Research on Secondary Metabolites
A more than 20-years period in the search for secondary metabolites from microorganisms provides the opportunity of looking back to the beginning of the research and summarizing an overview on the discovered and published novel natural compounds. First of all, I wish to acknowledge my appreciation of the outstanding work of my doctoral father and mentor Professor Hans Zähner, who founded in 1964 the Institute of Microbiology at the University of Tübingen. Both my diploma thesis in 1972 and my doctoral thesis in 1974 were performed under his supervision. The stimulating atmosphere in his huge team and the excellent technical facilities contributed mainly to the success of my own research. Hans Zähner’s retirement in 1994 was an encouragement to continue his fascinating scientific work in screening and discovery of novel biologically active secondary metabolites produced by new isolated members of the bacterial order Actinomycetales.
Looking back to the late 1980s, it was a promising decision to establish a natural product library based on high-performance liquid chromatography and diode-array monitoring (HPLC-DAD) with the aim to identify or to characterize secondary metabolite profiles of freshly isolated actinomycetes strains in culture filtrates and crude extracts. In 1984 the first fully automated HPLC systems equipped with a diode array monitoring system was introduced to the market. Hewlett-Packard (Waldbronn, Germany) was our main partner in optimizing hardware and software suitable for routine HPLC analysis. At the late 1980s, a first fast computing Pascal Workstation and hard disks having capacities of 10-20 MB (!) were available, even at a horrible price compared to the current market situation. This ’high-tech’ equipment permitted us to create an efficient data base on natural products which was built up in my group starting in 1988 (Fiedler, 1993). At this time no further coupling techniques with more efficient monitoring devices, e.g. mass spectrometry, were imaginable. Therefore, ortho-phosphoric acid was chosen as the most suitable acidic modifier added to the mobile phase, having optimal properties in separation selectivity and UV transmission. The separation was performed by a standardized linear gradient system from 0.1% aqueous ortho-phosphoric acid to 100% acetonitrile within 15 minutes. Nucleosil-100 C18 reversed-phase material (5-µm particles; Macherey & Nagel, Germany) was selected as stationary phase because of its less adsorptive properties, high resolution and stability of retention times. This system continued to work excellently today.
During the years 1988-1998 the main research of my group was sponsored by the Ministry of Research BMBF, who provided the fermentation and HPLC equipment. This period was the beginning of establishing my own group for isolating new actinomycetes strains from soil samples which were collected at various sites throughout the world, and resulted in the discovery of following new secondary metabolites in strains of the genus Streptomyces:
From a novel species of the genus Micromonospora, strain Tü 6368, the following new secondary metabolites were isolated and their structures elucidated (Antal et al., 2005; Ströch et al., 2005):
Within a scientific project funded by Novo Nordisk A/S, Denmark, specific attention was given to the actinomycete families Streptosporangiaceae and Micromonosporaceae. It was the beginning of a new area in the isolation and screening of less investigated actinomycete genera in my group with the aim to detect novel secondary metabolites. This work resulted in the discovery of:
BMBF and BASF AG (Ludwigshafen, Germany) funded a 6-year research project to investigate symbiotic microorganisms from arthropods as a source of novel bioactive secondary metabolites. The results were not very promising, but led to the discovery of the following novel antibiotics:
In addition, a multitude of partial novel natural products deriving from the primary metabolism were isolated and characterized, such as 3-hydroxyphenylacetic acid methylester, malonic acid phenylester N-acetyl-phenylalanine and N-acetyl-tryptophan.
A very stimulating and successful collaboration was started in 2000 together with the groups of Professor Michael Goodfellow from the University of Newcastle upon Tyne and Professor Alan T. Bull from the University of Kent (UK) that continued till my retirement in 2013. Both groups provided me with a huge series of dereplicated actinomycetes strains which were isolated from unique terrestrial and marine habitats including sediments of deep-sea trenches located in the Pacific and Atlantic Oceans. All strains were taxonomically characterised to the genus level that permitted an individual submerged cultivation in the screening stage, applying genus-adapted cultivation media. This collaboration resulted in the discovery of following metabolites:
A further series of novel secondary metabolites has been detected and isolated which are still under investigation.
The promising results coming out of the collaboration with Professor Goodfellow and Professor Bull was the reason that our consortium was funded by Boehringer Ingelheim Pharma (Biberach, Germany) in a 3-year scientific project starting from 2002 till 2004. The goal of the collaboration was to screen extracts from unique marine and some extremophilic terrestrial members of genera of the families Streptomycetaceae, Micromonosporaceae and Pseudonocardiaceae, and from selected members of mycolata as Nocardia, Rhodococcus, Gordonia and Tsukamurella for activities in various HTS assays. The first secondary metabolites coming out of this collaboration were:
Unfortunatelly, the Boehringer-Ingelheim Company decided in 2004 to drop their activities in natural products completely, and therefore, the planned second phase of the proposed project in metabolite isolation and structure elucidation of the numerous hit strains was not possible. Only a few of these strains could be investigated later on in my group by undergraduated students, resulting in following new metabolites:
Parallel to the NTK project sponsored by a pharmaceutical company, a further project was established with the aim to discover novel bioactive secondary metabolites from actinomycetes for clinical applications. Project ACTAPHARM was funded by the European Commission within the 5th framework program from 2001 till 2004, for a collaboration of the groups of Professor Liz Wellington (University of Warwick, UK), Professor Michael Goodfellow and Dr. Alan Ward (University of Newcastle, UK), Professor Lubbert Dujikhuizen (University of Groningen, The Netherlands), PD Dr. Joachim Vater (Technische Universität Berlin, Germany), Professor Amalia Karagouni (University of Athens, Greece), Dr. Flavia Marinelli (Biosearch Italia, Gerenzano, Italy), and Dr. Fotinos (Lavipharm Group, Athens, Greece) and my own group. Until today, following new secondary metabolites have been described:
A new research field was started in 2002 to investigate the interactions of soil streptomycetes with symbiotic fungi, pathogenic fungi and plants in the rhizosphere. The project was funded till 2010 by the Deutsche Forschungsgemeinschaft within the Graduate College ‘Infectious Biology’ at the University of Tübingen and was treated in close collaboration with the group of Professor Hampp, Institute of Physiological Ecology of Plants. The aim of our research was to characterize and identify the metabolites which act as signals in the communication between the different organisms. New metabolites coming out of this research were:
National and international activities with strains and secondary metabolites discovered by the HP Fiedler Group
Some of the discovered new secondary metabolites which showed an unusual structure or structural elements were attractive for various groups involved in the genetics of the biosynthetic pathways. The following metabolites were investigated regarding their biosynthetic gene cluster and mode of action:
The spirofungin producing strain Streptomyces violaceusniger Tü 4113 is investigared regarding volatiles in the group of Dr. Jeroen Dickschat, TU Braunschweig
Some of our secondary metabolites with interesting and unusual structural elements were subject for chemical total synthesis:
The production of microbial metabolites showing interesting and promising biological activities was optimized with the aim to increase the concentration per litre fermentation broth and to develop specific isolation techniques to obtain the products in a high amount of pure quality. These investigations were done mainly within doctoral theses under the guidance of the post-docs. Such drug candidates with an application potential were (i) nikkomycin Z, (ii) gallidermin, (iii) simocyclinone D8, and (iv) the microbial chelators EDDS and desferrioxamine E.
My special thanks are due to my doctoral students who were involved in the sceening for novel secondary metabolites and fermentation studies. Their names are in chronological order:
- Willi Kuhn (1993)
- Uwe Hartjen (1993)
- Uwe Pfefferle (1994)
- Sabine Blum (1995)
- Christoph Pfefferle (1997)
- Monika Richter (1997)
- Judith Schimana (1999)
- Markus Kempf (1999)
- Klaus Gebhardt (2001)
- Anke Dieter (2002)
- Marcelo Bertasso (2002)
- Noèmi Antal (2004)
- Julia Riedlinger (2006)
- Dirk Schulz (2009)
- Nadine Horlacher (2012)
- Marcell Wagner (2012)
Besides the doctoral students the following diploma students contributed to the progress in screening of secondary metabolites:
- Thomas Fritz, Jan Mirko Gutterer, Nicole Zwicker, Armin Maier, Antje Seidel, Miriam Langbecker, Elisabeth Zürn, Andreas Reicke, Tatiana Binder, Stefanie Baur, Grit Hanschel, Bettina Wieser, Andreas Gierth, Claudia Hohmann, Sonja Gern, Marcell Wagner, Philipp Jetter and Nicole Klüglein, Niko Manderscheid and Rebekka Jacek.
Without the support by my Post-Docs as the responsible persons, the various projects could not be realized. I appreciate in particular the work of Dr. Uwe Theobald, Dr. Johannes Müller, Dr. Judith Schimana and Dr. Christina Bruntner.
Last but not least, the assistance of the technical staff contributed greatly to the success of the research: Dip. Ing. Georg Grewe, Dipl. Ing. Andreas Kulik, Dipl. Ing. Jochen Schinko, Dipl. Biol. Barbara Holz, Mrs. Jutta Wachter, Mrs. Brigitte Maurer, Mr. Mulugeta Nega, Mr. Mete Bayraktar and Mr. Christian Tänzer.
National and international collaborations with collegues from various universities and chemical-pharmaceutical companies were essential in the screening of microbial strains and determining the biological activities and structures of the isolated metabolites.
Professor Dr. Michael Goodfellow and Professor Dr. Alan C. Ward from the University of Newcastle, Newcastle upon Tyne, UK, provided us with alkaliphilic, acidophilic and thermophilic actinomycetes strains from terrestrial soils and fresh water sediments collected at various sites in the UK. Halophilic actinomycetes from Egypt and marine actinomycetes isolated from sediments of deep sea trenches and various regions of the North Atlantic completed the spectrum of extreme habitats. Full taxonomic characterization was done in Newcastle in the case of antibiotic producing strains.
Professor Dr. Alan T. Bull from University of Kent, Canterbury, UK, provided us with marine actinomycetes from sediments of the Pacific and Atlantic Ocean including deep sea trenches, with terrestrial actinomycetes from Antarctica and with actinomycetes isolated from the Atacama Desert in Chile.
Professor Dr. Vikineswary from the University of Malaya, Kuala Lumpur, Malaysia, provided us with a huge collection of new isolated actinomycetes strains from pristine tropical habitats in Malaysia.
Dr. Patricia S.M. Sommer from University of Rio Grande do Norte, Natal, Brasil, provided us with actinomycetes strains which were isolated from Brazilian medicinal plants.
Professor Dr. Konrad Dettner from University of Bayreuth, Germany, provided us with endosymbiotic actinomycetes and fungi, which were isolated from the guts of a huge diversity of arthropods, especially insects and millipedes.
Professor Dr. Erko Stackebrandt and Professor Dr. Rainer-Michael Kroppenstedt supported the taxonomic characterization of new actinomycete isolates and in the specialist training of some of my doctoral students in taxonomical techniques.
With Dr. Ingrid Groth from Hans-Knöll-Institut für Naturstoff-Forschung, Jena, we had a fruitful long-term collaboration in taxonomic characterization of new isolated actinomycetes, resulting in various common publications.
Professor Dr. Rüdiger Hampp from the Department of Physiological Ecology of Plants and Professor Dr. Karl Poralla from the Institute of Microbiology of the University of Tübingen provided us with streptomycetes acting as mycorrhiza helper bacteria.
Without chemical characterization of the isolated metabolites the whole research in screening for new biologically active secondary metabolites would be only patchwork. Therefore, my deepest thanks are due to the groups of Professor Dr. Axel Zeeck, Dr. Jürgen Rohr and Dr. Stephanie Grond and their teams from the Organic Chemistry Department of the University of Göttingen – Professor Stephanie Grond moved recently to the Organic Chemistry Department, University of Tübingen –, Professor Dr. Günther Jung and his team from the Organic Chemistry Department of the University of Tübingen, Professor Dr. Gerhard Bringmann and his team from the Organic Chemistry Department of the University of Würzburg, Dr. Thomas Paululat from the Organic Chemistry Department of the University of Siegen, and last but not least my friend Professor Dr. Roderich Süssmuth from the Chemical Institute of the Technical University of Berlin, who started his career in Tübingen and moved in 2004 to Berlin. Roderich and his team of diploma and doctoral students were mainly involved during the last decade in structural elucidation of our isolated secondary metabolites. Without their enthusiasm for natural products our research could not be realized. But not only chemists from the various universities, also chemists from chemical-pharmaceutical companies contributed to the success of my group, as Dr. Jens Breinholt from Novo Nordisk, Denmark, Dr. Olivier Potterat and Dr. Carsten Puder from Boehringer-Ingelheim Pharma, Biberach, Germany.
Due to the limited facilities in determining the biological activities of the isolated new secondary metabolites, the collaboration in biological assays has been an essential part in my work. Professor Dr. Winfried Beil from the Medizinische Hochschule Hannover, Germany, performed antitumor assays to investigate activities against various human tumor cell lines. Professor Dr. Reto Brun from the Swiss Tropical Institute, Basel, took the part in screening for antiparasitic activities against various protozoan pathogens. Professor Dr. Johannes Imhoff from Leibnitz-Institut für Meereswissenschaften IFM-Geomar, Kiel, participates with testing our compounds in extended antibacterial, antifungal, antitumor and enzymatic assays.
Without financial support no reaserch would be possible. Therefore, my warmest thanks are due to following organisations and companies:
- The Deutsche Forschungsgemeinschaft contributed with a basic support for fermentation and HPLC equipment within SFB 323 during the years 1988-99.
- The Bundesministerium für Forschung und Technologie (BMFT) Germany, supported our research in the optimisation of the biotechnological manufacturing of secondary metabolites within the ‘Zentrales Schwerpunktprojekt Bioverfahrenstechnik’ from 1988-98.
- Novo Nordisk A/S, Bagsværd, Denmark, supported from 1995-98 our research in secondary metabolites from the families Streptosporangiaceae and Micomono¬sporaceae.
- The Bundesministerium für Bildung, Wissenschaft, Forschung und Technologie (BMBF) Germany together with BASF AG, Ludwigshafen, Germany, supported our research in secondary metabolites from endosymbiotic actinomycetes over a 6-year period from 1996-2001.
- The European Commission supported our research within the 5th framework, project ACTAPHARM in the screening for new bioactive secondary metabolites from various actinomycetes genera from 2001-2004.
- Boehringer Ingelheim Pharma GmbH, Biberach, Germany, supported our research in secondary metabolites from marine actinomycetes from 2002-2004.
- The Deutsche Forschungsgemeinschaft contributed from 2002-2011 within ’Graduiertenkolleg Infektionsbiologie’ with the support of three doctoral theses to our research on the interaction of soil streptomycetes, mycorrhization and plants.