #8405 several factors connected to the regulation of the biosynthesis were characterized and modified. In addition novel genetic methods for this particular genus were developed and successfully applied. 2 Introduction Introduction Natural products: history, sources, applications, and future potentials Natural products or so called secondary metabolites are by definition low molecular weight molecules which are produced by a biological source [1]. The tremendous chemical diversity generated throughout millions of years of evolution truly accounts for the certainty that natural products have been one of the most important sources for potential drug leads [2;3]. Besides their therapeutic use as for example antibiotics, antitumor or immunosuppressive medications, natural products are used in agriculture, serving as insecticides, herbicides, or fungicides. The biosynthesis of natural products comes along with notable expenses for the producer’s metabolism, thus supporting the current notion that literally all such secondary metabolites have a certain purpose. As a consequence of the metabolic effort that is needed, biosynthesis of those metabolites is strictly regulated. In the microbial world, secondary metabolites are secreted in order to interfere with competitor organisms [4;5], to facilitate interspecies communication [6;7], or to coordinate developmental processes [8-11]. The utilization of natural products by mankind is not a concept of the modern times; in fact the documented use of herbals to cure diseases dates far back to the 3rd millennium BC. The earliest record thereof was found on clay tablets in cuneiform from Mesopotamia dating from around 2600 BC. Those relicts describe the use of oils from trees like cypress or cedar and herbs like licorice and myrrh to treat coughs, colds and inflammations [12]. As plants have been widespread and readily accessible during the centuries, many effective therapies originate from people experimenting with their local flora, aiming to search for a cure to fight different diseases. To date, among the 270,000 terrestrial plants that were taxonomically classified a number of approximately 30,000 is used in medicinal applications [13], with salicin from Salix alba L. (analgesic), morphine from Papaver somniferum (analgesic, sedative), and digoxin from Digitalis lanata (treatment of heart conditions) being only some of the most prominent ones. In line with this, fungi are as well a noticeable source of important natural products, although their actual potential has long remained undiscovered. With the more recent advantages in microbiology and the ability to isolate and grow fungi they were recognized as valuable suppliers of natural products including pharmaceutically relevant molecules [14]. Without doubt, the discovery of penicillin from the fungus Penicillium notatum made by Alexander Fleming in 1929 [15] together with subsequent observations of its broad therapeutic applicability by Ernst Boris Chain and Howard Walter Florey ushered a new era in medicine (The Golden Age of Antibiotics) and won them the Nobel prize in 1945 [16]. Besides penicillin, other important natural products derived from fungi are the cyclosporines from Tolypocladium inflatum (immunosuppressant), 3 Introduction asperlicin from Aspergillus alliaceus (cholecystokinin receptor CCK antagonist), and mevinolin from Aspergillus terreus (cholesterol-lowering agent). The discovery of penicillin promoted the extensive investigation of microorganisms with the aim to detect more natural products of potential medicinal interest. During this time, mainly soil- derived bacteria came into the focus of natural product research. In 1939, René J. Dubos together with Rollin D. Hotchkiss discovered gramicidin, an antimicrobial peptide produced by Bacillus brevis that exhibits activity against gram positive bacteria [17-19]. It was the very first antibiotic that was used in clinics and it gained certain importance in treating infected wounds of soldiers during World War II. In the following decades, the identification of active compounds from microbes progressed rapidly. This impressive development is illustrated best by numbers: In 1940 only 10-20, in 1950 300-400, in 1960 around 800-1,000, and by the year 1970 already 2,500 antibiotics were known [20]. Thereafter the amount of identified bioactive
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